Correlation of lactulose-to-mannitol ratios in plasma and urine for intestinal permeability assessment in pigs.

OBJECTIVE: The lactulose-to-mannitol ratio test is a test to assess the disorders associated with gut permeability. The test requires an oral administration of the mixture of lactulose and mannitol and urine collection. The urinary ratio of lactulose to mannitol is an indicator of intestinal permeability. Due to the complexity of urine collection in animal studies, plasma exposure ratios of lactulose to mannitol compared to their urinary concentration ratios were evaluated following an oral administration of the sugar mixture in pigs. ANIMALS: 10 pigs were orally dosed with a solution of lactulose and mannitol mixture. PROCEDURES: Plasma samples were collected at predose, 10 and 30 minutes and 2, 4, and 6 hours postdosing, and cumulated urinary samples were collected at 6 hours for liquid chromatography-mass spectrometry analysis. The ratios of pharmacokinetic parameters of lactulose to mannitol and the plasma sugar ratios at a single time point or the mean values of several time points were compared to their urinary sugar ratios. RESULTS: The results revealed that the lactulose-to-mannitol ratios of AUC0-6h, AUCextrap, and Cmax were correlated to the urinary sugar ratios, and the plasma sugar ratios of a single time point at 2, 4, or 6 hours and the mean values of those time points were also appropriate to replace their urinary ratios in pigs. CLINICAL RELEVANCE: Following an oral administration of lactulose and mannitol mixture, blood collection, and assay can be an option for assessing intestinal permeability, especially in animal studies.

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.

Hepatic Flavin-containing Monooxygenase and Aldehyde Oxidase Activities in Male Domestic Pigs at Different Ages.

BACKGROUND: Age has a significant impact on activities of hepatic metabolizing enzymes in humans and animals. Flavin-containing Monooxygenase (FMO) and Aldehyde Oxidase (AO) are two important hepatic enzymes. Understanding of the impact of age on these two enzymes is still limited in pigs. OBJECTIVE: The aim of this work was to assess hepatic FMO and AO activities of male domestic pigs at five different ages of 1 day, 2, 5, 10 and 20 weeks. METHODS: Porcine liver microsomes and cytosol were prepared from the livers of male domestic pigs at ages of 1 day, 2, 5, 10 and 20 weeks. FMO activity was assessed using N-oxidation of benzydamine in porcine liver microsomes and AO activity was evaluated using oxidation of O6-benzylguanine in the porcine liver cytosol. RESULTS: Porcine hepatic FMO activity was substantial at the age of 1 day, rapidly increased in 2 weeks, and remained high afterwards. Porcine hepatic AO activity was minimal at the age of 1 day and gradually increased to the maximum in 5 weeks and remained relatively constant to the age of 20 weeks. Porcine hepatic FMO activity is higher than other species, including humans. Age-dependent FMO developmental pattern in porcine liver is different from porcine hepatic CYP450 and human hepatic FMO. Porcine hepatic AO activity is much lower than humans although their developmental patterns are similar. CONCLUSION: Age impact on hepatic activities of both FMO and AO is obvious in domestic male pigs although age patterns of both enzymes are different.

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.

Development of an in vitro screen for compound bioaccumulation in Haemonchus contortus.

The objective of the current study was to establish an in vitro screen and a highly sensitive analytical assay to delineate key physicochemical properties that favor compound bioaccumulation in the L3 life stage of a Haemonchus contortus isolate. Time-dependent studies revealed that absorption and elimination kinetics during the first 6 hr of exposure were sufficient to achieve maximum bioaccumulation for the majority of compounds tested. In subsequent studies, the larvae were incubated for 6 hr in a medium containing 146 compounds (5 µM initial concentration), including both human and veterinary medicines, characterized by a broad range of physicochemical properties. Bioaccumulation of the compounds by the nematodes was determined, and multiple physicochemical descriptors were selected for correlation. Data analysis using Bayes classification model and partial least-square regression revealed that clogD7.4, rotatable bond, E-state, and hydrogen bond donor each correlated with compound bioaccumulation in H. contortus L3. The finding that lipophilicity was critical for transcuticle compound permeation was consistent with previous studies in other parasitic species and in adult H. contortus . The finding of additional physicochemical properties that contribute to compound conformational flexibility, polarity, and electrotopological state shed light on the mechanisms governing transcuticle permeation. The relatively poor correlation between transcuticle and transmembrane permeation indicated the distinct mechanisms of compound permeation, likely due to the different constituents, and their contributions to overall transport function, of the lipid membranes and the porous collagen barrier of the nematode cuticle. Our study, for the first time, establishes a high-throughput screen for compound bioaccumulation in a parasitic nematode and further elucidates physicochemical factors governing transcuticular permeation of compounds. Application of this methodology will help explain the basis for discrepancies observed in receptor binding and whole organism potency assays and facilitate incorporation of drug delivery principles in the design of candidate anthelmintics.

Metabolism and pharmacokinetics of a novel Src kinase inhibitor TG100435 ([7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine) and its active N-oxide metabolite TG100855 ([7-(2,6-dichloro-phenyl)-5-methylbenzo[1,2,4]triazin-3-yl]-{4-[2-(1-oxy-pyrrolidin-1-yl)-ethoxy]-phenyl}-amine).

TG100435 ([7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine) is a novel multitargeted, orally active protein tyrosine kinase inhibitor. The inhibition constants (K(i)) of TG100435 against Src, Lyn, Abl, Yes, Lck, and EphB4 range from 13 to 64 nM. TG100435 has systemic clearance values of 20.1, 12.7, and 14.5 ml/min/kg and oral bioavailability of 74%, 23%, and 11% in mouse, rat, and dog, respectively. Four oxidation metabolites of TG100435 have been found in human, dog, and rat in vitro and in vivo. The ethylpyrrolidine N-oxide of TG100435 is the predominant metabolite (TG100855; [7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-{4-[2-(1-oxy-pyrrolidin-1-yl)-ethoxy]-phenyl}-amine) in human, dog, and rat. TG100855 is 2 to 9 times more potent than the parent compound. Flavin-containing monooxygenases are the primary enzymes mediating the biotransformation. Significant conversion of TG100435 to TG100855 has been observed in rat and dog after oral administration. Systemic exposure of TG100855 is 1.1- and 2.1-fold greater than that of TG100435 in rat and dog after oral dosing of TG100435. Since TG100435 is predominantly converted to the more potent N-oxide metabolite across species in vivo and in vitro, the overall tyrosine kinase inhibition in animal models may be substantially increased after oral administration of TG100435.