Drugs
Ciprofloxacin 500mg tablets manufactured in Nigeria were purchased from registered pharmacies within Awka metropolis, in Anambra State. The brands purchased are Ciprotab (Fidson healthcare plc, Lagos), Cipro-J (Juhel Nigeria Limited, Enugu), M & B Cipro tablets (May and Baker Nigeria Plc), Cipro-500 (Nuel pharmaceutical company, Ogun). Ciprotab was used as a reference drug because of previously published studies [15]. Ciprofloxacin internal standard (Zhejiang Guobang Pharmco limited, China) was obtained as gift from Pauco Pharmaceutical Limited, Nigeria.
Study Animal
Twelve healthy male rabbits (weight: 2.5–3.5 kg; Age: 6–10 month) were purchased from an animal farm in Nneobi, Anambra state. The Rabbits were allowed to acclimitize in its new environment during which they were closely and then, fasted for 12 hours with free access to water before the drug administration according to the methods of Venho and Eriksson, [16].
Ethical Clearance
An approval to conduct the study was obtained from the Faculty of Pharmaceutical sciences, Nnamdi Azikwe University, Awka.
Study Design
Balanced Incomplete Block Design (BIBD) with adequate washout period was adopted. In the first and second period, each of the animals was administered 500 mg of ciprofloxacin via oral route under direct observation.
Drug administration and sample collection
Five hundred milligram tablets of either the reference or test drug formulation were administered as whole tablet with the help of feeding trough with 5 mL of water. Mouth check was performed to ascertain that the animal has swallowed the medication before the water was given.
Following this, blood samples were collected from the great auricular vein of the rabbits at time interval of 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h. The samples were collected into sets of two different sample bottles (plain bottle; EDTA bottle). The blood sample in the plain bottles were allowed to stay for about 30 min to allow coagulation before been centrifuged at a speed of 3000 rpm for 10 min and the supernatant collected and stored at -180C in an eppendurf tube prior to biological assay. Meanwhile, the samples in the EDTA bottles were centrifuged immediately after collection and the supernatant plasma collected for u-v spectrophotometric analysis. Following the initial drug administration and sample collection from a particular group, a washout period of one week was allowed to pass before the second phase of drug administration and sample collection from the same group was carried out.
Drug analysis
Gradient concentrations of 0.2, 0.16, 0.12, 0.08, 0.04, 0.024, 0.016, and 0.008 µg/ml of ciprofloxacin were prepared with distilled water from the pure standard powder while the scan for the ciprofloxacin wavelength was run with distilled water as blank and these were used to prepare the calibration curve of ciprofloxacin at a scanned wavelength of 272.4 nm using the UV-Vis spectrophotometer. The plasma samples were then run on this UV-Vis spectrophotometer at the same wavelength 272.4nm with the readings taken in duplicate and the average was determined mathematically. The serum concentration of ciprofloxacin was determined using clinically isolated Staphylococcus aureaus samples from hospitalized patients. The nutrient agar was cooled to 50 oC and inoculated with a 24 hour incubated Staphylococcus aureus culture (0.1mL/100mL agar). Holes of 8 mm were punched out of the agar after its solidification. The punch-holes were filled with 100 µL of serum in duplicate for calibrators and samples. After the incubation at 37 oC for 18 hours, the inhibition zones were measured and the concentrations were determined.
Pharmacokinetic analysis
Pharmacokinetic parameters determination and calculation was done on concentration-time graphs with the use of WinNonlin software for pharmacokinetic studies. The area under the curve to the last measurable concentration (AUC0 − t) was determined directly from the WinNonlin software while the area under the curve extrapolated to infinity (AUC0−∞) was determined as AUC0–t + Ct/Ke, where Ct was the last measurable concentration and Ke, elimination constant (Walker, 2006). The elimination half-life (T1/2) was determined as 0.693/Ke. Maximal concentration of the drug (Cmax) and the time to attain the maximal concentration (tmax) were also determined.
Statistical analysis
Bioequivalence analysis utilizes mainly Cmax, AUC0 − tandAUC0−∞ as its pharmacokinetic parameters and it was determined by the estimation of 90% confidence interval (CI) for the ratio of the log-transformed data of the mean of the test to the reference products and also by the analysis of variance of the means of those parameters (FDA, 2003). These were done with SPSS version 20 with the difference between two means calculated using nonparametric paired t-test.