Association of pharmacokinetic profiles of lenalidomide in human plasma simulated using pharmacokinetic data in humanized-liver mice with liver toxicity detected by human serum albumin RNA.

Lenalidomide has been shown to be potentially teratogenic in thalidomide-sensitive animal species. Screening for thalidomide analogs devoid of teratogenicity/toxicity-attributable to drug metabolism and disposition, but having immunomodulatory properties-is a strategic pathway towards development of new anticancer drugs. Plasma concentrations of lenalidomide were investigated in immunodeficient control and humanized-liver mice following oral administration of lenalidomide (50 mg/kg). Plasma concentrations of lenalidomide (1-2 hr after administration) were slightly but significantly higher in humanized-liver mice than in control mice (p < 0.05). Human albumin mRNA, a liver-specific toxicity marker, was found in the blood of humanized-liver mice 24 hr after lenalidomide administration. Simulations of human plasma concentrations of lenalidomide were achieved with simplified physiologically-based pharmacokinetic models in control and humanized-liver mice or by the direct fitting analysis of reported human data, in accordance with reported lenalidomide concentrations after low dose administration in humans. The results indicate that pharmacokinetic profiles of lenalidomide, a compound resulting from introducing one aromatic amino group into thalidomide and removing one keto group, resulted in less species variation in in vivo pharmacokinetics in control and humanized-liver mice and that immunodeficient humanized-liver mice can serve as experimental model animals for human liver injury in drug development at high doses, with human albumin RNA analysis in plasma.

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

R-warfarin clearances from plasma associated with polymorphic cytochrome P450 2C19 and simulated by individual physiologically based pharmacokinetic models for 11 cynomolgus monkeys.

1. Cynomolgus monkey cytochrome P450 2C19 (formerly known as P450 2C75), homologous to human P450 2C19, has been identified as R-warfarin 7-hydroxylase. In this study, simulations of R-warfarin clearance in individual cynomolgus monkeys genotyped for P450 2C19 p.[(Phe100Asn; Ala103Val; Ile112Leu)] were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling. 2. Pharmacokinetic parameters and absorption rate constants, volumes of the systemic circulation, and hepatic intrinsic clearances for individual PBPK models were estimated for eleven cynomolgus monkeys. 3. One-way ANOVA revealed significant effects of the genotype (p < 0.01) on the observed elimination half-lives and areas under the curves of R-warfarin among the homozygous mutant, heterozygous mutant, and wild-type groups. R-Warfarin clearances in individual cynomolgus monkeys genotyped for P450 2C19 were simulated by simplified PBPK modeling. The modeled hepatic intrinsic clearances were significantly associated with the P450 2C19 genotypes. The liver microsomal elimination rates of R-warfarin for individual animals after in vivo administration showed significant reductions associated with the genotype (p < 0.01). 4. This study provides important information to help simulate clearances of R-warfarin and related medicines associated with polymorphic P450 2C19 in individual cynomolgus monkeys, thereby facilitating calculation of the fraction of hepatic clearance.

Simulation of human plasma concentration-time profiles of the partial glucokinase activator PF-04937319 and its disproportionate N-demethylated metabolite using humanized chimeric mice and semi-physiological pharmacokinetic modeling.

1. The partial glucokinase activator N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319) is biotransformed in humans to N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (M1), accounting for ∼65% of total exposure at steady state. 2. As the disproportionately abundant nature of M1 could not be reliably predicted from in vitro metabolism studies, we evaluated a chimeric mouse model with humanized liver on TK-NOG background for its ability to retrospectively predict human disposition of PF-04937319. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, hepatic intrinsic clearances normalized for liver weight, metabolite formation and liver to plasma concentration ratios were plotted against the replacement index by human hepatocytes and extrapolated to those in the virtual chimeric mouse with 100% humanized liver. 3. Semi-physiological pharmacokinetic analyses using the above parameters revealed that simulated concentration curves of PF-04937319 and M1 were approximately superimposed with the observed clinical data in humans. 4. Finally, qualitative profiling of circulating metabolites in humanized chimeric mice dosed with PF-04937319 or M1 also revealed the presence of a carbinolamide metabolite, identified in the clinical study as a human-specific metabolite. The case study demonstrates that humanized chimeric mice may be potentially useful in preclinical discovery towards studying disproportionate or human-specific metabolism of drug candidates.

Metabolic profiles of pomalidomide in human plasma simulated with pharmacokinetic data in control and humanized-liver mice.

1. Pomalidomide has been shown to be potentially teratogenic in thalidomide-sensitive animal species such as rabbits. Screening for thalidomide analogs devoid of teratogenicity/toxicity - attributable to metabolites formed by cytochrome P450 enzymes - but having immunomodulatory properties is a strategic pathway towards development of new anticancer drugs. 2. In this study, plasma concentrations of pomalidomide, its primary 5-hydroxylated metabolite, and its glucuronide conjugate(s) were investigated in control and humanized-liver mice. Following oral administration of pomalidomide (100 mg/kg), plasma concentrations of 7-hydroxypomalidomide and 5-hydroxypomalidomide glucuronide were slightly higher in humanized-liver mice than in control mice. 3. Simulations of human plasma concentrations of pomalidomide were achieved with simplified physiologically-based pharmacokinetic models in both groups of mice in accordance with reported pomalidomide concentrations after low dose administration in humans. 4. The results indicate that pharmacokinetic profiles of pomalidomide were roughly similar between control mice and humanized-liver mice and that control and humanized-liver mice mediated pomalidomide 5-hydroxylation in vivo. Introducing one aromatic amino group into thalidomide resulted in less species differences in in vivo pharmacokinetics in control and humanized-liver mice.

Combining Chimeric Mice with Humanized Liver, Mass Spectrometry, and Physiologically-Based Pharmacokinetic Modeling in Toxicology.

Species differences exist in terms of drug oxidation activities, which are mediated mainly by cytochrome P450 (P450) enzymes. To overcome the problem of species extrapolation, transchromosomic mice containing a human P450 3A cluster or chimeric mice transplanted with human hepatocytes have been introduced into the human toxicology research area. In this review, drug metabolism and disposition mediated by humanized livers in chimeric mice are summarized in terms of biliary/urinary excretions of phthalate and bisphenol A and plasma clearances of the human cocktail probe drugs caffeine, warfarin, omeprazole, metoprolol, and midazolam. Simulation of human plasma concentrations of the teratogen thalidomide and its human metabolites is possible with a simplified physiologically based pharmacokinetic model based on data obtained in chimeric mice, in accordance with reported clinical thalidomide concentrations. In addition, in vivo nonspecific hepatic protein binding parameters of metabolically activated 14C-drug candidate and hepatotoxic medicines in humanized liver mice can be analyzed by accelerator mass spectrometry and are useful for predictions in humans.

Human plasma concentrations of cytochrome P450 probe cocktails extrapolated from pharmacokinetics in mice transplanted with human hepatocytes and from pharmacokinetics in common marmosets using physiologically based pharmacokinetic modeling.

1. The pharmacokinetic data of cytochrome P450 probes in humans can be extrapolated from corresponding data in cynomolgus monkeys, dogs and minipigs using simplified physiologically based pharmacokinetic (PBPK) modeling. In this study, the modeling methodology was further adapted to estimate human plasma concentrations of P450 probes based on data from mice transplanted with human hepatocytes or based on data from marmosets. 2. Using known species allometric scaling factors, the observed plasma concentrations of caffeine, warfarin, omeprazole, metoprolol, and midazolam in chimeric TK-NOG mice with humanized liver were scaled to human oral monitoring equivalents. Using the same approach, the previously reported pharmacokinetics of the five P450 probes in marmosets were also scaled to reported equivalents in humans using in vitro metabolic clearance data. 3. Human plasma concentration profiles of the five P450 probes estimated by simplified human PBPK models based on the observed pharmacokinetics in mice with humanized liver and on the reported pharmacokinetics in marmosets were consistent with previously published pharmacokinetic data in Caucasians. 4. These results suggest that mice with humanized liver and/or marmosets could be suitable pharmacokinetic models for humans during research into new drugs, especially when used in combination with simple PBPK models.

Pollination of soybean (Glycine max L. Merril) by honeybees (Apis mellifera L.)

Este experimento teve como objetivo avaliar a polinização realizada pelas abelhas na produção e qualidade das sementes da soja (Glycine max L. Merril) na região de Maringá-PR. Os tratamentos constituíram de áreas demarcadas de livre visitação por insetos, áreas cobertas por gaiolas com uma colônia de abelhas (Apis mellifera) e plantas também cobertas por gaiolas que impediam a visitação por insetos. Todas as áreas possuíam 24 m2 (4 m x 6 m), com cinco repetições cada. A produção de sementes foi maior (P=0,0001) nas áreas cobertas com abelhas e de livre visitação com um incremento na produtividade de 50,64% e 57,73%, respectivamente, em relação à área coberta sem abelhas. Pode-se considerar que as abelhas A. mellifera foram responsáveis por 95,5% da polinização realizada pelos insetos no tratamento livre. O número de vagens no tratamento coberto com abelhas foi 61,38% maior (P=0,0002) do que no coberto sem abelhas. Onde as abelhas A. mellifera foram responsáveis pela polinização cruzada, houve um aumento de 58,86% no número de sementes em relação ao tratamento onde não foi permitida a polinização realizada por insetos. Entretanto, o peso médio de 100 sementes foi maior (P=0,0001) na área coberta sem abelhas, atingiu um peso médio de 17,80 g, mostrando que plantas com menor produção formaram sementes maiores. No tratamento livre, o peso médio de 100 sementes foi de 15,26 g e no coberto com abelhas foi de 15,37 g. O teor médio de proteína bruta no grão foi de 36,69% e a média do teor de óleo foi de 20,24%. O teste de germinação não mostrou diferenças entre as sementes nos diferentes tratamentos. Pode-se concluir que as abelhas A. mellifera foram eficientes no trabalho de polinização na soja, proporcionando um aumento considerável na produção de grãos e estes resultados reforçam a necessidade do uso das abelhas A. mellifera para elevar a produtividade da soja.