Dear Kushal,

The following publication entitled " Pharmacokinetic Study of Two Macrolide Antibiotic Oral Suspensions Using an Optimized Bioassay Procedure" contains a protocol for bioequivalence study of antibacterial agents:

Abstract

Purpose: Erythromycin (ERY) (CAS 114-07-8) is a macrolide antibacterial with a broad and essentially bacteriostatic action and Azithromycin (AZI) (CAS 83905-01-5) is a semi-synthetic, acid stable erythromycin derivative with an expanded spectrum of activity and improved tissue pharmacokinetic characteristics relative to ERY. The aim of the present study was to develop an optimized procedure for determination of macrolide antibiotics in human serum as well as to compare the bioequivalence of two commercial brands of Erythromycin and Azithromycin Suspensions in healthy Iranian volunteers.

Methods: Two brands of erythromycin ethylsuccinate and azithromycin oral suspensions were used. An equivalent 400-mg ERY suspension and 500-mg AZI suspension were given orally to each subject in two separate studies as a

single dose with 200 ml of water. ERY and AZI concentrations in serum were determined using an optimized agar well diffusion technique with Sarcina lutea (Micrococcus Luteus, ATCC 9341).

Results: Following administration of test and reference ERY products the mean values for Cmax (1168.5, 1115.0 ng/ml), Tmax (1.4, 1.38 hr), 0 t AUC (4021.4, 4010.0 ngh/ml), AUC0  (4852.6, 4787.4 ngh/ml) and T1/2 (3.64, 3.61hr)

were obtained. For AZI mean values for Cmax (468.4, 488.1 ng/ml) ,Tmax (1.96, 2.08 hr) , 0 t AUC (7575.4, 8046.6 ngh/ml), AUC0  (7990.7,8436.7 ngh/ml) and T1/2 (24.98,25.18 hr) were reported. A two-compartment model best described the

disposition of ERY and AZI after oral administration in human. The analytical method was validated in terms of linearity, accuracy and precision. The limit of quantitation for ERY and AZI were 50 and 40 ng/ml respectively.

Conclusion: From the results obtained it can be concluded that the optimized method introduced in this study could be successfully applied for the evaluation of pharmacokinetic parameters of both AZI and ERY. Moreover the results revealed that test preparations were equivalent with respective innovator products in terms of rate and extent of absorption.

Materials and Methods

Clinical protocol and study design

The study was an open-label, single-dose, single-blind, randomized study with a cross over design carried out in accordance with the guidelines of the Declaration of Helsinki (World Medical Assembly 1964) as revised in Edinburgh (2000). The protocol of this study was approved by the ethical committee of the Tabriz University

of Medical Sciences. For each drug twenty four male healthy volunteers were enrolled in the study. They were all Iranians, aged between 21 and 31 years (23.2 ± 1.7 years) and weight from 56 to 90kg (70.0 ± 9.3kg). The volunteers were informed about possible risks and adverse effects of taking the drug, and written consent

was obtained. The test products and reference were randomized and given to the volunteers. One-week and two-week washout periods were included between each administration of ERY and AZI preparations respectively. An equivalent 400 mg ERY suspension was given orally as a single dose with 200 ml of water. Five milliliters of blood were drawn at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 10 hours after each administration. In the second study an equivalent 500 mg AZI suspension was administered orally to each subject as a single dose with 200 ml of water. Five milliliters of blood were drawn at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 48 and 72 hours after administration. The blood samples were taken from subject’s forearm veins. All samples were centrifuged and the serum samples were separated and kept frozen at the temperature below -20°C for subsequent analysis. Bio-analysis

Optimized analytical procedures were used for determination of ERY and AZI in plasma. Microbiological assay of ERY and AZI was performed by an agar well diffusion procedure with Sarcina lutea (Micrococcus Luteus, ATCC 9341). Briefly the assay plate composed of two layer of antibiotic agar: the first one called base layer contained 21 ml of antibiotic agar I (PH 8.6 for AZI and 8.3 for ERY) and the

second layer known as the seed layer made of 4 ml of antibiotic agar I (PH 6.7) inoculated with the bacterial inoculums. After 24 h of incubation in 35°C, the diameter ofinhibition zone was measured. The method was validated by determination of the following operational characteristics: linearity, precision and accuracy. The linearity was evaluated by linear regression analysis, which was calculated by the least squares regression method in concentration level of 50, 100,

200, 400, 800 and 1600 ng/ml of spiked plasma. The precision of the assay was determined by repeatability (intra-assay) and intermediate precision (inter-assay) and was expressed as the relative standard deviation (RSD) of four quality control samples. The accuracy was determined by adding known amounts of ERY and AZI reference substance (Quality control samples) at the beginning of the process.

For ERY one milliliter of plasma and one hundred micro liter of concentrated phosphate buffer PH 8.3 were incubated for two hours at 40ºC to hydrolyze the ester linkage of the ERY ethylsuccinate. Two hundred micro liter of the resulting plasma samples were applied to the bioassay plates, duplicate of six clinical and standard samples (range 50-1600 and 40-800 ng/ml for ERY and AZI, respectively) were distributed over the sample plate. The plates were incubated at 35ºC for 24 h. Calibration graphs were constructed by plotting the diameters of the inhibition zones against the logarithm of the ERY or AZI concentrations.

Pharmacokinetic parameters and statistical analysis

The pharmacokinetic parameters for investigated macrolide antibiotics consisting of maximum plasma concentration (Cmax) and the time at which this concentration is reached (Tmax) were determined from the individual subject plasma concentration-time profiles by visual inspection and used as criteria of the rate of absorption. The area under the plasma concentration - time curve from time zero to t was calculated using linear trapezoidal rule. The apparent elimination rate constant (Kel) was determined by linear regression of log-transformed data in the terminal phase of plasma concentration-time profile. The constant Kel was used to extrapolate

AUC0-t and was calculated by the quotient of 0.693/kel (Zakeri-Milani et al., 2009; Zakeri-Milani et al., 2008; Valizadeh et al., 2009). The AUC0-  which means the area under the serum concentration-time curve extrapolated to infinity was calculated according to the following equation:

AUC0- = AUC0-t + Ct/Ke

Bioequivalence between the products was determined by calculating 90% confidence intervals (90% C.I.) for the ratio of Cmax, 0 t AUC and AUC0

 values for the test and reference products. Analysis of variance (ANOVA) was used to assess formulation, sequence, subjects and period effects (Zakeri-Milani et al., 2009; Zakeri-Milani et al., 2008; Valizadeh et al., 2009). Moreover a computer program (kinetic 5.0 PK/PD Analysis) was used for pharmacokinetic analyses.

Peroral data from each volunteer were fitted to one, two and three compartment models with first order absorption, with lag time and elimination from the central compartment.

To view the full publication, please use the following link:

http://www.omicsonline.org/pharmacokinetic-study-of-two-macrolide-antibiotic-oral-suspensions-using-an-optimized-bioassay-procedure-jbb.1000041.pdf

Hoping this will be helpful,

Rafik

 Thanks a lot... This would be very helpful for my study. Is there any study protocol for  Cephalosporin/penicillin group ?

Thank you Mr. Jun Shi this will help me a lot. 

When the elimination half-live is longer than 24 hours one can also opt for using trancated AUCs. I.e. take blood samples upto 72h after dosing. See FDA and EMEA guidelines on comparative bioavailability studies.

Dear  Kushal Biswas,

The following publication entitled "Comparative Bioequivalence Study of Three Formulations of Enrofloxacin in Sheep " contains a full, clear protocol for studying the Bioequivalence of Enrofloxacin.

Abstract:

A comparative pharmacokinetic study of three enrofloxacin injectable solutions was carried out in six healthy Barky rams after intramuscular injection according to a single dose, randomized, crossover experimental design. The three formulations were enrofloxacin 10% (Baytril®), enrofloxacin 10% plus bromhexine 1% (Mucotryl®) and enrofloxacin 10 % solution without bromhexine (Mucotryl without bromhexine). The three formulations were given a single intramuscular dose at a dose rate of 5 mg kg-1 b.wt. The concentrations of the drug in the serum were measured using highperformance liquid chromatography (HPLC) with fluorescence detection. The results indicate that there were no significant differences between the distibution rate constant (kab) and distribution half-life (t1/2ab), the maximum serum concentration (Cmax) and the time to peak concentration (Tmax) between Baytril and the other two formulations. There were significant differences between the elimination half life (t1/2el) and elimination rate constant (kel), for Baytril and enrofloxacin (the exact formulation of Mucotryl without bromhexine). Enrofloxacin was rapidly absorbed following IM administration of 5 mg kg-1 b. wt. The peak serum concentrations (Cmax) were 2.83, 2.45 and 3.12 μg ml-1 and were attained at (tmax) 1.15, 1.41 and 1.26 hours when given Mucotryl, Baytril and enrofloxacin (the exact formulation of Mucotryl without bromhexine) to sheep, respectively. The injectable formulations investigated were bioequivalent after their intramuscular injection to sheep at recommended dose rate. As our results showed that, values of Tmax, Cmax and AUC (Area under time curve concentration) determined for both Baytril, Mucotryl, and enrofloxacin (the exact formulation of Mucotryl without bromhexine ( reference ant test products) are within the acceptable range of 0.80-1.20, this means that the tested products product under investigation was bio-equivalent. These findings showed that the efficacy of the two test formulations was similar or possibly enhanced compared with Baytril based on the pharmacokinetic parameters obtained and due to concentration dependent activity of enrofloxacin ..

Please revise this important article; Drug Metabolism Letters, Volume 5, Number 2, April 2011, pp. 85-91(7)

Bioequivalence and Population Pharmacokinetic Modeling of Two Forms of Antibiotic, Cefuroxime Lysine and Cefuroxime Sodium, after Intravenous Infusion in Beagle Dogs

Longshan Zhao,  Qing Li, Xingang Li, 2 Ran Yin,  Xiaohui Chen,  Lulu Geng,  and Kaishun Bi 

To investigate the bioequivalence and the population pharmacokinetics of cefuroxime lysine and cefuroxime sodium in healthy beagle dogs. A randomized 2-period crossover design in 18 healthy beagle dogs after receiving 20, 40, and 80 mg/kg of cefuroxime lysine or cefuroxime sodium was conducted. A 3-compartment open model was used as the basic model for the population pharmacokinetic study. Both of the antibiotics exhibited dose-proportional pharmacokinetics over the dose range of 20–80 mg/kg. The mean relative bioavailability of cefuroxime lysine versus cefuroxime sodium was 1.05 (range, 0.71 to 1.42), with a significant difference between males and females. The estimates of population pharmacokinetic of CL, V1,Q2, V2, Q3, V3 were 3.74 mL/h, 1.70 mL, 29.5 mL/min, 3.58 mL, 0.31 mL/min, and 158 mL for cefuroxime lysine and 4.10 mL/h, 1.00 mL, 38.5 mL/min, 4.19 mL, 0.06 mL/min, and 13.6 mL for cefuroxime sodium, respectively. The inter-individual variability was determined to be less than 29.1%. A linear pharmacokinetic was revealed for cefuroxime lysine and cefuroxime sodium in dogs after intravenous infusion, and the bioequivalence of these forms of the antibiotic was observed with the significant gender-related differences in mean relative bioavailability of cefuroxime lysine versus cefuroxime sodium

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3403372/

Thank you all for your contribution. These papers and your advice will help me a lot.