Abstract: Background: Cefpirome is a broad-spectrum semi synthetic β-lactamase resistant fourth generation cephalosporin. Looking to potential for clinical use, pharmacokinetics of cefpirome following intravenous and intramuscular administration (10 mg kg-1) in cow calves was determined. Methods: Cefpirome concentration in plasma samples was determined by reverse-phase high performance liquid chromatography with mobile phase. The mobile phase was a mixture of 0.2 M sodium acetate (3.5%), acetronitrile (17.5%) and HPLC water (79%) with a 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. Results: Following intravenous administration of the cefpirome in goats, volume of distribution at steady-state (Vdss), elimination half-life (t1/2β) and total body clearance (ClB) were reported 0.33±0.01 L kg-1, 2.41±0.23 h and 2.36±0.18 mL min-1 kg-1, respectively. Following intramuscular administration of the drug, peak plasma concentration (Cmax), elimination half-life (t½β), apparent volume of distribution (Vdarea), total body clearance (ClB), bioavailability (F) were 13.27±0.19 μg mL-1, 3.61±0.12 h, 0.81±0.007 L kg-1, 2.83±0.08 mL min-1 kg-1 and 61±1%, respectively. Conclusion: Pharmacokinetic – pharmacodynamics integration indicates cefpirome can be useful in cow calves at dose rate of 10 mg kg-1 and repeated at interval of 12 h by intramuscular route.
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 calves6,7 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 assays8,9 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 (Cmax) of 13.27±0.19 μg mL–1 was achieved at 0.75 h (Tmax) 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 (t1/2α) 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 (t1/2β) ranged from 1.62 to 3.12 h with a mean of 2.41±0.23 h. Total body clearance (ClB) 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 (t½Ka) and elimination half-life (t½β) were 0.16±0.01 and 3.61±0.12 h, respectively. The mean apparent volume of distribution (Vdarea), total body clearance (ClB) 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.
DISCUSSION
Following single dose intravenous administration, elimination half life (t1/2β: 2.41±0.23 h) of cefpirome observed in cow calves is in agreement to the elimination half life of 2.14±0.02 h and 2.12±0.14 h reported in buffalo calves and goats6,8. However, shorter elimination half life of 0.19 h in mouse, 1.05 h in dogs, 1.17 h in monkeys and 1.48 h in rabbits5, have been reported.
The mean residence time calculated following single dose intravenous administration was 2.14±0.06 h in cow calves, which is in agreement with reported values of 2.89±0.01 and 2.72±0.11 h in buffalo calves and goats6,8. However, it was observed that, cefpirome principally eliminated by the kidney and 80-90% of the administered drug was recovered as unchanged form in the urine11. Body clearance (2.36±0.18 mL min–1 kg–1) of cefpirome in cow calves following intravenous route of administration in present study found to be higher than clearance values of 2.00±0.05 mL min–1 kg–1 in buffalo calves6, 2.13±0.05 mL min–1 kg–1 in goats8 and 3.2 mL min–1 kg–1 in dogs8 reported following intravenous administration of cefpirome. The low value of mean volume of distribution at steady state (Vdss: 0.33±0.01 L kg–1) in cow calves following cefpirome intravenous administration indicated the limited distribution of drug into various body fluids and tissues. Similar low value of Vdss (0.40±0.004 and 0.35±0.01 L kg–1) have been reported in buffalo calves and goats6,8. In addition to this limited distribution of other cephalosporins like cefepime was also reported in goat and sheep12,13,14,15.
Following intramuscular administration of cefpirome in cow calves, the peak plasma concentration (Cmax) of 13.27±0.19 μg mL–1 was observed at 0.75 h (Tmax). Similarly Cmax of 9.04±0.5 μg mL–1 at 0.5 h in buffalo calves6, 11.7 μg mL–1 at 0.42 h in monkeys, 9.2 μg mL–1 at 0.48 h in rats and 15.4 μg mL–1 at 0.70 h in dogs5 and 10.97±0.34 μg mL–1 in goats8 have been reported. Elimination half-life (t½β:3.61±0.12 h) obtained in present study is higher than reported elimination half-life of 2.09±0.08 h in goats8, 2.39±0.05 h in buffalo calves7, 1.38 h in dogs and 1.23 h in monkeys5. Clearance (2.83±0.08 mL min–1 kg–1) of the drug observed following intramuscular administration is similar to that reported in goat (2.88±0.10 mL min–1 kg–1)8. Moreover, value of mean apparent volume of distribution (Vdarea: 0.812±0.007 L kg–1) indicated good distribution of drugs in body tissues following intramuscular administration of cefpirome in cow calvess. Value of Vdarea for cow calves in present study is higher than reported values of 0.42±0.01 L kg–1 in buffalo calves11 and 0.52±0.02 L kg–1 in goats8. Systemic bioavailability (61±1%) following intramuscular administration of cefpirome in cow calves is higher than 35.3±3.1% reported in buffalo calves7 and in less than that reported in goats (75±4%)8. High systemic bioavailability and maintenance of therapeutic concentration up to 12 h following intramuscular injection suggests that cefpirome is suitable for intramuscular administration for the treatment for systemic bacterial infections in cow calves.
For β-lactam antibiotics, time for which serum drug concentration exceeds the MIC (T>MIC) of pathogens is considered as primary determinant of antibacterial efficacy16. In addition to this, for β-lactam antibiotics maximum killing was seen when the time above MIC is at least 70% of the dosing interval17. Minimum inhibitory concentration obtain for cefpirome sensitive bacteria like Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Bacillus subtilis, range from 0.1 to 0.5 μg mL–1 and similar finding were also reported buy other authors18,19. Integrating the cefpirome pharmacokinetic data and the MIC values a cefpirome, an intravenously or intramuscular dose of 10mg kg–1 repeated at 12 h interval is sufficient to maintain plasma concentration of the drug above the MIC. However, for Pseudomonas aeruginosa (whose MIC value is 2 μg mL–1), a cefpirome dose of 10 mg kg–1 is sufficient to maintain plasma concentration of the drug above the MIC when it is administered intravenously or intramuscularly at 8 h interval.
CONCLUSIONS
Pharmacokinetic-pharmacodynamics integration indicates cefpirome can be useful in cow calves at dose rate of 10 mg kg–1 and repeated at interval of 12 h by intramuscular route.
ACKNOWLEDGMENT
Authors are thankful to Dean, Vanbandhu Veterinary College, Navsari Agricultural University, for providing infrastructure facilities for completion of this study. The authors declare that they have no conflict of interest.