Characterization of Fasiglifam-Related Liver Toxicity in Dogs.

Fasiglifam, a potent and highly selective agonist of G protein-coupled receptor 40, was developed for the treatment of type 2 diabetes mellitus. However, phase III clinical programs were terminated owing to liver safety concerns. Fasiglifam-related liver toxicity was also observed in repeat-dose dog toxicology studies, characterized by granulomatous inflammation with crystal formation in the liver and/or bile ducts. These histopathological changes were not observed in rat toxicology studies. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of dog liver sections obtained from a repeat-dose toxicology study indicated that the crystalline material in the affected dog liver contained fasiglifam and fasiglifam glucuronide (fasiglifam-G). Nonclinical mechanistic studies indicated that after 14 days of repeated oral dosing with [14C]fasiglifam at 200 mg/kg per day to dogs, the concentrations of fasiglifam and fasiglifam-G in the bile exceeded the solubility limit of these compounds in the bile (approximately 3000 µg/ml). After single oral 2- and 200-mg/kg doses administered to rats and dogs, fasiglifam and fasiglifam-G concentrations in dog bile were 5- to 10-fold higher than those in rat bile for the same dose of fasiglifam, while the bile flow rate adjusted by body weight was 4- to 8-fold lower in dogs than in rats. High fasiglifam and fasiglifam-G concentrations in dog bile together with lower bile flow rate could cause crystal formation in dog bile, resulting in secondary granulomatous inflammation in the dog liver.

The effect of elevated α1-acid glycoprotein on the pharmacokinetics of TAK-272 (SCO-272), an orally active renin inhibitor, in rats.

The pharmacokinetics of TAK-272 (SCO-272), an orally active renin inhibitor, was investigated in rats with subcutaneously injected turpentine oil, which was an inflammation animal model. Following intravenous administration of TAK-272 to the turpentine-treated rats, the systemic clearance and volume of distribution decreased with the elevated plasma α1-acid glycoprotein (AGP) levels. The elevated plasma AGP levels were negatively correlated with the plasma unbound fraction of TAK-272 in the rats. Although the AUCs of total TAK-272 in the turpentine-treated rats were higher than those in the control rats after intravenous and oral administration, those of unbound TAK-272, which seem to directly contribute to the pharmacological effect and safety, were nearly equal between the turpentine-treated and control rats in the respective dose routes. TAK-272 has been shown to primarily bind to AGP in the human plasma. These results strongly suggested that the pharmacokinetic of TAK-272 in humans would also be affected by the variation in the plasma AGP levels and should be discussed with not only the total concentrations but also the unbound concentrations in the clinical trial for patients with elevated plasma AGP levels.

Differences in nonclinical pharmacokinetics between species and prediction of human pharmacokinetics of TAK-272 (SCO-272), a novel orally active renin inhibitor.

In the search for orally available drugs, the prediction of human pharmacokinetics (PK) is essential for successfully selecting compounds that will be clinically useful. This report describes the selection of TAK-272 (SCO-272), a novel orally active renin inhibitor, as a clinical candidate via the detailed investigation of nonclinical PK data and human PK prediction. The bioavailability (BA) of TAK-272 after oral administration to rats and monkeys was low, especially in fasted monkeys, and the systemic exposure of TAK-272 was highly variable in monkeys. The results of mass balance studies in animals suggested that the absorbed TAK-272 was largely eliminated by metabolism. In vitro studies revealed that TAK-272 was mainly metabolized by CYP3A4/5 in humans, and it was a P-glycoprotein substrate. PK analysis suggested that the factors responsible for the low BA were different in rats and monkeys. First-pass hepatic extraction was high in rats, while the fraction absorbed from the gastrointestinal tract (Fa * Fg ) was low in monkeys. It was predicted that humans would have a higher BA and a longer half-life in the plasma compared with the animals by a simple calculation using intrinsic hepatic clearance in monkeys, which correlates well with human values for CYP3A4 substrates, and Fa * Fg in rats, which correlates relatively well with human values. TAK-272 was finally selected as a clinical candidate based on the result of human PK prediction. The actual human PK after oral administration of TAK-272 was comparable to the predicted profile and was preferable for clinical usage.

Fasiglifam (TAK-875) Alters Bile Acid Homeostasis in Rats and Dogs: A Potential Cause of Drug Induced Liver Injury.

Fasiglifam (TAK-875), a Free Fatty Acid Receptor 1 (FFAR1) agonist in development for the treatment of type 2 diabetes, was voluntarily terminated in phase 3 due to adverse liver effects. A mechanistic investigation described in this manuscript focused on the inhibition of bile acid (BA) transporters as a driver of the liver findings. TAK-875 was an in vitro inhibitor of multiple influx (NTCP and OATPs) and efflux (BSEP and MRPs) hepatobiliary BA transporters at micromolar concentrations. Repeat dose studies determined that TAK-875 caused a dose-dependent increase in serum total BA in rats and dogs. Additionally, there were dose-dependent increases in both unconjugated and conjugated individual BAs in both species. Rats had an increase in serum markers of liver injury without correlative microscopic signs of tissue damage. Two of 6 dogs that received the highest dose of TAK-875 developed liver injury with clinical pathology changes, and by microscopic analysis had portal granulomatous inflammation with neutrophils around a crystalline deposition. The BA composition of dog bile also significantly changed in a dose-dependent manner following TAK-875 administration. At the highest dose, levels of taurocholic acid were 50% greater than in controls with a corresponding 50% decrease in taurochenodeoxycholic acid. Transporter inhibition by TAK-875 may cause liver injury in dogs through altered bile BA composition characteristics, as evidenced by crystalline deposition, likely composed of test article, in the bile duct. In conclusion, a combination of in vitro and in vivo evidence suggests that BA transporter inhibition could contribute to TAK-875-mediated liver injury in dogs.

Species differences of organic anion transporters involved in the renal uptake of 4-amino-3-chlorophenyl hydrogen sulfate, a metabolite of resatorvid, between rats and dogs.

Previous studies on the metabolic fate of resatorvid (TAK-242) have shown that species differences in the pharmacokinetics of 4-amino-3-chlorophenyl hydrogen sulfate (M-III), a metabolite of TAK-242, between rats and dogs are mainly attributable to the urinary excretion process. In the present study, the renal uptake mechanism of M-III was investigated using kidney slices and Xenopus laevis oocytes expressing rat organic anion transporter 1 (rOat1; Slc22a6) and rOat3 (Slc22a8). The uptake of p-aminohippuric acid (PAH), a substrate for Oats, by kidney slices from rats and dogs increased at 37 °C and M-III inhibited the uptake. The initial uptake clearance of M-III by rat kidney slices was 0.295 and 0.0114 ml/min/g at 37 °C and 4 °C, respectively. The Eadie-Hofstee plot of M-III uptake at 37 °C revealed two-component transport processes with K(m) values being 6.48 and 724 µmol/l. The uptake was inhibited by probenecid (PBC), PAH and benzylpenicillin (PCG). In contrast, in dog kidney slices, the initial uptake clearance of M-III was 8.70 × 10(-3) and 9.00 × 10(-3) ml/min/g at 37 °C and 4 °C, respectively, and the uptake was not inhibited by PBC. Furthermore, rOat1- and rOat3-expressing oocytes mediated M-III uptake and the uptake was inhibited by PAH and PCG, respectively. These results suggest that rOat1 and rOat3 are responsible for the renal uptake of M-III in rats. Moreover, it is speculated that Oat(s) is unable to transport M-III in dogs and that the difference in the substrate recognition of Oat(s) contributes to the species difference in the pharmacokinetics of M-III between rats and dogs.

Absorption of TAK-491, a new angiotensin II receptor antagonist, in animals.

The absorption process in animals of TAK-491, designed as ester-based prodrug with medoxomil moiety, was evaluated. In the plasma of rats and dogs, TAK-536, the pharmacologically active metabolite, was present as the main component with hardly detectable concentrations of TAK-491 after oral administration of TAK-491. In the rat portal plasma, TAK-536 was also present as the main component with hardly detectable concentrations of TAK-491 after jejunal loop injection of TAK-491, suggesting TAK-491 was absorbed from small intestine and hydrolyzed almost completely during absorption. Caco-2 study indicated the permeability of TAK-491 was improved by prodrug modification and the compound could be mainly transferred as TAK-491. This is well consistent with the facts that the AUC and T(max) of TAK-536 after oral administration of TAK-491 were higher and shorter than those after oral administration of TAK-536 in dogs Hydrolysis of TAK-491 is observed not only by the intestinal and hepatic S9 fraction, but also by plasma and human serum albumin. However, medoxomil alcohol wasn't detected during the hydrolysis of TAK-491. These metabolic features of TAK-491 were similar to olmesartan medoxomil, suggesting the hydrolytic pathway and enzymes for TAK-491 when catalyzing to TAK-536 would be the same as olmesartan medoxomil.

Marked impact of P-glycoprotein on the absorption of TAK-427 in rats.

The role of P-glycoprotein (P-gp, ABCB1) on the absorption process was investigated by drug-drug interaction studies of TAK-427 with P-gp inhibitors (erythromycin, ketoconazole or quinidine) in rats and by transport studies using rat multidrug resistance (MDR1) stably expressing cells and rat small intestine mounted in a Ussing-type chamber. TAK-427 showed high efflux activity with low permeability in rat MDR1a and MDR1b stably expressing cells and was revealed to be a typical substrate for P-gps. Although TAK-427 was mainly absorbed from the small intestine in rats, a large part of the dosed compound remained in the gastrointestinal tract. Orally co-administered P-gp inhibitors (50 mg/kg) increased the AUC of TAK-427 after a 5 mg/kg oral dose 5.4- to 18.3-fold, whereas orally administered P-gp inhibitors had a minor effect on the increase in the AUC of TAK-427 (1.3- to 2.2-fold) after a 0.5 mg/kg intravenous dose. Thus, the bioavailability of TAK-427 after oral administration in rats (7.3%) markedly increased when co-administered with P-gp inhibitors (28.6-57.6%). Moreover, the transport of TAK-427 was predominantly secretory throughout the rat small intestine and was inhibited by P-gp inhibitors. In conclusion, P-gp can markedly reduce the absorption of a typical P-gp substrate by its efflux activity throughout the absorption site.

Establishment and characterization of the transformants stably-expressing MDR1 derived from various animal species in LLC-PK1.

PURPOSE: Stable transformants expressing human multidrug resistance 1 (MDR1), monkey MDR1, canine MDR1, rat MDR1a, rat MDR1b, mouse mdr1a, and mouse mdr1b in LLC-PK1 were established to investigate species differences in P-glycoprotein (P-gp, ABCB1) mediated efflux activity. METHODS: The seven cDNAs of MDR1 from five animals were cloned, and their transformants stably expressing the series of MDR1 in LLC-PK1 were established. Transport studies of clarithromycin, daunorubicin, digoxin, erythromycin, etoposide, paclitaxel, propranolol, quinidine, ritonavir, saquinavir, verapamil, and vinblastine were performed by using these cells, and efflux activity was compared among the species. RESULTS: Except for propranolol, all compounds showed efflux activity in all transformants, and were judged to be substrates of P-gp. There were slight interspecies and interisoforms differences in the substrate recognition. However, the efflux ratio among the series of the MDR1 stably expressing cells showed good correlation as represented between human and monkey MDR1, and poor correlation as represented between human MDR1 and mouse mdr1a, and human and canine MDR1. CONCLUSIONS: Results in the present study indicate that all MDR1 stably expressing cells have efflux activity for various P-gp substrates, and that interspecies differences and similarities of the P-gp substrate efflux activity may exist.

Identification of domains participating in the substrate specificity and subcellular localization of the multidrug resistance proteins MRP1 and MRP2.

The human multidrug resistance protein MRP1 and its homolog, MRP2, are both thought to be involved in cancer drug resistance and the transport of a wide variety of organic anions, including the cysteinyl leukotriene C4 (LTC4) (Km = 0.1 and 1 microm). To determine which domain of these proteins is associated with substrate specificity and subcellular localization, we constructed various chimeric MRP1/MRP2 molecules and expressed them in polarized mammalian LLC-PK1 cells. We examined the kinetic properties of each chimeric protein by measuring LTC4 and methotrexate transport in inside-out membrane vesicles, sensitivity to an anticancer agent, etoposide, and subcellular localization by indirect immunofluorescence methods. The following results were determined in these studies: (i) when the NH2-proximal 108 amino acids of MRP2, including transmembrane (TM) helices 1-3, were exchanged with the corresponding region of MRP1, Km(LTC4) values of the chimera decreased approximately 4-fold and Km(methotrexate) values increased approximately 5-fold relative to those of wild-type MRP2 and MRP1, respectively, whereas resistance to etoposide increased approximately 3-fold; (ii) when the NH2-proximal region up to TM9 of MRP2 was exchanged with the corresponding region of MRP1, a further increase in etoposide resistance was observed, and subcellular localization moved from the apical to the lateral membrane; (iii) when two-thirds of MRP2 at the NH2 terminus were exchanged with the corresponding MRP1 region, the chimeric protein transported LTC4 with an efficiency comparable with that achieved by the wild-type MRP1; and (iv) exchange of the COOH-terminal 51 amino acids between MRP1 and MRP2 did not affect the localization of either of the proteins. These results provide a strong framework for further studies aimed at determining the precise domains of MRP1 and MRP2 with affinity for LTC4 and anticancer agents.

Trafficking and functional defects by mutations of the ATP-binding domains in MRP2 in patients with Dubin-Johnson syndrome.

Dubin-Johnson syndrome (DJS) is a hereditary disease characterized by hyperbilirubinemia. We investigated the consequences of 2 missense mutations, R768W and Q1382R, of nucleotide-binding domains (NBDs) of the multidrug resistance protein 2 (MRP2; ABCC2) that were previously identified in patients with DJS. Pulse chase analysis revealed that the precursor form of the wild-type and Q1382R MRP2 were converted to the mature form, which is resistant to endoglycosidase H (Endo H) in about 60 minutes. However, the precursor form of the R768W MRP2, which is sensitive to endoglycosidase H, was degraded within 120 minutes and did not mature to the fully glycosylated form. Proteasome inhibitors inhibited the degradation of the precursor form of the R768W MRP2. Unlike the R768W MRP2, the Q1382R MRP2 was mainly localized on the apical membrane in the wild-type form. However, efflux of glutathione monochlorobimane (GS-MCLB) and ATP-dependent leukotriene C(4) (LTC(4)) uptake into plasma membrane vesicles from cells expressing the Q1382R MRP2 were markedly reduced, suggesting that the Q1382R MRP2 on the apical membrane was nonfunctional. Vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP in the wild-type MRP2 was stimulated by estradiol glucuronide (E(2)17betaG) in a concentration-dependent manner but that in the Q1382R MRP2 was not. In conclusion, the R768W mutation causes deficient maturation and impaired sorting, and the Q1382R mutation does not affect maturation or sorting but impairs the substrate-induced ATP hydrolysis.