Identification of the Oxidative and Conjugative Enzymes Involved in the Biotransformation of Brivanib

Gong, Jiachang; Gan, Jinping; Iyer, Ramaswamy A.

doi:10.1124/dmd.111.042457

Cited by 12 publications

(15 citation statements)

References 40 publications

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“…In the absence of PAPS, almost no 3-GSH-NBP was observed in HLC or RLC. PAPS-dependence suggested the involvement of SULT (Gong et al, 2012;Yalcin et al, 2013).…”

Section: Resultsmentioning

confidence: 99%

Bioactivation of 3-n-Butylphthalide via Sulfation of Its Major Metabolite 3-Hydroxy-NBP: Mediated Mainly by Sulfotransferase 1A1

et al. 2014

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3-n-Butylphthalide (NBP) [(6)-3-butyl-1(3H)-isobenzofuranone] is an anti-cerebral-ischemia drug. Moderate hepatotoxicity has been observed in clinical applications. One of the major metabolites, 3-N-acetylcysteine-NBP, has been detected in human urine, indicating the formation of a reactive metabolite. We elucidated the formation mechanism of the reactive metabolite and its association with the hepatotoxicity of NBP. The in vitro incubations revealed that 3-glutathione-NBP (3-GSH-NBP) was observed only in fresh rat liver homogenate rather than in liver microsomes, liver cytosol, or liver 9,000g supernatant supplemented with NADPH and GSH. We also detected 3-GSH-NBP when 39-phosphoadenosine-59-phosphosulfate was added in GSH-fortified human liver cytosol (HLC). The formation of 3-GSH-NBP was 39.3-fold higher using 3-hydroxy-NBP (3-OH-NBP) as the substrate than NBP. The sulfotransferase (SULT) inhibitors DCNP (2,6-dichloro-4-nitrophenol) and quercetin suppressed 3-GSH-NBP formation in HLC by 75 and 82%, respectively, suggesting that 3-OH-NBP sulfation was involved in 3-GSH-NBP formation. Further SULT phenotyping revealed that SULT1A1 is the major isoform responsible for the sulfation. Dose-dependent toxicity was observed in primary rat hepatocytes exposed to 3-OH-NBP, with an IC 50 of approximately 168 mM. Addition of DCNP and quercetin significantly increased cell viability, whereas L-buthionine-sulfoximine (a GSH depleter) decreased cell viability. Overall, our study revealed the underlying mechanism for the bioactivation of NBP is as follows. NBP is first oxidized to 3-OH-NBP and further undergoes sulfation to form 3-OH-NBP sulfate. The sulfate spontaneously cleaves off, generating highly reactive electrophilic cations, which can bind either to GSH to detoxify or to hepatocellular proteins to cause undesirable side effects.

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“…In the absence of PAPS, almost no 3-GSH-NBP was observed in HLC or RLC. PAPS-dependence suggested the involvement of SULT (Gong et al, 2012;Yalcin et al, 2013).…”

Section: Resultsmentioning

confidence: 99%

Bioactivation of 3-n-Butylphthalide via Sulfation of Its Major Metabolite 3-Hydroxy-NBP: Mediated Mainly by Sulfotransferase 1A1

et al. 2014

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“…Incubations of brivanib and its enantiomer with cytosols were conducted following the procedures described previously (Gong et al, 2012). The kinetics were determined at nine substrate concentrations (1, 2, 5, 10, 20, 30, 60, 80, and 120 M).…”

Section: Downloaded Frommentioning

confidence: 99%

“…The incubation mixtures consisted of brivanib or its enantiomer (1-120 M), 3Ј-phosphoadenosine 5Ј-phosphosulfate (0.5 mM), MgCl 2 (0.5 mM), and cytosols from rat, monkey, and human (0.2 mg/ml protein) in phosphate buffer (100 mM, pH 7.4) with a final volume of 0.5 ml. After incubation, the concentrations of the sulfation metabolites were determined with a LC/MS/MS method reported previously (Gong et al, 2012).…”

Section: Downloaded Frommentioning

confidence: 99%

“…2), generated with an achiral LC/MS method, were qualitatively similar across species. The radioprofile of the incubation mixture with HLM has been described previously (Gong et al, 2012) and is also included here as a comparison. In addition to the hydroxy metabolite (M7) and the carboxylic acid metabolite (M26), a ketone metabolite (M31) was observed as a prominent metabolite in microsomal incubations in the presence of NADPH.…”

Section: Brivanib Chiral Inversion In Incubations Withmentioning

confidence: 99%

“…Sample analysis, conducted with achiral LC/MS methods, suggested that brivanib underwent both oxidative metabolism and sulfate conjugation. A ketone metabolite, which can be potentially reduced to brivanib and its enantiomer, was observed in incubations of brivanib with human liver microsomes (HLM) (Gong et al, 2012) and in rats, monkeys, and humans after oral doses of brivanib alaninate (Gong et al, 2011). This study describes the characterization of brivanib chiral inversion in incubations with liver subcellular fractions and its exposure in animals and humans after oral doses of brivanib alaninate.…”

Section: Introductionmentioning

confidence: 98%

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Metabolic Chiral Inversion of Brivanib and Its Relevance to Safety and Pharmacology

et al. 2012

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ABSTRACT:Brivanib alaninate is an orally administered alanine prodrug of brivanib, a dual inhibitor of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. It is currently in clinical trials for the treatment of hepatocellular carcinoma and colorectal cancer. Brivanib has a single asymmetric center derived from a secondary alcohol. The potential for chiral inversion was investigated in incubations with liver subcellular fractions and in animals and humans after oral doses of brivanib alaninate. Incubations of [ 14 C]brivanib alaninate with liver microsomes and cytosols from rats, monkeys, and humans followed by chiral chromatography resulted in two radioactive peaks, corresponding to brivanib and its enantiomer. The percentage of the enantiomeric metabolite relative to brivanib in microsomal and cytosolic incubations of different species in the presence of NADPH ranged from 11.6 to 15.8 and 0.8 to 3.1%, respectively. The proposed mechanism of inversion involves the oxidation of brivanib to a ketone metabolite, which is subsequently reduced to brivanib and its enantiomer. After oral doses of brivanib alaninate to rats and monkeys, the enantiomeric metabolite was a prominent drug-related component in plasma, with the percentages of area under the curve (AUC) at 94.7 and 39.7%, respectively, relative to brivanib. In humans, the enantiomeric metabolite was a minor circulating component, with the AUC <3% of brivanib. Pharmacological studies indicated that brivanib and its enantiomer had similar potency toward the inhibition of VEGF receptor-2 and FGF receptor-1 kinases. Because of low plasma concentration in humans, the enantiomeric metabolite was not expected to contribute significantly to target-related pharmacology of brivanib. Moreover, adequate exposure in the toxicology species suggested no specific safety concerns with respect to exposure to the enantiomeric metabolite.

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Identification and characterization of in vitro and in vivo metabolites of steroidal alkaloid veratramine

Lyu

Zhou

Zhang

et al. 2015

Biopharm & Drug Disp

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Veratramine, a steroidal alkaloid originating from Veratrum nigrum L., has demonstrated distinct anti-tumor and anti-hypertension effects, however, its metabolism has rarely been explored. The objective of the current study was to provide a comprehensive investigation of its metabolic pathways. The in vitro metabolic profiles of veratramine were evaluated by incubating it with liver microsomes and cytosols. The in vivo metabolic profiles in plasma, bile, urine and feces were monitored by UPLC-MS/MS after oral (20 mg/kg) and i.v. (50 µg/kg) administration in rats. Meanwhile, related P450s inhibitors and recombinant P450s and SULTs were used to identify the isozymes responsible for its metabolism. Eleven metabolites of veratramine, including seven hydroxylated, two sulfated and two glucuronidated metabolites, were characterized. Unlike most alkaloids, the major reactive sites of veratramine were on ring A and B instead of on the amine moiety. CYP2D6 was the major isozyme mediating hydroxylation, and substrate inhibition was observed with a Vmax , Ki and Clint of 2.05 ± 0.53 nmol/min/mg, 33.08 ± 10.13 µ m and 13.58 ± 1.27 µL/min/mg. SULT2A1, with Km , Vmax and Clint values of 19.37 ± 0.87 µ m, 1.51 ± 0.02 nmol/min/mg and 78.19 ± 8.57 µL/min/mg, was identified as the major isozyme contributing to its sulfation. In conclusion, CYP2D6 and SULT2A1 mediating hydroxylation and sulfation were identified as the major biotransformation for veratramine.

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Identification of the Oxidative and Conjugative Enzymes Involved in the Biotransformation of Brivanib

Cited by 12 publications

References 40 publications

Bioactivation of 3-n-Butylphthalide via Sulfation of Its Major Metabolite 3-Hydroxy-NBP: Mediated Mainly by Sulfotransferase 1A1

Bioactivation of 3-n-Butylphthalide via Sulfation of Its Major Metabolite 3-Hydroxy-NBP: Mediated Mainly by Sulfotransferase 1A1

Metabolic Chiral Inversion of Brivanib and Its Relevance to Safety and Pharmacology

Identification and characterization of in vitro and in vivo metabolites of steroidal alkaloid veratramine

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