SUB-CHRONIC EFFECT OF CO-ADMINISTRATION OF METHFORMINE AND AMILODIPINE ON SOME HAEMATOLOGICAL INDICES IN EXPERIMENTAL ANIMAL (WISTAR RATS)

TABLE OF CONTENT

Title page                                                                                                                   i

Declaration                                                                                                                  ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv

Acknowledgement                                                                                                      v

Table of content                                                                                                          vi

List of table                                                                                                                 vii

List of figures                                                                                                             viii

Abstract                                                                                                                      ix

CHAPTER ONE

1.0 Introduction                                                                                                          1

1.1 Background of study                                                                                            1

1.2 Statement of Problems                                                                                          2

1.3 Justifications                                                                                                         4

1.4 Aims                                                                                                                      4

1.5 Research Objectives                                                                                              5

1.6 Research Hypothesis                                                                                             5

1.7 Significance of research                                                                                        5

CHAPTER TWO

2.0 Literature review                                                                                                   6

2.1 Metformin                                                                                                             6

2.1.1 Mechanism of metformin                                                                                   7

2.1.2 Pharmacological properties of metformin                                                          10

2.1.3 Side effects and contra-indications of metformin                                             11

2.1.4 Therapeutic application of metformin                                                                13

2.2 Vitamin B12: biochemistry, deficiency and anaemia                                           15

2.3 Relationship between metformin and vitamin B12                                              22

2.4 Metformin and haemolytic anaemia                                                                     24

2.5 Diagnosis of anaemia                                                                                           25       

2.6 Efficacy of Metformin                                                                                          27

2.7 Amlodipine                                                                                                           31

2.7.1 Chemistry                                                                                                           31

2.7.2 Mechanism of action                                                                                          31

2.7.3 Side effects                                                                                                        32

2.7.4 Pharmacokinetics                                                                                               32

2.8 Angiotensin Converting Enzyme Inhibitors                                                         33

2.9 Reference range of haematological parameters of rats                                         34

2.10 Comparative haematology of rat and human                                                      36

CHAPTER THREE

3.1 Study Design                                                                                                        39

3.2 Preparation of animals                                                                                         40

3.3 Sample size determination                                                                                  40

3.4 Reagent Kits/Drug Preparation and Dosage                                                      40          

3.5 Dosage                                                                                                              41

3.6 Sample collection                                                                                              43

3.7 Measurement of variables                                                             .                  43

3.8 Ethical consideration                                                                                      46

3.9 Statistical analysis                                                                                           47

CHAPTER FOUR

4.1 Result                                                                                                             48

42.Differential white blood cell counts in controls and tests groups                 49               

CHAPTER FIVE

5.0 Discussion                                                                                                    58

5.1 Conclusion                                                                                                   61

5.2 Recommendation                                                                                         62

References                                                                                                         63

Appendix I                                                                                                        75

Appendix II                                                                                                      76

LIST OF TABLES

Table 1: Haematological parameters in control, Co-administration of Metformin and Amlodipine treated                                                                                                 39                                                                                                             

LIST OF FIGURES

Figure 1: Shows mechanism of action of metformin                                                              10

Figure 2: Shows Pie chat representation of Packed cell volume of control, Co-administration of metformin and amlodipine treated Wistar Rats                                                                      54

Figure 3: Shows Histogram representation of Hemoglobin and red blood cell count of control, Co-administration of metformin and amlodipine treated Wistar Rats                 55

Figure 4: Shows Histogram representation of red cell indicies of control, Co-administration of metformin and amlodipine treated Wistar Rats                                                                      56

Figure 5: Shows Histogram representation of platelet and white blood cell count of control, Co-administration of metformin and amlodipine treated Wistar Rats                             57

ABSTRACT

Metformin, which belongs to the biguanide class, is one of the most generally used oral hypoglycemic agents. It has been used for more than 50 years and was approved by the US Food and Drug Administration (FDA) in 1994 (American Diabetes Association, 2009) whereas Amlodipine is a long acting dihydropyridine calcium channel blocker, which is used in the treatment of angina to lower the BP (Blood pressure). the aim is to know the effect of co-administration of this two drugs in Wistar rats. To assess the MCV,MCH,MCHC level of experimental animal and that of control group after combined administration with amilodipine and metformin.  Animals were randomly grouped into Two (A and B) groups, each groups contains eight (8) animals. Group A was administered normal saline; Group B was administered combined administration with amilodipine (0.00264mg/ml/132g) and metformin (0.0438mg/ml/132g) once daily for 30days after 2 weeks of acclamatization. Each group of rats was allowed to have free access to water ad libitum and standard rat chow (SRC) throughout the experimental period. Blood was collected from the Jugular vein at the end of the experiment to determine the full blood count of each animal. There was a significant reduction (p≤0.05) in hemoglobin level, RBC, PCV and increase in WBC and decrease in PLT count, and with increase in MCV, MCH with no difference in MCHC after co-administration of Metformin and Amlodipine in Wistar Rat as compared to control. These findings suggests that Co-administration of Metformin and Amlodipine causes decrease in red cell dependent parameters gradually leading to anaemia with long term usage thus regarded as a hematotoxicity agent to the blood profile.  

Key words: Metformin, Amlodipine, haematological parameters, Wistar Rats.

CHAPTER ONE

1.0  INTRODUCTION

1.1 Background of the study

Metformin, which belongs to the biguanide class, is one of the most generally used oral hypoglycemic agents. It has been used for more than 50 years and was approved by the US Food and Drug Administration (FDA) in 1994 (American Diabetes Association, 2009). Currently, many clinical practice guidelines for patients with type 2 diabetes, including the American Diabetes Association (ADA), the European Association for the Study of Diabetes (EASD), and the Korean Diabetes Association (KDA), recommend that metformin treatment should begin at the time of diagnosis of diabetes with lifestyle modification in the absence of contraindications. Metformin is now the most widely used anti-diabetic drug, with almost all guidelines throughout the world recommending metformin as first-line treatment for patients with type 2 diabetes mellitus (T2DM). Metformin may also be used to treat other conditions involving insulin resistance, such as polycystic ovary syndrome (PCOS) (Boyle  et al., 2010). Metformin has beneficial effects on carbohydrate metabolism, weight loss, and vascular protection but also has important side effects. For example, patients on long-term metformin therapy were found to be at risk of anaemia (Maida et al., 2011). This may be due to a metformin related vitamin B12 reduction. It is reported that, 30% of patients receiving long-term metformin treatment experienced malabsorption of vitamin B12, with a decrease in serum vitamin B12 concentration of 14% to 30% (Burcelin, 2014).

Vitamin B12 is a vital nutrient for health. It plays an important role in the functioning of the brain and nervous system, and in the formation of red blood cells. In addition to anemia, vitamin B12 deficiency may increase the severity of peripheral neuropathy in patients with T2DM (Owen et al., 2000; Stephenne et al., 2011). Furthermore, because vitamin B12 participates in the most important pathway of homocysteine (Hcy) metabolism, a reduction in vitamin B12 would increase plasma concentrations of Hcy, which is strongly linked to cardiovascular disease in patients with T2DM and PCOS (Saeedi et al., 2008). Although some clinical studies have reported that metformin lowered vitamin B12 level, other studies have reported that it did not. To date, no consensus has been reached on whether metformin induces vitamin B12 reduction. It is therefore imperative to know the effect of metformin on the hematological parameters of experimental animal (Wistar Rats) so as to arrive at a conclusion if the vitamin B12 deficiency is as a result of Diabetes mellitus or due to metformin (anti-diabetic drug) (Leone et al., 2014).

On the other hand, co-administration of metformin and amilodipine have been used in patients with type 2 diabetes with concomitant hypertension (type 2 diabetes-induced hypertension) (Wang et al., 2009). Amlodipine (as besylate, mesylate or maleate) is a long acting calcium channel blocker (dihydropyridine class) used as an anti-hypertensive and in the treatment of angina (Viollet et al., 2012). Like other calcium channel blockers, amlodipine acts by relaxing the smooth muscle in the arterial wall, decreasing peripheral resistance and hence reducing blood pressure; in angina it increases blood flow to the heart muscle (Patade and Marita, 2014).

Amilodipine (an antihypertensive medication) have been found to be associated with a reduction in hemoglobin concentration with a long term exposure (Yamagduchi  et al., 2005). The magnitude of such a change is generally small, but in certain instances it can be extreme enough to produce a clinically significant degree of anemia (Zankat et al., 2015). The mechanistic basis for antihypertensive medication-related changes in hemoglobin concentration include hemodilution, hemolytic anemia, and suppression of red blood cell production, as this occurs most commonly with angiotensin- converting enzyme inhibitors and angiotensin receptor blockers (Lakshmi et al., 2015). Researchers are suspecting that reduction in hemoglobin concentration in a patient who is receiving treatment for hypertension and does not have an obvious source of blood loss should account for potential antihypertensive therapy involvement (Bogachus and Turcotte, 2010). To find solution to the un-going suspicions and hypothesis.

It was therefore imperative to investigate the effect of co-administration of metformin and amilodipine on some hematological parameters in experimental animal (Wistar Rats).

1.2 Statement of problem

Drug-drug interactions are a major problem in health facilities the world over. The prevalence of interactions is estimated to be between 1- 22% (Lakshmi et al., 2015). Underlying risk factors for drug-drug interactions include polypharmacy and co-morbid conditions. High blood pressure in patients with diabetes presents a major health problem because of increased risk of polypharmacy. Polypharmacy leads to prescribing drugs that may have drug interactions. The interactions can lead to life threatening situations, hospitalization, increased burden to patients, hematotocixitiy (anaemia either haemolytic or vitamin B12 deficiency) as well as suppression of bone marrow activity from calcium blocker mechanism of antihypertensive drugs and adjusted quality of life. A considerable number of the drug-drug interactions can be avoided if health workers involved in patient care have the right information. Various hospitals and clinics serves patients from various regions that visit the facility for various ailments including diabetes and hypertension which are among the conditions on the rise, thus availability of data for the study is essential.

1.3 Justification

There are no local studies on the hematotoxicity of potential drug-drug interactions among patients receiving both hypoglycemic and antihypertensives drug and thus the need to carry out the study. Many hypothesis and theory have been postulated by researchers that long term usage of metformin have the ability to induce Vitamin B12 deficiency as well as some institutions having the complain that metformin drug despite it’s world-wide acceptability as anti-diabetic drug causes haemolytic anaemia. This led to the need to bridge the knowledge gap by carry out the study. Also, hypothesis have been postulated that long term usage of anti-hypertensive drugs causes a decrease in haemoglobin in which the mechanism is not known yet. This led to the need to bridge the knowledge gap by carry out the study. The findings of this study will create awareness to the clinicians and pharmacists on the need for a better dosage or a better drug so as to prevent hematotoxicity effect of drug-drug interactions in the case of diabetics with concomitant hypertension.

1.4 Aim

This study aims at investigating the combine effect of Metformin and Amilodipine on haematological experimental animal (Wistar Rats)

1.5 Research objectives

1.      To assess the MCV level of experimental animal and that of control group after combined administration with amilodipine and metformin.

2.      To investigate the MCH level of experimental animal and that of control group after combined administration with amilodipine and metformin.

3.      To determine the MCHC level of experimental animal and that of control group after combined administration with amilodipine and metformin.

1.6 Research hypothesis (Null)

(a)    There is no significant difference in the level of MCV level after co-administration of amilodipine and metformin.

(b)   There is no significant difference in the level of MCH level after co-administration of amilodipine and metformin.

(c)    There is no significant difference in the level of MCHC level after co-administration of amilodipine and metformin.

1.7  Significance of research

        Findings from this study will help policy makers to determine if long term exposure to Amilodipine and Metformin causes anaemia thus thus creating awareness of the drug usage to prevent hematoxicity (another form of complication to diabetic-hypertensive situation). Also the findings from the effect of Metformin and Amilodipine on experimental animal (Wistar Rat) will further generate need to examine the other complications that arise from the combined drug usage.

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