pharmacokinetics, pharmacodynamics, metabolism, distribution, and excretion of carfilzomib in rats

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  • Pharmacokinetics, Pharmacodynamics, Metabolism, Distribution,and Excretion of Carfilzomib in RatsS

    Jinfu Yang, Zhengping Wang, Ying Fang, Jing Jiang, Frances Zhao, Hansen Wong,Mark K. Bennett, Christopher J. Molineaux, and Christopher J. Kirk

    Onyx Pharmaceuticals, Inc., South San Francisco, California

    Received March 4, 2011; accepted July 12, 2011

    ABSTRACT:

    Carfilzomib [(2S)-N-[(S)-1-[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethyl]-2-[(S)-2-(2-morpholinoac-etamido)-4-phenylbutanamido]-4-methylpentanamide, also knownas PR-171] is a selective, irreversible proteasome inhibitor that hasshown encouraging results in clinical trials in multiple myeloma. Inthis study, the pharmacokinetics, pharmacodynamics, metabolism,distribution, and excretion of carfilzomib in Sprague-Dawley ratswere characterized. After intravenous administration, the plasmaconcentration of carfilzomib declined rapidly in a biphasic manner.Carfilzomib displayed high plasma clearance [195319 ml/(min kg)],a short-terminal half-life (520 min), and rapid and wide tissue distri-bution in rats. The exposure to carfilzomib (Cmax and area under the

    curve) increased dose proportionally from 2 to 4 mg/kg but less thandose proportionally from 4 to 8 mg/kg. The high clearance was me-diated predominantly by extrahepatic metabolism through peptidasecleavage and epoxide hydrolysis. Carfilzomib was excreted mainly asmetabolites resulting from peptidase cleavage. Carfilzomib and itsmajor metabolites in urine and bile accounted for approximately 26and 31% of the total dose, respectively, for a total of 57% within 24 hpostdose. Despite the high systemic clearance, potent proteasomeinhibition was observed in blood and a variety of tissues. Togetherwith rapid and irreversible target binding, the high clearance mayprovide an advantage in that unnecessary exposure to the drug isminimized and potential drug-related side effects may be reduced.

    Introduction

    The proteasome is a multicatalytic enzyme complex that is the primarymeans for protein degradation in all cells (Kisselev and Goldberg, 2001;Ciechanover, 2005). Inhibition of the proteasome leads to the accumu-lation of misfolded proteins and the induction of apoptosis. The proteo-lytic activities of the proteasome are encoded by distinct, N-terminalthreonine-containing enzymes that function as chymotrypsin-like (CT-L),trypsin-like, and caspase-like proteases. Tumor cells are more suscep-tible than nontransformed cells to the cytotoxic effects of proteasomeinhibition, making the proteasome an attractive target for the treatment ofmalignant diseases (Adams, 2004). The approval of bortezomib (Vel-cade) by the U.S. Food and Drug Administration for the treatment ofmultiple myeloma (MM) and mantle cell lymphoma provided clinical

    validation of the proteasome as a therapeutic target in oncology (Adamset al., 1998; Bross et al., 2004; Chauhan et al., 2005; Goy et al., 2005;OConnor et al., 2005; Richardson et al., 2005). Bortezomib is a dipeptideboronic acid that reversibly inhibits the 20S proteasome by forming atetrahedral intermediate with the side chain hydroxyl group of the N-ter-minal threonine (Borissenko and Groll, 2007). Despite the encouragingclinical success of bortezomib, adverse effects including painful periph-eral neuropathy have been reported, and a significant fraction of patientsrelapse from or are refractory to treatment with bortezomib (Orlowski etal., 2002, 2007a,b; Richardson et al., 2003, 2005; Goy et al., 2005;OConnor et al., 2005). In addition, bortezomib is metabolized primarilyby cytochrome P450 3A4 (Uttamsingh et al., 2005), and coadministrationof cytochrome P450 3A4 inhibitors such as ketoconazole causes a sig-nificant change in bortezomib plasma levels in humans (Venkatakrishnanet al., 2009). In the past several years, the next generation of proteasomeinhibitors has been explored, with the aim to improve safety and efficacyprofiles. Three small molecules [carfilzomib, (4R,5S)-4-(2-chloroethyl)-1-((1S)-cyclohex-2-enyl(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione (NPI-0052), and[(1R)-1-[[(2S,3R)-3-hydroxy-2-[[(6-phenylpyridin-2-yl)carbonyl]

    J.Y. and Z.W. contributed equally to this work.Article, publication date, and citation information can be found at

    http://dmd.aspetjournals.org.doi:10.1124/dmd.111.039164.S The online version of this article (available at http://dmd.aspetjournals.org)

    contains supplemental material.

    ABBREVIATIONS: CT-L, chymotrypsin-like; MM, multiple myeloma; CEP-18770, [(1R)-1-[[(2S,3R)-3-hydroxy-2-[[(6-phenylpyridin-2-yl)carbonyl]amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid; NPI-0052, (4R,5S)-4-(2-chloroethyl)-1-((1S)-cyclohex-2-enyl(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione; PR-171 (carfilzomib), (2S)-N-[(S)-1-[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethyl]-2-[(S)-2-(2-morpholinoacetamido)-4-phenylbutanamido]-4-methylpentanamide; PR-054591, N-[(S)-1-[(S)-1-[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethylcarbamoyl]-3-methylbutyl]-5-(morpholinomethyl)isoxazole-3-carboxamide; ONX 0912, (2S)-3-methoxy-2-[(2S)-3-methoxy-2-[(2-methyl-1,3-thiazol-5-yl)formamido] propanamido]-N-[(2S)-1-[(2R)-2-methyloxiran-2-yl]-1-oxo-3-phenylpropan-2-yl]propanamide; IS, internalstandard; PK, pharmacokinetics; AUC, area under plasma concentration-time curve; CL, plasma clearance; t1/2, terminal half-life; Vss, volume of distribution atsteady state; LC-MS/MS, liquid chromatography tandem mass spectrometry; MRM, multiple ion reaction monitoring; AUClast, area under plasma concentra-tion-time curve calculated to the last measurable concentration; AUCinf, area under plasma concentration-time curve extrapolated to infinity; Css, steady-stateconcentration; RP, rat plasma; RU, rat urine; RB, rat bile; PD, pharmacodynamics.

    0090-9556/11/3910-18731882$25.00DRUG METABOLISM AND DISPOSITION Vol. 39, No. 10Copyright 2011 by The American Society for Pharmacology and Experimental Therapeutics 39164/3717903DMD 39:18731882, 2011 Printed in U.S.A.

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    http://dmd.aspetjournals.org/content/suppl/2011/07/13/dmd.111.039164.DC1.htmlSupplemental material to this article can be found at:

    at ASPE

    T Journals on D

    ecember 17, 2014

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    http://dmd.aspetjournals.org/content/suppl/2011/07/13/dmd.111.039164.DC1.htmlhttp://dmd.aspetjournals.org/

  • amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid (CEP-18770)] are currently in clinical development (Bennett and Kirk, 2008).

    Carfilzomib (Fig. 1) is a tetrapeptide epoxyketone analog of epox-omicin and eponemycin, a pair of related natural products that werediscovered initially as antitumor agents in animals and later shown toinhibit the CT-L activity of the proteasome (Hanada et al., 1992; Kimet al., 1999; Meng et al., 1999a,b; Groll et al., 2000). The epoxyketonepharmacophore forms a dual covalent morpholino adduct with theN-terminal threonine. Carfilzomib shows primary selectivity for theCT-L activity and displays little activity against the trypsin-like orcaspase-like activities of the proteasome (Demo et al., 2007). Uponthe basis of encouraging preclinical data, including the induction ofpotent proteasome inhibition in blood and tissues in rodents and

    greater antitumor activity compared with that of bortezomib in mousemodels of cancer (Demo et al., 2007; Kuhn et al., 2007), carfilzomibentered clinical testing in MM. Carfilzomib has shown encouragingresults in a broad clinical trial program in MM (Alsina et al., 2007;Jakubowiak et al., 2010a,b; Jagannath et al., 2010; Martin et al., 2010;Siegel et al., 2010; Singhal et al., 2010; Vij et al., 2010), including inpatients refractory to bortezomib. Carfilzomib is currently being de-veloped by Onyx Pharmaceuticals, Inc. for the treatment of MM andis in phase III clinical trials.

    The objective of the present study was to characterize the disposi-tion of carfilzomib in rats, a pharmacological and toxicological spe-cies used in preclinical evaluation of the compound. We show herethat, after intravenous administration to Sprague-Dawley rats, carfil-zomib was cleared rapidly from systemic circulation. In vitro and invivo studies showed that carfilzomib was metabolized extensively inblood and a variety of tissues. The major metabolites detected in ratplasma, urine, and bile were from peptidase cleavage and epoxidehydrolysis. Elimination occurred mainly via biliary and renal excre-tion in the form of metabolites. Despite rapid systemic clearance,carfilzomib generated potent proteasome inhibition in a variety oftissues due to irreversible inhibition of its target, demonstrating thewide tissue distribution of carfilzomib upon intravenous administra-tion. We finally show that a 30-min infusion administration re-sulted in a 28-fold lower maximum plasma concentration (Cmax)but with comparable levels of proteasome inhibition in blood andtissues as those of an intravenous bolus.

    Materials and Methods

    Materials. (2S)-N-[(S)-1-[(S)-4-Methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethyl]-2-[(S)-2-(2-morpholinoacetamido)-4-phenylbutanamido]-4-methylpentanamide (carfilzomib, also known as PR-171),N-[(S)-1-[(S)-1-[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethylcarbamoyl]-3-methylbutyl]-5-(morpholinomethyl)isoxazole-3-carboxamide (PR-054591), and (2S)-3-methoxy-2-[(2S)-3-methoxy-2-[(2-methyl-1,3-thiazol-5-yl)formamido] propanamido]-N-[(2S)-1-[(2R)-2-methyloxiran-2-yl]-1-oxo-3-phenylpropan-2-yl]propanamide (ONX 0912), tripep-tide analogs of carfilzomib shown in Fig. 1, and authentic standards of carfilzomibmetabolites (M14, M15, and M16; Fig. 2 and Table 3) were synthesized at Onyx

    FIG. 1. Chemical structures

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