Covey, Joseph M.

AbstractThe purpose of this study was to develop and validate a population pharmacokinetic model for Z‐endoxifen in patients with advanced solid tumors and to identify clinical variables that influence pharmacokinetic parameters. Z‐endoxifen‐HCl was administered orally once a day on a 28‐day cycle (±3 days) over 11 dose levels ranging from 20 to 360 mg. A total of 1256 Z‐endoxifen plasma concentration samples from 80 patients were analyzed using nonlinear mixed‐effects modeling to develop a population pharmacokinetic model for Z‐endoxifen. A 2‐compartment model with oral depot and linear elimination adequately described the data. The estimated apparent total clearance, apparent central volume of distribution, and apparent peripheral volume of distribution were 4.89 L/h, 323 L, and 39.7 L, respectively, with weight‐effect exponents of 0.75, 1, and 1, respectively. This model was used to explore the effects of clinical and demographic variables on Z‐endoxifen pharmacokinetics. Weight, race on clearance, and aspartate aminotransferase on the absorption rate constant were identified as significant covariates in the final model. This novel population pharmacokinetic model provides insight regarding factors that may affect the pharmacokinetics of Z‐endoxifen and may assist in the design of future clinical trials.

Abstract NSC 373981 is 1-ethanol, 2-nitro-naphtho[2,1-b]furan that has demonstrated activity in the NCI60 anticancer screen and in the mouse hollow-fiber assay. Growth inhibitory activity was also observed in several xenograft models. There is some evidence that antitumor activity is greater in vivo than would be predicted by the in vitro results, suggesting the possibility of activating metabolism. While the mechanism of anticancer activity is unknown, naphthofurans have been reported to modulate certain nuclear receptors. The purpose of this study was to characterize the plasma pharmacokinetics of NSC 373981 following i.v. and i.p. administration to athymic nude mice and to characterize potential metabolites of this compound in plasma, urine, and bile. We developed and validated suitable positive ionization LC/MS/MS methodology for measuring NSC 373981 that utilized liquid extraction with 1-chlorobutane and MS/MS detection with the 258>212 and 244>169 transitions, respectively, for NSC 373981 and NSC 329226, an analog that was used as the internal standard. Baseline separations were achieved on a Polaris C18 column under a mobile phase consisting of 70:30 methanol:water containing 0.1% formic acid. Standard curves were linear over the concentration range 2.5-1000 ng/mL with a lower limit of detection of 2.5 ng/mL. NSC 373981 was stable under acidic, neutral and basic conditions, as well as human and mouse plasma during 24-hr incubation at 37°C. NSC 373981 pharmacokinetics were characterized in male nu/nu mice after a 37.5 mg/kg i.v. dose or a 150 mg/kg i.p. dose. Plasma, bile, urine and feces were collected for 24-hr after drug administration. Following i.v. administration, the peak concentration was > 5.9 μg/mL and the terminal elimination half-life was 1.2 hours. Volume of distribution and plasma clearance values were 694 ml and 390 mL/hr, respectively. Following i.p. administration, NSC373981 reached a peak concentration of 2.1 μg/mL at 15 minutes and the terminal elimination half-life was 36.8 hours. The i.p. bioavailability of NSC373981 was 58%. In vivo metabolite profiles were characterized in plasma, urine, feces, and bile. We detected several Phase I metabolites in these samples including those produced by reduction of the aromatic nitro group, oxidation of the primary alcohol moiety on the reduced metabolite, oxidation of the primary alcohol moiety, benzylic hydroxylation, hydroxylation and dihydrodiol formation on the naphthalene moiety, and Phase II metabolites including glucuronidation of the carboxylic acids and a glutathione metabolite of hydroxylated NSC 373981. Supported by NCI contract N01-CM-2011-00014. Citation Format: Joel M. Reid, Sarah A. Buhrow, Renee M. McGovern, Joseph M. Covey, Matthew M. Ames. Pharmacokinetics and in vivo metabolism of the naphthofuran NSC373981 in male nu/nu mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2462. doi:10.1158/1538-7445.AM2013-2462

Abstract Batracylin (NSC 320846, BAT) is a heterocyclic arylamine and topoisomerase II inhibitor shown to be active in murine tumor models, but with demonstrated species differences in toxicity associated with metabolism to N-acetylbatracylin (NBAT) by N-acetyltransferase (NAT). BAT is a preferential substrate of human NAT2. Accordingly, production of NBAT should be subject to population variability as a function of NAT2 polymorphisms. A phase I clinical trial of BAT in patients with advanced cancer and slow acetylator NAT2 phenotypes to reduce the risk of NBAT-related toxicity found a dose-limiting toxicity (DLT) of cystitis/hemorrhagic cystitis. In subsequent in vitro and in vivo preclinical studies to elucidate metabolic pathways that might contribute to BAT toxicity, we found oxidative metabolites and thiol conjugates of BAT and NBAT (MM Ames et al, Cancer Res 2011;71(8 Suppl):Abstract nr 1303). Since these data are consistent with metabolic activation of BAT to potentially toxic metabolites, we assessed formation of reactive intermediates as measured by covalent binding (CB) to protein and DNA or by formation of glutathione conjugates. Radiolabeled BAT and NBAT were incubated with 3-MC-induced rat (r), human (h) and dog (d) liver microsomes (LM), as well as with recombinant human cytochromes P450 (CYPs) with or without added DNA. NADPH-dependent BAT protein and DNA CB were detected in all microsomal preparations (see table below). NADPH-dependent NBAT protein CB was detected in all microsomal preparations except hCYP1B1. NADPH-dependent NBAT DNA CB was detected in all microsomal preparations except 3MC-rLM. We also detected a glutathione conjugate of mono-hydroxy BAT by lc/ms/ms analysis when BAT was incubated with hLM in the presence of glutathione and NADPH. Thus, CYP-catalyzed oxidation of BAT yields reactive metabolites that that bind protein and DNA and may contribute to drug-related DLT. Supported in part by NCI Contract N01-CM-52206. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3769. doi:1538-7445.AM2012-3769

Abstract Batracylin (NSC 320846, BAT) is a heterocyclic arylamine and topoisomerase II inhibitor that was shown to be active in several murine tumor models. Pharmacology and toxicology studies demonstrated marked species differences in sensitivity to the toxic effects of BAT attributed to metabolism to N-acetylbatracylin (NacBAT) by N-acetyltransferase (NAT). It was subsequently shown that BAT is a preferential substrate for human NAT2; thus production of NacBAT is likely subject to population variability as a function of known NAT2 polymorphisms. A phase I clinical trial of BAT in patients with advanced cancer and slow acetylator NAT2 genotypes to reduce the risk of toxicity due to NacBAT is ongoing and a potentially dose-limiting toxicity of cystitis/hemorrhagic cystitis was observed in several patients. In order to determine if this toxicity is due to BAT, NacBAT, and/or other metabolites, we investigated the in vitro metabolism of BAT and NacBAT in human (h) and rat (r) liver microsomes (LM) and hepatocytes, and the in vivo metabolism of BAT and NacBAT in rats. 14C-BAT and differentially deuterated (d3- and d4-) BAT were provided to facilitate metabolic profiling. d3- and d4- NacBAT were generated in situ. BAT, NacBAT and their metabolites were detected and quantified by HPLC with diode array, fluorescence, radiochemical and mass spectrometry as appropriate. The goals of these investigations were to obtain a detailed metabolic profile for BAT in rats and humans and to elucidate pathways that might contribute to BAT toxicity. In NADPH-fortified microsomal preparations, loss of BAT was accompanied by formation of one hydroxylated metabolite in hLM and three hydroxylated metabolites in rLM. The single hydroxylated metabolite found in incubations of NacBAT with hLM was attributed to deacetylation by microsomal carboxlesterase. Six mono- and di- hydroxy NacBAT metabolites were found in incubations of NacBAT with rLM. Hydroxylation sites were identified with deuterated BAT and NacBAT. Incubation with recombinant cytochomes P450 (CYPs) identified rCYP1A1, rCYP1A2, hCYP1A1 and hCYP1B1 as the major CYP isoforms that metabolize BAT and NacBAT. Glucuronide conjugates of BAT and NacBAT were identified in rat and human hepatocyte incubations. NacBAT, hydroxylated BAT and NacBAT, and glucuronide conjugates were detected in in vivo metabolism and pharmacokinetic studies with BAT and NacBAT in rats. Finally, thiol-containing adducts, consistent with metabolic activation of BAT, were identified in kidney and urine samples from in vivo metabolism and pharmacokinetic studies with rats. In conclusion, we characterized the in vitro and in vivo metabolism of BAT and identified a pathway for metabolic activation of BAT to potential toxic metabolites. Supported by NCI Contract N01-CM-52206. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1303. doi:10.1158/1538-7445.AM2011-1303

Abstract Introduction Cytidine drugs, such as gemcitabine, undergo rapid, inactivating catabolism by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine drug metabolism. However, p.o. THU is at most 20% bioavailable (Beumer et al.), which limits its preclinical evaluation and clinical use. Therefore, the more lipophilic prodrug triacetyl THU (taTHU) was developed. We characterized THU pharmacokinetics after administration of taTHU to mice. Methods Mice were dosed 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters and p.o. bioavailability were calculated non-compartmentally and compartmentally. We assessed taTHU inhibition of metabolism of gemcitabine by recombinant CD. ResultsTHU parameterstaTHU i.v. 150 mg/kgtaTHU p.o. 150 mg/kgCmax (µg/mL)10611.8Tmax (min)560Half-life (min)235122AUC0-inf (mg/mL*min)7.553.36Vd/F (L/kg)4.495.26Cl/F (mL/min/kg)13.229.8F-THUi.v. (%)68.030.3% dose in 0-16 h urine405.6% dose in 0-24 h urine5512 THU, after 150 mg/kg taTHU i.v., had a 235 min terminal half-life and produced plasma THU concentrations >1 µg/mL, the concentration shown to inhibit CD, for 10 h. A multi-compartment model fit the data best. 150 mg/kg p.o. taTHU produced a concentration versus time profile with a plateau of approximately 10 µg/mL from 0.5-2 h, followed by a decline with a 122 min half-life. Approximately 68% of i.v. taTHU was converted to THU. Approximately 30% of p.o. taTHU reached the systemic circulation as THU. After i.v dosing, renal excretion accounted for 40-55% of the taTHU dose, 26-35% as THU and an additional 14-19% as acetylated forms of THU. After p.o. dosing, the renal excretion percentages were 6-12%, 5.2-11%, and 0.42-0.71%, respectively. taTHU did not inhibit recombinant CD. Conclusion The availability of THU after p.o. taTHU in mice is 30%, as compared to the 20% achieved after p.o. THU. Clinical studies are warranted to evaluate availability of THU from taTHU in humans. Support: N01-CM07106, N01-CM52202, P30-CA47904 from the NCI and P41-EB001978. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4549.