Effect of cytochrome P450 inhibition on pharmacokinetics and toxicity of diclofenac in chickens : unravelling toxicity in Gyps vultures

Abstract

The nonsteroidal anti-inflammatory drug (NSAID) diclofenac was responsible for the decimation of Gyps vulture species on the Indian subcontinent over the last two decades of the 20th century. For an unknown reason, Gyps vultures were extremely sensitive to diclofenac (LD50 ~ 0.1-0.2 mg/kg), with toxicity appearing to be linked to a metabolic deficiency, demonstrated by the long T1/2 (~12-17 h) and low Cl (0.0001-0.0002 L/h*kg). This was in striking comparison to other bird species such as the domestic chicken (Gallus gallus domesticus), where the LD50 is ~10 mg/kg, the T1/2 is ~1 h and the Cl values are ~0.1-0.2 ml/h*kg. The aim of this study was to determine if Cytochrome P450 2C9 (CYP2C9) homolog pharmacogenomic differences among avian species is driving diclofenac toxicity in Gyps vultures. For this evaluation, we exposed each of 10 CYP-inhibited (fluconazole) test group domestic chickens to a unique dose of diclofenac, centred on the LD50 of 9.8 mg/kg, as per OECD toxicity testing guidelines. The toxicity and pharmacokinetic results were compared to control group birds that received no fluconazole.

The birds showed typical clinical and post mortem signs of diclofenac toxicity; depression, lethargy and anorexia within 48 -56 h and visceral gout with varying degrees of nephrosis. Though no differences were noted in the LD50 values for each group (11.92 mg/kg in the CYP-inhibited test group and 11.58 mg/kg in the control group), the pharmacokinetic profile of the test group was suggestive of partial inhibition of CYP metabolism. This was evident in the geomean values for Cmax (0.61 vs. 0.41 ug/ml), AUClast (0.5 ug/ml*h vs. 0.4 ug/ml*h) and clearance (1.52 L/h*kg vs. 1.59 L/h*kg), despite CYP-inhibited birds at the two highest doses succumbing without a definable pharmacokinetic curve. In contrast both birds dosed at the two highest doses from the control group demonstrated high T1/2 and MRT values, consistent with expectations.

Evaluation of the metabolite peaks produced also suggested partial inhibition of CYP enzymatic metabolism in test group birds as they produced lower amounts of metabolites for one of the 3 peaks demonstrated and had higher diclofenac exposure. Furthermore, though the general trend was that birds that produced less metabolites and that died tended to be those dosed towards the higher end of the dose range, the results were not consistent. One bird in the test group, dosed at a much lower dose, exhibited very low metabolite production compared to birds in both treatment groups. This bird also exhibited pharmacokinetic data suggestive of metabolic constraint. These findings, coupled with the high variation in levels of metabolites produced across both treatment groups, indicates that there is a degree of natural variation in metabolism which is independent of dose in chickens, and which would also explain the higher LD50 in the chicken in comparison to the vulture.

This pilot study supports the hypothesis that CYP metabolism is varied among bird species and may explain the higher resilience to diclofenac in the chicken vs. Gyps vultures. Further studies using a larger sample size and a single dose of diclofenac may provide more conclusive results.