ABSTRACT:The pharmacokinetics and metabolism of the direct thrombin inhibitor dabigatran ( Antithrombotic therapy plays an important role in the prevention and treatment of thromboembolic disorders. However, currently available agents are subject to certain limitations. Oral vitamin K antagonists, such as warfarin, have unpredictable pharmacokinetics and show numerous drug and food interactions (Ansell et al., 2004), whereas unfractionated and low molecular weight heparins and fondaparinux require parenteral administration. An orally active direct thrombin inhibitor would offer a number of potential advantages over these agents (Weitz and Bates, 2005).Dabigatran is a reversible, competitive, direct thrombin inhibitor that has been shown to be an effective antithrombotic agent in animal models (Stassen et al., 2001; Wienen et al., 2001a,b) and to be efficacious and safe in the prevention of deep vein thrombosis in patients undergoing elective total hip or knee replacement (Eriksson et al., 2005). Dabigatran etexilate is currently in Phase III development for primary prevention of venous thromboembolism (VTE) in patients undergoing major orthopedic surgery, acute VTE treatment, and VTE secondary prevention, as well as stroke prevention in patients with atrial fibrillation. Pharmacokinetic studies in healthy volunteers and orthopedic surgery patients showed that dose-dependent concentrations of dabigatran are achieved after p.o. administration of dabigatran etexilate, with peak concentrations reached after approximately 2 h and with a slight delay up to 6 h on the day of surgery (Eriksson et al., 2004(Eriksson et al., , 2005Stangier et al., 2005).This article describes a series of in vivo and in vitro studies performed to investigate the pharmacokinetics and metabolism of dabigatran in humans. ABBREVIATIONS: Dabigatran, -alanine, N-[[2-[[[4-[[[(hexyloxy) Materials and Methods Reference

ABSTRACT:The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDPglucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

ABSTRACT:The pharmacokinetics and metabolism of linagliptin (BI1356, 8-(3R-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methylquinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione) were investigated in healthy volunteers. Type 2 diabetes mellitus (T2DM) accounts for 90 to 95% of all diabetes cases and its incidence is increasing (Wild et al., 2004). The high frequency of complications associated with the disease leads to a significant reduction in life expectancy. One relatively new therapeutic option is the inhibition of the enzyme dipeptidyl peptidase-4 (DPP-4), which is responsible for the rapid degradation of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide. After food intake, both hormones augment the action of insulin (Holst and Gromada, 2004;Mari et al., 2005;Drucker and Nauck, 2006;Drucker, 2007). The plasma half-life of GLP-1 is limited to a few minutes because of its rapid proteolytic degradation by DPP-4 (Graefe-Mody et al., 2009). Inhibitors of DPP-4 prolong the half-life of GLP-1 and glucose-dependent insulinotropic polypeptide, which leads to increases in glucose-dependent insulin secretion, inhibition of endogenous glucose production, decreased blood glucose, and the induction of feelings of satiety (Drucker, 2002;Nauck et al., 2003;Holst and Gromada, 2004).Linagliptin is a novel, orally active, highly specific, and potent inhibitor of DPP-4 that is currently in clinical development for the treatment of T2DM (Eckhardt et al., 2007;Fuchs et al., 2009b). Early clinical studies with linagliptin suggested a reduction in the glycated hemoglobin levels in patients with T2DM while maintaining a placebo-like safety and tolerability profile (Heise et al., 2009;Retlich et al., 2009). The pharmacokinetics of linagliptin were previously shown to be nonlinear due to target-mediated, concentrationdependent changes in binding to DPP-4 (Hüttner et al., 2008; Thomas et al., 2008a,b;Fuchs et al., 2009a;Heise et al., 2009).We report here a series of in vivo and in vitro studies performed to further establish the pharmacokinetics and metabolism of linagliptin in humans after intravenous and oral administration.Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.109.031476.ABBREVIATIONS: T2DM, type 2 diabetes mellitus; DPP-4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide-1; BI1355, 8-(3S-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione; CD1790, 7-but-2-ynyl-8-(3S-hydroxy-piperidin-1-yl)-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione; CD1789, 7-but-2-ynyl-8-(3R-hydroxy-piperidin-1-yl)-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione; CD10604, 7-but-2-yn-1-yl-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-8-(3-oxopiperidin-1-yl)-3, 7-dihydro-1H-purine-2,6-dione; LOQ, lower limit of quantification; LC-MS/MS, liquid chromatography-tandem mass spectrometry; HPLC, high-performance liquid chromat...

Following oral administration of empagliflozin (1000 mg/kg/day) to male and female CD-1 mice for 2 years, renal tubular injury was identified in male mice. Renal injury was not detected in male mice (≤300 mg/kg/day), in female mice (1000 mg/kg/day), or in male or female Han Wistar rats (700 mg/kg/day). Using transfected HEK293 cells and Xenopus oocytes, empagliflozin was found to be a substrate of various mouse and rat organic anion transporters (oat/Oat) and organic anion transporting polypeptide (oatp/Oatp) transporters: mouse oat3, rat Oat3, mouse oatp1a1, and rat Oatp1a1. However, using isolated kidney slices from male and female mice and rats, no sex-based difference in the extent of uptake of empagliflozin occurred. Metabolism studies using hepatic and renal microsomes from male and female mice, rats, and humans revealed a hemiacetal metabolite of empagliflozin (M466/2), predominantly formed in male mouse kidney microsomes. Formation of M466/2 in male mouse kidney microsomes was 31-fold higher compared to that in female mouse kidney microsomes and was ∼29- and ∼20-fold higher compared to that in male and female mouse liver microsomes, respectively. M466/2 is unstable and degrades to form a phenol metabolite (M380/1) and 4-hydroxycrotonaldehyde (4-OH CTA). Formed 4-OH CTA was trapped by reduced GSH, and the structure of the GSH adduct was confirmed by mass spectrometry. Stoichiometric formation of M380/1 from M466/2 was observed (93-96% at 24 h); however, formation of 4-OH CTA was considerably lower (∼17.5% at 40 h), which is consistent with 4-OH CTA being a highly reactive species. These data represent a highly selective tissue-, species-, and sex-specific lesion in male CD-1 mice associated with a cytotoxic metabolite product, 4-OH CTA. In humans, glucuronidation of empagliflozin is the most prevalent metabolic pathway, and oxidation is a minor pathway. Thus, renal toxicity due to the formation of 4-OH CTA from empagliflozin is not expected in humans.

Dabigatran etexilate, an oral, reversible, competitive, and direct thrombin inhibitor, is an in vitro and in vivo substrate of Pglycoprotein (P-gp). Dabigatran etexilate was proposed as an in vivo probe substrate for intestinal P-gp inhibition in a recent guidance on drug-drug interactions (DDI) from the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). We conducted transcellular transport studies across Caco-2 cell monolayers with dabigatran etexilate in the presence of various P-gp inhibitors to examine how well in vitro IC 50 data, in combination with mathematical equations provided by regulatory guidances, predict DDI likelihood. From a set of potential P-gp inhibitors, clarithromycin, cyclosporin A, itraconazole, ketoconazole, quinidine, and ritonavir inhibited P-gp-mediated transport of dabigatran etexilate over a concentration range that may hypothetically occur in the intestine. IC 50 values of P-gp inhibitors for dabigatran etexilate transport were comparable to those of digoxin, a well established in vitro and in vivo P-gp substrate. However, IC 50 values varied depending whether they were calculated from efflux ratios or permeability coefficients. Prediction of DDI likelihood of P-gp inhibitors using IC 50 values, the hypothetical concentration of P-gp inhibitors, and the cut-off value recommended by both the FDA and EMA were in line with the DDI occurrence in clinical studies with dabigatran etexilate. However, it has to be kept in mind that validity of the cut-off criteria proposed by the FDA and EMA depends on in vitro experimental systems and the IC 50 -calculation methods that are employed, as IC 50 values are substantially influenced by these factors.