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A THESIS SUBMITTED TO THE
IFTM UNIVERSITY For
THE AWARD OF DEGREE OF
Jaya Agnihotri Under the supervision of
IFTM UNIVERSITY
MORADABAD, INDIA 2015
APRIL 2015
DECLARATION
DEDICATED To
MY PARENTS AND
FAMILY
ACKNOWLEDGEMENT
ACKNOWLEDGEMENT
God, the almighty is acknowledged most for the courage, zest, dedication and
determination he granted me to make my work complete. He infused me the strength
and concentration to embark on and accomplish this research work.
I would like to express my deepest sense of gratitude and indebtedness towards
my esteemed guide Dr. Sobhna Singh, Associate Professor, Department of Pharmacy,
Faculty of Engineering & Technology, MJP Rohilkhand University, Bareilly (U.P.)
She had always been very understanding and helpful. Her invaluable guidance, patient
hearing and professional approach were largely responsible for the finalization of this
thesis.
During the course of my academic career, I have received constant support
from eminent professors. Prof. N K Jain, Prof.V K Dixit and Prof. D.V. Kohli, Deptt.
of Pharmaceutical Sciences, Dr. H.S. Gour Central University, Sagar (MP), who are
the assets to their students.
I am thankful to Dr. Anubha Khale, Co- Supervisor Principal H K College of
Pharmacy for providing all the facility at college level.
I am grateful to Prof. Shubhini Saraf, Professor and Head/Coordinator
(Deptt. Of Pharm. Sciences), Dean, School of Biosciences & Biotechnology,
Babasaheb Bhimrao Ambedkar University (A Central University), Lucknow for her
persistent interest, vital encouragement and critical evaluation.
Thanks seems to be small word for Dr. Papiya Bigonia, Principal at
Radharaman College of Pharmacy, Bhopal India , as she continuously encouraged me,
gave me support, guidance and showed interest during the study which really made me
to finish my work on time.
ACKNOWLEDGEMENT
I would also like to thank Dr. Vaibhav Sihorkar Principal Scientist (Assoc
Director) at Dr. Reddy's Laboratories for helping me during this long period of
research. His logical and scientific way of thinking, wide knowledge and dedication to
research have always been a source of inspiration.
I am thankful to Prof. Vandana B Patrawala Department of Pharmaceutical
Sciences and Technology, Institute of Chemical Technology and Lab staff for their
invaluable help in handling and operating the instruments in their laboratory.
I would like to take this opportunity to extend my thanks to Prof. A. K. Ghosh
Mr. Phoolchandra, Ms Neetu and all faculty members, teaching and non-teaching
staff of IFTM University for their help and cooperation at all stages of my research
work.
I am thankful to Dr. Kamal Vashi Manglam Drugs and Organics Limited
Mumbai for providing gift samples of Hydroxychloroquinine.
I am thankful to Mr. G. B Hadge, Technical Officer CIRCOT, Matunga,
Mumbai for scanning electron microscopy.
I would like to take this opportunity to extend my thanks to all faculty
members, teaching and non-teaching staff of H K College of Pharmacy for their help
and cooperation at all stages of my research work.
I cannot fail to mention the love, care and whole hearted support of my mother
in-laws, brothers, and kids all through. It is their blessings and sacrifices that
have placed me in the position where I find myself today. My extraordinary thanks to
my husband for encouraging me supporting me in every aspect of life. Many others
unnamed, who provided me information and support I convey my sincere thanks to
them.
(Mrs. Jaya Agnihotri)
ABSTRACT
ABSTRACT
Nanotechnology has provided the possibility of delivering drugs to specific cells using nano
particles. The overall drug consumption and side-effects may be lowered significantly by
using the concept of targeted drug delivery by depositing the active agent in the morbid
region only and in no higher dose than needed. When designed to avoid the body's defence
mechanisms, nanoparticles have beneficial properties that can be used to improve drug
delivery. The larger particles get cleared from the body; cells take up these nanoparticles
because of their size and do not clear them. This highly selective approach would reduce
costs and human suffering. Biodegradable cellular carrier systems, like bacterial ghost, stem
cells, fibroblast platelets, erythrosomes, nanoerythrosomes can be designed to improve the
pharmacological and therapeutic properties of drugs. The strength of drug delivery systems is
their ability to alter the pharmacokinetics and bio distribution of the drug.
Anchoring the drugs to RBC membranes with the help of nanotechnology is a
powerful method to load the drug that shows binding affinity towards the membrane protein
and remains effective during the long circulation lifetime of the carrier. Nano Erythrosomes
(NE) are small vesicles that are produced from red blood cells by hypotonic lysis method to
remove their haemoglobin content. Subsequently, these erythrocytes (ghosts) are extruded to
form small vesicles having a mean diameter of about 100 nm. In the present research
Artesunate (ART), Hydroxychloroquine (HCQ) and Pyrimethamine (PMA) drugs were
conjugated on nanoerythrosomes carriers for controlling drug delivery, to avoid drug leakage,
to improve the circulatory time of carrier, to increase the stability, to reduce cost and
toxicities of artemisinin combination therapy which will present a significant advantage over
many conventional systemically administered formulations. The present studies focuses on
development and optimization of nanoerythrosome based formulation of artesunate,
ABSTRACT
hydroxychloroquine, pyrimethamine as well as combined formulation of antimalarial drugs.
The formulations were prepared by extrusion technique and sonication technique and
subsequently drugs were loaded with help of spacer to optimize drug content and to avoid
drug leakage during the transit time. Fourier transform infrared (FTIR) spectroscopy reveals
that lipid chain order is not significantly affected in moderate conditions The developed
formulations were optimized for effective drug loading at variable drug concentration,
surface morphology, viscosity and sedimentation volume and in vitro release rate. The drug
loading was found to 25.20±1.3µg/ml for ART-NE, 28.09±1.3 µg/ml for HCQ-NE and 24.20
± 1.2 µg/ml for PMA-NE. The surface morphological characters revealed homogeneity and
integrity of preparation. The sedimentation volume was 1 which indicated excellent stability.
The formulations had viscosity 29.3±1.4 cps, 29.01±1.3 cps, and 30.02 ± 0.5 cps for ART-
NE, HCQ-NE and PMA-NE formulations respectively. The release mechanisms of artesunate
and hydroxychloroquine drugs as well as combined formulation of these two drugs were
accessed and percent release from all the respective formulations were found to be of zero
order as per kinetic study analysis. It was seen that percent release from formulations
increased with respect to time and formulations were capable to control the drug release up to
24 hrs when it was compared with pure drug. The in vitro release of pyrimethamine loaded
NE prepared by sonication method was accessed. It was found that 25.64 ± 0.5 µg/ml and
17.12±0.8 µg/ml pyrimethamine was released after 8 hours from pyrimethamine-loaded
nanoerythrosomes and free drug sample, respectively. It was found that the 98.51 ± 2.4% of
the free drug was released from the dialysis membrane in 20 hours; while only 22.57± 0.3%
drug was released in case of pyrimethamine loaded nano erythrosomes in 20 hours. The
decreased drug release rate can be ascribed to the cross-linking of drug to nanoerythrosomes
by gluteraldehyde. Sedimentation volumes in each case were unity in storage conditions
revealed excellent stability of the formulation. After giving turbulence shock to the each
ABSTRACT
reconstituted formulation it was found that only slight amount of drug leached out suggesting
formulation is quite stable when stored at 40C in lyophilized form. The formulations could
bear fairer amount of centrifugal stress of 2500 rpm in stability studies suggesting that
formulations had good stability. The in vivo studies were also performed. i.v. administration
of free Artesunate drug concentration after 24 hrs was found to be 1.5 µg/ml while it was 5.52
µg/ml from artesunate drug conjugates (ART-NE).In case of free HCQ, drug concentration
after 24 hrs was found17.02±0.3 µg/ml while it was 40.12µg/ml from hydroxychloroquine
drug conjugates (HCQ-NE).Free pyrimethamine drug concentration after 24 hrs was found
7.62 ± 1.5 µg/ml while it was 12.01±0.4 µg/ml from artesunate drug conjugates (PMA-NE).
Key words: Antimalarial; Artesunate; Cellular carrier; Erythrocytes; Hydroxychloroquine;
Pyrimethamine; Nanoerythrosomes; Targeting.
PREFACE
The success of formulation depends upon delivery of biologically active form of drug to the site
of action. Biodegradable nano cellular carrier fulfills many of the desired properties in achieving
effective long-term protection that is safe, economical and potentially more practicable on a
global scale.
There is a need to develop new and improved formulations for a range of diseases for which
current vaccines are either inadequate or non-existent.Current malaria therapies require strategies
capable of selectively delivering drugs to the cells infected by Plasmodium.The most critical
problem in facing the treatment of malaria is the development of resistance to classical quinoline
antimalarial compounds such as chloroquinine and antifolates. Emerging resistance to
antimalarial drugs poses the greatest threat to the National Drug Policy on malaria
The present investigation was aimed to develop Nanoerythrosomes based formulation of
antimalarial drugs, artesunate, hydroxychloroquine and pyrimethamine and to explore the
feasibility of the formulation. Artemisinins are effective not only against multi-resistant strains
of P. falciparum, but have broad stage specificity against the Plasmodium life cycle including
activity throughout the asexual blood stages and also the sexual gametocyte stages which may
reduce the spread of the disease in areas of low transmission.
The developed formulation will reduces the cost of drug, reduces the dose, increases circulation
time of carrier and most important it will reduce the resistance of drugs to malarial parasites.
Resistance of drug is a major challenge while treating with anti microbial drugs. Erythrocyte is a
suitable carrier for the preparation of novel biodegradable cellular carriers loaded with artesunate
PREFACE
hydroxychloroquine and pyrimethamine. The promising in vitro profiles of developed
formulation has prompted the call that the work can be up taken for further studies and pilot
scale study by pharmaceutical industry. The formulation can be up taken for more in-depth and
elaborative studies. In the present study, the overall aim was to develop stable, more effective,
fast acting injectable preparation of anti-malarial drugs as well as to find out the possibility of
combined biodegradable carrier based formulation.
In the introduction, chapter 1, shortcomings of existing drugs and drug delivery system used for
the treatment of malaria have been described. Treatment and control have become more difficult
with the spread of drug-resistant strains of parasites and insecticide-resistant strains of mosquito
vectors. Health education, better case management, better control tools and concerted action are
needed to limit the burden of the disease. In chapter 2; there is background of research study and
relevant reports available in literature. In chapter 3, discussion of drug profile available in
literature,their identification and authentification have been described. Methodology for
preformulation studies and analytical methods for the estimation of drug have been discussed.
Preformulation work was conducted to establish solubility and stability of drugs at different
climatic conditions. A simple, rapid, sensitive and statistically validated RP-HPLC method was
developed for simultaneous estimation of HCQ and ART as per ICH guideline. Methods adopted
for development and evaluation of nanoerythrosomes formulation of drugs ART, HCQ and PMA
have been described. . In chapter 4 results are presented for every test method and discussed with
respect to available literature. The HPLC method for simultaneous estimation of ART and HCQ
showed high percent of recovery and indicated that the method is free from interference and had
high accuracy. Hence developed method can be conveniently adopted for quality control analysis
PREFACE
of these drugs in combined dosage form and for clinical trials as well. Advances in nano
technology and increased knowledge on biology helped in development of formulation of anti-
malarial drugs individually as well as in combination. Optimization techniques were used to
optimize the formulation. With the promising in vitro results in vivo studies were conducted to
find out the possibility of localization of carriers and to find out plasma concentration of drug
with respect to time to establish them as a prolonged circulating vectors. The results were
interpreted and discussed in chapter V. Conclusion of results is mentioned in chapter VI. The
findings suggests that nanoerythrosome based combination formulation can be developed for the
treatment of malaria which will have benefits of: Complete cure; Control of severe malaria;
Prevention of death; Interruption of transmission; Minimizing risk of spread of drug resistance in
parasites because combination therapies provide improved efficacy over monotherapies due to
the synergistic activity of the combination drugs. Moreover, the presence of multiple drugs helps
in reducing the selection for resistance development against a specific agent and slows down the
process overall. Hence in depth studies of nanoerythrosomes formulation of anti malarial drugs
are warranted.
TABLE OF CONTENT
S. NO. CONTENT PAGE NO.
I Certificate
II Acknowledgement 1-2
III Abstract 3-5
IV Preface 6-8
V Table of Content 9
VI List of Tables 10-12
VII List of Figures 13-16
VIII List of abbreviations 17
1 Chapter 1 : Introduction 18-29
2 Chapter 2:Literature Search 30-81
3 Chapter 3: Materials and Methods 82-113
4 Chapter 4: Results and Discussion 114-179
5 Chapter 5:Conclusion 180-183
6 Chapter 6 Bibliography 184-205
7 Index 206-216
LIST OF TABLES
Table No. Table Content Page No.
1 Cell and cell ghost used for drug and gene delivery. 22
2 Malaria parasite species, their type. 55
3 Pharmacokinetic profile of Artesunate. 84
4 Parenteral dosage regimen of Artesunate. 86
5 Pharmacokinetic profile of Hydroxychloroquine 87
6 Proprietary Name of Hydroxychloroquine 89
7 Drug Interaction of Hydroxychloroquine 89
8 Pharmacokinetic Profile of Pyrimethamine. 91
9 Drug Interaction of Pyrimethamine. 92
10 Proprietary preparation of Pyrimethamine. 93
11 List of materials. 93-95
12 Physicochemical and U.V.characteristics of Hydroxychloroquine. 97
13 Lyophilization protocol of Nanoerythrosome. Formulation. 108
14 In vivo study protocol. 113
15 Solubility analysis of Artesunate in different solvent system. 120
16 Solid state stability of Artesunate. 121
LIST OF TABLES
17 Hydrolytic degradation of Artesunate in phosphate buffers pH 5.8 to 8.0. 124
18 Solubility analysis of Hydroxychloroquine Sulphate in different solvent system. 126
19 Photochemical degradation of HydroxychloroquineSulphate in solid state at 25ºC. 127
20 Compatibility studies of Hydroxychloroquine sulphate 128
21 Solubility analysis of pyrimethamine in different solvents 130
22 Stability studies of pyrimethamine 131
23 Preparation of standard curve of artesunate. 132
24 Preparation of standard curve of pyrimethamine in 0.1N-HCL-ethanol (IP) 133
25 Preparation of standard curve of hydroxychloroquine in water 134
26 Recovery studies for Artesunate (ART) and Hydroxychloroquine (HCQ) 138
27 Precision studies for Artesunate and Hydroxychloroquine. 139
28 Robustness studies for Artesunate and Hydroxychloroquine. 139
29 Summary of validation parameters for Artesunate and Hydroxychloroquine 140
30 Linearly Regressed Curve of ART and HCQ in Blood Plasma. 141
31 Vesicle size of nanoerythrosomes. 145
32 Poly dispersity index of nanoerythryosome formulations. 145
33 Effect of drug concentration on drug loading
153
34 Viscosity and sedimentation volume of nanoerythrosomes. 154
LIST OF TABLES
35 In vitro release rate study of combined formulations(F2 & F3). 155
36 In vitro release rate study of combined formulation. 156
37 In vitro release rate study of pyrimethamine formulation (F6). 157
38 Comparative release rate study from different drug loaded formulations. 158
39 Multiple coefficients determination data by using modeling software. 159
40 Stability studies of different nanoerythrosomes formulation at different temperature. 162
41 Effect of centrifugal force and turbulence shock on stability of artesunate drug loaded formulation. 162
42 Effect of centrifugal force and turbulence shock on stability of Hydroxychloroquine drug loaded formulation. 163
43 Effect of centrifugal force and turbulence shock on stability of pyrimethamine drug loaded formulation. 163
44 Treatment protocol for in vivo study. 164
45 Plasma concentration data of drug in artesunate treated groups. 165
46 Plasma clearance data of drug in hydroxyquine treated groups 166
47 Plasma concentration data of drug in pyrimethamine treated groups 167
LIST OF FIGURES
Figure No. Content Page No.
1 Schematic illustrations for methods of drug loading into erythrocytes. 40
2 Scheme represents the details of hypotonic dialysis method. 40
Mechanism for formation of nanoerythrosomes using extrusion
method. 45
4 Strategies for coupling therapeutic agents to nanoerythrosome surface. 46
5 Cross-linking of proteins with glutaraldehyde.
6 Malaria parasite life cycles. 56
7 Molecular structure of Artesunate. 83
8 Molecular structure of Hydroxychloroquine. 87
9 Molecular structure of Pyrimethamine. 90
10 UV Scan of the solution containing 10- 114
11 Chromatoplate of artesunate observed after exposure to anisaldehyde. 115
12 FTIR spectra of Artesunate. 115
LIST OF FIGURES
13 FTIR spectra of Hydroxychloroquine. 117
14 UV scan of the Pyrimethamine solution. 118
15 FT-IR spectra of Pyrimethamine. 119
16 Percent solubility analysis of Artesunate in different solvents and buffers. 121
17 Solid state stress testing of Artesunate at different temperature and humidity. 122
18 Solubility analysis of Pyrimethamine in different solvent system. 125
19 Compatibility studies of Hydroxychloroquine in different solvent system at 4ºC 129
20 Compatibility studies of Hydroxychloroquine in different solvent system at 37 ºC 129
21 Solubility analysis of Pyrimethamine in different solvent system 131
22 Standard curve of Artesunate in 50%v/v ethanol at 521nm. 132
23 Standard curve of Pyrimethamine in 0.1N-HCl-Ethanol 133
24 Standard curve of Hydroxychloroquine in water at 343 nm (USP 29) 134
25 Chromatogram of Hydroxychloroquine and Artesunate in 70% methanol (pH 3) at 222nm. 135
26 Chromatogram of Hydroxychloroquine and Artesunate in 40% acetonitrile and 60% buffer (pH 3) at 235nm.
136
LIST OF FIGURES
27 Chromatogram of Hydroxychloroquine and Artesunate in 50% acetonitrile and 50% buffer (pH 3) at 235nm. 136
28 Calibration curve of Artesunate. 137
29 Calibration curve of Hydroxychloroquine. 137
30 Calibration curve of Artesunate in blood Plasma. 141
31 Calibration curve of Hydroxychloroquine in blood Plasma. 142
32 Vesicle size distribution of Placebo (F1) (Extrusion method). 142
33 Vesicle size distribution of ART loaded Formulation (F2). 143
34 Vesicle size distribution of HCQ loaded Formulation (F3). 143
35 Vesicle size distribution of Placebo (F5) (Sonication method). 144
36 Vesicle size distribution of PMA loaded formulation (F6) (Sonication method). 144
37 Photomicrograph of erythrocytes ghosts. 146
38 Scanning Electron Photomicrograph of Placebo Nanoerythrosomes. 147
39 Scanning Electron Photomicrograph of Artesunate Nanoerythrosomes. 147
40 Scanning Electron Photomicrograph of Hydroxy Chloroquine loaded Nanoerythrosomes.
148
LIST OF FIGURES
41 Scanning Electron Photomicrograph of Pyrimethamine loaded nanoerythrosomes . 148
42 FTIR Spectra of Placebo Nanoerythrosomes 149
43 FTIR spectra of Artesunate loaded nanoerythrosomes. 150
44 FTIR of Hydroxychloroquine sulphate loaded Nanoerythrosoms 151
45 FTIR spectra of Pyrimethamine loaded nanoerythrosomes 152
46 Optimization of drug concentration. 153
47 Zero order plot for in vitro Release rate study of ART and HCQ nanoerythrosome formulations. 159
48 First order plot for In vitro Release rate study of ART and HCQ nanoerythrosome formulations. 160
49 Higuchi plot for In vitro release rate study of ART and HCQ nanoerythrosome formulations. 160
50 Cumulative percentage of drug released from pure drug solution and drug loaded formulations. 161
51 The blood plasma concentration of pure drug solution and loaded Artesunate nanoerythrosomes. 165
52 The blood plasma concentration of pure drug solution and loaded Hydrovychloroquinine nanoerythrosomes. 166
53 The blood plasma concentration of pure drug solution and Pyrimethamine-loaded nanoerythrosomes. 167
LIST OF ABBREVIATIONS
17
ART Artesunate
ART-NE Artesunate Loaded Nanoerythrosomes
BP British Pharmacopeia
FTIR Fourier Transform Infrared Microscopy
GA Gluteraldehyde
Hct Haematocrit
Hb Haemoglobin
HPLC High Performance Liquid Chromatography
HCQ Hydroxychloroquine
HCQ-NE Hydroxychloroquine Loaded Nanoerythrosomes
IP Indian Pharmacopeia
ICH International committee for Harmonization
LOD Limits of detection
LOQ Limits of quantification
Micro litre
Microgram
mg Milligram
ml Milliliter
NE Nanoerythrosomes
nm Nanometer
PBS Phosphate Buffer Saline
PMA Pyrimethamine
PMA-NE Pyrimethamine Loaded Nanoerythrosomes
RH Relative Humidity
RES Reticuloendothelial system
USP United State Pharmacopeia