Identification of the Human Enzymes Involved in the Oxidative Metabolism of Dasatinib: An Effective Approach for Determining Metabolite Formation Kinetics

Wang, Lifei; Christopher, Lisa J.; Cui, Donghui; Li, Wenying; Iyer, Ramaswamy A.; Humphreys, W. Griffith; Zhang, Donglu

doi:10.1124/dmd.107.020255

Cited by 67 publications

(42 citation statements)

References 17 publications

(21 reference statements)

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“…dmd.aspetjournals.org multiple metabolites through HPLC separation, fraction collection, and radioactivity counting, analyzing the large number of samples with this method from a detailed kinetic experiment is a very timeconsuming process (Zhao et al, 2009). Therefore, the formation kinetics (K m values) of M13 and M23 were determined by an LC/ MS/SRM method to analyze the relative metabolite formation using a nonlabeled substrate as described recently by Wang et al (2008). The V max values for formation of these metabolites were calculated from the predetermined K m values and the reaction rates from a limited number of incubations with the radiolabeled material.…”

Section: Discussionmentioning

confidence: 99%

CYP3A4-Mediated Ester Cleavage as the Major Metabolic Pathway of the Oral Taxane 3′-tert-Butyl-3′-N-tert-butyloxycarbonyl-4-deacetyl-3′-dephenyl-3′-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183)

Zhang

Ly²,

Lago³

et al. 2009

Drug Metab Dispos

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3-tert-Butyl-3-N-tert-butyloxycarbonyl-4-deacetyl-3-dephenyl-3-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183)is an orally available taxane analog that has the potential to be used as an oral agent to treat cancers. The compound is similar to the two clinically intravenously administered taxanes, paclitaxel and docetaxel, in that it contains a baccatin ring linked to a side chain through an ester bond. Unlike the other taxanes, the hydrolysis of this ester bond leads to formation of a free baccatin core (M13) that was the major metabolism pathway in incubations of Paclitaxel (Taxol, Bristol-Myers Squibb Co., Wallingford, CT), initially isolated in 1967 from the bark of the Pacific yew tree, Taxus brevifolia, showed an antitumor activity for a broad range of rodent tumors (Wall and Wani, 1995). Paclitaxel has become a widely used and effective chemotherapy agent to treat patients with lung, ovarian, and breast cancer and an advanced form of Kaposi's sarcoma (Saville et al., 1995;Rose et al., 2001). Together with docetaxel, paclitaxel forms the drug category of the taxanes. Taxanes, as well as recently marketed epothilone classes (Goel et al., 2008), stop cell division by inhibition of the microtubule function through stabilizing GDP-bound tubulin in the microtubule. Paclitaxel and docetaxel are administered to patients intravenously because of their poor oral bioavailability. Oral treatment with this class of chemotherapy agents would increase convenience to patients, reduce cost of administration, and allow the use of chronic treatment regimens.BMS-275183 was synthesized as a part of a program to identify taxanes with potential for oral use in the treatment of cancers (Bröker et al., 2007). The compound is a C-4 methyl carbonate analog of paclitaxel that contains additional modifications in the side chain where the two phenyl groups of paclitaxel were replaced by t-butyl groups (Bröker et al., 2007). BMS-275183 exhibited good oral bioavailability in both rat and dog and showed antitumor activity comparable with intravenous paclitaxel (Frapolli et al., 2006).[ 14 C]BMS-275183 was metabolized to oxidative metabolites in liver microsomes of animals and humans and in bile duct-cannulated rats Kim-Kang et al., 2002). Only trace amounts of conjugated metabolites were detected in rat bile. The major in vitro metabolites were identified as M13 (hydrolysis metabolite), M20 and M20B (a carbamate metabolite resulting from hydroxylation of the oxycarbonyl t-butyl group followed by cyclization or a lactol metabolite resulting from hydroxylation of the C3Ј t-butyl groups followed by cyclization), M21 (␥-lactone metabolite), M22 (a carboxylic acid metabolite), and M23 (an oxycarbonyl t-butyl hydroxylated metabolite). In contrast to 6␣-hydroxylation of the baccatin ring of paclitaxel, the major metabolic pathways in BMS-275183 mainly occurred by oxidation and hydrolytic cleavage of the side chain.Article, publication date, and citation information can be found at

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Section: Discussionmentioning

confidence: 99%

CYP3A4-Mediated Ester Cleavage as the Major Metabolic Pathway of the Oral Taxane 3′-tert-Butyl-3′-N-tert-butyloxycarbonyl-4-deacetyl-3′-dephenyl-3′-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183)

Zhang

Ly²,

Lago³

et al. 2009

Drug Metab Dispos

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“…In recent years, numerous laboratories have reported the use of high-throughput bioanalytical procedures for the quantification of antileukemia drugs [5,6], a small number of analytical methods have been reported for dasatinib based on high-performance thin-layer chromatography (HPTLC) or high-performance liquid chromatography (HPLC) or radioactive labeling methods [7][8][9] and a liquid chromatography-mass spectrometry (LC-MS) method for plasma determination of this agent [10][11][12][13]. As with most approaches used to quantify pharmaceuticals, analytical methods for measuring tyrosine kinase inhibitor levels have focused on each agent in isolation and generally have been applied to pharmaceutical analysis or plasma determination of the agent.…”

Section: Dasatinib (Bms-354825) N-(2-chloro-6-methylphenyl)-2-[[6-[4mentioning

confidence: 99%

Gradient elution LC-MS determination of dasatinib in rat plasma and its pharmacokinetic study

Wen¹,

Zhang²,

He³

et al. 2015

Acta Chromatographica

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Summary.A sensitive and simple liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for determination of dasatinib in rat plasma using one-step protein precipitation was developed. After addition of carbamazepine as internal standard (IS), protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on an SB-C18 (2.1 mm × 150 mm, 5 μm) column with methanol-0.1% formic acid as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used to quantification using target fragment ions m/z 488.2 for dasatinib and m/z 338.7 for the IS. Calibration plots were linear over the range of 10-1000 ng mL −1 for dasatinib in rat plasma. Lower limit of quantification (LLOQ) for dasatinib was 10 ng mL −1 . Mean recovery of dasatinib from plasma was in the range 82.2%-93.6%. Relative standard deviation (RSD) of intra-day and inter-day precision were both less than 8%. This developed method is successfully used in pharmacokinetic study of dasatinib in rats.

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“…Flavin-containing monooxygenase can catalyze the formation of an N-oxide on the piperazine ring, and an unidentified cytosolic oxidoreductase converts the hydroxyethyl group to an acid. Additional secondary metabolites were detected as phase II metabolites due to sulfation or glucuronidation (Christopher et al, 2008a,b;Kamath et al, 2008;Wang et al, 2008).…”

mentioning

confidence: 99%

Characterization of Dasatinib and Its Structural Analogs as CYP3A4 Mechanism-Based Inactivators and the Proposed Bioactivation Pathways

He²,

Ruiz³

et al. 2009

Drug Metab Dispos

107

105

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ABSTRACT:Dasatinib was approved in 2006 for the treatment of imatinibresistant chronic myelogenous leukemia and functions primarily through the inhibition of BCR-ABL and Src kinase. Dasatinib is extensively metabolized in humans by CYP3A4. In this study, we report that the bioactivation of dasatinib by CYP3A4 proceeds through a reactive intermediate that leads to CYP3A4 inactivation with K I ‫؍‬ 6.3 M and k inact ‫؍‬ 0.034 min ؊1. The major mechanism of inactivation proceeds through hydroxylation at the para-position of the 2-chloro-6-methylphenyl ring followed by further oxidation, forming a reactive quinone-imine, similar to the reactive intermediates formed by acetaminophen and diclofenac. Formation of a reactive imine-methide was also detected but appears to be a minor pathway. When glutathione was added to human liver microsomal incubations, dasatinib-glutathione adducts were detected. Numerous dasatinib analogs were synthesized in an effort to understand what modifications would block the formation of reactive intermediates during dasatinib metabolism. It is interesting to note that blocking the site of hydroxylation with a methyl group was not effective because a reactive imine-methide was formed, nor was blocking the site with fluorine because the fluorine was removed through an oxidative defluorination mechanism and the reactive quinone-imine was still formed. Numerous analogs are presented that did effectively block the formation of glutathione adducts and prevent the inactivation of CYP3A4.

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Identification of the Human Enzymes Involved in the Oxidative Metabolism of Dasatinib: An Effective Approach for Determining Metabolite Formation Kinetics

Cited by 67 publications

References 17 publications

CYP3A4-Mediated Ester Cleavage as the Major Metabolic Pathway of the Oral Taxane 3′-tert-Butyl-3′-N-tert-butyloxycarbonyl-4-deacetyl-3′-dephenyl-3′-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183)

CYP3A4-Mediated Ester Cleavage as the Major Metabolic Pathway of the Oral Taxane 3′-tert-Butyl-3′-N-tert-butyloxycarbonyl-4-deacetyl-3′-dephenyl-3′-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183)

Gradient elution LC-MS determination of dasatinib in rat plasma and its pharmacokinetic study

Characterization of Dasatinib and Its Structural Analogs as CYP3A4 Mechanism-Based Inactivators and the Proposed Bioactivation Pathways

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