Rao N.V.S. Mamidi

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 [14C]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 [14C]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.

Effects of rifampin, cyclosporine A, and probenecid on the pharmacokinetic profile of canagliflozin, a sodium glucose co-transporter 2 inhibitor, in healthy participants.

Objective: Canagliflozin, a sodium-glucose co-transporter 2 inhibitor, approved for the treatment of type-2 diabetes mellitus (T2DM), is metabolized by uridine diphosphate-glucuronosyltransferases (UGT) 1A9 and UGT2B4, and is a substrate of P-glycoprotein (P-gp). Canagliflozin exposures may be affected by coadministration of drugs that induce (e.g., rifampin for UGT) or inhibit (e.g. probenecid for UGT; cyclosporine A for P-gp) these pathways. The primary objective of these three independent studies (single-center, open-label, fixed-sequence) was to evaluate the effects of rifampin (study 1), probenecid (study 2), and cyclosporine A (study 3) on the pharmacokinetics of canagliflozin in healthy participants. Methods: Participants received; in study 1: canagliflozin 300 mg (days 1 and 10), rifampin 600 mg (days 4 – 12); study 2: canagliflozin 300 mg (days 1 – 17), probenecid 500 mg twice daily (days 15 – 17); and study 3: canagliflozin 300 mg (days 1 – 8), cyclosporine A 400 mg (day 8). Pharmacokinetics were assessed at pre-specified intervals on days 1 and 10 (study 1); on days 14 and 17 (study 2), and on days 2 – 8 (study 3). Results: Rifampin decreased the maximum plasma canagliflozin concentration (Cmax) by 28% and its area under the curve (AUC) by 51%. Probenecid increased the Cmax by 13% and the AUC by 21%. Cyclosporine A increased the AUC by 23% but did not affect the Cmax. Conclusion: Coadministration of canagliflozin with rifampin, probenecid, and cyclosporine A was well-tolerated. No clinically meaningful interactions were observed for probenecid or cyclosporine A, while rifampin coadministration modestly reduced canagliflozin plasma concentrations and could necessitate an appropriate monitoring of glycemic control.

Carcinogenicity in rats of the SGLT2 inhibitor canagliflozin

The carcinogenicity potential of canagliflozin, an inhibitor of SGLT2, was evaluated in a 2-year rat study (10, 30, and 100 mg/kg). Rats showed an increase in pheochromocytomas, renal tubular tumors, and testicular Leydig cell tumors. Systemic exposure multiples at the highest dose relative to the maximum clinical dose were 12- to 21-fold. Pheochromocytomas and renal tubular tumors were noted in both sexes at 100 mg/kg. Leydig cell tumors were observed in males in all dose groups and were associated with increased luteinizing hormone levels. Hyperplasia was increased in the adrenal medulla at 100 mg/kg, but only a limited increase in simple tubular hyperplasia was observed in the kidney of males at 100 mg/kg. Hyperostosis occurred and was accompanied by substantial effects on calcium metabolism, including increased urinary calcium excretion and decreased levels of calcium regulating hormones (1,25-dihydroxyvitamin D and parathyroid hormone). A separate study with radiolabeled calcium confirmed that increased urinary calcium excretion was mediated via increased calcium absorption from the gastrointestinal tract. It was hypothesized that, at high doses, canagliflozin might have inhibited glucose absorption in the intestine via SGLT1 inhibition that resulted in glucose malabsorption, which increased calcium absorption by stimulating colonic glucose fermentation and reducing intestinal pH. Pheochromocytomas and adrenal medullary hyperplasia were attributed to altered calcium homeostasis, which have a known relationship in the rat. In conclusion, Leydig cell tumors were associated with increased luteinizing hormone levels and pheochromocytomas were most likely related to glucose malabsorption and altered calcium homeostasis. Renal tubular tumors may also have been linked to glucose malabsorption.

LC-ESI-MS/MS quantification of 4β-hydroxycholesterol and cholesterol in plasma samples of limited volume.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay was developed and qualified for analyzing 4β-hydroxycholesterol and cholesterol in 5 μl of human and mouse plasma. Stable isotope-labeled d7-analogs of both analytes were used as internal standards and 4.2% (w/v) human serum albumin in phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and QCs. The assay is capable of quantification of 4β-hydroxycholesterol and cholesterol from 5 to 500 ng/ml and 50 to 2000 μg/ml, respectively, with acceptable accuracy and precision following evaluation of recovery of analytes, autosampler stability and potential contribution of chemical oxidation to the formation of 4β-hydroxycholesterol. The final reconstituted solution was diluted for quantification of cholesterol typically present at 1000 fold higher concentration than 4β-hydroxycholesterol in the same samples used for 4β-hydroxycholesterol quantification. The successful quantification using a low plasma volume was achieved by quantification of total forms (free and conjugated) of both analytes after alkaline hydrolysis, followed by derivatization to form electrospray ionization-sensitive picolinyl esters, which upon collision-induced dissociation gave high mass precursor-product ion pair for selective detection by multiple reaction monitoring. In addition, chromatographic separation using a 16-min reversed phase gradient elution on a 1.9 μm particle size, C18 column, overcame interference from other isobaric plasma oxysterols during detection by multiple-reaction monitoring. This assay was compared to an orthogonal enzymatic assay for cholesterol and all samples, but one, provided values that were within 10% of each other. In addition, this assay passed the incurred sample tests for both analytes in human and mouse plasma samples according to reported acceptance criteria for incurred sample reanalysis. The quantification of both analytes permitted the determination of 4β-hydroxycholesterol compared to its ratio to cholesterol as an endogenous biomarker for CYP3A4/5 activity. The LC-ESI-MS/MS assay was also successfully applied to quantification of 4β-hydroxycholesterol and cholesterol in plasma samples from untreated human and mice including FRG™ KO C57Bl/6 chimeric mice with humanized livers. The preliminary data indicated that the plasma 4β-hydroxycholesterol concentrations or their ratio to cholesterol from mice including chimeric mice were higher than those from human.

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 [14C]‐canagliflozin microdose infusion in nine healthy men. Pharmacokinetics of canagliflozin, [14C]‐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 [14C]‐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 [14C]‐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 [14C]‐canagliflozin and metabolites accounted for about 57% and 43% of the plasma total [14C] radioactivity AUC∞, respectively. For total [14C] 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 [14C] radioactivity. The absolute oral bioavailability of canagliflozin was 65% (90% confidence interval: 55.41; 76.07). Overall, oral canagliflozin 300 mg coadministered with intravenous [14C]‐canagliflozin (10 µg) was generally well‐tolerated in healthy men, with no treatment‐emergent adverse events.

Carbohydrate malabsorption mechanism for tumor formation in rats treated with the SGLT2 inhibitor canagliflozin

Canagliflozin is an SGLT2 inhibitor used for the treatment of type 2 diabetes mellitus. Studies were conducted to investigate the mechanism responsible for renal tubular tumors and pheochromocytomas observed at the high dose in a 2-year carcinogenicity study in rats. At the high dose (100mg/kg) in rats, canagliflozin caused carbohydrate malabsorption evidenced by inhibition of intestinal glucose uptake, decreased intestinal pH and increased urinary calcium excretion. In a 6-month mechanistic study utilization of a glucose-free diet prevented carbohydrate malabsorption and its sequelae, including increased calcium absorption and urinary calcium excretion, and hyperostosis. Cell proliferation in the kidney and adrenal medulla was increased in rats maintained on standard diet and administered canagliflozin (100mg/kg), and in addition an increase in the renal injury biomarker KIM-1 was observed. Increased cell proliferation is considered as a proximal event in carcinogenesis. Effects on cell proliferation, KIM-1 and calcium excretion were inhibited in rats maintained on the glucose-free diet, indicating they are secondary to carbohydrate malabsorption and are not direct effects of canagliflozin.

De-Risking Bio-therapeutics for Possible Drug Interactions Using Cryopreserved Human Hepatocytes

Inflammatory diseases such as rheumatoid arthritis and psoriasis are characterized by increases in circulating cytokines, which play an important role in modulation of the disease state. Several marketed bio-therapeutics target cytokines and act as effective treatment strategies. Previous in-vitro and in-vivo studies have suggested that cytokines may have both direct and indirect effects on drug metabolizing enzyme levels in the liver. Few studies have characterized models to evaluate the risk of potential drug interactions that might be mediated by changes in cytokine levels. In the present studies the potential of three cytokines (IL-2, IL-6 and TNF-α) to modulate gene expression and activity of the major human cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4) in cryopreserved human hepatocytes (CHH) was investigated. Significant decreases in the activity of all 6 CYP isoforms occurred in hepatocytes incubated with TNF-α or IL-6 (17-85%; and 22-76% of untreated control values, respectively). TNF-α down-regulated the gene expression of CYP1A2, 2D6 and 3A4 only, whereas IL-6 down-regulated gene expression of all of the tested CYP isoforms except 2D6. IL-2 had only mild effects on CYP activity and mRNA levels of examined isoforms. In CHH exposed to TNF-α, changes in CYP activity were not always paralleled by gene expression alterations for three of the examined CYP isoforms. These studies highlight several potential pitfalls in using isolated human hepatocytes for determination of drug interactions by bio-therapeutics including lack of correlation of mRNA and activity measurements for some CYP isoforms when using single time point determinations, and appropriateness of the model for indirect acting cytokine and cytokine modulators.

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.

Effect of canagliflozin on the pharmacokinetics of glyburide, metformin, and simvastatin in healthy participants

Drug–drug interactions between canagliflozin, a sodium glucose co‐transporter 2 inhibitor, and glyburide, metformin, and simvastatin were evaluated in three phase‐1 studies in healthy participants. In these open‐label, fixed sequence studies, participants received: Study 1‐glyburide 1.25 mg/day (Day 1), canagliflozin 200 mg/day (Days 4–8), canagliflozin with glyburide (Day 9); Study 2‐metformin 2,000 mg/day (Day 1), canagliflozin 300 mg/day (Days 4–7), metformin with canagliflozin (Day 8); Study 3‐simvastatin 40 mg/day (Day 1), canagliflozin 300 mg/day (Days 2–6), simvastatin with canagliflozin (Day 7). Pharmacokinetic parameters were assessed at prespecified intervals. Co‐administration of canagliflozin and glyburide did not affect the overall exposure (maximum plasma concentration [Cmax] and area under the plasma concentration–time curve [AUC]) of glyburide and its metabolites (4‐trans‐hydroxy‐glyburide and 3‐cis‐hydroxy‐glyburide). Canagliflozin did not affect the peak concentration of metformin; however, AUC increased by 20%. Though Cmax and AUC were slightly increased for simvastatin (9% and 12%) and simvastatin acid (26% and 18%) following coadministration with canagliflozin, compared with simvastatin administration alone; however, no effect on active 3‐hydroxy‐3‐methyl‐glutaryl‐CoA (HMG‐CoA) reductase inhibitory activity was observed. There were no serious adverse events or hypoglycemic episodes. No drug–drug interactions were observed between canagliflozin and glyburide, metformin, or simvastatin. All treatments were well‐tolerated in healthy participants.

Successful integration of nonclinical and clinical findings in interpreting the clinical relevance of rodent neoplasia with a new chemical entity.

Canagliflozin, a sodium glucose co-transporter 2 (SGLT2) inhibitor, has been developed for the treatment of adults with type 2 diabetes mellitus (T2DM). During the phase 3 program, treatment-related pheochromocytomas, renal tubular tumors, and testicular Leydig cell tumors were reported in the 2-year rat toxicology study. Treatment-related tumors were not seen in the 2-year mouse study. A cross-functional, mechanism-based approach was undertaken to determine whether the mechanisms responsible for tumorigenesis in the rat were of relevance to humans. Based on findings from nonclinical and clinical studies, the treatment-related tumors observed in rats were not deemed to be of clinical relevance. Here, we describe the scientific and regulatory journey from learning of the 2-year rat study findings to the approval of canagliflozin for the treatment of T2DM.