INTRODUCTION

Cefpirome is a broad-spectrum semi synthetic β-lactamase resistant fourth generation cephalosporin used in severely ill patients in the intensive care, oncology and transplantation units¹. It has increased affinity for PBPs², reduce susceptibility to extended-spectrum β-lactamase and have ability to interfere with PBPs mediated cell wall synthesis ultimately leads to cell lysis³. It has potent bactericidal activity against a broad range of gram-negative and gram-positive organisms, including Pseudomonas aeruginosa and methicillin susceptible Staphylococcus spp., Haemophillus influenzae type B and many members of the Enterobacteriaceae family⁴. The disposition kinetics of cefpirome have been investigated in rabbits, dogs, mice, rat and monkey⁵, buffalo calves^6,7 and goats⁸. However, there is no information available on the pharmacokinetic of cefpirome in cow calves following intravenous and intramuscular routes of administration. Looking to potential for clinical use and possibility for species difference, the study was undertaken on pharmacokinetics of cefpirome following intravenous and intramuscular administration in cow calves.

MATERIALS AND METHODS

Experimental animals: The experiment was conducted on six healthy crossbred (Kankrej X H.F), weighing 65-105 kg. Each animal was housed in a separate pen and provided standard ration with ad libitum water. Cow calves were kept under constant observation for two weeks before the commencement of the experiment and subjected to clinical examination to exclude possibility of any diseases. The experimental protocol was approved by Institutional Animal Ethics Committee.

Drug and chemical: Cefpirome technical grade powder was procured from Orchid Pharma Ltd., Chennai. Cefpirome sulpahte powder (1 g Ceforth®; Biochem pharmaceutical Industries Ltd., Mumbai) was purchased from local pharmacy. Water, acetonitrile, acetic acid (HPLC grade), sodium acetate and perchloric acid (AR grade) were purchased from S.D. Fine Chem Ltd., Merck India Ltd. and Sisco Research Laboratories Pvt. Limited, Mumbai, India.

Drug administration and sample collection: All six animals were randomly allocated to receive either an intravenous or intramuscular injection of Cefpirome at the dose rate of 10 mg kg^–1. A washout period of two weeks was observed between treatments. An intravenous injection of Cefpirome was administered in the left jugular vein. Blood samples (3 mL) were collected through an intravenous catheter (Venflon, 22x0.9x25 mm) fixed in the contra lateral jugular vein in glass test tubes, prior to injection and at 2, 5, 10, 15, 30 min and 1, 2, 4, 8, 12 and 18 h after intravenous administration. Following intramuscular injection of Cefpirome in the left deep gluteal muscle, blood samples (3 mL) were collected before administration and at 5, 10, 15, 30 min and 1, 2, 4, 8, 12, 18 and 24 h. Cow calves were monitored for any adverse reactions during the entire study period. Blood samples were centrifuged at 4116 g for 10 min at 4°C and plasma was transferred to cryo-vials (2 mL) and, stored at -20°C. Samples were analyzed within 48 h to quantify cefpirome concentration using high performance liquid chromatography.

Analytical assay of cefpirome and pharmacokinetic analysis: Cefpirome concentration in plasma samples was determined by reverse-phase High Performance Liquid Chromatography (HPLC) after extraction, using reported assays^8,9 with minor modifications. The High Performance Liquid Chromatography (HPLC) apparatus of Adept Cecil comprised of binary gradient delivery pump (model 4901), UV detector (model 4902) and reverse phase C18 column (4.6x100 mm ID). Pharmacokinetic data integration was performed using software “Power Stream” (Version 4.2). Plasma (500 μL) was deproteinized by addition of perchloric acid (0.8 M) and methanol (50:50) and vortexed for one minute. This was followed by centrifugation at 4116 g for 10 min. An aliquot of supernatant was collected in clean auto sampler vial. The mobile phase was a mixture of 0.2 M sodium acetate (3.5%), acetronitrile (17.5%) and HPLC water (79%). 0.2M acetic acid was used to adjust pH of 5.1. Mobile phase was filtered by 0.45 μ filters and pumped into column at a flow rate of 1.0 mL min^–1 at ambient temperature. The effluent was monitored at 258 nm wavelength.

Calibration curve was prepared daily for drug concentration ranging from 0.8-200 μg mL^–1. The assay was sensitive (LLOD: 0.8 μg mL^–1) and reproducible and linearity was observed from 0.8-200 μg mL^–1 (r² = 0.99). Precision and accuracy were determined using Quality Control (QC) samples at concentrations 1.6, 50, 200 μg mL^–1 (5 replicates each day). Intraday and interday co-efficients of variability for five QC samples were satisfactory with the relative deviations (RSD) of less than 6.54%. An absolute recovery of cefpirome was measured up to 96.30%. Various pharmacokinetic parameters were calculated from serum concentration of cefpirome using software PK solution (version 2.0). The bioavailability (F) was calculated using following equation:

Determination of minimal inhibitory concentrations of levofloxacin: Minimal inhibitory concentrations were determined against different organism Streptococcus pyogenes (ATCC:8668), Staphylococcus aureus (ATCC:25923), Escherichia coli (ATCC:25922), Salmonella typhimurium (ATCC:23564), Pseudomonas aeruginosa (ATCC:27853), Proteus mirabilis (NCIM:2241) and Bacillus subtilis (ATCC:9372). Which were procured form National Collection of Industrial Microorganism (NCIM), National Chemical Laboratory, Pune. The MIC of cefpirome was determined by micro broth dilution method in triplicates¹⁰.

Statistical analysis: Cefpirome serum concentration and pharmacokinetic parameters of different treatment groups were compared by students’ “t” test using SPSS software (version 12.0.1).

RESULTS

Following single dose intravenous and intramuscular administration of cefpirome in the cow calves, adverse reactions were not observed. Pharmacokinetic parameters (Mean±SE) calculated for both route of drug administration have been depicted in Table 1.

Following intravenous administration of cefpirome, therapeutic concentration of cefpirome = 0.5 μg mL^–1 was maintained in plasma from upto 8 h. Following intramuscular administration of the cefpirome, mean peak plasma drug concentration (C_max) of 13.27±0.19 μg mL^–1 was achieved at 0.75 h (T_max) which declined rapidly to 8.27±0.14 μg mL^–1 at 2 h. The drug concentration of 1.06±0.05 μg mL^–1 in plasma was detected at 12 h and thereafter drug was not detected in plasma samples collected beyond 12 h post intramuscular administration in cow calves.

Following intravenous administration of the drug in cow calves, distribution half-life (t_1/2α) ranged between 0.46 and 0.64 h with a mean of 0.53±0.03 h. The mean values of volume of distribution at steady-state (Vdss) were calculated to be 0.30±0.01 L kg^–1.

The elimination half-life (t_1/2β) ranged from 1.62 to 3.12 h with a mean of 2.41±0.23 h. Total body clearance (Cl_B) of the drug was 2.36±0.18 mL min^–1 kg^–1 with Mean Residence Time (MRT) of 2.14±0.06 h. Following intramuscular administration of the drug, absorption (t_½Ka) and elimination half-life (t½β) were 0.16±0.01 and 3.61±0.12 h, respectively. The mean apparent volume of distribution (Vd_area), total body clearance (Cl_B) and Mean Residence Time (MRT) were 0.81±0.07 L kg^–1, 2.83±0.08 mL min^–1 kg^–1 and 4.88±0.13 h, respectively. The bioavailability (F) of the drug following intramuscular administration ranged from 59-64% with an average of 61±1%.

The minimal inhibitory concentration of cefpirome were 0.13, 0.25, 0.5, 0.5, 1, 2 and 8 μg mL^–1 for Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes, Pseudomonas aeruginosa and Proteus mirabilis, respectively.