Herein, the in vitro metabolism of tyrosine kinase inhibitor cabozantinib, the drug used for the treatment of metastatic medullary thyroid cancer and advanced renal cell carcinoma, was studied using hepatic microsomal samples of different human donors, human recombinant cytochromes P450 (CYPs), flavin-containing mono-oxygenases (FMOs) and aldehyde oxidase. After incubation with human microsomes, three metabolites, namely cabozantinib N-oxide, desmethyl cabozantinib and monohydroxy cabozantinib, were detected. Significant correlations were found between CYP3A4 activity and generation of all metabolites. The privileged role of CYP3A4 was further confirmed by examining the effect of CYP inhibitors and by human recombinant enzymes. Only four of all tested human recombinant cytochrome P450 were able to oxidize cabozantinib, and CYP3A4 exhibited the most efficient activity. Importantly, cytochrome b5 (cyt b5) stimulates the CYP3A4-catalyzed formation of cabozantinib metabolites. In addition, cyt b5 also stimulates the activity of CYP3A5, whereas two other enzymes, CYP1A1 and 1B1, were not affected by cyt b5. Since CYP3A4 exhibits high expression in the human liver and was found to be the most efficient enzyme in cabozantinib oxidation, we examined the kinetics of this oxidation. The present study provides substantial insights into the metabolism of cabozantinib and brings novel findings related to cabozantinib pharmacokinetics towards possible utilization in personalized medicine.

Central European Institute of Technology Brno University of Technology Purkynova 656 123 61200 Brno Czech Republic

Department of Biochemistry Faculty of Science Charles University Albertov 6 12800 Prague 2 Czech Republic

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 61300 Brno Czech Republic

Zobrazit více v PubMed

Wilkinson G.R. Drug metabolism and variability among patients in drug response. N. Engl. J. Med. 2005;352:2211–2221. doi: 10.1056/NEJMra032424. PubMed DOI

Fujita K. Cytochrome P450 and anticancer drugs. Curr. Drug Metab. 2006;7:23–37. doi: 10.2174/138920006774832587. PubMed DOI

Kurzrock R., Sherman S.I., Ball D.W., Forastiere A.A., Cohen R.B., Mehra R., Pfister D.G., Cohen E.E.W., Janisch L., Nauling F., et al. Activity of XL184 (Cabozantinib), an oral tyrosine kinase inhibitor, in patients with medullary thyroid cancer. J. Clin. Oncol. 2011;29:2660–2666. doi: 10.1200/JCO.2010.32.4145. PubMed DOI PMC

Tolaney S.M., Nechushtan H., Ron I., Schöffski P., Awada A., Yasenchak C.A., Laird A.D., O’Keeffe B., Shapiro G.I., Winer E.R. Cabozantinib for metastatic breast carcinoma: Results of a phase II placebo-controlled randomized discontinuation study. Breast Cancer Res. Treat. 2016;160:305–312. doi: 10.1007/s10549-016-4001-y. PubMed DOI PMC

Tolaney S.M., Ziehr D.R., Guo H., Ng M.R., Barry W.T., Higgins M.J., Isakoff S.J., Brock J.E., Ivanova E.V., Paweletz C.P., et al. Phase II and biomarker study of cabozantinib in metastatic triple-negative breast cancer patients. Oncologist. 2017;22:25–32. doi: 10.1634/theoncologist.2016-0229. PubMed DOI PMC

Kelley R.K., Verslype C., Cohn A.L., Yang T.S., Su W.C., Burris H., Braiteh F., Vogelzang N., Spira A., Foster P., et al. Cabozantinib in hepatocellular carcinoma: Results of a phase 2 placebo-controlled randomized discontinuation study. Ann. Oncol. 2017;28:528–534. doi: 10.1093/annonc/mdw651. PubMed DOI PMC

Abou-Alfa G.K., Meyer T., Cheng A.L., El-Khoueiry A.B., Rimassa L., Ryoo B.Y., Cicin I., Merle P., Chen Y.H., Park J.W., et al. Cabozantinib in patients with advanced and progressing hepatocellular carcinoma. N. Engl. J. Med. 2018;379:54–63. doi: 10.1056/NEJMoa1717002. PubMed DOI PMC

Drilon A., Wang L., Hasanovic A., Suehara Y., Lipson D., Stephens P., Roos J., Miller V., Ginsberg M., Zakowski M.F., et al. Response to Cabozantinib in patients with RET fusion-positive lung adenocarcinomas. Cancer Discov. 2013;3:630–635. doi: 10.1158/2159-8290.CD-13-0035. PubMed DOI PMC

Nokihara H., Nishio M., Yamamoto N., Fujiwara Y., Horinouchi H., Kanda S., Horiike A., Ohyanagi F., Yanagitani N., Nguyen L., et al. Phase 1 Study of Cabozantinib in Japanese Patients with Expansion Cohorts in Non–Small-Cell Lung Cancer. Clin. Lung Cancer. 2019;20:e317–e328. doi: 10.1016/j.cllc.2018.12.018. PubMed DOI

Smith D.C., Smith M.R., Sweeney C., Elfiky A.A., Logothetis C., Corn P.G., Vogelzang N.J., Small E.J., Harzstark A.L., Gordon S., et al. Cabozantinib in patients with advanced prostate cancer: Results of a phase II randomized discontinuation trial. J. Clin. Oncol. 2013;31:412–419. doi: 10.1200/JCO.2012.45.0494. PubMed DOI PMC

Dai J., Zhang H., Karatsinides A., Keller J.M., Kozloff K.M., Aftab D.T., Schimmoller F., Keller E.T. Cabozantinib inhibits prostate cancer growth and prevents tumor-induced bone lesions. Clin. Cancer Res. 2014;20:617–630. doi: 10.1158/1078-0432.CCR-13-0839. PubMed DOI PMC

Choueiri T.K., Escudier B., Powles T., Mainwaring P.N., Rini B.I., Donskov F., Hammers H., Hutson T.E., Lee J.L., Peltola K., et al. Cabozantinib versus everolimus in advanced renal-cell carcinoma. N. Engl. J. Med. 2015;373:1814–1823. doi: 10.1056/NEJMoa1510016. PubMed DOI PMC

Choueiri T.K., Escudier B., Powles T., Tannir N.M., Mainwaring P.N., Rini B.I., Hammers H.J., Donskov F., Roth B.J., Peltola K., et al. Cabozantinib versus everolimus in advanced renal cell carcinoma (METEOR): Final results from a randomised, open-label, phase 3 trial. Lancet Oncol. 2016;17:917–927. doi: 10.1016/S1470-2045(16)30107-3. PubMed DOI

Hage C., Rausch V., Giese N., Giese T., Schönsiegel F., Labsch S., Nwaeburu C., Mattern J., Gladkich J., Herr I. The novel c-Met inhibitor cabozantinib overcomes gemcitabine resistance and stem cell signaling in pancreatic cancer. Cell Death Dis. 2013;4:e627. doi: 10.1038/cddis.2013.158. PubMed DOI PMC

Yakes F.M., Chen J., Tan J., Yamaguchi K., Shi Y., Yu P., Qian F., Chu F., Bentzien F., Cancilla B., et al. Cabozantinib (XL184), a novel MET and VEGFR2 inhibitor, simultaneously suppresses metastasis, angiogenesis, and tumor growth. Mol. Cancer Ther. 2011;10:2298–2308. doi: 10.1158/1535-7163.MCT-11-0264. PubMed DOI

Bentzien F., Zuzow M., Heald N., Gibson A., Shi Y., Goon L., Yu P., Engst S., Zhang W., Huang D., et al. In vitro and in vivo activity of cabozantinib (XL184), an inhibitor of RET, MET, and VEGFR2, in a model of medullary thyroid cancer. Thyroid. 2013;23:1569–1577. doi: 10.1089/thy.2013.0137. PubMed DOI PMC

Xiang Q., Chen W., Ren M., Wang J., Zhang H., Deng D.Y.B., Zhang L., Shang C., Chen Y. Cabozantinib suppresses tumor growth and metastasis in hepatocellular carcinoma by a dual blockade of VEGFR2 and MET. Clin. Cancer Res. 2014;20:2959–2970. doi: 10.1158/1078-0432.CCR-13-2620. PubMed DOI

Lacy S., Hsu B., Miles D., Aftab D., Wang R., Nguyen L. Metabolism and disposition of cabozantinib in healthy male volunteers and pharmacologic characterization of its major metabolites. Drug Metab. Dispos. 2015;43:1190–1207. doi: 10.1124/dmd.115.063610. PubMed DOI

Gerendash B.S., Creel P.A. Practical management of adverse events associated with cabozantinib treatment in patients with renal-cell carcinoma. OncoTargets Ther. 2017;10:5053. doi: 10.2147/OTT.S145295. PubMed DOI PMC

FDA Center for Drug Evaluation and Research Cabometyx Full Prescribing Information. [(accessed on 20 September 2020)]; Available online: https://www.accessdata.fda.gov/drugsatfda_docs/label/2016/208692s000lbl.pdf.

Stiborová M., Indra R., Mizerovská M., Frei E., Schmeiser H.H., Kopka K., Philips D.H., Arlt V.M. NADH:Cytochrome b5 reductase and cytochrome b5 can act as sole electron donors to human cytochrome P450 1A1-mediated oxidation and DNA adduct formation by benzo[a]pyrene. Chem. Res. Toxicol. 2016;29:1325–1334. doi: 10.1021/acs.chemrestox.6b00143. PubMed DOI PMC

Kotrbova V., Mrazova B., Moserova M., Martinek V., Hodek P., Hudecek J., Frei E., Stiborova M. Cytochrome b5 shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy. Biochem. Pharmacol. 2011;82:669–680. doi: 10.1016/j.bcp.2011.06.003. PubMed DOI

Šulc M., Indra R., Moserová M., Schmeiser H.H., Frei E., Arlt V.M., Stiborová M. The impact of individual cytochrome P450 enzymes on oxidative metabolism of benzo[a]pyrene in human livers. Environ. Mol. Mutagen. 2016;57:229–235. doi: 10.1002/em.22001. PubMed DOI PMC

Stiborová M., Borek-Dohalska L., Aimova D., Kotrbova V., Kukackova K., Janouchova K., Rupertova M., Ryslava H., Hudecek J., Frei E. Oxidation pattern of the anticancer drug ellipticine by hepatic microsomes—Similarity between human and rat systems. Gen. Physiol. Biophys. 2006;25:245–261. PubMed

Rendic S., DiCarlo F.J. Human cytochrome P450 enzymes: A status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab. Rev. 1997;29:413–480. doi: 10.3109/03602539709037591. PubMed DOI

Stiborová M., Martínek V., Rýdlová H., Hodek P., Frei E. Sudan I is a potential carcinogen for humans: Evidence for its metabolic activation and detoxication by human recombinant cytochrome P450 1A1 and liver microsomes. Cancer Res. 2002;62:5678–5684. PubMed

Stiborová M., Martínek V., Rýdlová H., Koblas T., Hodek P. Expression of cytochrome P450 1A1 and its contribution to oxidation of a potential human carcinogen 1-phenylazo-2-naphthol (Sudan I) in human livers. Cancer Lett. 2005;220:145–154. doi: 10.1016/j.canlet.2004.07.036. PubMed DOI

Yamazaki H., Nakano M., Omak Y., Ueng Y.F., Guengerich F.P., Shimada T. Roles of cytochrome b5 in the oxidation of testosterone and nifedipine by recombinant cytochrome P450 3A4 and by human liver microsomes. Arch. Biochem. Biophys. 1996;325:174–182. doi: 10.1006/abbi.1996.0022. PubMed DOI

Porter T.D. The roles of cytochrome b5 in cytochrome P450 reactions. J. Biochem. Mol. Toxicol. 2002;16:311–316. doi: 10.1002/jbt.10052. PubMed DOI

Stiborová M., Indra R., Frei E., Kopečková K., Schmeiser H.H., Eckschlager T., Adam V., Heger Z., Arlt V.M., Martínek V. Cytochrome b5 plays a dual role in the reaction cycle of cytochrome P450 3A4 during oxidation of the anticancer drug ellipticine. Monatsh. Chem. 2017;148:1983–1991. doi: 10.1007/s00706-017-1986-9. PubMed DOI PMC

Indra R., Pompach P., Martínek V., Takácsová P., Vavrová K., Heger Z., Adam V., Eckschlager T., Kopečková K., Arlt V.M., et al. Identification of Human Enzymes Oxidizing the Anti-Thyroid-Cancer Drug Vandetanib and Explanation of the High Efficiency of Cytochrome P450 3A4 in its Oxidation. Int. J. Mol. Sci. 2019;20:3392. doi: 10.3390/ijms20143392. PubMed DOI PMC

Schenkman J.B., Jansson I. The many roles of cytochrome b5. Pharmacol. Ther. 2003;97:139–152. doi: 10.1016/S0163-7258(02)00327-3. PubMed DOI

Achira M., Ito K., Suzuki H., Sugiyama Y. Comparative studies to determine the selective inhibitors for P-glycoprotein and cytochrome P 4503A4. AAPS Pharmsci. 1999;1:14–19. doi: 10.1208/ps010418. PubMed DOI PMC

Greenblatt D.J., Zhao Y., Venkatakrishnan K., Duan S.X., Harmatz J.S., Parent S.J., Court M.H., von Moltke L.L. Mechanism of cytochrome P450-3A inhibition by ketoconazole. J. Pharm. Pharmacol. 2011;63:214–221. doi: 10.1111/j.2042-7158.2010.01202.x. PubMed DOI

Walsky R.L., Obach R.S., Hyland R., Kang P., Zhou S., West M., Geoghegan K.F., Helal C.J., Walker G.S., Goosen T.C., et al. Selective mechanism-based inactivation of CYP3A4 by CYP3cide (PF-04981517) and its utility as an in vitro tool for delineating the relative roles of CYP3A4 versus CYP3A5 in the metabolism of drugs. Drug Metab. Dispos. 2012;40:1686–1697. doi: 10.1124/dmd.112.045302. PubMed DOI

Nguyen L., Holland J., Miles D., Engel C., Benrimoh N., O’Reilly T., Lacy S. Pharmacokinetic (PK) drug interaction studies of cabozantinib: Effect of CYP3A inducer rifampin and inhibitor ketoconazole on cabozantinib plasma PK and effect of cabozantinib on CYP2C8 probe substrate rosiglitazone plasma PK. J. Clin. Pharmacol. 2015;55:1012–1023. doi: 10.1002/jcph.510. PubMed DOI

Lin Q.M., Li Y.H., Lu X.R., Wang R., Pang N.H., Xu R.A., Cai J.P., Hu G.X. Characterization of Genetic Variation in CYP3A4 on the Metabolism of Cabozantinib in Vitro. Chem. Res. Toxicol. 2019;32:1583–1590. doi: 10.1021/acs.chemrestox.9b00100. PubMed DOI

Li J., Zhao M., He P., Hidalgo M., Baker S.D. Differential metabolism of gefitinib and erlotinib by human cytochrome P450 enzymes. Clin. Cancer Res. 2007;13:3731–3737. doi: 10.1158/1078-0432.CCR-07-0088. PubMed DOI

Van Erp N.P., Gelderblom H., Karlsson M.O., Li J., Zhao M., Ouwerkerk J., Nortier J.W., Guchelaar H.J., Baker S.D., Sparreboom A. Influence of CYP3A4 inhibition on the steady-state pharmacokinetics of imatinib. Clin. Cancer Res. 2007;13:7394–7400. doi: 10.1158/1078-0432.CCR-07-0346. PubMed DOI

Kim A., Balis F.M., Widemann B.C. Sorafenib and sunitinib. Oncologist. 2009;14:800. doi: 10.1634/theoncologist.2009-0088. PubMed DOI PMC

Jackson K.D., Durandis R., Vergne M.J. Role of cytochrome P450 enzymes in the metabolic activation of tyrosine kinase inhibitors. Int. J. Mol. Sci. 2018;19:2367. doi: 10.3390/ijms19082367. PubMed DOI PMC

Zanger U.M., Schwab M. Cytochrome P450 enzymes in drug metabolism: Regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol. Ther. 2013;138:103–141. doi: 10.1016/j.pharmthera.2012.12.007. PubMed DOI

Voice M.W., Zhang Y., Wolf C.R., Burchell B., Friedberg T. Effects of Human Cytochrome b5on CYP3A4 Activity and Stability in Vivo. Arch. Biochem. Biophys. 1999;366:116–124. doi: 10.1006/abbi.1999.1192. PubMed DOI

Stiborová M., Sejbal J., Bořek-Dohalská L., Aimová D., Poljaková J., Forsterová K., Rupertová M., Wiesner J., Hudeček J., Wiessler M., et al. The anticancer drug ellipticine forms covalent DNA adducts, mediated by human cytochromes P450, through metabolism to 13-hydroxyellipticine and ellipticine N2-oxide. Cancer Res. 2004;64:8374–8380. doi: 10.1158/0008-5472.CAN-04-2202. PubMed DOI

Jushchyshyn M.I., Hutzler J.M., Schrag M.L., Wienkers L.C. Catalytic turnover of pyrene by CYP3A4: Evidence that cytochrome b5 directly induces positive cooperativity. Arch. Biochem. Biophys. 2005;438:21–28. doi: 10.1016/j.abb.2005.02.027. PubMed DOI

Henderson C.J., McLaughlin L.A., Scheer N., Stanley L.A., Wolf C.R. Cytochrome b5 is a major determinant of human cytochrome P450 CYP2D6 and CYP3A4 activity in vivo. Mol. Pharmacol. 2015;87:733–739. doi: 10.1124/mol.114.097394. PubMed DOI

Ingelman-Sundberg M., Sim S.C., Gomez A., Rodriguez-Antona C. Influence of cytochrome P450 polymorphisms on drug therapies: Pharmacogenetic, pharmacoepigenetic and clinical aspects. Pharmacol. Therap. 2007;116:496–526. doi: 10.1016/j.pharmthera.2007.09.004. PubMed DOI

Yoo S.E., Yi M., Kim W.Y., Cho S.A., Lee S.S., Lee S.J., Shin J.G. Influences of cytochrome b5 expression and its genetic variant on the activity of CYP2C9, CYP2C19 and CYP3A4. Drug Metab. Pharmacokinet. 2019;34:201–208. doi: 10.1016/j.dmpk.2019.03.001. PubMed DOI

Zhang H., Gao N., Liu T., Fang Y., Qi B., Wen Q., Zhou J., Jia L., Qiao H. Effect of cytochrome b5 content on the activity of polymorphic CYP1A2, 2B6, and 2E1 in human liver microsomes. PLoS ONE. 2015;10:e0128547. doi: 10.1371/journal.pone.0128547. PubMed DOI PMC

Takahashi K., Oda Y., Toyoda Y., Fukami T., Yokoi T., Nakajima M. Regulation of cytochrome b5 expression by miR-223 in human liver: Effects on cytochrome P450 activities. Pharm. Res. 2014;31:780–794. doi: 10.1007/s11095-013-1200-7. PubMed DOI

Sacco J.C., Trepanier L.A. Cytochrome b5 and NADH cytochrome b5 reductase: Genotype-phenotype correlations for hydroxylamine reduction. Pharmacogenet. Genom. 2010;20:26. doi: 10.1097/FPC.0b013e3283343296. PubMed DOI PMC

Ruiz J.N., Belum V.R., Creel P., Cohn A., Ewer M., Lacouture M.E. Current practices in the management of adverse events associated with targeted therapies for advanced renal cell carcinoma: A national survey of oncologists. Clin. Genitourin. Cancer. 2014;12:341–347. doi: 10.1016/j.clgc.2014.04.001. PubMed DOI PMC

Ferrer F., Solas C., Giocanti M., Lacarelle B., Deville J.L., Gravis G., Ciccolini J. A simple and rapid liquid chromatography-mass spectrometry method to assay cabozantinib in plasma: Application to therapeutic drug monitoring in patients with renal cell carcinoma. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2020;1138:121968. doi: 10.1016/j.jchromb.2020.121968. PubMed DOI

Miles D., Jumbe N.L., Lacy S., Nguyen L. Population pharmacokinetic model of cabozantinib in patients with medullary thyroid carcinoma and its application to an exposure-response analysis. Clin. Pharmacokinet. 2016;55:93–105. doi: 10.1007/s40262-015-0295-x. PubMed DOI