1. Linezolid (ZYVOX), the first of a new class of antibiotics, the oxazolidinones, is approved for treatment of Gram-positive bacterial infections. 2. The aim was to determine the absorption, distribution, metabolism and excretion (ADME) of linezolid in mouse, rat and dog in support of preclinical safety studies and clinical development. 3. Conventional replicate study designs were employed in animal experiments, and biofluids were assayed by HPLC or HPLC-MS. 4. Linezolid was rapidly absorbed after p.o. dosing with an p.o. bioavailability of > 95% in rat and dog, and > 70% in mouse. Twenty-eight-day i.v./p.o. toxicokinetic studies in rat (20-200mg kg(-1) day(-1)) and dog (10-80 mg kg(-1) day(-1)) revealed neither a meaningful increase in clearance nor accumulation upon multiple dosing. 5. Linezolid had limited protein binding (<35%) and was very well distributed to most extravascular sites, with a volume of distribution at steady-state (V(ss)) approximately equal to total body water. 6. Linezolid circulated mainly as parent drug and was excreted mainly as parent drug and two inactive carboxylic acids, PNU-142586 and PNU-142300. Minor secondary metabolites were also characterized. In all species, the clearance rate was determined by metabolism. 7. Radioactivity recovery was essentially complete within 24-48 h. Renal excretion of parent drug and metabolites was a major elimination route. Parent drug underwent renal tubular reabsorption, significantly slowing parent drug excretion and allowing a slow metabolic process to become rate-limiting in overall clearance. 8. It is concluded that ADME data were relatively consistent across species and supported the rat and dog as the principal non-clinical safety species.

ABSTRACT: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.

ABSTRACT: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 > 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.Metabolism-mediated drug interactions are an important consideration during preclinical drug lead optimization. Inhibitors of drugmetabolizing enzymes such as cytochromes P450 (P450) may be capable of decreasing the clearance of coadministered drugs when their clearance is metabolic. Therefore, it is important to evaluate new chemical entities as substrates and inhibitors of P450. To speed up this evaluation process, in vitro-in vivo extrapolation methods aimed at predicting metabolic drug interactions have continued to evolve. For instance, the choice of inhibition values (e.g., K i versus unbound K i ) (Brown et al., 2006), inhibitor absorption rates (Kanamitsu et al., 2000;Brown et al., 2005), P450 induction , and the choice of in vivo concentrations of P450 inhibitors (Brown et al., 2005;Obach et al., 2005) have all been studied with respect to in vitro-based drug-drug interaction (DDI) predictions. Irreversible enzyme inhibition mechanisms, although recognized for some time, have increasingly been added to in vitro-based drug interaction extrapolation methods with assumptions about the turnover rates of P450 isoforms (Mayhew et al., 2000;Venkatakrishnan and Obach, 2007). Several of these in vitro findings or approaches have even been integrated into several commercial software packages as biology continues to advance toward more physiolog...

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1. Metabolites of the cyclic bisphosphonate ester, 4-[2,2'-bis-(5,5- dimethyl-1,3,2-dioxaphosphorinan-2-yl)] butanoyl-3-fluoro-benzene (PNU-91638) in bile or urine of the male Sprague-Dawley rat were characterized by mass spectrometry. The chronically bile duct/duodenal-cannulated male rats received a single oral dose of 100 mg/kg [13C] [14C]PNU-91638. Bile and urine samples were analysed for radioactivity and profiled by hplc with radiometric and UV detection. 2. The 0-28-h bile and urine accounted for 46.0 and 19.7% of dose respectively. The structures of radioactive peaks were investigated by ionspray and liquid secondary ion mass spectrometry (LSIMS) and LSIMS/MS analysis. 3. Major metabolites in urine included two regioisomeric phenol glucuronides, a gem methyl hydroxylated metabolite of the bisphosphonate heterocycle, a phenol metabolite, parent drug and a benzylic alcohol metabolite. Additional metabolites in bile included an unstable phenol/glutathione adduct (from a putative epoxide intermediate) with several minor isobaric regioisomers, and a carboxylic acid derived from the gem methyl hydroxylated bisphosphonate ring. 4. The structures proposed have not been confirmed by nmr due to discontinuation of the development of PNU-91638.