Evaluation of the Subacute and Delayed Toxicity of Artemether-Lumefantrine Combination in Rats

ABSTRACT 

Despite the reported rapid increase in demand for artemisinin and its derivatives, there is little independent collaboration of the toxicity patterns of artemether-lumefantrine combination besides those documented by the manufacturer in its application for approval. It is however, pertinent to conduct an independent evaluation of the subacute and delayed toxicity of artemether-lumefantrine combination (ALC). In this study, rats in groups I-VI were administered varying concentrations of oral artemether-lumefantrine combination as a single dose for 7 days. The animals in groups I-III were sacrificed while groups IV-VI were maintained on water and normal diet until the 15th day. On the 15th day, these animals were sacrificed to assess for delayed effects. The animals in group VII served as control and received only sterilized water and commercial feeds. Results indicated that ALC significantly (p0.05) effects on serum electrolytes (Na+ , K+ , Cl- and HCO3 - ) level, blood urea, bilirubin, cholesterol, and liver enzymes (ALT, AST and ALP). Histopathological studies revealed no consistent evidence of significant toxicities on liver, kidney and the heart. Hepatic vascular congestion and lymphocytic infiltration as well as granular and eosinophilic hyaline casts in renal tubules were also observed, in addition to an isolated case of acute tubular necrosis with organizing fibrinous pericarditis were observed. The administration of increasing doses of oral artemether- lumefantrine combination given once daily to rats over an interval of one week, produced varying subacute and delayed hematological, biochemical and histopathological effects. ALC showed hyperglycemic effect as well as stimulatory effects on haemopoeisis and immunity but no significant subacute and delayed toxic effects on the liver, kidneys and heart. The isolated cases of acute tubular necrosis and organizing fibrinous pericarditis need further evaluation.

TABLE OF CONTENTS

CONTENTS

PAGES

TITLE

PAGE...………….……………………………………………………………………..……..………………………….....................i

DECLARATION……………………………………………………………………………………..…….……….……………..………….....ii

CERTIFICATION……….….……………………………………………………………………..….….………..................................iii

DEDICATION...………………………………………………………………………………….…….………….…..............................iv

ACKNOWLEDGEMENT..............................................................................................................................v

TABLE OF CONTENTS...............................................................................................................................vi

LIST OF FIGURES……………………………………………………………………………………….……..……..........……………….…xi

LIST OF TABLES…………………………………………………………………………………………………..……….…………………....xi

LIST OF PLATES………………………………………………………………………………………….………..…….........................xii

ABSTRACT………………………………………………………………………………………………………..……….….....................xiv

1. CHAPTER ONE: INTRODUCTION 1

1.1. Historical aspects of artemether-lumefantrine combination (ALC) 1

1.2. Pharmacology of ALC 3

1.2.1. Description 3

1.2.2. Composition and formulation 4

1.2.3. Indications and usage 4

1.2.4. Dosage and administration 4

1.2.4.1. Treatment in semi-immunes 5

1.2.4.2. Treatment in multi-drug resistant areas and non-immunes 5

1.2.5. Pharmacodynamics 6

1.2.6. Pharmacokinetics 7

1.2.6.1. Artemether 7

1.2.6.2. Lumefantrine 7

1.2.7. Precautions and contra-indications 8

1.3. Review of literature on ALC 8

1.4 Toxicity 12

1.4.1 Acute toxicity 13

1.4.2 Subacute toxicity 13

1.4.3 Delayed toxicity 14

1.4.4 Adverse drug reaction 14

1.5 Drug induced toxicity 15

1.6 Drug induced hepatotoxicity 15

1.6.1 Mechanisms of drug induced hepatotoxicity 16

1.6.2 Susceptibility to drug-induced hepatotoxicity 20

1.6.2.1 Biochemical patterns of drug-induced liver disease 21

1.6.2.2 Histopathological patterns of liver injury 25

1.6.2.2.1 Zonal necrosis 25

1.6.2.2.2 Hepatitis 25

1.6.2.2.3 Cholestasis 26

1.6.2.2.4 Steatosis 26

1.6.2.2.5 Granuloma 27

1.6.2.2.6 Vascular lesions 27

1.6.2.2.7 Neoplasm 27

1.7 Drug induced nephrotoxicity 27

1.7.1 Mechanisms of drug-induced nephrotoxicity 27

1.7.1.1 Pre-renal effects 30

1.7.1.2 Obstructive uropathy 30

1.7.1.3 Allergic or immunological damage 31

1.7.1.4 Direct nephrotoxicity 32

1.8 Drug-induced cardiotoxicity 33

1.8.1 Mechanisms of drug-induced cardiotoxicity 34

1.8.1.1 Cellular events involved in cardiotoxicity 34

1.8.1.1.1 Myocardial apoptosis 34

1.8.1.1.2 Myocardial necrosis 35

1.8.1.1.3 Myocardial adaptation 36

1.8.1.1.4 Molecular mechanisms involved in cardiotoxicity 36

1.8.1.1.5 Mitochondrial factors in myocardial cell death 37

1.8.1.1.6 Cytokines and other factors in myocardial death signaling 39

1.8.1.1.7 Sarcoplasmic reticulum pathway leading to apoptosis 39

1.8.1.1.8 Role of calcium in cardiotoxicity 40

1.8.1.1.9 Protein kinase c (PKC) and the myocardial signaling pathway 40

1.8.1.1.10 Oxidative stress and mitogen-activated protein kinases (MAPK) 40

1.9 Statement of problem 42

1.10 Aim and objectives of study 45

1.10.1 Aim 45

1.10.2 Objectives 45

2. CHAPTER TWO: MATERIALS AND METHODS 46

2.1. Materials 46

2.1.1 Animals 46

2.1.2 Drug source 46

2.1.3 Reagents 46

2.1.4 Exposure to ALC 47

2.2 Methods /Experimental design 47

2.2.2 Subacute and delayed toxicity test 47

2.2.2 Calculated dosage formula 48

2.2.3 Clinical assessment 49

2.2.4 Biochemical studies 49

2.2.5 Hematological studies 51

2.2.6 Histopathological studies 51

2.2.7 Statistical analysis 51

3. CHAPTER THREE: RESULTS 52

1.1. Hematological effects of ALC 52

3.1.1 Effect on hemoglobin concentration (Hb) 52

3.1.2 Effect on packed cell volume (PCV) 52

3.1.3 Effect on reticulocyte count (Retics count) 53

3.1.4 Effect on white blood cell total count (WBC count) 53

3.1.5 Effect on white blood cell differential count (N, L count) 53

3.2 Biochemical effects of ALC 53

3.2.1 Effects of ALC on serum electrolyte concentrations (Na+, K+, Cland HCO3-) 53

3.2.2 Effects of ALC on blood urea level 53

3.2.3 Effects of ALC on random blood sugar 56

3.2.4 Effects of ALC on liver enzymes 57

3.2.5 Effects of ALC on serum cholesterol 57

3.3 Histopathological effect of artemether-lumefantrine combination 60

3.3.1 Subacute toxicity 60

3.3.2 Delayed toxicity 61

4. CHAPTER FOUR: DISCUSSION AND CONCLUSION 77

4.1. DISCUSSION 77

4.2. CONCLUSION 81

1. Appendix 83

2. References 92