ABSTRACT

Globally, an estimated 3.4 billion people are at risk of malaria, including Nigerians. The current World Health Organization (WHO) Guideline for the treatment of malaria recommends the use of artemisinin combination therapy (ACT) for the treatment of uncomplicated malaria. Presently, the available formulations of ACTs are administered through the oral route. Rectal administration, which formed the basis of this work, is an alternative route of administration which overcomes the limitations of oral and parenteral administration of drugs. Differential Scanning Calorimetry (DSC) was employed to study the compatibility between artesunate, amodiaquine and the suppository bases and to determine the optimum ratio of the excipients. Three batches of structured suppositories of artesunate and amodiaquine (ACT) were formulated by the melt moulding (fusion) technique, evaluated for various parameters including physical characterization, weight variation, drug content, morphology and in vitro drug release. The drug release data were analyzed by zero order and Higuchi models. The suppositories were further evaluated in mice against virulent rodent malaria parasite Plasmodium berghei using Peter’s 4 days suppressive protocol. Data were expressed as mean ± SD. The parasitemia of the different groups were statistically assessed by ANOVA. The compatibility results proved that artesunate and amodiaquine are compatible. The optimum excipient ratio as determined by DSC was 4:4:3 for Phospholipon®90G-Softisan®154 matrix, Castor oil, and PEG 4000 respectively. The colours of the prepared suppositories ranged from light yellow to deep yellow; no fissures, fat blooming, exudation or migration of active ingredients was observed. The weight variation of all the suppositories met the BP Standards, with uniform drug content. The release profile for artesunate ranged from 99.21 % to 81.31 % and from 99.89 % to 28.81 % for amodiaquine. The developed suppositories reduced parasitemia to undetectable levels 28 days after drug administration. The survival rate of animals treated with E and PS suppositories was 100 %, while the survival of animals treated with P was 80 %, 55 days after drug administration. The results of the hemoglobin and hematocrit tests 55 days after drug administration revealed that the blood levels were normal for animals in the treatment groups. Stability studies revealed that the suppositories stored at 4 o C were more stable than the suppositories stored at 27 o C

TABLE OF CONTENTS

Title i

Certification ii

Dedication Page iii

Acknowledgement iv

Table of contents v

List of tables xv

List of figures xvi

Abstract xx

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1

1.1 General Introduction 1

1.2 Malaria 1

1.2.1 Epidemiology of malaria 1

1.2.2 Plasmodium species responsible for malaria 2

1.2.3 Malaria infection and the life cycle of the mosquito 5

1.2.4 Severe malaria 7

1.2.4.1 Pathogenic characteristics of severe malaria 7

1.2.4.2 Specific complications of severe malaria infection 12

1.2.5 Diagnosis of malaria 13

1.2.5.1 Laboratory diagnosis of malaria 13

1.2.5.2 Clinical diagnosis of malaria 15

1.2.6 Pharmacological treatment of malaria 15

1.2.7 Properties of the ideal anti-malarial drug 16

1.2.8 The artemisinin – based combination therapy 16

1.2.8.1 Amodiaquine 19

1.2.8.1.2 General Characteristics of amodiaquine 19

1.2.8.1.3 Therapeutic indications 19

1.2.8.1.4 Mechanism of action 19

1.2.8.1.5 Therapeutic index and toxicity 20

1.2.8.1.6 Physicochemical properties 20

1.2.8.1.7 Pharmacokinetic properties 20

1.2.8.1.8 Dosage form 21

1.2.8.2 Artesunate 22

1.2.8.2.1 Chemistry and synthesis 22

1.2.8.2.2 Therapeutic indications 22

1.2.8.2.3 Mechanism of action 23

1.2.8.2.4 Therapeutic index and toxicity 24

1.2.8.2.5 Pharmacokinetic properties 24

1.3 Rectal drug administration 25

1.3.1 Anatomy and physiology of the rectum 25

1.3.2 Absorption of drugs from the rectum 25

1.3.3 Types of rectal preparations 28

1.3.4 Suppositories 28

1.3.4.1 Properties of an ideal suppository 28

1.3.4.2 Advantages of suppositories as pharmaceutical dosage forms 29

1.3.4.3 Limitations of rectal dosing 29

1.3.4.4 Types of suppository bases and excipients 30

1.3.4.5 Methods of preparation of suppositories 33

1.3.4.6 Compatibility and stability studies of suppositories 33

1.4 Rationale and objectives of the study 34

CHAPTER TWO : MATERIALS AND METHODS 36

2.1 Materials 36

2.1.1 Drugs 36

2.1.2 Excipients 36

2.1.3 Chemicals 36

2.1.4 Reagents 36

2.2 Methods 37

2.2.1 UV spectrophotometric analysis of amodiaquine HCl and artesunate 37

2.2.1.1 UV spectrophotometric analysis of amodiaquine HCl 37

2.2.1.2 UV spectrophotometric analysis of artesunate 37

2.3 Solubility studies of artesunate and amodiaquine HCl in the excipients 38

2.4 Simultaneous UV spectrophotometric analysis of amodiaquine HCl and artesunate 39

2.4.1 UV spectrophotometric analysis of amodiaquine HCl in the presence of artesunate 39

2.4.2 UV spectrophotometric analysis of artesunate in the presence of amodiaquine HCl 40

2.5 Pre-formulation studies 40

2.6 Differential scanning calorimetric analysis 41

2.6.1 DSC on selection of excipient ratio for suppository formulations 41

2.6.2 DSC analysis for compatibility studies 43

2.7 Preparation of artesunate and amodiaquine HCl suppositories 43

2.7.1 Calibration of suppository moulds 43

2.7.2 Calculation of displacement value 44

2.7.3 Artesunate and amodiaquine suppositories prepared with PEG 4000/castor oil 44

2.7.3.1 Determination of displacement value of artesunate and amodiaquinein PEG 4000/castor oil in artesunate and amodiaquine HCl 44

2.7.3.2 Preparation of artesunate and amodiaquine suppositories with PEG 4000 and castor oil 46

2.7.4 Artesunate and amodiaquine suppositories prepared with Phospholipon ® 90G/ Softisan® 154 /castor oil 47

2.7.4.1 Determination of displacement value of artesunate and amodiaquine in Phospholipon ®90G/ Softisan ®154/ castor oil 47

2.7.4.2 Preparation of artesunate and amodiaquine suppositories with Phospholipon ®90G/ Softisan ®154/ castor oil 49

2.7.5 Artesunate and amodiaquine suppositories prepared with Phospholipon ®90G/ Softisan ®154/PEG 4000/ castor oil 49

2.7.5.1 Determination of displacement value of artesunate and amodiaquine in Phospholipon ®90G/ Softisan ®154/PEG 4000/ castor oil 49

2.7.5.2 Preparation of artesunate and amodiaquine suppositories containing Phospholipon ®90G/ Softisan ®154/PEG 4000/ castor oil 51

2.8 Evaluation of artesunate and amodiaquine suppositories 52

2.8.1 Visual characterization 52

2.8.2 Weight uniformity test 52

2.8.3 Content uniformity test 52

2.8.4 Softening time test 53

2.8.5 In vitro drug release studies 53

2.8.5.1 In vitro release of artesunate and amodiaquine from suppositories prepared with PEG 4000 and castor oil 55

2.8.5.2 In vitro release of artesunate and amodiaquine from suppositories prepared with Phospholipon® 90G/Softisan ®154 /castor oil 55

2.8.5.3 In vitro release of artesunate and amodiaquine from suppositories prepared with Phospholipon ®90G, Softisan ®154/PEG 4000/ castor oil 56

2.8.5.4 Determination of the kinetics and mechanism of drug release 56

2.8.6 Morphology study 56

2.8.7 Pharmacodynamic Evaluation ( In vivo antimalarial efficacy test in Plasmodium berghei-infected mice) 57

2.8.7.1 Experimental animals 57

2.8.7.2 Parasite inoculation 57

2.8.7.3 Experimental protocols 58

2.8.7.4 The parasite count 59

2.8.7.5 Survival trend of experimental animals 60

2.8.7.6 Hemoglobin estimation 60

2.8.7.7 Hematocrit estimation 60

2.9 Stability studies 60

2.9.1 Statistical analysis 61

CHAPTER THREE: RESULTS AND DISCUSSION 62

3.1 UV spectrophotometric analysis result of amodiaquine HCl/ artesunate 62

3.2 Result of solubility tests of amodiaquine and artesunate in the excipients 68

3.3 Ultra-violet spectrophotometric analysis result of drug combination 68

3.4 Result of differential scanning calorimetry (DSC) for the choice of ratios of excipients 69

3.5 Results of compatibility studies of amodiaquine hydrochloride, artesunate and the lipid matrix 79

3.6 Results of physical characterization of formulated amodiaquine and artesunate suppositories 87

3.6.1 Colour 87

3.6.2 Surface characteristics 87

3.7 Result of weight uniformity test 87

3.8 Results of drug content uniformity test 88

3.9 Results of softening time test 88

3.10 In vitro drug release results 94

3.10.1 In vitro drug release from artesunate and amodiaquine suppositories prepared with PEG 4000 and castor oil 94

3.10.2 In vitro drug release from artesunate and amodiaquine suppositories prepared with Phospholipon® 90 G / Softisan® 154/ castor oil (PS) 94

3.10.3 In vitro drug release from artesunate and amodiaquine suppositories prepared with Phospholipon® 90 G / Softisan® 154/ PEG 4000/castor oil (E) 95

3.11 Kinetics of drug release from formulated amodiaquine and artesunate suppositories 100

3.12 Morphology analysis result 113

3.13 Pharmacodynamic evaluation result 113

3.13.1 Suppressive effect of suppositories on Plasmodium berghei 113

3.13.2 Percent activity of suppositories 117

3.13.3 Survival trend of Plasmodium berghei –infected mice 122

3.13.4 Result of hemoglobin estimation 122

3.13.5 Result of hematocrit estimation 123

3.14 Result of stability studies 127

CHAPTER FOUR: SUMMARY AND CONCLUSION 128

REFERENCES 130