Pharmacokinetic study of flunixin and its interaction with enrofloxacin after intramuscular administration in calves

The Pharmacokinetic aspects of flunixin (FL) administered alone and in combination with enrofloxacin (EN), were studied in clinically healthy calves. The experiments were performed on two groups: FL alone {2.2 mg/kg, intramuscular (IM)}, and combination of FL (2.2 mg/kg, IM) and EN {2.5 mg/kg, IM}. Plasma concentrations of FL were determined using High Performance Liquid Chromatography (HPLC) method. Moreover, the effects of FL alone or in combination on liver and kidney functions were also assessed. Flunixin was rapidly absorbed intramuscularly with a half-life of absorption (t ) of 0.094 h and the peak plasma concentration (C ) was 1.27 g/mL was attained after 1/2ab max 0.49 h (T ). Enrofloxacin significantly altered the pharmacokinetics of FL by delaying its absorption and accelerate its max elimination from body. Significant increases (32%) in the area under the curve (AUC) and (37%) in the elimination rate constant (K ) from the central compartment and a significant decrease (27%) in the elimination half-life (t ) of FL el 1/2el were found following coadministration with EN, compared with administration of FL alone. The maximum plasma drug concentration (C ) showed significant increase (28%) following the coadministration of EN with FL as max compared to that following the administration of FL alone. It was concluded that the combination of FL and EN negatively altered the kinetics of FL and exaggerated the adverse effect on hepato-renal function in calves consequently; the concomitant use of FL and EN should be avoided in calves.


Introduction
concentration, and high bioavailability following oral or parenteral administration (Dorfman, et al., 1995), Flunixin (FL) is nonsteroidal anti-inflammatory with long serum half-life, thus it used once or twice drug (NSAID) inhibiting cycloxygenase enzymes in daily (Abo- EL-Sooud, 2003).NSAIDs are frequently the arachidonic acid cascade, thus block the formation coadministered to enhance the rate of recovery in of cycloxygenase derived eicosanoid inflammatory combination with antimicrobial drugs in calves mediators (Landoni et al., 1995;Cheng et al., 1998).
suffering from endotoximia, pneumonia and other Due to its anti-inflammatory, analgesic, and antipyretic viral and bacterial respiratory diseases.However, it effects (Mckellar et al., 1989;Beretta et al., 2005), FL has been reported that the distribution and elimination is widely used in veterinary medicine, particularly in of antimicrobials are altered when they are ruminant to treat the musculoskeletal conditions, coadministered with NSAIDs (Whittem et al., 1996 & endotoxic shock, acute mastitis, endotoxemia, and calf EL-Banna, 1999).Further, previous workers reported pneumonia (Anderson et al., 1991 (2) to determine pharmacokinetic parameters for FL 1998).It has bactericidal activity at relatively low after co-administration with EN injected intramus-reported by Odensvik & Johansson, (1995).Briefly, cularly at the dose rate recommended for calves to One ml of plasma was mixed with 2.0 ml of potassium establish any interaction between the drugs and (3) to phosphate buffer (potassium dihydrogenate phosphate evaluate the effects of FL alone and FL + EN on the anhydrous, 0.3 M, pH 3.5).Then FL was extracted by liver and kidney functions.
an addition of 5 ml distilled diethyl ether, after that the tubes were agitated for 30 minutes.Then the tubes

Materials and Methods
were centrifuged for 10 minutes at 500-x g using cool centrifuge.The organic phase transferred to new tube performed with a flow rate at 0.8 ml/min and a run time Animals were fed on barseem, barely, and of 10 min.FL was detected by UV absorption at a concentrated mixture in a pellet and water was adwavelength of 254 nm.The retention time for FL was libitum.Calves were kept indoors under good hygienic 7.4 min.The limit of quantification for the method conditions and under direct observation for a month was.Specificity of the method was assessed with no before the start of experiment to insure complete observed any interference with endogenous clearance from any previous drug residues.The study compounds or with the anticoagulant or with EN or was reviewed and approved by the Animals Ethics ciprofloxacin, while the accuracy of our method was Committee of University of Science and Technology, 98.61%.The mean absolute recovery of FL in plasma Irbid, Jordan.
was found to be 93.75%.Experimental design: The animals were randomly Method validation: The precision and accuracy of divided into two group's five calves each.All the method were evaluated by repetitive analysis of the injections were made at zero time into the thigh plasma samples (n=12) spiked with different known muscles.The doses were selected on the bases that this concentrations of FL.The percentage recoveries were is the recommended dose rate for use in calves.
determined by comparing the peak height of blank Animals of the first group received a single dose of FL samples spiked with different amounts of drug and at 2.2 mg/kg intramuscularly into the left gluteal treated as any sample, with the peak height of the same muscles.Those of group two were injected FL (2.2 standards prepared in phosphate buffer (n=6).mg/kg IM) in the left gluteal muscle, one h before the Intra-assay variations were determined by injection of EN at a dose of 2.5 mg/kg in the right gluteal muscle.
measuring six replicates (n=6) of three standard samples used for calibration curves.The intra-assay Sampling: Ten ml venous whole blood samples were variation coefficient was < 2.0.Inter-assay precisions collected from the right jugular vein into 10 mL were determined by assaying the three standard heparinized Vacutainers (Becton Dickinson samples on three separate days.The inter-assay vacutainers Systems) at 0 (blank sample), 0.166, 0.33, variation coefficient was < 3.7.Recovery of FL from 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72 and 96 h after plasma was found to be 97%.The limit of quantitreatment.All the blood samples were centrifuged at fication (LOQ) of FL in plasma was 39 ng/mL.The 3000 g for 15 min to separate the plasma.The plasma standard curve in calves' plasma was linear between samples were divided into two parts, the first part for 2 0.01 and 10 µg/mL with correlation coefficients (r ) estimation of hepato-renal functions directly and the 0 second one was frozen at -70 C until analysed by was 0.99.The peak height ratios of an unknown HPLC for FL assay.
specimen (peak height of difloxacin/peak height of Flunixin Assay: The concentration of FL in plasma internal standard) were compared with that of the samples were assayed by HPLC method previously standard.alanine transaminase (ALT) and creatinine and urea concentrations were estimated in plasma of all animals for 5 consecutive days from the drug administrations.Determination of AST, ALT, urea and creatinine in plasma were measured colorimetrically using Bio Merieux Kits (Vitek, Inc., USA).following coadministration with EN, compared with life (t ) were calculated as ln2/K or ln2/K , 1/2ab el ab administration of FL alone.The maximum plasma respectively.The areas under the concentration-time drug concentration (C ) showed significant increase max curves (AUC) were calculated by the trapezoidal rule (28%) following the coadministration of EN with FL and further extrapolated to infinity.The mean as compared to that following the administration of FL residence time (MRT) was calculated as AUMC/AUC, alone.Transaminases (AST, ALT) activities, urea and where AUC is as defined previously and AUMC is the creatinine were significantly increased till the 48 h and area under the first moment curve (Gibaldi & Perrier, after that they are returning into the normal level 96 h 1982).
after in both groups (Tables 2&3).Although, the Statistical analysis: Data are presented as mean ± concurrent administration of EN with FL was SE obtained from different five animals.An unpaired significantly exaggerated the hepato-renal functions student's t-test was used for statistical analysis.P value in marked manner than FL alone.(*<0.05,**< 0.01, ***< 0.001) compared with control values was considered statistically significant.Data were analyzed by Graphpad Prism software version 4.0 (San Diego, California, USA).

Mean (±SE) concentrations of FL in plasma after
the administration of FL and FL+EN intramuscularly are presented in Figure 1.After both FL and FL+EN administrations, plasma concentration-time curves were best fitted to an absorption and one-compartment elimination model in all animals.The pharmacokinetic parameters of FL in plasma after administration of FL and FL + EN combination were presented in Table 1.
Enrofloxacin significantly altered the pharmacokinetics of FL by delaying its absorption and accelerate its elimination from body.Significant increases (32%) in the area under the curve (AUC) and central compartment and a significant decrease (27%)

Discussion
previous study (Landoni et al., 1995).Pharmacokinetic interactions of fluoroquinolones Pharmacokinetic interactions between NSAIDs commonly occur with other drugs, usually by altering and antimicrobial drugs have received little attention their hepatic biotransformation.Some quinolones in veterinary medicine, in spite of their frequent use in preferentially inhibit cytochrome (CYP1A2), which is combination.However, pharmacokinetic interactions partially responsible for drug metabolism (Gillum et between phenylbutazone and the antibiotics benzylal., 1993).Although the mechanisms and scope of penicillin and gentamicin have been studied in horses these interactions are becoming well characterized, (Whittem et al., 1996).Phenylbutazone increased the the remaining challenge is how to best inform the serum concentrations of penicillin in one study but clinician, so that the undesirable consequences of there was no effect of phenylbutazone on gentamicin interactions may be prevented.NSAIDs are pharmacokinetics.
commonly used in combination with antimicrobials The pharmacokinetics of FL after IM adminis- (Kopcha et al., 1992).The concurrent administration tration at a dosage of 2.2 mg/kg was described by a of NSAIDs with fluoroquinolones; such as fenbufen one-compartment model preceded by absorption, with enoxacin has been associated with seizures in confirming the findings of Landoni et al. (1995).human beings, but other NSAIDs with other member Absorption of FL from the IM injection site was rapid, of fluoroquinolones have not developed seizures as indicated by the short absorption half-life (t = (Wolfsan & Hooper, 1991).
1/2ab 0.094 h) and time to attain maximum concentration The pharmacokinetics behavior of FL has been (T = 0.49 h).Similar values were reported for FL in a previously studied in calves after intravenous max

Figure- 1 :
Figure-1: Semilogarithmic plot of plasma concentration time (37%) in the elimination rate constant (K ) from the el

Table - 1: Mean ± SE of pharmacokinetic Evaluation of liver and kidney functions: The parameters of FL in plasma after administration activity
of plasma aspartate transaminase (AST) and