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).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.
Key words: Metformin, Amlodipine, haematological parameters, Wistar Rats.
TABLE OF CONTENT
Title page i
Table of content vi
List of table vii
List of figures viii
1.0 Introduction 1
1.1 Background of study 1
1.2 Statement of Problems 2
1.3 Justifications 4
1.5Research Objectives 5
1.6Research Hypothesis 5
1.7 Significance of research 5
2.0 Literature review 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 anaemia25
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
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
4.1 Result 48
42.Differential white blood cell counts in controls and tests groups 49
5.0 Discussion 58
5.1 Conclusion 61
5.2 Recommendation 62
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
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.
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.
This study aims at investigating thecombine effect of Metformin and Amilodipine on haematologicalexperimental animal (Wistar Rats)
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). 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..