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Identification and profiling of GDC-0810 metabolites from admistration of a single oral dose of 14C GDC-0810 to intact and bile duct cannulated (BDC) rats
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Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
Co., Ltd., Ibaraki, Japan; 3 Central Institute for Experimental Animals, Kawasaki, Japan; 4 Pfizer Inc., Cambridge, MA, USA; 5 Showa Pharmaceutical University, Machida, Japan When metabolites formed uniquely or disproportionately higher in humans than in experimental animals are found at a certain level in the circulating plasma, safety of these metabolites is requested to be demonstrated before entering the large-scale clinical study (the Metabolites in Safety Testing (MIST) and International Conference on Harmonization M3(R2) guidance). A partial glucokinase activator, N,N-dimethyl-5-((2methyl-6-((5-methylpyrazin-2-yl)carbamoyl) benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319), reportedly generated 16 metabolites in incubation mixtures with rat, dog and human hepatocytes, and 9 metabolites of them were produced by human hepatocytes. However, an N-demethylated metabolite (M1) accounted for 50 to 65% of total drugrelated exposure in the phase I study, which necessitated several activities to meet the MIST guidance. In the present study, we gave PF-04937319 orally to chimeric mice with humanized livers and found that plasma concentration of M1 was comparable to the unchanged drug while that in a control mouse was relatively low. Then pharmacokinetic analyses were performed after intravenous administration of PF-04937319 and M1 to the same animal individuals. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, pharmacokinetic parameters including metabolic clearance normalized by liver weight were plotted against the replacement index by human hepatocytes and extrapolated to those in the virtual chimeric mouse with 100% humanized liver. Simultaneously, conversion ratios from PF-04937319 to M1 in mouse and human hepatocytes in vivo were estimated by analyses of the PK data and also by in vitro experiments using mouse and human liver microsomes. Results of these experiments suggested that higher plasma level of M1 in chimeric mice was attributed to higher formation ratio of M1 in human hepatocytes and increased liver volume, but not to prolonged elimination processes of the metabolite. On the other hand, this plotting method was deemed to be applicable to estimate liver to plasma concentration ratio in humans which was practically almost impossible to measure. Moreover, when the plasma clearance value of PF-04937319 in 100% chimeric mice was multiplied by liver to body weight ratio in humans, this value was found to be comparable to that in humans in a phase I studies if oral bioavailability could be assumed as 50%. In addition, administration studies of PF-04937319 and M1 to other humanized chimeric mice revealed that a minor human-specific metabolite (~4% of total drug-related exposure), assumed to be a carbinolamide derivative of M1, was produced from PF-04937319 taking M1 as an intermediate metabolite. P175 HUMAN PLASMA CONCENTRATION-TIME PROFILES OF TROGLITAZONE AND TROGLITAZONE SULFATE ESTIMATED BY IN VIVO EXPERIMENTS WITH CHIMERIC MICE WTH HUMANIZED LIVERS Yosuke Yamamoto 1, Satoshi Ito 2, Hiroyuki Chijiwa 1, Takeshi Okuzono 1, Tomohiro Ishiguro 1, Miyuki Kuronuma 3, Hiroshi Suemizu 3, Hidetaka Kamimura 2, Shin-ichi Ninomiya 1, Hiroshi Yamazaki 4. 1 Sekisui Medical Co., Ltd., Ibaraki, Japan; 2 Sekisui Medical Co., Ltd., Tokyo, Japan; 3 Central Institute for Experimental Animals, Kawasaki, Japan; 4 Showa Pharmaceutical University, Machida, Japan For the prediction of human pharmacokinetics (PK), various approaches such as allometric scaling, in vitro-in vivo extrapolation and PBPK modeling have been employed. Prediction of human PK of troglitazone after p.o. administration by allometric scaling was reported1). This approach includes p.o. and i.v. administration of troglitazone to mice, rats, dogs and monkeys, and fairly good description of plasma concentration-time profiles of the unchanged drug in a clinical study was observed. Chimeric mice with humanized livers are expected as a tool for predicting human PK. Single-species allometric scaling demonstrated favorable agreement between plasma clearances (CLp) predicted from chimeric mice and observed values in humans2). However, the predicted CLp value of diazepam was 17.5 times greater than that observed in humans, which was considered due to remnant murine metabolism. Moreover these studies were dealt with the unchanged drugs only, predictions of their metabolites
were sometimes important in relation to the metabolite in safety testing (MIST) guidance. In the present study, troglitazone and its major metabolite, troglitazone sulfate, were administrated intravenously to chimeric mice with humanized livers. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, CLp normalized by liver weight was plotted against the replacement index by human hepatocytes, and extrapolated to that in the virtual chimeric mouse with 100% humanized liver. As the results CLp of troglitazone in 100% chimeric mice was about one third of that in control mice while CLp of troglitazone sulfate was very low and no difference between control and chimeric mice was observed. When CLp of troglitazone in 100% chimeric mice expressed as mL/hr/g liver was extrapolated to humans by multiplying it by the liver to body weight ratio in humans, the calculated CLp value was consistent with CLp predicted by Izumi et al.1). Moreover, this plotting method was applicable to estimate liver to plasma concentration ratio (Kp,h) in humans which was practically almost impossible to measure. Kp,h of troglitazone sulfate was ~20 and 4 in control and 100% chimeric mice, respectively. However, there was no differences in Kp,h of troglitazone between these animals. 1. Izumi et al.: Prediction of the human pharmacokinetics of troglitazone, a new and extensively metabolized antidiabetic agent, after oral administration, with an animal scale-up approach. J Pharmacol Exp Ther 277:16301641, 1996. 2. Sanoh et al.: Predictability of plasma concentration-time curves in humans using single-species allometric scaling of chimeric mice with humanized liver. Xenobiotica 45:605-614, 2015. P176 IDENTIFICATION AND PROFILING OF GDC-0810 METABOLITES FROM ADMISTRATION OF A SINGLE ORAL DOSE OF 14C GDC-0810 TO INTACT AND BILE DUCT CANNULATED (BDC) RATS Chenghong Zhang, Donglu Zhang, Edna F. Choo, Kevin DeMent, Scott Savage, S. Cyrus Khojasteh. Drug Metabolism and Pharmacokinetics, Genentech, Inc., South San Francisco, CA, USA GDC-0810 (also known as ARN-810 and RO7056118) is a novel, selective estrogen receptor-alpha (ERa) antagonist and inducer of ERa degradation that is being developed as an oral anti-cancer drug for use as treatment for estrogen receptor-positive (ER+) breast cancers. The objectives of this study were to understand the route of excretion and identify circulating metabolites of GDC-0810 in female rat following single oral administration of 75 mg/kg 14C-GDC-0810 (approximately 400 mCi/kg). Total dose recovery was 89.2% and 56.9% from intact and BDC animals, respectively. Samples (plasma, bile, urine and feces) were prepared by either centrifugation or organic extraction and injected on to LC-MS for analysis with fraction collecting for radioactive measurement. The extraction recovery is >90%. In plasma from intact animals, unchanged drug was the most abundant component and accounted for an average of 93.7% of the sample radioactivity for a 0-24 hour AUC pooled sample. The only circulating metabolite was an acyl-glucuronide by radioactivity, which accounted for an average of 4.9% of the sample radioactivity in plasma. Little acyl-glucuronide migration was detected. Urine contained low levels of radioactivity (< 0.1% dose), therefore no LC radio-chromatographic profiles of rat urine were acquired. In feces, unchanged drug was the most abundant component and accounted for 71.8% and 56.4% of the administered dose from intact and BDC rat, respectively. There were no metabolites detected by radioactivity in BDC rat feces, but in intact rat feces, oxidation and glucuronidation metabolites accounted for 4.79% and 10.5% of the administered dose, respectively. In bile, unchanged drug was a very minor component and accounted for 0.2% of the administered dose. The most abundant metabolite in bile was an acyl-glucuronide, which accounted for 10.2% of the administered dose. Several minor mono-oxidative glucuronides (sum 2.4% of the dose) and one monooxidative metabolite (0.3% of the dose) were also detected. We were trying to use retention time to predict N or O- glucuronides verse acylglucuronides. In summary, unchanged drug was the major circulating component. The elimination of GDC-0810 was through bile and feces. Glucuronidation was the metabolic pathway that is responsible for clearance in female rat.
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
Co., Ltd., Ibaraki, Japan; 3 Central Institute for Experimental Animals, Kawasaki, Japan; 4 Pfizer Inc., Cambridge, MA, USA; 5 Showa Pharmaceutical University, Machida, Japan When metabolites formed uniquely or disproportionately higher in humans than in experimental animals are found at a certain level in the circulating plasma, safety of these metabolites is requested to be demonstrated before entering the large-scale clinical study (the Metabolites in Safety Testing (MIST) and International Conference on Harmonization M3(R2) guidance). A partial glucokinase activator, N,N-dimethyl-5-((2methyl-6-((5-methylpyrazin-2-yl)carbamoyl) benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319), reportedly generated 16 metabolites in incubation mixtures with rat, dog and human hepatocytes, and 9 metabolites of them were produced by human hepatocytes. However, an N-demethylated metabolite (M1) accounted for 50 to 65% of total drugrelated exposure in the phase I study, which necessitated several activities to meet the MIST guidance. In the present study, we gave PF-04937319 orally to chimeric mice with humanized livers and found that plasma concentration of M1 was comparable to the unchanged drug while that in a control mouse was relatively low. Then pharmacokinetic analyses were performed after intravenous administration of PF-04937319 and M1 to the same animal individuals. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, pharmacokinetic parameters including metabolic clearance normalized by liver weight were plotted against the replacement index by human hepatocytes and extrapolated to those in the virtual chimeric mouse with 100% humanized liver. Simultaneously, conversion ratios from PF-04937319 to M1 in mouse and human hepatocytes in vivo were estimated by analyses of the PK data and also by in vitro experiments using mouse and human liver microsomes. Results of these experiments suggested that higher plasma level of M1 in chimeric mice was attributed to higher formation ratio of M1 in human hepatocytes and increased liver volume, but not to prolonged elimination processes of the metabolite. On the other hand, this plotting method was deemed to be applicable to estimate liver to plasma concentration ratio in humans which was practically almost impossible to measure. Moreover, when the plasma clearance value of PF-04937319 in 100% chimeric mice was multiplied by liver to body weight ratio in humans, this value was found to be comparable to that in humans in a phase I studies if oral bioavailability could be assumed as 50%. In addition, administration studies of PF-04937319 and M1 to other humanized chimeric mice revealed that a minor human-specific metabolite (~4% of total drug-related exposure), assumed to be a carbinolamide derivative of M1, was produced from PF-04937319 taking M1 as an intermediate metabolite. P175 HUMAN PLASMA CONCENTRATION-TIME PROFILES OF TROGLITAZONE AND TROGLITAZONE SULFATE ESTIMATED BY IN VIVO EXPERIMENTS WITH CHIMERIC MICE WTH HUMANIZED LIVERS Yosuke Yamamoto 1, Satoshi Ito 2, Hiroyuki Chijiwa 1, Takeshi Okuzono 1, Tomohiro Ishiguro 1, Miyuki Kuronuma 3, Hiroshi Suemizu 3, Hidetaka Kamimura 2, Shin-ichi Ninomiya 1, Hiroshi Yamazaki 4. 1 Sekisui Medical Co., Ltd., Ibaraki, Japan; 2 Sekisui Medical Co., Ltd., Tokyo, Japan; 3 Central Institute for Experimental Animals, Kawasaki, Japan; 4 Showa Pharmaceutical University, Machida, Japan For the prediction of human pharmacokinetics (PK), various approaches such as allometric scaling, in vitro-in vivo extrapolation and PBPK modeling have been employed. Prediction of human PK of troglitazone after p.o. administration by allometric scaling was reported1). This approach includes p.o. and i.v. administration of troglitazone to mice, rats, dogs and monkeys, and fairly good description of plasma concentration-time profiles of the unchanged drug in a clinical study was observed. Chimeric mice with humanized livers are expected as a tool for predicting human PK. Single-species allometric scaling demonstrated favorable agreement between plasma clearances (CLp) predicted from chimeric mice and observed values in humans2). However, the predicted CLp value of diazepam was 17.5 times greater than that observed in humans, which was considered due to remnant murine metabolism. Moreover these studies were dealt with the unchanged drugs only, predictions of their metabolites
were sometimes important in relation to the metabolite in safety testing (MIST) guidance. In the present study, troglitazone and its major metabolite, troglitazone sulfate, were administrated intravenously to chimeric mice with humanized livers. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, CLp normalized by liver weight was plotted against the replacement index by human hepatocytes, and extrapolated to that in the virtual chimeric mouse with 100% humanized liver. As the results CLp of troglitazone in 100% chimeric mice was about one third of that in control mice while CLp of troglitazone sulfate was very low and no difference between control and chimeric mice was observed. When CLp of troglitazone in 100% chimeric mice expressed as mL/hr/g liver was extrapolated to humans by multiplying it by the liver to body weight ratio in humans, the calculated CLp value was consistent with CLp predicted by Izumi et al.1). Moreover, this plotting method was applicable to estimate liver to plasma concentration ratio (Kp,h) in humans which was practically almost impossible to measure. Kp,h of troglitazone sulfate was ~20 and 4 in control and 100% chimeric mice, respectively. However, there was no differences in Kp,h of troglitazone between these animals. 1. Izumi et al.: Prediction of the human pharmacokinetics of troglitazone, a new and extensively metabolized antidiabetic agent, after oral administration, with an animal scale-up approach. J Pharmacol Exp Ther 277:16301641, 1996. 2. Sanoh et al.: Predictability of plasma concentration-time curves in humans using single-species allometric scaling of chimeric mice with humanized liver. Xenobiotica 45:605-614, 2015. P176 IDENTIFICATION AND PROFILING OF GDC-0810 METABOLITES FROM ADMISTRATION OF A SINGLE ORAL DOSE OF 14C GDC-0810 TO INTACT AND BILE DUCT CANNULATED (BDC) RATS Chenghong Zhang, Donglu Zhang, Edna F. Choo, Kevin DeMent, Scott Savage, S. Cyrus Khojasteh. Drug Metabolism and Pharmacokinetics, Genentech, Inc., South San Francisco, CA, USA GDC-0810 (also known as ARN-810 and RO7056118) is a novel, selective estrogen receptor-alpha (ERa) antagonist and inducer of ERa degradation that is being developed as an oral anti-cancer drug for use as treatment for estrogen receptor-positive (ER+) breast cancers. The objectives of this study were to understand the route of excretion and identify circulating metabolites of GDC-0810 in female rat following single oral administration of 75 mg/kg 14C-GDC-0810 (approximately 400 mCi/kg). Total dose recovery was 89.2% and 56.9% from intact and BDC animals, respectively. Samples (plasma, bile, urine and feces) were prepared by either centrifugation or organic extraction and injected on to LC-MS for analysis with fraction collecting for radioactive measurement. The extraction recovery is >90%. In plasma from intact animals, unchanged drug was the most abundant component and accounted for an average of 93.7% of the sample radioactivity for a 0-24 hour AUC pooled sample. The only circulating metabolite was an acyl-glucuronide by radioactivity, which accounted for an average of 4.9% of the sample radioactivity in plasma. Little acyl-glucuronide migration was detected. Urine contained low levels of radioactivity (< 0.1% dose), therefore no LC radio-chromatographic profiles of rat urine were acquired. In feces, unchanged drug was the most abundant component and accounted for 71.8% and 56.4% of the administered dose from intact and BDC rat, respectively. There were no metabolites detected by radioactivity in BDC rat feces, but in intact rat feces, oxidation and glucuronidation metabolites accounted for 4.79% and 10.5% of the administered dose, respectively. In bile, unchanged drug was a very minor component and accounted for 0.2% of the administered dose. The most abundant metabolite in bile was an acyl-glucuronide, which accounted for 10.2% of the administered dose. Several minor mono-oxidative glucuronides (sum 2.4% of the dose) and one monooxidative metabolite (0.3% of the dose) were also detected. We were trying to use retention time to predict N or O- glucuronides verse acylglucuronides. In summary, unchanged drug was the major circulating component. The elimination of GDC-0810 was through bile and feces. Glucuronidation was the metabolic pathway that is responsible for clearance in female rat.