Stability of Ketoprofen Methylester in Plasma of Different Species.

BACKGROUND: Pharmacokinetic and pharmacodynamic assessment of ester-containing drugs can be impacted by hydrolysis of the drugs in plasma samples post blood collection. The impact is different in the plasma of different species. OBJECTIVE: This study evaluated the stability of a prodrug, ketoprofen methylester (KME), in commercially purchased and freshly collected plasma of mouse, rat, dog, cat, pig, sheep, cattle and horse. METHODS: KME hydrolysis was determined following its incubation in commercially purchased and freshly collected plasma of those species. Different esterase inhibitors were evaluated for prevention of the hydrolysis in rat, dog and pig plasma. RESULTS: KME was rapidly hydrolyzed in both commercially purchased and freshly collected plasma of mouse, rat, and horse. The hydrolysis was initially quick and then limited in cat plasma. KME hydrolysis was minimum in commercially purchased plasma of dog, pig, sheep and cattle but substantial in freshly collected plasma of those species. Different esterase inhibitors showed different effects on the stability of KME in rat, dog and pig plasma. CONCLUSION: These results indicate that plasma of different species has different hydrolytic activities to estercontaining drugs. The activities in commercially purchased and freshly collected plasma may be different and species-dependent. Esterase inhibitors have different effects on preventing hydrolysis of the ester-containing drugs in the plasma of different species.

Assessment of Inhibition of Bovine Hepatic Cytochrome P450 by 43 Commercial Bovine Medicines Using a Combination of In Vitro Assays and Pharmacokinetic Data from the Literature.

BACKGROUND: There has been a lack of information about the inhibition of bovine medicines on bovine hepatic CYP450 at their commercial doses and dosing routes. OBJECTIVE: The aim of this work was to assess the inhibition of 43 bovine medicines on bovine hepatic CYP450 using a combination of in vitro assay and Cmax values from pharmacokinetic studies with their commercial doses and dosing routes in the literature. METHODS: Those drugs were first evaluated through a single point inhibitory assay at 3 µM in bovine liver microsomes for six specific CYP450 metabolisms, phenacetin o-deethylation, coumarin 7- hydroxylation, tolbutamide 4-hydroxylation, bufuralol 1-hydroxylation, chlorzoxazone 6-hydroxylation and midazolam 1'-hydroxylation. When the inhibition was greater than 20% in the assay, IC50 values were then determined. The potential in vivo bovine hepatic CYP450 inhibition by those drugs was assessed using a combination of the IC50 values and in vivo Cmax values from pharmacokinetic studies at their commercial doses and administration routes in the literature. RESULTS: Fifteen bovine medicines or metabolites showed in vitro inhibition on one or more bovine hepatic CYP450 metabolisms with different IC50 values. Desfuroylceftiour (active metabolite of ceftiofur), nitroxinil and flunixin have the potential to inhibit one of the bovine hepatic CYP450 isoforms in vivo at their commercial doses and administration routes. The rest of the bovine medicines had low risks of in vivo bovine hepatic CYP450 inhibition. CONCLUSION: This combination of in vitro assay and in vivo Cmax data provides a good approach to assess the inhibition of bovine medicines on bovine hepatic CYP450.

Pharmacokinetics and brain distribution of amitraz and its metabolites in rats.

Amitraz is an acaricide and insecticide widely used in agriculture and veterinary medicine. Although central nervous system (CNS) toxicity is one of major toxicities following oral ingestion of amitraz, the understanding of the cause of the toxicity is limited. This study evaluated the systemic and brain exposure of amitraz and its major metabolites, BTS27271, 2',4'-formoxylidide, and 2,4-dimethylaniline following administration of amitraz in male Sprague-Dawley rats. Significant metabolism of amitraz was observed following the intravenous and oral administration. Amitraz related metabolites were majority of the total exposure observed, especially following oral administration. BTS27271 had higher brain exposure than amitraz and its other metabolites, which was due to low plasma protein binding but high brain tissue binding of BTS27271. Since BTS27271 has similar or higher toxicity and α2-adrenoreceptor agonist potency than amitraz, its exposure in brain tissues may be the major cause of CNS toxicity of amitraz in animals and humans.

Assessment of inhibition of porcine hepatic cytochrome P450 enzymes by 48 commercial drugs.

Drug interactions due to inhibition of hepatic cytochrome P450 (CYP450) enzymes are not well understood in veterinary medicine. Forty-eight commercial porcine medicines were selected to evaluate their potential inhibition on porcine hepatic CYP450 enzymes at their commercial doses and administration routes. Those drugs were first assessed through a single point inhibitory assay at 3 µM in porcine liver microsomes for six specific CYP450 metabolisms (phenacetin o-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, bufuralol 1-hydroxylation, chlorozoxazone 6-hydroxylation and midazolam 1'-hydroxylation). When the inhibition was > 10% in the single point inhibitory assay, IC50 values (inhibitory concentrations that decrease biotransformation of selected substrate by 50%) were determined. Overall, 17 drugs showed in vitro inhibition on one or more porcine hepatic CYP450 metabolisms with different IC50 values. The potential in vivo porcine hepatic CYP450 inhibition by those drugs was assessed by combining the in vitro data and in vivo Cmax (maximum plasma concentrations from pharmacokinetic studies of the porcine medicines at their commercial doses and administration routes). Three drugs showed high potential inhibition to one or two porcine hepatic CYP450 isoforms at their commercial doses and administration routes, while seven drugs had medium risk and seven had low risk of such in vivo inhibition. These data are useful to prevent potential drug interactions in veterinary medical practice.

Current cytochrome P450 phenotyping methods applied to metabolic drug-drug interaction prediction in dogs.

Recombinant cytochrome P450 (P450) phenotyping, different approaches for estimating fraction metabolized (f(m)), and multiple measures of in vivo inhibitor exposure were tested for their ability to predict drug interaction magnitude in dogs. In previous reports, midazolam-ketoconazole interaction studies in dogs have been attributed to inhibition of CYP3A pathways. However, in vitro phenotyping studies demonstrated higher apparent intrinsic clearances (CL(int,app)) of midazolam with canine CYP2B11 and CYP2C21. Application of activity correction factors and isoform hepatic abundance to liver microsome CL(int,app) values further implicated CYP2B11 (f(m) >or= 0.89) as the dog enzyme responsible for midazolam- and temazepam-ketoconazole interactions in vivo. Mean area under the curve (AUC) in the presence of the inhibitor/AUC ratios from intravenous and oral midazolam interaction studies were predicted well with unbound K(i) and estimates of unbound hepatic inlet inhibitor concentrations and intestinal metabolism using the AUC-competitive inhibitor relationship. No interactions were observed in vivo with bufuralol, although significant interactions with bufuralol were predicted with fluoxetine via CYP2D and CYP2C pathways (>2.45-fold) but not with clomipramine (<2-fold). The minor caffeine-fluvoxamine interaction (1.78-fold) was slightly higher than predicted values based on determination of a moderate f(m) value for CYP1A1, although CYP1A2 may also be involved in caffeine metabolism. The findings suggest promise for in vitro approaches to drug interaction assessment in dogs, but they also highlight the need to identify improved substrate and inhibitor probes for canine P450s.

Evaluation of Escherichia coli membrane preparations of canine CYP1A1, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 with coexpressed canine cytochrome P450 reductase.

The preparation of bacterial membranes ("Bactosomes") containing expressed canine (beagle) hepatic cytochromes P450 (P450s) is described. cDNAs from seven canine P450s were subcloned into inducible expression plasmids and, for the first time, cotransformed and expressed with a canine P450 oxidoreductase in Escherichia coli to produce active, full-length, native sequence P450s. Enzyme expression levels, although variable, were generally sufficient to enable short incubation times and to limit the total protein present in enzyme incubations. Steady-state kinetics of CYP1A1, 2C21, and 2D15 Bactosomes demonstrated similarities with dog liver microsomes or Baculosomes. However, 3A12 lacked substrate inhibition in the formation of 1'-OH midazolam, and 2B11 displayed non-Michaelis-Menten kinetics, suggesting possible differences in protein interaction effects. In monitoring the metabolites of common P450 substrates, phenacetin deethylation, temazepam demethylation, and bufuralol 1'-hydroxylation were shown to be relatively selective reactions catalyzed by CYP1A1, 2B11, and 2D15, respectively. 1'-OH midazolam was formed in higher quantities by CYP2B11 and 2C21 than by 3A12, raising questions about the use of midazolam as a CYP3A12 probe in vivo. In summary, a panel of recombinant P450s was produced to make up for the lack of commercially available canine P450 isoforms. The Bactosomes are expected to facilitate reaction phenotyping and metabolic drug-drug interaction assessment in canine drug development and to enable the study of interspecies differences in P450-mediated drug metabolism.