INTERACTION OF ANTIMALARIAL DRUGS (PYRIMETHAMINE AND SULPHADOXINE) WITH NORMAL AND SICKLE HAEMOGLOBINS: A UV-VISIBLE STUDY

ABSTRACT

Crude haemoglobins were extracted from blood samples of identified individuals of normal (AA), sickle trait carrier (AS), and sickle (SS) by employing centrifugation techniques. The crude haemoglobins were dialysed at 4^oC for 12hr against 50mM Tris-HCl buffer of pH 7.2. The effects of pyrimethamine and sulphadoxine on the haemoglobins in the presence and absence of sodium dodecyl sulphate (SDS) were studied at pH 5.0 and 7.2 with uv-visible titration spectrophotometry. The study showed that sodium dodecyl sulphate at pH 5.0 unfolded the studied proteins. These can be related to destabilization of haemoglobin structure by proteases such as plasmepsins and falcipains in the acidic environment of malaria parasite food vacuole due to malaria parasite infection. Pyrimethamine and sulphadoxine at pH 5.0 and 7.2 decreased the concentration of oxyhaemoglobin and increased the concentrations of methaemoglobin and deoxyhaemoglobin of the studied proteins. The results also show how haemoglobins are deoxygenated due to interaction with sodium dodecyl sulphate. Deoxygenation of haemoglobin as a result of their interaction with SDS can be likened to pathological condition whereby malaria parasites infection reduced the oxygen tension of erythrocytes of their host. HbS had the highest interaction with sulphadoxine and pyrimethamine followed by HbAS while HbA had the least interaction. Formation of methaemoglobin is associated with lipid oxidation. Increase in absorbance at 275 nm observed in this study refers to dynamic motion of the studied proteins and their deviation from normal structure and function. The interaction of haemoglobins with sulphadoxine-pyrimethamine combination at pH 5.0 caused a large perturbation of the protein conformation that was reflected in modest spectral shift of the soret band.

TABLE OF CONTENT

Title     –               –           –           –           –           –           –           –           –           i

Certification    –    –           –           –           –           –           –           –           –           ii

Dedication      –               –           –           –           –           –           –           –           iii

Acknowledgements    –    –           –           –           –           –           –           –           iv

Abstract          –             –           –           –           –           –           –           –           v

Table of Content          –           –           –           –           –           –           –           vi

List of Figures               –           –           –           –           –           –           –           x

List of Tables  –       –           –                       –           –           –           xiii

CHAPTER ONE: INTRODUCTION

1.1 Malaria      –           –           –           –           –           –           –           –           2

1.1.1 A health problem                –           –           –           –           –           –           2

1.1.2 History of malaria                  –           –           –           –           –           2

1.1.3 The life cycle of the malaria parasite –     –           –           –           2

1.1.4 Signs and symptoms of malaria      –           –           –           –           –           4

1.1.5 Diagnosis and treatment of malaria       –           –           –           5

1.2 Antifolates     –           –           –           –           –           –           –           –           6

1.2.1 Combination of DHPS and DHFR inhibitors         –           –           –           7

1.2.2 Sulphadoxine-pyrimethamine combination therapy    –           –           8

1.2.3 Sulphadoxine and its pharmacological classification  –           –           8

1.2.4 The mechanism of action of sulphonamides      –           –           –           9

1.2.5 Physico-chemical properties of sulphadoxine   –           –           9

1.2.6 Pyrimethamine and its pharmacological classification    –           10

1.2.7 The mechanism of action of diaminopyrimidines        –           10

1.2.8: Binding mechanism of DHFR inhibitors     –           –           10

1.2.9 Physico-chemical properties of pyrimethamine          –           12

1.2.10 Clinical uses and adverse effects of the combination            12

1.3 Chloroquine     –           –           –           –           –           –           –           –           13

1.4 Lumefantrine            –           –           –           –           –           –           –           14

1.5 Artemisinins and synthetic peroxides        –           –           –           –           15

1.5.1 Artemether       –           –           –           –           –           –           –           15

1.6 Oxygen-carrying protein in the blood: Haemoglobin        –           17

1.6.1 Haemoglobin variants     –           –           –           –           –           –           18

1.6.2 Haemoglobin digestion by malaria parasite: Role of multiple proteases           –           18

1.6.3 Oxidative stress generated by haemoglobin degradation   –           19

1.7 Haem detoxification pathways of the malaria parasite   –           –           20

1.8 Haemozoin:The antimalarial drug target         –           –           21

1.8.1 Mechanism of haemozoin formation in the malaria parasite –           22

1.9 Spectral properties of haemoglobin           –           –           –           –           23

1.10 Aim and Objectives of Study           –           –           –           –           –           25

1.10.1 Aim of the Study       –           –           –           –           –           –           25

1. Specific Objectives of the study        –           –           –           –           25

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials   –         –           –           –           –           –           –           –           26

2.1.1 Chemicals              –           –           –           –           –           –           –           26

2.1.2 Equipment     –           –           –           –           –           –           –           26

2.2 Methods         –           –           –           –           –           –           –           26

2.2.7 Collection of blood samples         –           –           –           –           –           26

2.2.8 Isolation and purification of haemoglobin            –           –           –           27

2.2.9 UV – Visible titration             –           –           –           –           –           –           27

2.2.10 Data analysis    – –                       –           –           –           –           –           28

CHAPTER THREE: RESULTS

3.1 Absorption spectra of haemoglobin A                –           –           –           29

3.1.1 Absorption spectra of haemoglobin A in varying concentrations of pyrimethamine-   29

3.1.2 Absorption spectra of haemoglobin A in varying concentrations of

pyrimethamine, in the presence of SDS         –           –           –           29

3.1.3 Absorption spectra of haemoglobin A in varying concentrations of

pyrimethamine and constant concentration of sulphadoxine  –           29

3.1.4 Absorption spectra of haemoglobin A in varying concentrations of pyrimethamine and constant concentration of sulphadoxine in the presence of SDS- 33

3.1.5 Absorption spectra of haemoglobin A in varying concentrations of sulphadoxine-      33

3.1.6 Absorption spectra of haemoglobin A in varying concentrations of sulphadoxine in the presence of SDS     –           –           –           –           33

3.1.7 Absorption spectra of haemoglobin A in varying concentrations of sulphadoxine and constant concentration of pyrimethamine  –           33

3.1.8 Absorption spectra of haemoglobin A in varying concentrations of sulphadoxine and constant concentration of pyrimethamine –           38

3.2 Absorption spectra of haemoglobin AS         –           –           –           –           40

3.2.1 Absorption spectra of haemoglobin AS in varying concentrations of pyrimethamine-             –           –           –           –           –           –           40

3.2.2 Absorption spectra of haemoglobin AS in varying concentrations of pyrimethamine in the presence of SDS  –  –           –           –           –           40

3.2.3 Absorption spectra of haemoglobin AS in varying concentrations of pyrimethamine and constant concentration of sulphadoxine          –           40

3.2.4 Absorption spectra of haemoglobin AS in varying concentrations of pyrimethamine and constant concentration of sulphadoxine in the presence of SDS- 40       

3.2.5 Absorption spectra of haemoglobin AS in varying concentrations of sulphadoxine-   45

3.2.6 Absorption spectra of haemoglobin AS in varying concentration of sulphadoxine in the presence of SDS       –           –           –           –           45

3.2.7 Absorption spectra of haemoglobin AS in varying concentrations of sulphadoxine and constant concentration of pyrimethamine        –           45

3.2.8 Absorption spectra of haemoglobin AS in varying concentrations of sulphadoxine and constant concentration of pyrimethamine in the presence of SDS- 45

3.3 Absorption spectra of haemoglobin S               –           –           –           50

3.3.1 Absorption spectra of haemoglobin S in varying concentrations of pyrimethamine-    50

3.3.2 Absorption spectra of haemoglobin S in varying concentrations of pyrimethamine in the presence of SDS  –           –           –           –           –           –           50

3.3.3 Absorption spectra of haemoglobin S in varying concentrations of pyrimethamine and constant concentration of sulphadoxine         –           50

3.3.4 Absorption spectra of haemoglobin S in varying concentrations of pyrimethamine and constant concentration of sulphadoxine in the presence of SDS- 50

3.3.5 Absorption spectra of haemoglobin S in varying concentration of sulphadoxine-        55

3.3.6 Absorption spectra of haemoglobin S in varying concentrations of sulphadoxine in the presence of SDS    –            –           55

3.3.7 Absorption spectra of haemoglobin S in varying concentration of sulphadoxine and constant concentration of pyrimethamine   –           55

3.3.8 Absorption spectra of haemoglobin S in varying concentrations of sulphadoxine and constant concentration of pyrimethamine in the

presence of SDS             –           –           –           –           –           –           55

CHAPTER FOUR: DISCUSSION

Discussion         –           –           –           –           –           –           –           –           60

Conclusion              –           –           –           –           –           –           –           62

References               –           –           –           –           –           –           –           63

Appendices               –           –           –           –           –           –           –           73

LIST OF FIGURES

Figure 1.1: The life cycle of malaria parasite –            –           –           4

Figure 1.2: The folate pathway (simplified) showing the targets of the antifolates  –           7

Figure 1.3: The chemical structure of sulphadoxine   –       –           –           9

Figure 1.4: Binding of pyrimethamine to Plasmodium falciparum DHFR (PfDHFR)-       11

Figure 1.5: The chemical structure of pyrimethamine –   12

Figure 1.6: The Chemical Structure of Chloroquine  –        –           –           14

Figure 1.7: The chemical structure of Lumefantrine –         –           –           15

Figure 1.8: The chemical structure of artemether      –       –           –           16

Figure 1.9: Three dimensional structure of haemoglobin   –           –           17

Figure 1.10: Proposed pathway for haemoglobin degradation in P. falciparum       –           19

Figure 1.11: Proposed structure of haemozoin                 –           –           22

Figure 1.12: The proposed mechanism of haemozoin formation in digestive

vacuole of malaria parasite –           –           –           –           –           23       

Figure 3.1: Absorption spectra of haemoglobin A in varying concentrations

(0-0.025mM) of pyrimethamine, at (a) pH 5.0 and (b) pH 7.2     30

Figure 3.2:Absorption spectra of haemoglobin A in varying concentrations (0-0.025mM) of pyrimethamine, in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2   –           –           –           –           –           –           –           –           –           31

Figure 3.3: Absorption spectra of haemoglobin A in varying concentrations(0-0.025mM) of pyrimethamine and constant concentration (0.008mM) of sulphadoxine, at (a) pH 5.0 and (b) pH 7.2             32

Figure 3.4: Absorption spectra of haemoglobin A in varying concentrations(0-0.025mM) of pyrimethamine and constant concentration (0.008mM) of sulphadoxine in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2     –           34

Figure 3.5: Absorption spectra of haemoglobin A in varying concentrations(0-0.012mM) of sulphadoxine, at (a) pH 5.0 and (b) pH 7.2         –           –           35

Figure 3.6: Absorption spectra of haemoglobin A in varying concentrations(0-0.012mM) of sulphadoxine in the presence of SDS, at (a) pH 5.0 and(b) pH 7.2   –             –           –           –           –           –           36

Figure 3.7: Absorption spectra of haemoglobin A in varying concentrations(0-0.012mM) of sulphadoxine and constant concentration(0.017mM) of pyrimethamine, at (a) pH 5.0 and (b) pH 7.2          –           –           37

Figure 3.8: Absorption spectra of haemoglobin A in varying concentrations(0-0.012mM) of sulphadoxine and constant concentration (0.017mM) of pyrimethamine in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2   –           –           –           –           –           –           –           –           –           39

Figure 3.9: Absorption spectra of haemoglobin AS in varying concentrations(0-0.025mM) of pyrimethamine, at (a) pH 5.0 and (b) pH 7.2      –           –           41

Figure 3.10: Absorption spectra of haemoglobin AS in varying concentrations (0-0.025mM) of pyrimethamine in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2   –           –           –           –           –           –           –           –           –           42

Figure 3.11: Absorption spectra of haemoglobin AS in varying concentrations(0-0.025mM) of pyrimethamine and constant concentration(0.008mM) of sulphadoxine, at (a) pH 5.0 and (b) pH 7.2            –           –           43

Figure 3.12: Absorption spectra of haemoglobin AS in varying concentrations(0-0.025mM) of pyrimethamine and constant concentration (0.008mM) of sulphadoxine in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2   –           –           –           –           –           –           –           –           –           44

Figure 3.13: Absorption spectra of haemoglobin AS in varying concentrations (0-0.012 mM) of sulphadoxine, at (a) pH 5.0 and (b) pH 7.2        –           –           46

Figure 3.14: Absorption spectra of haemoglobin AS in varying concentration (0-0.012 mM) of sulphadoxine in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2   –           –           –           –           –           –           –           –           –           47

Figure 3.15: Absorption spectra of haemoglobin AS in varying concentrations(0-0.012mM) of sulphadoxine and constant concentration (0.017mM) of pyrimethamine, at (a) pH 5.0 and (b) pH 7.2           48

Figure 3.16: Absorption spectra of haemoglobin AS in varying concentrations(0-0.012mM) of sulphadoxine and constant concentration (0.017mM) of pyrimethamine in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2  –           –           –           –           –           –           –           –           –           49

Figure 3.17: Absorption spectra of haemoglobin S in varying concentrations (0-0.025mM) of pyrimethamine, at (a) pH 5.0 and (b) pH 7.2           51

Figure 3.18: Absorption spectra of haemoglobin S in varying concentrations (0-0.025mM) of pyrimethamine in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2  –           –           –           –           –           –           –           –           –           52

Figure 3.19: Absorption spectra of haemoglobin S in varying concentration s(0-0.025mM) of pyrimethamine and constant concentration (0.008mM) of sulphadoxine, at (a) pH 5.0 and (b) pH 7.2           –           –           53

Figure 3.20: Absorption spectra of haemoglobin S in varying concentrations(0-0.025mM) of pyrimethamine and constant concentration (0.008mM) of sulphadoxine in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2  –           –           –           –           –           –           –                       –           54

Figure 3.21: Absorption spectra of haemoglobin S in varying concentrations(0-0.012mM) of sulphadoxine, at (a) pH 5.0 and (b) pH 7.2       –           –           56

Figure 3.22: Absorption spectra of haemoglobin S in varying concentrations(0-0.012mM) of sulphadoxine in the presence of SDS, at (a) pH 5.0 and(b) pH 7.2  –           –             –           –           –           –           57

Figure 3.23: Absorption spectra of haemoglobin S in varying concentrations(0-0.012mM) of sulphadoxine and constant concentration (0.017 mM) of pyrimethamine, at (a) pH 5.0 and (b) pH 7.2     –           58

Figure 3.24: Absorption spectra of haemoglobin S in varying concentrations(0-0.012 mM) of sulphadoxine and constant concentration (0.017 mM) of pyrimethamine in the presence of SDS, at (a) pH 5.0 and (b) pH 7.2             –           –           –           –           –           –           –           –           59

LIST OF TABLES

Table 1: Titration of haemoglobin with varying concentrations (0-0.025 mM) of pyrimethamine at pH 5.0 and 7.2    –      –           –           –           –           74

Table 2: Titration of Haemoglobin with varying concentrations (0-0.012 mM) of Sulphadoxine at pH 5.0 and 7.2         –           –           –           –           74

Table 3: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.025 mM) of pyrimethamine in the absence of SDS     –           75

Table 4: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.025 mM) of pyrimethamine in the presence of SDS    –           75

Table 5: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.012 mM) of sulphadoxine in the absence of SDS       –           75

Table 6: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.012 mM) of sulphadoxine in the presence of SDS      –           76

Table 7: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.025 mM) of pyrimethamine in the absence of SDS     –           76

Table 8: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.025 mM) of pyrimethamine in the presence of SDS    –           76

Table 9: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.012 mM) of sulphadoxine in the absence of SDS       –           76

Table 10: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.012 mM) of sulphadoxine in the presence of SDS      –           77

Table 11: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.025 mM) of pyrimethamine in the absence of SDS     –           77

Table 12: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.025 mM) of pyrimethamine in the presence of SDS    –           77

Table 13: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.012 mM) of sulphadoxine in the absence of SDS       –           78

Table 14: Changes in haemoglobin absorbance at 275 nm and 415 nm in varying concentrations (0-0.012 mM) of sulphadoxine in the presence of SDS      –           78

CHAPTER ONE

INTRODUCTION

One of the main causes of death today is malaria, especially in numerous parts of Asia, Sub-Saharan Africa and the America (Esparza, 2005). Of the four Plasmodia that cause malaria, Plasmodium falciparum is responsible for the majority of illness and death in mankind (Duraisingh and Refour, 2005; Idro et al., 2005; Okie, 2005; Worrall et al., 2005). In Sub-Saharan Africa, this disease has a profound impact on children and infants, whilst millions have already died from AIDS (Acquired Immunodeficiency Syndrome) (Esparza, 2005; Harms and Feldmeier, 2005). In addition to this, malaria adds in mortality while the spread of chloroquine resistant strains of the plasmodium parasites across Africa increases (Farooq and Mahajan, 2004; Mahajan et al., 2005). Approximately, three million people, of whom more than half are children, die of malaria caused by P. falciparum annually (Duraisingh and Refour, 2005). Mortality and morbidity increase every year with over 500 million people infected with P. falciparum, presenting clinical symptoms of mild to severe malaria.

There exist several reasons for the increase in the occurrence of malaria including:

i An increase of the protozoan parasite’s resistance to anti-malarial drugs,

ii The development of the anopheles mosquito vectors’ resistance to numerous insecticides

iii The growth and the widespread migration of vulnerable populations to vastly endemic areas (Abdel-Hameed, 2003; Gregson and Plowe, 2005).

Malaria can be treated with various anti-malarial drugs. Sulphadoxine and pyrimethamine combination therapy was introduced into clinical practice for the prevention and treatment of malaria in the late 1960s as a follow-up on the drug chloroquine (Schultz et al., 1994). The first product by the name of Fansidar® containing sulphadoxine and pyrimethamine in combination was produced by Roche in 1971. Since the expiration of the Fansidar® patent, numerous generic products have been produced worldwide, contributing towards cheaper anti-malaria therapy.

1.1 Malaria

1.1.1 A Health Problem

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