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Sub-Chronic Effect Of Co-Administration Of Methformine And Amilodipine On Some Haematological Indices In Experimental Animal (Wistar Rats)

Metformin, which belongs to the biguanide class, is one of themost generally used oral hypoglycemic agents. It has been used formore than 50 years and was approved by the US Food and DrugAdministration (FDA) in 1994 (American Diabetes Association, 2009) whereas Amlodipine is a long acting dihydropyridine calcium channel blocker, which is used in thetreatment of angina to lower the BP (Blood pressure).

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Description

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

1.4Aims                                                                                                                       4

1.5Research Objectives                                                                                               5

1.6Research Hypothesis                                                                                              5

1.7 Significance of research                                                                                        5

CHAPTER TWO

2.0 Literature review                                                                                                   6

2.1Metformin                                                                                                              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 volumeof 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 themost generally used oral hypoglycemic agents. It has been used formore than 50 years and was approved by the US Food and DrugAdministration (FDA) in 1994 (American Diabetes Association, 2009) whereas Amlodipine is a long acting dihydropyridine calcium channel blocker, which is used in thetreatment 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 dailyfor 30days after 2 weeks of acclamatization. Each group of rats was allowed to have free access to waterad libitumand 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.

CHAPTER ONE

  • INTRODUCTION

1.1 Background of the study

Metformin, which belongs to the biguanide class, is one of themost generally used oral hypoglycemic agents. It has been used formore than 50 years and was approved by the US Food and DrugAdministration (FDA) in 1994 (American Diabetes Association, 2009). Currently, many clinicalpractice guidelines for patients with type 2 diabetes, including theAmerican Diabetes Association (ADA), the European Associationfor the Study of Diabetes (EASD), and the Korean DiabetesAssociation (KDA), recommend that metformin treatment shouldbegin at the time of diagnosis of diabetes with lifestyle modificationin the absence of contraindications.Metformin is now the most widely used anti-diabetic drug, withalmost all guidelines throughout the world recommendingmetformin as first-line treatment for patients with type 2 diabetesmellitus (T2DM). Metformin may also be used to treat otherconditions involving insulin resistance, such as polycystic ovarysyndrome (PCOS) (Boyleet al., 2010). Metformin has beneficial effects oncarbohydrate metabolism, weight loss, and vascular protection but also has important side effects. For example, patients onlong-term metformin therapy were found to be at risk of anaemia (Maidaet al., 2011). This may be due to a metformin related vitamin B12reduction. It is reported that, 30% of patients receiving long-termmetformin treatment experienced malabsorption of vitamin B12,with a decrease in serum vitamin B12 concentration of 14% to30% (Burcelin, 2014).

Vitamin B12 is a vital nutrient for health. It plays an importantrole in the functioning of the brain and nervous system, and in theformation of red blood cells. In addition to anemia, vitamin B12deficiency may increase the severity of peripheral neuropathy inpatients with T2DM (Owenet al., 2000; Stephenne et al., 2011). Furthermore, because vitamin B12participates in the most important pathway of homocysteine (Hcy)metabolism, a reduction in vitamin B12 would increase plasmaconcentrations of Hcy, which is strongly linked to cardiovasculardisease in patients with T2DM and PCOS (Saeediet al., 2008).Although some clinical studies have reported that metforminlowered vitamin B12 level, other studies have reported that it didnot. To date, no consensus has been reached on whethermetformin 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) (Leoneet 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 longactingcalcium channel blocker (dihydropyridine class)used as an anti-hypertensive and in the treatment ofangina (Violletet al., 2012). Like other calcium channel blockers, amlodipineacts by relaxing the smooth muscle in the arterial wall,decreasing peripheral resistance and hence reducing bloodpressure; in angina it increases blood flow to the heartmuscle (Patade and Marita,2014).

Amilodipine (an antihypertensive medication) have been found to be associatedwith a reduction in hemoglobin concentration with a long term exposure (Yamagduchi et al., 2005).The magnitude of such a change is generallysmall, but in certain instances it can be extremeenough to produce a clinically significant degreeof anemia (Zankat et al., 2015). The mechanistic basis for antihypertensivemedication-related changes in hemoglobinconcentration include hemodilution, hemolyticanemia, and suppression of red blood cell production,as this occurs most commonly with angiotensin-converting enzyme inhibitors and angiotensinreceptor blockers (Lakshmi et al., 2015). Researchers are suspecting that reduction in hemoglobinconcentration in a patient who is receivingtreatment for hypertension and does not have anobvious source of blood loss should account forpotential 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 assuppression 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.

1.4Aim

This study aims at investigating thecombine effect of Metformin and Amilodipine on haematologicalexperimental animal (Wistar Rats)

1.5Research 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)

  • There is no significant difference in the level of MCV level after co-administration of amilodipine and metformin.
  • There is no significant difference in the level of MCH level after co-administration of amilodipine and metformin.
  • There is no significant difference in the level of MCHC level after co-administration of amilodipine and metformin.
    • 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). Alsothe 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.

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Metformin

Metformin (1,1-dimethylbiguanide) is the most widely used drug to treat type 2 diabetes, and is one of only two oral anti-diabetic drugs on the World Health Organization (WHO) list of essential medicines (American Diabetes Association, 2009). Its history can be traced back to the use of Galega officinalis as a herbal medicine in medieval Europe. Its name derives from gale (milk) and ega (to bring on), as Galega has been used as a galactogogue in small domestic animals (hence the name “Goat’s rue”) (Patade and Marita,2014). Studies in the late 1800s indicated that Galega officinalis was rich in guanidine and in 1918 guanidine was shown to possess hypoglycaemic activity in animals (Chen and Anderson, 1947). Jean Sterne was the first to investigate dimethylbiguanide (Metformin) for clinical development and proposed the name ‘Glucophage’ (glucose eater) and published his results in 1957 (Sterne, 1957). Metformin have therapeutic potential in other conditions in which insulin resistance constitutes part of the pathogenesis, including obesity, prediabetes, polycystic ovary disease, non-alcoholics fatty liver and premature pubarche (Violletet al., 2012). Epidemiological studies have shown a decrease in cancer incidence in Metformin-treated patients, suggesting a potential application of the drug as an anti-cancer agent (Leoneet al., 2014).

2.1.1Mechanism of action of metformin

Although metformin is prescribed and used extensively since the end of the precise molecular (or biochemical) mechanism/s of action remain incompletely understood. It acts by countering insulin resistance, particularly in liver and skeletal muscle (Viollet et al., 2009). It suppresses hepatic gluconeogenesis, increases peripheral insulin sensitivity in insulin sensitive tissues such as muscle and adipose tissue, and enhances peripheral glucose utilization (Zhouet al., 2001). Metformin Decreases:

  1. Hyperinsulinemia, Prediabetes ( IFG, IGT), T2DM
  2. Decreased Glucose uptake, increased blood glucose, prediabetes, T2DM.
  3. Increased gluconeogenesis, decreased glucose uptake increased blood glucose, increased lipogenesis, fatty liver.
  4. Increased androgen secretion, hirsutism polycystic ovary disease. 5. Increased androgen secretion, premature pubarche.(Yamagduchi et al., 2005; Bogachus and Turcotte, 2010).

Metformin is the most widely prescribed drug to treat hyperglycemia in individuals with T2DM and is recommended, in conjunction with lifestyle modification (diet, weight control and physical activity), as a first line oral therapy in the recent guidelines of the American Diabetes Association and European Association of the Study of Diabetes (American Diabetes Association, 2009). It is effective anti-hyperglycaemic agent that inhibits hepatic glucose production and increases peripheral glucose uptake. Metformin also exerts beneficial effects on circulating lipids and exhibits cardio-protective features in obese patients (Saeediet al., 2008). Clinical trials suggest that Metformin, that is effective in treating T2DM, may alsoHowever, have some main effect for example it appears to be decreasing hepatic glucose production through a mild inhibition of the mitochondrial respiratory-chain complex (American Diabetes Association, 2009).

This transient decrease in cellular energy status promotes activation of adenosine monophosphate-activated protein (AMPK), a well-known cellular energetic sensor. AMPK is a protein kinase ubiquitously expressed in mammalian tissues and it is involved in regulating energy balance (Stephenneet al., 2011). Activation of AMPK stimulates adenosine triphosphate (ATP)-producing catabolic pathways, while inhibiting ATP-consuming anabolic pathways, thereby, maintaining cellular energy stores (Owenet al., 2000; Stephenne et al., 2011). In skeletal muscle, activation of AMPK increases glucose uptake and lipid oxidation. In adipose tissue, activation of AMPK reduces both lipolysis and lipogenesis (Stephenneet al., 2011).Metformin regulates glucose transporter 4 (GLUT4) translocation through AMP-activated Protein Kinase (AMPK)-mediated Cbl/CAP Signaling. It enhances insulin signaling in insulin-dependent and -independent pathways (Collieret al., 2006). In the liver, activation of AMPK inhibits gluconeogenesis and lipid synthesis but increases lipid oxidation (Burcelin, 2014). The activated AMPK decreases flux of free fatty acids and inhibits lipolysis, which may indirectly improve insulin sensitivity through reduced lipotoxicity (reduces hepatic lipogenesis) and exert an indirect effect on hepatic insulin sensitivity to control hepatic glucose output (Milleret al., 2013). In the heart, Metformin increases fatty acids uptake and oxidation, and increases glucose uptake and glycolysis. Metformin can also antagonize the action of glucagon, thus reducing fasting glucose levels (Burcelin, 2014).

Additional action of Metformin action is through induction of a profound shift in the faecal microbial community profile in diabetic mice and it has also been proposed that this may contribute to its mode of action possibly through an effect on Glucagon-like peptide-1 (GLP-1) secretion (Boyleet al., 2010; Maidaet al., 2011). Moreover, Metformin enhances the expression of the genes encoding the receptors for both GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) in mouse islets and also increases the effects of GIP and GLP-1 on insulin secretion from beta cells (Cho and Kieffer, 2011). These incretin-sensitising effects of Metformin appear to be mediated by a peroxisome proliferator-activated receptor α-dependent pathway, as opposed to the more commonly ascribed pathway of Metformin action involving AMP-activated protein kinase (Cho and Kieffer, 2011).

The protective effect on the cardiovascular system cannot be fully explained by its blood glucose-lowering properties (Stephenne et al., 2011). These effects may be partly mediated via beneficial effects on circulating markers of endothelial function (vascular cell adhesion molecule-1 [VCAM-1], E-selectin), fibrinolysis (plasminogen activator inhibitor-1 [PAI-1]) and chronic inflammation (C-reactive protein [CRP]) (Bristol-Myers, 2008). These mechanisms work together to reduce the levels of circulating glucose, increase insulin sensitivity, and reduce the hyperinsulinemia associated with insulin resistance (Cho and Kieffer, 2011).

Figure 1: diagram showing Metformin mechanism of action in various organs of the body (Bristol-Myers, 2008)…

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