formulation and evaluation of controlled release ...i, b.jaykar certify that the thesis entitled...
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FORMULATION AND EVALUATION OF CONTROLLED RELEASE MICROSPHERES CONTAINING SELECTED
ACID-RESISTANCE POLYMERS
Thesis submitted in partial fulfillment for the award of
DEGREE OF DOCTOR OF PHILOSOPHY In
Pharmacy By
A.PASUPATHI, M.Pharm.,
Under the Guidance of
Prof. Dr. B. JAYKAR, M.Pharm, Ph.D.,
VINAYAKA MISSIONS UNIVERSITY SALEM-636 308
TAMIL NADU, INDIA
JULY - 2016
I, B.JAYKAR certify that the thesis entitled FORMULATION AND
EVALUATION OF CONTROLLED RELEASE MICROSPHERES
CONTAINING SELECTED ACID-RESISTANCE POLYMERS submitted
for the award of Degree of Doctor of Philosophy by Mr. A.Pasupathi is
the record of research work carried out by him during the period from
January 2011 to July 2016 under my guidance and supervision and this
work has not formed the basis for the award of any other degree,
diploma, associateship, fellowship or any other titles in this university or
any other university or Institution of higher learning.
Place: Prof. Dr. B. Jaykar,
Date: Principal, Vinayaka Mission’s College of Pharmacy, Salem.
CERTIFICATE
I, A.PASUPATHI declare that the thesis entitled FORMULATION
AND EVALUATION OF CONTROLLED RELEASE MICROSPHERES
CONTAINING SELECTED ACID-RESISTANCE POLYMERS submitted
by me for the award of Degree of Doctor of Philosophy is the record of
research work carried out by me during the period from January 2011 to July
2016 under the guidance of Prof.Dr.B.Jaykar, M.Pharm, Ph.D., Principal,
Vinayaka Mission’s College of Pharmacy, Salem and this work has not formed
the basis for the award of any other degree, diploma, associateship, fellowship
or any other titles in this university or any other university or Institution of
higher learning.
Place:
Date: A.Pasupathi
DECLARATION
On the occasion of presenting this thesis, I express deep gratitude,
Sincere and heartful thanks to my research guide Prof.Dr.B.Jaykar,
M.Pharm,Ph.D., principal, Vinayaka Mission’s College of Pharmacy, who
have provided excellent guidance, knowledge on the subjects and regular
observation. He gave me valuable advices, support and shared intelligent
thoughts, Inculcated discipline and encouraged me in every step to complete
my thesis work. I am highly indebted to him for his valuable suggestion and
guidance from his busy schedule, which helped me to complete this work
successfully.
All praise to ‘Almighty’ who enabled me to accomplish this research
work. I am highly obliged to Prof.Dr.B.S.Venkateswarlu, M.Pharm, Ph.D.,
Head, Department of Pharmaceutics, Vinayaka Mission’s College of
Pharmacy, Salem for his kind guidance, deep inspiration and co-operative
nature with due respect in my heart. I thank for his never ending willingness to
render generous help whenever needed.
I express my profound and sincere gratitude to Prof.Dr.R.Margret
Chandra, M.Pharm,Ph.D., Professor, Department of Pharmaceutics for their
valuable help throughout this research work.
ACKNOWLEDGEMENT
I render my sincere thanks to Mr.V.Muruganantham,M.Pharm.,
Mr.R.Saravanan,M.Pharm., Mr.G.R.Vijaya Sankar, M.Pharm., Asst.Professors
and Mr.P.Palanisamy, M.Pharm., Mr.K.Somu, B.Pharm, M.B.A.,
Mr.S.Gunasekaran, B.Pharm., Lecturers, Department of Pharmaceutics for
their valuable support throughout this research work.
I am very much grateful and thankful to Dr.A.Nallathambi, M.L.I.S.,
Librarian for providing the most necessary materials and journals.
It is my pleasure to express my deep sense of gratitude and
thankfulness to my beloved parents, my beloved wife and my daughter who
always covered me under the shade of their love and blessing for their
valuable moral support directly or indirectly, the spirit and cooperation for the
timely completion of my research work.
The journey towards achievement of Ph.D Degree is obviously not
possible without the personal support of my friends and family members.
A.Pasupathi
S. NO. TITLE PAGE
NO.
1 INTRODUCTION 1
1.1 Oral route for drug delivery system 1
1.2 Controlled drug delivery system 3
1.3 Targeted drug delivery system 7
1.4 Microspheres as drug delivery carrier 35
2 AIM AND OBJECTIVE OF PRESENT STUDY 60
3 REVIEW OF LITERATURES 63
4 DRUG AND EXCIPIENTS PROFILE 143
4.1 Drug profile: Balsalazide 143
4.2 Excipients profile 154
5 EXPERIMENTS 179
5.1 Plan of work 179
5.2 Chemicals, reagents and equipments 181
5.3 Experimental Methods 184
5.3.1 Standard curve of Balsalazide using UV
Spectroscopy
184
CONTENTS
S. NO. TITLE PAGE
NO.
5.3.2 Drug polymer interaction 184
5.3.2.1 FTIR study 185
5.3.2.2 Differential scanning calorimetry
(DSC)
185
5.3.3 Preparation of ALG-CHI PEC microspheres 186
5.3.4 Characterization of PEC microspheres 189
5.3.4.1 Micromeritic properties 189
5.3.4.2 General characterization of PEC
microspheres
192
5.3.4.3 In vitro drug release study of
microspheres
196
5.3.4.4 Mathematical modeling of drug
release profile
197
5.3.4.5 Stability studies of microspheres 200
5.3.5 Preparation of enteric coating solution 200
5.3.6 Enteric coating of selected batch M6 201
5.3.7 Preparation of matrix tablets of enteric coated
microspheres
201
5.3.7.1 Sieve analysis of prepared granules 202
S. NO. TITLE PAGE
NO.
5.3.7.2 Preparation of tablets 203
5.3.8 Characterization of matrix tablets 203
5.3.8.1 General evaluation of tablets 203
5.3.8.2 In vitro dissolution studies of tablets 206
5.3.8.3 Stability studies of tablets 208
5.3.9 Statistical analysis 208
6 RESULTS AND DISCUSSION 210
6.1 Standard curve of Balsalazide 210
6.2 Drug Polymer Interaction 211
6.2.1 FTIR spectra 212
6.2.2 DSC 214
6.3 Preparation of microspheres 215
6.4 Comparative characterization of microspheres 216
6.4.1 Micromeritic characterization 217
6.4.2 General characterization of microspheres 223
6.4.3 In vitro drug release studies of microspheres 232
6.4.4 Stability analysis for selected batch (M6) 247
S. NO. TITLE PAGE
NO.
6.5 Preparation of tablet with enteric coated
microspheres (M6)
249
6.5.1 Enteric coating of microspheres 250
6.5.2 Sieve analysis of granules 250
6.5.3 Preparation of matrix tablet of enteric coated
microspheres
252
6.6 Evaluation of tablet of enteric coated microspheres 254
6.6.1 General evaluation of enteric coated tablets 254
6.6.2 Stability analysis of enteric coated tablets 263
7 SUMMARY AND CONCLUSION 266
8 BIBLIOGRAPHY 270
LIST OF TABLES
S. NO. TITLE PAGE
NO.
1 List of drug and chemicals used 181-182
2 List of equipments used 182-183
3 Different batches of PEC microspheres 188
4 Data for plot of standard curve of Balsalazide 210
5 Micromeritic properties of microspheres 221
6 General characterization of microspheres 226
7A In vitro drug release profile of microspheres 233-234
7B Data for zero order plot (Cumul. Quantity
released Vs Time) 237
7C Data for First order plot (Log Cumul. Drug
Remained Vs Log Time) 240
7D Data for Higuchi plot (Cum. Drug Released Vs.
Square root of Time) 242
7E Data for Korsemeyer-Peppas plot (Log Cumul.
Drug Released Vs Log Time) 244
8 Kinetic interpretation of drug release data 246
S. NO. TITLE PAGE
NO.
9 Stability analysis of selected batch of
microspheres (M6) 248
10 Sieve analysis of granules 251
11 General evaluation of tablet 255
12 Dissolution study of tablets of enteric coated
microspheres (M6) 258
13 Kinetic analysis of tablet drug release data 260
14 Stability analysis of enteric coated microsphere
tablet 264
LIST OF FIGURES
S. NO. TITLE PAGE
NO.
1 Anatomy of colon 12
2 Structural formula of Balsalazide disodium 143
3 Structural formula of Chitosan 155
4 Structural formula of Sodium alginate 160
5 Structural formula of Eudragit S100 164
6 Structural formula of SPAN 80 167
7 Structural formula of HPMC 168
8 Structural formula of Lactose monohydrate 171
9 Structural formula of Micro crystalline cellulose 172
10 Structural formula of Magnesium stearate 174
11 Standard curve of Balsalazide 211
12 FTIR Spectra of Drug, Polymers and their
combination as PEC complex 213
13 DSC Thermogram of Drug, Polymers and PEC
complex with drug 214
S. NO. TITLE PAGE
NO.
14 SEM of prepared batches of microspheres with an
enlarged image of selected batch M6 231
15 Plot of In vitro drug release profile of different
batches of microspheres 235
15A In vitro drug release profile of microspheres (zero
order plot) 238
16 In vitro drug release profile of microspheres (First
order plot) 241
17 In vitro drug release profile of microspheres (Higuchi
plot) 243
18 In vitro drug release profile of microspheres
(Korsemeyer- Peppas plot) 245
19 SEM of Eudragit S100 coated microspheres (M6) 250
20 Plot for sieve analysis of granules 252
21 Dissolution rate analysis of tablet of uncoated and
enteric coated microspheres 261
ABBREVIATION
S.No Abbreviation Expanded form
1 DDS Drug delivery system
2 5-ASA 5 Amino salicylic acid
3 IBD Inflammatory bowel disease
4 UC Ulcerative colitis
5 ALG Sodium alginate
6 CM Chitosan microspheres
7 AM Alginate microspheres
8 PEC Polyeletrolyte complex
9 HPMC Hydroxy propyl methyl cellulose
10 PEG Poly ethylene glycol
11 PLA Polylactic acid
12 API Active pharmaceutical ingredients
13 HIV Human immunodeficiency virus
14 FTIR Fourier transform Infra-red
15 SEM Scanning electron microscopy
S.No Abbreviation Expanded form
16 PLGA Poly (lactic acid co glycolic acid)
17 GIT Gastro intestinal tract
18 HPMCP HPMC phthalate
19 IPA Isopropyl alcohol
20 DCM Dichloromethane
21 BSA Bovine serum albumin
22 SGF Simulated gastric fluid
23 SIF Simulated intestinal fluid
24 SCF Simulated colonic fluid
25 ICH International conference on harmonization
26 RNA Ribo nucleic acid
27 DSC Differential scanning calorimetry
28 DNA Deoxyribonucleic acid
29 RH Relative humidity
30 UV Ultraviolet
S.No Abbreviation Expanded form
31 AUC Area under the curve
32 FDA Food and drug administration
33 MCC Micro crystalline cellulose
34 USP-NF United states pharmacopoeia-national
formularies
35 BP British pharmacopoeia
36 JP Japanese pharmacopoeia
37 CTDDS Colon targeted drug delivery system
38 et al., and coworkers
39 Hrs Hours
40 Fig. Figure
1
1. INTRODUCTION
1.1. Oral route for drug delivery system
With gradual advancement detected in the field of
biopharmaceutics, several useful corners have been evolved for
discussion on designing and fabrication of drug delivery systems.
Several useful information collected upto date directed modern
research to have accuracy and rationality with sufficing every possible
need of pharmaceutical technology. Dosage form development has
rendered some new useful aspects of reliable drug carrier system with
their conventionally popular counterpart. Of several developed drug
administration methods, oral route has found its way to prove potential
convenience to offer the greatest potential for more effective
therapeutics, but they do not facilitate drug that easily cross mucosal
surfaces and biological membranes; they are easily denatured or
degraded, prone to rapid clearance in the liver and other body tissues
and require precise dosing. At present, susceptible drugs are usually
administered by injection but this route is less pleasant and also poses
problems of oscillating blood drug concentrations. Despite the barriers
for successful drug delivery that exist in the gastrointestinal tract (such
as acid-induced hydrolysis in the stomach, enzymatic degradation
throughout the gastrointestinal tract by several proteolytic enzymes,
2
bacterial fermentation in the colons), the oral route is still the most
intensively investigated as it offers advantages of convenience and
cheapness of administration, and potential manufacturing cost
savings. The design of oral control drug delivery systems (DDS)
should be primarily aimed to achieve more predictable and increased
bioavailability [Chawla et al., 2003]. Historically, oral drug
administration has been recognized as the predominant route for drug
delivery. During the past two decades, numerous oral delivery systems
have been developed to act as drug reservoirs from which the active
substance can be released over a defined period of time at a
predetermined and controlled rate. From a pharmacokinetic point of
view, the ideal sustained and controlled release dosage form should
be comparable with an intravenous infusion, which supplies
continuously the amount of drug needed to maintain constant plasma
levels once the steady state is reached. Nowadays most of the
pharmaceutical scientists sre involved in developing the ideal system
that should have advantage of single dose for the whole duration of
treatment and it should deliver the active drug directly at the specific
site. Scientists have succeeded to develop a system and it
encourages the scientists to develop control release systems. Control
release implies the predictability and reproducibility to control the drug
3
release, drug concentration in target tissues and optimization of the
therapeutic effect of a drug by controlling its release in the body with
lower and less frequent dose [Shivkumar et al., 2004]. However the
oral route of drug administration presents its own unique set of
problems and constraints. The time frame, or “window,” for absorption
is limited to the total GI residence time. Taking into account gastric
emptying and small and large intestine transit time, it would seem that
a reasonable duration in the GI tract is approximately 24 hours. The
absorption, distribution and elimination of drugs are normally simplified
by considering them all to be simple first-order processes. Given the
average 24-hour residence time and high individual variability in the GI
tract, only drugs with relatively short elimination half-lives should be
considered for membrane-controlled reservoir systems.
1.2. Controlled drug delivery system
Controlled drug release and subsequent biodegradation are
important for developing successful formulations of targeted and/or
controlled drug delivery system. The principles, theories and devices
in chemical engineering can be modified and further developed to
meet the challenges in the design of drug delivery systems. Therefore,
controlled drug delivery can become a major possibility for chemical
engineering to make significant contributions to human health care
4
[Raval et al., 2010]. Novel approach for drug delivery is the method by
which a drug is delivered can have a significant effect on its efficacy.
Some drugs have an optimum concentration range within which
maximum benefit is achieved and concentrations above or below this
range can be toxic or produce no therapeutic benefit at all, on the
other hand, the very slow progress in the efficacy of the treatment of
severe diseases, has suggested a growing need for a multidisciplinary
approach to the delivery of therapeutics to targets in tissues. From
this, new ideas on controlling the pharmacokinetics,
pharmacodynamics, non-specific toxicity, immunogenicity,
biorecognition and efficacy of drugs were generated. These new
strategies, often called drug delivery systems (DDS), are based on
interdisciplinary approaches that combine polymer science,
pharmaceutics, bioconjugate chemistry, and molecular biology. Novel
drug delivery system uses both physical and biochemical mechanism
[Vyas and Khar, 2008].
Controlled-release dosage forms are gaining rapid popularity in
clinical medicine. The more sophisticated systems are used to alter
the pharmacokinetic behavior of drugs in order to provide twice- or
once-a-day dosage. Other applications include enteric coatings for the
protection of drugs from degradation within the GI tract or the
5
protection of the stomach from the irritating effects of the drug, and the
delivery of drugs to absorption windows or specific targets within the
GI tract, particularly the colon. Potential release mechanisms involve:
(i) desorption of surface-bound /adsorbed drugs; (ii) diffusion through
the carrier matrix; (iii) diffusion through the carrier wall; (iv) carrier
matrix erosion and (v) a combined erosion /diffusion process. The
mode of delivery can be the difference between a drug’s success and
failure, as the choice of a drug is often influenced by the way the
medicine is administered[Kopecek,2003].All these mechanisms
employ physical transformation of constituents involved in the system
when they are put into a biological environment. Although there are
feasible chemically driven drug delivery systems, they involve
chemical modifications with active agents and carrier vehicles for
which regulatory approval and adequate toxicology and safety profiles
are needed before reaching final application. For such reasons,
simpler systems with approved active agents and excipients are often
utilized in the preparation of the controlled drug delivery systems used
for medical applications. Sustained (or continuous) release of a drug
involves polymers that release the drug at a controlled rate due to
diffusion, out of the polymer or by degradation of the polymer over
time. Pulsatile release is often the preferred method of drug delivery,
6
as it closely mimics the way by which the body naturally produces
hormones such as insulin. It is achieved by using drug-carrying
polymers that respond to specific stimuli (e.g., exposure to light,
changes in pH or temperature) [Torchilin, 2001]. Mathematical
modeling of the release kinetics of specific classes of controlled-
release systems may be used to predict solute release rates from and
solute diffusion behavior through polymers and to elucidate the
physical mechanisms of solute transport by simply comparing the
release data to mathematical models.The development of polymeric
controlled release systems introduced a new concept in drug
administration to treat numerous diseases.The purpose of controlled
release systems is to maintain an adequate drug concentration in the
blood or in target tissues at a desired value as long as possible and,
for this, they are able to control drug release rate [Langer, 1990 &
Pillai et al., 2001]. These systems are less complicated with high
stability. Biodegradable polymers have been used in controlled drug
delivery for many years as a means of prolonging the action of
therapeutic agents in the body. Oral controlled release formulations for
the small intestine and colon have received considerable attention in
the past 25 years for a variety of reasons including Pharmaceutical
superiority and clinical benefits derived from the drug release pattern
7
that are not achieved with traditional immediate or sustained release
products[Banker ,2002].
1.3. Targeted drug delivery system
Targeted drug delivery is an advanced method of delivering
drugs to the patients in such a targeted sequences that increases the
concentration of delivered drug to the targeted body part of interest
only (organs/tissues/ cells) which in turn improves efficacy of treatment
by reducing side effects of drug administration. Basically, targeted
drug delivery is to assist the drug molecule to reach preferably to the
desired site to direct the drug loaded system to the site of interest.
Thus targeted drug delivery is a method of delivering medication to a
patient in a manner that increases the concentration of the medication
in some parts of the body relative to others [Vyas and Khar, 2008].
Targeted drug delivery system is preferred over conventional drug
delivery systems due to three main reasons. The first being
pharmaceutical reason conventional drugs have low solubility and
more drug instability in comparison to targeted drug delivery systems.
Secondly conventional drugs also have poor absorption, shorter half-
life and require large volume of distribution. These constitute its
pharmacokinetic properties. The third reason constitutes the
pharmacodynamic properties of drugs. The conventional drug delivery
8
systems have low specificity and low therapeutic index as compared to
targeted drug delivery system. The targeted or site- specific delivery of
drugs is indeed a very attractive goal because this provides one of the
most potential ways to improve the therapeutic index of the drugs. To
minimize drug degradation and loss, to prevent harmful side-effects
and to increase drug bioavailability and the fraction of the drug
accumulated in the required zone, various drug delivery and drug
targeting systems are currently under development [Rani and Paliwal ,
2014]. Targeted drug delivery seeks to concentrate the medication in
the tissues of interest while reducing the relative concentration of the
medication in the remaining tissues. This improves efficacy of the drug
while reducing side effects. Drug targeting is the delivery of drugs to
receptors or organs or any other specific part of the body to which one
wishes to deliver the drugs exclusively [Gupta and Sharma, 2011].
Targeting is the ability to direct the drug-loaded system to the site of
interest. Two major mechanisms can be distinguished for addressing
the desired sites for drug release: (i) passive and (ii) active targeting. A
strategy that could allow active targeting involves the surface
functionalization of drug carriers with ligands that are selectively
recognized by receptors on the surface of the cells of interest as
second order targeting whereas for third order intracellular molecules
9
are more specifically targeted [Kannagi et al., 2004]. Passive targeting
refers to the accumulation of drug or drug carrier system at a specific
site such as anti-cancerous drug whose explanation may be attributed
to physicochemical or pharmacological factors of the disease. An
example of passive targeting is the preferential accumulation of
chemotherapeutic agents in solid tumors as a result of the enhanced
vascular permeability of tumor tissues compared with healthy tissue.
Since ligand–receptor interactions can be highly selective, this could
allow a more precise targeting of the site of interest [Gref et al., 1994].
Targeted drug delivery is a kind of smart drug delivery system
which is miraculous in delivering the drug to a patient. This
conventional drug delivery system is done by the absorption of the
drug across a biological membrane, whereas the targeted release
system is that drug is released in a dosage form [Allen and Cullis,
2004]. Carrier is one of the special molecule or system essentially
required for effective transportation of loaded drug up to the pre-
selected sites. These are engineered vectors which retain drug inside
or onto them either via encapsulation and/ or via spacer moiety and
transport or deliver it into vicinity of target cell [Gujral and Khatri,
2013].Drug action can be improved by developing new drug delivery
system, such as the mucoadhesive microsphere drug delivery system.
10
These systems remain in close contact with the absorption tissue, the
mucous membrane, releasing the drug at the action site leading to a
bioavailability increase and both local and systemic effects The oral
route of drug administration constitutes the most convenient and
preferred means of drug delivery to systemic circulation [Carvalho et
al., 2010].
1.3.1. Colon targeted drug delivery system
1.3.1.1. Colon: Anatomy and Physiology
The entire colon is about 5 feet (150 cm) long, and is divided in
to five major segments. Peritoneal folds called as mesentery which is
supported by ascending and descending colon. The right colon
consists of the cecum, ascending colon, hepatic flexure and the right
half of the transverse colon.
The left colon contains the left half of the transverse colon,
descending colon, splenic flexure and sigmoid. The rectum is the last
anatomic segment before the anus (Fig. 1). Histologically the colon
can be divided into four layers: mucosa, submucosa, muscularis
externa and serosa. The mucosa is composed by the epithelium,
lamina propria and muscularis mucosae.It has a simple columnar
epithelium shaped into straight tubular crypts, which are short
invaginations of mucosal epithelium and provide protected pockets for
11
special cellular functions. There are no villi. In cellular composition, the
epithelium resembles that of the small intestine, but with a higher
proportion of goblet cells interspersed among the absorptive cells
(enterocytes). Goblet cells are specialized for secretion of mucus,
which facilitates passage of material through the bowel, while
enterocytes are specialized for absorption of nutrients across the
apical plasma membrane and export of these same nutrients across
the basal plasma membrane. The crypt epithelium also includes stem
cells which replenish the epithelium every few days, enteroendocrine
cells, and Paneth cells (secretory epithelial cells located at the ends of
intestinal crypts. The function for these cells is secretion of anti-
bacterial proteins into the crypt lumen, thereby providing protection for
the stem cells which line the crypt walls). The crypts are separated by
conspicuous lamina propria, the loose connective tissue in a mucosa.
Lamina propria supports the delicate mucosal epithelium, allows the
epithelium to move freely with respect to deeper structures, and
provides for immune defense, it is composed by connective tissue
infiltrated by many white blood cells, with capillaries and thin strands
of smooth muscle. The muscularis mucosa of the lower tract forms a
thin layer (only a few muscle fibers in thickness) beneath the deep
ends of the crypts. The submucosa is a connective tissue layer deep
12
to and supporting the mucosa. The muscularis externa of the colon
has the standard layers of inner circular and outer longitudinal smooth
muscle. The outer layer of the colon is a serosa attached to
mesentery, ordinary connective tissue with a surface of mesothelium.
The major function of the colon is the creation of suitable environment
for the growth of colonic microorganisms, storage reservoir of faecal
contents, expulsion of the contents of the colon at an appropriate time
and absorption of potassium and water from the lumen [Bajpai et al.,
2003].
Fig. 1: Anatomy of Colon
13
1.3.1.2. Colon Diseases and treatments
Site specific drug delivery to the colon is important for the
treatment of diseases associated with the colon, reducing the side
effects of the drug and reducing the administered dose. The most
important colon-associated diseases are: inflammatory bowel disease
(Crohn’s disease and ulcerative colitis), colon cancer, irritable bowel
syndrome, diverticulitis and amoebiasis of which inflammatory bowel
disease remains most important matter of concern for present study.
The term inflammatory bowel disease (IBD) covers a group of
disorders in which the intestine become inflamed, probably as a result
of an immune reaction of the body against its own intestinal tissue.
Two major types of IBD have been described: ulcerative colitis (UC)
and Crohn's disease (CD). As the name suggests, ulcerative colitis is
limited to the colon (large intestine), although Crohn's disease can
involve any part of the gastrointestinal tract from the mouth to the
anus; it most commonly affects the small intestine and/or the colon
[Hibi and Ogata, 2006]. Because inflammatory bowel disease is a
chronic disease (lasting a long time), it goes through periods in which
the disease flares up and is considered to be in an active stage and
severe inflammation; these periods are followed by remission, in which
14
symptoms disappear or decrease and normal conditions return .
Symptoms may range from mild to severe and generally depend upon
the part of the intestinal tract involved. They include the following:
abdominal cramps and pain, bloody diarrhea, severe urgency to have
a bowel movement, fever, loss of appetite, weight loss, anemia (due to
blood loss). Researchers do not yet know what causes inflammatory
bowel disease [Strober et al., 2007]. Therefore, IBD is called an
idiopathic disease (disease with an unknown cause). An unknown
factor/agent (or a combination of factors) triggers the body’s immune
system to produce an inflammatory reaction in the intestinal tract that
continues without control. As a result of the inflammatory reaction, the
intestinal wall is damaged leading to bloody diarrhea and abdominal
pain. Genetic, infectious, immunologic, and psychological factors have
all been implicated in influencing the development of IBD. There is a
genetic predisposition (or perhaps susceptibility) to the development of
IBD. However, the triggering factor for activation of the body’s immune
system has yet to be identified. Factors that can turn on the body’s
immune system include an infectious agent, an immune response to
an antigen, or an autoimmune process. Genetic susceptibility is
influenced by the luminal microbiota, which provides antigens and
adjuvants that stimulate either pathogenic or protective immune
15
responses. Environmental triggers are necessary to initiate or
reactivate disease expression. In inflammatory bowel disease, the
well-controlled balance of the intestinal immune system is disturbed
[Xavier and Podolsky, 2007].
Standard treatment for ulcerative colitis depends on extent of
involvement and disease severity. The goal is to induce remission
initially with medications, followed by the administration of
maintenance medications to prevent a relapse of the disease.
Aminosalicylate, corticosteroids, immunosuppressive drugs and TNF
inhibitors are commonly used in the treatment of IBD levels [Baumgart
and Sandborn, 2007].
Aminosalicylate:
5-ASA compounds (mesalazine, osalazine, sulfasalazine,
balsalazide) [Campieri, 2002] have been shown to be useful in the
treatment of mild-to-moderate Crohn's disease and ulcerative colitis
and as maintenance therapy.
I. Corticosteroids:
They are a class of anti-inflammatory drug that are used
primarily for treatment of moderate to severe IBD. The most commonly
16
prescribed oral steroid is prednisone, but the following corticosteroids
are also used as immune system suppressants in treatment of
ulcerative colitis: cortisone, hydrocortisone and budesonide [Xu et al.,
2004].
II. Immunosuppressive drugs:
They inhibit the immune system generally. These include the
cytostatic drugs that inhibit cell division, including the cloning of white
blood cells that is a part of the immune response. Immunosuppressive
drugs used with ulcerative colitis include: mercaptopurine (6-MP, it is a
cytostatic drug that is an antimetabolite, it mimics purine, which is
necessary for the synthesis of DNA, with mercaptopurine present,
cells are not able to make DNA, and cell division is inhibited);
azathioprine (which metabolises to 6-MP) and methotrexate (which
inhibits folic acid) [Choi andTargan, 1994].
III. TNF inhibitors:
They are monoclonal antibodies that inhibit the proinfla-mmatory
cytokine tumour necrosis factor (TNF). The most important are
infliximab and adalimubab [Noble et al., 2008].
17
IV. Antibiotics:
Metronidazole and ciprofloxacin are antibiotics which are used to
treat IBD. They are also used for treatment of complications, including
abscesses and other infections.
5-Aminosalicylic acid (5-ASA), also known as mesalazine or
mesalamine, is an anti-inflammatory drug used to treat inflammation of
the digestive tract, ulcerative colitis and Crohn's disease (Inflammatory
Bowel Disease, IBD). It is a bowel specific aminosalicylate drug that
acts locally in the gut and has its predominant actions there. Blockage
of the lipooxygenase pathway has also been shown [Stenson, 1990]
inhibiting both 5-lipooxygenase and 5-lipooxygenase-activating
protein. It is also one of the most potent known free radical scavengers
and antioxidants [McKenzie et al., 1999]. Many of the effects of 5-ASA
may also be explained by inhibition of activation of nuclear factor-αB
(NF-αB), a central transcription regulatory factor involved in mediating
the initiation and perpetuation of inflammatory processes [Punchard et
al.,1992]. Activated NF-αB has been detected in macrophages and
epithelial cells in inflamed mucosa from Crohn’s disease and
ulcerative colitis. 5-ASA is rapidly and completely absorbed from the
upper intestine when administered orally, [Zhou et al., 1999].
18
1.3.1.3. Colon microflora: Role in metabolism
The absorptive capacity is very high, each about 2000ml of fluid
enters the colon through the ileocecal valve from which more than
90% of the fluid is absorbed. On average, it has been estimated that
colon contains only about 220 gm of wet material equivalent to just 35
gm of dry matter. The majority of this dry matter is bacteria. About 400
bacterial species have been found in the colon and some fungi. The
important bacteria present in the colon such as Bacteroides,
Bifidobacterium, Eubacterium, Peptococcus, Lactobacillus, Clostridium
secrete a wide range of reductive and hydrolytic enzymes such as β-
glucuronidase, β-xylosidase, β-galactosidase, α-arabinosidase,
nitroreductase, azoreductase, deaminase and urea hydroxylase.
These enzymes are responsible for degradation of di-, tri- and
polysaccharides [Sinha and Rachna, 2003]. In the small intestine, the
microflora is mainly aerobic, but in the large intestine it is anaerobic.
Most bacteria inhabit in the proximal areas of the large intestine,
where energy sources are greatest. This resident microflora could also
affect colonic performance via metabolic degradation of the drug. The
presence of colonic microflora has formed a basis for development of
colon-specific drug delivery systems [Wang et al., 1993]. The resident
microflora could also affect colonic performance via metabolic
19
degradation of the drug. Interest has focused primarily on azo
reduction and hydrolysis of glycoside bonds. However, the colonic
microflora varies substantially between and within individuals,
reflecting diet, age and disease [Ibekwe et al., 2008].
1.3.1.4. Colon specific Drug delivery approaches
In the stomach pH ranges between 1 and 2 during fasting but
increases after meal. The pH is about 6.5 in the proximal small
intestine and about 7.5 in the distal small intestine. From the ileum to
colon, pH declines significantly. It is about 6.4 in the caecum.
However, pH values as low as 5.7 have been measured in the
ascending colon in healthy volunteers. The pH in the transverse colon
is 6.6, in the descending colon 7.0. Use of pH dependent polymers is
based on these differences in pH levels [Sangalli et al., 2001]. The
polymers described as pH-dependent in colon specific drug delivery
are insoluble at low pH levels but become increasingly soluble as pH
rises. There are various problems with this approach. The pH in the
gastrointestinal tract varies between and within individuals. In
ulcerative colitis pH values between 2.3 and 4.7 have been measured
in the proximal parts of the colon. Although a pH dependent polymer
can protect a formulation in the stomach and proximal small intestine,
Very common physiological factor which is considered in the design of
20
delayed release colonic formulations is pH gradient of the
gastrointestinal tract. Lower surface area and relative tightness of the
junctions in the colon can also restrict drug transport across the
mucosa and in the systemic circulation [Kumar et al., 2010]. In normal
healthy subjects, there is a progressive increase in luminal pH from
the duodenum (pH is 6.6±0.5) to the end of themileum (pH is 7.5 ±
0.4), a decrease in the cecum (pH is 6.4± 0.4) and then a slow rise
from the right to the left colon with a final value of 7.0 ± 0.7. Some
reports suggested that alterations in gastrointestinal pH profiles may
occur in patients with inflammatory bowel disease, which should be
considered in the development of delayed release formulations [Anil
and Philip, 2010]. It may start to dissolve even in the lower small
intestine and the site-specificity of formulations can be poor. As a site
for drug delivery, the colon offers a near neutral pH, reduced digestive
enzymatic activity, a long transit time and increased responsiveness to
absorption enhancers; however, the targeting of drugs to the colon is
very complicated. Due to its location in the distal part of the alimentary
canal, the colon is particularly difficult to access. In addition to that the
wide range of pH values and different enzymes present throughout the
gastrointestinal tract, through which the dosage form has to travel
before reaching the target site, further complicate the reliability and
21
delivery efficiency [Kimura and Higaki, 2002]. Successful delivery
through this site also requires the drug to be in solution form before it
arrives in the colon or alternatively, it should dissolve in the luminal
fluids of the colon, but this can be a limiting factor for poorly soluble
drugs as the fluid content in the colon is much lower and it is more
viscous than in the upper part of the GI tract [Kumar et al., 2012]. Drug
targeting to colon is useful when a delay in drug absorption is desired
from the therapeutic point of view, such as treatment of diseases that
have peak symptoms in the early morning, like nocturnal asthma,
angina or arthritis1. By definition, an oral colonic delivery system
should retard drug release in the stomach and small but allow
complete release in the colon. Colonic delivery refers to targeted
delivery of drugs into the lower gastrointestinal tract, which occurs
primarily in the large intestine (i.e. colon). The site specific delivery of
drugs to lower parts of the gastro intestinal tract is advantageous for
localized treatment of several colonic diseases, mainly inflammatory
bowel disease (Crohn’s disease and ulcerative colitis), irritable bowel
syndrome, and colon cancer[Ravi et al., 2008]. The luminal pH of the
distal intestine in patients with inflammatory bowel disease (IBD) or
ulcerative colitis can be lower than that seen in healthy volunteers as
found in previous study involving six patients with ulcerative colitis, the
22
colonic pH of three patients varied from 5.0 to 7.0, whereas in case of
other three subjects very low pH of 2.3, 2.9 and 3.4 were observed
[Fallingborg et al., 1993]. More importantly natural Killer cells,
macrophages and so on are largely accumulated in inflamed region of
colon. It has been reported that microspheres and nanoparticles could
be efficiently taken up by these macrophages [Lamprecht et al., 2001]
that also make a reasonable basis for using microspherical drug
carrier. It has also gained increased importance not just for the
delivery of drugs for the treatment of local diseases, but also potential
site for the systemic delivery of therapeutic proteins and peptides
which are being delivered by injections. These delivery systems when
taken orally, allow drugs to release the drug from the delivery system
once the delivery system arrives into the colon. Other potential
applications of colonic delivery include chronotherapy, prophylaxis of
colon cancer and treatment of nicotine addiction [Bajpai et al., 2003].
Lower surface area and relative‘tightness’ of the junctions in the colon
can also restrict drug transport across the mucosa and into the
systemic circulation. Therefore formulations for colonic delivery are
also suitable for delivery of drugs which are polar and/or susceptible to
chemical and enzymatic degradation in the upper gastrointestinal tract,
highly affected by hepatic metabolism, in particular, therapeutic
23
proteins and peptides [Ratna et al., 2010]. Drug absorption is often
found unsatisfactory and highly variable among and between
individuals, despite excellent in vitro release patterns. The reasons for
this are essentially physiological and usually affected by the
gastrointestinal (GI) transit of the form, especially its gastric residence
time (GRT), which appears to be one of the major causes of the
overall transit time variability. Colon targeted drug delivery would
ensures direct treatment at the disease site, lower dosing and less
systemic side effects. In addition to restricted therapy, the colon can
also be utilized as a portal for the entry of drugs into the systemic
circulation [Rajguru et al., 2011]. Overall, there is less free fluid in the
colon than in the small intestine and hence, dissolution could be
problematic for poorly water-soluble drugs. In such instance, the drug
may need to be delivered in a presolubilized form either entrapped in
carrier or encapsulated by polymeric release controlling membrane
[Sarasija and Hota, 2000]. Failure of enteric-coated dosage forms,
especially single unit dosage forms, because of lack of disintegration
has been reported. The decline in pH from the end of the small
intestine to the colon can also result in problems. Lengthy lag times at
the ileo-caecal junction or rapid transit through the ascending colon
can also result in poor site-specificity of enteric-coated single-unit
24
formulations. Eudragit products are pH-dependent methacrylic acid
polymers containing carboxyl groups. The number of esterified
carboxyl groups affects the pH level at which dissolution takes place.
Eudragit S is soluble above pH 7 and Eudragit L above pH 6. Eudragit
S coatings protect well against drug liberation in the upper parts of the
gastrointestinal tract and have been used in preparing colon-specific
formulations. When sites of disintegration of Eudragit S-coated single-
unit tablets were investigated using a gamma camera they were found
to lie between the ileum and splenic flexure. Site specificity of Eudragit
S formulations, both single and multiple units, is usually poor. Eudragit
S coatings have been used to target the anti-inflammatory drug of 5-
aminosalicylic acid (5-ASA) in single-unit formulations on the large
intestine. Eudragit L coatings have been used in single unit tablets to
target 5-ASA on the colon in patients with ulcerative colitis or Crohn’s
disease [Danda and Brahma, 2013].
1.3.1.5. Lectins: Roles in Colon targeting
It is a well-known fact that the surface of the mammalian or
microbial cells contains carbohydrate moieties in abundance mainly
oligosaccharides associated with membrane lipids, proteins or peptide
glycans. This membrane associated carbohydrate-rich material is
referred to as extracellular matrix or glycocalyx [Sihorkar and Vyas,
25
2001]. Colon cells usually have a well-developed glycocalyx. Being
attached to the external surface of colon cells carbohydrate domains
of glycoproteins and glycolipids might be used as targets for colon-
specific delivery. It is important that nature already developed a
powerful targeting moiety for these molecules—lectins. Lectins are
proteins of non-immunological origin, capable of recognizing and
binding to glycoproteins expressed on cell surface. Lectins interaction
with certain carbohydrate is very specific. This interaction is as specific
as the enzyme–substrate, or antigen– antibody interactions. Lectins
may bind with free sugar or with sugar residues of polysaccharides,
glycoproteins, or glycolipids which can be free or bound (as in cell
membranes). Some lectins are expressed on the surface of human
cells, and therefore, can be used as a target for colon-specific drug
delivery. Lectins are naturally occurring proteins that play a
fundamental role in biological recognition phenomena involving cells
and proteins. For example, some bacteria use lectins to attach
themselves to the cells of the host organism during infection [Wirth et
al., 1998]. Lectins belong to a group of structurally diverse proteins
and glycoproteins that can bind reversibly to specific carbohydrate
residues [Tamara, 2004]. While the majority of lectins used and
studied are from plant or microbial origin, it has become clear in recent
26
years that there exist numerous animal lectins. In the gut, it was
known that certain bacteria expressed glycan containing molecules in
their cell walls that bound to the epithelial surfaces via lectin
interactions, indicating that there were endogenous lectins exposed on
epithelial cell surfaces which could be targeted by sugar bearing drug
formulations [Biesa et al., 2004]. In the 1980s, synthetic polymers
bearing pendant sugar moieties were synthesised as potential drug
carriers and these were tested for interaction with the GI tract.
Different sugars gave different profiles of interaction with gut tissue,
with galactose bearing polymers showing greater interaction in
proximal regions of the gut, while fucose bearing polymers consistently
showed the greatest interaction and were more specific for distal gut
regions [Bridges et al., 1998]. In vertebrates, two broad classes of
lectins have been identified [Perillo et al., 1998]. The C-type lectins,
such as selectins and pentraxins, require calcium for carbonate
binding. The S-type lectins, now known as the galectins, are calcium
independent and are found in species ranging from C. elegans to
humans. Well-studied galectins-1 and -3 are expressed on normal
colon cells and overexpressed in colon cancer cells [Schoeppner et
al., 1995]. Direct lectin targeting or glycotargeting relies on carrier
molecules possessing carbohydrates that are recognized and
27
internalized by cell surface mammalian lectins whereas reverse lectin
targeting approach utilizes exogenous lectins as targeting moieties
that target whole DDS to glycoproteins or glycolipids overexpressed
on the surface of colon cells. Potential use of wheat germ agglutinin
(WGA) and Solanum tuberosum lectin (STL) as auxiliary excipients for
targeting drugs to colonocytes was also analyzed and found that WGA
and STL, due to specific and sufficient adhesion and internalization by
colon cells, are anticipated as targeting moieties in lectin-mediated
DDS utilizing reverse lectin targeting [ Wirth et al., 1998 & Wirth et al.,
2002].
1.3.1.6. Natural polymers for colon targeted drug delivery system
The use of natural polymers and polymethacrylates as drug
carriers is one of the main objectives of researchers dealing with long
acting dosage forms [Tiwari and Shukla, 2009].Natural
polysaccharides are extensively used for the development of solid
dosage forms. These polymers of monosaccharide (sugars) are
inexpensive and available in a variety of structures with a variety of
properties. They are highly stable, safe, non-toxic, and hydrophilic and
gel forming in nature. Pectin, starch, guar gum, amylase and karaya
gum are a few polysaccharides commonly used in dosage forms. Non-
starch, linear polysaccharides remain intact in the physiological
28
environment of the stomach and the small intestine, but are degraded
by the bacterial inhabitants of the human colon which make them
potentially useful in targeted delivery systems to the colon [Cummings
et al., 1979]. Over the past few years, the use of natural polymers in
the design of drug delivery formulation has received much attention
due to their excellent biocompatibility and biodegradability. Several
such reported functional natural polymers are described briefly stating
their potential importance.
I. Pectin:
Pectins are nonstarch linear complex polysaccharides that
consist of α-1, 4 D-galacturonic acid and 1, 2 D-rhamnose with D-
galactose and D-arabinose side chains having average molecular
weights between 50,000 to 150,000. They are heterogeneous moieties
with respect to chemical structure and molecular weight and can be
classified into low methoxy (LM), high methoxy (HM) and amidated
pectins [BeMiller, 1986]. Pectin tends to produce lower viscosities than
other plant gums. It is refractory to host gastric and small intestinal
enzymes but is almost completely degraded by the colonic bacterial
enzymes to produce a series of soluble oligalactorunates. Pectins
have a number of pharmaceutical applications and are presently
29
considered as promising biodegradable carriers for colon-specific drug
delivery [Subudhi et al., 2015].
II. Alginates:
Alginates are linear polymers that have 1-4’linked β-D-
mannuronic acid and α-L-guluronic acid residue arranged as blocks of
either type of unit or as a random distribution of each type. A Eudragit
L-30D–coated calcium alginates bead for colonic delivery of 5-
aminosalicylic acid has been reported. Different enteric as well as
sustained release polymers were applied as coat on calcium alginate
beads. A system was prepared by coating calcium alginate beads with
Aquacoat® that is a pH-independent polymer followed by 2 % w/w
coating of Eudragit L-30D [Shun and Ayres, 1992]. When drug-loaded
calcium alginate beads swell sufficiently (osmotic gradient) to exceed
the strength of outer sustained released coat, the film bursts to release
the drug. Such a system delivers drug to the distal intestine with
minimal initial leak and provides sustained release in the colon.
III. Starches:
It is the principal form of carbohydrate reserve in green plants
and especially present in seeds and underground organs. Starch
occurs in the form of granules (starch grains), the shape and size of
30
which are characteristic of the species, as is also the ratio of the
content of the principal constituents, amylose and amylopectin. A
number of starches are recognized for pharmaceutical use. These
include maize (Zea mays), rice (Oryza sativa) , wheat ( Triticum
aestivum ), and potato (Solanum tuberosum ). To deliver proteins or
peptidic drugs orally, microcapsules containing a protein and a
proteinase inhibitor were prepared. Starch/bovine serum albumin
mixed-walled microcapsules were prepared using interfacial cross-
linking with terephthaloyl chloride [McIntosh et al., 2005].
IV. Xanthan gum:
Xanthan gum is a high molecular weight extra cellular
polysaccharide produced by the fermentation of the gram-negative
bacterium Xanthomonas campestris. The anionic character of this
polymer is due to the presence of both glucuronicacid and pyruvic acid
groups in the side chain. In one of the trials, xanthan gum showed a
higher ability to retard the drug release than synthetic
hydroxyproylmethylcellulose [Bhardwaj et al., 2000].
V. Guar gums:
It is a naturally occurring galactomannan polysaccharide;
consists of chiefly high molecular weight hydrocolloidal
31
polysaccharide, composed of galactan and mannan units combined
through glycosidic linkages and shows degradation in the large
intestine due the presence of microbial enzymes. Guar gum is
hydrophilic and swells in cold water, forming viscous colloidal
dispersions or sols. This gelling property retards release of the drug
from the dosage form, making it more likely that degradation will occur
in the colon. Guar gum was found to be a colon-specific drug carrier in
the form of matrix and compression-coated tablets as well as
microspheres [Al-Saidan et al., 2005].
VI. Chitosan:
Chitin after alkaline deacetylation is dissolved in acid, filtered
and the precipitate formed is washed and dried to get amine free
chitosan. Chitosan is a high molecular weight polycationic
polysaccharide derived from naturally occurring chitin by alkaline
deacetylation. Chemically, it is a poly (N-glucosamine). Chitosan has
favourable biological properties such as nontoxicity, biocompatibility
and biodegradability. Similar to other polysaccharides it also
undergoes degradation by the action of colonic microflora and hence
poses its candidature for colon targeted drug delivery developed
colon-specific insulin delivery with chitosan capsules. A pH-sensitive
drug delivery carrier has also been reported for chitosan-based
32
hydrogels [Tozaki et al., 2002]. Chitosan is a weak base and is
insoluble in water and organic solvents, however, it is soluble in dilute
aqueous acidic solution (pH < 6.5), which can convert the glucosamine
units into a soluble form R–NH3+ [Chandy and Sharma, 1990]. It gets
precipitated in alkaline solution or with polyanions and forms gel at
lower pH. Chitosan has been noted for its film-forming properties and
is gaining increasing importance due to its good biocompatibility,
biodegradability and due to their favourable toxicological properties.
Chitosan may provide improved drug delivery via a paracellular route
through neutralization of fixed anionic sites within the tight junctions
between mucosal cells.It has also been shown to enhance drug
absorption via mucoadhesive mechanism.Chitosan has been shown to
possess mucoadhesive properties [Shimoda et al., 2001 & Kockisch et
al., 2003] and this pharmaceutically useful property comprised with
molecular attractive forces formed by electrostatic interaction between
positively charged chitosan and negatively charged mucosal surfaces
may be attributed to: (a) strong charges [Dodane et al., 1999] ; (b)
sufficient chain flexibility [He et al., 1998] (c) surface energy properties
favoring spreading into mucus (Lue en et al., 1994] (d) high molecular
weight [ Kotze et al., 1998] and (e) strong hydrogen bonding groups
like –OH, –COOH (Schipper et al., 1997]. However, the success of the
33
Chitosan (obtained by deacetylation of chitin) is a cationic polymer that
has been proposed for use in microsphere systems by a number of
authors [Dubey and Parikh, 2004]. Chitosan was selected as a
polymer in the preparation of mucoadhesive microspheres because of
its good mucoadhesive and biodegradable properties.
1.3.2. Bioadhesion in targeted drug delivery systems
Bioadhesive drug delivery systems are used as a delivery device
within the human to enhance drug absorption in a site-specific
manner. In this approach, bio adhesive polymers are used and they
can adhere to the epithelial surface in the stomach. Thus, they
improve the prolongation of gastric retention. The basis of adhesion in
that a dosage form can stick to the mucosal surface by different
mechanism [Wittaya-Areekul et al., 2006].These mechanisms are as
follows:
I. The wetting theory which is based on the ability of bioadhesive
polymers to spread and develop intimate contact with the
mucous layers.
II. The diffusion theory which proposes physical entanglement of
mucin strands the flexible polymer chains, or an interpenetration
of mucin strands into the porous structure of the polymer
substrate.
34
III. The absorption theory suggests that bioadhesion is due to
secondary forces such as Vander Waal forces and hydrogen
bonding.
IV. The electron theory which proposes attractive electrostatic
forces between the glycoprotein mucin net work and the bio
adhesive material.
Materials commonly used for bioadhesion are poly acrylic acid,
chitosan, cholestyramine, sodium alginate, hydroxypropyl
methylcellulose (HPMC), sucralfate, tragacanth, dextrin, polyethylene
glycol (PEG) and polylactic acids etc. Polymer has been used for the
specific delivery of insulin to the colon. The chitosan capsules were
coated with enteric coating (Hydroxy propyl methyl cellulose phthalate)
and contained, apart from insulin, various additional absorption
enhancer and enzyme inhibitor. It was found that capsules specifically
disintegrated in the colonic region. It was suggested that this
disintegration was due to either the lower pH in the ascending colon as
compared to the terminal ileum or to the presence bacterial enzyme,
which can degrade the polymer [Chaurasia and Jain, 2003]. The term
bioadhesion refers to any bond formed between two biological
surfaces or a bond between a biological and a synthetic surface. In
case of bioadhesive drug delivery, the term bioadhesion is used to
35
describe the adhesion between polymers, either synthetic or natural
and soft tissues or the gastrointestinal mucosa.
1.4. Microspheres as drug delivery carrier
Microspheres are the carrier linked drug delivery system in which
particle size is ranges from 1-1000 μm range in diameter having a
core of drug and entirely outer layers of polymer as coating material
and are defined as Monolithic sphere or therapeutic agent distributed
throughout the matrix either as a molecular dispersion of particles” (or)
can be defined as structure made up of continuous phase of one or
more miscible polymers in which drug particles are dispersed at the
molecular or macroscopic level [Mathew Sam et al., 2008].
Microencapsulation for oral use has been employed to sustain the
drug release, and to reduce or eliminate gastrointestinal tract irritation.
In addition, multiparticulate delivery systems spread out more
uniformly in the gastrointestinal tract. This results in more reproducible
drug absorption and reduces local irritation when compared to single-
unit dosage forms such as no disintegrating, polymeric matrix tablets.
Microspheres are sometimes referred to as microparticles
.Microspheres can be manufactured from various natural and synthetic
materials. Glass microspheres, polymer microspheres and ceramic
microspheres are commercially available. Similarly in pharmaceutical
36
field different types of microspheres such as magnetic microsphere
floating microsphere, polymeric microsphere, bioadhesive
microsphere, biodegradable microsphere etc. are extensively tried for
different controlled release dosage forms [Moy et al., 2011].Solid and
hollow microspheres vary widely in density and, therefore, are used for
different applications. Over the past three decades, the pursuit and
exploration of devices designed to be retained in the upper part of the
GI tract has advanced consistently in terms of technology and
diversity, encompassing a variety of systems and devices such as
floating systems, raft systems, expanding systems, swelling systems,
bioadhesive systems and low-density systems. Microspheres are
limited owing to their short residence time at the site of absorption. It
would, therefore, be advantageous to have means for providing an
intimate contact of the drug delivery system with the absorbing
membranes [Schaefer and Singh, 2000]. This can be achieved by
coupling bioadhesion characteristics to microspheres and developing
bioadhesive microspheres. Bioadhesive microspheres have
advantages such as efficient absorption and enhanced bioavailability
of drugs owing to a high surface-to-volume ratio, a much more intimate
contact with the mucus layer, and specific targeting of drugs to the
absorption site [Chowdary et al., 2003 & Carvalho et al., 2010].
37
1.4.1 Method of preparation of microspheres
There exist several methods to prepare microsphere that differ
as per the characteristics of polymers used, nature of microspheres,
and nature of manufacturing condition. These are described as
following:
I. Spray Drying:
In Spray Drying the polymer is first dissolved in a suitable volatile
organic solvent such as dichloromethane, Acetone, etc. The drug in
the solid form is then dispersed in the polymer solution under high-
speed homogenization. This dispersion is then atomized in a stream of
hot air. The atomization leads to the formation of the small droplets or
the fine mist from which the solvent evaporate instantaneously leading
the formation of the microspheres in a size range 1-100μm. Micro
particles are separated from the hot air by means of the cyclone
separator while the trace of solvent is removed by vacuum drying. One
of the major advantages of process is feasibility of operation under
aseptic conditions this process is rapid and this leads to the formation
of porous microparticles [Mathew Sam et al., 2008 & Ghulam et
al.,2009].
38
II. Solvent Evaporation:
The processes are carried out in a liquid manufacturing vehicle.
The microcapsule coating is dispersed in a volatile solvent which is
immiscible with the liquid manufacturing vehicle phase. A core material
to be microencapsulated is dissolved or dispersed in the coating
polymer solution. With agitation the core material mixture is dispersed
in the liquid manufacturing vehicle phase to obtainthe appropriate size
microcapsule. The mixture is then heated if necessary to evaporate
the solvent for the polymer of the core material is disperse in the
polymer solution, polymer shrinks around the core. If the core material
is dissolved in the coating polymer solution, matrix – type
microcapsules are formed. The core materials may be either water
soluble or water in soluble materials. Solvent evaporation involves the
formation of an emulsion between polymer solution and an immiscible
continuous phase whether aqueous (o/w) or non-aqueous. The
comparison of mucoadhesive microspheres of hyaluronic acid,
Chitosan glutamate and a combination of the two prepared by solvent
evaporation with microcapsules of hyaluronic acid and gelating
prepared by complex coacervation were made [Trivedi et al., 2008 &
Kannan et al., 2009].
39
III. Hot Melt Microencapsulation:
The polymer is first melted and then mixed with solid particles of
the drug that have been sieved to less than 50 μm. The mixture is
suspended in a non-miscible solvent (like silicone oil), continuously
stirred, and heated to 5°C above the melting point of the polymer.
Once the emulsion is stabilized, it is cooled until the polymer particles
solidify. The resulting microspheres are washed by decantation with
petroleum ether. The primary objective for developing this method is to
develop a microencapsulation process suitable for the water labile
polymers, e.g. poly anhydrides. Microspheres with diameter of 1- 1000
μm can be obtained and the size distribution can be easily controlled
by altering the stirring rate. The only disadvantage of this method is
moderate temperature to which the drug is exposed [Owen and Anne,
2003].
IV. Single emulsion technique:
The micro particulate carriers of natural polymers of natural
polymers i.e. those of proteins and carbohydrates are prepared by
single emulsion technique. The natural polymers are dissolved or
dispersed in aqueous medium followed by dispersion in non-aqueous
medium like oil. Next cross linking of the dispersed globule is carried
40
out. The cross linking can be achieved either by means of heat or by
using the chemical cross linkers. The chemical cross linking agents
used are glutaraldehyde, formaldehyde, acid chloride etc. Heat
denaturation is not suitable for thermolabile substances. Chemical
cross linking suffers the disadvantage of excessive exposure of active
ingredient to chemicals if added at the time of preparation and then
subjected to centrifugation, washing, separation. The nature of the
surfactants used to stabilize the emulsion phases can greatly influence
the size, size distribution, surface morphology, loading, drug release,
and bio performance of the final multiparticulate product [Pradesh et
al., 2005].
V. Double emulsion technique:
Double emulsion method of microspheres preparation involves
the formation of the multiple emulsions or the double emulsion of type
w/o/w and is best suited to water soluble drugs, peptides, proteins and
the vaccines. This method can be used with both the natural as well as
synthetic polymers. The aqueous protein solution is dispersed in a
lipophilic organic continuous phase. This protein solution may contain
the active constituents. The continuous phase is generally consisted of
the polymer solution that eventually encapsulates of the protein
contained in dispersed aqueous phase. The primary emulsion is
41
subjected then to the homogenization or the sonication before addition
to the aqueous solution of the poly vinyl alcohol (PVA). This results in
the formation of a double emulsion. The emulsion is then subjected to
solvent removal either by solvent evaporation or by solvent extraction.
a number of hydrophilic drugs like leutinizing hormone releasing
hormone (LH-RH) agonist, vaccines, proteins/peptides and
conventional molecules are successfully incorporated into the
microspheres using the method of double emulsion solvent
evaporation/ extraction [Dandagi et al., 2007].
VI. Polymerization techniques:
The polymerization techniques conventionally used for the
preparation of the microspheres are mainly classified as:
I. Normal polymerization
II. Interfacial polymerization. Both are carried out in liquid phase.
Normal polymerization is carried out using different techniques
as bulk, suspension, precipitation, emulsion and micellar
polymerization processes. In bulk, a monomer or a mixture of
monomers along with the initiator or catalyst is usually heated to
initiate polymerization. Polymer so obtained may be moulded as
microspheres. Drug loading may be done during the process of
42
polymerization. Suspension polymerization also referred as bead or
pearl polymerization. Here it is carried out by heating the monomer or
mixture of monomers as droplets dispersion in a continuous aqueous
phase. The droplets may also contain an initiator and other additives.
Emulsion polymerization differs from suspension polymerization as
due to the presence initiator in the aqueous phase, which later on
diffuses to the surface of micelles. Bulk polymerization has an
advantage of formation of pure polymers [Gohel, 2005].
Interfacial polymerization involves the reaction of various
monomers at the interface between the two immiscible liquid phases
to form a film of polymer that essentially envelops the dispersed phase
[Li et al., 1998].
VII. Phase separation coacervation technique:
This process is based on the principle of decreasing the
solubility of the polymer in organic phase to affect the formation of
polymer rich phase called the coacervates. In this method, the drug
particles are dispersed in a solution of the polymer and an
incompatible polymer is added to the system which makes first
polymer to phase separate and engulf the drug particles. Addition of
non-solvent results in the solidification of polymer. Poly lactic acid
43
(PLA) microspheres have been prepared by this method by using
butadiene as incompatible polymer. The process variables are very
important since the rate of achieving the coacervates determines the
distribution of the polymer film, the particle size and agglomeration of
the formed particles. The agglomeration must be avoided by stirring
the suspension using a suitable speed stirrer since as the process of
microspheres formation begins the formed polymerize globules start to
stick and form the agglomerates. Therefore the process variables are
critical as they control the kinetic of the formed particles since there is
no defined state of equilibrium attainment. Microparticles can also be
prepared by complex co acervation, Sodium alginate, sodium CMC
and sodium polyacrylic acid can be used for complex coacervation
with CS to form microspheres. These microparticles are formed by
interionic interaction between oppositely charged polymers solutions
and KCl & CaCl2 solutions. The obtained capsules were hardened in
the counter ion solution before washing and drying [Ghulam et al.,
2009].
VIII. Spray drying and spray congealing:
These methods are based on the drying of the mist of the
polymer and drug in the air. Depending upon the removal of the
solvent or cooling of the solution, the two processes are named spray
44
drying and spray congealing respectively. The polymer is first
dissolved in a suitable volatile organic solvent such as
dichloromethane, acetone, etc. The drug in the solid form is then
dispersed in the polymer solution under high speed
homogenization.This dispersion is then atomized in a stream of hot air.
The atomization leads to the formation of the small droplets or the fine
mist from which the solvent evaporates instantaneously leading the
formation of the microspheres in a size range 1-100 μm. Microparticles
are separated from the hot air by means of the cyclone separator while
the traces of solvent are removed by vacuum drying. One of the major
advantages of the process is feasibility of operation under aseptic
conditions. The spray drying process is used to encapsulate various
penicillins. Thiamine mononitrate and sulpha ethylthiadizole are
encapsulated in a mixture of mono- and diglycerides of stearic acid
and palmiticacid using spray congealing. Very rapid solvent
evaporation, however leads to the formation of porous microparticles
[Li et al., 1988].
IX. Solvent extraction:
Solvent evaporation method is used for the preparation of
microparticles, involves removal of the organic phase by extraction of
the organic solvent. The method involves water miscible organic
45
solvents such as isopropanol. Organic phase is removed by extraction
with water. This process decreases the hardening time for then
microspheres. One variation of the process involves direct addition of
the drug or protein to polymer organic solution. The rate of solvent
removal by extraction method depends on the temperature of water,
ratio of emulsion volume to the water and the solubility profile of the
polymer [Agusundaram et al., 2009].
X. Thermal cross-linking:
Citric acid, as a cross-linking agent was added to 30 mL of an
aqueous acetic acid solution of chitosan (2.5% wt/vol) maintaining a
constant molar ratio between chitosan and citric acid (6.90 × 10−3 mol
chitosan: 1 mol citric acid). The chitosan cross-linker solution was
cooled to 0°C and then added to 25 mL of corn oil previously
maintained at 0°C, with stirring for 2 minutes. This emulsion was then
added to 175 mL of corn oil maintained at 120°C, and cross-linking
was performed in a glass beaker under vigorous stirring (1000 rpm) for
40 minutes. The microspheres obtained were filtered and then washed
with diethyl ether, dried, and sieved [Orienti et al., 1996].
XI. Glutaraldehyde cross linking:
A 2.5% (w/v) chitosan solution in aqueous acetic acid was
prepared. This freshdispersed phase was added to continuous phase
46
(125 mL) consisting of light liquid paraffin and heavy liquid paraffin in
the ratio of 1:1 containing 0.5% (wt/vol) Span 85 to form a water in oil
(w/o) emulsion. Stirring was continued at 2000 rpm using a 3- blade
propeller stirrer). A drop-by-drop solution of a measured quantity (2.5
mL each) of aqueous glutaraldehyde (25% v/v) was added at 15, 30,
45, and 60 minutes.Stirring was continued for 2.5 hours and separated
by filtration under vacuum and washed, first with petroleum ether
(60°C- 80°C) and then with distilled water to remove the adhered liquid
paraffin and glutaraldehyde, respectively. The microspheres were then
finally dried in vacuum desiccators [Thanoo et al., 1992].
1.4.2. Applications of microspheres
Solid microspheres have numerous applications depending on
what material they are constructed of and what size they are. Some of
the applications of microspheres are mentioned as following: -
1. Controlled and sustained release dosage forms.
2. Microsphere can be used to prepare enteric-coated dosage
forms, so that the medicament will be selectively absorbed in
the intestine rather than the stomach.
3. It has been used to protect drugs from environmental hazards
such as humidity, light, oxygen or heat. Microsphere does not
47
yet provide a perfect barrier for materials, which degrade in
the presence of oxygen, moisture or heat, however a great
degree of protection against these elements can be provided.
For example, vitamin A and K have been shown to be
protected from moisture and oxygen through microsphere.
4. The separations of incompatible substances, for example, the
popular pharmaceutical eutectics have been achieved by
encapsulation. This is a case where direct contact of
materials brings about liquid formation. The stability
enhancement of incompatible aspirin chlorpheniramine
maleate mixture is accomplished by microencapsulating both
of them before mixing.
5. Microsphere can be used to decrease the volatility. An
encapsulated volatile substance can be stored for longer
times without substantial evaporation.
6. Microsphere has also been used to decrease potential danger
of handling of toxic or noxious substances. The toxicity
occurred due to handling of fumigants, herbicides,
insecticides and pesticides have been advantageously
decreased after microencapsulation.
48
7. The hygroscopic properties of many core materials may be
reduced by microsphere.
8. Many drugs have been microencapsulated to reduce gastric
irritation [Meena et al., 2011].
9. Microsphere method has also been proposed to prepare
intrauterine contraceptive device.
10. Therapeutic magnetic microspheres are used to deliver
chemotherapeutic agent to liver tumour. Drugs like proteins
and peptides can also be targeted through this system.
11. Mucoadhesive microspheres exhibit a prolonged residence
time at the site of application and causes intimate contact with
the absorption site and produces better therapeutic action.
12. Radioactive microspheres are used for imaging of liver,
spleen, bone marrow, lung etc and even imaging of thrombus
in deep vein thrombosis can be done [Moy et al., 2011].
1.4.3. Mucoadhesive microspheres as carrier for colon targeting
Mucoadhesion can be defined as a state in which two
components, of which one is of biological origin are held together for
extended periods of time by the help of interfacial forces. Intimate
contact between a bioadhesive and a membrane (wetting or swelling
phenomenon) and penetration of the bioadhesive into the tissue or into
49
the surface of the mucous membrane (interpenetration) are the two
proposed mechanisms for mucoadhesion [Chowdary and Srinivas,
2000 & Alexander et al., 2011]. In biological systems, bioadhesion can
be classified into 3 types.
I. Adhesion between two biological phases, for example, platelet
aggregation and wound healing.
II. Adhesion of a biological phase to an artificial substrate, for
example tissue, cell adhesion to culture dishes and biofilm
formation on prosthetic devices and inserts.
III. Adhesion of an artificial substance to a biological substrate, for
example, adhesion of synthetic hydrogels to soft tissues [Shaikh
et al., 2011].
1.4.3.1. Mucoadhesive microspheres
Mucoadhesive microspheres include microparticles and
microcapsules (having a core of drug) consisting either entirely of a
Mucoadhesive polymer or having an outer coating of it, respectively.
Microspheres, in general, have the potential to be used for targeted
and controlled release drug delivery; but coupling of bioadhesive
properties to microspheres has additional advantages e.g. efficient
absorption and bioavailability of the drugs due to high surface to
volume ratio, a much more intimate contact with the mucous layer,
50
specific targeting of drugs to the absorption site. Microspheres vary
widely in quality, sphericity, uniformity of particle and particle size
distribution. The appropriate microsphere needs to be chosen for each
unique application [Parmar et al., 2010 & Thanoo et al., 1992].
Mucoadhesive microsphere form carrier systems and are made from
the biodegradable polymers in sustained drug delivery. Recently,
dosage forms that can precisely control the release rates and target
drugs to a specific body site have made an enormous impact in the
formulation and development of novel drug delivery systems.
Microspheres form an important part of such novel drug delivery
system. They have varied applications and are prepared using
assorted polymers [Kaurav et al., 2012]. Formulation development has
been to improve therapeutic efficacy and reduce the severity of
gastrointestinal adverse effects through altering the dosage forms by
modifying the release of the formulations to optimize drug delivery
system. One such approach is using mucoadhesive polymeric
microspheres as carriers of drugs [Sachan and Bhattacharya, 2009].
Mucoadhesive formulations orally would achieve a substantial
increase in the length of stay of the drug in GI tract stability problem in
the intestinal fluid can be improved [Brahmaiah et al., 2013]. The
inability of GIT enzyme to digest certain plant polysaccharide is taken
51
advantage of to develop a colon specific drug delivery system.
Biodegradable polymer matrix core embeds the drug by compressing
the blend of active drug, a biodegradable polymer and additives.
Various polysaccharides are being evaluated for colon targeting, like
pectin, guar gum, gum ghatti, dextran, chitosan and xylan [ Bhardwaj
et al., 2000].Microspheres form an important part of such novel drug
delivery systems [ Capan et al., 2003].They have varied applications
and are prepared using assorted polymers [Vasir et al., 2003].
1.4.3.2. Advantages of mucoadhesive microspheres
Following advantages of mucoadhesive microspheres drug
delivery system have been identified: (1) As a result of adhesion and
intimate contact, the formulation stays longer at the delivery site
improving API bioavailability using lower API concentrations for
disease treatment. (2) The use of specific bioadhesive molecules
allows for possible targeting of particular sites or tissues, for example
the gastrointestinal (GI) tract. (3) Increased residence time combined
with controlled API release may lead to lower administration
frequency.(4) Offers an excellent route, for the systemic delivery of
drugs with high first-pass metabolism, there by offering a greater
bioavailability [Punitha et al., 2010]. (5) Additionally significant cost
reductions may be achieved and dose-related side effects may be
52
reduced due to API localization at the disease site [Gavin et al.,
2009].(6) Better patient compliance and convenience due to less
frequent drug administration.(7) Uniform and wide distribution of drug
throughout the gastrointestinal tract which improves the drug
absorption.(8) Prolonged and sustained release of drug.(9)
Maintenance of therapeutic plasma drug concentration.(10) Better
processability (improving solubility, dispersibility, flowability).(11)
Increased safety margin of high potency drugs due to better control of
plasma levels.(12) Reduction in fluctuation in steady state levels and
therefore better control of disease condition and reduced intensity of
local or systemic side effects [Venkateshwaramurthy et al., 2010]. (13)
Drugs which are unstable in the acidic environment are destroyed by
enzymatic or alkaline environment of intestine can be administered by
this route.Oral administration of most of the drugs in conventional
dosage forms has short-term limitations due to their inability to restrain
and localize the system at gastro-intestinal tract. Microspheres
constitute an important part of these particulate drug delivery systems
by virtue of their small size and efficient carrier capacity.
1.4.4. Chitosan based polyelectrolyte complexes as Microspheres
Microsphere based systems may increase the life span of active
constituents and control the release of bioactive agents. Being small in
53
size, microspheres have large surface to volume ratios and can be
used for controlled release of insoluble drugs. Extensive research is
being carried out to exploit chitosan as a drug carrier to attain the
desirable drug release profile. Chitosan possesses no toxicity and can
be applied onto the epithelium. It swells and forms a gel like layer in
aqueous environment (by absorbing water from the mucous layer),
which is favorable for interpenetration of polymer and glycoprotein
chains into mucous. The positive charge on chitosan polymer gives
rise to strong electrostatic interaction with mucus or negatively
charged sialic acid residues on the mucosal surface [Illum, 1998].
Chitosan also shows good bioadhesive characteristics and can reduce
the rate of clearance of drug from the site thereby increasing the
bioavailability of drugs incorporated in it [Soane et al., 1999]. Chitosan
microspheres are used to provide controlled release of many drugs
and to improve the bioavailability of degradable substances such as
protein, as well as to improve the uptake of hydrophilic substances
across the epithelial layers [Tomolin et al., 1989]. These microspheres
are being investigated both for parenteral and oral drug delivery.
Chitosan microcores containing drug (sodium diclofenac) can be
coated with acrylic polymers, namely, Eudragit L100 and Eudragit
S100 and even with Eudragit P-4135 F, a new pH sensitive polymer
54
was used to prepare microparticles that shown degradation at above
pH 7.2 [Lamprecht et al., 2005].These systems provide an intimate
contact with the negatively charged (due to sialic acid or carboxyl or
sulphate groups in the mucus glycoprotein) mucus membrane due to
polyvalent adhesive interaction or electrostatic attraction, H-bond
formation, van-der-Waal forces and other [Lehr et al., 1993]. The
system has an additional advantage of protecting acid sensitive drugs
against acid degradation and offers effective drug diffusion across the
mucus layer.Enhancement of mucosal delivery may also be obtained
through the use of appropriate cytoadhesives that can bind to mucosal
surfaces. The most widely investigated of such systems in this respect
are lectins. Chitosan microspheres are the most widely studied drug
delivery systems for the controlled release of drugs viz., antibiotics,
antihypertensive agents, anticancer agents, proteins, peptide drugs
and vaccines [Sinha et al., 2004]. Chitosan complexes have been
used in a wide range of pharmaceutical applications and complexes
formed between chitosan and anionic polymers have been
investigated for use as biosensors, scaffolds in tissue engineering, for
waste-water treatment and for drug delivery in different forms
[Bernabe et al., 2005]. The negatively charged carboxylic acid groups
of manuronic and guluronic acid units in alginate interact
55
electrostatically with the positively charged amino groups of chitosan
to form a polyelectrolyte complex. The selected pH values ensured an
increased charge density on each polymer and led to intense cross-
linking during polyelectrolyte complex formation and consequently
beads with small micropores were formed.
Polyelectrolyte complexes (PEC) are formed by the ionic
interactions as ionically cross-linked networks when two oppositely
charged polyelectrolytes bind each other in an aqueous solution. 9e
net charge Exed on the complex, which is an important factor
determining the swelling and the induced volume change of the PEC,
is affected by pH value of ambient solution due to the variation in the
degree of ionization of functional groups [Prado et al., 2012]. Thus the
nature of highly pH-sensitive swelling brings PEC to the application of
oral drug delivery because the pH varies at each organs or the
diseased part of human body [Li et al., 2013]. Alginate-chitosan
hydrogels (ALG-CHI) have been proposed as drug delivery system in
the past decade, due to their attractive combination of pHsensitivity,
bio-compatibility and adhesiveness, requiring relative mild gelation
conditions for the network formation [Berger et al., 2004 a].Chitosan
based polyelectrolyte complexes have been developed' for local or
systemic administration of drugs and biodrugs. In particular recent
56
trends in research has been focused on the study of'different dosage
forms for oral, buccal, nasal, vaginal and intravenous administration
of'drugs with unfavourable biopharmaceutical properties (peptide and
protein, nucleic acids, antipsychotic substances and antihypertensive
drugs). Great attention has been given to the choice of (1)suitable poly
electrolyte complex: chitosan deacetylation degree and molecular
weight, kind of polyanion (hyaluronic acid, alginic acid, pectin and
gelatin), chitosan/polyanion molar ratio;(2)preparative technologies
(spray drying,freeze drying, film casting and coacervation);(3)
functional properties of the carriers comprising morphological aspect,
size distribution, loading efficiency, swelling ability, mucoadhesion
properties, site specificity, drug release kinetics and drug permeation
across biological membranes [Michaels and Miekka, 1961]. Mixing
oppositely charged polyelectrolytes in solution will result in their self
assembly or spontaneous association due to the formation of strong,
but reversible electrostatic links. These direct interactions between the
polymeric chains lead to the formation of polyelectrolyte complex
networks with non-permanent structures while avoiding the use of
covalent cross-linkers. In general, these polymeric networks or
hydrogels are well tolerated, biocompatible and are more sensitive to
changes in environmental conditions [Berger et al., 2004 a]. The drug
57
release was expected to take place after dissolution of the enteric
coating in the small intestine and biodegradation of the chitosan in the
colon due to presence of polysaccharides in the colonic contents. In
order to prevent early loss of drug from microspheres, the chitosan
was cross linked with glutaraldehyde [Fatima et al., 2006]. The cationic
amino groups on the C2 position of the repeating glucopyranose units
of chitosan can interact electrostatically with the anionic groups
(usually carboxylic acid groups) of other polyions to form
polyelectrolyte complexes. Many different polyanions from natural
origin (e.g. pectin, alginate, carrageenan, xanthan gum, carboxymethyl
cellulose, chondroitin sulphate, dextran sulphate, hyaluronic acid) or
synthetic origin (e.g., poly acrylic acid), polyphosphoric acid, poly (L-
lactide) have been used to form polyelectrolyte complexes with
chitosan in order to provide the required physicochemical properties
for the design of specific drug delivery systems [Berger et al., 2004 b].
Today the stress is on patient compliance and to achieve this objective
there is a spurt in the development of novel and targeted Drug
Delivery System. Use of different polymeric complexes either as to
prepare mucoadhesive microspheres and as polymeric binding ligand
like lectins and agglutinins for colon specific targeting. As the Natural
Polysaccharides are promising biodegradable materials, these can be
58
chemically compatible with the excipients in drug delivery systems. In
addition Natural Polysaccharides are non-toxic, freely available, and
less expensive compared to their synthetic counterparts. They have a
major role to play in pharmaceutical industry. Therefore, in the years to
come, there is going to be continued interest in the natural
polysaccharides to have better materials for drug delivery systems.
Several recent approaches such as pH dependent spray dried
microspheres based on chitosan/pectin complexes for colon delivery
of vancomycin [Bigucci et al., 2008], freeze dried inserts based on
chitosan/hyaluronic acid complexesfornasal delivery of vancomycin
and insulin, freeze dried inserts based on chitosan/pectin complexes
for nasal delivery of chlorpromazine [Luppi et al.,2010], chitosan,
chitosan/tripolyphosphate and chitosan/hyaluronic acid nanoparticles,
obtained by simple and complex coacervation for siRNA delivery
[Luppi et al., 2009], chitosan/gelatine films obtained by film casting for
buccal delivery of propranolol[Abruzzo et al., 2012], freeze dried
inserts based on chitosan/alginate for vaginal delivery of chlorhexidine
and some works in progress like chitosan/hyaluronate films for
transdermal delivery of thiocolchicoside, chitosan / carboxy methyl
cellulose inserts for vaginal delivery of chlorhexidine [Abruzzo et al.,
2013], chitosan nanoparticles for nasal delivery of sodium
59
chromoglicate are definitely recognized as platform for future works in
this field. There are many factors that determine the drug release
behavior from chitosan microspheres. these include molecular weight
and concentration of the chitosan, the cross linking agent used and it’s
concentration, process variables like stirring speed, type of oil,
additives, cross linking process used, drug chitosan ratio, etc. Various
kinetic models have been proposed for the release of drugs from
chitosan microspheres. It was observed that the best fit for release of
drug from chitosan microspheres was obtained by Higuchi equation. It
was also reported that when the release data was subjected to simple
power law equation, the mode of release was foundto be non-fickian
and super case II type [Nair et al., 2009].
60
2. AIM AND OBJECTIVE OF PRESENT STUDY
Oral controlled release formulations for the small intestine and
colon have received considerable attention in the past 25 years for a
variety of reasons including Pharmaceutical superiority and clinical
benefits derived from the drug release pattern that are not achieved
with traditional immediate or sustained release products [Banker,
2002]. Colonic drug delivery has gained increased importance not just
for the delivery of the drugs for the treatment of local diseases
associated with the colon but also for its potential for the delivery of
proteins and therapeutic peptides. Natural polysaccharides have been
used as a tool to deliver the drugs specifically to the colon.
Mucoadhesives must interact with mucin layer during the process of
attachment. The mucous layer is the first surface encountered by
particulate system and its complex structure offers many opportunities
for the development of adhesive interaction with small polymeric
particles either through non specific or specific interaction between
complimentary structures. Colon specific diseases are not efficiently
managed by oral delivery system, because most orally adminsterd
drugs are absorbed before arriving in the colon, therefore colon
specific drug delivery system which can deliver the drug to the lower
gastrointestinal tract without releasing them in the upper GI tract can
61
be expected to increase the patient compliance [Lee et al., 2000].
Therefore microsphere formulations facilitates accurate delivery of
drug to the target site, reduced drug concentration at the sites other
than target organ or tissue, protection of labile compound before and
after administration and prior to appearance at the site of action,
provides sustained release and increase therapeutic effect. This novel
drug delivery system offers vital role in various diseases. Providing
microspheres mucoadhesive property using polymeric enteric coating
makes it more specific in colon targeted and sustained drug effect.
Among different polymers, chitosan is gaining increasing importance in
medical and pharmaceutical applications due to its good
mucoadhesion and absorption enhancing ability, moreover chitosan
shows the ability to form hydrogels able to control the rate of drug
release from the delivery system as well as protect the drug from
chemical and enzymatic degradation in the administration site. In
particular when chitosan is cross linked or complexed with an
oppositely charged polyelectrolyte, a three dimensional network is
formed in which the drug can be incorporated in order to control its
release. By adjusting factors that cause the swelling properties of Poly
Electrolyte Complex (PEC), it is possible to precisely modulate the
drug release to the target site. Because of mucoadhesive properties of
62
chitosan, chitosan-based PEC might give added advantage to
enhance the intestinal absorption of drugs, to prevent the presystemic
metabolism of peptides and to increase the residence time of the
delivery system. Mucoadhesive microspheres, in general, have the
potential to be used for sustained release drug delivery, but coupling
of mucoadhesive properties to microspheres has additional
advantages, e.g. efficient absorption and enhanced bioavailability of
the drugs due to a high surface to volume ratio, a much more intimate
contact with the mucus layer. Mucoadhesive microspheres can be
tailored to adhere to any mucosal tissue including those found in
stomach, thus offering the possibilities of localized as well as systemic
controlled release of drugs. Mucoadhesive microspheres are widely
used because they release the drug for prolonged period, reduce
frequency of drug administration.
Present study comprises elaborate investigation on the effect of
different polymeric combination in form of chitosan alginate
polyelectrolyte complex on preparation and efficacy of mucoadhesive
microspheres as matrix tablet for colon specific delivery of
“balsalazide” to achieve sustained therapeutic profile, targeting colon
for both local and systemic effect.
63
3. REVIEW OF LITERATURES
To achieve desired goal in scientific research never involve any
short cut way to present fate of experiments. Rather it is a long journey
rendering continuous efforts to render real and rational shape from
scientific thoughts arrised in our curious mind comprised with
systematic adoption of all possible standard methods using modern
sophisticated and validated instruments in order to find out exactness
of any hypothesis generated before design of any feasible experiment.
Fulfillment of the purpose needs strong scientific background support
that can be obtained only after gathering huge potential information
backed with previous research work done in the field under study.
Robustness of presentation of new experimental results and their
corresponding discussion can avail perfection only when these are
well supported by evidence of earlier experimental outcomes.
Therefore elaborate, untidy and vivid searching is required to get
proper background support in allied area of colon targeted drug dlivery
system as present investigation in order to reach a successful end
point. Several scientific literatures suggesting different important
aspects needful for present study are arranged in accordance with the
year starting from the recent and cited below as a necessary part of
thesis.
64
Yang et al., (2015) formulated new enrofloxacin microspheres
and examined their physical properties, lung-targeting ability, and
tissue distribution in rats. The microspheres had a regular and round
shape. The mean diameter was 10.06 µm, and the diameter of
89.93% of all microspheres ranged from 7.0 µm to 30.0 µm. Tissue
distribution of the microspheres was evaluated along with a
conventional enrofloxacin preparation after a single intravenous
injection (7.5 mg of enrofloxacin/kg bw). The results showed that the
elimination half-life (t1/2β) of enrofloxacin from lung was prolonged
from 7.94 h for the conventional enrofloxacin to 13.28 h for the
microspheres. Area under the lung concentration versus time curve
from 0 h to ∞ (AUC0-∞) was increased from 11.66 h·µg/g to 508.00
h·µg/g. The peak concentration (Cmax) in lung was increased from
5.95µg/g to 93.36µg/g. Three lung-targeting parameters were further
assessed and showed that the microspheres had remarkable lung-
targeting capabilities.
Kurnool et al., (2015) carried out preparation and evaluation of
microspheres of naturally occurring xanthan gum and guar gum in the
view of effectiveness, biodegradable, easy of availability, cost
effectiveness and drug release rate controlling agent with Lamivudine
as model drug. Lamivudine is an active anti-retroviral drug having
65
biological half life of 4-6 hours and 86% bioavailability and licensed for
the treatment of HIV and chronic Hepatitis B. Compatibility study was
carried out by using FTIR Spectra at the range of 800cm-1 to 3800cm-
1 and shows no significant change in the characteristic peaks of
Lamuvidine and excipients in all the formulation. Microspheres of
Lamuvidine were prepared by solvent evaporation technique using
xanthan gum and guar gum as rate controlling agent. In-vitro drug
release rate was carried out by USP dissolution rate apparatus type-II
and data was subjected to various kinetic models. Microspheres thus
obtained were found to be pale yellow color and free flowing. The
Scanning Electron Microscopy (SEM) studies inferred the spherical
shape and size range of 100μm to 200μm for the total of 9
formulations. In-vitro drug release shows decreases as concentration
of xanthan gum increases and release rate was zero order and Fickian
diffusion controlled. Stability studies were carried out which indicate
that selected formulation was stable. From the results, we conclude
that microspheres offer a practical and suitable approach to prepare
controlled release of Lamuvidine with natural occurring xanthan gum
as rate controlling agent to enhance bioavailability and reduction in
dose frequency.
66
Prabhakar et al., (2015) prepared microspheres of ciprofloxacin
hydro-chloride from biodegradable polymer PLGA (75:25) by using
spray drying technique.Various studies have been reported on
different pulmonary drug delivery system but pulmonary microspheres
are one of the convenient drug delivery system due to its efficiency to
deliver drug. These formulations were studied and compared on the
basis of their polymeric concentration with the help of different
evaluation parameters. Study suggested that by using optimum
concentration of polymers in the formulations of pulmonary
microspheres the alveolar level for drug deposition can be achieved
and spray drying was considered as one of the convenient methods to
formulate pulmonary microspheres.
Subudhi et al., (2015) prepared Eudragit S100 coated Citrus
Pectin Nanoparticles (E-CPNs) for the colon targeting of 5-Fluorouracil
(5-FU). As per their report citrus pectin also acted as a ligand for
galectin-3 receptors that are over expressed on colorectal cancer
cells. Nanoparticles (CPNs and E-CPNs) were characterized for
various physical parameters such as particle size, size distribution,
and shape etc. In vitro drug release studies revealed selective drug
release in the colonic region in the case of E-CPNs of more than 70%
after 24 h. In vitro cytoxicity assay (Sulphorhodamine B assay) was
67
performed against HT-29 cancer cells and exhibited 1.5 fold greater
cytotoxicity potential of nanoparticles compared to 5-FU solution. In
vivo data clearly depicted that Eudragit S100 successfully guarded
nanoparticles to reach the colonic region wherein nanoparticles were
taken up and showed drug release for an extended period of time.
Therefore, a multifaceted strategy is introduced here in terms of
receptor mediated uptake and pH-dependent release using E-CPNs
for effective chemotherapy of colorectal cancer with uncompromised
safety and efficacy.
Pramod et al., (2014) prepared and evaluated colon specific
microspheres of indomethacin for the treatment of colorectal cancer.
Sodium alginate microspheres are prepared by ionotropic gelation
method using different ratios of indomethacin and sodium alginate
(1:1, 1:2, 1:3, 1:4, 2:1, 2:3 & 4:1). Eudragit S-100 coating of
indomethacin and sodium alginate microspheres are performed by
coacervation phase separation technique. The microspheres were
characterized by shape, particle size, size distribution, Entrapment
efficiency, in vitro drug release and stability studies. The outer surface
of core and coated microspheres which was spherical in shape, were
rough and smooth respectively. The size of the core microspheres
ranged from 20 -50 µm and the size of the coated microspheres
68
ranged from 107 – 124 µm. The core microspheres sustained the
release for 10 hrs in a pH progression medium mimicking the condition
of GIT. The release studies of coated microspheres were performed in
a similar dissolution medium as mentioned above. In acidic medium
the release rate was much slower, however the drug was released
quickly at pH 7.4 and their release was sustained upto 24 hrs. It is
concluded from the present investigation that Eudragit coated sodium
alginate microspheres are promising controlled release carriers for
colon targeted delivery of indomethacin.
Hariyadi et al., (2014) investigated effect of polymer and cross
linking agent on the characteristics of ovalbumin-loaded alginate
microspheres. Ovalbumin was selected as a protein model antigen;
barium chloride and calcium chloride were used as cross linking agent.
Ionotropic gelation using aerosolisation and drop technique were
applied in this study. The microspheres produce were characterized
for the size, morphology, encapsulation efficiency, protein loading,
yield and in vitro release. Release of the protein was also studied.
Ovalbumin-loaded alginate microspheres were successfully produced
by aerosolisation with maximum encapsulation efficiency and loadings
of about 89%. Smooth and spherical microspheres were shown for
both alginate microspheres produced using Ca2+ and Ba2+ of the
69
aerosolisation method with average sizes from 12 to 30μm. In case
drop technique, bigger microspheres size was produced of around 1-3
mm. The in vitro release study revealed that protein release decreased
by decreasing alginate concentration, whereas no significant
differences of ovalbumin release by decreasing calcium chloride
concentration. Interestingly, alginate microspheres produced using
barium chloride resulted burst and faster release behaviour of
ovalbumin in HCl pH 1.2 and PBS pH 7.4 release medium. This result
suggested that modification of cross linking agent and polymer
concentration were important for sustained release characteristics of
ovalbumin-loaded alginate microspheres.
Birch and Schiffman (2014) in their recent research work
stated that chronic wounds continue to be a global healthcare concern.
Thus, the development of new nanoparticle-based therapies that treat
multiple symptoms of these “non-healing” wounds without encouraging
antibiotic resistance was imperative. They proposed one potential
solution as to use chitosan, a naturally antimicrobial polycation, which
can spontaneously form polyelectrolyte complexes when mixed with a
polyanion in appropriate aqueous conditions. The requirement of at
least two different polymers opened up the opportunity for all to form
chitosan complexes with an additional functional polyanion. In their
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study, chitosan: pectin (CS: Pec) nanoparticles were synthesized
using an aqueous spontaneous ionic gelation method. Systematically,
a number of parameters, polymer concentration, addition order, mass
ratio, and solution pH, were explored and their effect on nanoparticle
formation was determined. The size and surface charge of the
particles were characterized, as well as their morphology using
transmission electron microscopy. The effect of polymer concentration
and addition order on the nanoparticles was found to be similar to that
of other chitosan: polyanion complexes. The mass ratio was tuned to
create nanoparticles with a chitosan shell and a controllable positive
zeta potential. The particles were stable in a pH range from 3.5 to 6.0
and lost stability after 14 days of storage in aqueous media. Due to the
high positive surface charge of the particles, the innate properties of
the polysaccharides used, and the harmless disassociation of the
polyelectrolytes, they suggested that the development of these CS:
Pec nanoparticles offers great promise as a chronic wound healing
platform.
Zheng et al., (2014) formulated Chitosan-pectin (CS-PT)
microspheres using inverse phase suspension method, with liquid
paraffin and Span 80 as the oil phase, chitosan-pectin acetic acid as
aqueous solution and glutaraldehyde as cross-linker. Based on the
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theory of inverse emulsion polymerization, the optimal conditions were
studied by single-factor test: the reaction temperature was 60 °C,
chitosan-pectin solution of acetic acid was 0.025 g/mL, mCS: mPT =
4:1, the oil were liquid paraffin and a mixture of Span 80, the volume
ratio of oil phase to aqueous was 2:1, the dosage of cross-linker was
0.30 mL, the dosage of Span 80 was 0.6 g, the time of reacting was 3
h. The synthesized chitosan-pectin microspheres are dark yellow and
have smoother appearance and the diameter is about 50 microns. The
structure of microspheres was characterized by FT-IR, Bio-optical
microscope and X-ray diffraction studies. The adsorption of chitosan-
pectin microspheres was good in the solution of methylene blue.
Rani and Paliwal (2014) cited several important aspects
regarding Targeted drug delivery system stating it’s anadvanced
method of delivering drugs to the patients in such a targeted
sequences that increases the concentration of delivered drug to the
targeted body part of interest only (organs/tissues/ cells) which in turn
improves efficacy of treatment by reducing side effects of drug
administration. They also suggested that basically, targeted drug
delivery is to assist the drug molecule to reach preferably to the
desired site. The inherent advantage of this technique leads to
administration of required drug with its reduced dose and reducedits
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side effect. They also mentioned that the inherent advantage of
targeted drug delivery system is under high consideration of research
and development in clinical and pharmaceutical fields as backbone of
therapeutics & diagnostics too. Various drug carriers which can be
used in this advanced delivery system are soluble polymers,
biodegradable microsphere polymers (synthetic and natural),
neutrophils, fibroblasts, artificial cells, lipoproteins, liposomes, micelles
and immune micelle. They concluded with the statement of goal of a
targeted drug delivery system is to prolong, localize, target and have a
protected drug interaction with the diseased tissue.
Jana et al., (2014) investigated the influence of polyelectrolyte
complexes composed of chitosan and pectin on the release behaviour
of aceclofenac. Polyelectrolyte complexes between chitosan and
pectin were prepared at different ratios by mixing solutions of chitosan
and pectin with same ionic strength. The drug entrapment efficiency of
these polyelectrolyte complex microparticles was found 30.29±1.82%
to 77.64±1.85% and their average particle sizes were ranged from
440.75 ± 28.54 to 548.73 ± 41.34 μm. FT-IR spectra were analysed to
study the degree of interactive strength between polyions. The in-vitro
drug release from these aceclofenac-loaded chitosan-pectin
polyelectrolyte complex microparticles showed sustained release of
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aceclofenac over 8 hours and followed the Korsmeyer-Peppas model
(R2= 0.9832–0.9856) with anomalous (non-Fickian) diffusion as drug
release mechanism.
Rathore et al., (2013) formulated and evaluated enteric coated
tablets for Ilaprazole to reduce the gastrointestin al tract side effects.
Four formulations of core tablets were prepared and one who shows
rapid disintegration (near around three mi nutes) was selected for
enteric coating. Ilaprazole which have an irritant effect on the stomach
was coated with a substance that will only dissolve in the small
intestine. Enteric coat was optimized using two different polymers such
as HPMCP 50 and Eudragit L 100 in different concentrations. The
prepared tablets were evaluated in terms of their pre-compression
parameters, physical characteristics and in-vitro release study. 2.5%
seal coating on core tablets was optimized and 9% enteric coating on
seal coated tablets was performed using HPMCP 50 (60%), triethyl
citrate (10%) and IPA: DCM (60:40) which gave the highest dissolution
release profile and f2 value. Seal coating trial was taken on core tablet
of F3 batch. 2.5% seal coating of core tablet was taken as optimize
percentage coating of seal coat as compared to 2% and 3%. Enteric
coating was performed by two different polymers, HPMCP 50 and
Eudragit L100. It was concluded after study that HPMCP 50 was more
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effective as enteric coating polymer at same concentration than
Eudragit L 100 along with 10% Triethyl citrate and 9% enteric coating
on seal coated tablet. As concentration of enteric coating polymer
increases in formulation, acid resistance increases. Moreover 9%
enteric coating on seal coated tablet was optimum to protect core
tablet from acidic environment of stomach in-vivo. Based on f2 value
of optimized batch EC5 when compared with reference product and
developed formulation of delayed release tablet of Ilaprazole was
similar with reference product. From the stability result they made
conclusion that there was no change in the formulation after 1 month
accelerated stability study and prepared delayed release tablet of
proton pump inhibitor was stable.
Ratnaparkhi et al., (2013) prepared Lactose-based placebo
tablets and coated using various combinations of Eudragit L100 and
Eudragit S100, by spraying from aqueous systems. The Eudragit L100
Eudragit S100 combinations (w/w) studied were 1:0, 4:1, 3:2, 1:1, 2:3,
1:4, 1:5 and 0:1. The coated tablets were tested in vitro for their
suitability for pH dependent colon targeted oral drug delivery. The
same coating formulations were then applied on tablets containing
ornidazole as a model drug and evaluated for in vitro dissolution rates
under various conditions. The disintegration data obtained from the
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placebo tablets demonstrate that disintegration rate of the studied
tablets is dependent on: (i) the polymers combination used to coat the
tablets, (ii) pH of the disintegration media, and (iii) the coating level of
the tablets. Dissolution studies performed on the ornidazole tablets
further confirmed that the release profiles of the drug could be
manipulated by changing the Eudragit L100 and Eudragit S100 ratios
within the pH range of 6.0 to 7.0 in which the individual polymers are
soluble respectively, and a coating formulation consisting of a
combination of the two copolymers can overcome the issue of high
gastrointestinal (GI) pH variability among individuals. The results also
demonstrated that a combination of Eudragit L100 and Eudragit S100
can be successfully used from aqueous system to coat tablets for
colon targeted delivery of drugs. For colon targeted delivery of drugs
the proposed combination system is superior to tablets coated with
either Eudragit L100 or Eudragit S100 alone.
Pandey et al., (2013) prepared a polyelectrolyte complex (PEC)
between chitosan (polycation) & pectin (polyanion) and developed
enteric coated tablets for colon delivery using the PEC.These were
prepared using different concentrations of chitosan and pectin. Drug
loaded enteric coated tablets were prepared by wet granulation
method using PEC to sustain the release at colon and coating was
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done with Eudragit S 100 to prevent the early release of the drug in
stomach and intestine. Two independent variable, % PEC
(chitosan/pectin) and % coating were optimized by 32 full factorial
design. Statistical models were also used to supplement the
optimization. DSC was performed to confirm the interaction between
the polyions. Developed formulations were evaluated for physical
appearance, weight variation, thickness, hardness, friability, %
swelling, assay, in-vitro and ex-vivo drug release studies to investigate
the PEC's ability to deliver the drug to colon. Ex-vivo release study
using rat caecal content was also carried out on optimized formulation.
DSC results confirmed chitosan/pectin interaction and subsequent
formation of PEC. The optimized formulation containing 1.1% of PEC
and 3% of coating showed highest swelling and release in alkaline pH
mechanism of which was found to be microbial enzyme dependent
degradation established by ex-vivo study using rat caecal content.
Ofokansi and Kenechukwu (2013) in their study, prepared
tablets by wet granulation based on the IPECs using various
interpolyelectrolyte complexes (IPECs), formed between Eudragit
RL100 (EL) and chitosan (CS) by nonstoichiometric method.They
evaluated as potential oral CTDDSs for ibuprofen (IBF). Colon-
targeted drug delivery systems (CTDDSs) could be useful for local
77
treatment of inflammatory bowel diseases (IBDs). Results obtained
showed that the tablets conformed to compendial requirements for
acceptance and that CS and EL formed IPECs that showed pH-
dependent swelling properties and prolonged the in vitro release of
IBF from the tablets in the following descending order: 3 : 2 > 2 : 3 > 1 : 1
ratios of CS and EL. An electrostatic interaction between the carbonyl
(–CO–) group of EL and amino (–NH2–) group of CS of the tablets
formulated with the IPECs was capable of preventing drug release in
the stomach and small intestine and helped in delivering the drug to
the colon. Kinetic analysis of drug release profiles showed that the
systems predominantly released IBF in a zero-order manner. IPECs
based on CS and EL could be exploited successfully for colon-
targeted delivery of IBF in the treatment of IBDs.
Cunben et al., (2013) prepared a chitosan-carrageenan
polyelectrolyte complex (PEC) by salt induced impeding of polyplex
formation method and it encapsulated bovine serum albumin (BSA) to
study the potential to be tailored to the pH responsive oral delivery of
protein drugs. The FTIR spectra showed the successful formation of
the PEC under the experimental condition. The release kinetics of
BSA from the PEC was studied in the simulated gastrointestinal fluids
with and without digestive enzymes. The prepared PEC showed the
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nature of pH-sensitivity. A typical controlled release of BSA from the
PEC (180 μg of BSA from 3 mg of PEC) was obtained in the simulated
intestinal fluid (SIF, pH 7.5), which was due to the significant swelling
and disintegration of PEC, but little amount of BSA was released
(11 μg of BSA from 3 mg of PEC) in the simulated gastric fluid (SGF,
pH 1.2), confirming acidic stability of the prepared PEC. The presence
of digestive enzymes was found not to affect the response of PEC to
ambient pH value, but to speed up the release of BSA from carriers.
Badhana et al., (2013) prepared, characterized and evaluated
the colon-targeted microspheres of mesalamine for the treatment and
management of ulcerative colitis (UC). Microspheres were prepared by
the ionicgelation emulsification method using tripolyphosphate (TPP)
as cross linking agent. The microspheres were coated with Eudragit S-
100 by the solvent evaporation technique to prevent drug release in
the stomach. The prepared microspheres were evaluated for surface
morphology, entrapment efficiency, drug loading, micromeritic
properties and in-vitro drug release. The microspheres formed had
rough surface as observed in scanning electron microscopy. The
entrapment efficiency of microspheres ranged from 43.72%-82.27%,
drug loading from 20.28%-33.26%. The size of the prepared
microspheres ranged between 61.22-90.41μm which was found to
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increase with increase in polymer concentration. All values are
statistically significant as p<0.05. Micromeritic properties showed good
flow properties and packability of prepared microspheres. The drug
release of mesalamine from microspheres was found to decrease as
the polymer concentration increases. The release profile of
mesalamine from eudragit-coated chitosan microspheres was found to
be pH dependent. It was observed that Eudragit S100 coated chitosan
microspheres gave no release in the simulated gastric fluid, negligible
release in the simulated intestinal fluid and maximum release in the
colonic environment. It was concluded from the study that Eudragit-
coated chitosan microspheres were promising carriers for colon-
targeted delivery of Mesalamine.
Mehta et al., (2013) prepared matrix tablets of naproxen using a
hydrophobic polymer, i.e., Eudragit RLPO, RSPO, and combination of
both, by wet granulation method. The tablets were further coated with
different concentrations of Eudragit S-100, a pH-sensitive polymer, by
dip immerse method. In vitro drug release studies of tablets were
carried out in different dissolution media, i.e., 0.1 N HCl (pH 1.2),
phosphate buffers pH 6.8 and 7.4, with or without rat cecal content.
The swelling studies of the optimized formulation were carried out. The
physicochemical parameters of all the formulations were found to be in
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compliance with the pharmacopoeial standards. The effect of
dissolution medium on the surface of matrix tablet was determined by
using Scanning Electron Microscopy technique. The stability studies of
all formulations were performed as per ICH guidelines. The results
demonstrated that the tablets coated with Eudragit S-100 (2% w/v)
showed a sustained release of 94.67% for 24 h, but drug release
increased to about 98.60% for 24 h in the presence of rat cecal
content while the uncoated tablets released the drug within 5 h. With
regard to release kinetics, the data were best fitted with the Higuchi
model with non-Fickian drug release kinetics mechanism. The stability
studies of tablets showed less degradation during accelerated and
room temperature storage conditions for 6 months. The enteric-coated
Eudragit S-100 coated matrix tablets of naproxen showed promising
site-specific drug delivery in the colon region.
Wajid et al., (2013) described in controlled drug delivery
systems where the drug level in the blood following the profile,
remained constant, between the desired maximum and minimum, for
an extended period of time. They mentioned three primary
mechanisms by which active agents can be released from a delivery
system which includes diffusion, degradation, and swelling. Their
investigation was aimed at using these inexpensive, naturally
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occurring and abundantly available polysaccharides for colon delivery
of 5-fluorouracil. An attempt was made to formulate a dosage form
which consisted of biodegradable polysaccharides as the main
constituent, showed minimal release of 5-fluorouracil in the tracts of
the upper GIT and rapid release in the tracts of the colon. 5-
Fluorouracil is a pyrimidine analogue and is the drug of choice for
colon cancer, it inhibited RNA function and processing and synthesis
of thymidylate. It is administered parenterally since absorption after
ingestion was unpredictable and incomplete. Targeting of 5-
fluorouracil to the colon in cases of colon cancer would not only
reduce the systemic toxicity of the drug but would also show the
desired action in a lesser dose. Nine batches of 5-fluorouracil matrix
tablets were prepared by wet granulation method with different drug-
polymer ratios (1:0.5, 1:1, and 1:1.5) by using guar gum, pectin and
combination guar gum and pectin gum. The prepared formulations
were given enteric coating using Eudragit L-100, S-100 and
combination of both Eudragit L-100 and S-100 (1:2). The tablets were
evaluated with different physicochemical evaluations. The results
indicated the good physicochemical characteristics for matrix tablets.
Singh and Khanna (2012) had their exhaustive review
describing that the oral route of drug administration is the most
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convenient and important method of administering drugs for systemic
effect. Nearly 50% of the drug delivery systems available in the market
are oral D.D.S. and these systems have more advantages due to
patient acceptance and ease of administration. During the last decade
there has been interest in developing site-specific formulations for
targeting drug to the colon. Day by day there appeared new
developments in field of colon specific drug delivery system. Colonic
drug delivery gained increased importance not just for the delivery of
the drugs for the treatment of local diseases associated with the colon
like Crohn’s disease, ulcerative colitis, etc. but also for the systemic
delivery of proteins, therapeutic peptides, anti-asthmatic drugs,
antihypertensive drugs and anti-diabetic agents. New systems and
technologies developed for colon targeting and to overcome pervious
method’s limitations. Colon targeting held a great potential and still
need more innovative work. This review article also discussed, in brief,
introduction of colon along with the novel and emerging technologies
for colon targeting of drug molecule. Colonic drug delivery gained
increased importance not just for the delivery of the drugs for the
treatment of local diseases associated with the colon like Crohn’s
disease, ulcerative colitis, irritable bowel syndrome and constipation
but also for the systemic delivery of proteins, therapeutic peptides,
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antiasthmatic drugs, antihypertensive drugs and antidiabetic
agents.There are various methods or techniques through which colon
drug targeting can be achieved, for example, formation of prodrug,
coating with pH sensitive polymers, coating with biodegradable
polymers, designing formulations using polysaccharides, timed
released systems.
Chunyin et al., (2012) formulated near monodispersed
ibuprofen-loaded superparamagnetic alginate (AL/IBU/FeO)
nanoparticles with particles size less than 200 nm via the facile
heterogeneous coprecipitation of the superparamagnetic FeO
nanoparticles, sodium alginate (AL) and the model drug ibuprofen
(IBU) from the aqueous dispersion. Then the chitosan multilayers were
self-assembled onto the AL/IBU/FeO nanoparticles to produce novel
magnetic-targeted controlled release drug delivery system, with
chitosan as the polycation (CS) and the carboxymethyl chitosan
(CMCS) as the polyanion. The drug controlled releasing behaviors of
the AL/IBU/FeO nanoparticles and the CS multilayers encapsulated
ibuprofen-loaded superparamagnetic alginate ((AL/IBU/FeO)/(CS-
CMCS)) nanoparticles were compared in the different pH media. In
media with the same pH value, the encapsulated vessels exhibited the
slower releasing rate.
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Saravana Kumar et al., (2012) described that development of
new drug molecule is expensive and time consuming. Improving the
safety efficacy ratio of old drugs has been attempted using different
methods such as individualizing drug therapy and therapeutic drug
monitoring. Delivering drug at controlled rate, slow delivery, and
targeted delivery are other very attractive methods and have been
pursued very vigorously. Their work envisaged to reduce the dosing
frequency and improve patient compliance by designing and
evaluating sustained release mucoadhesive microspheres of
Naproxen sodium for effective control of rheumatoid arthritis.
Microspheres were prepared by Ionic gelation technique using sodium
alginate, carbopol 974, and hydroxyl propyl methyl cellulose K15 M
(HPMC) as a mucoadhesive polymers. Microspheres prepared were
found discrete, spherical and free flowing and exhibited good drug
entrapment efficiency. Naproxen sodium release from these
microspheres was slow and extended and dependent on the type of
polymer used. The data obtained thus suggested that mucoadhesive
microspheres could be successfully designed for sustained delivery of
Naproxen sodium and to improve patient compliance.
Gawde et al., (2012) developed Mucoadhesive Microsphere
with Deflazacort as a model drug for Ulcerative colitis of Colon.
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Microspheres were small spherical particles with diameter in
micrometer. Mucoadhesive Microspheres provided prolong residence
time at the site of absorption and facilitated firm contact with the
mucous lining and thus improved the therapeutic performance of the
drug. Deflazacort used for the treatment of Ulcerative Colitis, Crohn’s
disease, Leukaemia. Microsphere delivery of Deflazacort by coating it
with polymer chitosan and cross linked with Glutraldehyde was
improved bioavailability of the drug. The objective of this study was to
protect the drug from prior degradation by converting it into
microspheres and thus achieved sustained release of the drug and to
have maximum therapeutic effect.
Satheesh Madhav et al., (2012) demonstrated in their opinion
that the oral mucosa is an appropriate route for drug delivery systems,
as it can evade first-pass metabolism, enhance drug bioavailability and
thus provides the means for rapid drug transport to the systematic
circulation. This delivery system offers a more comfortable and
convenient delivery route compared with the intravenous route.
Although numerous drugs have been evaluated for oral mucosal
delivery, few of them are available commercially due to limitations
such as the high costs associated with developing such drug delivery
systems. The present review covered recent developments and
86
applications of oral transmucosal drug delivery systems. More
specifically, the review focused on the suitability of the oral soft palatal
site as a new route for drug delivery systems and stated that the
novelistic oral soft palatal platform is important as a promising
mucoadhesive site for delivering active pharmaceuticals, both
systemically and locally, and it can also serve as a smart route for the
targeting of drugs to the brain.
Kaurav et al., (2012) reviewed microspheres that constitute an
important part of novel drug delivery system by virtue of their small
size and efficient carrier capacity. Due to their short residence time,
bioadhesive characteristics can be coupled to microspheres to
develop mucoadhesive microspheres. Bioadhesion can be defined as
the state in which two materials, at least one of which is biological in
nature, are held together for a prolonged time period by means of
interfacial forces. Microspheres were defined as the carrier linked drug
delivery system in which particle size is ranges from 1-1000 μm range
in diameter having a core of drug and entirely outer layers of polymer
as coating material. Mucoadhesive microspheres showed advantages
like efficient absorption and enhanced bioavailability of the drugs due
to a high surface to volume ratio, a much more intimate contact with
the mucus layer, controlled and sustained release of drug from dosage
87
form and specific targeting of drugs to the absorption site. Their report
aimed to provide an overview of various aspects of mucoadhesive
microsphere based on various polymers, methodology of preparation
of mucoadhesive microspheres, method of evaluation and their
applications in drug delivery.
Anbinder et al., (2011) encapsulated natural extracts from this
South American herb, Yerba mate (Ilex paraguariensis) containing a
high amount of polyphenols associated with antiradical activity and
possible benefits for preventing degenerative diseases in calcium
alginate and calcium alginate-chitosan beads to be incorporated as an
additive in food products. The interactions between the active
compound and the polymers were evaluated by Scanning Electron
Microscopy (SEM), thermal analysis (Thermo Gravimetric Assays,
TGA, and Differential Scanning Calorimetry, DSC) and Fourier
Transform Infrared Spectrometry (FT-IR) studies. Also, the effect of
these interactions on extract release in a gastrointestinal model
system was evaluated. Results showed the interactions between the
calcium alginate matrix and the chitosan external layer. Also,
interactions between the natural extract and each polymer were
observed. In both encapsulation systems the highest polyphenol
content was released in simulated gastric fluid. However, capsules
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coated with chitosan allowed releasing a higher amount of polyphenols
into the simulated intestinal fluid. This fact was attributed to both the
protection of the chitosan barrier and the strong interaction between
yerba mate extract and chitosan.
Gupta and Sharma (2011) revewed targeted drug delivery
stating that it is a method of delivering medication to a patient in a
manner that increases the concentration of the medication in some
parts of the body relative to others. Targeted drug delivery seeks to
concentrate the medication in the tissues of interest while reducing the
relative concentration of the medication in the remaining tissues. This
improves efficacy of the while reducing side effects. It is very difficult
for a drug molecule to reach its destination in the complex cellular
network of an organism. They also added that targeted delivery of
drugs, as the name suggests, is to assist the drug molecule to reach
preferably to the desired site. The inherent advantage of this technique
has been the reduction in dose & side effect of the drug. As per their
report research related to the development of targeted drug delivery
system is now a day is highly preferred and facilitating field of
pharmaceutical world. A quantum dot is a semiconductor
nanostructure which is particularly significant for optical applications
due to their theoretically high quantum yield. They mentioned
89
Transdermal devices that allow for pharmaceuticals to be delivered
across the skin barrier. Molecules as diverse as small radiodiagnostic
imaging agents to large DNA plasmid formulations have successfully
been delivered inside FR-positive cells and tissue.
Moy et al., (2011) described in their review that there are
various departments of medicine like cancer, pulmonary, cardiology,
radiology, gynaecology, and oncology etc, numerous drugs are used
and they are delivered by various types of drug delivery system.
Among them microspheric drug delivery system has gained enormous
attention due to its wide range of application as it covers targeting the
drug to particular site to imaging and helping the diagnostic features. It
was also mentioned to have advantage over various other dosage
forms like we know for lungs disease now a days aerolised drugs are
used for local delivery of drugs but it has disadvantage of shorter
duration of action so for sustained release and reducing side effects
and hence to achieve better patient compliance microspheres can be
used. It also has advantage over liposomes as it is physicochemically
more stable. Moreover the microspheres were defined as of micron
size so they can easily fit into various capillary beds which are also
having micron size. The purpose of the review was to compile various
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types of microspheres, different methods to preparation, its
applications and also various parameters to evaluate their efficiency.
Tiwari et al., (2011) developed satranidazole loaded calcium-
pectinate microbeads by ionotropic gelation method. The in-vitro drug
release studies exhibited low drug release at gastric pH, however
continuous release of drug was observed from the formulation at
colonic pH. Further, the release of drug from formulation was found to
be higher in the presence of rat cecal contents, indicating the effect of
colonic enzymes on the calcium pectinate microbeads.
Mahesh et al., (2011) formulated matrix tablets of indomethacin
by wet granulation method using Guar gum as a carrier, 10% starch
paste, HPMC, citric acid and the mixture of talc and magnesium
stearate at 2:1 ratio. Coating was carried out by using 10% Eudragit L
100. All the prepared formulations were evaluated for hardness, drug
content uniformity, stability study and were subjected to in vitro drug
release studies in rat caecal contents. The highest in vitro dissolution
profile at the end of 24 h was shown by IF6 followed by IF7, IF8. The
other formulation IF4, IF3, IF2 and IF1 were failed to target in colon
and these formulation releases the majority of drug within 10 h of
study. It may be due to the less proportion of guar gum to retard the
drug release. The colon targeted matrix tablet of Indomethacin showed
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no change either in physical appearance, drug content or in dissolution
pattern after storage at 30° C/ 65±5 % RH for 2 months.
Giri Prasad et al., (2011) prepared aceclofenac loaded alginate
microspheres by the Ionotropic gelation technique using CaCl2 as
cross-linking agent. The process induced the formation of
microspheres with the incorporation efficiency of 86% to 97%. The
effect of Sodium Alginate, Carbopol, Hydroxy Propyl Methyl Cellulose,
Chitosan concentration and curing time was evaluated with respect to
entrapment efficiency, particle size, surface characteristics and In-vitro
release behaviors. Infrared spectroscopic study confirmed the
absence of any drug-polymer interaction. Differential scanning
calorimetric analysis revealed that the drug was molecularly dispersed
in the Alginate Microspheres matrices showing rough surface, which
was confirmed by Scanning Electron Microscopy Study. The mean
particle size and Entrapment Efficiency were found to be varied by
changing various formulation parameters. The In-vitro release profile
could be altered significantly by changing various formulation
parameters to give a controlled release of drug from the microspheres.
The release data from all the microspheres was found to fit in Power
law of expression (Mt/M∞ = Ktn) and the mechanism of drug release
changed from case-II (or) anomalous transport mechanism to non-
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fickian transport as the alginate was replaced in matrix with other
polymer (or) coated with Chitosan. It was concluded that the release of
the Aceclofenac could be prolonged using binary mixtures (or) coating
alginate microspheres with Chitosan.
Kataria et al., (2011) reviewed microspheres that were
characteristically free flowing powders consisting of proteins or
synthetic polymers having a particle size ranging from 1-1000 μm. The
range of Techniques for the preparation of microspheres offered a
Variety of opportunities to control aspects of drug administration and
enhance the therapeutic efficacy of a given drug. There were various
approaches in delivering a therapeutic substance to the target site in a
sustained controlled release fashion. One such approach was using
microspheres as carriers for drugs also known as microparticles. It is
the reliable means to deliver the drug to the target site with specificity,
if modified, and to maintain the desired concentration at the site of
interest.Microspheres received much attention not only for prolonged
release, but also for targeting of anticancer drugs. In future by
combining various other strategies, microspheres will find the central
place in novel drug delivery, particularly in diseased cell sorting,
diagnostics,gene & genetic materials, safe, targeted and effective in
93
vivo delivery and supplements as miniature versions of diseased
organ and tissues in the body.
Tangri et al., (2011) provided brief idea regarding bioadhesive
delivery systems based on hydrogels to biological surfaces that are
covered by mucus. Techniques that are frequently used to evaluate
the mucoadhesive drug delivery systems are discussed.
Mucoadhesion was defined as a state in which two components, of
which are of biological origin held together for extended periods of
time by the help of interfacial forces. Mucoadhesion is a complex
phenomenon which involved wetting, adsorption and interpenetration
of polymer chains. The concept of mucoadhesion in drug delivery was
introduced in the early 1980s. Thereafter, several researchers have
focused on the investigations of the interfacial phenomena of
mucoadhesion with the mucus. Mucoadhesive drug delivery systems
was described as one of the most important novel drug delivery
systems with its various advantages with a lot of potential in
formulating dosage forms for various chronic diseases.
Senthil et al., (2011) formulated and evaluated the
mucoadhesive microsphere of Glipizide using Hydroxyl Propyl Methyl
Cellulose K4M and Carboxy Methyl Cellulose as polymers. Glipizide
microspheres were prepared by simple emulsification phase
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separation technique using glutaraldehyde as a cross linking agent.
Twenty preliminary trial batches, F1-F20 batches of microspheres
were prepared by using different volume 10 to 70 ml of glutaraldehyde
as cross linking agent, cross linking time 1 to 4 hours and 3:1 ratio of
polymerto- drug with two different polymers. From these twenty
batches of each polymer, the optimized formulation was selected
based on the percentage of mucoadhesion, drug entrapment efficiency
and sphericity of microspheres. A 32 full factorial design was employed
to study the effect of independent variables, polymer-to-drug ratio
(X1), and stirring speed (X2) on dependent variables percentage of
mucoadhesion, drug entrapment efficiency, swelling index and invitro
drug release study. The drug polymer compatibility studies were
carried out using FTIR and the stability studies were conducted for the
optimized formulation. Among the two polymers, the best batch was
Hydroxy propyl methyl cellulose K4M exhibited a high drug entrapment
efficiency of 69% and a swelling index 1.16 % mucoadhesive after
1hour is 70% and the drug release was also sustained for more than
12 hours. The polymer-to-drug ratio had a more significant effect on
the dependent variables.
Mythri et al., (2011) focusesd on polymers used in mucosal
delivery of therapeutic agents. The mucoadhesive drug delivery
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system was reported as a popular novel drug delivery method
because mucous membranes are relatively permeable, allowing for
the rapid uptake of a drug into the systemic circulation and avoiding
the first pass metabolism.They also noted that mucoadhesive
polymers have been utilized in many different dosage forms in efforts
to achieve systemic delivery of drugs through the different mucosa.
These dosage forms include tablets, patches, tapes, films, semisolids
and powders. The objective of this review was to study about novel
mucoadhesive polymers and to design improved drug delivery
systems. They concluded that mucoadhesive drug delivery systems
are gaining popularity day by day in the global pharma industry and a
burning area of further research and development. Extensive research
efforts throughout the world have resulted in significant advances in
understanding the various aspects of mucoadhesion. The research on
mucoadhesives, however, is still in its early stage, and further
advances need to be made for the successful translation of the
concept into practical application in controlled drug delivery system
(CDDS). They also added that there is no doubt that mucoadhesion
has moved into a new area with these new specific targeting
compounds (lectins, thiomers, etc.) with researchers and drug
companies looking further into potential involvement of more smaller
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complex molecules, proteins and peptides, and DNA for future
technological advancement in the ever-evolving drug delivery arena.
Vasconcellos et al., (2011) described in recent years,
biodegradable And biocompatible polymeric microparticles have been
widely studied as potential carriers for controlled delivery of drugs.
Chitosan was reported as a deacetylated derivate of chitin, present in
crustaceans shells shuch as crab and shrimp. Due to its
biocompatibility with human tissues and organs, this material was
considered for several biomedical and pharmaceutical aplications.
Chitosan presented wound healing properties and the incorporation of
other drugs can improve such qualities. Papain was an enzyme that
presented anti-inflammatory and antibacterial properties. Therefore, it
could act improving the healing of injured epithelial tissues. In their
present work, chitosan microparticles were prepared using spraying
and coagulation process. Chitosan microparticles were modified with
papain and crosslinked with glutaraldehyde and sodium
tripoliphosphate. The objective of this work was to evaluate papain
immobilization in chitosan microparticles using different crosslinking
agents. Morphology and spectral structure of microparticles were
studied using Fourier transform infrared spectroscopy (FTIR-ATR),
scanning electron microscopy (SEM) and the amount of release
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papain in pH 7.4 phosphate buffer was measured with (UV)
spectrophotometer.
Asane et al., (2011) investigated the applicability of matrix type
mucoadhesive oral multiple unit systems (MUS) for sustaining the
release of ornidazole in the gastrointestinal tract (GIT).The MUS were
prepared by ionotropic gelation method using chitosan and
hydroxypropyl methyl cellulose K4M (HPMC K4M) according to 32
factorial designs and were evaluated in vitro and in vivo. The particle
size length ranged from 0.78 to 1.30 mm and breadth from 0.76 to
1.30 mm, respectively. The entrapment efficiency was in range of 80
to 96%. The rapid wash-off test was observed faster at intestinal pH
6.8 as compared to acidic pH 1.2. The fluoroscopic study revealed the
retention of MUS in GIT for more than 5 hours. The pharmacokinetic
parameters Cmax, Tmax, mean residence time (MRT) and area under
curve (AUC) of developed MUS were found to be improved
significantly (p<0.05) when compared with marketed immediate
release tablets each containing 500 mg of drug. This study
demonstrated that the MUS could be a good alternative to immediate
release tablets to deliver ornidazole and expected to be less irritating
to gastric and intestinal mucosa.
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Nayak et al., (2011) designed slow release enteric coated solid
formulations to avoid drug release in stomach and upper small
intestine but slowly to build up required drug concentration in the colon
as 5-Fluorouracil being recommended as a chemotherapeutic agent
for colorectal cancer, suffered from severe systemic toxicity and so
needs site-specific delivery.In this context Chitosan microspheres
were prepared by emulsification method using gluteraldehyde as cross
linking agent and then coated with Eudragit S100 by emulsion solvent
evaporation method. The coated microspheres were characterized for
particle size, entrapment efficiency and surface characteristics. In-vitro
drug release profile was studied by changing pH media as per USP
protocol and the data was subjected to kinetic interpretations. The
optimized microspheres showed particle size in the range of 62 to 65
μm with 65 ± 2% drug entrapments. Eudragit coated chitosan
microspheres showed particle size increase upto 390 ± 2 μm with
nearly spherical shape and smooth surface. In vitro drug release
profile of uncoated microspheres was typical like conventional dosage
forms with 38 %, 62 % and 88 % drug release at the end of 2 hrs, 6
hrs and 10 hrs respectively. Coated microspheres showed no drug
release in SGF (2hrs), negligible release (8 %) in 6hrs but substantial
release of 95% in 24 hours in simulated colon media. Drug distribution
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in GI following oral administration of coated microspheres in wistar rats
showed 84% of the drug accumulation in colon.
Arora et al., (2011) in their study, designed novel chitosan-
alginate polyelectrolyte complex (CS-ALG PEC) nanoparticles of
amoxicillin and optimized for various variables such as pH and mixing
ratio of polymers, concentrations of polymers, drug and surfactant,
using 33 Box-Behnken design.The study was undertaken to apply the
concept of nanoparticulate mucopenetrating drug delivery system for
complete eradication of Helicobacter pylori (H. pylori), colonised deep
into the gastric mucosal lining as most of the existing drug delivery
systems have failed on account of either improper mucoadhesion or
mucopenetration and no dosage form with dual activity of adhesion
and penetration has been designed till date for treating H. pylori
induced disorders. Various studies like particle size, surface charge,
percent drug entrapment, in-vitro mucoadhesion and in-vivo
mucopenetration of nanoparticles on rat models were conducted. The
optimised FITC labelled CS-ALG PEC nanoparticles shown
comparative low in-vitro mucoadhesion with respect to plain chitosan
nanoparticles, but excellent mucopenetration and localization was
observed with increased fluorescence in gastric mucosa continuously
over 6 hours, which clinically can help in eradication of H. pylori.
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Suksamran et al., (2011) developed chitosan/alginate
microparticles for the mucosal delivery of allergen from dust mite
(Dermatophagoides pteronyssinus). Chitosan/alginate microparticles
were prepared by ionotropic gelation. The effects of polymer content,
crosslinking agent, and preparation method on the physicochemical
characteristics of the microparticles as well as their in vitro cytotoxicity
were investigated.The microparticles were small (1 - 17 μm) and
spherical in shape. The highest allergen content (0.30 ± 0.07 mg/g)
was obtained with 2.5 % initial allergen loading in chitosan-
triphosphate (CS-TPP) microparticles. Sustained allergen release
(approx. 50 % over 24 h) was observed from alginate-coated chitosan
microparticles. Allergen incorporation method and initial drug-loading
could be varied to obtain optimum particle size with high allergen-
loading and sustained release. The cytotoxicity of various microparticle
formulations did not differ significantly (p > 0.05), as cell viability
values were close to 100 % Conclusion: This study indicates that
alginate and alginate-coated chitosan microparticles are safe and can
be further developed for mucosal allergen delivery.
Malviya and Srivastava (2011) carried out investigation with
aim to synthesize chitosan–alginate polyelectrolyte complex, their
characterization and then formulation of phenytoin sodium fast
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dispersible tablet using polyelectrolyte as active excipient. In this
study, polyelectrolyte complex was formed by ionic cross-linking of
polymers. Dried complex was evaluated for micromeritic properties
and flow behaviour. Tablets were prepared for six batches based on
different proportion of complex viz 5%, 10%, 20%, 30%, 40%, 50%
and 60%. Tablets were evaluated for hardness, friability, thickness, in
vitro disintegration time, in vitro dissolution study and stability study.
Results of micromeritic study and flow behaviour predicted that
complex could be used as an efficient excipient. Hardness, friability,
thickness all were in acceptable limit. Release studies showed that
tablets release drug up to 99.97%. Batch showed 14 sec of invitro
disintegration time. Stability study easily predicted that formulation
characteristics didn’t change during the whole period of study. From
the findings it was con-cluded that chitosan-alginate polyelectrolyte
complex is efficient excipient for fast dispersible formulation especially
required in case of epilepsy and chronic diseases.
Zeng et al., (2011) proposed Controlled release of neurotrophic
factors to target tissue via microsphere-based delivery systems is
critical for the treatment strategies of diverse neurodegenerative
disorders. The present study aimed to investigate the feasibility of the
controlled release of bioactive nerve growth factor (NGF) with ionically
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cross-linked chitosan microspheres (NGF–CMSs). The microspheres
were prepared by the emulsionionic cross-linking method with sodium
tripolyphosphate (STPP) as an ionic cross-linking agent. The size and
distribution of the microspheres, SEM images, Fourier transform infra
red spectroscopy (FT-IR), encapsulation efficiency, in vitro release
tests and bioactivity assay were subsequently evaluated. We found
that the microspheres had relatively rough surfaces with mean sizes
between 20 and 31 µm. FT-IR results provided evidence of ionic
interaction between amino groups and phosphoric groups of chitosan
and STPP. The NGF encapsulation efficiency ranged from 63% to
88% depending on the concentration of STPP. The in vitro release
profiles of NGF from NGF–CMSs were influenced by the concentration
of STPP. NGF–CMSs which were cross-linked with higher
concentration of STPP showed slower but sustained release of NGF.
In addition, the released NGF from NGF–CMSs was capable of
maintaining the viability of PC12 cells, as well as promoting their
differentiation. Findings suggested that NGF–CMSs are capable of
releasing bioactive NGF over 7 days, thus having potential application
in nerve injury repair.
Rajguru et al., (2011) reviewed microspheres stating its
importance in colonic drug delivery that has gained increased
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importance not just for the delivery of the drugs for the treatment of
local diseases associated with the colon but also for its potential for
the delivery of proteins and therapeutic peptides. They informed that
the natural polysaccharides have been used as a tool to deliver the
drugs specifically to the colon. Formulation coated with enteric
polymers releases drug, when pH move towards alkaline range while
as the multicoated formulation passes the stomach, the drug is
released after a lag time of 3-5 hours that is equivalent to small
intestinal transit time. Drug coated with a bioadhesive polymer that
selectively provides adhesion to the colonic mucosa may release drug
in the colon. Historically, the clinical applications of colonic drug
delivery have been limited to the local treatment of inflammatory bowel
disease with little consideration of the possibility for systemic
absorption.The physiology and environmental conditions in the colon
extremely low surface area due to lack of villi and lack of fluid would
seem to support this view. Nevertheless, other local diseases of the
large intestine could benefit from topical delivery to the colonic
mucosa. The potential of the colon for systemic delivery of drugs
including vaccines, proteins and peptides, is gaining renewed interest.
The review was aimed at understanding pharmaceutical approaches
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to colon targeted drug delivery systems for better therapeutic action
without compromising on drug degradation or its low bioavailability.
Gowda et al., (2011) conducted study to minimise the unwanted
side effects of Clozapine (CZ) drug by kinetic control of drug release, it
was entrapped into gastro resistant, biodegradable waxes such as
beeswax (BW) microspheres using meltable emulsified dispersion
cooling induced solidification technique utilizing a wetting agent. Solid,
discrete, reproducible free flowing microspheres were obtained. The
yield of the microspheres was up to 92.4%. The microspheres had
smooth surfaces, with free flowing and good packing properties,
indicating that the obtained angle of repose, % Carr’s index and
tapped density values were well within the limit. More than 95.0% of
the isolated spherical microspheres were in the particle size range of
315-328 μm which were further confirmed by scanning electron
microscopy (SEM) photographs. The drug loaded in microspheres was
stable and compatible, as confirmed by DSC and FTIR studies. The
release of drug was controlled for more than 8 h. Intestinal drug
release from microspheres was studied and compared with the
release behaviour of commercially available formulation Syclop®. The
release kinetics followed different transport mechanisms. The drug
release performance was greatly affected by the materials used in
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microsphere preparations, which allows absorption in the intestinal
tract.
Wu et al., (2010) investigated the factors influencing in-vitro
release characteristics of a model drug 5-fluorouracil from
hydroxypropylmethycellulose (HPMC) compression-coated tablets.
Study revealed that release of drug from the formulations began after
a time delay as a result of hydrogel swelling/retarding effect, followed
by zero-order release. HPMC viscosity, lactose content, and overall
coating weight of outer shell all had significant effect on release lag
time (Tlag) and release rate (k). Increase in HPMC viscosity, lactose
content, and coating weight all lead to increase in Tlag and decrease in
k. Hardness of the compression-coated tablets and pHs of the release
media had little effect on drug release profile. It was concluded that
The HPMC compression coated tablets achieved a release lag time
that was applicable for colon-specific drug delivery of 5-fluorouracil.
Yurdasiper and Sevgi (2010) reviewed different prepared
formulation types of microparticulate systems such as beads,
microbeads, microspheres and microsponges using to special
attention chitosan, alginate and eudragit RS 100. They described that
Chitosan and alginates are natural, anionic or cationic, biocompatible,
biodegradable and non-toxic polymers. They have excellent potential
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for pharmaceutical and biopharmaceutical applications. As per their
information Eudragit RS is an acrylic copolymer and it has well-
established mucoadhesive characteristics. It is still being used as a
sustained release coating materials in pharmaceutical field. Various
techniques used for preparing chitosan, alginate and eudragit RS
microparticles have also been reviewed. This review also included non
steoridal anti-inflammatory drug (NSAID) microparticle formulations
which have been prepared with these polymers to minimize side
effects and to obtain controlled release drug delivery systems.
Moreover, literatures and patents underlined a widespread use of
alginate, chitosan and eudragit RS were covered in this paper.
Parmar et al., (2010) described mucoadhesion as a topic of
current interest in the design of drug delivery system. The oral route of
drug administration constitutes the most convenient and preferred
means of drug delivery to systemic circulation in the body. However
oral administration of most of the drugs in conventional dosage forms
has short-term limitations due to their inability to restrain and localize
the system at gastro-intestinal tract.They also mentioned that
mucoadhesive microsphere can exhibit a prolonged residence time at
the site of application and thereby facilitate an intimate contact with the
underlying absorption surface and thus contribute to improved or
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better therapeutic performance of drug. As per their review
mucoadhesive drug delivery systems promises several advantages
that arise from localization at a given target site, prolonged residence
time at the site of drug absorption and an intensified contact with the
mucosa increasing the drug concentration gradient. Hence, uptake
and consequent bioavailability of the drug is increased and frequency
of dosing can be reduced with the result that patient compliance is
improved. In recent years such Mucoadhesive microspheres have
been developed for oral, buccal, nasal, ocular, rectal and vaginal for
either systemic or local effects. The principles underlying the
development of mucoadhesive microsphere and research work carried
out on these systems were reviewed in their work.
Raval et al., (2010) outlined utilization of biodegradable
polymers for controlled drug delivery has gained immense attention in
the pharmaceutical and medical device industry to administer various
drugs, proteins and other biomolecules both systematically and locally
to cure several diseases. The efficacy and toxicity of this local
therapeutics depends upon drug release kinetics, which will further
decide drug deposition, distribution, and retention at the target site.
Drug Eluting Stent (DES) presently possesses clinical importance as
an alternative to Coronary Artery Bypass Grafting due to the ease of
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the procedure and comparable safety and efficacy. Many models have
been developed to describe the drug delivery from polymeric carriers
based on the different mechanisms which control the release
phenomenon from DES. Advanced characterization techniques
facilitate an understanding of the complexities behind design and
related drug release behavior of drug eluting stents, which aids in the
development of improved future drug eluting systems. This review
discusses different drug release mechanisms, engineering principles,
mathematical models and current trends that are proposed for drug-
polymer coated medical devices such as cardiovascular stents and
different analytical methods currently utilized to probe diverse
characteristics of drug eluting devices.
Dash et al., (2010) demonstrated that over the past few
decades, significant medical advances have been made in the area of
drug delivery with the development of controlled release dosage
forms. There are large variety of formulations devoted to oral
controlled drug release, and also the varied physical properties that
influenced drug release from these formulations. In their discussion
the release patterns were divided into those that release drug at a
slow zero or first order rate and those that provide an initial rapid dose,
followed by slow zero or first order release of sustained component. In
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this work they reviewed the mathematical models used to determine
the kinetics of drug release from drug delivery systems. The
quantitative analysis of the values obtained in dissolution/release rates
was found easier when mathematical formulae were used to describe
the process. The mathematical modeling ultimately helped to optimize
the design of a therapeutic device to yield information on the efficacy
of various release models. The purpose of the controlled release
systems is to maintain drug concentration in the blood or in target
tissues at a desired value as long as possible. In other words, they
were able to exert a control on the drug release rate and duration.
Morris et al., (2010) in their review described that chitosans and
pectins are natural polysaccharides which show great potential in drug
delivery systems. As per their report Chitosans are a family of strongly
polycationic derivatives of poly-N-acetyl-D-glucosamine. This positive
charge is very important in chitosan drug delivery systems as it plays a
very important role in mucoadhesion (adhesion to the mucosal
surface). Other chitosan based drug delivery systems involved
complexation with ligands to form chitosan nanoparticles which can be
used to encapsulate active compounds. Pectins were made of several
structural elements the most important of which were the
homogalacturonan (HG) and type I rhamnogalacturonan (RG-I)
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regions often described in simplified terms as the “smooth” and “hairy”
regions respectively. Pectin HG regions consisted of poly-glacturonic
acid residues which can be partially methyl esterified. Pectins with a
degree of methyl esterification (DM) > 50% are known as high
methoxyl (HM) pectins and consequently low methoxyl (lM) pectins
had a DM < 50%. Additionally low methoxyl pectins were reported as
polymer of particular interest in drug delivery as they can form gels
with calcium ion (Ca2+) which has potential applications especially in
nasal formulations.
Grabnar et al., (2010) in their study designed pectin-chitosan
polyionic nanocomplexes, which form through interactions between
the carboxyl groups of pectin and the amine groups of
chitosan.Pectins were reported as anionic, soluble polysaccharides
extracted from the primary cell walls of plants. They formed gels by
controlled calcium-mediated interchain association to give an
extended, uniformly regular junction zone, presumably similar to that
depicted in the eggbox model proposed for calcium alginate. Chitosan
was described as the cationic deacetylated form of chitin obtained
from exoskeletons of marine arthropods and is widely used in NP
preparation.The main scope of the current study was the design,
formulation and physicochemical characterization of pectin-chitosan
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NPs. Such complexes are suitable for protein drug incorporation and
mucosal delivery. Protein ovalbumin (OVA) was used as a model drug.
Recent advances in nanotechnology applied to proteins were directed
towards safer and simpler methods of preparation, using naturally
occurring polymers such as alginate, pectin and chitosan. In this study,
pectin-chitosan nanoparticles (NPs) were designed by the mild
process of polyelectrolyte complexation, which occurs at room
temperature without using sonication or organic solvents. NPs with a
mean diameter between 300 and 400 nm and 45 to 86% protein
association efficiency were obtained by varying the pectin: chitosan
mass ratio and initial protein concentration. A prolonged release profile
without burst effect of investigated ovalbumin from pectin-chitosan
NPs was determined.
Hamman (2010) described their review work stating Chitosan as
the subject of interest for its use as a polymeric drug carrier material in
dosage form design due to its appealing properties such as
biocompatibility, biodegradability, low toxicity and relatively low
production cost from abundant natural sources. However, one
drawback of using this natural polysaccharide in modified release
dosage forms for oral administration was its fast dissolution rate in the
stomach. Since chitosan is positively charged at low pH values (below
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its pKa value), it spontaneously associated with negatively charged
polyions in solution to form polyelectrolyte complexes. It was the aim
of this review to describe complexation of chitosan with selected
natural and synthetic polyanions and to indicate some of the factors
that influence the formation and stability of these polyelectrolyte
complexes. These chitosan based polyelectrolyte complexes exhibited
favourable physicochemical properties with preservation of chitosan’s
biocompatible characteristics. As per their opinion these complexes
were therefore good candidate excipient materials for the design of
different types of dosage forms. Furthermore, recent investigations
into the use of these complexes as excipients in drug delivery systems
such as nano- and microparticles, beads, fibers, sponges and matrix
type tablets were briefly described.
Dhawale et al., (2010) reported the treatment of colon cancer
has been aimed by approaches of oral drug administration and for 5-
Fluorouracil as a candidate to be delivered orally to the colon they
used pH - sensitive polymers Eudragit S 100 and L 100 to prepare
microspheres by a simple oil /water emulsification process. Process
parameters were analyzed in order to optimize the drug loading and
release profiles. In further attempts mixtures with Eudragit S100 and
L100 were prepared to prolong drug release. Scanning electron
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microscopy permitted a structural analysis. The solvent extraction was
preferable over solvent evaporation with a view to the encapsulation
rate (extraction: 37%; evaporation: 19%) due to the hydrophilic
character of the drug while release pattern were nearly unchanged.
Eudragit S100, pure or in mixture, was found to retain drug release at
pH 4.5 lower than 41% within 6 h. At pH 7.4, nearly immediate release
(within 30 min) was observed for pure S100, while mixtures enabled to
prolong the release slightly. Analysis of the morphology led to an
inhomogeneous polymer distribution of S100 and L 100 throughout the
particle core. However, the formulation proved its applicability in-vitro
as a promising device for pH-dependent colon delivery of 5-
fluorouracil. The whole study was aimed to develop the porous
microspheres, which can control the drug release up to 6 h and hence
it can prevent the acid decomposition in stomach.
Aberu et al., (2010) formulated Alginate-chitosan (ALG-CHI)
microspheres by polyelectrolyte complexation are pH-sensitive,
biocompatible and adhesive, and are excellent candidates for the
delivery of drugs, proteins and peptides in the human body. A wide
variety of methods for the production of these polymeric complexes
was provided. The water-in-oil emulsion was a complex production
method, but generally enhances the control of particle size and particle
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size distribution of the microspheres, extremely necessary for
obtaining repeatable controlled release behavior. In this work, a novel
and facile water-in-oil emulsion method for the ALG-CHI
polyelectrolyte complexes was discussed. The method proposed
produced ALG-CHI microspheres with improved morphology and
enhanced drug loading in comparison with the aqueous medium
method. The drug loading in the water-in-oil emulsion was over 30%
higher than in the aqueous medium. It was an indication that the new
method proposed the common drug leaching during the microspheres’
preparation was avoided, being an interesting alternative to
encapsulate drugs of hydrophilic nature.
Deore et al., (2009) prepared Ketoprofen microspheres by
solvent diffusion technique using Aerosil as an inert dispersing carrier
to improve the dissolution rate of ketoprofen, and Eudragit RS as a
retarding agent to control the release rate. The microspheres were
found to be spherical. The average diameters were about 104-108μm
and the drug contents in the microspheres were 62-96%. The
concentration of Eudragit affects the release rate of ketoprofen and as
concentration of eudragit increased the release rate of ketoprofen
decreased. Dissolution profile showed that the release followed
Higuchi matrix model kinetics. The results of X-ray diffraction and
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thermal analysis reveal the conversion of crystalline drug to
amorphous. These results indicated that ketoprofen microspheres
could be prepared providing a sustained release property.
Jin et al., (2009) established a method for preparing protein
microspheres for oral administration. Using bovine serum albumin
(BSA) as a model protein and alginate and chitosan as carrier
materials, BSA loaded alginate/chitosan microspheres were prepared
by a modified emulsifying-gelatinization method. The infuence of the
preparation conditions on the encapsulation eficiency, drug loading
and yield of the microspheres was investigated by an orthogonal
design method and the optimal process parameters were obtained.
The in vitro release of BSA from the alginate/chitosan microspheres
was investigated in 0.1 M HCl solution (pH 1.2) and PBS (pH 7.4) as
the release media. The optimal process parameters for the preparation
of BSA loaded alginate/chitosan microspheres were obtained. The
concentrations of alginate solution, CaCl2 solution and chitosan
solution were 1.5% (w/v), 6% (w/v) and 2% (w/v), respectively, and the
cross-linking time was 10 min. The mean particle size of the
microspheres was 3 μm, the encapsulation efficiency was 81.4 ±
1.5%, and the drug loading was 6.4% ± 0.1%. The in vitro release of
BSA from the alginate/chitosan microspheres prepared with the
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optimal process showed that the initial burst release was marked both
in 0.1 M HCl solution (pH 1.2) and PBS (pH 7.4). The release in PBS
(pH 7.4) was faster than that in 0.1 M HCl solution (pH 1.2). The
modified emulsifying-gelatinization method is suitable for the
preparation of protein-loaded microspheres for oral administration.
Prabu et al., (2009) performed formulation and evaluation of
oral sustained release of Diltiazem Hydrochloride using rosin as matrix
forming material.Rosin being a natural resin was used as a
hydrophobic matrix material for the controlled release of diltizem HCL.
Matrix tablets were prepared by direct compression method using
rosin as matrix forming material in different ratio. The rosin was useful
in developing sustained release matrix tablets and it prolonged the
release of water soluble drug up to 24h.
Dashora and Jain (2009) in their study prepared a novel
microparticulate formulation of prednisolone, which was adequate for
the treatment of ulcerative colitis.The formulations prepared were
evaluated in vitro. Two types of pectin microspheres containing
prednisolone named, pectin-prednisolone microspheres (PPMS) and
pectin prednisolone eudragit microspheres (PPEMS), were prepared
by an emulsion-dehydration technique and o/o solvent evaporation
method respectively with some modifications. Various process
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variables as stirring speed, stirring time, as well as formulation
variables i.e.polymer concentration and emulsifier concentration were
optimized to get small uniform and spherical discrete microspheres. In
vitro drug release studies were performed in presence of simulated
gastric fluid, simulated gastric and intestinal fluid, and simulated
intestinal fluid respectively, in presence or absence of rat caecal
content. By coating the microspheres with eudragit S100 pH
dependent release profiles were obtained The cumulative percent drug
release of prednisolone from pectin microspheres in SGF and SIF
after 4 hrs were varied from 30- 45% and from eudragit coated
microspheres after 4 hrs it varied from 6.25 to 8.95% respectively.
Further, the release of drug was observed higher in the presence of rat
caecal contents, indicating the susceptibility of pectin to colonic
enzymes released from rat caecal content.
Argin-Soysal et al., (2009) formulated Polyelectrolyte hydrogels
by xanthan gum and chitosan can be used for encapsulation and
controlled release of food ingredients, cells, enzymes, and therapeutic
agents. In this study, xanthan–chitosan microcapsules were formed by
complex coacervation.The effects of initial polymer concentration and
chitosan solution pH on the crosslinking density of xanthan–chitosan
network were investigated by swelling studies and modulated
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differential scanning calorimetry (MDSC) analysis. The crosslinking
density was found to be less dependent on chitosan solution
concentration than xanthan solution concentration and chitosan pH.
The capsules were completely crosslinked at all conditions studied
when initial xanthan solution concentration was 1.5% (w/v). The
changes in the conformation of chitosan chains as chitosan pH
approaches 6.2 were found to be important in achieving capsule
network structures with different crosslinking densities. These findings
indicated that the parameters studied cannot be viewed as
independent parameters, as their effects on the degree of swelling are
interdependent.
Patil et al., (2009) prepared Mucoadhesive microspheres by an
interpolymer complexation poly(acrylic acid) (PAA) with poly(vinyl
pyrrolidone) (PVP) to increase gastric residence time and a solvent
diffusion method. The complexation between poly(acrylic acid) and
poly(vinyl pyrrolidone) as a result of hydrogen bonding was confirmed
by the shift in the carbonyl absorption bands of poly(acrylic acid) using
FT-IR. A mixture of ethanol/water was used as the internal phase, corn
oil was used as the external phase of emulsion, and span 80 was used
as the surfactant. Spherical microspheres were prepared with the
particle size increased as the content of water was increased. The
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mean particle size increased with the increase in polymer
concentration. The adhesive force of microspheres was equivalent to
that of Carbopol. The release rate of atenolol from the complex
microspheres was slower than the PVP microspheres at pH 2.0 and
6.8.In conclusion they demonstrated mucoadhesive microsphere
prepared by a solvent evaporation and interpolymer complexation
method characterized the dissolution rate of the complex
microspheres was significantly retarded when compared with that of
the PVP microspheres, particularly at pH 2.0. The results of this study
indicated that it might be feasible to use PAA/PVP mucoadhesive
microspheres as a gastric retentive drug delivery system for
antihypertensive action. The release rate of the Beta-blockers agents
could be retarded due to the slower dissolution rate of the complex
polymer.
Quiros et al., (2009) conducted a multicenter, double-blind
study to evaluate the safety, efficacy and pharmacokinetics of
balsalazide in pediatric patients with mild-to-moderate UC.In this study
sixty-eight patients, 5 to 17 years of age, with mild-to-moderate active
UC based on the modified Sutherland UC activity index (MUCAI), were
randomized to receive oral balsalazide 2.25 or 6.75 g/day for 8 weeks.
The primary endpoint was clinical improvement (reduction of the
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MUCAI score by ≥3 points from baseline). Clinical remission (MUCAI
score of 0 or 1 for stool frequency) and histological improvement after
8 weeks were also assessed. Pharmacokinetic parameters for
balsalazide, 5-aminosalicylic acid, and N-acetyl-5-aminosalicylic acid
were determined at 2 weeks. Adverse events and laboratory changes
were monitored throughout the study.Clinical improvement was
achieved by 45% and 37% of patients and clinical remission by 12%
and 9% of patients receiving 6.75 and 2.25 g/day, respectively.
Improvement in histologic grade was achieved by 8 of 16 (50%) and 3
of 10 (30%) patients receiving 6.75 and 2.25 g/day, respectively. No
significant differences were seen in efficacy. Pharmacokinetics in 12
patients were characterized by large inter-subject variability and low
systemic exposure. Adverse events were similar between the
treatment groups, the most common being headache and abdominal
pain.No clinically significant changes were observed in laboratory
values, including those indicative of hepatic or renal toxicity.They
concluded that Balsalazide is well-tolerated and improves the signs
and symptoms of mild-to-moderate active UC in pediatric patients 5 to
17 years of age.
Rassu et al., (2008) formulated ketoprofen spray-dried
microspheres that were affected by the long drug recrystallization time.
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Polymer type and drug–polymer ratio as well as manufacturing
parameters affect the preparation. The purpose of this work was to
evaluate the possibility to obtain ketoprofen spray-dried microspheres
using the Eudragit® RS and RL; the influence of the spray-drying
parameters on morphology, dimension, and physical stability of
microspheres was studied. Ketoprofen microspheres based on
Eudragit® blend was prepared by spray-drying and the nebulization
parameters did not influence significantly particle properties;
nevertheless, they could be affected by drying and storage methods.
No effect of the container material was found.
Nunthanid et al., (2008) developed colonic drug delivery based
on a combination of time-, pH-, and enzyme-controlled system. A
combination of Spray-dried chitosan acetate (CSA) and hydroxypropyl
methylcellulose (HPMC) was used as compression-coats for 5-
aminosalicylic acid (5-ASA) tablets. Factors affecting in-vitro drug
release, i.e. % weight ratio of coating polymers, enzyme activity, pH of
media, and excipients in core tablets, were evaluated. The tablets
compression-coated with HPMC:CSA at 60:40 and 50:50% weight
ratio providing lag times about 5–6 h were able to pass through the
stomach (stage I, 0.1 N HCl) and small intestine (stage II, pH 6.8,
Tris–HCl). The delayed release was time- and pH-controlled owing to
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the swelling with gradual dissolving of CSA and HPMC in 0.1 N HCl
and the less solubility of CSA at higher pH. After reaching the colon
(stage III, pH 5.0, acetate buffer), the dissolution of CSA at low pH
triggered the drug release over 90% within 14 h. Furthermore, the
degradation of CSA by b-glucosidase in the colonic fluid enhanced the
drug release.
Quan et al., (2008) aimed their study using Eudragit–cysteine
conjugate to coat on chitosan microspheres (CMs) for developing an
oral protein drug delivery system, having mucoadhesive and pH-
sensitive property. Bovine serum albumin (BSA) as a protein model
drug was loaded in thiolated Eudragit-coated CMs (TECMs) to study
the release character of the delivery system. After thiolated Eudragit
coating, it was found that the release rate of BSA from BSA-loaded
TECMs was observably suppressed at pH 2.0 PBS solution, while at
pH 7.4 PBS solution the BSA can be sustainingly released for several
hours. The structural integrity of BSA released from BSA-loaded
TECMs was guaranteed by sodium dodecylsulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and circular dichroism (CD)
spectroscopy. The mucoadhesive property of TECMs was evaluated
and compared with CMs and Eudragit-coated chitosan microspheres
(ECMs). It was confirmed that after coating thiolated Eudragit, the
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percentage of TECMs remained on the isolated porcine intestinal
mucosa surface was significantly higher than those of CMs and ECMs.
Likewise, gamma camera imaging of Tc-99m labeled microsphere
distribution in rats after oral administration also suggested that TECMs
had comparatively stronger mucoadhesive characters. The results
indicated that TECMs have potentials to be an oral protein drug
carrier.
Zhao et al., (2008) investigated Colon-specific drug delivery
systems (CDDS) can improve the bioavailability of drug through the
oral route. A novel formulation for oral administration using pH-enzyme
Di-dependent chitosan mcirospheres (MS) and 5-Fu as a model drug
for colon-specific drug delivery by the emulsification/chemical
crosslinking and coating technique, respectively. The influence of
polymer concentration, ratio of drug to polymer, the amount of
crosslinking agent and the stirring speed on the encapsulation
efficiency, particle size in microspheres were evaluated. The best
formulation was optimized by an orthogonal design. Drug release
studies under conditions mimicking stomach to colon transit have
shown that the drug was protected from being released in the
physiological environment of the stomach and small intestine. The
plasma concentrations of 5-Fu after oral administration of coated
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chitosan MS to rats were determined and compared with that of 5-Fu
solution. The in vivo pharmacokinetics study of 5-Fu loaded pH-
enzyme Di-dependent chitosan MS showed sustained plasma 5-Fu
concentration–time profile. The in vitro release correlated well with the
pharmacokinetics profile. The results clearly demonstrated that the
pH-enzyme Di-dependent chitosan MS is potential system for colon-
specific drug delivery of 5-Fu.
Saether et al., (2008) formulated Polyelectrolyte complexes
(PECs) of alginate and chitosan by addition of 0.1% alginate solution
(pH 6.5) to 0.1% chitosan solution (pH 4.0), and by adding the
chitosan solution to the alginate solution under high shearing
conditions. Variations in the properties of the polymers and the
preparation procedure were studied, and the resultant PEC size, zeta
potential (Zp), and pH were determined using dynamic light scattering
(DLS), electrophoresis and by measuring turbidity and pH. Tapping
mode atomic force microscopy (AFM) was used to examine some of
the complexes. The particle size was decreased as the speed and
diameter of the dispersing element of the homogenizer was increased.
The net charge ratio between chitosan and alginate, and the molecular
weights (MW) of both the alginate and chitosan samples were the
most significant parameters that influenced the particle size, Zp, and
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pH. The mixing order also influenced the size of the PECs however
the Zp and pH were not affected by the mixing order. The stability of
the complexes was investigated by incubation at an elevated
temperature (37oC), storage for one month at 4 oC, alteration of the pH
of the PEC mixture, and addition of salt to physiological ionic strength
(0.15 M NaCl). The properties of the PEC could be affected according
to the molecular properties of the polyelectrolytes selected and the
preparation procedures used. The resultant PEC sizes and properties
of the complex were rationalised using a core-shell model for the
structure of the complexes.
Patil and Moss (2008) informed in their review that 5-
aminosalicylates remain the first-line treatment for patients with
ulcerative colitis. As per their report, numbers of formulations are
available for the treatment of active ulcerative colitis, including
encapsulated mesalazine and mesalazine in combination with other
molecules. Balsalazide was described as an aminosalicylate prodrug
that releases mesalazine in the colon, thus exerting its multiple anti-
inflammatory effects in areas of colitis. This review examined the
pharmacological and therapeutic features of balsalazide as an anti-
inflammatory agent in ulcerative colitis including the introduction of
novel aminosalicylate formulations and an appreciation of their
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molecular mode of action, and thus renewed interest in these agents
in both maintenance of disease remission and cancer prevention.
Ma et al., (2008) reported the development of
microencapsulated unit of bacteriophage Felix O1 for oral delivery
using a chitosan-alginate-CaCl2 system. In vitro studies were used to
determine the effects of simulated gastric fluid (SGF) and bile salts on
the viability of free and encapsulated phage. Free phage Felix O1 was
found to be extremely sensitive to acidic environments and was not
detectable after a 5-min exposure to pHs below 3.7. In contrast, the
number of microencapsulated phage decreased by 0.67 log units only,
even at pH 2.4, for the same period of incubation. In this study the
viable count of microencapsulated phage decreased only 2.58 log
units during a 1-h exposure to SGF with pepsin at pH 2.4. After 3 h of
incubation in 1 and 2% bile solutions, the free phage count decreased
by 1.29 and 1.67 log units, respectively, while the viability of
encapsulated phage was fully maintained. Encapsulated phage was
completely released from the microspheres upon exposure to
simulated intestinal fluid (pH 6.8) within 6 h. The encapsulated phage
in wet microspheres retained full viability when stored at 4°C for the
duration of the testing period (6 weeks). With the use of trehalose as a
stabilizing agent, the microencapsulated phage in dried form had a
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12.6% survival rate after storage for 6 weeks. The current
encapsulation technique enabled a large proportion of bacteriophage
Felix O1 to remain bioactive in a simulated gastrointestinal tract
environment, which indicated that these microspheres may facilitate
delivery of therapeutic bacteriophage to the gut.
Lawrie et al., (2007) investigated alginate−chitosan
polyelectrolyte complexes (PECs) in the form of a film, a precipitate,
as well as a layer-by-layer (LbL) assembly with the focus to fully
characterize, using the complementary techniques of Fourier
transform infrared (FTIR) spectroscopy and X-ray photoelectron
spectroscopy (XPS) in combination with solution stability evaluation,
the interactions between alginate and chitosan in the PECs. In the
FTIR spectra, no significant change was noticed in the band position
of the two carbonyl vibrations from alginate occurs upon interaction
with different ionic species. However, protonation of the carboxylate
group caused a new band to appear at 1710 cm-1, as anticipated.
Partial protonation of the amine group of chitosan caused the
appearance of one new band (∼1530 cm-1) due to one of the −NH3+
vibrational modes (the other mode overlaps the amide I band).
Importantly, the position of the two main bands in the spectral region
of interest in partly protonated chitosan films was not dependent on
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the extent of protonation. XPS N 1s narrow scans can, however, used
to assess the degree of amine protonation. In our alginate−chitosan
film, precipitate, and LbL assembly, the bands were observed in the
FTIR corresponding to the species −COO- and −NH3+, but their
position was not different from each of the single components. They
stated in the conclusion of the study that FTIR cannot be used directly
to identify the presence of PECs. However, in combination with XPS
(survey and narrow N 1s scans) and solution stability evaluation, a
more complete description of the structure can be obtained. This
conclusion challenged the assignment of FTIR spectra in the previous
literature.
Bonartsev et al., (2007) formulated Novel biodegradable
microspheres on the base of poly(3-hydroxybutyrate) (PHB) designed
for controlled release of antithrombotic drug, namely dipyridamole
(DPD), and kinetically studied. The profiles of release from the
microspheres with different diameters 4, 9, 63, and 92 µm presented
the progression of nonlinear and linear stages. Diffusion kinetic
equation describing both linear (PHB hydrolysis) and nonlinear
(diffusion) stages of the DPD release profiles from the spherical
subjects was written down as the sum of two terms: desorption from
the homogeneous sphere in accordance with diffusion mechanism and
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the zero-order release. In contrast to the diffusivity dependence on
microsphere size, the constant characteristics (k) of linearity were
scarcely affected by the diameter of PHB microparticles. The view of
the kinetic profiles as well as the low rate of DPD release were in
satisfactory agreement with kinetics of weight loss measured in vitro
for the PHB films. Taking into account kinetic results, they supposed
that the degradation of both films and PHB microspheres was
responsible for the linear stage of DPD release profiles. As per their
prediction in the nearest future, combination of biodegradable PHB
and DPD as a representative of proliferation cell inhibitors will give
possibility to elaborate the novel injectable therapeutic system for a
local, long-term, antiproliferative action.
Jain et al., (2007) developed multiparticulate system, hydrogel
beads, combining the pH-sensitive property of enteric polymers as well
as the biodegradability of chitosan in the colon for targeting delivery of
satranidazole for the treatment of amoebiasis. Chitosan hydrogel
beads were prepared by the crosslinking method followed by enteric
coating with Eudragit S100. The amount of the drug released after 24h
from the formulation was found to be 97.67% in the presence of
extracellular enzymes as compared with 64.71% and 96.52% release
of drug after 3 and 6 days of enzyme induction, respectively, in the
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presence of 4% cecal content. Degradation of the chitosan hydrogel
beads in the presence of extracellular enzymes as compared with rat
cecal and colonic enzymes indicated the potential of this
multiparticulate system to serve as a carrier to deliver macromolecules
specifically to the colon and could be offered as a substitute in-vitro
system for performing degradation studies. Studies demonstrated that
orally administered chitosan hydrogel beads can be used effectively
for the delivery of drug to the colon.
Rahman et al., (2006) formulated Core microspheres of alginate
with 5-fluorouracil by modified emulsification method in liquid paraffin
followed by cross linking with calcium chloride. These core
microspheres were coated with Eudragit S-100 by solvent evaporation
technique. Drug release was sustained for upto 20 hours in
formulations with core microspheres to Eudragit coat ratio of 1:7 and
no change in size, shape and drug content were observed.
Kim and Pack (2006) in their famous book chapter described
about the development of precision particle fabrication (PPF)
technology that allowed the production of uniform microspheres and
double-wall microspheres capable of efficiently encapsulating model
drugs. Of these primary importance was the ability of monodisperse
microsphere formulations to eliminate initial drug burst while
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modulating the onset of steady drug release. Modified PPF technology
had also been established as a single-step method for producing
uniform polymeric microcapsules of controllable size and shell
thickness. Monodisperse system defined particle size distributions can
be achieved while maintaining the desired polymeric shell thickness.
Exact control of the volumetric flow-rates of the core and shell
materials also allowed the formation of particle populations exhibiting
discretely or incrementally increasing shell thickness. Controlled
release systems, especially those comprising biodegradable polymer
microparticles heavily studied and thus reached the clinic in several
cases. However, notable limitations especially in controlling delivery
rates were mentioned. Monodisperse PPF microspheres and core-
shell microparticles offered advantages in reproducibility, control, and
consistency that may provide valuable assistance in designing
advanced drug delivery systems. The alternative electro-hydrodynamic
method called flow-limited field-injection electrostatic spraying
technique that provided a simple and robust technique for fabricating
devices with a precisely defined nano-structure from a broad range of
biocompatible polymeric materials. It was established as capable of
producing nanometer-scale solid particles as small as 10 nm or even
smaller, and may be applicable to fabrication of nanocapsules.
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However, further refined and development were proposed as need to
achieve precise control of the particle size and reproducibly fabricate
nanocapsules, the technology. The release of macromolecules
typically exhibited an initial “burst” of drug, which was as much as 10–
50% of the drug load, followed by a “lag” phase exhibiting slow release
and finally a period of steady release.
Bernabé et al., (2005) prepared membranes of the
polyelectrolyte complex between chitosan and pectin by precipitating
the complex from a mixture of both polysaccharides. It was shown that
the swelling kinetics of these membranes to follow a Fickean behavior.
The membranes were heated at 120 °C in order to convert the –NH3 + -
OOC- salt bonds into amide bonds. The thermally treated membranes
were stable in strongly acid and basic media. The extent of amide
bond formation was followed by FTIR spectroscopy. It was found that
as the reaction time increased, both the absorbance ratio A1744/A1082
and the maximum swelling of the membrane decreased. The surface
morphology of the membranes did not vary appreciably with the
thermal treatment.
Ribeiro et al., (2005) prepared Alginate microspheres by
emulsification/internal gelation were chosen as carriers for a model
protein, hemoglobin (Hb).Reinforced chitosan-coated microspheres
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were obtained by an uninterrupted method, in order to simplify the
coating process, minimize protein losses during production and to
avoid Hb escape under acidic conditions. Microspheres recovery was
evaluated as well as its morphology by determination of Hb
encapsulation efficiency and microscopic observation, respectively.
The formation of chitosan membrane made of it interaction with
alginate was assessed by DSC (differential scanning calorimetry) and
FT-IR (Fourier-transform infrared spectrometry) studies. Spherical
uncoated microspheres with a mean diameter of 20µm and
encapsulation efficiency above 89% were obtained. Coated
microspheres provided similar encapsulation efficiency but a higher
mean diameter was obtained due to microspheres clumping during the
coating step. Protein loss occurred mainly during emulsification rather
than recovery. FT-IR and DSC together indicated electrostatic
interactions between alginate carboxylate and chitosan ammonium
groups as the main forces for complex formation.Hb release from
microspheres showed a pH-dependent profile and was affected by
chitosan coating. Under simulated gastric conditions, a total Hb burst
release fromuncoatedmicrosphereswas decreasedwith one-stage and
two-stage chitosan coatings (68%and 28%, respectively).At pH 6.8,
the Hb release from coated microspheres was fast but incomplete.
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These results suggested an optimization of the coating method to
protect Hb under acidic conditions and to permit a complete but
sustained release of Hb.
Sinha et al., (2004) in their exhaustive review described that
chitosan is a biodegradable natural polymer with great potential for
pharmaceutical applications due to its biocompatibility, high charge
density, non-toxicity and mucoadhesion. It was shown not only to
improve the dissolution of poorly soluble drugs but also to exert a
significant effect on fat metabolism in the body. Gel formation was
obtained by interactions of chitosans with low molecular counterions
such as polyphosphates, sulphates and crosslinking with
glutaraldehyde. This gelling property of chitosan allowed a wide range
of applications such as coating of pharmaceuticals and food products,
gel entrapment of biochemicals, plant embryo, whole cells,
microorganism and algae. This review was an insight into the
exploitation of the various properties of chitosan to microencapsulate
drugs. Various techniques for preparing chitosan microspheres and
evaluation of these microspheres were also reviewed. Moreover this
review also included the factors affecting the entrapment efficiency
and release kinetics of drugs from chitosan microspheres.
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Chourasia et al., (2004) combined pH dependent and
biodegradable approach for colon-targeted delivery of metronidazole.
The multiparticulate system was prepared by coating cross-linked
chitosan microspheres with Eudragit L-100 and S-100 as pH-sensitive
polymers. In-vitro drug-release studies were performed in conditions
simulating stomach-to-colon transit in presence and absence of rat
caecal contents. No release was observed at acidic pH; however,
when it reached the colon pH where Eudragit starts solublizing there
was continuous release of drug from the formulation. Due to the
susceptibility of chitosan matrix to colonic enzymes release of drug
was found to be higher in the presence of rat caecal contents.
Cheng et al., (2004) developed Time- and pH-dependent colon-
specific drug delivery systems (CDDS) for orally administered
diclofenac sodium (DS) and 5-aminosalicylic acid (5-ASA). DS tablets
and 5-ASA pellets were coated by ethylcellulose (EC) and methacrylic
acid copolymers (Eudragit® L100 and S100), respectively. Release
profile of time-dependent DS coated tablets was not influenced by pH
of the dissolution medium. On the contrary release profile of pH
dependent 5-ASA coated pellets was significantly governed by pH.
The absorption kinetic studies of the DS coated tablets in dogs
demonstrated that in-vivo lag time of absorption was in a good
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agreement with in-vitro lag time of release. It was concluded that on
using regular coating techniques also colon specific drug delivery can
be obtained.
Sandborn et al., (2004) determined the pharmacokinetic
parameters of 5-aminosalicylic acid and : N-acetyl-5-aminosalicylic
acid from equimolar doses of 5-aminosalicylic acid administered as
Asacol and balsalazide as existing pharmacokinetic data were
insufficient to determine whether a delayed-release formulation of
mesalamine (Asacol) results in greater systemic exposure to 5-
aminosalicylic acid and its major metabolite N-acetyl-5-aminosalicylic
acid than a prodrug (balsalazide). Nineteen healthy volunteers
completed an open-label, single-dose: randomized, crossover study
comparing the pharmacokinetics of 5-aminosalicylic acid and N-acetyl-
5-aminosalicylic acid from equimolar doses of 5-aminosalicylic acid
(800 mg) administered as Asacol (800 mg) and balsalazide (2250 mg).
Plasma and urine samples were analysed for 5-aminosalicylic acid, N-
acetyl-5-aminosalicylic acid, and balsalazide (urine only) using high-
performance liquid chromatography methods with mass spectrometric
detection. Pharmacokinetic parameters assessed for 5-aminosalicylic
acid and N-acetyl-5-aminosalicylic acid included: percentage of dose
excreted in urine (Ae%), area under the plasma concentration–time
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curve (AUCtlast); and maximum plasma concentration (Cmax).The
geometric mean total (5-aminosalicylic acid and : N-acetyl-5-
aminosalicylic acid) urinary excretion values (Ae%) of Asacol and
balsalazide were 19.26 and 19.31% (P = 0.98). The geometric mean
Ae% values of 5-aminosalicylic acid for Asacol and balsalazide were
0.39 and 0.37% (P = 0.78); the geometric mean Ae% values of N-
acetyl-5-aminosalicylic acid for Asacol and balsalazide were 18.78 and
18.83% (P = 0.98). The geometric mean 5-aminosalicylic acid
AUC(tlast) values for Asacol and balsalazide were 3295 and
3449 ng h/mL (P = 0.85); the geometric mean N-acetyl-5-amino
salicylic acid AUC(tlast) values for Asacol and balsalazide were 15 364
and 16 050 ng h/mL (P = 0.69). The geometric mean 5-5-
aminosalicylic acid Cmax values for Asacol and balsalazide were 319
and 348 ng/mL (P = 0.80); the geometric mean N-acetyl-5-
aminosalicylic acid Cmax values for Asacol and balsalazide 927 and
1009 ng/mL (P = 0.67). They demonstrated in conclusions that the
systemic absorption of 5-aminosalicylic acid and : N-acetyl-5-
aminosalicylic acid from Asacol and balsalazide are comparable based
upon plasma pharmacokinetic parameters and urinary excretion
values.
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Simsek-Ege et al., (2003) in their study revealed the interaction
between chitosan and alginate at different pH values by means of a
particular method for Fourier transform infrared (FTIR) studies. A
previously reported disagreement between the yield of the complexes
in weight and density of the interacting functional groups was
explained through this method.They explained various uses of
Chitosan and alginate as two important polyelectrolytes viz. as
thickening agents in the food industry, in drug-release systems in
pharmaceutical applications as biomaterials in wound healing, and cell
culture applications, or as ion exchange material for the removal of
heavy metal ions from industrial wastewaters. These two
polysaccharides can also be used together to form a polyelectrolyte
complex, especially to encapsulate proteins, cells, and enzymes.
Although there are many applications of these polyions, few
publications explained the interaction between their functional groups.
This is mostly because of the difficulty of following ionic interaction in
an interface of macromolecules, especially since they altered much
with the reaction conditions such as pH. The results were supported
with the morphological studies of the polyelectrolyte beads prepared at
different pH values. Freeze-dried beads of both alginate and chitosan-
coated alginate beads could be viewed after hexamethyl disilazane
(HMDS) treatment.
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Sinha et al., (2002) carried out the study to develop a single
unit, site-specific drug formulation allowing targeted drug release in the
colon. Tablets were prepared using polysaccharides or synthetic
polymer as binders. These included xanthan gum, guar gum, chitosan
and Eudragit E. Indomethacin was used as a model drug. The
prepared tablets were enteric-coated with Eudragit L100 to give
protection in the stomach. The coated tablets were tested in vitro for
their suitability as colon-specific DDS. The drug release studies were
carried out in simulated stomach environment (pH 1.2) for 2 h followed
by small intestinal environment at pH 6.8. The study shows that
chitosan could be successfully used as a binder, for colon targeting of
water insoluble drugs in preference to guar gum when used in the
same concentration. In addition, it was also found that aqueous
dispersions of Eudragit L100–Eudragit S100 combinations can
efficiently be used for coating tablets for colon targeted delivery of
drugs, and that the formulation can be adjusted to deliver drug(s) at
any other desirable site of the intestinal region of the GI tract in which
pH of the fluid is within the range 6.0–7.0. For colon targeted delivery
of drugs, the proposed combination system was superior to tablets
coated with either Eudragit L100 or Eudragit S100 alone.
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Gonza´lez-Rodriguez et al., (2002) prepared Alginate/chitosan
particles by ionotropic gelation (Ca2+ and Al3+) for the sodium
diclofenac release. The drug encapsulation yield was more than 98%,
and the efficacy was neither affected by the alginate amount nor the
crosslinking ion used. Thus, this method was useful to encapsulate
ionic drugs with high water solubility. The use of Ca2+ resulted
acceptable sphericity and a notable surface porosity. The morphology
of the particulates prepared with Al3+ ions didn’t exhibited spherical
morphology the particles were flattened, disk-shaped with a collapsed
center. The trivalent ions caused more points of aggregation between
two contiguous alginate chains, binding them strictly and quickly that
they can’t be in spherical forms during their formation. The
neutralization between oppositely charged alginate and chitosan
decreased the solubility of the alginate/chitosan particles. This
mechanism gave rise to the high efficiency of the microsphere
formation in depleting diclofenac from the solution. At pH 6.4, a rapid
increase of the release rate was observed because the deprotonation
of the alginic acid causes the disintegration of the microsphere
systems and the increasing deprotonation of chitosan weakened the
extent of the interactions inside the microspheres.
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Muijsers et al., (2002) described in their eminent report that the
aminosalicylate balsalazide is a prodrug which is metabolised by
bacterial azo reductases in the colon to release its therapeutically
active moiety mesalazine [mesalamine (US) or 5-aminosalicylic acid]
and an inert carrier molecule. The systemic absorption of balsalazide
and its metabolites is not required for the therapeutic efficacy of the
drug, and has been demonstrated to be limited.Data from well
designed trials with a duration of 8 to 12 weeks show that oral
balsalazide 6.75 g/day is as effective as (two trials) or more effective
than (one trial) oral delayed-release (pH-dependent) mesalazine 2.4
g/day and appears to be as effective as oral sulfasalazine 3 g/day in
the treatment of active mild-to-moderate ulcerative colitis. In addition,
balsalazide appears to have a faster onset of action than
mesalazine.Furthermore, balsalazide was as effective as delayed-
release mesalazine (dosages used were 1.2 and 1.5 g/day, where 1.6
g/day is recommended) and oral sulfasalazine 2 g/day (recommended
dosage) in the prevention of relapse in ulcerative colitis in remission
after 6 to 12 months of treatment; the balsalazide dosage was 3 g/day
versus mesalazine and 2 g/day versus sulfasalazine. Although not well
established, additional benefits may be achieved with balsalazide
dosages up to 6 g/day.Whereas data from well designed, 2- to 12-
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month trials show that balsalazide is well tolerated by patients with
ulcerative colitis in both acute and maintenance indications, and is
better tolerated than standard formulations of sulfasalazine at
therapeutically relevant dosages. Their conclusion mentioned that
Balsalazide is a well tolerated and effective first-line therapeutic option
for patients with ulcerative colitis, both for the treatment of active mild-
to-moderate disease and as maintenance therapy to prevent disease
relapse.
Uekama et al., (1994) prepared cross linked chitosan
microspheres of 5 -Fluorouracil and studied for their suitability to colon
specific delivery. Polysaccharide based systems underwent enzymatic
degradation in colon and chitosan is selected here as the matrix
material to deliver the drug to colon. Eudragit S-100 was used as an
enteric coating material to keep the microspheres intact and not to
release the drug in stomach and or upper intestine. In contrast to
single unit systems like tablets for oral use, multiple unit systems like
microspheres were administered here as they show marked
advantages like spreading over a large area in colon and avoiding
exposure of high drug concentrations to a confined part of colonic
mucosa.
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4. DRUG AND EXCIPIENTS PROFILE
4.1. Drug profile: Balsalazide [Colazide Monograph, 1998]
4.1.1. Structure and General information
Fig. 2: Structural formula of Balsalazide disodium
Balsalazide disodium has the chemical name (E)-5-[[-4-[[(2-
carboxyethyl) amino] carbonyl] phenyl] azo]-2-hydroxybenzoic acid,
disodium salt, dihydrate. Its structural formula is:
Molecular Weight: 437.32
Molecular Formula: C17H13N3O6Na2 ·2H2O
Balsalazide disodium is a stable, odorless orange to yellow
microcrystalline powder. It is freely soluble in water and isotonic saline,
sparingly soluble in methanol and ethanol, and practically insoluble in
all other organic solvents.
4.1.2. Clinical Pharmacology
Balsalazide disodium is delivered intact to the colon where it is
cleaved by bacterial azoreduction to release equimolar quantities of
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parent drug mesalamine, which is the therapeutically active portion of
the molecule, and 4-aminobenzoyl-ß-alanine. The recommended dose
of 6.75 grams/day, for the treatment of active disease, provides 2.4
grams of free 5-ASA to the colon. The 4-aminobenzoyl-ß-alanine
carrier moiety released when balsalazide disodium is cleaved is only
minimally absorbed and largely inert. The mechanism of action of 5-
ASA is unknown, but appears to be local to the colonic mucosa rather
than systemic mucosal production of arachidonic acid metabolites,
both through the cyclooxygenase pathways, i.e., prostanoids, and
through the lipoxygenase pathways, i.e., leukotrienes and
hydroxyeicosatetraenoic acids, is increased in patients with chronic
inflammatory bowel disease, and it is possible that 5-ASA diminishes
inflammation by blocking production of arachidonic acid metabolites in
the colon. High-dose balsalazide is superior to low-dose balsalazide
and to low-dose delayed-release 5-ASA for maintenance of remission
in ulcerative colitis [Kruis et al., 2001] and has similar efficacy to
sulfasalazine for this indication [Mansfield et al., 2002]
4.1.3. Pharmacokinetics
Balsalazide disodium is insoluble in acid and designed to be
delivered to the colon as the intact prodrug. Upon reaching the colon,
bacterial azoreductases cleave the compound to release 5-ASA, the
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therapeutically active portion of the molecule, and 4-aminobenzoyl-ß-
alanine. 5-ASA is further metabolized to yield N-acetyl-5-aminosalicylic
acid (N-Ac-5-ASA), a second key metabolite.
I. Absorption: The maximum plasma concentration (Cmax) and the
time at which Cmax is observed (tmax) were obtained by visual
inspection. The lag time before the onset of drug absorption (tlag) was
determined as the time-point prior to the occurrence of the first
quantifiable concentration. The terminal exponential half-life (t1/2,z) was
calculated as 0.693/m, where m is the slope of the natural log
concentration–time plots. The terminal slope, m, was determined by
visual inspection of concentration–time data plotted on a semi-log
scale. The area under the plasma concentration–time curve from time
0 to the last quantifiable concentration (AUCtlast) was determined from
time 0 to the last quantifiable concentration using the linear trapezoidal
rule. The area under the curve from time 0 to infinity was calculated as
sum of AUC t-last and AUC extrapolated. AUCextrapolated was calculated by
dividing the last quantifiable plasma concentration by the slope of the
natural log-concentration–time plots. The cumulative percentage of
dose excreted in urine (Ae%) was calculated by adding the percentage
of the dose excreted in each interval [ Johnson et al., 2003].The
plasma pharmacokinetics of balsalazide and its key metabolites from a
146
crossover study in healthy volunteers. In this study, a single oral dose
of balsalazide was administered to healthy volunteers as intact
capsules (3 x 750 mg) under fasting conditions, as intact capsules (3 x
750 mg) after a high-fat meal, and unencapuslated (3 x 750 mg) as
sprinkles on applesauce.A relatively low systemic exposure was
observed under all three administered conditions (fasting, fed with
high-fat meal, sprinkled on applesauce), which reflects the variable,
but minimal absorption of balsalazide disodium and its metabolites.
The data indicated that both Cmax and AUClast were lower, while tmax
was markedly prolonged, under fed (high-fat meal) compared to fasted
conditions. Moreover, the data suggested that dosing balsalazide
disodium as a sprinkle or as a capsule provides highly variable, but
relatively similar mean pharmacokinetic parameter values. No
inference was made as to how the systemic exposure differences of
balsalazide and its metabolites in this study might predict the clinical
efficacy under different dosing conditions (i.e., fasted, fed with high-fat
meal, or sprinkled on applesauce) since clinical efficacy after
balsalazide disodium administration is presumed to be primarily due to
the local effects of 5-ASA on the colonic mucosa [Sandborn et al.,
2003]. In a study of patients with mild-to-moderate active ulcerative
colitis receiving three 750-mg Balsalazide capsules 3 times daily (6.75
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g/day) for 8 weeks, steady state was reached within 2 weeks. In a
separate study of ulcerative colitis, patients received balsalazide, 1.5
grams twice daily, for over 1 year. Systemic drug exposure, based on
mean AUC values, was up to 60 times greater (8 ng.hr/mL to 480
ng.hr/mL) after equivalent multiple doses of 1.5 grams twice daily
when compared to healthy subjects who received the same dose.
II. Distribution: The binding of balsalazide to human plasma proteins
was 99%.
III. Metabolism: The products of the azoreduction of this compound,
5-ASA and 4-aminobenzoyl-ß-alanine, and their N-acetylated
metabolites have been identified in plasma, urine and feces.
IV. Elimination: After cleavage of the balsalazide azo-bond by
bacterial azo-reductase enzymes, most of the free 5-ASA released
into the colonic lumen is then absorbed into the colonic epithelium,
where it undergoes extensive metabolism to N-acetyl-5-ASA (N-Ac-5-
ASA) by the enzyme N-acetyltransferase 1 (NAT 1) [Allgayer et al.,
1989]. From the colonic epithelium, N-Ac-5-ASA is either absorbed
systemically into the blood and then excreted in the urine, or secreted
back into the lumen by the membrane-bound drug efflux pump P-
glycoprotein and excreted in the faeces [Zhou et al., 1999]. Following
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single-dose administration of 2.25 gms Balsalazide (three 750-mg
capsules) under fasting conditions in healthy subjects, mean urinary
recovery of balsalazide, 5-ASA, and N-Ac-5-ASA was 0.20%, 0.22%
and 10.2%, respectively. In a multiple dose study in healthy subjects
receiving a Balsalazide dose of two 750 mg capsules twice daily (3
g/day) for 10 days, mean urinary recovery of balsalazide, 5-ASA, and
N-Ac-5-ASA was 0.1%, 0%, and 11.3%, respectively. During this
study, subjects received their morning dose 0.5 hours after being fed a
standard meal and subjects received their evening dose 2 hours after
being fed a standard meal [Green et al., 2002]. In a study with 10
healthy volunteers, 65% of a single 2.25 gram dose of Balsalazide was
recovered as 5-ASA, 4-aminobenzoyl-ß-alanine, and the N-acetylated
metabolites in feces, while <1% of the dose was recovered as parent
compound. In a study that examined the disposition of balsalazide in
patients who were taking 3-6 grams of Balsalazide daily for more than
one year and who were in remission from ulcerative colitis, less than
1% of an oral dose was recovered as intact balsalazide in the urine.
Less than 4% of the dose was recovered as 5-ASA, while virtually no
4-aminobenzoyl-ß-alanine was detected in urine. The mean urinary
recovery of N-Ac-5-ASA and N-acetyl-4-aminobenzoyl-ß-alanine
comprised <16% and <12% of the balsalazide dose, respectively. No
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fecal recovery studies were performed in this population [Chan et al.,
1983].
4.1.4. Indications and Usage
Balsalazide is indicated for the treatment of mildly to moderately
active ulcerative colitis. Safety and effectiveness of Balsalazide
beyond 12 weeks has not been established.
I.Therapeutic indications
Balsalazide is indicated for treatment of mild-to-moderate active
ulcerative colitis and maintenance of remission. 2.25g Balsalazide
disodium three times daily (6.75g daily) until remission or for 12 weeks
maximum. Rectal or oral steroids can be given concomitantly if
necessary.
II. Maintenance treatment
The recommended starting dose is 1.5g Balsalazide disodium (2
capsules) twice daily (3g daily). The dose can be adjusted based on
each patient's response; there may be an additional benefit with a
dose upto 6g daily. For elderly patients no dose adjustment is
anticipated but for Children Balsalazide is not recommended. But
Balsalazide is well-tolerated and improves the signs and symptoms of
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mild-to-moderate active Ulcerative colitis (UC) in pediatric patients 5 to
17 years of age [Quiros et al., 2009]. Balsalazide has a reduced side
effect profile relative to that observed with sulfasalazine. Following 8
weeks of treatment, balsalazide has been shown to be more effective
and more rapid in onset than mesalamine in improving signs and
symptoms of acute UC [Mc Intyre et al., 1998]. In January, 2007, the
Food and Drug Administration (FDA) approved balsalazide for
treatment of mild-to-moderate active UC in pediatric patients 5 to 17
years of age.
4.1.5. Contraindications
Balsalazide is contraindicated in patients with hypersensitivity to
salicylates or to any of the components of Balsalazide capsules or
balsalazide metabolites.
4.1.5.1. Precautions
Of the 259 patients treated with Balsalazide 6.75 grams/day in
controlled clinical trials of active disease, exacerbation of the
symptoms of colitis, possibly related to drug use, has been reported by
3 patients.
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I. General: Patients with pyloric stenosis may have prolonged gastric
retention of Balsalazide capsules.
II. Renal: At doses up to 2000 mg/kg (approximately 21 times the
recommended 6.75 grams/day dose on a mg/kg basis for a 70 kg
person), Balsalazide had no nephrotoxic effects in rats or dogs. Renal
toxicity has been observed in animals and patients given other
mesalamine products. Therefore, caution should be exercised when
administering Balsalazide to patients with known renal dysfunction or a
history of renal disease [Green et al., 2001].
4.1.6. Drug Interactions
No drug interaction studies have been conducted for
Balsalazide, however, the use of orally administered antibiotics could,
theoretically, interfere with the release of mesalamine in the colon.
Formal interaction studies have not been performed with Balsalazide.
Available data suggest that the systemically available amounts of
balsalazide and its metabolites may be increased if balsalazide is
administered in the fasting as compared with the fed state. Therefore,
balsalazide should preferably be administered with food. The
acetylated metabolites of balsalazide are actively secreted in the renal
tubule to a high degree. Therefore, plasma levels of co-prescribed
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drugs also eliminated by this route may be raised and this should be
noted in the case of those with a narrow therapeutic range, such as
methotrexate. Pharmacodynamic interactions have not been studied.
However, while balsalazide, mesalazine, and N-acetylmesalazine are
salicylates chemically, their properties and kinetics make classical
salicylate interactions such as those found with acetylsalicylic acid
very unlikely [Salix Pharmaceuticals, 2000].
4.1.7 Adverse reactions
Vital signs (blood pressure, temperature, pulse and respiration)
were obtained at screening and daily for 4 days following each
administration of study drug. Adverse events were monitored
throughout the study and were classified according to the COSTART
dictionary [COSTART, 1995]. Over 1000 patients received treatment
with Balsalazide in domestic and foreign clinical trials. In four
controlled clinical trials, patients receiving a Balsalazide dose of 6.75
grams/day most frequently reported the following events (reporting
frequency 3%), headache (8%), abdominal pain (6%), diarrhoea (5%),
nausea (5%), vomiting (4%), respiratory infection (4%), and arthralgia
(4%). Withdrawal from therapy due to adverse events was comparable
among patients on Balsalazide and placebo [Levine et al., 2002].
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4.1.8. Dosage and Administration
For Treatment of Active Ulcerative Colitis, the usual dose in
adults is three 750 mg Balsalazide capsules to be taken three times a
day for a total daily dose of 6.75 grams for duration of 8 weeks. Some
patients in the clinical trials required treatment for upto 12
weeks.Balsalazide capsules may also be administered by carefully
opening the capsule and sprinkling the capsule contents on
applesauce.
4.1.9. Overdosage
No case of overdose has occurred with Balsalazide. A 3-year-old
boy is reported to have ingested 2 grams of another mesalamine
product. He was treated with ipecac and activated charcoal with no
adverse reactions. If an overdose occurs with Balsalazide use,
treatment should be supportive, with particular attention to correction
of electrolyte abnormalities. A single oral dose of balsalazide disodium
at 5 grams/kg or 4-aminobenzoyl-ß-alanine, a metabolite of
balsalazide disodium, at 1 gram/kg was non-lethal in mice and rats. No
symptoms of acute toxicity were seen at these doses [Mansfield et al.,
2002].
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4.2. Excipients profile
A brief description of different important excipients used in
present study was provided in order to cite a complete picture showing
possibility of fruitful combination in design of oral dosage form.
4.2.1. Chitosan
4.2.1.1. Structure and properties of chitosan
Chitosan is (1-4)-2-amino-2-deoxy-B-D glucon. It has similar
structural characteristics as that of glucosaminoglycans. It is tough,
biodegradable and nontoxic. Chitin, poly-B-(1-4) linked N acetyl –D-
glucosamine is a highly hydrophobic material that is insoluble in water
and most ordinary solvents. This property of chitin restricts its use to
application that do not require solubilization of the polymer.
Considering chitosan as a weak base, a certain minimum amount of
acid is required to transform the glucosamine units into the positively
charged, water soluble form. After deacetylation of chitin, the chitosan
obtained is dissolved in acid, filtered and the precipitate is washed and
dried to get amine free chitosan. The structural formula of chitosan is
presented in Fig. 3.
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Fig. 3: Structural formula of Chitosan
At neutral pH most chitosan molecules will lose their charge and
precipitate from solution. Chitosan is soluble in dilute organic acids like
formic, acetic, propionic, oxalic, malonic, succinic, adipic, lactic,
pyruvic, malic, tartaric and citric. Chitosan is a cationic polymer, which
is the second most abundant polymer in nature after cellulose. Chitin
is the primary structural component of chitos and also found in many
other species such as molluscs, insects and fungi. The most
commonly obtained form of chitosan is the shrimp shell wastes
[George and Abraham, 2006]. Chitosan is also soluble in dilute nitric
and hydrochloric acids, marginally soluble in 0.5% phosphoric acid
and insoluble in sulfuric acid at room temperature. Formic acid is the
best solvent, overall good solutions being obtained in aqueous
systems containing 0.2 to 100% of this acid. Acetic acid has been
selected as the standard solvent for solution property measurement.
Chitosan readily dissolves in 3:1 glycerol water when the mixture
contains 1% acetic acid, resulting in clear colorless and very viscous
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solution [Rao and Sharma, 1997]. Solutions of Chitosan in 10% w/v
aqueous oxalic acid show thermo reversible gel property. A solution
containing more than 7 % chitosan will gel in less than a day and 3%
solution will gel in about 3 weeks. The chitosan films were cross-linked
by glutaraldehyde vapors in a closed chamber for 24 hrs at ambient
temperature. This process was done to retard the chitosan
degradation rate. The decrease in degradation rate of cross linked
chitosan was probably due to the retarded hydrolysis of Schiff’s bases
induced by the glutaraldehyde cross linked of chitosan’s amino
groups. Chitosan a linear polyelectrolyte at acidic pH, is soluble in
variety of acids and interacts with polyanionic counterions. It forms
gels with a number of multivalent anions and also with glutaraldehyde.
It has a high charge density i.e. one charge per glucosamine unit.
Since many minerals carry negative charges, the positive charge of
chitosan interacts strongly with negative surfaces. Chitosan is a linear
polyamine where amino groups are readily available for chemical
reactions and salt formation with acids. The important characteristics
of chitosan are its molecular weight, viscosity, deacetylation degree
(DA) crystallinity index, number of monomeric units (n), water retention
value, pka and energy of hydration.
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4.2.1.2. Biological and chemical properties of chitosan
Biocompatibility (e.g. Nontoxic, biodegradable, natural),
bioactivity, wound healing acceleration, reduced blood cholesterol
levels, and immune system stimulant effect. Biomedical properties,
biological activity and biodegradation of chitosan are stated by
Knapczyk [Knapczyk, 1993]. Muzzarellia [Muzzarellia et al., 2004]
explained the chemical behavior of chitosan and modified chitosan
whereas Sandford summarized the chemical and biological properties
of chitosan that relate to applications [Sandford, 2003].
4.2.1.3. Pharmaceutical requirements of chitosan
Particle size < 30 μm, density between 1.35 and 1.40 g/cc, pH
6.5-7.5, insoluble in water, and partially soluble in acids. Chitosan can
also be characterized in terms of its quality, intrinsic properties and
physical forms. The quality characteristics of chitosan are levels of
heavy metals and proteins, pyrogenicity and degree of deacetylation
are the intrinsic properties.
4.2.1.4. Mucoadhesive properties of chitosan
Lehr first evaluated mucoadhesive properties of chitosan. A
number of characteristics are necessary for mucoadhesion (a) strong
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hydrogen bonding groups (-OH, -COOH), (b) strong anionic charges,
(c) high molecular weight, (d) sufficient chain flexibility, and (e) surface
energy properties favoring spreading on to mucus. However, chitosan
is a poly-cationic polymer and does not have any anionic charge.
Instead, a positively charged hydrogel is formed in acidic environment
that could develop additional molecular attractive forces by
electrostatic interactions with negatively charged mucosal surfaces or
the negatively charged sialic acid groups of the mucus network. High
molecular weight chitosan gave the best mucoadhesive properties
[Lehr et al. 1992].
4.2.1.5. Toxicological studies of chitosan
In-vivo toxicity tests indicated that chitosan is non toxic, inert and
sterilized films were free of pyrogens. LD 50 and oral toxicity levels of
chitosan were estimated in both rats and mice. Lack of cute oral
toxicity to chitosan was noticed as evidenced by an oral LD 50, 10g/
kg in mice. Acute systemic toxicity tests in mice did not show any
significant toxic effects of chitosan [Rao and Sharma, 1997]. Also
Chitosan is a biodegradable, hydrophilic, biocompatible and natural
linear biopolyaminosaccharide with good potential for pharmaceutical
applications due to its high charge density, non-toxicity and
mucoadhesion. In addition, chitosan was studied as a carrier for
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microsphere drug delivery. Reacting chitosan with controlled amounts
of multivalent anion results in crosslinking between chitosan
molecules. This crosslinking has been used extensively for the
preparation of chitosan microspheres. They are the mos systems for
the controlled release of drugs such as antibiotics, antihypertensive
agents, anticancer agents, proteins, peptide drugs and vaccines
[Biswas et al., 2014]. A number of anionic polysaccharides deserved
immediate consideration, namely alginic acid, polygalacturonic acid,
carboxymethyl cellulose, carboxymethyl guaran, acacia gum, 6-
oxychitin, xanthan, hyaluronic acid, pectin, k-carrageenan, and
guargun, that represent a selection of anionic polyelectrolytes capable
of reacting with chitosan and currently studied in the food,
pharmaceutical and medical fields [Muzzarellia et al., 2004].
4.2.2. Alginate
Alginate is a natural, linear, unbranched, biodegradable
polysaccharide consisting of 1, 4-linked ∞-D-mannuronic acid and ∞-L-
guluronic acid monomers in varying proportions (Fig. 4). Alginates are
extracted from brown seaweeds and marine algae such as Laminaria
hyperborea, Ascophyllum nodosum and Macrocystis pyrifera [Beneke
et al., 2009]. Alginate is a naturally occurring biopolymer that is finding
increasing applications in the biotechnology industry. Alginate has
160
been used successfully for many years in the food and beverage
industry as a thickening agent, a gelling agent and a colloidal
stabilizer.
Fig. 4: Structural formula of Sodium alginate
Alginate also has several unique properties that have enabled it
to be used as a matrix for the entrapment and/or delivery of a variety
of proteins and cells. These properties include: (i) a relatively inert
aqueous environment within the matrix; (ii) a mild room temperature
encapsulation process free of organic solvents; (iii) a high gel porosity
which allows for high diffusion rates of macromolecules; (iv) its ability
to control this porosity with simple coating procedures and (v)
dissolution and biodegradation of the system under normal
physiological conditions.
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4.2.2.1. Sources of alginate
Commercial alginates are extracted primarily from three species
of brown algae (kelp). These include Laminaria hyperborea,
Ascophyllum nodosum, and Macrocystis pyrifera. Other sources
include Laminaria japonica, Eclonia maxima, Lesonia negrescens and
Sargassum species [Lehr et al., 1992]. In all of these algae, alginate is
the primary polysaccharide present and it may comprise up to 40% of
the dry weight.
4.2.2.2. Extraction and preparation of alginate
To commercially prepare alginates, the algae is mechanically
harvested and dried before further processing except for M. pyrifera
which is processed when wet. Alginate is then extracted from dried
and milled algal material after treatment with dilute mineral acid to
remove or degrade associated neutral homopolysaccharides such as
laminarin and fucoidin. Concurrently, the alkaline earth cations are
exchanged for H+. The alginate is then converted from the insoluble
protonated form to the soluble sodium salt by addition of sodium
carbonate at a pH below 10. After extraction, the alginate can be
further purified and then converted to either a salt or acid .Alginates,
which are naturally occurring substances found in brown seaweed and
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algae, have received much attention for use in pharmaceutical dosage
forms, particularly as a vehicle for controlled drug delivery. The
formation of a matrix upon hydration causes a gelatinous layer which
can act as a drug diffusion barrier. Alginate is a family of
polysaccharides composed of α-L-guluronic acid and β-D-mannuronic
acid residues, arranged in homopolymeric blocks of each type and in
heteropolymeric blocks [Liew et al., 2006]. Alginates form hydrogels in
the presence of divalent cations like Ca2+. It can be ionically
crosslinked by the addition of divalent cations in aqueous solution. The
native alginate is mainly present as an insoluble Ca2+ crosslinked gel.
The viscosity of alginate solutions depends primarily on the molecular
weight of the material. The divalent cations bind to the α-L-guluronic
acid blocks in a highly cooperative manner and the size of the
cooperative unit is more than 20 monomers. Complex coacervation of
oppositely charged poly electrolytes has been commonly used as a
method for preparing microbeads. In the alginate–chitosan system, the
complex is formed by spraying a sodium alginate solution into the
chitosan solution. The resultant alginate–chitosan microbeads are
mechanically strong and stable over a wide pH range [Ching et al.,
2008].
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4.2.2.3. Pharmaceutical use of alginate
Alginate is an anionic, biodegredable and biocompatible natural
polymer and alginate gels have been used to encapsulate other
delivery systems including microspheres and liposomes. They could
potentially be useful as an oral delivery system for micro- or
nanoparticles. Also alginate is a bioadhesive polymer which can be
advantageous for the site specific delivery to mucosal tissues. When it
is taken orally, it protects the mucous membrane of the upper
gastrointestinal tract from the irritation of chemicals. The alginate
monomer composition is reported to have a major impact on the drug
release properties of the different formulation systems [Sudhakar et
al., 2006]. When exposed to low pH, it can therefore undergo a
reduction in alginate molecular weight which results in faster
degradation and release of a molecule when the gel is reequilibrated
in a neutral pH solution. The release of the drug is dependent on both
dissolution of the gel and diffusion of the drug into the GI fluid. The
release of macromolecules from alginate beads in low pH solutions is
also significantly reduced which could be advantageous in the
development of an oral delivery system using a crosslinked alginate
matrix delivery system. Alginate gels have been used to encapsulate
other delivery systems including microspheres. As research and
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development continues with alginate polymeric delivery systems, we
expect to see many innovative and exciting applications in the future
[Lee et al., 1998].
4.2.3. Eudragit S100
EUDRAGIT® S 100 are anionic copolymers based on
methacrylic acid and methyl methacrylate. Its chemical/IUPAC name is
Poly(methacylic acid-co-methyl methacrylate) 1:2 (fig. 6). It is a solid
substance in form of a white powder with a faint characteristic odour
showing dissolution in pH 7.0. Eudragit is trademark of Rohm GmbH &
Co. KG. Darmstadt in Germany, first marketed in 1950s. Eudragit
prepared by the polymerization of acrylic and methacrylic acids or their
esters. Eudragit introduced in USPNF, BP, PhEur, Hand book of
pharmaceutical excipients [Rowe et al., 2000].
Fig. 5: Structural formula of Eudragit S100
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The need for gastroretentive dosage forms has led to extensive
efforts in both academia and industry towards the development of
such drug delivery systems. These efforts resulted in gastroretentive
dosage forms that were designed, in large part, based on the following
approaches,Low density form of the dosage form that causes
buoyancy in gastric fluid, High density dosage form that is retained in
the bottom of the stomach, Bioadhesion to stomach mucosa, Slowed
motility of the gastrointestinal tract by concomitant administration of
drugs or pharmaceutical excipients, Expansion by swelling or
unfolding to a large size which limits emptying of the dosage form
through the pyloric sphincter . All these techniques we can achieved
with different grades of eudragit [Garg and Gupta, 2008]. The
microspheres of eudragit S100 were found to float continuously in the
acidic solution and successfully release drug in a predetermined rate
[Kale and Tayade, 2007].
Sustained intestine delivery of drugs was developed that could
bypass the stomach and release the loaded drug for long periods into
the intestine by coating of eudragit polymer. Eudragit L & Eudragit S
are two forms of commercially available enteric acrylic resins.Both of
them produce films resistant to gastric fluid. Eudragit L & S are soluble
in intestinal fluid at pH 6 & 7 respectively. Eudragit L is available as an
166
organic solution (Isopropanol), solid or aqueous dispersion. Eudragit S
is available only as an organic solution (Isopropanol) and solid.
Colonic drug delivery is a relatively recent approach for the treatment
of diseases like ulcerative colitis, Crohn's disease, and irritable bowel
syndrome. pH-sensitive polymers that dissolve, or above pH 7 used
for colonic drug delivery [Jain et al., 2005 ]. In another experiment
Tegaserod maleate was used as a drug for irritable bowel syndrome,
whereas Eudragit L 100 and S100 mixture (1:1, 1:2, and 1:3) were
used [Venkatesh et al., 2009]. Granulation of drug substances in
powder form for controlled release can be used to prepare tablets.
Effective and stable enteric coatings with a fast dissolution in the
upper Bowel also possible with these polymers.Site specific drug
delivery in intestine by combination with EUDRAGIT® S grades can be
achieved.Variable release profiles can be observed.
4.2.4. SPAN 80
Sorbitol is a white, sweetish, hygroscopic, crystalline sugar
alcohol of six carbons. The term sorbitan describes the anhydride form
of sorbitol, whose fatty acids are lipophilic whereas sorbitol body is
hydrophilic. Span 80 is a nonionic surfactant with Fatty acid
composition: Oleic acid (C18:1) ≤ 60%; balance primarily linoleic
(C18:2), linolenic (C18:3) and palmitic (C16:0) acids (Fig. 6). It is
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Yellowish brown water immiscible luquid having HLB value of 4.3 and
Flash point at 140oC. It has reported density of 0.994 g/mL at 20 °C
and it is Stable, Combustible and incompatible with strong oxidizing
agents. SPAN80 is a low HLB surfactant suggested for use as a w/o
emulsifier or as an o/w emulsifier.
Fig. 6: Structural formula of SPAN 80
This bifunctionality in one molecule provides the basic properties
useful in cleaners, detergents, polymer additives, and textile industry
as emulsifiers, wetting agents, and viscosity modifiers. Sorbitan esters
are rather lipophilic (or hydrophobic) surfactants exhibiting low HLB
(Hydrophilic-Lipophilic Balance) values; having an affinity for, tending
to combine with, or capable of dissolving in lipids (or water-insoluble).
While the ethoxylated sorbitan esters are hydrophilics exhibiting high
HLB values having an affinity for water; readily absorbing or dissolving
in water. The type of fatty acid and the mole number of ethylene oxide
provides diverse HLB values for proper applications. Span 80 has
been used in a study to assess transfersomes as a transdermal
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delivery system for sertraline. It has also been used in a study to
investigate the dominant factors affecting the stability of
nanoemulsions through the use of artificial neural networks (ANNs).
4.2.5. Hydroxy propyl methyl cellulose (HPMC)
Hydroxypropylmethyl cellulose is available under the trade name
Methocel from DOW Chemicals Company.Hydroxypropylmethyl
cellulose also known as hypromellose is white, yellowish-white powder
or granules, odorless, tasteless and hygroscopic after drying.
Fig. 7: Structural formula of HPMC
HPMC is a semisynthetic, inert, viscoelastic polymer used as an
ophthalmic lubricant, as well as an excipient and controlled-delivery
component in oral medicaments, found in a variety of commercial
products [Williams et al., 2001].Hypromellose is a solid, and is a
slightly off-white to beige powder in its outer appearance and may be
formed into granules. The compound forms colloids when dissolved in
water. This non-toxic ingredient is combustible and can react
vigorously with oxidising agents.Hypromellose has approved
169
regulatory status as per USP-NF, Ph.Eur. JP and BP. HPMC is GRAS
listed excipient. Hypromellose is soluble in cold water, forming a
viscous colloidal solution, practically insoluble in chloroform, ethanol
and ether. It in an aqueous solution, unlike methylcellulose, exhibits a
thermal gelation property. When the solution heats up to a critical
temperature, the solution congeals into a non-flowable but semi-
flexible mass. Typically, this critical (congealing) temperature is
inversely related to both the solution concentration of HPMC and the
concentration of the methoxy group within the HPMC molecule (which
in turn depends on both the degree of substitution of the methoxy
group and the molar substitution. That is, the higher the concentration
of the methoxy group, the lower the critical temperature. The
inflexibility/viscosity of the resulting mass, however, is directly related
to the concentration of the methoxy group (the higher the
concentration, the more viscous or less flexible the resulting mass
is).In addition to its use in ophthalmic liquids, hypromellose has been
used as an excipient in oral tablet and capsule formulations, where,
depending on the grade, it functions as controlled release agent to
delay the release of a medicinal compound into the digestive tract. It is
also used as a binder and as a component of tablet coatings [Sarfaraz,
2009].
170
Hydroxypropylmethyl cellulose is used as an excipient in a wide
range of pharmaceutical products, including oral tablets and
suspensions and topical gel preparations. It is available in several
grades with viscosity ranging from 3cps to 100000 cps. As tablet
binders, it is used in concentrations 2-5% w/w, for film coating 2-20%
w/w, depending on the type of release required above 20% depending
on the grade. Hypromellose can be stored with normal precautions of
storage.
4.2.6 Lactose monohydrate
Lactose monohydrate [CAS no. 64044-51-5]. Lactose
monohydrate is available under the trade name Supertab 30.The
product has approved regulatory status as per USP-NF, Ph.Eur., IP,
JP and BP. Lactose monohydrate occurs as white, odourless, free
flowing powder slightly sweet is taste. It is a natural disaccharide,
obtained from milk, which consists of one glucose and one galactose
moiety (Fig. 8).
Lactose monohydrate, spray dried lactose and anhydrous
lactose are widely used as diluent in tablets and capsule formulations.
It produces a hard tablet and the tablet hardness increases on
storage. Disintegrant is usually needed in lactose containing tablets.
171
Drug release rate is usually not affected. It is usually non reactive,
except for discoloration when formulated with amines and alkaline
materials due to maillard reaction. It contains approximately 5% water.
Fig. 8: Structural formula of Lactose monohydrate
It needs high compression pressures in order to produce hard
tablets. Lactose monohydrate (SuperTab® 30) is produced by fluid
bed granulation and has very good flow properties. It shows consistent
compaction over a wide range of humidity. Mould growth may occur
under humid conditions. Lactose should be stored in a well closed
container and stored in cool dry place.
4.2.7. Microcrystalline cellulose
Microcrystalline cellulose is available under the brand name
Avicel PH 101 and Avicel 102 from FMC Biopolymer USA.
Microcrystalline cellulose is purified, partially depolymerised cellulose
that occurs as a white, odorless, tasteless, crystalline powder
172
composed of porous particles. Microcrystalline cellulose is a purified
partially depolymerized cellulose prepared by treating alpha-cellulose,
obtained as a pulp from fibrous plant material, with mineral acids. The
degree of polymerization is typically less than 400. Not more than 10%
of the material has a particle size of less than 5 µm. It is GRAS listed
excipient. It is commercially available in different particle sizes and
moisture grades that have different properties and applications. It has
approved regulatory status as per BP, JP, IP, Ph. Eur. and USP NF. It
is available in many brand names as Avicel, empirical formula as
(C6H10O5) n ≈ 36000, where n≈ 220 (Fig. 9). Microcrystalline
cellulose is a commonly used excipient in the pharmaceutical industry.
It has excellent compressibility properties and is used in solid dose
forms, such as tablets. Tablets can be formed that are hard, but
dissolve quickly.
Fig. 9: Structural formula of Micro crystalline cellulose
173
Microcrystalline cellulose is the same as cellulose, except that it
meets USP standards. It is also found in many processed food
products, and may be used as an anti-caking agent, stabilizer, texture
modifier, or suspending agent among other uses.According to the
Select Committee on GRAS Substances, microcrystalline cellulose is
generally regarded as safe when used in normal quantities [F.A.O,
U.N Doc Repository].Microcrystalline cellulose is widely used in
pharmaceuticals, primarily as a binder/diluent in oral tablet and
capsule formulations where it is used in both wet granulation and
direct compression processes. Microcrystalline cellulose should be
used in the ratio of 20-90% as tablet binder/diluent, 5-15% as tablet
disintegrant. Avicel PH 101 has a bulk density of approx. 0.32% and
tapped density of 0.45%. Avicel PH 101 has the particle size of 50 μm
(60 mesh < 1.0% and 200 mesh < 30%) with moisture content of
<5.0% and Avicel PH 102 has nominal mean particles size of 100 μm
(60 mesh < 8.0% and 200 mesh < 45%) with moisture content of <
5%. Microcrystalline cellulose (MCC) is one of the most important
tableting excipients thanks to its outstanding dry binding properties,
enabling the manufacture of tablets by direct compression (DC). DC
remains the most economical technique to produce large batches of
tablets, however its efficacy is directly impacted by the raw material
174
attributes [Thoorens et al., 2014].Microcrystalline cellulose is slightly
soluble in 5% w/v sodium hydroxide solution, practically insoluble in
water, dilute acids and most organic solvents. Avicel PH 101 and 102
have a specific surface area of 1.06 -1.12m2/g and 1.21-1.3m2/g
respectively. Microcrystalline cellulose is a stable though hygroscopic
material170. The bulk material should be stored in well closed
container in a cool, dry place.
4.2.8. Magnesium stearate
Magnesium stearate [CAS no. 557-04-0] is official in Ph. Eur.,
USP-NF, BP and JP. Magnesium stearate is a white and solid at room
temperature. It has the chemical formula Mg (C18H35O2)2 (Fig. 10). It is
the salt containing two equivalents of stearate (the anion of stearic
acid) and one magnesium cation (Mg++). Magnesium stearate melts
at about 88oC, is not soluble in water and is generally considered safe
for human consumption at levels below 2500 mg/kg [FDA’s SCOGS
database, 1979].
Fig. 10: Structural formula of Magnesium stearate
175
Magnesium stearate exists as a salt form and is useful for it's
lubricating properties for capsules and tablets in industry. It is used to
help prevent pharmaceutical ingredients from adhering to industry
equipment [Dave, 2008]. Magnesium stearate may be derived from
both plant and animal sources. Magnesium stearate is used as a
diluent in the manufacture of tablets, capsules and powders. It has
lubricating properties, preventing ingredients from sticking to
manufacturing equipment during compression into solid tablets.
Studies have shown that magnesium stearate may affect the release
time of the active ingredients in the tablets. Magnesium stearate is
manufactured from both animals and vegetables.
4.2.9. Talc
Talc is a mineral composed of hydrated magnesium silicate with
the chemical formula H2Mg3(SiO3)4 or Mg3Si4O10(OH)2. Talc is a tri-
octahedral layered mineral; its structure is similar to that of
pyrophyllite, but with magnesium in the octahedral sites of the
composite layers [Deer et al., 1992]. In loose form, it is the widely used
substance known as baby powder (aka talcum). It occurs as foliated to
fibrous masses, and in an exceptionally rare crystal form. It has a
perfect basal cleavage, and the folia are non-elastic, although slightly
flexible. It is the softest known mineral and listed as 1 on the Mohs
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hardness scale. As such, it can be easily scratched by a fingernail. It
has a specific gravity of 2.5–2.8, a clear or dusty luster, and is
translucent to opaque. Talc is not soluble in water, but it is slightly
soluble in dilute mineral acids. Its color ranges from white to grey or
green and it has a distinctly greasy feel. Its streak is white. Talc
powder is a household item, sold globally for use in personal hygiene
and cosmetics. Some suspicions have been raised about the
possibility that its use contributes to certain types of diseases, mainly
cancers of the ovaries and lungs (it is in the same 2B category in the
IARC listing as mobile phones and coffee) although the relationship is
still largely a hypothesis and not scientifically cited and proven
[Hollinger, 1990].
Talc is used in many industries such as paper making, plastic,
paint and coatings, rubber, food, electric cable, pharmaceuticals,
cosmetics, ceramics, etc. A coarse grayish-green high-talc rock is
soapstone or steatite and has been used for stoves, sinks, electrical
switchboards, crayons, soap, etc. Talc is also used as food additive or
in pharmaceutical products as a glidant. In medicine talc is used as a
pleurodesis agent to prevent recurrent pleural effusion or
pneumothorax. In the European Union the additive number is E553b.
Due to its low shear strength, talc is one of the oldest known solid
177
lubricants. There is also a limited use of talc as friction-reducing
additive in lubricating oils [Rudenko and Bandyopadhyay, 2013].
4.2.10. Calcium chloride
Calcium chloride is created from the ionic bonds that form
between calcium cations and chloride anions. Chloride ions have a
charge of -1, while calcium ions have a charge of +2. The molecule for
calcium chloride has two chloride ions and one calcium ion, which
means that the overall charge for the molecule is 0, or neutral.
Calcium chloride salts can also form crystals based on these same
ionic properties. Positive calcium ions can orient themselves so that
they are close to the negative chloride ions in another molecule.
Calcium chloride can serve as a source of calcium ions in an aqueous
solution, as calcium chloride is soluble in water. The anhydrous salt is
deliquescent; it can accumulate enough water in its crystal lattice to
form a solution. Drying tubes are frequently packed with calcium
chloride. Kelp is dried with calcium chloride for use in producing
sodium carbonate. Anhydrous calcium chloride has been approved by
the FDA as a packaging aid to ensure dryness [SIDS, 2002].
As an ingredient, it is listed as a permitted food additive in the
European Union for use as a sequestrant and firming agent with the E
178
number E509. It is considered as generally recognized as safe
(GRAS) by the U.S. Food and Drug Administration. Its use in organic
crop production is generally prohibited under US National Organic
Program's National List of Allowed and Prohibited Substances. The
average intake of calcium chloride as food additives has been
estimated to be 160–345 mg/day for individuals [The Innovation
Group, 2005]. It is injected to treat internal hydrofluoric acid burns. It
can be used to treat magnesium intoxication. Calcium chloride
injection may antagonize cardiac toxicity as measured by
electrocardiogram. It can help to protect the myocardium from
dangerously high levels of serum potassium in hyperkalemia. Calcium
chloride can be used to quickly treat calcium channel blocker toxicity,
from the side effects of drugs such as diltiazem (Cardizem)-helping
avoid potential heart attacks [Jana and Samanta, 2011]. Aqueous
calcium chloride is used in genetic transformation of cells by
increasing the cell membrane permeability, inducing competence for
DNA uptake (allowing DNA fragments to enter the cell more readily). It
is extensively used as cross linking agent in preparation of drug
loaded microcarriers made of alginates [Chan et al., 2002 & Hariyadi
et al., 2014].
179
5. EXPERIMENTS
5.1. Plan of work
• Pre formulation study
o Standard calibration curve of Drug
o Drug polymer interaction study using FT-IR & DSC.
• Formulation of ALG-CHI PEC microspheres
• Micromeritic properties of PEC microspheres
o Bulk and Tapped density
o Hausner’s ratio
o Carr’s index
o Angle of repose
o Compressibility index
• General Characterization of PEC microspheres o Percentage drug entrapment
o Percentage yield
o Mucoadhesive property
o Swelling index
o Surface morphology
o Zeta potential
o In-vitro Drug release study of microspheres using
simulated colonic fluid(pH 7.4)
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• Mathematical modelling of Drug Release profile
o Zero order kinetic model
o First order kinetics
o Higuchi model
o Korsemeyer –Peppas model
• In vitro stability study was performed for selected batch.
• Preparation of enteric coating solution
• Coating of microspheres
• Preparation of matrix tablet of enteric coated microspheres
• Evaluation of tablets
o weight variation
o Thickness
o Hardness
o Friability
o Disintegration time and dissolution studies
• Stability studies of Optimized batch.
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5.2. Chemicals, reagents and equipments
Drug and other necessary Chemical either purchased or gifted
from different renound suppliers are listed below in Table 1. Double
distilled water was used in all the preparations. All other articles
procured were of analytical grade with certified purity in order to obtain
optimum results. Complete list of equipments and instruments used
are listed in Table 2.
Table 1: List of drug and chemicals used
Sl.no Name Manufacturer
1 Balsalazide Krebs Pharma, Chennai.
2 Chitosan Sigma Aldrich, USA.
3 Sodium Alginate HiMedia Laboratories Pvt. Ltd, Mumbai
4 Calcium chloride SD Fine Chemicals Ltd., Mumbai
5 Hydrochloric acid SD Fine Chemicals Ltd., Mumbai
6 Disodium hydrogen phosphate
SD Fine Chemicals Ltd., Mumbai
7 Potassium dihydrogen phosphate
SD Fine Chemicals Ltd., Mumbai
8 Eudragit S 100 Evonik Industries ,Mumbai
9 Span 80 Loba Chemie Pvt. Ltd.,Mumbai
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Table 1: List of drug and chemicals used (Contd...)
Sl.No Name Manufacturer
10 Ethyl Alcohol SD Fine Chemicals Ltd., Mumbai
11 Acetone SD Fine Chemicals Ltd., Mumbai
12 HPMC (K4M) Zydus Cadila, Ahmedabad
13 Lactose SD Fine Chemicals Ltd., Mumbai
14 MCC HiMedia Laboratories Pvt. Ltd, Mumbai
15 Magnesium stearate Loba Chemie Pvt. Ltd.,Mumbai
16 Talc Loba Chemie Pvt. Ltd.,Mumbai
Table 2: List of equipments used
Sl.No. Name Manufacturer
1 UV/Visible spectrophotometer (UV Pharmaspec 1700)
Shimadzu Corporation, Kyoto, Japan
2 Scanning electron microscopy (JSM 6100)
Jeol, Tokyo, Japan
3 I R Spectrophotometer Hitachi-270-30 IR Spectrophotometer, Japan
4 Differential Scanning Calorimetry
(TA–60)
Shimadzu Corporation, Kyoto, Japan
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Table 2: List of equipments used (Contd...)
Sl.No. Name Manufacturer
5 USP Dissolution Apparatus II(DS 1800)
Labindia Analytical instruments pvt. Ltd, Mumbai
6 Tablet Disintegration Apparatus (DS8000)
Labindia Analytical instruments pvt. Ltd, Mumbai
7 Hardness tester (TH1050M)
Labindia Analytical instruments pvt. Ltd, Mumbai
8 Friability Tester Remi equipments, Mumbai
9 Coating Pan Premier Engineering Works, New Delhi
10 Desiccator Labindia analytical instruments pvt. Ltd, Mumbai
11 Stability Chamber Labindia analytical instruments pvt. Ltd, Mumbai
12 Single pan Balance Dhona, Kolkata
13 Hot air oven Spencers,Delhi
14 Digital balance Afcoset Digital Balance,Mumbai
15 Zetasizer nano ZS apparatus
Malvern,UK
16 Optical microscope eclipse TE 2000-U with digital camera DXM 1200 F.
Nikon
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5.3. Experimental Methods
5.3.1 Standard curve of Balsalazide using UV Spectroscopy
This method is based on the measurements of absorbance of
Balsalazide at its λmax was found to be 358 nm. Stock solution of
Balsalazide (0.5 – 5 ml of 200 μg /ml) were transferred into 50ml
volumetric flask and made upto mark with distilled water.
0.2,0.4,0.6,0.8,1.0 ml of the stock were transferred to 10 ml volumetric
flask volume adjusted and the absorbances of resulting solutions
were measured at 358 nm using water as blank. Calibration curve was
plotted by using concentration versus absorbance data with proper
regression [Beckett and Stenlake, 2002].
5.3.2. Drug polymer interaction
Drug is needed to have distinct and known reactivity profile with
other main excipients used in formulation for designing favorable
dosage forms sufficing specific requirements for proper delivery of
drug. Drug polymer interaction study is essential to know the nature of
reaction and its severity affecting design of formulation. Nonreactive
combination of drug and polymer is considered as only the eligible
candidate for design of safe and optimum drug delivery. DSC and
FTIR Spectroscopy are two well known methods to detect drug-
185
polymer and polymer- polymer interaction [Gomathi et al., 2014;
Jelvehgari et al., 2011]. In present section these two methods were
adopted with fulfillmen of all possible requirements.
5.3.2.1. FTIR study
IR spectroscopy can be performed by Fourier transform infrared
spectrophotometer. Infrared (IR) spectra of alginate, chitosan, and
alginate–chitosan complex were recorded with a spectrometer using
the attenuated total reflection (ATR) method. The pellets of drug and
potassium bromide were prepared by compressing the powders at 20
psi for 10 min on KBr-press and the spectra were scanned in the wave
number range of 4000- 600 cm-1at a resolution of 4 cm−1. FTIR study
was carried on pure drug, physical mixture, formulations and empty
microspheres [Lamprechta et al., 2005].
5.3.2.2. Differential scanning calorimetry (DSC)
Differential scanning calorimetric (DSC) analysis of the
Balsalazide and polymer were carried out by using differential
scanning calorimeter equipped with computer. DSC Thermogram was
used to determine a shift of the alginate endothermic peak or the
appearance of exothermic peaks and consequently detect interactions
between chitosan and alginate. Sodium alginate and chitosan acetate
186
were obtained by lyophilization of aqueous solution 0.3% (w/v) and an
aqueous acetic acid solution 0.3% (w/v), respectively. Physical mixture
was prepared by mixing (1/1, w/w) lyophilized sodium alginate with
chitosan acetate. Chitosan–alginate (CS–ALG) complexes were
obtained by adding 10ml of chitosan solution 0.3% (w/v) at pH 6.4 to
10ml of alginate solution 0.3% (w/v) at pH 4.5 under agitation for
20min followed by lyophilization. Microspheres were isolated by
filtration, washed with water and lyophilized. Samples (3-7 mg) were
heated under nitrogen atmosphere on an aluminum pan at a heating
rate of 10 °C/min over the temperature range of 30-300oC [Ribeiro et
al., 2005].
5.3.3. Preparation of ALG-CHI PEC microspheres
Alginate Chitosan (ALG-CHI) microspheres were produced in
W/O emulsion, as described in earlier work [Arora et al., 2011] using
span80 as surfactant and CaCl2 as crosslinker, resulting in nine
different formulations in emulsion. ALG-CHI microspheres were
produced in aqueous medium. Polymer complexes were prepared
using different proportions, in order to obtain hydrogels with polymer
ratio. Though the condition previously optimized in previous articles
[Abreu et al., 2010 & Ma et al., 2008], variable combinations using
different concentrations of two polymers were included for preparation
187
and subsequent comparison of microspheres. The model drug
balsalazide was added using different mass ratio forming Polymer:
Drug of 0.5:1,1:1,2:1,3:1,4:1, 5:1 6:1,8:1and 10:1 All nine experimental
formulations (M1-M9)were produced in triplicate. Aqueous solution of
ALG was prepared and diluted differently to a final concentration of
0.1%, 0.2% and 0.3% w/v using distilled water. Similarly CHI solution
was prepared by dissolving in an acetic acid and magnetically stirred
(100 rpm) the solution overnight at 4 oC. Solutions were further diluted
to obtain required concentration with distilled water. The pH of the
alginate solution was 6.5, and the pH of the chitosan chloride solution
was 4.0 after the dissolution. These pHs ensured that the alginate was
fully deprotonated and that the chitosan was fully protonated. The
polyelectrolyte complexes (PECs) were prepared at room temperature.
The solution of CHI and ALG were placed in separate tubes and 2 mM
CaCl2 solution was added into the tube with Chitosan solution and
homogenized.
188
Table 3: Different batches of PEC microspheres
Formula Alginate W/W
Chitosan W/W
Polymer complex-Drug ratio
Span 80
AM 0.2% 1:1 1%
CM 0.2% 1:1 1%
M1 0.1% 0.1% 0.5:1 0.2%
M2 0.1% 0.2% 1:1 0.4%
M3 0.1% 0.3% 2:1 0.6%
M4 0.2% 0.1% 3:1 0.8%
M5 0.2% 0.2% 4:1 1.0%
M6 0.2% 0.3% 5;1 1.5%
M7 0.3% 0.1% 6:1 2.0%
M8 0.3% 0.2% 8:1 2.5%
M9 0.3% 0.3% 10:1 3.0%
The surfactant Span80 was added in each tube in a varying
concentration of 0.2%, 0.4%, 0.6%, 0.8%.1.0% 1.5%, 2.0%, 2.5%and
3.0% w/v respectively and homogenized in an ultrasonic bath for 25
min. Formula batches were designed as per predetermined protocol
cited in Table 3. Each formula of ALG-CHI–Ca+2 microspheres was
189
prepared by mixing both solutions by carefully adding to a vessel
containing liquid paraffin in a volume ratio 6:1 paraffin: aqueous
phase. The mixture was vigorously sonicated with an ultrasonic probe
for 3 min producing a stable emulsion, and then replaced in the
ultrasonic bath for additional 20 min.The emulsion was centrifuged at
3500 rpm for 30 min for aqueous and oil-phase separation. The
aqueous-phase was again centrifuged and the solid obtained was
finally dried under vacuum. For efficient comparison two additional
batches were prepared with only Alginate (AM) and only Chitosan
(CM) adopting similar method as cited above.
4.3.4. Characterization of PEC microspheres
Nine batches of microspheres were prepared with varying
polymer drug combination to make a wide variety in formula for
effective comparison and final selection of the potential batch.
Different parameters were monitored to characterize all batches using
standard and reported methods as described below.
5.3.4.1. Micromeritic properties
Micromeritic properties were evaluated for prepared
microspheres to describe their comparative nature to assess suitability
of pharmaceutical formulation.Several official methods were adopted
as derived previously [Martin, 2001].
190
5.3.4.1.1. Particle size measurement
The particle size of the PEC microparticles was measured using
a stage micrometer scale. For optical microscopy the microspheres
were directly observed under magnification. Instrument was calibrated
and found that 1 unit of eyepiece micrometer was equal to 12.5μm.
Dry microspheres (3 mg) were suspended in distilled water and
ultrasonicated for 10 minutes. A drop of suspension was placed on a
clean glass slide, and the microspheres were counted under optical
microscopy. A minimum of 100 microspheres was counted per batch
with a magnification of 45X [Giri Prasad et al., 2011 & Huang et al.,
2000]. The average size of 100 particles was determined by the given
equation(s) [Vajpayee et al., 2011]:
Size of individual particle (μm) = Number of division on eye piece ×
Calibration factor
Average Particle Size (μm) = Sum of Size of Individual Particles / 100
Results obtained after optical microscopy was closely matched
with that of SEM to ensure symmetry in size measurement.
191
5.3.4.1.2. Determination of bulk density (ρB)
Bulk density of the formulations was calculated by volume (Vb)
of 5 gm of microspheres observed in a 10 ml measuring cylinder and
dividing Weight of sample (W) by Vb using the following formula: ρB
=W/Vb
5.3.4.1.3. Determination of tapped density (ρT)
Tapped density is used to investigate packing properties of
microcapsules. The tapped density was measured by employing the
conventional tapping method using a 10mL measuring cylinder and
the number of tappings was 100 as sufficient to bring a plateau
condition. Tapped volume (Vt) was observed. Tapped density was
calculated dividing Weight of sample (W) by Vt using the following
formula: ρT =W/Vt
5.3.4.1.4. Determination of angle of repose
Angle of repose of the microspheres was determined by passing
microspheres through the glass funnel on a horizontal surface. The
height (h) of the heap formed was measured and the radius (r) of the
cone base was also observed and calculated. The angle of repose (θ)
was calculated as: tan θ = h/r
192
5.3.4.1.5. Determination of Hausner's ratio
The Hausner’s ratio (H) is a number that is correlated to the
flowability of a powder or granular material. The ratio of tapped density
(ρT) to bulk density (ρB) of the powders is called the Hausner's ratio. It
is another parameter for measuring flowability of the microcapsules
and was calculated by the equation: H= ρT/Ρb
5.3.4.1.6. Determination of compressibility index
It is indirect measurement of bulk density, size and shape,
surface area, moisture content, and cohesiveness of materials since
all of them can influence the consolidation index. It is also called as
compressibility index or Carr’s Index. It is denoted by (Ci) and is
calculated using the formula: Carr’s Index (%) = (ρT- ρB) / ρT X 100
5.3.4.2. General characterization of PEC microspheres
5.3.4.2.1. Surface morphology of microspheres
The morphology of the PEC microspheres was examined by
field emission scanning electron microscopy (SEM). Microsphere
features such as shape and existence of aggregates was examined
after isolation by using an optical microscope. Scanning electron
microscopy (SEM) was utilized to compare microspheres morphology,
193
especially the surface characteristics of microspheres. The sample
was mounted on to an aluminum stub and sputter-coated for 120
minutes with platinum particles in an argon atmosphere. Prior to SEM
examination, lyophilized microspheres were mounted on mental stubs
using double-sided tape and coated with a 150 Å layer of gold under
the reduced pressure (0.001mm of Hg). The voltage was used is 5KV
[Kim et al., 2008].
5.3.4.2.2. Determination of Zeta Potential
The zeta potential is representative of particle charge. Zeta
potentials were measured by electrophoresis. Phosphate buffer with
pH 7.4 (0.001 M) was used as environment. The microspheres were
suspended in buffer by ultrasonication for 30 minutes. The
concentration of the suspension was 2% w/v. The cell was filled with a
measured amount of sample and inserted with its integral gold
electrodes close to the lid [Fischer et al., 2004].
5.3.4.2.3. Percentage Yield
Suitability of preparation under variable influencing factors yield
of product must be considered as an important parameter [Gowda et
al., 2011]. The microspheres were evaluated for percentage yield. For
different batches individual weight of drug to be loaded and other used
194
excipients were measured. After preparation and subsequent drying of
microspheres final weight was measured in triplicate. The yield was
calculated as per equation below.
Percentage Yield= (Total Weight of Microspheres/Total Weight of
Excipients and Drug) × 100
5.3.4.2.4. Entrapment efficiency
Entrapment efficiency is the percentage of drug entrapped in
Balsalazide loaded microspheres related to the initial quantity of the
drug used in the formulation. Analogous to previous method [Jin et al.,
2009]100mg of microspheres were taken and crushed in a glass
mortar pestle. In a 100mL volumetric flask, the grounded microsphere
powder equivalent to 10mg of Balsalazide was dissolved in 20mL
methanol/water(1:2) and volume made up to 100mL with pH 7.0
phosphate buffer with 0.5%SLS. The Solution was filtered through
Whatmann filter paper no. 41 to obtain the stock Solution. 1mL of the
Stock Solution was further diluted to10mL to obtain several working
dilutions. The Absorbance of the resulting solution was measured at
wavelength maximum of 358nm using double beam UV-Visible
Spectrophotometer with 1cm path length sample cells. Results were
presented as mean± SEM of three observations (n=3) at
195
p=0.5.Entrapment efficiency was calculated using the following
formula:
% Entrapment = Actual content/Theoretical content x 100.
5.3.4.2.5. Swelling Index
Swelling index was determined by measuring the extent of
swelling of microspheres in the given buffer.To ensure the complete
equilibrium, exactly weighed amount of microspheres were allowed to
swell in given buffer having pH7.4. The excess surface adhered liquid
drops were removed by blotting and the swollen microspheres were
weighed by using microbalance. The hydrogel microspheres were then
dried in an oven at 60° for 5 hours until there was no change in the
dried mass of sample as noted in earlier reports [Fandueanu et al.,
2004 & Kulkarni et al., 2004].Data was presented as mean±SEM of
three observations calculated at 95% confidence level (p=0.5).It was
calculated using the formula:
Swelling index= (mass of swollen microspheres - mass of dry
microspheres/mass of dried microspheres) X 100.
196
5.3.4.2.6. Mucoadhesive property by In vitro wash-off test
The mucoadhesive properties of the ALG-CHI microspheres
were evaluated by the in vitro wash-off test as reported by Lehr et al.
[Lehr et al., 1990]. A 4cm x 4cm piece of goat intestine mucosa was
tied onto a glass slide using thread. Microspheres were spread onto
the wet, rinsed, tissue specimen and the prepared slide was hung on
to one of the groves of a USP tablet disintegrating test apparatus. The
disintegrating test apparatus was operated such that the tissue
specimen was given regular up and down movements in the beakers
containing the simulated intestinal fluid at pH 7.4. At the end of 30
minutes, 1 hour, and at hourly intervals upto 10 hours, the number of
microspheres still adhering on to the tissue was counted. Results were
presented as mean± SEM of three observations (n=3) at p=0.5.using
following equation:
Mucoadhesion property = (No of microspheres adhered/ No of
microspheres applied) X 100
5.3.4.3. In vitro drug release study of microspheres
To overcome the limitations of conventional dissolution testing
for evaluating the performance of colon specific drug delivery systems
triggered by colon specific bacteria, rat caecal contents has been
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utilized as an alternative dissolution medium, called rat caecal content
medium or simulated colonic fluid(SCF). 4% of caecal contents were
used as simulated colonic fluid which contains 6.8 g of potassium
dihydrogen phosphate and 190 mL of 0.2 M sodium hydroxide in 1 L of
deionized water [Li et al., 2013]. The suspension was filtered through
cotton wool and sonicated (50 watt) for 20min at 4OC to disrupt the
bacterial cells. After sonication, the mixture was centrifuged at 2000
rpm for 20 min. Microspheres equivalent to 20 mg of balsalazide
sodium were weighed accurately and suspended in 20 mL of prepared
medium. The mixture was stirred at 37 °C using a magnetic stirrer at a
stirring speed of 50 rpm for 3 h. At specified time intervals, samples
were withdrawn (2 mL) and replaced with the same volume of fresh
media. The withdrawn samples were centrifuged at 3000 rpm for 10
min and were then filtered and diluted with phosphate buffer pH 7.4.
The drug content was measured by taking supernatant absorbance
using a UV/Visible spectrophotometer [Dashora and Jain, 2009].
5.3.4.4. Mathematical modeling of drug release profile
The cumulative amount of balsalazide released from the
formulated tablets at different time intervals were fitted to zero order
kinetics, first order kinetics, Higuchi model and Koremeyer –Peppas
model to characterize mechanism of drug release.
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5.3.4.4.1. Zero order kinetic model
It describes the system in which the drug release rate is
independent of its concentration.Qt = Qo + Ko t Where Qt= Amount of
drug dissolved in time t and the Qo = Initial amount of drug in the
solution, which is often zero and Ko is the zero order release constant.
If the zero order drug release kinetic is obeyed then a plot of Qt versus
t will give a straight line with a slope of Ko and an intercept at zero.
5.3.4.4.2. First order kinetic model
It describes the drug release from the systems in which the
release rate is concentration dependent. log Qt = log Qo + kt / 2.303
Where Qt is the amount of drug released in time t. Qo is the initial
amount of drug in the solution and k is the first order release constant
If the first order drug release kinetic is obeyed, then a plot of log (Qo-
Qt) versus t will be straight line with a slope of kt/ 2.303 and an
intercept at t=0 of log Qo [Moore and Flanner, 1996]
5.3.4.4.3. Higuchi model
It describes the fraction of drug release from a matrix is
proportional to square root of time.Mt / M∞ = kHt1/2 where Mt and M∞
are cumulative amounts of drug release at time t and infinite time and
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kH is the Higuchi dissolution constant i.e. reflection of formulation
characteristics. If the Higuchi model of drug release (i.e. Fickian
diffusion) is obeyed, then a plot of Mt / M∞ versus t1/2 will be straight
line with slope of k H [Higuchi, 1963].
5.3.4.4.4. Korsmeyer-Peppas model (Power Law)
The power law describes the drug release from the polymeric
system in which release deviates from Fickian diffusion, as expressed
in equation: Mt / M∞ = ktn and log [Mt / M∞ =] = log k + n log t where Mt
and M∞ are cumulative amounts of drug release at time t and infinite
time (i.e. fraction of drug release at time t), k is the constant
incorporating structural and geometrical characteristics of Controlled
Release system, and n is a diffusional release exponent indicative of
the mechanism of drug release for drug dissolution. To characterize
the release mechanism, the dissolution data were evaluated. A plot of
log {Mt / M∞} versus log t will be linear with slope of n and intercept
gave the value of log k. Antilog of log k was the value of k. This
equation has two distinct physical realistic meaning in the two special
cases of n = 0.5 (indicating diffusion- controlled drug release) and n =
1. n between 0.5 and 1 can be regarded as an indicator for the
superposition of both phenomena (anomalous transport) [ Wagner,
1969].
200
5.3.4.5. Stability studies of PEC microspheres
Selected batch of microspheres were subjected to stability
studies under accelerated storage conditions according to the
International Conference of Harmonization (ICH) guidelines.
Microspheres were divided into 2 sets wrapped in aluminium foil
laminated on the inside with polyethylene and placed in a glass vial.
The samples were these stored at elevated temperature and humidity
conditions of 40 ± 2°C/ 75% ± 5% RH and a control sample was
placed at an ambient condition [ICH, 2003] in a stability chamber. Both
test and control samples were withdrawn. A the end of Real time
stability studies were performed by periodical testing of the entrapment
efficiency, percent mucoadhesion index and in-vitro drug release at pH
7.4 at intervals of 0, 30, 90 and 180 days during 6 months [Chawla et
al., 2012].
5.3.5. Preparation of enteric coating solution
The enteric coating solution was prepared by simple solution
method. It was prepared by 6% W/W of Eudragit S100 as an enteric
coating polymer, PEG 1.5% w/w as plasticizer and a mixture of
acetone and Ethanol (2:1) was used as solvent. This mixture was
constantly stirred for 1h with paddle mechanical stirrer at the rate of
201
1000 rpm and the stirred coating solution was again filtered through
muslin cloth, a coating solution was obtained [Neelam et al.,
2011].Coating solution was freshly prepared prior to coating operation
to get satisfactory result.
5.3.6. Enteric coating of selected batch M6
The optimized chitosan microspheres were coated with Eudragit
S–100 by formerly used emulsion solvent evaporation method [Nayak
et al., 2011]. The microspheres (batch M6) were suspended in 10 ml
of organic solvent (acetone: ethanol = 2:1) of Eudragit S–100 that was
previously prepared. The above organic dispersion was then
emulsified in 100 ml of liquid paraffin containing 3 % span 80 and
stirred at 1000 rpm for 3 hours at room temperature to remove the
solvents by evaporation. The Eudragit coated microspheres were
separated, rinsed with n–Hexane to remove residual traces of liquid
paraffin, dried and stored in vacuum desiccators.
5.3.7.Preparation of matrix tablets of enteric coated microspheres
Tablets were prepared by wet granulation technique [Ofokansi
and Kenechukwu, 2013] using microspheres equivalent to 70% of total
quantity of balsalazide (50 mg /tablet). Remaining 30% of drug (15
mg) was mixed with binder and thereby kept outside the
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microspherical barrier for instant release from disintegrated tablet
mass. The damp mass containing lactose (15 mg/tablet) as bulking
agent, HPMC (5mg/tablet) as binder and 70% of total MCC(15 mg) as
disintegrant formed was then forced through sieve no. 10 (1.7 mm
mesh) and was dried at 50°C for about 1 h until all the moisture was
removed. The dry mass was also forced through sieve no. 16 (1.0 mm
mesh) and was stored in a desiccator until used.
5.3.7.1. Sieve analysis of prepared granules
Dried sample of granules of microspheres that weighs about 150
gm were taken for sieve analysis. A stack of sieves was prepared (#s
4 and 200 were included) in such way that sieves having larger
opening sizes (i.e lower numbers) are placed above the ones having
smaller opening sizes (i.e higher numbers). A pan is placed under the
stack to collect the portion of granules passing #200 sieve. Then the
stack was placed on the sieve shaker and fixed the clamps and the
shaking time was adjusted to 15 minutes. Mass of granules retained
on each sieve was measured carefully in triplicate from the difference
between mass of each sieve with retained mass and empty sieve.
Average was taken into account. Calculation on the basis of % of
sample retained on each sieve was done to make a plot of % retained
Vs size of sieve [Meral and Kandemir, 2008] was done using formula:
203
% Retained = (Mass of sample retained on sieve / Total mass of
sample) X 100
5.3.7.2. Preparation of Tablets
The dried granules of nearly uniform size distribution were
passed through sieve no. 20 and were retained on sieve no. 44.
Magnesium stearate (2mg/tablet) as lubricant, talc (2mg/tablet) as
glidant and remaining 30% MCC (6mg/tablet) and remaining amount
of fines (separated from granules) were added to the granules. Tablets
were compressed using 4 mm biconvex punches in a eight station
rotary tablet compression machine fitted with 4.5 mm circular, flat
punches at a pressure of 50 kg/cm2 [ Rathore et al., 2013].
5.3.8. Characterization of matrix tablets
5.3.8.1. General evaluation of tablets
General evaluation involved determination of some useful
parameters like appearance, weight variation, thickness, diameter,
hardness, friability and assay to characterize overall performance of
prepared tablet facilitating requirements of delivery system [Bhanja et
al., 2010 &Rathore et al., 2013].
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5.3.8.1.1. Appearance
Twenty tablets of each formulation were taken to check any
discoloration or degradation of drug in the tablets by visual method. If
any discoloration or black spots appears, it shows the degradation or
decomposition of the drug in the tablet formulation.
5.3.8.1.2. Weight Variation
For the determination of weight variation of each batch, tablets
were randomly sampled and individual weight of 20 tablets was taken
in analytical balance. Observation was repeated three times and result
was tabulated as Mean ±SEM calculated at p=0.5.
5.3.8.1.3. Thickness
From randomly sampled tablets, thickness of 10 tablets was
measured individually using digital vernier caliper. Then the result was
cited as mean ± SEM (p=0.5; n=3).
5.3.8.1.4. Hardness
Hardness of 10 tablets was measured individually using pre-
calibrated Monsanto hardness tester. Then mean ± SEM (n=3) was
calculated.
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5.3.8.1.5. Friability
20 tablets were weighted in a balance having readability of 1
mg. These tablets were transformed into Roche friabilator set 100
revolutions. After the completion of revolution dust was removed
completely, weighted again in the same balance and percentage loss
was calculated. Mean of triplicate result was tabulated as mean ± SEM
(p=0.5).
5.3.8.1.6. Assay
20 tablets were weighed and its average weight was taken which
was crushed in motor and pestle. The powder weight equivalent to
single tablet i.e. 75 mg was dissolved in 10 ml water in a 100 ml
volumetric flask and allowed to stand for 10 minutes. To that 75 ml of
methanol was added initially followed by addition of sufficient methanol
to produce 100 ml which was then filtered through Whatmann filter
paper. 5 ml of this resulting solution was further diluted to 50 ml with
7.4 pH phosphate buffer: methanol (1:1). Again 5 ml was diluted to 50
ml by the same solvent. The absorbance of each of the standard and
sample solution were taken in UV-visible spectrophotometer at 358 nm
using equal volumes of 7.4 pH phosphate buffer and methanol as
blank. Result was presented as mean ± SEM (p=0.5) from observation
made in triplicate (n=3).
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5.3.8.1.7. Disintegration time
Disintegration testing of core and coated tablets was carried out
in the six tablet basket rack USP disintegration apparatus. One tablet
was introduced into each tube of the basket rack assembly of the
disintegration apparatus without disc. The assembly was positioned in
the beaker containing disintegration media maintained at
37±2°C.Medium pH was maintained at 7.4 which contains 6.8 g of
potassium dihydrogen phosphate and 190 mL of 0.2 M sodium
hydroxide in 1 L of deionized water, was prepared with and without 10
g of pancreatin separately and disintegration time was noted in each
medium [Nelson et al., 1961]. Process was repeated three times and
the result was presented as mean ± SEM (p=0.5).
5.3.8.2. In vitro dissolution studies of tablets
In vitro release of balsalazide from the enteric coated tablets
was performed using USP (Dissolution Apparatus 1-basket method) at
37 ± 0.5°C and 100 rpm in three different release media (SGF pH 1.2,
SIF pH 6.8, and SCF pH 7.4). Medium pH was maintained at 1.2,
using simulated Gastric fluid(SGF) which contains 7 mL of
hydrochloric acid (37.4%), 2 g of sodium chloride in 1 L of deionized
water prepared with and without 3.2 g of pepsin.Simulated Intestinal
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Fluid (SIF) pH 6.8 consisted of KH2PO4 (6.8 g), 0.2N NaOH (190 mL),
and pancreatin (10.0 g) [Vajpayee et al., 2011] and simulated caecal
fluid (SCF) of pH 7.4 which contains 6.8 g of potassium dihydrogen
phosphate and 190 mL of 0.2 M sodium hydroxide in 1 L of deionized
water, was prepared with and without 10 g of pancreatin
separately[Nelson et al., 1961]. Each tablet was placed in the
cylindrical basket of a dissolution apparatus attached to the rotating
spindle suspended in the dissolution medium of volume of 900 mL (pH
1.2) and continued for 2 hours as symmetric as the gastric transit time.
Then equipment was switched off and basket was unscrewed out to
rinse properly with previous medium after removal of tablet. Second
medium of pH6.8 was emptied into the one-litre cylindrical plastic
container and experiment was continued for another 4 hours to get
simulation with average intestinal transit time. After elapse of four
hours same process was repeated to wash and replace the dissolution
media with third one having pH7.4 and continued until nearly all drug
was released from the tablet [Obitte et al., 2010]. The time of tablet
addition was noted and 2 ml of sample was withdrawn at an interval of
20 minutes for first hour and 30 minutes for second hour and 1hour for
remaining period for each release media, 5 ml of dissolution medium
was replaced into dissolution vessel after each sampling in order to
208
maintain sink condition. Control batch of tablet (uncoated M6) was
treated similarly as test. The absorbance of each of the standard and
sample solution were taken in UV-visible spectrophotometer at 358 nm
using phosphate buffer of suitable pH as corresponding blank [ Roa et
al., 2007]. Results were presented after getting three observations
(n=3) and data was prepared as mean ± SEM (p=0.5).
5.3.8.3. Stability studies of tablets
Stability studies were performed as per the ICH guidelines.
Selected formulations of Balsalazide sodium tablet were sealed in
aluminum foil cover and stored at (40 ± 2 °C / 75 ± 5 % RH) for a
period of 6 months. Samples from each formulation which are kept for
examination were withdrawn at definite time intervals. The withdrawn
samples were evaluated for hardness, drug content, disintegration
time and time to release 80% of drug(T80) following reported method
[Singh et al.,2009]. Each parameter was evaluated in triplicate and
results were tabulated as mean±SEM at 95% confidence level.
5.3.9. Statistical analysis
As found in earlier works reported no experiment is complete
without statistical approaches to present results. In present
investigation entire process was also comprised with several
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interbatch comparisons of different parameters monitored. Data was
generated after suitable mathematical calculations and corresponding
statistical interpretation accessed from MS Excel 2007. The results
were expressed in mean ± SEM. whereas comparison between two
means was performed for studying the statistical significance using
unpaired student ‘t’ test in Microsoft office excel 2007.In each aspect
values of p= 0.05 were considered to be significant.
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6. RESULTS AND DISCUSSION
6.1. Standard curve of Balsalazide
Data sufficing need of all analytical estimation of the drug
Balsalazide was generated using UV Spectrophotometry and
tabulated as below to make standard curve with proper regression
(Table 4). This was utilized in all further works relating to quantitative
estimation of the drug.
Table 4: Data for plot of standard curve of Balsalazide
Concentration
(µg/ml)
Absorbance at
λmax = 358 nm
0.00 0.00
0.2 0.017
0.4 0.029
0.6 0.041
0.8 0.052
1.0 0.064
1.5 0.093
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Absorbance values were obtained using very dilute solution of
the drug so as to obey Beer Lambert’s law properly [Beer, 1852]. Data
was plotted to yield standard curve (Fig. 11) characterizing absorption
pattern measured at λmax of 358 nm. as followed in earlier
research[Anandakumar et al., 2008 & Hussain et al., 2000].
Fig. 11: Standard curve of Balsalazide
6.2. Drug Polymer Interaction
Results of FTIR Spectroscopy and DSC thermogram of
individual polymer, drug and their complex were depicted placing their
individual spectrum closely to make a clear distinction between them
to predict their interaction before and after formation of complex in
order to present their contribution whatever obtained in the selected
batch of microspheres.
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6.2.1. FTIR spectra
The FT-IR spectra of Balsalazide, chitosan, alginate and their
complex forming microspheres were shown in Fig. 12. In the FT-IR
spectrum of chitosan, a broad absorption band at 3430 cm−1
corresponded to the stretching vibration of –NH2 and –OH groups. The
peaks at 2930 and 2848 cm−1 were typical of C–H stretch vibration,
while the peaks at 1636, 1596 and 1313 cm−1 corresponded to
amides I, II and III, respectively. The sharp peaks at 1426 and 1380
cm−1 attributed to the CH3 symmetrical deformation mode and 1155
and 1078 cm−1 were indicative of C–O stretching vibrations [-- (C–O–
C)]. The absorption band at 896 cm−1 was characteristic of
saccharide structure of chitosan [Yuan et al., 2010]. From Alginate
microspheres spectrum (Fig. 12), it was found that the peak of 1616
cm-1, 1417 cm−1 and 1030 cm-1, which implied that hydrogen
bonding was significantly enhanced. The peak of –COOH bending
vibration shifted from 1640 cm−1 to 1560 cm−1. This result was similar
to the one reported by Knaul et al. [Knaul et al., 1999]. For drug
Balsalazide some characteristic peaks at 3445 cm-1,2783 cm-1 2245
cm-1 and for 5ASA structure some identifying peaks at 1650 cm-1
1620 cm-1, 1356 cm-1,838 cm-1 and 785 cm-1. In PEC complex
formed also showed all identifying peaks characterizing presence of
213
drug, alginate and chitosan with a favorable interaction to form stable
microspheres rendering required swelling nature adequate
mucoadhesion and satisfactory sustained release profile.
Fig. 12: FTIR Spectra of Drug, Polymers and their combination as
PEC complex.
214
6.2.2 DSC
Previous works utilized DSC as a useful tool for identification of
drug - polymer interaction [Prasad et al., 2011 & Gowda et al., 2011].In
present study DSC thermogram of alginate, chitosan and their
complex containing drug Balsalazide was depicted in Fig. 13 In this
observation characteristic exothermic peak at 75.9 for alginate, 71.8
for chitosan, 62.3 for ALG-CHI polyelectrolyte complex, 276.2 for
Balsalazide and two peaks at 64.1 and 272.4 found for drug loaded
PEC.
Fig. 13: DSC Thermogram of Drug, Polymers and PEC complex
with drug
215
All transition temperatures limit for change of state detected in
each compound had close symmetry with corresponding reference
compound. After careful comparison, it was revealed that in complex
there was no adverse interaction between drug and polymer
combination found in formed complex that could be explored for
designing suitable controlled release colon targeted microspheres.
6.3. Preparation of microspheres
Alginate-chitosan hydrogels (ALG-CHI) have been proposed as
drug delivery system in the past decade, due to their attractive
combination of pH sensitivity, bio-compatibility and adhesiveness,
requiring relative mild gelation conditions for the network formation
[Berger et al., 2004a].But One of the limitations of these hydrogels is
the drug leaching during their preparation [George and Abraham,
2006] which can be reduced by controlling the reaction conditions
[Wittaya-areekul et al., 2006]. In present study microencapsulation
process of the drug balsalazide in alginate, chitosan microspheres and
alginate/chitosan polyelectrolyte mixres were investigated. In this
direction, complex was formed using the external ionotropic
gelatinization method in which the encapsulated material (wall
material) was a complex system of polyelectrolytes formed from
alginate and chitosan in different ratios. Polyelectrolyte Complex
216
(PEC) was formed as a result of the electrostatic interactions between
the protonated amino group cations of chitosan and the carboxylate
anions of alginate. Calcium chloride as ionic and covalent crosslinkers
was added to the ALG-CHI system for improving the properties and
thereby causing a reduction in the hydrogel porosity [Kim et al., 2008].
This process was powerfully influenced by the pH value because the
chitosan has the value of pKa = 6.5 and the alginate has the value of
pKa = 3.6, pH was adjusted for the two reactants namely alginate at
pH 6.5 and for chitosan at pH 4.0 to favor optimum protonation and
thus the complexation reaction was performed at pH = 5. In this
environment the two polyelectrolytes have an ionization degree large
enough for the maximum interactions to form most potent microsphere
with all satisfactory performances [Becheran-Maron et al., 2004].
Based on all earlier data support in tabulated data several formulae for
such batches (M1-M9) with alginate alone (AM) and chitosan alone
(CM) control batches for efficient comparison was made to depict
overall efficiency PEC microspheres.
6.4. Comparative characterization of microspheres
Different parameters monitored for each batch of microspheres
were carefully compared with corresponding control and other batches
having nearest possible value with statistical method and each data
217
was generated after calculating mean of three repeated readings and
result was presented as mean ± SEM taken at 5% significant level.
Several tabulated data with possible explanations for each case was
cited as follows:
6.4.1. Micromeritic characterization
Results for comparative evaluation of all micromeritic parameters
such as Particle size, Bulk density, Tapped density, Hausner’s Ratio,
Angle of repose and Carr’s Index were performed and arranged in
Table 5. It showed a comparative picture featuring the effect of
proportion of chitosan and alginate in variable ratio to form PEC as
different batches with additional two batches prepared using alginate
and chitosan alone.
6.4.1.1. Particle size analysis
The average particle size in all the formulations was observed in
between 71.07±1.35μm for batch CM to 101.41±1.33 μm for the batch
M9 (Table 5). By keeping all the variables constant except polymer
concentration, slightly increased particle size was observed with the
increase in polymer concentration. A higher concentration of polymer
used in formula batch M7-M9 produced a more viscous dispersion,
which formed larger droplets and consequently larger microspheres
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(83.21±1.16 to101.41±1.33 μm). The particle size of chitosan and
alginate only microspheres (AM and CM) were found to be significantly
smaller size compared to M7-M9 batch of microspheres (p=0.5).
Whereas the batch M6 was found to have an optimum size of
78.15±1.01μm that was higher compared to the control groups
(p=0.5). These results were of close symmetry with that obtained in
SEM (Fig. 14).The higher concentration of polymer (more than 0.75
%) lead to irregular shape of microspheres was observed. Though
several factors could be monitored to determine their influence,
present study confined the span of investigation within specific limit as
most of the variables were monitored in earlier works [Abreu et al.,
2010] and revealed the influence of polymer concentration and
specially their molecular proportion at particular pH affected a lot on
the appearance of microspheres [Badhana et al., 2013].
6.4.1.2. Bulk and Tapped density
Bulk and tapped density of different grades of raw materials
were measured and the results are presented in Table 5. It was found
that the batch CM had lower bulk density (0.292±0.19gm/cc)and
tapped densities (0.329±0.19gm/cc)as compared to that of the batch
M6 having bulk density of 0.374±0.06 gm/cc and corresponding
tapped density of 0.458±0.09 gm/cc. Also it was found that the
219
measurements were highly dependent on amount of sample and since
the volumetric measurements are obtained visually, it is highly variable
from analyst to analyst. Above result cited a prominent picture of
comparative analysis of compactness present in the samples of
microspheres. It clearly dictated optimum size distribution and uniform
structural pattern rendering sphericity in microspheres that could be a
factor controlling uniformity in drug release profile. These results found
well symmetry with earlier results [Hancock et al., 2004].
6.4.1.3. Flow properties
Powder flow is a key requirement for pharmaceutical
manufacturing process. Tablets are often manufactured on a rotary
multi-station tablet press by filling the tablet die with powders or
granules based on volume. Thus, the flow of powder from the hopper
into the dies often determines weight, hardness, and content
uniformity of tablets. In case of capsules manufacturing, similar
volume filling of powders or granules is widely used. Understanding of
powder flow is also crucial during mixing, packaging, and
transportation. And thus, it becomes essential to measure the flow
properties of these materials prior to tabletting or capsule filling [USP
2007]. The angle of repose, a traditional characterization method for
pharmaceutical powder flow, is also used in other branches of science
220
(i.e. geology) to characterize solids. The height of the granules forming
the cone, h and the radius, r of the base were measured [Cooper and
Gunn, 1986]. The angle of repose (θ) was calculated and shown in
Table 5 for different batches of PEC microspheres. Flowability can be
indicated based on the angle of repose.A value of <30° indicates
‘excellent’ flow whereas >56° indicates ‘very poor’ flow. The
intermediate scale indicates ‘good’ (θ between 31–35°), ‘fair’ (θ
between 36–40°), ‘passable which may hang up’ (θ between 41–45°),
and ‘poor which must be agitated or vibrated’ (θ between 46–55°)
[Hancock et al., 2004]. In present investigation the angle of repose
value is 12.76±0.55° for batch M4 and the maximum value
24.71±0.44° shown for batch M7.These values were within the range
of good flow character whereas the batch M6 had a promising value of
16.53±0.41°. Also the above micromeritic studies predicted that the
prepared microcapsules were spherical, non-aggregated and uniform
size that supported all other related micromeritic properties revealing a
promising and acceptable performance of that particular batch keeping
similarity with earlier findings [Navaneetha et al., 2006].
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Table 5: Micromreitic properties of microspheres
Batch Avg. Size
(µm) Bulk
Density(gm/cc) Tapped
Density(gm/cc) Hausner’s
Ratio Angle of
Repose (o) Carr’s Index
AM 79.08±1.03 0.321±0.07 0.353±0.11 1.09±0.07 18.53±0.41 9.19±0.19
CM 71.07±1.35 0.292±0.19 0.329±0.19 1.13±0.03 23.15±0.17 11.28±0.34
M1 74.11±1.17 0.324±0.01 0.346±0.02 1.067±0.02 16.33±0.49 6.26±0.22
M2 86.09±1.09 0.357±0.03 0.382±0.05 1.070±0.04 13.98±0.23 6.49±0.27
M3 82.29±0.99 0.364±0.06 0.414±0.07 1.137±0.01 19.44±0.91 12.02±0.31
M4 88.25±1.11 0.388±0.09 0.440±0.01 1.134±0.07 12.76±0.55 11.67±0.53
M5 80.03±0.79 0.358±0.02 0.423±0.06 1.181±0.05 20.29±0.86 15.29±0.44
M6 78.15±1.01 0.374±0.06 0.458±0.09 1.224±0.03 16.53±0.41 18.33±0.21
M7 83.21±1.16 0.333±0.04 0.383±0.01 1.150±0.02 24.71±0.44 12.93±0.73
M8 98.35±1.21 0.349±0.02 0.411±0.04 1.177±0.07 19.99±0.16 15.01±0.18
M9 101.41±1.33 0.352±0.04 0.409±0.03 1.162±0.06 20.03±0.22 13.92±0.19 *Mean ± SD, (n=3)
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6.4.1.4. Compressibility Index
The Carr’s compressibility index (CI) and Hausner ratio (HR) were
calculated based on the equations for different batches of microspheres.
These were calculated in accordance with density measurements. CI is
a measure of powder bridge strength and stability, and the Hausner ratio
(HR) is a measure of the interparticulate friction character is rated based
on compressibility index and Hausner ratio. Lower CI or lower Hausner
ratios of a material indicate better flow properties than higher ones. A
Carr’s CI of <10 or HR of <1.11 is considered ‘excellent’ flow where as
CI>38% or HR>1.60 is considered ‘very very poor’ flow. There are
intermediate scales for CI between 11–15% or HR between 1.12–1.18 is
considered ‘good’ flow, CI between 16–20 % or HR between 1.19–1.25
is considered ‘fair’ flow, CI between 21–25 %or HR between 1.26–1.34
is considered passable flow, CI between 26–31 or HR between 1.35–
1.45 is considered ‘poor’ flow, and CI between 32–37 %or HR between
1.46–1.59 is considered ‘very poor’ flow [ Hausner , 1967]. If powders
are readily compressed by tapping, their flow energy requirement
increases. Based on the results obtained, flow of sample was rated as
‘poor’, that of it was rated as ‘very poor’, and it was considered to be
‘very very poor’ in terms of its flow based on CI and HR values [ Carr
,1965]. Present study resulted as shown in Table 5 that the HR value
223
was minimum in the batch M1 i.e. 1.067±0.02 and maximum value
obtained for the batch M6 was 1.224±0.03 but in case of Carr’s Index
(CI) was found minimum for M1 showing value of 6.26±0.22 and
maximum for M6 of 18.33±0.21 showing the compressibility of
microspheres characterizing convenience of formulation aspect.
Suitability of formulation was potentially supported by these tabulated
results showing effect of formulation variables on the micromeritic
properties that perfectly supported earlier analogous works [Deore et al.,
2009].
6.4.2. General characterization of microspheres
Parameters like % yield, drug entrapment, swelling index,
mucoadhesive nature, surface charge(zeta potential) of each batch of
prepared microspheres were comparatively monitored with the purpose
to define effectiveness of selected batch that could affect further stages
of investigation.Results of the parameters evaluated for comparison
between different batches of microspheres was depicted in Table 6.
6.4.2.1. Percentage yield
This parameter was monitored to find out efficiency of reproducible
and feasible and pharmaceutically adequate formulation using minimum
input showing usefulness of particular batch. The percentage yield was
224
between 69.49± 0.64 % to 89.97±0.66% of all the formulations which
was found to be directly proportional to number of drop of solution which
fell into calcium chloride solution. [Arora et al., 2011] Without changing
any formulation variability, only as the concentration of polymer
increased, a slight decrease in the percentage yield was
observed.(Table 6) But in case of formula M7-M9 no such significant
change was noticed when compared to control batches (p=0.5). These
findings described significant effect of polymeric combination together
with their proportion in complex on the yield of product as higher
proportion had no additional advantage due to lack of optimum
interaction between polymers used. Almost every related research works
investigated this parameter and stated its importance in their results
[Sinha et al., 2004 & Gawde et al., 2012].
6.4.2.2. Entrapment efficiency
Entrapment of drug in carrier system is considered as important
criteria for selection of suitable batch formula as amount of drug retained
in carrier indicates the overall efficiency of drug delivery system showing
sustainability and ability to prolong drug availability in site of action
[Anande et al., 2008]. The percentage of entrapment efficiency was
increased with the increase in polymer concentration as shown in
present study (Table 6). The drug entrapment efficiency of ALG-CHI
225
Polyelectrolyte complex microspheres were found to be more than
control batches i.e. AM and CM microspheres. Drug entrapment capacity
also had a strong dependence on particular proportion of polymeric
complex and drug ratio where higher ratio as used in formula batches
M7-M9 was not significantly higher than optimum batch i.e.M6 that
showed a value of 78.83±0.52% that was well above (p=0.5) than other
batches from M1-M5 including control batches (AM and CM).These
variation in drug entrapment was not directly proportional to increasing
polymer drug ratio. Hence above an optimum level there was no
significant increase in drug retaining property in microspheres as found
in previous discussions [Mi et al., 2002 & Jose et al., 2011].
6.4.2.3 Swelling Index
Ability of microspheres to swell in presence of suitable medium is
also a matter of prime importance to determine its capacity to liberate
entrapped drug into release medium. Swelling behavior of microspheres
predict drug release profile facilitating the requirement of optimum drug
action [Quan et al., 2008].
226
Table 6: General characterization of microspheres
Batch Drug entrapment Swelling index % Mucoadhesion
After 10 hours Zeta potential (mv) % yield
AM 47.99±0.76 0.581±0.04 77.98±1.17 -45.15±0.97 69.49±0.64
CM 51.28±0.49 0.673±0.07 68.43±1.11 +42.33±1.03 73.91±0.88
M1 54.12±0.16 0.693±0.03 84.22±0.79 +45.04±1.11 76.44±0.32
M2 58.44±0.32 0.671±0.02 82.91±0.62 +41.09±0.46 82.27±0.44
M3 63.07±0.53 0.591±0.01 87.09±0.99 +45.91±0.85 77.22±0.29
M4 66.71±0.73 0.598±0.05 83.77±1.02 +40.44±1.17 81.88±0.18
M5 63.11±0.44 0.610±0.09 81.13±0.91 +43.29±0.49 87.91±0.49
M6 78.83±0.52 0.701±0.02 89.63±0.82 +54.09±0.73 89.97±0.66
M7 80.09±0.69 0.700±0.08 85.45±1.08 +51.22±1.15 86.15±0.51
M8 71.47±0.42 0.691±0.04 85.27±1.15 +43.33±0.87 86.09±0.33
M9 77.81±0.47 0.707±0.09 86.66±0.95 +50.97±0.46 84.38±0.27 *Mean ± SD, (n=3)
227
As shown in Table 6 swelling behavior was found to be a variable
depending on the nature of polymer used, their surface charges, degree
of interaction to form complex, available porosity after swelling etc. The
preliminary experimental studies revealed that the release of the
balsalazide depends on the swelling degree of ALG-CHI microspheres.
Therefore, the result presented a study regarding the variation of the
swelling degree of microspheres in relation with the pH and the
temperature. Table 6 presented the swelling degrees of the eleven types
of microspheres in different conditions of temperature and pH after a
period of five hours. Data revealed that variation in alginate and chitosan
ratio in PEC affected a lot the degree of swelling of each batch of
microsphere and more distinctly the range of swelling Index of
0.581±0.04 to 0.707±0.09 where it was found to increase the value with
change in polymer proportion in formula and above 0.3% w/w of alginate
and 0.2%w/w of chitosan was not so sharp as shown in batch M7-M9.
However a satisfactory value of 0.701±0.02 was found for the batch M6
that was considered optimum because efficient control of electrostatic
charges on the surface achieved by using optimum proportion of
polymers for controlled interaction at predetermined pH 5.0. These
findings had well conformity with earlier work. [Dima et al., 2013 &
Obeidat and Price, 2006] revealing variation in all influencing factors
toward performance of formed microspheres.
228
6.4.2.4. Mucoadhesive property
Mucoadhesive property of microspheres being explored for
targeting purpose is considered as a prime parameter for evaluation of
performance as mucoadhesion and its durability both can predict the
degree of sustainability and duration of drug availability at the desired
site [Grabovac et al., 2008]. Present in vitro wash off study also
determined the effect of variation in polymer concentration in formed
complex on their mucoadhesive nature. The adsorption of ALG-CHI
microspheres on rat small intestine was found to attach more to the
mucosal tissue whereas only a few of the AM and CM microspheres
were adsorbed to the tissue. This is the further evidence for the strong
interaction between PEC microspheres and mucus glycoprotein and/or
mucosal surfaces. Results presented in Table 6 described comparative
aspects of % of mucoadhesion after 10 hours in colon pH. It was found
to have a range of 68.43±1.11 for the batch CM to 89.63±0.82 % for the
batch M6. Moreover the degree of mucoadhesion was not so much
changed with increase in polymer proportion above 0.3% 0f alginate as
shown in the formula batches M7-M9. It clearly dictated a possibility of
getting a rough idea about optimum proportion used to formulate PEC
microspheres with satisfactory mucoadhesion as proposed earlier
[Barger et al., 2004a & Craig, 1997]. Optimum combination of reacting
229
polymers could predict optimum rate of electrostatic interaction between
them and thereby enhanced interaction with mucosal cells in the colon
probably through sialic acid and cellular lectin conjugation following
reverse targeting pathway as proposed in earlier findings.[Wirth et
al.,1998 & Tamara,2004] Controlled net surface charges available on the
microspheres could be the possible reason for achievement of potential
mucoadhesive nature in PEC microspheres as found in M6.
6.4.2.5. Determination of Zeta potential
As a potential parameter to determine surface electrostatic nature
of microspherical drug carrier system Zeta potential measurement has
its long known importance. Present study investigated surface charge on
each batch of microspheres. From the results shown in Table 6 it had a
range of -45.15±0.97 mv found in batch AM whereas maximum charge
of +54.09±0.73mv was found for batch M6.This variation in surface
charge strictly relied on the interaction behavior of alginate and chitosan
forming complex at different proportion used to make different batches of
microspheres supporting analogous finding [Subudhi et al., 2015]. Like
other parameters zeta potential also control overall interaction of
polymers thereby predict their size, shape, stability in liquid medium.
Generally an ideal range between +35 mv and +58 mv of zeta potential
present in PEC microspheres dictate optimum stability and satisfactory
230
mucoadhesion [Fischer et al., 2004]. As shown in tabulated data zeta
potential increased gradually with addition of more and more chitosan to
form complex and after a concentration of 0.3% of chitosan and 0.2% of
alginate forming microsphere of batch M6 in comparison to the control
batches and other batches with greater proportion of chitosan
used(p=0.5). It dictated role of chitosan to provide sufficient +ve charge
on the surface of microspheres that could interact with mucosal tissue to
provide optimal bioadhesion and colon targeting efficiency conforming to
earlier experimental data [Dhawan et al., 2004].
6.4.2.6. Surface Morphology
The results obtained from SEM studies (Fig. 14) confirmed the
porous and spherical structure of microspheres. Moreover, morphology
of microspheres revealed that degree of porosity of microspheres was
dependant on the composition of alginate and chitosan in optimum
molecular combination present in the microspheres. High content of
Chitosan led to less porosity probably due to excessive electrostatic
interaction. There was less porosity appearing in PEC microspheres with
0.3% of chitosan in formulation, whereas greater than the optimum
content of chitosan resulted in loss of spherical structure and mechanical
strength. The spherical shape of microspheres may be attributed to a
high degree of cross-linking occurring in each case. As per earlier report
231
externally made alginate microspheres morphology is frequently related
to a disc-like geometry due to its heterogeneous cross-linked structure,
which is higher at the outer surface. Photomicrograph revealed the
absence of drug crystals on the surface of microsphere, indicating
uniform distribution of the drug within the microspheres with no event of
aggregation resuted from controlled surface charges yielding optimum
zeta potential [Ribeiro et al., 2005]. The rate of solvent removal from the
microspheres exerts an influence on the morphology of the final product.
The collapse of microspheres at high magnification may be attributed to
a rapid and extensive dehydration upon high-energy incidence as
occurred in case of PEC microspheres that perfectly corroborated with
earlier finding in allied field [Abreu et al., 2010].
232
6.4.3. In vitro drug release studies of microspheres
The formulations targeted to the colon should not only protect the drug
from being released on the physiological environment of stomach and
small intestine, but also release the drug in the colon after enzymatic
degradation by colonic bacteria. Hence invitro drug release studies were
carried out in SCF (pH 7.4 phosphate buffer containing 4%w/v of rat
caecal contents [Anande et al, 2008]. Results shown in Table 7A and
corresponding Fig. 15 revealed at the end of the 24 hr of testing that
percentage drug released from the Alginate microspheres (AM) was
found to be 99.88%, within 12 hours and for Chitosan batch (CM) it was
99.08% within 12 hours.
233
Table 7A: In vitro drug release profile of microspheres
Time Hour
AM %
CDR
CM %
CDR
M1 %
CDR
M2 %
CDR
M3 %
CDR
M4 %
CDR
M5 %
CDR
M6 %
CDR
M7 %
CDR
M8 %
CDR
M9 %
CDR 0 0 0 0 0 0 0 0 0 0 0 0 0.25 7.56
±1.19 9.91 ±1.7
4.21 ±2.22
6.36 ±2.33
4.14 ±1.9
4.14 ±2.86
4.14 ±3.01
3.37 ±2.73
4.99 ±4.22
7.56 ±2.68
4.09 ±3.77
0.5 18.78 ±3.93
21.44 ±4.22
10.98 ±4.31
13.09 ±5.11
8.29 ±4.89
5.39 ±4.22
5.39 ±5.98
7.18 ±4.87
11.31 ±5.98
17.78 ±3.77
10.32 ±4.09
0.75 26.34 ±4.11
34.19 ±3.09
29.43 ±3.98
20.11 ±3.04
14.75 ±5.98
18.22 ±3.91
18.22 ±4.99
11.25 ±3.01
21.09 ±5.68
21.34 ±4.09
14.38 ±7.07
1 39.29 ±2.73
45.51 ±2.77
42.43 ±2.05
28.42 ±4.9
25.03 ±2.74
27.31 ±2.68
27.31 ±3.97
16.19 ±5.88
28.26 ±3.27
26.49 ±3.07
22.09 ±3.23
2 56.56 ±4.87
53.33 ±2.18
51.88 ±4.22
34.93 ±3.13
37.22 ±5.18
42.29 ±3.77
38.29 ±8.13
22.53 ±6.61
37.44 ±4.09
31.56 ±3.23
34.16 ±5.99
3 68.56 ±3.01
65.63 ±1.99
60.25 ±3.04
52.16 ±5.99
48.86 ±3.87
57.93 ±4.09
47.93 ±3.08
30.84 ±4.01
40.71 ±3.07
33.56 ±5.99
42.23 ±3.87
4 77.78 ±6.88
78.09 ±2.04
76.11 ±5.99
68.88 ±3.17
61.49 ±4.02
64.41 ±3.07
54.41 ±4.18
39.18 ±6.32
53.28 ±6.23
40.78 ±3.87
55.04 ±7.61
*Mean ± SD, (n=3)
234
Table 7A: In vitro drug release profile of microspheres (Contd...)
Time Hour
AM %
CDR
CM %
CDR
M1 %
CDR
M2 %
CDR
M3 %
CDR
M4 %
CDR
M5 %
CDR
M6 %
CDR
M7 %
CDR
M8 %
CDR
M9 %
CDR 4 77.78
±6.88 78.09 ±2.04
76.11 ±5.99
68.88 ±3.17
61.49 ±4.02
64.41 ±3.07
54.41 ±4.18
39.18 ±6.32
53.28 ±6.23
40.78 ±3.87
55.04 ±7.61
6 90.18 ±3.61
86.74 ±5.57
88.37 ±3.09
75.37 ±7.87
68.31 ±8.29
79.02 ±6.23
59.02 ±8.22
48.04 ±3.03
65.53 ±5.99
45.18 ±6.61
61.9 ±4.01
8 98.64 ±4.01
93.23 ±3.4
95.42 ±5.45
82.02 ±3.09
80.04 ±3.54
84.66 ±5.99
64.66 ±7.97
56.39 ±5.97
78.2 ±3.87
58.64 ±4.01
77.11 ±6.77
10 99.46 ±2.32
95.17 ±5.61
97.29 ±2.97
88.43 ±5.91
87.48 ±4.28
90.17 ±3.87
76.17 ±8.64
66.26 ±3.29
90.41 ±8.61
79.87 ±6.72
89.03 ±4.84
12 99.88 ±3.03
99.08 ±2.17
99.06 ±8.03
90.28 ±5.18
91.17 ±3.75
97.25 ±3.87
88.25 ±4.09
70.91 ±8.09
92.13 ±8.04
95.67 ±4.84
96.14 ±4.11
22 99.89 ±5.97
99.41 ±5.93
99.84 ±4.11
98.11 ±4.72
99.21 ±6.27
99.18 ±3.61
99.18 ±6.27
88.99 ±3.75
94.08 ±8.99
97.14 ±4.11
96.36 ±7.09
23 99.93 ±2.98
99.87 ±9.33
98.79 ±8.79
99.48 ±4.09
99.43 ±4.01
99.43 ±4.88
92.14 ±6.27
94.11 ±3.09
97.49 ±7.09
97.13 ±3.03
24 99.24 ±8.24
99.67 ±3.28
99.79 ±6.77
99.49 ±6.91
95.08 ±4.09
94.64 ±5.45
98.06 ±4.09
97.68 ±5.97
*Mean ± SD, (n=3)
235
Fig. 15: Plot In vitro drug release profile of different batches of
microspheres
This release profile indicated earlier swelling and corresponding
faster rate of drug release at the colon pH. In case of other test batches
of PEC microspheres drug release rate was somewhat restricted by the
process of controlled swelling of microspheres in colon. However after
careful comparison within the test batches (M1-M9) drug release rate
was more efficiently controlled and sustained as found in the batch M6
showing more uniform rate of drug release that was maintained
uninterruptedly up to 24 hours as a continuous pattern showing 48.04%
after 6 hours, 66.26% after 10 hours, 88.99% after 22 hours and finally
liberated 95.08% after 24 hours. This eventually demonstrated a
-20
0
20
40
60
80
100
120
0 5 10 15 20 25 30
% C
UM
. DRU
G R
ELEA
SED
TIME (HOUR)
PLOT FOR DRUG RELEASE DATA OF MICROSPHERES
AM
CM
M1
M2
M3
M4
M5
M6
M7
M8
M9
236
capacity of specific molecular combination of reacting polymers to
contribute controlled swelling, profound mucoadhesion due to more
uniform surface charge distribution, porosity and controlled viscous
interior environment that cumulatively rendered the microspheres to
possess enormous potential to release drug in a predetermined,
controlled and reproducible way [Khamanga et al., 2012].
237
Table 7B: Data for Zero order plot (Cumul. Quantity released Vs Time)
Time (Hour)
AM CDR
CM CDR
M1 CDR
M2 CDR
M3 CDR
M4 CDR
M5 CDR
M6 CDR
M7 CDR
M8 CDR
M9 CDR
0 0 0 0 0 0 0 0 0 0 0 0
0.25 1.512 1.982 0.842 1.272 0.828 0.828 0.828 0.674 0.998 1.512 0.818
0.5 3.756 4.288 2.196 2.618 1.658 1.078 1.078 1.436 2.262 3.556 2.064
0.75 5.268 6.838 5.886 4.022 2.95 3.644 3.644 2.25 4.218 4.268 2.876
1 7.858 9.102 8.486 5.684 5.006 5.462 5.462 3.238 5.652 5.298 4.418
2 11.312 10.666 10.376 6.986 7.444 8.458 7.658 4.506 7.488 6.312 6.832
3 13.712 13.126 12.05 10.432 9.772 11.586 9.586 6.168 8.142 6.712 8.446
4 15.556 15.618 15.222 13.776 12.298 12.882 10.882 7.836 10.656 8.156 11.008
6 18.036 17.348 17.674 15.074 13.662 15.804 11.804 9.608 13.106 9.036 12.38
8 19.728 18.646 19.084 16.404 16.008 16.932 12.932 11.278 15.64 11.728 15.422
10 19.892 19.034 19.458 17.686 17.496 18.034 15.234 13.252 18.082 15.974 17.806
12 19.976 19.816 19.812 18.056 18.234 19.45 17.65 14.182 18.426 19.134 19.228
22 19.978 19.882 19.968 19.622 19.842 19.836 19.836 17.798 18.816 19.428 19.272
23 19.986 19.974 19.758 19.896 19.886 19.886 18.428 18.822 19.498 19.426
24 19.848 19.934 19.958 19.898 19.016 18.928 19.612 19.536
238
Cumulative Drug Release (CDR) vs Time data was used for zero
order plot (Table 7B and Fig. 15A) whereas for first order plot Drug
remaining vs Time (Table 7C and Fig. 16), for Higuchi plot CDR vs
square root of time (Table 7D and Fig. 17) and logarithm of CDR VS
logarithm of time for Korsemeyer-Peppas plot (Table 7E and Fig. 18)
were adopted following previously reported articles [Soppimath et al.,
2001].
Fig. 15A: In vitro drug release profile of microspheres (zero order
plots)
It was also observed that maximum batches of microspheres followed
release pattern that were very close to first order model and more
distinctly the batch M6 that followed Higuchi model withhighest
0
5
10
15
20
25
0 5 10 15 20 25 30
CUM
. QTY
. OF
DRU
G R
ELEA
SED
TIME (HOUR)
ZERO ORDER PLOT FOR DRUG RELEASE DATA
AM
CM
M1
M2
M3
M4
M5
M6
M7
M8
M9
239
correlation co-efficient of 0.992 (Table 8) that predicted uniform release
through spherical matrix following diffusion method [Higuchi, 1963]. As
per data generated the batch M6 had appreciable correlation with first
order plot (R2=0.985) and simultaneously corroborated Higuchi drug
release profile (R2=0.992) presented a mixed drug release pattern
probably due to this controlled swelling behavior unlike other PEC
batches of microspheres.As per data fitting with Korsemeyer-Peppas
model value of n for each batch was calculated and found to be above
0.684 for M6 describing presence of both Fickian and non-Fickian drug
release mechanism [ Bonartsev et al., 2007]. It was observed, from table
7 and Fig. 18 that the swelling degree varied together with the proposed
pH in close relation with the microsphere composition.
240
Table 7 C: Data for First order plot (Log Cumul. Drug Remained Vs Log Time)
Log Time
(Hour)
AM Log
CD Rem
CM Log
CD Rem
M1 Log
CD Rem
M2 Log
CD Rem
M3 Log
CD Rem
M4 Log
CD Rem
M5 Log
CD Rem
M6 Log
CD Rem
M7 Log
CD Rem
M8 Log
CD Rem
M9 Log
CD Rem
-0.60206 1.26689 1.255707 1.28235 1.272491 1.282667 1.282667 1.282667 1.286142 1.278799 1.26689 1.282894
-0.30103 1.210693 1.196231 1.250518 1.2401 1.263447 1.276967 1.276967 1.268672 1.248905 1.216007 1.253726
-0.12494 1.168262 1.119322 1.14965 1.203522 1.231724 1.213677 1.213677 1.249198 1.198162 1.196784 1.233605
0 1.08429 1.037347 1.061226 1.155822 1.175918 1.162505 1.162505 1.224326 1.156791 1.167376 1.192623
0.30103 0.93892 0.970068 0.983356 1.114411 1.098851 1.062281 1.091386 1.190164 1.097327 1.13634 1.11952
0.477121 0.798513 0.83721 0.900367 0.980821 1.009791 0.925003 1.017618 1.140885 1.074011 1.12346 1.062732
0.60206 0.647774 0.641672 0.679246 0.79407 0.886604 0.852358 0.9599 1.085076 0.970533 1.073498 0.953856
0.778151 0.293141 0.423574 0.36661 0.692494 0.801952 0.622835 0.913602 1.016699 0.838471 1.039969 0.881955
0.90309 -0.56543 0.131619 -0.0381 0.55582 0.601191 0.486855 0.849297 0.940616 0.639486 0.917611 0.660676
1 -0.96658 -0.01502 -0.266 0.364363 0.398634 0.293584 0.678154 0.829175 0.282849 0.604874 0.341237
1.079181 -1.61979 -0.73518 -0.72584 0.288696 0.246991 -0.25964 0.371068 0.764774 0.197005 -0.06248 -0.11238
1.342423 -1.65758 -0.92812 -1.49485 -0.42251 -0.80134 -0.78516 -0.78516 0.342817 0.073352 -0.2426 -0.13787
1.361728 -1.85387 -1.58503 -0.61618 -0.98297 -0.9431 -0.9431 0.196453 0.071145 -0.2993 -0.24109
1.380211 -0.81816 -1.18046 -1.37675 -0.9914 -0.007 0.030195 -0.41117 -0.33348
241
Both molecular combination of alginate and chitosan and their pH
induced optimum interaction affected much to control drug release
pattern. Thus, for pH 4.0 the swelling degree gets higher as the chitosan
content increases.
Fig. 16: In vitro drug release profile of microspheres (First order plot)
As previous research already detailed about this relation as
resulted from the total protonation of the amino groups that happened at
the pH decreased (pH≤4) and that was responsible to repulsion between
the polycation’s bonds thus favoured the water diffusion. At lower pH,
the chitosan and the alginate become partially protonated, and the
electrostatic forces, manifested between the ammonium and carboxylate
ions to which are added the hydrogen bounds and hydrophobic
interactions, make the matrix network become more compact and less
permissive for the water molecules [Saether et al., 2008].
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
0 5 10 15 20 25 30
LOG
QTY
. OF
DRU
G R
EMAI
NIN
G
TIME (HOUR)
FIRST ORDER PLOT FOR DRUG RELEASE DATA
AM
CM
M1
M2
M3
M4
M6
M7
M8
M9
242
Table 7D: Data for Higuchi plot (Cum. Drug Released Vs. Square root of Time)
Sq Root Time
(Hour)
AM CDR
CM CDR
M1 CDR
M2 CDR
M3 CDR
M4 CDR
M5 CDR
M6 CDR
M7 CDR
M8 CDR
M9 CDR
0 0 0 0 0 0 0 0 0 0 0 0 0.5 1.512 1.982 0.842 1.272 0.828 0.828 0.828 0.674 0.998 1.512 0.818 0.707107 3.756 4.288 2.196 2.618 1.658 1.078 1.078 1.436 2.262 3.556 2.064 0.866025 5.268 6.838 5.886 4.022 2.95 3.644 3.644 2.25 4.218 4.268 2.876 1 7.858 9.102 8.486 5.684 5.006 5.462 5.462 3.238 5.652 5.298 4.418 1.414214 11.312 10.666 10.376 6.986 7.444 8.458 7.658 4.506 7.488 6.312 6.832 1.732051 13.712 13.126 12.05 10.432 9.772 11.586 9.586 6.168 8.142 6.712 8.446 2 15.556 15.618 15.222 13.776 12.298 12.882 10.882 7.836 10.656 8.156 11.008 2.44949 18.036 17.348 17.674 15.074 13.662 15.804 11.804 9.608 13.106 9.036 12.38 2.828427 19.728 18.646 19.084 16.404 16.008 16.932 12.932 11.278 15.64 11.728 15.422 3.162278 19.892 19.034 19.458 17.686 17.496 18.034 15.234 13.252 18.082 15.974 17.806 3.464102 19.976 19.816 19.812 18.056 18.234 19.45 17.65 14.182 18.426 19.134 19.228 4.690416 19.978 19.882 19.968 19.622 19.842 19.836 19.836 17.798 18.816 19.428 19.272 4.795832
19.986 19.974 19.758 19.896 19.886 19.886 18.428 18.822 19.498 19.426
4.898979
19.848 19.934 19.958 19.898 19.016 18.928 19.612 19.536
243
At higher pH, the swelling degree of alginate microspheres grows
abruptly as a result of the network destruction manifested through the
calcium ions detachment and the permeation of water within the
microsphere [Becheran-Maron et al., 2004]. For higher values of pH, the
solubility of the chitosan decreases and the prepared microspheres
becomes rigid, almost impermeable for water.
Fig. 17: In vitro drug release profile of microspheres (Higuchi plot)
The temperature favours the swelling degree for all types of
microspheres. As the temperature rises, the pH influence over the
swelling degree of microspheres is less noticed. The release of the
coriander oil is correlated with the swelling degree, which means that the
main release mechanism is diffusion [Dima et al., 2013]. For pH 5, the
chitosan and the alginate become partially protonated, and the
electrostatic forces, manifested between the ammonium and carboxylate
ions to which are added the hydrogen bounds and hydrophobic
interactions, make the matrix network become more compact and less
permissive for the water molecules.
0
10
20
30
0 1 2 3 4 5 6
CUM
. QTY
. OF
DRU
G
RELE
ASED
SQUARE ROOT OF TIME (HOUR)
HIGUCHI PLOT FOR DRUG RELEASE DATA
AM
CM
M1
M2
M3
244
Table 7E: Data for Korsemeyer-Peppas plot (Log Cumul. Drug Released Vs Log Time)
Log Time
AM Log
CM Log
M1 Log
M2 Log
M3 Log
M4 Log
M5 Log
M6 Log
M7 Log
M8 Log
M9 Log
(Hour) CD R CD R CD R CD R CD R CD R CD R CD R CD R CD R CD R
-0.60206 0.179552 0.297104 -0.07469 0.104487 -0.08197 -0.08197 -0.08197 -0.17134 -0.00087 0.179552 -0.08725
-0.30103 0.574726 0.632255 0.341632 0.41797 0.219585 0.032619 0.032619 0.157154 0.354493 0.550962 0.31471
-0.12494 0.721646 0.834929 0.76982 0.604442 0.469822 0.561578 0.561578 0.352183 0.625107 0.630224 0.458789
0 0.895312 0.959137 0.928703 0.754654 0.699491 0.737352 0.737352 0.510277 0.752202 0.724112 0.645226
0.30103 1.053539 1.028002 1.01603 0.844229 0.871806 0.927268 0.884115 0.653791 0.874366 0.800167 0.834548
0.477121 1.137101 1.118132 1.080987 1.018368 0.989983 1.063934 0.981637 0.790144 0.910731 0.826852 0.926651
0.60206 1.191898 1.193625 1.182472 1.139123 1.089834 1.109983 1.036709 0.894094 1.027594 0.911477 1.041708
0.778151 1.25614 1.239249 1.247335 1.178229 1.135514 1.198767 1.072029 0.982633 1.11747 0.955976 1.092721
0.90309 1.295083 1.270586 1.280669 1.21495 1.204337 1.228708 1.111666 1.052232 1.194237 1.069224 1.188141
1 1.298678 1.27953 1.289098 1.24763 1.242939 1.256092 1.182814 1.122281 1.257246 1.203414 1.250566
1.079181 1.300509 1.297016 1.296928 1.256622 1.260882 1.28892 1.246745 1.151737 1.265431 1.281806 1.283934
1.342423 1.300552 1.29846 1.300335 1.292743 1.297585 1.297454 1.297454 1.250371 1.274527 1.288428 1.284927
1.361728
1.300726 1.300465 1.295743 1.298766 1.298547 1.298547 1.265478 1.274666 1.28999 1.288383
1.380211
1.297717 1.299594 1.300117 1.298809 1.279119 1.277105 1.292522 1.290836
245
From the data shown in Table 8 meaningful correlation for zero
order (R2 = 0.910) found for M6 showing a possibility of concentration
independence to drug release simulating matrix erosion whereas for
most batches had well correlation specially for M3 highest correlation
found for first order plot (R2 = 0.994) showing diffusion controlled drug
release pattern and more importantly in Higuchi plot highest correlation
found (R2 = 0.992) for batch M6 as porosity and swelling was controlled
efficiently.
Fig. 18: In vitro drug release profile of microspheres (Korsemeyer-
Peppas plot)
These findings predicted a well defined drug release profile with
preferential diffusion gradual erosion of drug retaining matrix as
predominant mechanism for each batch of PEC microspheres.Data also
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
-1 -0.5 0 0.5 1 1.5
LOG
CU
M. Q
TY. O
F D
RUG
REL
EASE
D
LOG TIME (HOUR)
KOSEMEYER PEPPAS PLOT FOR DRUG RELEASE DATA AM
CM
M1
M2
M3
M4
M5
M6
M7
M8
M9
246
corroborated earlier report describing controlled drug release profile that
could be obtained by providing effective control over selection of proper
ratio of interacting polymers with their suitable combination and proper
method of preparation in order to formulate potential carrier system for
colon targeting [Costa and Sousa, 2001].
Table 8: Kinetic interpretation of drug release data
Formula
Batch
Zero Order
R2
First Order
R2
Higuchi Model
R2
Korsemeyer-Peppas
R2 n
AM 0.597 0.875 0.849 0.881 0.555
CM 0.598 0.947 0.836 0.865 0.453
M1 0.598 0.976 0.830 0.810 0.605
M2 0.720 0.991 0.903 0.920 0.558
M3 0.755 0.994 0.925 0.913 0.652
M4 0.690 0.982 0.959 0.862 0.669
M5 0.823 0.976 0.883 0.887 0.652
M6 0.910 0.985 0.992 0.972 0.684
M7 0.743 0.900 0.915 0.913 0.578
M8 0.860 0.972 0.953 0.955 0.506
M9 0.767 0.930 0.927 0.933 0.643
247
6.4.4. Stability analysis for selected batch (M6)
Stability study was performed mandatorily in previous
investigations cited [Brahmaiah et al., 2013 & Zezin and Rogacheva,
1973] to assess the capability of microsphere to withstand all wear and
tear during accelerated storage condition as per ICH guidelines. In this
evaluation several parameters were monitored. Result of such
investigation was shown in Table 9. Drug content as prime parameter
was monitored at several predetermined sampling periods and data
showed that selected batch M6 could maintain drug content well above
99 % after 45 days and then decreased to 98.05±0.95% at the end of
study showing very slow rate of decrease noted from 90 to 180 days that
could be considered insignificant compared to initial value (p=0.5).
Similarly % mucoadhesion was also shown a decrease from 89.18±0.88
% to 87.95±0.85 % after 180 days rendering an insignificant change
compared to the initial data.
248
Table 9: Stability analysis of selected batch of microspheres (M6)
Stability
condition
Sampling Time
(days)
Drug content
% (n=3)
% Mucoadhesion
After 10 hours
At pH7.4 (n=3)
Drug release profile(n=3)
T50 (hour)
T80 (hour)
T90 (hour)
40 ±2 0C
75 ± 5% RH
0 99.87±1.01 89.18±0.88 6.02±0.08 10.04±0.13 22.41±0.11
15 99.83±0.99 89.06±0.78 5.97±0.03 10.17v0.08 21.87±0.09
30 99.85±1.12 89.11±0.91 6.06±0.02 10.22±0.15 22.09±0.04
45 99.72±0.87 88.87±1.03 6.19±0.06 11.05±0.03 22.31±0.13
90 98.50±0.82 88.62±0.99 5.88±0.04 10.39±0.04 21.17±0.07
120 98.27±0.88 88.09±0.68 5.91±0.01 10.33±0.05 20.37±0.02
140 98.12±1.01 89.16±0.82 6.01±0.06 9.84±0.13 21.19±0.12
160 98.01±1.09 88.22±1.05 5.74±0.04 11.04±0.02 22.02±0.11
180 98.05±0.95 87.95±0.85 6.11±0.09 10.84±0.05 22.07±0.06
*Mean ± SD, (n=3)
249
Moreover drug release profile 50%, 80% and 90% also shown a
change of 6.02±0.08 to 6.11±0.09 hours, from10.04±0.13 to 10.84±0.05
hours and from 22.41±0.11 to 22.07±0.06 hours respectively with some
nominal fluctuation in between .These cumulative observation revealed
overall satisfactory maintenance of quality even after 6 months of
exaggerated environment which made symmetry with previous
experimental outcome [Rahman et al., 2006 & Vaidya et al., 2009].
6.5. Preparation of tablet with enteric coated microsphere (M6)
Matrix tablet of enteric coated microspheres was prepared using
official method maintaining all needful steps like coating of microspheres
granule preparation, their characterization including size distribution.
Mixing of fines and portion of disintegrating agent and lubricant etc. was
also monitored so as to ensure tablet prepared with dose and weight
uniformity and proper size and thickness suitable for compression into
tablet [ Ansel and Poppovich ,1995 ].
250
6.5.1. Enteric coating of microspheres (M6):
Fig. 19 SEM of Eudragit S100 coated microspheres (M6)
Microsspheres were coated with proper thickness of enteric polymer so
as to obtain satisfactory level of protection against gastric pH.
Additionally rough and irregularities if any on surface of microspheres
were also sufficiently covered to yield uniform size distribution of
prepared granules (Fig. 19).
6.5.2. Sieve analysis of granules
Sieve analysis data obtained for prepared microsphere granules
(150 gm)were in the size range of 0.075 to 4.75 mm and 2.67% to 4.00
% of total wt of sample were retained on sieve with a maximum value of
25.34% retained on sieve no 30 (size 0.6mm).
251
Table 10: Sieve analysis of granules
Sieve No Size (mm)
Wt of sample Retained(gm)
% Retained
200 0.075 4 2.67
100 0.15 11 7.34
50 0.3 28 18.67
40 0.425 25 16.67
30 0.6 38 25.34
16 1.18 22 14.67
8 2.36 16 10.67
4 4.75 6 4.00
It was observed that the average size of the microsphere granules
ranged between 500 to 600μm as presented in Table 10. It was
prominent from the Fig. 20 that size distribution was also normal
showing a possibility of normal distribution of granules of different size
ranges retained on each sieve. From the pattern shown in the diagram
major % of granules were retained on sieve no 16 and 50 with maximum
proportion retained on sieve no 30.These range was found to be within
official working range of granulation for matrix tablet. These uniformity
predicted possibility of well compact tablet with dosage and weight
uniformity as obtained in previous research [Ofokans and Kenechukwu,
2013; Bruce et al., 2003].
252
Fig. 20: Plot for sieve analysis of granules
6.5.3. Preparation of matrix tablet of enteric coated microspheres
Stemming from previously reported results [Jadhav et al., 2013 &
Nagaich et al., 2010] wet granulation method was adopted for
preparation of matrix tablet using microsphere of selected batch M6.
HPMC was used as binder as it had long known potential as strong
binder at lowest possible concentration. Lactose was used as easily
available and low cost inert diluents and MCC played a role as a
potential disintegrant. Magnesium stearate and talc rendered their
253
specific function as lubricating agent and glidant respectively. Following
official method 20% of MCC was used before compression of granular
mass to form matrix tablet whereas 80% disintegrant was used during
preparation of granules to avail facility of complete breakdown of
granules to liberate microspheres to release drug [Mahesh et al., 2011].
Proper size distribution of granules including fractional portion of fines
inside contributed much toward uniform filling of granules into die cavity
to ensure uniform drug content in each tablet. Moreover drug quantity
was adjusted in such way that 30% of drug remained outside
microsphere to facilitate immediate release and remaining 70% was kept
inside microspheres to achieve sustained release at colon satisfying
targeting purpose [Kuksal et al., 2006].
Coating process ensured uniform thickness of enteric coating film
of Eudragit S100 on microspheres. Three times consecutive coating with
sufficient time provided for drying rendered microspheres enough
protection from gastric environment and facilitate easy and complete
disintegration in colon to liberate drug at highest rate and extent of drug
availability thus sufficing effective colon targeting [Ofokansi and
Kenechukwu, 2013].
254
6.6. Evaluation of tablet of enteric coated microspheres
Overall performance of tablet dosage forms depends upon their
mechanical strength, dose uniformity, weight and thickness, drug
content mode of disintegration at desired pH condition and most
importantly their reproducible drug release profile according to official
allowances provided. In present investigation each parameters were
monitored with comparison to uncoated counterpart to have a distinct
idea about efficiency of enteric coating to achieve the goal of colon
targeting.
6.6.1 General evaluation of enteric coated tablets
In this section all parameters relating generalized evaluation of
uncoated tablet was presented to monitor tablet appearance, size,
shape, weight and strength eligible for coating. Result of all necessary
parameters monitored for prepared enteric coated tablet was cited in
Table 11.
6.6.1.1. Weight variation test
The weight variation test was conducted as per I.P and the results
are shown in Table 11. Average weight of tablet was 99.54±1.28 mg.
255
Table 11: General Evaluation of Tablet
Sl.No Parameters evaluated Observed Value
1 Avg. Weight(mg) 99.54±1.28
2 Thickness (mm) 2.11±0.09
3 Hardness (Kg/cm2) 5.5±0.11
4 Friability (%) 0.35±0.02
5 Assay (%) 98.78±1.42
6 Disintegration Time 43.22 ±1.32 minutes
*Mean ± SD, (n=3)
The weight variation test for prepared tablet complied with the IP
limit (± 10%). This weight of tablet was used for coating to get a
satisfactory weight to facilitate proper packaging. Proper size distribution
of granules and uniform size of microspheres were found to be
responsible for uniform weight as mentioned earlier [Srivastava et al.,
2010].
6.6.1.2 Hardness test
The adequate tablet hardness is necessary requisite for consumer
acceptance and handling .The measured mean hardness of the tablets
was found to be 5.5±0.11 Kg/cm2 and the results are shown in Table 11.
This value was found optimum to withstand wear and tear during further
handling. HPMC used as binder in the formula increased mechanical
strength of prepared tablet [Sherimeier and Schmidt, 2002].
256
6.6.1.3 Friability test
The friability test for all the formulations were done as per the
standard procedure I.P. The results of the friability test were tabulated in
Table 11 and it presented as 0.35±0.02 %.The data indicated that the
friability was less than 1% ensuring their mechanically stability. Coating
rendered the tablet more strength. Binder selection could be a reason for
achieving potential to resist mechanical and frictional stress [Nagaich et
al., 2010].
6.6.1.4. Thickness
The thickness of the tablets was found to be almost uniform in
tablet. The mean thickness was found to be 2.11±0.09 mm. None of
individual tablet showed a deviation. Hence, it was concluded that all the
formulations complied the thickness test and the results are shown in
Table11. Satisfactory size of granular microspheres with accepted
compressibility and proper selection of punches in tableting machine
were probably responsible for such uniformity in thickness.
6.6.1.5. Drug content
The drug content of tablet was evaluated as per the standard
protocol and the results are shown in the Table 11. The results showed
a mean value of 98.78±1.42 % indicating the percentage of drug content
257
to be in the official range 95.00% to 101.00% complying with the
acceptable limits as per Indian Pharmacopoeia i.e.± 5%.Spherical size of
microsphere loaded granules and their proper size distribution could be
the reason for such uniformity.
6.6.1.6. Disintegration Time
Capacity of uncoated tablet to disintegrate completely was
evaluated with official apparatus and data cited in Table 11 shown the
mean value of 43.22 ±1.32 minutes. It demonstrated the performance of
tablet without any external protection in variable biological environment
[Sharma et al., 2014 & Raghuram et al., 2003].
6.6.1.7. Dissolution rate analysis of tablet
Result of drug dissolution rate was depicted in Table12. Over the
past three decades, dissolution testing has evolved into a powerful tool
for characterizing the quality of oral pharmaceutical products. The term
dissolution can be defined as a process in which a known amount of
drug dissolves in a given medium per unit time under standardized
conditions [O.C.S., 2007 & Kanfer, 2010]. Result of entire drug release
profile of tablet was depicted in Table 12 as three separate phases. In
first phase of study the rate of release was compared with subsequent
uncoated microsphere tablet as control.
258
Table 12: Dissolution study of tablets of enteric coated microspheres (M6) Time
(Hour) Uncoated
Microsphere %CDR(U)
Enteric coated Microsphere
%CDR(E)
% Drug Remaining (U)
% Drug Remaining (E)
Log %CDR (U) mean± SEM
Log %CDR (E) mean± SEM
0 0 0 100 100 2 2 0.25 9.78 0.03 90.22 99.97 1.95±0.28 1.99±0.31 0.5 11.34 0.12 88.66 99.88 1.95±0.21 1.99±0.33 0.75 18.29 0.28 81.71 99.72 1.91±0.29 1.99±0.39
1 24.06 0.59 75.94 99.41 1.88±0.18 1.99±0.24 2 33.56 0.86 66.44 99.14 1.82±0.18 1.99±0.26 3 40.78 3.43 59.22 96.57 1.77±0.18 1.98±0.21 4 48.45 8.04 51.55 91.96 1.71±0.22 1.96±0.23 6 62.64 14.29 37.36 85.71 1.57±0.19 1.93±0.19 8 80.06 25.11 19.94 74.89 1.29±0.35 1.87±0.28
10 89.15 38.03 10.85 61.97 1.04±0.22 1.79±0.32 18 99.26 69.14 0.74 30.86 -0.13±0.03 1.49±0.11 20 99.29 76.74 0.71 23.26 -0.15±0.05 1.37±0.16 22 99.32 81.53 0.68 18.47 -0.17±0.01 1.27±0.17 24 99.57 86.18 0.43 13.82 -0.36±0.01 1.14±0.19
*Mean ± SD, (n=3)
259
Data shown in table 12 was plotted in fig. 21 A to represent overall
performance of enteric coating to control site specific release of drug in
colonic region after oral administration as matrix tablet. Kinetic behavior
of the release pattern was also analyzed kinetically to reveal possible
underlying release mechanism as per shown in table 13. It demonstrated
that uncoated microspheres released maximum of 33.56±0.83% in first 2
hours in SGF (pH 1.2), 62.64±1.02 % in next 4 hours in SIF (pH 6.8) and
99.57±0.91% in next 18 hours in SCF (pH 7.4) whereas Enteric coated
microspheres furnished only 0.86±0.08% drug release in first 2 hours in
SGF, 14.29±0.26% in next 4 hours in SIF and 86.18±1.01% in next 18
hours in SCF.
260
Table 13: Kinetic analysis of tablet drug release data
Batch Dissolution Medium
Time period (total 24 hours)
Maximum % CDR
Regression Equation (First order plot)
R2
Tablet of
Uncoated
microspheres
SGF
(pH1.2)
First 2
Hours
33.56±0.83 Y=-
0.086X+1.984
0.958
SIF
(pH 6.8)
Next 4
Hours
62.64±1.02 Y=-
0.065X+1.956
0.994
SCF
(pH7.4)
Next 18
Hours
99.57±0.91 Y=-
0.111X+2.159
0.967
Tablet of
Eudragit
S100 Coated
microspheres
SGF
(pH1.2)
First 2
Hours
0.86±0.08 Y=-
0.002X+2.000
0.956
SIF
(pH 6.8)
Next 4
Hours
14.29±0.26 Y=-
0.016X+2.030
0.993
SCF
(pH7.4)
Next 18
Hours
86.18±1.01 Y=-
0.043X+2.217
0.991
*Mean ± SD, (n=3)
Data also demonstrated that enteric coating of Eudragit S100
facilitated microspheres to restrict drug release showing insignificant
extent compared to control (p=0.5). After being introduced to SIF enteric
coating shown minimal but significant erosion of coating releasing
substantial amount (14.29±0.26%) of drug from the region exterior to
261
microspheres in tablet mass for immediate release in comparison to
uncoated counterpart that showed more than 50% release in next 4
hours (Table 13 and Fig. 21). These findings simulated earlier research
with acceptable evidences [Crotts and Sheth, 2000 & Prajapati and
Patel, 2010].
Fig. 21 : Dissolution rate analysis of tablet of uncoated and enteric coated microspheres
Moreover in last phase in SCF enteric coated microspheres were
disintegrated completely to release of remaining drug from sustained
release microsphere for maintenance of further continuous and
controlled drug release pattern keeping symmetry with previous research
262
[Kuksal et al., 2006 ; Brahmaiah et al, 2013; Chaurasia and Jain, 2003 ;
Arora et al., 2011]. Kinetic interpretation of these release profile as
depicted in Table 13 and corresponding Fig. 21A revealed first order
drug release profile was most conveniently adopted in both cases
showing higher correlation calculated for all three phases.
In present study drug released was solely dependent on capacity
of microspheres in the tablet because being enteric coated it was able to
deliver themselves to colon where they could release drug through a
controlled release mechanisn as shown in previous section (section
6.4.3). It was only the amount of drug at the exterior of microspheres in
the tablet responsible to release initial drug in first phase of study. Tablet
dissolution rate was analysed to comply first order model assuming the
fate of a simple tablet after getting disintegrated in favourable dissolution
media i.e. SCF where tablet breakdown and subsequent drug release
could rationally follow concentration dependent rate as described in first
order kinetics. As per data in first 2 hours (Fig. 21B) uncoated
microspheres in tablet released considerable portion of drug
(33.56±0.83%) compared to insignificant release found for enteric coated
microspheres (0.86±0.08%). Moreover uncoated microspheres shown
highest correlation(R2 =0.994) releasing maximum drug in SCF detected
after 4 hours whereas highest R2 value was detected (R2=0.993) in next
263
4 hours in SCF (Fig. 21C)and maintained almost similar drug release
pattern even up to last 18 hours(R2=0.991) measured in pH 7.4 (Fig.
21D)compared to that observed for uncoated counterpart (p=0.5)
predicting possibility of higher rate of sustained and controlled drug
release resulted from fast disintegration of tablet followed by gradual
disruption of Eudragit S100 coating on the surface of microspheres
rendering controlled swelling, porosity and sustained residence of
mucoadhesive microspheres in colon probably through endogenous
lectin conjugation mechanism [Costa et al.,2001 & Obitte et al.,
2010].Stemming from earlier research evidences cited above careful
comparison between two experimental batches could be concluded with
potential capacity of the enteric coating to deliver controlled release
balsalazide microspheres uninterruptedly to the distal region of colon
where enhanced mucoadhesion and controlled swelling rendered
sufficient time period for releasing drug in a sustained manner to ensure
pharmaceutically recognized mechanism best identified for oral route of
drug administration designed for this purpose.
6.6.2. Stability analysis of enteric coated tablets
Enormous studies were undertaken in the investigation of stability
of solid oral dosage forms to support post formulation strategies as per
ICH guide line [ICH, 1996]. From the results of the accelerated stability
264
study (Table 14) of tablet of enteric coated microspheres for 6 months, it
was concluded that with storage conditions no significant changes were
found in the sample. In this aspect several parameters viz hardness,
Disintegration time, Drug content and T80 were monitored to evaluate its
capacity to withstand accelerated condition.
Table 14: Stability analysis of enteric coated microspheres tablet
Stability Condition
Sampling Time (days)
Hardness (kg/cm2) n=3
Disintegration Time (minutes) n=3
Drug content (%) n=3
T80(hours) pH7.4 n=3
40 ± 2OC
75±5 % RH
0 5.5±0.05 43.22 ±1.32 98.81±1.25 12.18±0.29
15 5.5± 0.04 43.19±1.08 98.81±1.11 12.04±1.02
30 5.5± 0.09 43.38±1.11 98.80±1.01 11.87±0.94
60 5.5±0.01 44.06±1.23 98.79±0.89 12.08±0.88
120 6.0±0.03 44.11±1.09 97.89±1.07 11.90±1.01
180 6.0±0.11 44.16±1.21 97.75±0.99 12.06±1.12
*Mean ± SD, (n=3)
265
Hardness was found to increase by 0.5 kg/cm2 after a period of
120 days, Disintegration time increased to 44.16±1.21 minutes after 120
days, whereas % drug content decreased to 97.89±1.07 after 120 days
and Time to release 80% of drug decreased to 11.90±1.01hours.These
findings cumulatively directed us to conclude that there was no
significant change found as compared to their corresponding initial
values (p=0.5) in the properties even after 6 months in accelerated
environment. These data corroborated well with previous researches
[Battu et al., 2007 & Bi et al., 1996].
266
7. SUMMARY AND CONCLUSSION
Colon targeted drug delivery system has gained enormous
popularity due to its ever increasing contribution towards the aspect of
being an effective mean to deliver drug uninterruptedly to colon to
facilitate effective treatment of both local as well as systemic disorders.
Microsphere as a carrier for colon specific drug delivery has a long
known importance due to their variable drug release profiles with easy
and reproducible adoption of several pharmaceutical manipulation
techniques. Out of several members of drug, 5-ASA family used to treat
colon infection and other disorders such as IBD etc. Balsalazide has a
well known importance as prodrug for excellent pharmacokinetic profile.
These potential sources of information created a strong platform to
undertake present study that investigated formation of ALG-CHI
Polyelectrolyte complex in which influence of several polymeric
combinations of two naturally polymers namely Alginate and Chitosan.
These were kept in individual predetermined pH environment in order to
achieve controlled protonation to promote sufficient interaction between
them.
Drug polymer interaction study performed by FTIR spectra and
DSC Thermogram revealed nonreacttivity and feasibility of formulations.
Different batches of complex were formulated by changing their
267
molecular ratio and subsequent batches of microspheres were prepared
using w/o emulsion method through ionotropic gelation mechanism.
Nine(9) experimental batches of microspheres loaded with drug
Balsalazide(M1 to M9) with two control batches with Alginate (AM) and
Chitosan (CM) were prepared and comparatively evaluated for several
characterizing parameters like Micromritic properties such as Angle of
repose,Compressibility Index, Hausner’s Ratio along with Particle size,
percentage yield, percentage drug entrapment, percentage
Mucoadhesion , Surface morphology, Zeta potential by adopting
previously established methods. Particle size of each microspheres was
found to have controlled average diameter ranging from 70 to 100 µm
with micromeritic characters comprising bulk density between
0.292±0.19 and 0.388±0.09 gm/cc and tapped density from 0.329±0.19
to 0.458±0.09 gm/cc, Hausner’s ratio between 1.070±0.04 and
1.224±0.03, angle of repose between 12.76±0.55 º and 24.71±0.44 º
indicating that microspheres were with in the pharmacopieal
specification. Percentage yield was between 69.49± 0.64 % to
89.97±0.66%, Swelling index of 0.5 to 0.7, 75 to 90 % mucoadhesion
and a satisfactory level of drug entrapment capacity within 50 to 80 %
range; all of which indicated a result of varying degree of individual
performance due to optimum polymeric interaction, surface morphology
268
showing SEM images and zeta potential between - 45.15 ± 0.97 mv
found in batch AM whereas maximum charge of +54.09±0.73mv found in
batch M6 proving acceptability relating to selection of optimized formula.
All formulated batchs were evaluated for in-vitro drug release rate in
simulated colonic environment, Accelerated stability studies. Preliminary
data showed the percentage cumulative drug was released in 24 hours
study was 50% in 8 hours ,80% in 22 hours and 95% after 24 hours for
M6 proving efficiency toward control of drug release and it was further
treated for kinetic analysis to investigate release pattern and release
mechanism, it followed nonfickian release mechanism. After careful
comparison, batch M6 having all promising evidences of performances
evaluated because this batch of ALG-CHI microspheres were prepared
from 0.2 wt% of alginate and 0.3% chitosan in polymer solution having
the polymer mass ratio ALG/CHI = 35/65 and these conditions were
responsible for good particle stability and properties reported previously
[Abreu et al., 2009]. Therefore it was selected for further part of study
featuring enteric coating of microspheres, preparation and evaluation
matrix tablet using M6 batch of microspheres.
The optimized batch (M6) microspheres were selected for entric
coating and formulated to prepare matrix tablets by wet granulation
method. The prepared tablets were evaluated for physical parameters
269
such as weight variation, thickness, hardness, friability, percentage of
assay and rate of drug release by invitro dissolution method. The results
showing that average weight of 99.54±1.28 mg , Thickness of 2.11±0.09
mm, Hardness of 5.5±0.11 kg/cm2 , Friability of 0.35±0.02%, Assay
98.78±1.42%. indicated the formulated tablets were complied as per
pharmacopoeial spefcifications. The dissolution profile showed that the
rate of drug release was 0.86±0.08 % after 2 hours in SGF, 14.29±0.26
% after next 4 hours in SIF, 86.18±1.01% after next 18 hours in SCF
rendered sufficiently controlled drug release pattern following first order
sustainable release and satisfactory in vitro stability all of which were
considered as a potential candidate that could be explored further to
design of matrix tablet with enteric coated microsphere for colon
targeting purpose.
The stability studies indicated that there was no significant change
in hardness, disintegration time and drug content after the period of 6
months. Hance the prepared formulation were stable. Present
investigation was aimed to provide additional valuable information to
support future research in this ever popular field of colon targeted drug
delivery system and contribute little more area for extended scientific
and robust critical aspects due for more refinement, development and
growth.
270
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LIST OF PUBLICATIONS
S.No
Title of the Paper Authors Name
Journal Name Month & Year of Publication Volume. No & Issue. No Page. No
1 Formulation and
evaluation of
controlled release
microspheres
containing acid
resistant polymers
A.Pasupathi
and
B.Jaykar
Journal of
Chemical and
Pharmaceutical
Science
January -
March 2016,
Volume. No.
9,
Issue No.1,
Page No: 1- 7
2 Formulation and
evaluation of colon
targeted controlled
drug delivery
system for
balsalazide
disodium
A.Pasupathi
and
B.Jaykar
World Journal of
Pharmaceutical
Research
Decmber-
2015,
Volume. No.
4,
Issue No.12,
Page No:
775-790