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