Sub-chronic Effect Of Co-administration Of Methformine And Amilodipine On Some Haematological Indices In Experimental Animal
[A CASE STUDY OF WISTAR RATS]

CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Study design
Animals were randomly divided into two (2) groups. Animals were randomly shared into the two groups with each groups containing equal number of animals.
Group A(1) (n=8) were administered normal saline for 30 days.
Group B(2) (n=8) were co-administered 0.0438mg/ml/132g body weight of metformin and 0.00264mg/ml/132g body weight Amilodipine besylate for 30 days.
Each group of rats will be allowed to have free access to water ad libitum and standard rat chow (SRC) for 30days with 2 weeks of acclimatization. The weight of each rat will be measured and computed daily. Furthermore, each rat will be examined daily for possible behavioral and gross morphological or physical changes.

3.2 Preparation of animals
The animals were housed in hanging stainless-steel wire cages kept in an isolated room at a controlled temperature (23–25oC) and humidity (40–60%). Lighting was maintained on a 12-h cycle (lights on from 07.00 to 19.00h).
3.3 Sample size determination
The sample size was calculated by "resource equation method" described by Festing et al, (2002). In this method a value E is calculated based on decided sample size.
Where E = Total number of animals - Total number of groups
In this animal study 2 groups of animals were formed having a about 6-9 animals for each different interventions, therefore the total animals was 18 (9 × 2). Hence E was
E = 18-2 = 16
E (16) was used for this study.
3.4 Reagent Kits/Drug Preparation and Dosage
All assay reagent kit was from AGAPPE Laboratories Ltd and was purchased from Nums diagnostic services Abuja. Amlodipine and Metformin manufactured by Pfizer Pharmaceutical Company was purchased at a pharmaceutical outlet in Ilorin. Amlodipine and metformin was dissolved at room temperature (RT) in distilled water, under sterile conditions and vortexed to obtain transparent solution. Drug doses were guided by the literature and the rats were administered intra-peritoneally with mg/kg/day (Begum and Akhter, 2007).
3.5 Dosage
The dosage of each drug was calculated using the dose by factor approach according to Anroop and Shery (2016).
    In the dose by factor approach, the number of observed adverse effect level NOAEL of drugs is scaled by making use of allometry to derive the maximum recommended starting dose MRSD for clinical studies. This simple empirical approach considers the size of individuals species based on body surface area which is related to metabolic rate of an animal that is established through evolutionary adaptation of animals to their size (Kearny and White, 2014).
Dose is equally related to body weight but it is not the lone factor which influences the scaling for dose calculation. The correction factor known as the km is estimated by dividing the average body weight (kg) of species to its body surface area (m2).