Prediction of the drug-drug interaction potential of the α1-acid glycoprotein bound, CYP3A4/CYP2C9 metabolized oncology drug, erdafitinib.

Erdafitinib is a potent oral pan-fibroblast growth factor receptor inhibitor being developed as oncology drug for patients with alterations in the fibroblast growth factor receptor pathway. Erdafitinib binds preferentially to α1-acid glycoprotein (AGP) and is primarily metabolized by cytochrome P450 (CYP) 2C9 and 3A4. This article describes a physiologically based pharmacokinetic (PBPK) model for erdafitinib to assess the drug-drug interaction (DDI) potential of CYP3A4 and CYP2C9 inhibitors and CYP3A4/CYP2C9 inducers on erdafitinib pharmacokinetics (PK) in patients with cancer exhibiting higher AGP levels and in populations with different CYP2C9 genotypes. Erdafitinib's DDI potential as a perpetrator for transporter inhibition and for time-dependent inhibition and/or induction of CYP3A was also evaluated. The PBPK model incorporated input parameters from various in vitro and clinical PK studies, and the model was verified using a clinical DDI study with itraconazole and fluconazole. Erdafitinib clearance in the PBPK model consisted of multiple pathways (CYP2C9/3A4, renal, intestinal; additional hepatic clearance), making the compound less susceptible to DDIs. In poor-metabolizing CYP2C9 populations carrying the CYP2C9*3/*3 genotype, simulations shown clinically relevant increase in erdafitinib plasma concentrations. Simulated luminal and enterocyte concentration showed potential risk of P-glycoprotein inhibition with erdafitinib in the first 5 h after dosing, and simulations showed this interaction can be avoided by staggering erdafitinib and digoxin dosing. Other than a simulated ~ 60% exposure reduction with strong CYP3A/2C inducers such as rifampicin, other DDI liabilities were minimal and considered not clinically relevant.

Metabolism and disposition in rats, dogs, and humans of erdafitinib, an orally administered potent pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor.

This article describes in vivo biotransformation and disposition of erdafitinib following single oral dose of 3H-erdafitinib and 14C-erdafitinib to intact and bile duct-cannulated (BC) rats (4 mg/kg), 3H-erdafitinib to intact dogs (0.25 mg/kg), and 14C-erdafitinib to humans (12 mg; NCT02692677). Peak plasma concentrations of total radioactivity were achieved rapidly (Tmax: animals, 1 h; humans, 2-3 h). Recovery of drug-derived radioactivity was significantly slower in humans (87%, 384 h) versus animals (rats: 91-98%, 48 h; dogs: 81%, 72 h). Faeces was the primary route of elimination in intact rats (95%), dogs (76%), and humans (69%); and bile in BC rats (48%). Renal elimination of radioactivity was relatively low in animals (2-12%) versus humans (19%). Unchanged erdafitinib was major component in human excreta (faeces, 17%; urine, 11%) relative to animals. M6 (O-desmethyl) was the major faecal metabolite in humans (24%) and rats (intact, 46%; BC, 11%), and M2 (O-glucuronide of M6) was the prevalent biliary metabolite in rats (14%). In dogs, besides M6, majority of radioactive dose in faeces was composed of multiple minor metabolites. In humans, unchanged erdafitinib was the major circulating entity. O-demethylation of erdafitinib was the major metabolic pathway in humans and animals.

In vitro and physiologically-based pharmacokinetic based assessment of drug-drug interaction potential of canagliflozin.

AIMS: Canagliflozin is a recently approved drug for use in the treatment of type 2 diabetes. The potential for canagliflozin to cause clinical drug-drug interactions (DDIs) was assessed. METHODS: DDI potential of canagliflozin was investigated using in vitro test systems containing drug metabolizing enzymes or transporters. Basic predictive approaches were applied to determine potential interactions in vivo. A physiologically-based pharmacokinetic (PBPK) model was developed and clinical DDI simulations were performed to determine the likelihood of cytochrome P450 (CYP) inhibition by canagliflozin. RESULTS: Canagliflozin was primarily metabolized by uridine 5'-diphospho-glucuronosyltransferase 1A9 and 2B4 enzymes. Canagliflozin was a substrate of efflux transporters (P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein-2) but was not a substrate of uptake transporters (organic anion transporter polypeptide isoforms OATP1B1, OATP1B3, organic anion transporters OAT1 and OAT3, and organic cationic transporters OCT1, and OCT2). In inhibition assays, canagliflozin was shown to be a weak in vitro inhibitor (IC50 ) of CYP3A4 (27 µmol l -1 , standard error [SE] 4.9), CYP2C9 (80 µmol l -1 , SE 8.1), CYP2B6 (16 µmol l-1 , SE 2.1), CYP2C8 (75 µmol l -1 , SE 6.4), P-glycoprotein (19.3 µmol l -1 , SE 7.2), and multidrug resistance-associated protein-2 (21.5 µmol l -1 , SE 3.1). Basic models recommended in DDI guidelines (US Food & Drug Administration and European Medicines Agency) predicted moderate to low likelihood of interaction for these CYPs and efflux transporters. PBPK DDI simulations of canagliflozin with CYP probe substrates (simvastatin, S-warfarin, bupropion, repaglinide) did not show relevant interaction in humans since mean areas under the concentration-time curve and maximum plasma concentration ratios for probe substrates with and without canagliflozin and its 95% CIs were within 0.80-1.25. CONCLUSIONS: In vitro DDI followed by a predictive or PBPK approach was applied to determine DDI potential of canagliflozin. Overall, canagliflozin is neither a perpetrator nor a victim of clinically important interactions.

In vitro metabolism of canagliflozin in human liver, kidney, intestine microsomes, and recombinant uridine diphosphate glucuronosyltransferases (UGT) and the effect of genetic variability of UGT enzymes on the pharmacokinetics of canagliflozin in humans.

O-glucuronidation is the major metabolic elimination pathway for canagliflozin. The objective was to identify enzymes and tissues involved in the formation of 2 major glucuronidated metabolites (M7 and M5) of canagliflozin and subsequently to assess the impact of genetic variations in these uridine diphosphate glucuronosyltransferases (UGTs) on in vivo pharmacokinetics in humans. In vitro incubations with recombinant UGTs revealed involvement of UGT1A9 and UGT2B4 in the formation of M7 and M5, respectively. Although M7 and M5 were formed in liver microsomes, only M7 was formed in kidney microsomes. Participants from 7 phase 1 studies were pooled for pharmacogenomic analyses. A total of 134 participants (mean age, 41 years; men, 63%; white, 84%) were included in the analysis. In UGT1A9*3 carriers, exposure of plasma canagliflozin (Cmax,ss , 11%; AUCτ,ss , 45%) increased relative to the wild type. An increase in exposure of plasma canagliflozin (Cmax,ss , 21%; AUCt,ss , 18%) was observed in participants with UGT2B4*2 genotype compared with UGT2B4*2 noncarriers. Metabolites further delineate the role of both enzymes. The pharmacokinetic findings in participants carrying the UGT1A9*3 and UGT2B4*2 allele implicate that UGT1A9 and UGT2B4 are involved in the metabolism of canagliflozin to M7 and M5, respectively.

Absorption, metabolism, and excretion of oral ¹4C radiolabeled ibrutinib: an open-label, phase I, single-dose study in healthy men.

The absorption, metabolism, and excretion of ibrutinib were investigated in healthy men after administration of a single oral dose of 140 mg of ¹4C-labeled ibrutinib. The mean (S.D.) cumulative excretion of radioactivity of the dose was 7.8% (1.4%) in urine and 80.6% (3.1%) in feces with <1% excreted as parent ibrutinib. Only oxidative metabolites and very limited parent compound were detected in feces, and this indicated that ibrutinib was completely absorbed from the gastrointestinal tract. Metabolism occurred via three major pathways (hydroxylation of the phenyl (M35), opening of the piperidine (M25 and M34), and epoxidation of the ethylene on the acryloyl moiety with further hydrolysis to dihydrodiol (PCI-45227, and M37). Additional metabolites were formed by combinations of the primary metabolic pathways or by further metabolism. In blood and plasma, a rapid initial decline in radioactivity was observed along with long terminal elimination half-life for total radioactivity. The maximum concentration (Cmax) and area under the concentration-time curve (AUC) for total radioactivity were higher in plasma compared with blood. The main circulating entities in blood and plasma were M21 (sulfate conjugate of a monooxidized metabolite on phenoxyphenyl), M25, M34, M37 (PCI-45227), and ibrutinib. At Cmax of radioactivity, 12% of total radioactivity was accounted for by covalent binding in human plasma. More than 50% of total plasma radioactivity was attributed to covalently bound material from 8 hours onward; as a result, covalent binding accounted for 38% and 51% of total radioactivity AUC(0-24 h) and AUC(0-72 h), respectively. No effect of CYP2D6 genotype was observed on ibrutinib metabolism. Ibrutinib was well-tolerated by healthy participants.

Absolute oral bioavailability and pharmacokinetics of canagliflozin: A microdose study in healthy participants.

Absolute oral bioavailability of canagliflozin was assessed by simultaneous oral administration with intravenous [(14) C]-canagliflozin microdose infusion in nine healthy men. Pharmacokinetics of canagliflozin, [(14) C]-canagliflozin, and total radioactivity, and safety and tolerability were assessed at prespecified timepoints. On day 1, single-dose oral canagliflozin (300 mg) followed 105 minutes later by intravenous [(14) C]-canagliflozin (10 µg, 200 nCi) was administered. After oral administration, the mean (SD) Cmax of canagliflozin was 2504 (482) ng/mL at 1.5 hours, AUC∞ 17,375 (3555) ng.h/mL, and t1/2 11.6 (0.70) hours. After intravenous administration, the mean (SD) Cmax of unchanged [(14) C]-canagliflozin was 17,605 (6901) ng/mL, AUC∞ 27,100 (10,778) ng.h/mL, Vdss 83.5 (29.2) L, Vdz 119 (41.6) L, and CL 12.2 (3.79) L/h. Unchanged [(14) C]-canagliflozin and metabolites accounted for about 57% and 43% of the plasma total [(14) C] radioactivity AUC∞ , respectively. For total [(14) C] radioactivity, the mean (SD) Cmax was 15,981 (2721) ng-eq/mL, and AUC∞ 53,755 (15,587) ng-eq.h/mL. Renal (34.5% in urine) and biliary (34.1% in feces) excretions were the major elimination pathways for total [(14) C] radioactivity. The absolute oral bioavailability of canagliflozin was 65% (90% confidence interval: 55.41; 76.07). Overall, oral canagliflozin 300 mg coadministered with intravenous [(14) C]-canagliflozin (10 µg) was generally well-tolerated in healthy men, with no treatment-emergent adverse events.

Metabolism and excretion of canagliflozin in mice, rats, dogs, and humans.

Canagliflozin is an oral antihyperglycemic agent used for the treatment of type 2 diabetes mellitus. It blocks the reabsorption of glucose in the proximal renal tubule by inhibiting the sodium-glucose cotransporter 2. This article describes the in vivo biotransformation and disposition of canagliflozin after a single oral dose of [(14)C]canagliflozin to intact and bile duct-cannulated (BDC) mice and rats and to intact dogs and humans. Fecal excretion was the primary route of elimination of drug-derived radioactivity in both animals and humans. In BDC mice and rats, most radioactivity was excreted in bile. The extent of radioactivity excreted in urine as a percentage of the administered [(14)C]canagliflozin dose was 1.2%-7.6% in animals and approximately 33% in humans. The primary pathways contributing to the metabolic clearance of canagliflozin were oxidation in animals and direct glucuronidation of canagliflozin in humans. Unchanged canagliflozin was the major component in systemic circulation in all species. In human plasma, two pharmacologically inactive O-glucuronide conjugates of canagliflozin, M5 and M7, represented 19% and 14% of total drug-related exposure and were considered major human metabolites. Plasma concentrations of M5 and M7 in mice and rats from repeated dose safety studies were lower than those in humans given canagliflozin at the maximum recommended dose of 300 mg. However, biliary metabolite profiling in rodents indicated that mouse and rat livers had significant exposure to M5 and M7. Pharmacologic inactivity and high water solubility of M5 and M7 support glucuronidation of canagliflozin as a safe detoxification pathway.

Cytotoxicity of amino alcohols to rat hepatoma-derived Fa32 cells.

Amino alcohols are used as emulsifying agents in dry-cleaning soaps, wax removers, cosmetics, paints and insecticides. The cytotoxicities of 12 amino alcohols, which differed in chain length, position of the amino and alcohol groups, and the presence of an additional phenyl group, were determined by the neutral red uptake inhibition assay with normally cultured, glutathione-depleted or antioxidant-enriched Fa32 rat hepatoma-derived cells. Glutathione depletion and antioxidant enrichment were achieved by including 50(M L-buthionine-S,R-sulphoximine (BSO) or 100(M (-tocopherol acetate (vitamin E) in the culture medium for 24 hours before and during the assay. The cytotoxicity of the amino alcohols observed after treatment for 24 hours was expressed as the concentration of compound needed to induce a 50% reduction in neutral red uptake (NI50). The observed NI50 values ranged from 3mM to 30mM. The individual stereoisomers and a racemic mixture of 1-amino-2-propanol exhibited similar cytotoxicities (with normally cultured Fa32 cells, and vitamin E- and BSO-treated cultures). Similar NI50 values for D-(+)-2-amino-1-propanol, 3-amino-1-propanol and the L-, D- or DL- forms of 1-amino-2-propanol, indicated that the position of the amino group had little influence on the cytotoxicities of the amino alcohols. In contrast, the position of the hydroxyl group appeared to play an important role for the toxicity of the compound, as indicated by the significantly different NI50 values for 4-amino-1-butanol and 4-amino-2-butanol. An additional phenyl group greatly increased the cytotoxicity of 2-amino-1,3-propanediol. For most of the compounds, cytotoxicity increased when GSH was depleted, and decreased when the cells were enriched with vitamin E. This indicated that most of the tested chemicals interact with GSH, either directly or indirectly, by processes which generate oxygen free-radicals. Decreased toxicity was found for most of the chemicals administered to vitamin E-enriched cells, indicating that reactive oxygen species could be involved in the toxicity of the amino alcohols.

Development of an in vitro test battery for the estimation of acute human systemic toxicity: An outline of the EDIT project. Evaluation-guided Development of New In Vitro Test Batteries.

The aim of the Evaluation-guided Development of new In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R(2) = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that the development within the EDIT target organ programme should initially be focused on the nervous system.

Cystathionine pathway-dependent cytotoxicities of diethyl maleate and diamide in rat and human hepatoma-derived cell cultures.

Glutathione (GSH) plays a role in many toxicologically important metabolic processes. It was previously established that L-buthionine S,R-sulphoximine (BSO), a specific inhibitor of (- glutamylcysteine synthetase, reduces the GSH content more efficiently in rat (Fa32) than in human (HEp-G2) hepatoma-derived cells. We therefore investigated whether the cystathionase inhibitor propargylglycine (PPG) could further decrease the BSO-induced GSH depletion in HEp-G2 cells. The influence of the cystathionine precursors N-acetylmethionine, methionine and homocysteine on the cytotoxicity of diethyl maleate (DEM) and diamide [1,1'-azobis(N,N-dimethylformamide)] was also investigated. PPG reduced the GSH content in both cell lines. A further GSH decrease in HEp-G2 was obtained when using a BSO + PPG combination containing relatively high concentrations of PPG. BSO diminished the toxicity of PPG. Homocysteine was the most efficacious of the tested cystathionine precursors in increasing the GSH content and reducing the cytotoxicity of DEM and diamide in Fa32 and HEp-G2 cells.

Neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells to predict human toxicity.

The cytotoxicity of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) reference chemicals was investigated by measuring the neutral red uptake inhibition in adhered and adhering rat hepatoma-derived Fa32 cells. The adhered cells were seeded and then treated and the adhering cells were treated simultaneously upon seeding. Five of the 44 test chemicals were twofold more toxic in adhering cells; ethylene glycol was 28-fold more toxic and mercuric chloride was 5.2-fold more toxic than in adhered cells. The cytotoxicity of dithiothreitol was altered in the same way as that of ethylene glycol, probably by interacting with calcium. When the neutral red uptake inhibition was compared with human toxicity, the correlation coefficient for adhering cells was almost identical to that obtained previously in human hepatoma-derived Hep G2 cells and slightly higher for adhered cells. The Hep G2 assay was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. An obviously better correlation was obtained when the strong intoxicant mercuric chloride was withdrawn from the comparison, both for the adhered and the adhering cells. Altogether, the results can be integrated very well with the basal cytotoxicity concept.