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 Wister rats.

To assess the MCV,MCH,MCHC level of experimental animal and that of control group after combined administration with amlodipine 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 amlodipine (0.00264mg/ml/132g) and metformin (0.0438mg/ml/132g) once daily for 30days after 2 weeks of acclimatization.

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 Westar 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 anemia with long term usage thus regarded as a hepatotoxicity agent to the blood profile.
Key words: Metformin, Amlodipine, hematological parameters, Westar 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 (Wister 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 amlodipine have been used in patients with type 2 diabetes with concomitant hypertension (type 2 diabetes-induced hypertension) (Wang et al., 2009).

Amlodipine (as baseplate, maculate 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 (Parade and Marital, 2014).
Amlodipine (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 (Zakat 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 (Bacchus and Truscott, 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 amlodipine 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 (anemia either hemolytic 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 hepatotoxicity of potential drug-drug interactions among patients receiving both hypoglycemic and antihypertensive 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 hemolytic anemia.

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 hemoglobin 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 hepatotoxicity 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 Amlodipine on hematological 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 amlodipine and metformin.
2. To investigate the MCH level of experimental animal and that of control group after combined administration with amlodipine and metformin.
3. To determine the MCHC level of experimental animal and that of control group after combined administration with amlodipine and metformin.
1.6 Research hypothesis (Null)
(a) There is no significant difference in the level of MCV level after co-administration of amlodipine and metformin.
(b) There is no significant difference in the level of MCH level after co-administration of amlodipine and metformin.
(c) There is no significant difference in the level of MCHC level after co-administration of amlodipine and metformin.
1.7 Significance of research
Findings from this study will help policy makers to determine if long term exposure to Amlodipine and Metformin causes anemia thus thus creating awareness of the drug usage to prevent hem toxicity (another form of complication to diabetic-hypertensive situation).

Also the findings from the effect of Metformin and Amlodipine on experimental animal (Wistar Rat) will further generate need to examine the other complications that arise from the combined drug usage.