Pharmacokinetics, Pharmacodynamics, Metabolism, Distribution,and Excretion of Carfilzomib in Rats□S

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 [195–319 ml/(min � kg)],a short-terminal half-life (5–20 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;O’Connor 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;O’Connor 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.

0090-9556/11/3910-1873–1882$25.00DRUG METABOLISM AND DISPOSITION Vol. 39, No. 10Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 39164/3717903DMD 39:1873–1882, 2011 Printed in U.S.A.

1873

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

dmd.aspetjournals.org

Dow

nloaded from

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 of carfilzomib (PR-171), PR-054591 (IS), and ONX 0912.

FIG. 2. Major metabolic pathways of carfil-zomib from metabolite profiling in rat plasma,urine, and bile samples.

1874 YANG ET AL.

at ASPE

T Journals on D

ecember 17, 2014

dmd.aspetjournals.org

Dow

nloaded from

Pharmaceuticals, Inc. PR-054591 was used as the internal standard (IS) for all ofthe quantitative analyses. Rat whole blood and rat and monkey plasma in sodiumheparin were from Bioreclamation (East Meadow, NY). Tissue homogenates wereprepared in house. All of the other reagents were obtained from commercialsources with an appropriate quality.

Pharmacokinetics Studies. All of the animal studies were conducted inaccordance with the Guide for the Care and Use of Laboratory Animals(Institute of Laboratory Animal Resources, 1996). Male Sprague-Dawley rats,weighing approximately 250 to 350 g and containing cannulas in the jugularvein and carotid artery, were obtained from Charles River Laboratories, Inc.(Wilmington, MA) and used for all of the pharmacokinetics (PK) studies.Animals were allowed to acclimate for a minimum of 3 days before experi-mentation and allowed food and water ad libitum.

Carfilzomib was formulated in 10% (w/v) sulfobutyl ether �-cyclodextrin(Captisol; Cydex Pharmaceuticals Inc., Lenexa, KS) in 10 mM citrate (pH 3.5)at a concentration of 2 mg/ml. For intravenous bolus administration, four tofive animals per dose level received a single dose of carfilzomib at 2, 4, or 8mg/kg, respectively. Blood samples (�0.4 ml) were collected before andimmediately after dosing (within 10 s) and at 1, 2, 5, 15, 30, and 60 minpostdose. For intravenous infusion administration, four rats received carfil-zomib via a 30-min constant infusion at 8 mg/kg. Blood samples werecollected before dosing, at 15 min after the start of infusion, end of infusion,and at 2, 5, 15, 30, 60, and 120 min postdose.

To assess the effects of target binding, the PK profiles of carfilzomib fromtwo groups of male Sprague-Dawley rats (three per group) were compared.One group was pretreated with an intravenous bolus dose of 10 mg/kg ONX0912 (also known as PR-047), a tripeptide analog of carfilzomib with potentirreversible proteasome inhibition (Zhou et al., 2009), whereas the other groupwas pretreated with the formulation vehicle. Thirty minutes after pretreatment,carfilzomib was administered via an intravenous bolus at 2 mg/kg. Bloodsamples were taken before and immediately after the carfilzomib dose and at1, 2, 5, 15, 30, and 60 min after the carfilzomib dose.

A PK study also was conducted for M16, the diol of carfilzomib derivedfrom epoxide hydrolysis via an intravenous bolus administration to maleSprague-Dawley rats at 5 mg/kg. M16 was formulated at 2 mg/ml in Captisolcontaining 10 mM citrate (pH 3.5). Blood samples were collected beforedosing and at 2, 5, 10, 15, 30, 60, 120, and 360 min postdose.

The PK of carfilzomib also was assessed in non-naive male cynomolgusmonkeys with an average weight of 3 kg. The in-life portion of the study was

conducted at SNBL USA (Everett, WA). Carfilzomib was formulated in 5%hydroxypropyl-�-cyclodextrin (w/v) in 50 mM sodium citrate (pH 3.35) at aconcentration of 1 mg/ml. Three animals received a single intravenous bolusdose of carfilzomib at 1 mg/kg. Blood samples were collected before dosingand at 2, 5, 15, 30, and 60 min postdose.

All of the blood samples were collected in tubes containing sodium heparinas the anticoagulant. Blood samples were kept on ice during sample collectionand centrifuged at 3000g for 10 min at 4°C to obtain plasma. Plasma sampleswere stored in cryotubes at �70°C until analysis.

Calculation of PK Parameters. PK parameters were calculated by non-compartmental analysis using WinNonlin 5.2 (Pharsight, Mountain View,CA). Plasma concentrations reported as below the lower limit of quantificationwere treated as zero for PK analysis. Area under plasma concentration-timecurve (AUC) values were estimated using the linear trapezoidal method.Plasma clearance (CL) was calculated from dose/AUC extrapolated to infinity(AUCinf). Terminal half-life (t1/2) values were calculated as ln(2)/k, where krepresents the terminal elimination rate constant obtained from the slope of thesemilogarithmic plot of the concentration-time profile. Mean residence timewas estimated by moment analysis. Volume of distribution at steady state (Vss)was calculated from mean residence time � CL. Cmax was recorded asobserved. The amount of M16 generated in rats after carfilzomib dosing wascalculated by multiplying the AUCinf of M16 by the clearance of M16 obtainedin the PK study for M16.

Pharmacodynamic Study. Blood and tissue (adrenal, heart, liver, and lung)samples were collected from Sprague-Dawley rats receiving 8 mg/kg carfil-zomib by either an intravenous bolus or a 30-min infusion administration.Blood samples were collected at predose and 0.25, 2, and 24 h postdose. Forintravenous infusion, blood samples also were taken at 0.25 h after the start ofinfusion. Tissue samples were collected at 2 and 24 h postdose. ProteasomeCT-L activity was measured in lysates from cells and blood with Leu-Leu-Val-Tyr-AMC (LLVY-AMC) as the substrate as described previously (Demoet al., 2007).

Excretion Study. Excretion of carfilzomib was determined in maleSprague-Dawley rats with bile duct cannulation after a single intravenousbolus administration at 2 mg/kg. Bile was collected cumulatively at 0 to 4 and4 to 8 h postdose, no bile samples were collected at 8 to 24 h postdose, andurine was collectively 0 to 4, 4 to 8, and 8 to 24 h postdose. Volumes of thebile and urine samples were measured. Acetonitrile was added to the originalurine sample containers after the first thawing to achieve a final organic

FIG. 3. PK and PD of carfilzomib after a sin-gle intravenous bolus or a 30-min intravenousinfusion administration to male Sprague-Daw-ley rats. A, plasma concentration-time profilesof carfilzomib after a single intravenous bolusadministration at 2, 4, and 8 mg/kg. Plasmasamples were collected before dose, immedi-ately after dose (within 10 s), and at 1, 2, 5, 15,30, and 60 min postdose. B, plasma concentra-tion-time profile of carfilzomib after a 30-minintravenous infusion at 8 mg/kg. Plasma sam-ples were collected before dose, at 15 min afterthe start of infusion, at the end of infusion, andat 2, 5, 15, 30, 60, and 120 min postdose. C,proteasome inhibition and recovery after a sin-gle intravenous bolus or a 30-min intravenousinfusion of carfilzomib to Sprague-Dawley rats.Rats received a single intravenous bolus (opencolumn) or a 30-min intravenous infusion (solidcolumn) of carfilzomib at 8 mg/kg. ProteasomeCT-L activity was determined with Leu-Leu-Val-Tyr-AMC (LLVY-AMC) as the substratein lysates prepared from whole blood or tissuesat 2 and 24 h postdose. For whole blood, CT-Lactivity was also measured at 15 min after thestart of infusion (infusion only) and 15 minpostdose. Columns, mean activity relative tovehicle controls; bars, S.D. (n � 3 per dosegroup).

1875PHARMACOKINETICS OF CARFILZOMIB IN RATS

at ASPE

T Journals on D

ecember 17, 2014

dmd.aspetjournals.org

Dow

nloaded from

content of 20% (v/v). This was done to ensure full recovery of carfilzomibfrom the container. Plasma samples also were collected in this study before andimmediately after dosing and at 1, 2, 5, 15, 30, 60, and 120 min postdose intotubes containing sodium heparin. The urine, bile, and plasma samples werestored at �70°C until analysis.

Blood Plasma Partitioning. The whole blood to plasma ratio of carfil-zomib was determined in vitro. Carfilzomib was spiked into prewarmed ratblood at nominal concentrations of 0.2, 2, or 10 �M. The samples were mixedgently by inverting the tubes several times and then incubated at 37°C. At 15min, an aliquot of 100 �l of blood sample was taken and immediatelyquenched into 300 �l of acetonitrile containing 0.5 �M IS. The remainingblood samples were centrifuged at 3000g for 10 min at 4°C to obtain plasma.

Metabolite Profiling in Rat Plasma, Urine, and Bile Samples. Rat plasmasamples collected in a 6-month toxicity study to support toxicokinetic evalu-ation of carfilzomib were used to search for circulating metabolites. Carfil-zomib was given to three groups of rats at dose levels of 1, 2, or 4 mg/kg forsix 28-day cycles with intravenous bolus dosing on days 1, 2, 8, 9, 15, and 16.Plasma samples were collected on the first day of cycles 1, 3, and 6 (days 1,57, and 142, respectively) before dose and at 5, 15, 30, and 60 min postdose.Aliquots of plasma samples were pooled across animals, dose levels, collectiondays, and collection time points to generate one pooled sample for metaboliteprofiling. A pooled predose plasma sample was used as a control. The pooledsamples were extracted multiple times with three volumes of acetonitrile torecover carfilzomib and metabolites. The supernatants were combined andevaporated to dryness under a gentle stream of nitrogen. The residues werereconstituted with 0.5 ml of acetonitrile/water (1:1, v/v) and analyzed by liquidchromatography tandem mass spectrometry (LC-MS/MS).

The urine and bile samples used in metabolite profiling were obtained fromthe excretion study as described in the preceding section. Aliquots of bilesamples or acetonitrile-treated urine samples were pooled across animals togenerate one pooled bile or urine sample per time interval. The pooled urineand bile samples were subjected directly to LC-MS/MS analyses.

LC-MS/MS analyses were done in positive ionization mode using precursorion and neutral loss scans on LTQ mass spectrometer (Thermo Fisher Scien-tific, Waltham, MA) and multiple ion reaction monitoring (MRM) on an API4000 QTrap LC/MS/MS System (AB SCIEX, Foster City, CA) using a LunaC18(2) column (150 � 4.6 mm, 3-�m particle size; Phenomenex, Torrance,CA) and gradient elution. The mobile phases were 0.4% formic acid in wateradjusted to pH 3.2 with ammonium hydroxide (A) and acetonitrile (B). Theflow rate was 0.6 ml/min with solvent A held at 100% for 5 min, followed byramping solvent B from 0 to 70% in 55 min and 70 to 100% B in 2 min. Thestructures of the metabolites were proposed based on the molecular ion massesand fragmentation patterns generated by product ion scans. Semiquantificationwas done in MRM mode by comparing the peak area of each metabolite to thatof carfilzomib.

Metabolism Study in Rat Blood and Tissue Homogenates from Adre-nal, Blood, Heart, Kidney, Liver, and Lung. The metabolism of carfilzomibwas evaluated in rat blood and tissue homogenates (adrenal, heart, kidney,liver, and lung) harvested from untreated male Sprague-Dawley rats. Theisolated organs were chopped into small pieces, washed several times with

phosphate-buffered saline (pH 7.4), and then weighed into 2-ml Eppendorftubes. Dulbecco’s phosphate-buffered saline (pH 7.4) then was added to thetubes to achieve a 1:5 ratio of tissue weight to Dulbecco’s phosphate-bufferedsaline volume. The tissues were homogenized using a TissueLyser (Retsch,Inc., Newtown, PA) at a frequency of 20 s�1. Aliquots of 300 �l of rat bloodor tissue homogenates were preincubated at 37°C for 5 min before the additionof 5 �l of carfilzomib working solution at a concentration of 60 �g/ml inacetonitrile. The final concentration of carfilzomib in the incubation was 1�g/ml. The mixture was incubated at 37°C with gentle agitation. An aliquot of40 �l of reaction mixture was taken at 2, 10, 20, 30, 60, and 90 min andquenched with 120 �l of acetonitrile containing 0.5 �M IS. After centrifuga-tion, the supernatants were used to measure the disappearance of carfilzomiband the formation of selected metabolites, M14, M15, and M16. The relativerates of carfilzomib metabolism in rat adrenal and liver were evaluated in aseparate experiment under the same experimental conditions described above.

Quantitative Determination of Carfilzomib and Its Metabolites inBlood, Plasma, Urine, and Bile. Concentrations of carfilzomib and its me-tabolites (M14, M15, and M16) in blood, plasma, urine, and bile samples weredetermined by LC-MS/MS. Plasma, urine, and bile samples were stored at�70°C immediately after collection. Stability testing demonstrated that the ana-lytes were stable under the storage condition, during the extraction process, and forat least three freeze/thaw cycles. Carfilzomib and its metabolites were quantifiedby comparing the peak area ratios of the analyte to IS in study samples to those ofthe standard calibration samples prepared in the same manner. To determine theconcentration of carfilzomib in blood samples from an in vitro partitioning study,the standard calibration samples were prepared by spiking carfilzomib into bloodprequenched with three volumes of acetonitrile containing 0.5 �M IS. An aliquot(40 �l) of plasma, acetonitrile-treated urine, or bile sample was mixed with 80 �lof acetonitrile containing 0.5 �M IS to extract carfilzomib and its metabolites. Theextracted samples were vortexed and centrifuged. The supernatants were filteredthrough a 96-well filter plate (AcroPrep 96-well Filter Plate, 0.45 �m; PallCorporation, East Hills, NY) into a 96-well microplate, and 10 �l was injected forLC-MS/MS analysis.

The LC-MS/MS system comprised either an API 3000 or an API 3200 massspectrometer (AB SCIEX) equipped with two Shimadzu LC-10ADvp pumpsand a Shimadzu SIL-HTc autosampler with controller system (Shimadzu,Kyoto, Japan). Chromatographic separation was achieved on an ACE C18column (2.1 � 50 mm, 3.5-�m particle size; Advanced ChromatographyTechnology, Aberdeen, Scotland) using gradient elution. The mobile phaseswere 0.1% formic acid in water (A) and methanol (B). Mass spectrometricdetection was accomplished using the turbo ion spray interface in the positiveionization mode by MRM of the selective m/z transitions: 720.5 3 100 forcarfilzomib, 307.3 3 100 for M14, 420.4 3 100 for M15, 738.5 3 100 forM16, and 626.3 3 199.0 for IS. The lower limit of quantification was 2.00ng/ml with calibration ranges of 2.00 to 3000 ng/ml for blood and plasmasamples and 2.00 to 1000 ng/ml for urine and bile samples.

Results

Pharmacokinetics. After a single intravenous bolus dose of 2, 4, or8 mg/kg, carfilzomib plasma concentration-time curves display a verysteep initial phase (Fig. 3A). The first plasma sampling was done

TABLE 1

PK parameters of carfilzomib obtained from single intravenous bolus (2, 4, and8 mg/kg) and 30-min intravenous infusion (8 mg/kg) administrations to male

Sprague-Dawley rats

ParametersBolus Dose (mg/kg i.v.) Infusion

(mg/kg i.v.)

2 4 8 8

First sampling time (min) 0.1 0.1 0.1 15t1/2 (min) 5 � 0 17 � 9 17 � 3 10 � 6Cmax or Css (�M) 16.4 � 4.5 39.9 � 3.2 42.9 � 4.5 1.6 � 0.4AUClast �(min � �mol)/l� 12.6 � 2.9 28.5 � 1.8 37.6 � 2.8 35.8 � 7.3AUCinf �min � �mol/l� 12.7 � 2.9 28.6 � 1.8 37.8 � 2.8 35.9 � 7.2AUCinf/D

�(min � kg � �mol)/(l � �mol)�

4.6 � 1.1 5.1 � 0.3 3.4 � 0.3 3.2 � 0.7

CL (ml � min�1 � kg�1) 229 � 55 195 � 12 296 � 22 319 � 66Vss (l/kg) 0.3 � 0.1 0.3 � 0.1 0.6 � 0.1 2.0 � 0.5

TABLE 2

PK parameters of carfilzomib obtained from a single intravenous bolus dosing ofcarfilzomib (2 mg/kg) to Sprague-Dawley rats pretreated with ONX 0912 or

formulation vehicle

PK Parameters Pretreatment withVehicle

Pretreatment withONX 0912

First sampling time (min) 0.1 0.1t1/2 (min) 4 � 1 4 � 3Cmax (�M) 20.8 � 8.5 19.8 � 3.2AUClast (min � �mol/l) 15.2 � 5.6 14.7 � 2.0AUCinf (min � �mol/l) 15.2 � 5.6 14.8 � 2.1AUCinf/D

�(min � kg � �mol)/(l � �mol)�5.5 � 2.0 5.3 � 0.7

CL (ml � min�1 � kg�1) 199 � 69 190 � 27Vss (l/kg) 0.2 � 0.1 0.2 � 0.0

1876 YANG ET AL.

at ASPE

T Journals on D

ecember 17, 2014

dmd.aspetjournals.org

Dow

nloaded from

TABLE 3

Summary of carfilzomib metabolites identified in rat plasma, urine, and bile samples

Metabolite Code Proposed Structure Molecular Mass Retention Time Source

Da min

M1*H2N

O

OH 131 10.4 RP, RU, and RB

M2* H2N

O O

OHO

201 13.1 RP, RU, and RB

M5*

NH2

OHO

OH 181 13.1 RP, RU, and RB

M7*

NH2

OHO

165 15.9 RP and RU

M8*

H2N

OH

OHO

189 17.2 RP, RU, and RB

M9*

H2N

HOH2C

NHO

OH

O

OH

310 16.7 RP and RB

M10 OH

NH2

O

OH195 19.0 RU

M11*H2N N

H

O

O

O

318 20.2 RP, RU, and RB

M12*H2N

HN

O

O

OH

OH

294 21.2 RP, RU, and RB

M13* H2NHN

O

O

OH 278 25.1 RB

1877PHARMACOKINETICS OF CARFILZOMIB IN RATS

at ASPE

T Journals on D

ecember 17, 2014

dmd.aspetjournals.org

Dow

nloaded from

TABLE 3—Continued

Metabolite Code Proposed Structure Molecular Mass Retention Time Source

Da min

M14*

NHN

O

OOOH

306 25.9 RP, RU, and RB

M15*N

HN

NH

O

OOO

OH

419 33.0 RP, RU, and RB

M16*N

HN

NH

HN

NH

O

O

O

OO O

OHOH

737 43.8 RP, RU, and RB

M17N

HN

NH

HN

NH

O

HOH2C

O

O

OO O

O

735 51.1 RP and RU

M18N

HN

NH

HN

NH

O

O

O

OO O

OHOH

735 51.1 RU and RB

M19N

HN

NH

HN

NH

O

O

O

OO O

O

717 59.3 RB

M20N

HN

NH

HN

NH

O

O

O

OO O

O

OH

733 46.2 RU and RB

1878 YANG ET AL.

at ASPE

T Journals on D

ecember 17, 2014

dmd.aspetjournals.org

Dow

nloaded from

within 10 s after dosing to accurately measure the PK of carfilzomibin rats; plasma levels at 2 min were �5% of those immediately afterdosing (Fig. 3A, inset). The t1/2 value ranged from 5 to 20 min, andCL values were 195 to 296 ml/(min � kg) (Table 1). Both AUC andCmax increased dose proportionally, and the CL values were constantfrom 2 to 4 mg/kg. When the dose level was increased to 8 mg/kg,however, AUC and Cmax increased less than dose proportionally, andthe CL value increased. Rapid clearance and short half-life also wereobserved in cynomolgus monkeys after intravenous administration at1 mg/kg (Supplemental Fig. S1).

Carfilzomib is a rapidly binding and irreversible inhibitor of theproteasome, a ubiquitously expressed target that accounts for approx-imately 1% of total cellular protein (Ciechanover 2005; Demo et al.,2007). To determine whether the high level of target expressionaffects its PK, carfilzomib was administered to two groups of rats: onepretreated with an intravenous bolus dose of the vehicle (controlgroup) and the other pretreated with an intravenous bolus dose ofONX 0912 (formerly known as PR-047), a tripeptide analog of

carfilzomib that displays potent and irreversible inhibition of the 20Sproteasome (Zhou et al., 2009). A dose of 10 mg/kg was chosen forONX 0912 to completely inhibit the primary target of carfilzomib, theCT-L activity of the proteasome, in blood and available tissues (J.Jiang and C. Kirk, unpublished observations). Thirty minutes afterpretreatment, carfilzomib was administered as an intravenous bolus at2 mg/kg to both groups of animals. No significant differences in thePK parameters between the two groups were noted, suggesting thattarget binding is not a major mechanism of clearance for carfilzomib(Table 2).

Pharmacodynamics. The level of proteasome inhibition could bea function of peak plasma levels or total exposure to carfilzomib.When administered as a 30-min infusion at 8 mg/kg, steady state wasachieved essentially within 15 min, which is the first plasma samplingduring the infusion (Fig. 3B). The steady-state concentration (Css) ofcarfilzomib is 28-fold lower than the Cmax in the bolus group at thesame dose level (Table 1). The other PK parameters, including AUC,were equivalent. The level of proteasome inhibition in blood and

TABLE 3—Continued

Metabolite Code Proposed Structure Molecular Mass Retention Time Source

Da min

M21N

HN

NH

HN

NH

O

O

O

OO O

O

OH

733 50.1 RU and RB

M22 NHN

NH

HN

NH

O

O

O

OO O

O

+2O-2H

749 42.5 RB

M23 NHN

NH

HN

NH

O

O

O

OO O

OHOH

OH

753 39.1 RB

M24 NHN

NH

HN

NH

O

O

O

OO O

O

OH

735 44.4 RU

RP, rat plasma; RU, rat urine; RB, rat bile.* These metabolites were also observed in monkey plasma samples obtained in a 9-month toxicity study.

1879PHARMACOKINETICS OF CARFILZOMIB IN RATS

at ASPE

T Journals on D

ecember 17, 2014

dmd.aspetjournals.org

Dow

nloaded from

tissues was also equivalent between the two groups (Fig. 3C). Thesedata demonstrate that the level of target inhibition achieved in vivo isa function of the total dose administered but not Cmax. The potentproteasome inhibition in a variety of tissues also demonstrated rapidand wide tissue distribution of carfilzomib after a single intravenousbolus or infusion administration.

In Vitro Blood Partitioning. The ratios of whole blood concentra-tions to plasma concentrations were 0.82, 0.74, and 0.63 at the nominalblood concentrations of 0.2, 2, and 10 �M carfilzomib, respectively.Because the calibration standards were prepared by spiking carfilzomibinto prequenched whole blood, the measured whole blood levels do notrepresent the fraction bound to the drug target.

Metabolism. All of the predictable metabolites were searched in ratplasma, urine, and bile using the LC-MS/MS method described underMaterials and Methods. A total of 21 metabolites were identifiedbased on molecular ion masses and fragmentation patterns (Table 3).Although a few oxidative metabolites were observed, most metabo-lites were derived from peptide and epoxide hydrolysis. Semiquanti-fication in MRM mode, assuming that carfilzomib and its metabolitesexhibited similar responses under analytical conditions, has shownthat morpholino-homophenylalanine (M14), morpholino-homopheny-lalanine-leucine (M15), and the diol of carfilzomib (M16) were themost abundant metabolites and that the oxidative metabolites wereminor. Authentic standards of these three metabolites were synthe-sized to further confirm their structures and used for quantitativedetermination of their levels in plasma and excreta. Amino acids,naturally occurring dipeptides, and a low level of the dipeptide ep-oxyketone (M11) resulting from hydrolysis of carfilzomib also wereobserved. In an in vitro metabolism study in rat hepatocytes using[3H]carfilzomib with the label present in the aromatic ring of thephenylalanine, radioactive phenylalanine indeed was detected as arelatively abundant metabolite (J. Jiang and C. Kirk, unpublishedobservations). However, when nonradiolabeled carfilzomib was ad-ministrated to rats as conducted in the present study, these amino acidsand dipeptides could not be differentiated from endogenous ones.Therefore, they were not assessed quantitatively in this study.

As shown in Fig. 4, all three metabolites, M14, M15, and M16,formed rapidly, with measurable levels immediately postdose andpeak levels within 5 min of dosing. M14 is the most abundantcirculating metabolite, with a terminal half-life longer than that of theparent drug. The AUC ratios (metabolite/carfilzomib) were 2.7, 0.33,and 0.12 for M14, M15, and M16, respectively. As determined in aseparate PK study, plasma clearance of M16 was rapid [110 ml/(min �kg)] (Supplemental Table S1). Using this clearance value and theobserved AUCinf of M16 after intravenous bolus administration of

carfilzomib, we estimated that approximately 6% of the carfilzomibadministered to rats was biotransformed to the diol.

Carfilzomib was also metabolized rapidly in vitro after incubation withrat tissue homogenates, including adrenal, heart, kidney, liver, and lung.The half-life of carfilzomib disappearance was 4 to 39 min in blood andtissues (Fig. 5; Supplemental Fig. S2). Consistent with observations in thein vivo samples, the peptide hydrolysis products M14 and M15 were theabundant metabolites. M16 was also detected, but at a lower level. Takentogether, these data suggest that carfilzomib is metabolized rapidly in ratsmainly via peptidase cleavage and epoxide hydrolysis. The peptidasecleavage product M14 was the most abundant metabolite in both sys-temic circulation and excreta. Although M14 can be a secondary metab-olite of M16 as depicted in Fig. 2, conversion of M16 to M14 is not likelythe main route of M14 formation in rats because only approximately 6%of carfilzomib was converted to M16.

Excretion. Carfilzomib and its major metabolites were quantifiedin the collected urine and bile samples. Less than 1% of the intrave-nously dosed carfilzomib was excreted intact (Table 4). The productsof peptide cleavage (M14 and M15) were the main forms of excretion.The majority of the metabolites were excreted within 4 h after dosingof carfilzomib. Renal and biliary excretion of carfilzomib and metab-olites accounted for 26 and 31% of the dose, respectively, for a totalof 57% recovered within 24 h postdose.

FIG. 4. Plasma concentration-time profiles of carfilzomib and metabolites (M14,M15, and M16). Male Sprague-Dawley rats received 2 mg/kg carfilzomib via asingle intravenous bolus. Plasma samples were collected before dose, immediatelyafter dose, and at 1, 2, 5, 15, 30, and 60 min postdose.

FIG. 5. In vitro stability of carfilzomib in rat blood and tissue homogenates (heart,kidney, liver, and lung). Incubations were done at 37°C with a carfilzomib concen-tration of 1 �g/ml. Aliquots of the incubation mixture were taken at 2, 10, 20, 30,60, and 90 min to determine the level of carfilzomib remaining.

TABLE 4

Percentage (%) of carfilzomib dose detected in rat urine and bile following asingle intravenous bolus administration of carfilzomib at 2 mg/kg

Analyte Time

Mean � S.D.

Percentage (%)of Dose (Urine)

Percentage (%)of Dose (Bile)

h

Carfilzomib 0–4 0.032 � 0.021 0.017 � 0.0194–8 0.005 � 0.007 BQL8–24 0.003 � 0.004 NA

M14 0–4 18.6 � 10.6 12.9 � 2.34–8 3.86 � 2.75 0.230 � 0.1088–24 3.29 � 1.29 NA

M15 0–4 0.164 � 0.127 14.7 � 2.14–8 0.027 � 0.034 0.076 � 0.0568–24 0.045 � 0.006 NA

M16 0–4 0.127 � 0.100 2.64 � 3.094–8 0.027 � 0.045 0.013 � 0.0108–24 0.010 � 0.006 NA

Total 0–24 26 � 12 31 � 5

NA, not applicable (bile samples were not collected from 8 to 24 h); BQL, below thequantitation limit.

1880 YANG ET AL.

at ASPE

T Journals on D

ecember 17, 2014

dmd.aspetjournals.org

Dow

nloaded from

Discussion

In this study, we characterized the PK, pharmacodynamics (PD),metabolism, distribution, and excretion of carfilzomib, an irreversibleproteasome inhibitor, in rats, the rodent species used in pharmacologyand toxicology studies of the drug candidate. The results of these studiessuggest that carfilzomib displays several properties that may help toexplain the favorable clinical safety profile of this agent (Vij et al., 2010).

After intravenous bolus administration, carfilzomib was clearedrapidly from systemic circulation with a t1/2 value of �20 min. Theclearance is more rapid than that reported for bortezomib in preclin-ical and clinical models (Papandreou et al., 2004; Hemeryck et al.,2007). Furthermore, plasma clearance at all of the dose levels wasmuch higher than the reported hepatic blood flow for rats [55 ml/(min � kg)] (Davies and Morris, 1993), suggesting that, unlike bort-ezomib, carfilzomib clearance is mediated largely by extrahepaticmechanisms. We determined that the rapid clearance of carfilzomibwas not due to irreversible binding of the proteasome because pre-treatment with another irreversible proteasome inhibitor (ONX 0912)did not affect the clearance of carfilzomib. Furthermore, the clearanceof M16, which contains the peptide backbone of carfilzomib but lacksthe ability to irreversibly bind to the proteasome, was also higher thanthe hepatic blood flow in rats. In addition, the high plasma clearancewas not due to partitioning of carfilzomib to blood cells because thein vitro whole blood/plasma partitioning ranged from 0.64 to 0.82across a concentration range from 0.2 to 10 �M. Because carfilzomibis an irreversible inhibitor, target inhibition is a function of protea-some turnover and persists even after the drug is cleared systemically.Indeed, rapid clearance may prevent its ability to inhibit nonprotea-somal targets and thus reduce potential idiosyncratic toxicities.

The major metabolites in rat plasma, urine, and bile samples werefrom peptide and epoxide hydrolysis; oxidative metabolites wereobserved only at low levels (�0.1% of total dose in urine and bilesamples). In vitro, carfilzomib was metabolized rapidly in a variety oftissues and blood also via hydrolysis. Together with the rapid (�10 s)formation of these hydrolysis metabolites in vivo, these data suggestthat metabolism of carfilzomib occurs immediately upon administra-tion and likely occurs systemically throughout the body. These me-tabolites lack the epoxyketone pharmacophore and therefore have noactivity as proteasome inhibitors. Their further processing to individualamino acids also suggests that they have little toxicological impact invivo. A minor metabolite (Phe-Leu-Epoxyketone, M11), resulting frompeptidase cleavage, was detected in rat plasma, urine, and bile samples.The low abundance of M11 may be attributed to rapid secondary metab-olism to phenylalanine (M7), tyrosine (M5), leucine diol (M8), and a lowlevel of oxidated leucine epoxyketone (M2). We showed recently thatdipeptide epoxyketone, morpholino-Phe-Leu-epoxyketone, is not an ac-tive proteasome inhibitor and has no activity against serine proteases inan activity-based profiling assay (Zhou et al., 2009; Arastu-Kapur et al.,2011). Therefore, the low level of M11 is unlikely to contribute to PD orinduce off-target toxicity. Because peptide epoxyketones are highly se-lective for proteasome active sites, these findings suggest that the PDeffect of carfilzomib is being driven by the parent compound (Mirabellaet al., 2011). We also have noted rapid clearance and extrahepaticmetabolism in the form of peptide and epoxide hydrolysis in humans, andsimilar to rats, the peptidase cleavage product M14 is the most abundantcirculation and excretion metabolite in humans (Z. Wang, J. Yang, and C.Kirk, manuscript in preparation). Therefore, exposure to carfilzomib isunlikely to be altered in patients with hepatic impairment or takingconcomitant medications that alter cytochrome P450 activity.

Carfilzomib also distributes rapidly to tissues after intravenous admin-istration as demonstrated by potent proteasome inhibition in a variety of

tissues. Potent proteasome inhibition in different tissues (except brain)after intravenous bolus administration has been described previously(Demo et al., 2007). In this study, we determined that a 30-min infusionof carfilzomib at 8 mg/kg resulted in equivalent levels of proteasomeinhibition in blood and tissues despite a 28-fold lower Cmax (1.6 versus42.9 �M) than that with intravenous bolus administration at the samedose. Therefore, we conclude that the in vivo potency of this agent is aconsequence of total dose administered but not Cmax. As has beendescribed with other classes of covalent inhibitors, these data indicate thatthe time course of target inhibition by carfilzomib is not determineddirectly by its plasma exposure (Singh et al., 2011). Furthermore, wefound that bolus administration of carfilzomib at 8 mg/kg resulted in amortality rate of approximately 44% (14 of 32 animals), whereas a30-min infusion of the same dose was well tolerated generally and did notresult in mortality (0 of 24 animals; J. Jiang and C. Kirk, unpublishedobservations). The higher incidence of mortality with bolus administra-tion is likely associated with the high Cmax. Thus, intravenous infusionmight be an alternative route of administration to bolus injection toimprove the therapeutic window of carfilzomib. We have demonstratedcurrently that carfilzomib can be administered safely up to 56 mg/m2 inMM patients with intravenous infusion, which is more than 2-fold themaximum tolerated dose for an intravenous bolus administration (Papa-dopoulos et al., 2010).

Despite the wide tissue distribution of carfilzomib, the apparent Vss

values for carfilzomib were relatively small (Table 1), 0.3, 0.3, and0.6 l/kg at 2, 4, and 8 mg/kg, respectively, when administered via anintravenous bolus. With intravenous infusion administration at 8 mg/kg, the measured Vss was 2 l/kg. It appears that the Vss value waslower at low doses (2 and 4 mg/kg). At 8 mg/kg, Vss obtained fromintravenous infusion was higher than that from bolus administration.The specific reason for this observation was not clear. However, theapparent Vss values may not truly reflect the tissue distribution of anirreversible inhibitor. Moreover, as described in the preceding sec-tions, carfilzomib is highly metabolized in a variety of tissues bypeptidase and epoxide hydrolase. This portion of carfilzomib wascounted as elimination rather than as distribution.

Previously and in this study, we have noted that the liver was relativelyinsensitive to proteasome inhibition compared with adrenal, blood, heart,and lung (Demo et al., 2007). However, radioactivity in the liver at 0.5 hpostdose is quite high (Demo et al., 2007). These disparate findings maybe attributed to the competition between drug-metabolizing enzymes andbinding to the proteasome within the liver. Indeed, the rate of carfilzomibdisappearance in liver was more rapid relative to those in blood and otherorgans (adrenal, heart, and lung), as demonstrated in the in vitro study (Fig.5; Supplemental Fig. S2). Therefore, we believe that a large portion of theradioactivity observed in rat livers represents radioactive metabolites.

Less than 1% of carfilzomib was excreted intact in urine and bile within24 h postdose in rats. Carfilzomib is eliminated mainly in the form ofmetabolites, particularly peptidase cleavage products M14 and M15. In aprevious report, high levels of radioactivity in bile and urine at 0.5 h postdosein the quantitative whole-body autoradiography study were noted (Demo etal., 2007), which likely represents the rapid elimination of radiolabeledmetabolites. However, the radioactivity did not represent M14 and M15because these two major metabolites do not contain the 3H-labeled phenyl-alanine group. The total recoveries of carfilzomib and monitored metabolites(M14, M15, and M16) in rat bile and urine and bile were 26 and 31% of thedose, respectively, for a total of 57% within 24 h postdose in the present studyusing nonradiolabeled carfilzomib (Table 4). The incomplete recovery of theadministrated carfilzomib may represent the incorporation of the amino acidsfrom the peptide backbone of the drug into biosynthetic pathways; retentionof carfilzomib bound to proteasomes in enucleated cells (erythrocytes andplatelets), which cannot turnover their proteasomes; and the observed dipep-

1881PHARMACOKINETICS OF CARFILZOMIB IN RATS

at ASPE

T Journals on D

ecember 17, 2014

dmd.aspetjournals.org

Dow

nloaded from

tides, minor metabolites, or undiscovered metabolites that were not quantifiedin the present study. Further studies are needed to better understand thecomplete fate of administered carfilzomib.

In summary, carfilzomib was cleared rapidly from systemic circulationafter intravenous administration to rats largely due to extrahepatic metabo-lism via peptidase cleavage and epoxide hydrolysis. Despite rapid clearance,carfilzomib showed potent proteasome inhibition in a variety of tissues,demonstrating widespread tissue distribution. Elimination occurred mainlyvia biliary and renal excretion in the form of metabolites rather than intactdrug. Together with the irreversible and rapid onset of target binding, the highclearance may provide an advantage in that unnecessary exposure to the drugis minimized and potential drug-related side effects may be reduced. Fur-thermore, the major metabolic pathways of carfilzomib result in toxicologi-cally insignificant metabolites (modified peptides and amino acids). Carfil-zomib is currently in late-stage clinical development, with encouraging safetyand efficacy profiles demonstrated with intravenous bolus and infusion ad-ministrations. The studies presented here help to shed light on the dispositionof this novel class of irreversible proteasome inhibitors.

Authorship Contributions

Participated in research design: Yang, Wang, Fang, Jiang, Bennett, Mo-lineaux, and Kirk.

Conducted experiments: Yang, Fang, Wang, Jiang, Zhao, and Kirk.Performed data analysis: Yang, Wang, Fang, Jiang, Zhao, Wong, and Kirk.Wrote or contributed to the writing of the manuscript: Yang, Wang, Wong,

and Kirk.

References

Adams J (2004) The proteasome: a suitable antineoplastic target. Nat Rev Cancer 4:349–360.Adams J, Behnke M, Chen S, Cruickshank AA, Dick LR, Grenier L, Klunder JM, Ma YT,

Plamondon L, and Stein RL (1998) Potent and selective inhibitors of the proteasome:dipeptidyl boronic acids. Bioorg Med Chem Lett 8:333–338.

Alsina M, Trudel S, Vallone M, Molineaux C, Kunkel L, and Goy A (2007) Phase 1 single agentantitumor activity of twice weekly consecutive day dosing of the proteasome inhibitorcarfilzomib (PR-171) in hematologic malignancies. Blood (ASH Annual Meeting Abstracts)110:411.

Arastu-Kapur S, Anderl JL, Kraus M, Parlati F, Shenk KD, Lee SJ, Muchamuel T, Bennett MK,Driessen C, Ball AJ, et al. (2011) Nonproteasomal targets of the proteasome inhibitorsbortezomib and carfilzomib: a link to clinical adverse events. Clin Cancer Res 17:2734–2743.

Bennett MK and Kirk CJ (2008) Development of proteasome inhibitors in oncology andautoimmune diseases. Curr Opin Drug Discov Devel 11:616–625.

Borissenko L and Groll M (2007) 20S proteasome and its inhibitors: crystallographic knowledgefor drug development. Chem Rev 107:687–717.

Bross PF, Kane R, Farrell AT, Abraham S, Benson K, Brower ME, Bradley S, Gobburu JV,Goheer A, Lee SL, et al. (2004) Approval summary for bortezomib for injection in thetreatment of multiple myeloma. Clin Cancer Res 10:3954–3964.

Chauhan D, Hideshima T, Mitsiades C, Richardson P, and Anderson KC (2005) Proteasomeinhibitor therapy in multiple myeloma. Mol Cancer Ther 4:686–692.

Ciechanover A (2005) Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat RevMol Cell Biol 6:79–87.

Davies B and Morris T (1993) Physiological parameters in laboratory animals and humans.Pharm Res 10:1093–1095.

Demo SD, Kirk CJ, Aujay MA, Buchholz TJ, Dajee M, Ho MN, Jiang J, Laidig GJ, Lewis ER,Parlati F, et al. (2007) Antitumor activity of PR-171, a novel irreversible inhibitor of theproteasome. Cancer Res 67:6383–6391.

Goy A, Younes A, McLaughlin P, Pro B, Romaguera JE, Hagemeister F, Fayad L, Dang NH,Samaniego F, Wang M, et al. (2005) Phase II study of proteasome inhibitor bortezomib inrelapsed or refractory B-cell non-Hodgkin’s lymphoma. J Clin Oncol 23:667–675.

Groll M, Kim KB, Kairies N, Huber R, and Crews CM (2000) Crystal structure of epoxomicin:20S proteasome reveals a molecular basis for selectivity of alpha’,beta’-epoxyketone protea-some inhibitors. J Am Chem Soc 122:1237–1238.

Hanada M, Sugawara K, Kaneta K, Toda S, Nishiyama Y, Tomita K, Yamamoto H, Konishi M,and Oki T (1992) Epoxomicin, a new antitumor agent of microbial origin. J Antibiot45:1746–1752.

Hemeryck A, Geerts R, Monbaliu J, Hassler S, Verhaeghe T, Diels L, Verluyten W, vanBeijsterveldt L, Mamidi RN, Janssen C, et al. (2007) Tissue distribution and depletion kineticsof bortezomib and bortezomib-related radioactivity in male rats after single and repeatedintravenous injection of 14 C-bortezomib. Cancer Chemother Pharmacol 60:777–787.

Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of LaboratoryAnimals, 7th ed, Institute of Laboratory Animal Resources, Commission on Life Sciences,National Research Council, Washington DC.

Jagannath S, Vij R, Kaufman JL, Martin T, Niesvizky R, Gabrail NY, Alsina M, Wong AF, LeMH, McCulloch L, et al. (2010) Long-term treatment and tolerability of the novel proteasomeinhibitor carfilzomib (CFZ) in patients with relapsed and/or refractory multiple myeloma (R/RMM). Blood (ASH Annual Meeting Abstracts) 116:1953.

Jakubowiak AJ, Martin T, Singhal SB, Wang M, Vij R, Jagannath S, Lonial S, Kukreti V, Buadi

F, Bray L, et al. (2010a) Responses and survival are not affected by cytogenetics in patientswith relapsed and refractory multiple myeloma (R/R MM) treated with single-agent carfil-zomib. Blood (ASH Annual Meeting Abstracts) 116:1942.

Jakubowiak AJ, Dytfeld D, Jagannath S, Vesole DH, Anderson TB, Nordgren BK, Lebovic D,Stockerl-Goldstein KE, Griffith KA, Hill MA, et al. (2010b) Carfilzomib, lenalidomide, anddexamethasone in newly diagnosed multiple myeloma: initial results of phase I/II MMRC trial.Blood (ASH Annual Meeting Abstracts) 116:862.

Kim KB, Myung J, Sin N, and Crews CM (1999) Proteasome inhibition by the natural productsepoxomicin and dihydroeponemycin: insights into specificity and potency. Bioorg Med Chem Lett9:3335–3340.

Kisselev AF and Goldberg AL (2001) Proteasome inhibitors: from research tools to drugcandidates. Chem Biol 8:739–758.

Kuhn DJ, Chen Q, Voorhees PM, Strader JS, Shenk KD, Sun CM, Demo SD, Bennett MK, vanLeeuwen FW, Chanan-Khan AA, et al. (2007) Potent activity of carfilzomib, a novel,irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models ofmultiple myeloma. Blood 110:3281–3290.

Martin T, Singhal SB, Vij R, Wang M, Stewart AK, Jagannath S, Lonial S, Jakubowiak AJ,Kukreti V, Bahlis NJ, et al. (2010) Baseline peripheral neuropathy does not impact the efficacyand tolerability of the novel proteasome inhibitor carfilzomib (CFZ): results of a subsetanalysis of a phase 2 trial in patients with relapsed and refractory multiple myeloma (R/RMM). Blood (ASH Annual Meeting Abstracts) 116:3031.

Meng L, Kwok BH, Sin N, and Crews CM (1999a) Eponemycin exerts its antitumor effectthrough the inhibition of proteasome function. Cancer Res 59:2798–2801.

Meng L, Mohan R, Kwok BH, Elofsson M, Sin N, and Crews CM (1999b) Epoxomicin, a potentand selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc Natl AcadSci USA 96:10403–10408.

Mirabella AC, Pletnev AA, Downey SL, Florea BI, Shabaneh TB, Britton M, Verdoes M,Filippov DV, Overkleeft HS, and Kisselev AF (2011) Specific cell-permeable inhibitor ofproteasome trypsin-like sites selectively sensitizes myeloma cells to bortezomib and carfil-zomib. Chem Biol 18:608–618.

O’Connor OA, Wright J, Moskowitz C, Muzzy J, MacGregor-Cortelli B, Stubblefield M, StrausD, Portlock C, Hamlin P, Choi E, et al. (2005) Phase II clinical experience with the novelproteasome inhibitor bortezomib in patients with indolent non-Hodgkin’s lymphoma andmantle cell lymphoma. J Clin Oncol 23:676–684.

Orlowski RZ, Stinchcombe TE, Mitchell BS, Shea TC, Baldwin AS, Stahl S, Adams J, EsseltineDL, Elliott PJ, Pien CS, et al. (2002) Phase I trial of the proteasome inhibitor PS-341 inpatients with refractory hematologic malignancies. J Clin Oncol 20:4420–4427.

Orlowski RZ, Nagler A, Sonneveld P, Blade J, Hajek R, Spencer A, San Miguel J, Robak T,Dmoszynska A, Horvath N, et al. (2007a) Randomized phase III study of pegylated liposomaldoxorubicin plus bortezomib compared with bortezomib alone in relapsed or refractory multiplemyeloma: combination therapy improves time to progression. J Clin Oncol 25:3892–3901.

Orlowski RZ, Stewart K, Vallone M, Molineaux C, Kunkel L, Gericitano J, and O’Connor OA(2007b) Safety and antitumor efficacy of the proteasome inhibitor carfilzomib (PR-171) dosedfor five consecutive days in hematologic malignancies: phase 1 results. Blood (ASH AnnualMeeting Abstracts) 110:409.

Papadopoulos K, Siegel DS, Singhal SB, Infante JR, Sausville EA, Gordon MS, Kauffman M,Woo T, Lee S, Bui L, et al. (2010) Phase 1b evaluation of the safety and efficacy of a30-minute IV infusion of carfilzomib in patients with relapsed and/or refractory multiplemyeloma. Blood (ASH Annual Meeting Abstracts) 116:3024.

Papandreou CN, Daliani DD, Nix D, Yang H, Madden T, Wang X, Pien CS, Millikan RE, Tu SM,Pagliaro L, et al. (2004) Phase I trial of the proteasome inhibitor bortezomib in patients with advancedsolid tumors with observations in androgen-independent prostate cancer. J Clin Oncol 22:2108–2121.

Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin D, Rajkumar SV,Srkalovic G, Alsina M, Alexanian R, et al. (2003) A phase 2 study of bortezomib in relapsed,refractory myeloma. N Engl J Med 348:2609–2617.

Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA, Facon T, Harousseau JL,Ben-Yehuda D, Lonial S, Goldschmidt H, et al. (2005) Bortezomib or high-dose dexameth-asone for relapsed multiple myeloma. N Engl J Med 352:2487–2498.

Siegel DS, Martin T, Wang M, Vij R, Jakubowiak AJ, Jagannath S, Lonial S, Kukreti V, BahlisNJ, Alsina M, et al. (2010) Results of PX-171-003-A1, an open-label, single-arm, phase 2 (Ph2) study of carfilzomib (CFZ) in patients (pts) with relapsed and refractory multiple myeloma(MM). Blood (ASH Annual Meeting Abstracts) 116:985.

Singh J, Petter RC, Baillie TA, and Whitty A (2011) The resurgence of covalent drugs. Nat RevDrug Discov 10:307–317.

Singhal SB, Siegel DS, Martin T, Vij R, Wang M, Jakubowiak AJ, Lonial S, Kukreti V, ZonderJA, Wong AF, et al. (2010) Pooled safety analysis from phase (Ph) 1 and 2 studies ofcarfilzomib (CFZ) in patients with relapsed and/or refractory multiple myeloma (MM). Blood(ASH Annual Meeting Abstracts) 116:1954.

Uttamsingh V, Lu C, Miwa G, and Gan LS (2005) Relative contributions of the five major humancytochromes P450, 1A2, 2C9, 2C19, 2D6, and 3A4, to the hepatic metabolism of theproteasome inhibitor bortezomib. Drug Metab Dispos 33:1723–1728.

Venkatakrishnan K, Rader M, Ramanathan RK, Ramalingam S, Chen E, Riordan W, TrepicchioW, Cooper M, Karol M, von Moltke L, et al. (2009) Effect of the CYP3A inhibitorketoconazole on the pharmacokinetics and pharmacodynamics of bortezomib in patients withadvanced solid tumors: a prospective, multicenter, open-label, randomized, two-way crossoverdrug-drug interaction study. Clin Ther 31:2444–2458.

Vij R, Kaufman JL, Jakubowiak AJ, Stewart AK, Jagannath S, Kukreti V, McDonagh KT, AlsinaM, Bahlis NJ, Belch A, et al. (2010) Carfilzomib: high single agent response rate with minimalneuropathy even in high-risk patients. Blood (ASH Annual Meeting Abstracts) 116:1938.

Zhou HJ, Aujay MA, Bennett MK, Dajee M, Demo SD, Fang Y, Ho MN, Jiang J, Kirk CJ, LaidigGJ, et al. (2009) Design and synthesis of an orally bioavailable and selective peptideepoxyketone proteasome inhibitor (PR-047). J Med Chem 52:3028–3038.

Address correspondence to: Christopher J. Kirk, Onyx Pharmaceuticals, 249E Grand Ave., South San Francisco, CA 94080. E-mail: ckirk@onyx-pharm.com

1882 YANG ET AL.

at ASPE

T Journals on D

ecember 17, 2014

dmd.aspetjournals.org

Dow

nloaded from