microscopic evaluation of novel topical formulation for treatment of arthritis chitkara college of...
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Microscopic evaluation of novel topical formulation for treatment of Arthritis
CHITKARA COLLEGE OF PHARMACY CHITKARA UNIVERSITY, PUNJAB - 140401
Mohit
ARTHRITIS
Osteoarthritis
Rheumatoid Arthritis
Psoriatic Arthritis
ARTHRITIS
Psoriasis of Skin Joint Affected by Osteoarthritis
ARTHRITIS(Cont…….)
Factors Involved in Autoimmune Disorders
Genetic disposition,
Environmental factors,
Endocrinological factors
Immune dysfunction
In indvidual with a susceptible genotype,
exposure to above factors initiate an
autoimmune response to self and foreign
antigen through modulation of cytokines
production and effector cell function
ETIOLOGY
Gender
Genetics
Immune System
ETIOLOGY
Unknown antigen initiates the immune response resulting in rheumatoid arthritis
Women get arthritis 2-3 times more often than men Remission when they get pregnant.Women have higher absolute number of CD4 lymphocytes relative to men,.
Found in 20% of general population Not a diagnostic tool, many people who have the marker either do not have or will never get rheumatoid arthritis.
Infection
Bacterial infections are most important eg. septic arthritis, reactive arthritis, osteomyletis and osteitisViral for eg. rubella virus, human parvovirus, hepatitis B virusFungal eg. Candida
DIAGNOSIS OF RHEUMATOID ARTHRITIS
LAB TEST
Complete blood Count- Low WBC count suggests felty’s syndrome
Platelet count is elevated in severe inflammation
Erythrocyte sedimentation rate (ESR) 60% of people have an elevated ESR
C-reactive protein
Rheumatoid factor (RF) +ive seropositive & -ive seronegative
Imaging studies Swelling of soft tissues and loss of bone density around the joints (X-ray) , MRI - Detect early inflammation before it is visible on X-rays, Joint ultrasound and bone densitometry -Measuring bone density used primarily to detect osteoporosis
DIAGNOSIS
PHYSICAL EXAMINATION
Joint swelling &tenderness
Malignancy
Loss of motion in joints
MEDICAL HISTORY
CONVENTIONAL THERAPY
CORTICOSTERIODS
Conventional therapy
NSAID’S
(Used in early weeks)
BIOLOGIC RESPONSE MODIFIERS
Remicade
DMARD’SMethotrexate (MTX) , GoldHydroxychloroquineSulfasalazineCyclosporine ,Azathioprine
Approved in 1996, Enbrel (etanercept) is the first biologic response modifier to receive FDA approval for patients with moderate to severe rheumatoid arthritis
SYNOVIAL REPLISHNERS
(Glucosamine )
LIMITATIONS OF ARTHRITIS THERAPYNSAID’S
About 80% of patient experience gastrointestinal side effect including gastric ulcer, perforation and
hemorrhage etc
Colchicine
Poor solubility leads to high variability in oral bioavailability (e.g. Celecoxib, Colchicine have variable
oral bioavailability from 24 to 88%) Short biological half life (e.g. Colchicine has only 20 min.)
Systemic side effects
High systemic side effects (e.g. Rofecoxib showed cardiotoxic and renal side effects leading to its
withdrawal from market, Methotrexate has shown prominant hepatotoxic and bone marrow depression
Cost
High cost of treatment (e.g. Methotrexate and TNF-α)
Every year 1.5% of patient with rheumatoid arthritis are hospitalized with gastrointestinal problems
PROBLEMS IN PRESENT CONVENTIONAL THERAPY
Several dose dependent toxic effect
Can’t maintain a constant plasma level
Conventional therapy
Short biological half life
Low oral bioavailability
Minimum patient compliance
Cost of treatment is high
Decrease efficacy of dugs during
chronic use
Also affect normal cells of body
Deliver a steady state infusion for prolong period of time
Reduce the adverse effect and toxicity
Improve the therapeutic utility of drug by reducing the problems like
First pass metabolism
GI irritation
GI decomposition
Low absorption
Increase the half life of drug
Reduce the frequency of administration
Improved patient compliance
Self administration is possible
Drug input can be terminated at any time
ADVANTAGES OF TOPICAL DRUG DELIVERY
STRUCTURE AND FUNCTION OF HUMAN SKIN
Most extensive organ of body covering area of 2 m2 . Receive approximately one third of blood supply
For the purpose of transdermal/topical drug delivery, we can examine the structure and function of human skin categorized into four main layers:Stratum corneum (Stratum corneum is the rate limiting barrier)EpidermisDermisHypodermis
PROBLEM IN PRESENT ANTI-ARTHRITIC THERAPY AND PROPOSED STRATEGY FOR SITE-SPECIFIC DRUG DELIVERY
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0 2 4 6 8 10 12 14
Time (hr)
Conc
entra
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(g/
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Carrier
ELASTIC LIPOSOMES/ FATTY ACIDS AS CARRIER SYSTEM
Modified lipid carriers that enable drug to reach deeper skin layers.
Colloidal particles, typically consisting of phospholipids and surfactant molecules.
Pass through skin pores of size less than their own diameter.
Serve as rate limiting membrane barrier for systemic absorption of drugs.
Accommodate both hydrophilic and lipophilic drugs.
Liposomal surfactants are biodegradable and biocompatible.
Prolong the drug release.
Aqueous Cavity
Lipid Bilayers
IN VIVO MODELS FOR ARTHRITIS
Antigen-Adjuvant induced model for arthritis
Intraarticular injections of soluble antigen (same mice previously immunized to the same antigen )
Water in oil emulsion by combining one volume of FCA with one volume of aqueous antigen solution
Interaction with relevant cells
Acute arthritis
FCA enhances antibody production primarily because of the depot effectNonspecific immunopotentiation of macrophages by surfactant and the mycobacterium
The adjuvant is a mixture of non-metabolizable oil (mineral oil), a surfactant (Aracel.A) and mycobacterium (M.tuberculosis or M.butyricum) is considered to be one of the most effective adjuvant
DRUG INTRODUCTION
Methotrexate (MTX) is a folic acid antagonist preferably used for long-term therapy of rheumatoid arthritis
Available in oral tablet and injectable form
Poor bioavailability &systemic use of this drug may provoke any of a number of side effects mainly hepatotoxicity and bone marrow suppression agranulocytosis and thrombocytopenia
Chemical structure of methotrexate
DRUG INTRODUCTION
The daily oral dose requirement of glucosamine is 1500mg/dayAvailable in oral tablet and injectable form
Oral bioavailability of drug molecule is just 26%(subjected to uptake and degradation by the liver )
Chemical structure of glucosamine
A major problem in topical administration of these proposed drugs is its hydro-solubility and dissociation at physiological pH so its capacity for passive diffusion is thus limited.
ARTHRITIC DRUGS MARKET
The major players in the arthritis drug market include
Abbott Laboratories
Johnson & Johnson
Amgen
Roche
Pfizer
As of 2008, Abbott Laboratories' Humira, which was approved by the FDA in 2003, is the
highest selling drug in the arthritis market, with sales growing 50% from 2007 to 2010 to $8.5
billion
THE OBJECTIVES OF THE PROPOSED RESEARCH WORK
TO DEVELOP NOVEL DRUG DELIVERY SYSTEM, WHICH PROVIDE SUSTAINED AND TARGETED DELIVERY OF DMR’D TO THEIR TARGET SITE (JOINTS).
TO PREPARE, CHARACTERIZE AND OPTIMIZE DIFFERENT VESICULAR FORMULATIONS(,FATTY ACID VESICLES, NIOSOMES, ELASTIC LIPOSOMES)
TO CARRY OUT STABILITY AND SKIN PERMEATION STUDIES OF OPTIMIZED VESICULAR FORMULATION.
TO COMPARE IN VIVO ANTI-ARTHRITIC ACTIVITY OF DEVELOPED VESICULAR FORMULATIONS WITH MARKETED FORMULATION.
METHODLOGY
Identification and characterization of drug
Estimation of drugs in buffers and biological fluids by spectroscopy
Preparation of proposed vesicular system
Microscopic studies and characterization of proposed system
I.Phase contrast microscopy
II.Transmission electron microscopy
III.Scanning electron microscopy
In vitro characterization of vesicular system
Shape
Size and size distribution studies ( Dynamic light scattering methods)
Entrapment efficiency (Minicolumn centrifugation methods)
Degree of deformability (Extrusion method)
Zeta potential ( Zeta meter)
Turbidity measurement (Nephalometer)
No. of vesicles per cubic mm (Hemocytometer)
Phospholipid-ethanol interaction study (Differential Scanning Calorimetry)
In vitro skin permeation and deposition study (Using Diffusion Cell)
Stability study of the optimized formulation
In vivo studyFluorescence microscopy of rat viable skin to determine the extend of penetration of
vesicular formulation (Qualitative)
Confocal laser scanning Microscopy (CLSM) of rat viable skin to determine the rate and
extend of penetration (Quantitative)
Histopathological study of inflamed joint
METHODLOGY(cont….)
IMPORTANCE OF PROPOSED RESEARCH INVESTIGATION (National &International market status…)
Prevalence of arthritis in India increased drastically for last one decade
Dramatic increase in the demand of anti-arthritis drug
The market for rheumatoid arthritis therapeutics is estimated to reach over $20B in
2011
Proposed novel formulations will selectively deliver the drug to the targeted
inflamed joint
Naturally taken up by cells of mononuclear phagocytic system (MPS)
Biocompatible and biodegradable as they are made from natural phospholipid
Reducing the dose of the drug by minimizing the systemic exposure of drug and
increasing the deposition in deeper layer of skin
Easy to scale up, as procedure is simple, do not involve lengthy procedure and
unnecessary use of pharmaceutically unacceptable additives
EXPERIMENTAL WORKEXPERIMENTAL WORK DONE DONE IDENTIFICATION (Drug selected for study)IDENTIFICATION (Drug selected for study) Ultraviolet Absorption Maxima (Ultraviolet Absorption Maxima (Max)Max)
Methotrexate and glucosamine :- 100µg /ml stock solution in distilled water Methotrexate and glucosamine :- 100µg /ml stock solution in distilled water Scanned:- Between 200-400nm exhibits maxima at 267 nmScanned:- Between 200-400nm exhibits maxima at 267 nm
Results are concordant with the value given in the official books (Merck Index, 1996).Results are concordant with the value given in the official books (Merck Index, 1996).
Infrared Spectral AssignmentInfrared Spectral Assignment The IR spectra of MTX was recorded using (Perkin Elmer, IR Spectrophotometer).The IR spectra of MTX was recorded using (Perkin Elmer, IR Spectrophotometer).
Nuclear Magnetic ResonanceNuclear Magnetic Resonance The NMR The NMR spectra of MTX was recorded using (Bruker, NMR). of MTX was recorded using (Bruker, NMR).
S. NO. SOLVENTS SOLUBILITY
1. Distilled Water 25 mg /mL
2. Phosphate Buffer Saline (PBS) pH 7.4
24.2 mg /ml
3. Methanol 65mg/ml
PREFORMULATION STUDIESPREFORMULATION STUDIES
Table :- Solubility Profile of MTX in Different Solvents
S. No. SOLVENTSYSTEM
PARTITIONCOEFFICIENT
1. n-octanol : Distilled Water
1.2220.13
2. n-octanol: PBS (pH 74)
1.1270.15
Table :- Partition coefficient data of MTX
PARTITION COEFFICIENTPARTITION COEFFICIENT SOLUBILITY STUDIESSOLUBILITY STUDIES
PREFORMULATION STUDIESPREFORMULATION STUDIES S. NoS. No.. ParameterParameter StandardStandard ObservatioObservatio
nn
11 I.R. spectrumI.R. spectrum
22 0.002% w/v solution in HPLC 0.002% w/v solution in HPLC grade methanol observed grade methanol observed spectrophotometrically.spectrophotometrically.
Exhibit maxima at 302 Exhibit maxima at 302 nm. nm.
Exhibit maxima at Exhibit maxima at 302 nm 302 nm
33 Retention time Retention time methanol/acetonitrile/pH 5.4 methanol/acetonitrile/pH 5.4
buffer solution ) as mobile phase buffer solution ) as mobile phase using C 18 column at the flow rate using C 18 column at the flow rate
of 1 ml/min of 1 ml/min
RT for MTX was RT for MTX was reported as 9.5 min. reported as 9.5 min. (Pereira (Pereira et. al.et. al., 2000; , 2000;
Seki Seki et al.,et al., 1991) 1991)
RT for MTX was RT for MTX was found 9.8 min. found 9.8 min.
44 Melting range Melting range 255°C 255°C 253-255°C 253-255°C
55 Solubility Solubility Sparingly soluble in Sparingly soluble in water, slightly soluble in water, slightly soluble in
chloroform and have chloroform and have good solubility in good solubility in
methanol and practically methanol and practically insoluble in ether. insoluble in ether.
Sparingly soluble Sparingly soluble in water, slightly in water, slightly
soluble in soluble in chloroform, chloroform, soluble in soluble in
methanol and methanol and practically practically
insoluble in ether insoluble in ether
Table:- PREFORMULATION STUDIES
STANDARD CURVE OF MTX IN DISTILLED STANDARD CURVE OF MTX IN DISTILLED WATER BY UV SPECTROPHOTOMETRIC WATER BY UV SPECTROPHOTOMETRIC
METHODMETHOD
S . No. Concentration (g/ml)
Absorbance RegressedAbsorbance
Statistical Parameter
1. 2 0.0839 0.0839 Y= 0.0739 x – 0.1339 R2 =0.9993
2. 4 0.1608 0.157
3. 6 0.2339 0.2324
4. 8 0.3098 0.3078
5. 10 0.3879 0.3832
6. 12 0.4579 0.4586
7. 14 0.5294 0.5341
8. 16 0.6074 0.6094
9. 18 0.6852 0.6848
10. 20 0.7637 0.7602
Table :- Standard Curve of MTX In Distilled Water at Max 302 nm
S. No. Concentration (g/ml)
Absorbance Regressed Absorbance
Statistical Parameter
1. 2 0.0796 0.0709 Y=0.0343 X + 0.0017R2 = 0.9962. 4 0.1410 0.1393
3. 6 0.2076 0.2077
4. 8 0.2758 0.2761
5. 10 0.3428 0.3445
6. 12 0.4082 0.4129
7. 14 0.4747 0.4813
8. 16 0.5499 0.5497
9. 18 0.6317 0.6181
10. 20 0.6886 0.6865
Table :- Standard Curve of MTX in PBS (pH 7.4) at Max 302 nm
STANDARD CURVE OF MTX IN PBS (pH 7.4) STANDARD CURVE OF MTX IN PBS (pH 7.4) BY UV SPECTROPHOTOMETRIC METHODBY UV SPECTROPHOTOMETRIC METHOD
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 2 4 6 8 10 12 14 16 18 20
Concentration (g/ml)
Ab
so
rba
nc
e
Fig.:- Standard Curve of MTX in PBS (pH 7.4) at Max 302 nm
HPLC ASSAY OF MTXHPLC ASSAY OF MTX
S. No. Concentration(g/ml)
Peak area StatisticalParameter
1 0.0 0.0 y = 148370x + 288443
R2 = 0.99042 0.2 305375
3 0.4 360342
4 0.8 403575
5 1.2 475400
6 2.0 580025
Table :- Standard Curve of MTX in Distilled Water by HPLC Method
y = 148370x + 288443
R2 = 0.9904
0
100000
200000
300000
400000
500000
600000
700000
0 0.5 1 1.5 2 2.5
Concentration (g/ml)
Pe
ak
are
a
Fig.:- Standard Curve of MTX In Distilled Water by HPLC Method
DEVELOPMENT OF VESICULAR CARRIERS -BASIC PRINCIPLE
PC + SURFACTANT
Applied always nonocclusively
Transfersomes
PC + SURFACTANT PC + CHOLESTEROL
Partially dehrdrate Skin surface
“Transdermal osmotic gradient”
Stratum corneum (15% Water)
Dermal layer (75% Water)
Transfersomes prevent complete dehydration
Due to deformability transfersomes pass through narrow pores in the skin
Resulting in better skin permeation
Liposomes are less deformable therefore they dehydrate completely and fuse
LiposomesFatty acid vesicles
PC + FATTY ACID
Table1 Size and entrapment efficiency of the prepared oleic acid vesiclesUF-1,2,3-ufasomes with different molar ratio of drug
VISUALIZATION OF ELASTIC LIPOSOMES
TEM ( X 1,80, 000) Photomicrograph of liposomal formulation
Optical microscopy ( X 450) Photomicrograph liposomal
formulation
Formulation code
Oleic acid: 5-MTX (Molar ratio)
Entrapment efficiency
Particle Size(nm)
PDI
UF-1 9:1 (39.4±2.1) 505 ± 15 0.367 ± 0.037
UF-2 8:2 (45.4±2.1) 523±12 0.468 ± 0.037
UF-3 7:3 (51.0±4.2%), 632±17 0.262 ± 0.037
UF-4 6:4 (49.4±2.7) 531±16 0.489 ± 0.037
UF-5 5:5 (48.4±2.4) 404±13 0.581 ± 0.037
Size and entrapment efficiency of the prepared oleic acid vesiclesUF-1,2,3-ufasomes with different molar ratio of drug
Formulation code
Oleic acid: Glucosamine (Molar ratio)
Entrapment efficiency
Particle Size(nm)
PDI
UF-1 9:1 (37.4±1.1) 525 ± 15 0.367 ± 0.027
UF-2 8:2 (55.4±1.1) 553±12 0.368 ± 0.017
UF-3 7:3 (49.0±3.2%), 632±17 0.262 ± 0.036
UF-4 6:4 (49.4±2.7) 631±16 0.489 ± 0.033
UF-5 5:5 (48.4±2.4) 414±23 0.681 ± 0.047
Size and entrapment efficiency of the prepared oleic acid vesiclesUF-1,2,3-ufasomes with different molar ratio of drug
Fig. 2. Differential scanning calorimetry traces of oleic acid (a), oleic acid –MTX vesicles (b), oleic acid –MTX 8:2 vesicles (c) and oleic acid –MTX 9:1 vesicles (d).
Optimized with span 20,conc of oleic acid 80%,ph 7.4,80mM
A B
figure –( A)-Optimized MTX, (B) Glucosamine ufasomal formulation
pH8.5
pH 5.5
pH 6.5
pH7.4
Figure 4. Photomicrograph of oleic acid vesicles dispersion incubated at different pH (400× magnification).
Conclusion • The results of the present study demonstrated that proposed vesicular formulation possess
great potential for skin accumulation, prolonging release, improving site specific delivery and reducing the skin toxicity of glucosamine and methotrexate . This formulation seems to represents an attractive strategy for site-specific sustained delivery of glucosamine and methotrexate. In addition they are cost effective and therapeutically viable. Sustained release behavior and drug retention in the deeper part of skin might be beneficial for the longterm effects of drugs. The oleic acid vesicles seemingly fuse with the skin and release the contents. They are seen to penetrate intact and to form drug depots in the skin. The fatty acid in addition may serve as a penetration enhancer, thus by circumventing the stratum corneum barrier potential they may lead to better permeation of the drug molecules.
Acknowledgement
1. Dr. Sandeep Arora, Director, Chitkara College of Pharmacy, Chitkara
University, India
2. Dr Arvind Sharma, Associate Professor, Chitkara College of Pharmacy, Chitkara
University, India