1521-009X/44/1/115–123$25.00 http://dx.doi.org/10.1124/dmd.115.066290DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:115–123, January 2016Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Human Intestinal Raf Kinase Inhibitor Protein (RKIP) CatalyzesPrasugrel as a Bioactivation Hydrolase s

Miho Kazui, Yuji Ogura, Katsunobu Hagihara, Kazuishi Kubota, and Atsushi Kurihara

Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd. (M.K., K.H., A.K.), and Daiichi Sankyo RDNovare Co., Ltd. (Y.O., K.K.), Tokyo, Japan

Received July 14, 2015; accepted November 4, 2015

ABSTRACT

Prasugrel is a thienopyridine antiplatelet prodrug that undergoesrapid hydrolysis in vivo to a thiolactone metabolite by humancarboxylesterase-2 (hCE2) during gastrointestinal absorption. Thethiolactone metabolite is further converted to a pharmacologicallyactive metabolite by cytochrome P450 isoforms. The aim of thecurrent study was to elucidate hydrolases other than hCE2 involvedin the bioactivation step of prasugrel in human intestine. Using size-exclusion column chromatography of a human small intestinal S9fraction, another peak besides the hCE2 peak was observed to haveprasugrel hydrolyzing activity, and this protein was found to have amolecularweight of about 20 kDa. This prasugrel hydrolyzingproteinwas successfully purified from a monkey small intestinal cytosolicfraction by successive four-step column chromatography andidentified as Raf-1 kinase inhibitor protein (RKIP) by liquid

chromatography-tandem mass spectrometry. Second, we evalu-ated the enzymatic kinetic parameters for prasugrel hydrolysis usingrecombinant humanRKIP and hCE2 and estimated the contributionsof these two hydrolyzing enzymes to the prasugrel hydrolysisreaction in human intestine, which were approximately 40% forhRKIP and 60% for hCE2. Moreover, prasugrel hydrolysis wasinhibited by anti-hRKIP antibody and carboxylesterase-specificchemical inhibitor (bis p-nitrophenyl phosphate) by 30% and 60%,respectively. In conclusion, another protein capable of hydrolyzingprasugrel to its thiolactone metabolite was identified as RKIP, andthis protein may play a significant role with hCE2 in prasugrelbioactivation in human intestine. RKIP is known to have diversefunctions inmany intracellular signaling cascades, but this is the firstreport describing RKIP as a hydrolase involved in drug metabolism.

Introduction

Prasugrel (marketed as Effient in the United States and as Efient inEurope, Japan, and other countries) is a third-generation thienopyridineantiplatelet prodrug (Angiolillo et al., 2008) indicated for the reductionof thrombotic cardiovascular events in patients with acute coronarysyndrome who are being treated by percutaneous coronary intervention.Prasugrel was not detected in the plasma, urine, or feces after oraladministration of prasugrel to humans because it is rapidly absorbedand extensively metabolized in human (Farid et al., 2007). Prasugrelundergoes rapid hydrolysis in vivo to a thiolactone metabolite, which isfurther converted to a pharmacologically active metabolite by cyto-chrome P450 isoforms (Fig. 1). This rapid hydrolysis reaction ofprasugrel may contribute to the rapid onset and the low individualvariability of pharmacologic effect of prasugrel compared with otherthienopyridine antiplatelet agents such as clopidogrel (Tcheng andMackay, 2012).We previously reported, based on in vitro experiments, that prasugrel

was converted to a thiolactone metabolite by human carboxylesterase 2(hCE2) and human carboxylesterase 1 (hCE1) (Williams et al., 2008).

The hydrolysis of prasugrel was at least 25 times greater with hCE2than hCE1. Prasugrel hydrolysis by hCE2 exhibited substrate inhibitionat high substrate concentration, although this in vitro observationdid not translate to in vivo relevance (Williams et al., 2008). Alinear relationship has been shown between the prasugrel dose and theplasma exposure to thiolactone metabolite in human (Asai et al., 2006).Therefore, it was suggested that the formation of thiolactone metabolitefrom prasugrel was catalyzed by not only hCES but other unknownenzymes. The aim of this study was to identify the hydrolysis enzymesother than hCE2 involved in the hydrolysis of prasugrel in the intestine.Another aim was to determine the contribution of hCE2 and the newlyidentified enzyme to the hydrolysis of prasugrel to the thiolactonemetabolite in human intestine by estimating enzyme kinetic parametersand enzyme inhibition.

Materials and Methods

Materials. Prasugrel, thiolactone metabolites (racemate), and R-135766(internal standard for thiolactone assay) were synthesized by Ube Industries, Ltd.(Ube, Japan) (Supplemental Fig. S1). Protease inhibitor cocktail (cOmplete mini:mixture of serine, cysteine and metaloprotease inhibitors) was purchased fromRoche Applied Science (Mannheim, Germany). Bis p-nitrophenyl phosphate(BNPP) was purchased from Sigma-Aldrich Corporation (St. Louis, MO).Laemmli sample buffer, Flamingo fluorescent gel stain, and 5%–20% SDSacrylamide gel were purchased from Bio-Rad Laboratories, Inc. (Richmond,

The study was sponsored by Daiichi-Sankyo Co., Ltd, Tokyo, Japan.dx.doi.org/10.1124/dmd.115.066290.s This article has supplemental material available at dmd.aspetjournals.org.

ABBREVIATIONS: BNPP, bis p-nitrophenyl phosphate; CES, carboxylesterase; DMSO, dimethylsulfoxide; GFP, green fluorescent protein; hCE,human carboxylesterase; HRP, horseradish peroxidase; LC-MS/MS, liquid chromatography-tandem mass spectrometry; PVDF, polyvinylidenedifluoride; R-135766, (2Z)-(1-{2-[(2,2,3,3-2H4)cyclopropyl]-1-(2-fluorophenyl)-2-oxoethyl}-4-[(

2H3)methylsulfanyl]piperidin-3-ylidene)ethanoic acid;RKIP, Raf1-kinase inhibitor protein; thiolactone metabolite, 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone; TTBS, Tris-buffered saline containing 0.05% Tween 20.

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CA). Bovine serum albumin was purchased from Thermo Scientific Pierce(Rockford, IL).

Biologic Samples. Pooled mixed-gender human intestinal S9 was purchasedfrom Xeno Tech, LLC (Lenexa, KS), and four individual human small intestinalS9 (HIS-063-S3: 16.6 mg protein/ml, HIS-067-S3: 12.6 mg protein/ml, HIS-084-S3: 19.9 mg protein/ml, HIS-111-S3: 5.7 mg protein/ml) were purchasedfrom the nonprofit Human and Animal Bridging Research Organization (Chiba,Japan). Ethical approval was obtained from the ethics committee of DaiichiSankyo Co., Ltd. (Tokyo, Japan). FreeStyle 293F cells, cell culture media, and293fectin were purchased from Invitrogen (Carlsbad, CA). FLAG-M2 agarosewas purchased from Sigma-Aldrich. The mouse anti-human Raf Kinase InhibitorProtein (hRKIP) antibody was obtained from Invitrogen. For immunodepletion,rabbit anti-hRKIP antibodies and the control rabbit IgG were produced byImmuno-Biologic Laboratories Co., Ltd (Gunma, Japan).

A rabbit polyclonal antibody to RKIP (RKIP FL-187: sc-28837) waspurchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rabbitmonoclonal antibody to hCE2 was constructed in Immuno-Biologic Laborato-ries Co., Ltd. (Minneapolis, MN) Amersham (Piscataway, NJ) ECL-anti-rabbitIgG, horseradish peroxidase (HRP)–linked species-specific whole antibody[ECL-anti-rabbit IgG-HRP linked] and Amersham ECL Advance WesternBlotting Detection Kit were purchased from GEHealthcare UK Limited. PeptideN-glycosidase F (PNGase F, Lot 0360910) was obtained from New EnglandBiolabs Japan, Inc. (Tokyo, Japan).

Preparation of Monkey Small Intestinal Subcellular Fractions. Allexperimental procedures were performed in accordance with the in-houseguidelines of the Institutional Animal Care and Use Committee of DaiichiSankyo Co., Ltd. All preparation was conducted at 4�C. The cynomolgus monkey(HAMURI Co., Ltd., Ibaragi, Japan) was euthanized, and the small intestine wasremoved. The small intestine was homogenized in 9 volumes of homogenizationbuffer [10 mM HEPES-NaOH (pH 7.0), 0.25 M sucrose, and protease inhibitorcocktail] using a polytron. The homogenate was centrifuged at 9000g for30 minutes at 4�C, and the supernatant was used as the S9 fraction. Additionally,the S9 fraction was ultracentrifuged at 105,000g for 1 hour at 4�C, and thesupernatant and precipitate fractions were used as the cytosolic and the microsomefraction, respectively. The subcellular fractions were stored at 280�C until use.

Gel Filtration of Human Small Intestine S9 and Monkey Small intestineCytosol. Two hundred microliters of human small intestinal S9 fraction (10 mg/ml)was loaded onto a gel filtration column (Superdex 75; GEHealthcare, Pittsburgh,PA) and eluted with 20 ml of 100 mM HEPES (pH 7.0) at 0.5 ml/min with afraction size of 0.5 ml. Similarly, 200 ml of monkey small intestinal cytosolicfraction (10 mg/ml) was separated by the same experimental conditions.

Purification of Endogenous Enzyme. The cytosolic fraction equivalent to5 g of monkey small intestine was dialyzed against 20 mM sodium acetate(pH 6.0). The dialysate was loaded onto a 20-ml HiPrep heparin column(GE Healthcare) and eluted with a 20-ml linear gradient of 0–0.5 M NaCl. Eachfraction was tested for prasugrel hydrolysis activity and analyzed by immuno-blotting with the monoclonal antibody to hCE2. The flow-through fractions(no. 5–10) were pooled and applied to a 1-ml mono S 5/50 GL column (GEHealthcare). The bound proteins were eluted with a 30-ml linear gradient of0–0.5MNaCl. Each fraction was tested for prasugrel hydrolysis activity, and theactive fractions were pooled and dialyzed against 20 mM Tris-HCl (pH 9.0)buffer. The dialyzed sample was applied to a 1-ml mono Q 5/50 GL column (GEHealthcare), and proteins were eluted with 30 ml linear gradient of 0–0.5 MNaCl. Each fraction was tested for the prasugrel hydrolysis activity, and a part offractions was loaded on SDS-PAGE. The active fractions were dialyzed against20 mM sodium acetate (pH 6.0) and applied to a 0.24-ml mini S PC 3.2/3 column

(GE Healthcare). The column was eluted with a 7.2-ml linear gradient of0-0.35 M NaCl, and the presence of purified enzyme in eluted fractions wasconfirmed by prasugrel hydrolysis activity and SDS-PAGE.

Prasugrel Hydrolase Assay for Subcellular Localization, Gel Filtration,and Endogenous Enzyme Purification. The subcellular fraction at a finalconcentration of 1 mg of protein/ml or 30 ml of fractions from endogenousenzyme purification was mixed with prasugrel dimethylsulfoxide (DMSO)solution at a final concentration of 6 mM in a final volume of 50 ml of 100 mMHEPES buffer (pH 7.0). The mixture was incubated at 37�C for 15 minutesfollowed by adding 100 ml of methanol to terminate the reaction. The sampleswere filtrated by an Ultrafree-MC 0.45 mm PVDF membrane filter unit(Millipore, Billerica, MA), and 2 ml of the filtrate was injected into a liquidchromatography (LC)-mass spectrometry (MS) system to determine theconcentrations of the thiolactone metabolite.

In case of fractions of gel filtration separation, 30 ml of each fraction wastested as described with slight modification; that is, fractions were tested with orwithout 1 mM BNPP, a specific inhibitor of human carboxyl esterase (hCE);incubation time was extended to 60 minutes.

Electrophoresis. The monoQ and miniS active fractions were resolvedby 5%–20% SDS-PAGE, and the gel was stained with Flamingo (Bio-RadLaboratories, Inc.) and scanned by molecular imager FX (Bio-Rad Laboratories,Inc.) system.

Protein Identification by LC-MS/MS. For protein separation, the activefractions from the mini S PC 3.2/3 column were loaded on a 5%–20% SDS-PAGE gel. After staining of the gel with the Flamingo staining, each gel piecewas excised from the gel. The gel piece was subjected to in-gel reduction andalkylation, followed by trypsin digestion (modified trypsin; Promega, Madison,WI). The resulting peptides were extracted and sequenced with LC-MS/MS on aDiNa nano-flow liquid chromatography system (KYA Tech, Tokyo, Japan)coupled to a LTQ-Orbitrap (Thermo Fisher Scientific). The mass spectrometerwas operated in the data-dependent mode to automatically switch betweenOrbitrap-MS and LTQ MS/MS acquisitions. Orbitrap MS full scans wereacquired in the Orbitrap analyzer in using lock mass recalibration in real time.Resolution in the Orbitrap MS acquisition was set to r = 15,000. The tandem

Fig. 1. Simplified metabolic pathway for prasugrel. Figure does notillustrate all metabolites of prasugrel that have been identified. *Anasymmetric center. Prasugrel is a racemate, and thiolactone and theactive metabolite are the mixture of four stereoisomers.

Fig. 2. Localization of prasugrel hydrolase in monkey small intestine. Comparisonof hydrolysis activity of respective fraction. The small intestine subcellular fractionswere incubated with 6 mM prasugrel for 15 minutes at 37�C.

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mass spectra of the six most intense peptide ions with charge states $2 werecollected. Data from LC-MS/MS measurements were searched against theSwiss-Plot database using the Mascot algorithm (Matrix Science, Boston, MA)with the following parameters: trypsin specificity, two missed cleavage, cysteinecarbamidomethylation (fixed), protein N-term acetylated, methionine oxidationand asparagine, glutamine deamidated (variable), and electrospray ionization-iontrap fragmentation. The maximum allowed mass deviation for MS and MS/MSscans was 5 ppm and 0.8 Da, respectively. The resulting files were summarizedand rearranged by an in-house developed software iMAP2.

Expression and Purification of Recombinant Proteins. The expressionvector of hCE2 fused with carboxyl terminal FLAG tag was prepared asdescribed (Ishizuka et al., 2013). The expression vector of the N-terminal FLAG-tagged enhanced green fluorescent protein (GFP) was kindly provided byDr. Keisuke Fukuchi from Daiichi Sankyo Co., Ltd. Human RKIP was clonedfrom cDNA library of human 293F cells, and the expression vector was constructedwith N-terminal FLAG tag under cytomegalovirus promoter as described(Kubota et al., 2015). GFP, RKIP, and hCE2 expression vectors (30 mg) were

transfected into 293F cells (3 � 107 cells) using 293fectin according to themanufacturer’s protocol, respectively. The transfected cells were cultured for72 hours. The cell culture was centrifuged, and the collected cells were suspendedfor 5minutes on ice in lysis buffer [20mMTris-HCl (pH 7.5), 500mMNaCl, 0.1%NP-40]. The cell extracts were centrifuged, and the supernatant was used as a celllysate. Proteins were purified using their FLAG-tag by affinity chromatography.Five microliters of anti-FLAGM2 agarose was added to 300ml of the lysate. After2-hour incubation at 4�C, the resin was collected by centrifuge and washed threetimes with 500ml of lysis buffer. Proteins bound to the gel were eluted with 100mlof lysis buffer containing 0.1 mg/ml FLAG peptide. Each purified recombinantprotein (i.e., hCE2, RKIP, GFP as control) was subjected to 5%–20% SDS-PAGEand visualized by Flamingo staining, which confirmed more than 90% purity(Supplemental Fig. S2). Additionally, we evaluated prasugrel hydrolysis activitywith each recombinant protein.

Determination of the Enzymatic Kinetic Parameters for ThiolactoneFormation from Prasugrel. The assay was performed by using recombinantRKIP and hCE2. The incubation mixture contained 1 mg of protein per milliliter

Fig. 3. Chromatography of human small intestinal S9 and monkeysmall intestinal cytosolic fraction on Superdex 75 gel filtrationcolumn. We applied 200 ml of human small intestine S9 fraction (A)or monkey small intestine cytosolic fraction (B) to a Superdex 75 gelfiltration column. Each fraction was incubated with 6 mM prasugrelfor 60 minutes at 37�C in the presence or absence of BNPP. Thepositions of the native molecular weight markers are indicated at thetop.

Fig. 4. Chromatography of monkey small intestinal cytosol fractionon HiPrep Heparin column and Western blotting of each elution.Heparin fractions were assayed for prasugrel hydrolysis activity.Each fraction was incubated with 6 mM prasugrel at 37�C for60 minutes. (A) Hydrolysis activity was found in fractions flow-through (1–12) and bound (19–28). (B) Each fraction was analyzedby immunoblotting with the indicated antibodies. IN, input.

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of recombinant RKIP or 0.25 mg of protein per milliliter of recombinant hCE2and 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320, and 640 mM prasugrel in afinal volume of 200 ml of 0.1N HEPES buffer (pH 7.0). A mixture withoutprasugrel was preincubated at 37�C for 5 minutes, and the reaction was startedby the addition of 2 ml of a solution of prasugrel in DMSO. After incubation at37�C for 2 minutes, 50 ml of the incubation mixture was collected and added to100 ml of acetonitrile and 50 ml of a solution of R-135766 as the internalstandard (2 mM in acetonitrile) to terminate the reaction. The mixture wascentrifuged at 15,000 rpm at 4�C for 3 minutes, and 5 ml of the supernatant wasinjected into LC-MS/MS system to determine the concentration of thethiolactone metabolite.

Western Blot Analysis. Western blotting was performed to determine theamount of RKIP and hCE2 in four individual human small intestinal S9.

Assay of the Contents of RKIP in Human Small Intestinal S9.Recombinant RKIP (100 mg/ml) was diluted with 0.1N HEPES buffer (pH 7.0)and prepared at the concentration of 2.5, 5, 10, 15, and 20 mg/ml. Each humansmall intestinal S9 (n= 4)was prepared at the concentration of 0.8mgof protein permilliliter in the same way. These prepared samples and GFP as control werediluted twice with Laemmli sample buffer containing 5% mercaptoethanoland boiled for 5 minutes at 100�C. Ten microliters of boiled samples was loadedonto commercially available 12.5% SDS-PAGE (Ready Gel J, Bio-Rad Labora-tories, Inc.), respectively. The electrophoresis was performed at 140 V for80 minutes. The proteins were electroblotted onto a polyvinylidene difluoride(PVDF) membrane (Bio-Rad Laboratories, Inc.) using a blotter (Trans-blot, Bio-Rad Laboratories, Inc.) at 15 V for 40 minutes. The PVDFmembrane was blocked with blocking buffer (Tris-buffered saline containing0.05%Tween 20 (TTBS) and 2% skimmedmilk) for 1 hour at room temperature.After washing the PVDF membrane with TTBS, the PVDF membrane wasincubated with the antibody to RKIP (dilution 1: 400) for 1 hour at roomtemperature. After washing the PVDF membrane with TTBS for 30 minutes,the PVDF membrane was incubated with ECL anti-rabbit IgG-HRP-linked(dilution 1: 5000) for 1 hour at room temperature. After washing with TTBSand water, the PVDF membrane was incubated with coloring reagent(Lumigen TMA-6, Western blotting kits) for 5 minutes and analyzed usingLumino-Image analyzer (LAS-4000UVmini; Fujifilm Co., Ltd., Tokyo,Japan).

Assay of the Contents of hCE2 in Human Small Intestinal S9. PNGaseF-treated recombinant hCE2 (50mg/ml) was diluted with Laemmli sample buffercontaining 5% mercaptoethanol and prepared at the concentration of 1, 2.5, 5,7.5, and 10 mg/ml. Each human small intestinal S9 (n = 4) was prepared at theconcentration of 1.6 mg protein/ml with 0.1N HEPES buffer (pH 7.0). Thediluted human small intestinal S9 was treated with PNGase F. PNGase F-treatedhuman small intestinal S9 samples were diluted twice with Laemmli samplebuffer containing 5% mercaptoethanol and boiled for 5 minutes at 100�C. Tenmicroliters of boiled samples was loaded onto commercially available 7.5%

SDS-PAGE, respectively. The details of the Western blot analysis are describedherein.

In this Western blotting, the monoclonal antibody to hCE2 (dilution 1:5000)was used as the primary antibody and an ECL anti-rabbit IgG-HRP-linkedantibody (dilution 1: 20000) was used as the secondary antibody.

Determination of RKIP and hCE2 Contents using Lumino-ImageAnalyzer. The expression protein (ng/mg S9) of RKIP and hCE2 in humansmall intestinal S9 was determined using LAS-4000UVmini. The amount ofchemiluminescence (AU) of each enzyme was computed using Multi Gaugeversion 3.0 software (Fujifilm Corp.). The calibration curve was constructed bylinear least squares regression, plotting the amount of chemiluminescenceagainst each recombinant concentration. The calibration curve range was from12.5 ng/10 ml to 100 ng/10ml for RKIP and from 10.0 ng/10 ml to 100 ng/10mlfor hCE2.

Inhibition Study. Human small intestine S9 fractions (20 mg) wereincubated with the anti-RKIP antibody or the control rabbit antibody (20 mg)in TNN buffer (20 mM Tris (pH 8.0), 100 mM NaCl, 1% NP-40) overnight at4�C. The immune complexes were removed by incubation with 10ml of proteinG-Sepharose (GE Healthcare). Depletion of RKIP was monitored by immu-noblotting with anti-RKIP antibodies. The depleted S9 fractions (10 ml each)were mixed with Laemmli sample buffer containing 5% b-mercaptoethanoland subjected to 5%–20% SDS-PAGE and transferred to a PVDF membraneusing the iBlot dry blotting system (Invitrogen). The PVDF membrane wastreated with TBS-T (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1%Tween 20) containing 10% Aqua block (Rockland) for 1 hour at roomtemperature. The blot was sequentially incubated with anti-RKIP antibody(1:5000 dilution) or anti-b-actin antibody (1:5000 dilution) as primaryantibody and anti-mouse IgG antibody conjugated with horseradish peroxidaseas secondary antibodies (1:50,000 dilution; GE Healthcare). The membranewas visualized with the ECL plus or advance (GE Healthcare) and developedusing a NightOWL imaging system (Berthold Technologies GmbH, BadWildbad, Germany).

After that, the incubationmixture contained each depleted S9 fraction (i.e., therabbit IgG-depleted S9 fraction, RKIP-depleted S9 fraction) (0.2 mg/ml) in afinal volume of 100ml of 50 mMHEPES buffer (pH 7.0) with or without 0.1 mM

A B

Fig. 5. Purification of prasugrel hydrolase. (A) Prasu-grel activity on the mono Q column. Mono Q fractionswere incubated with 6 mM prasugrel for 60 minutes at37�C. Hydrolase activity was found in fractions 6–14.The mono Q fractions were also resolved by 5%–20%SDS-PAGE, and the gel was stained with Flamingo andscanned by molecular imager FX. The band of 21 kDa(indicated by arrows) was associated with the activity.(B) The mini S active fractions were resolved by SDS-PAGE, and the gel was stained with Flamingo. MiniS fractions were incubated with 6 mM prasugrel for60 minutes at 37�C.

Fig. 6. Identified amino acid sequence as prasugrel hydrolase using monkey smallintestinal cytosolic fraction. The active fractions from mini S were subjected to SDS-PAGE, and the band of 21 kDa was subjected to LC-MS/MS analysis. The peptidesfound to be identical to monkey RKIP are shown in bold (GenBank accession no.P48737). The identical peptides covered 96% of RKIP amino acid sequence.

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BNPP. A mixture without prasugrel was preincubated at 37�C for 2 minutes, anda reaction was started by adding 40 mM prasugrel in a DMSO solution. Thereaction mixtures were incubated at 37�C for 20 minutes. A 10-ml aliquot wasremoved and mixed with 90 ml of methanol/H2O (1:1, v/v) to terminate thereaction; 25 ml of this sample was diluted with 75 ml of methanol/ H2O (1:1, v/v)and injected into the LC-MS/MS system to determine the concentration ofthiolactone metabolite.

Assay of Thiolactone Metabolites by LC-MS/MS System. Thiolactonemetabolites and R-135766 were analyzed using the LC-MS/MS system asdescribed as follows Yeung et al., 2001. Separation by high-performance liquidchromatography was conducted using an Alliance 2695 Separations Modulehigh-performance liquid chromatography system (Waters Corp., Milford, MA)with an Octa Decyl Silyl column (Inertsil ODS-3, 150 � 2.1 mm ID, 5.0 mm;GL Sciences, Inc., Tokyo, Japan) at a flow rate of 0.2 ml/min with amobile phase consisting of methanol, distilled water, and trifluoroacetic acid[570/430 /0.5 (v/v/v) or 520/480 /0.5 (v/v/v)]. The column temperature was set at40�C, and the injection volume was 2 or 5 ml. The mass spectra were determinedusing a Quattro LC-MS/MS system (Micromass UK Ltd., Wilmslow, UK) inpositive-ion detection mode at the electrospray ionization interface. Theprecursor ions of the thiolactone [M + H]+ atm/z 332 and R-135766[M + H]+ atm/z 548 (for the internal standard) were obtained in the first quadrupole. Afterthe collision-induced fragmentation in the second quadrupole, the product ionof the thiolactone metabolite [M + H]+ at m/z 149 and R-135766[M + H]+ atm/z 206 was monitored in the third quadrupole. The peak area ratio of eachcompound to the internal standard was computed using MassLynx version 4.0SP4 (Waters Corp.) software.

Enzyme Kinetic Analysis. The reaction rate (V, pmol/min per microgram ofprotein) and V/the substrate concentration (S, mM) were calculated usingMicrosoft Office Excel 2003 (version SP2, Microsoft Corp.) according to eq. 1and eq. 2:

Vðpmol=min=mg  proteinÞ¼ generated  concentrationðmMÞ � 10001  or  0:25  mg  protein=mL� incubation  time ðminÞ

ð1Þ

VS¼ the  reaction  rateðpmol=min=mg proteinÞ

substrate  concentration  ðmMÞ ð2Þ

Initially the obtained data were analyzed by an Eadie-Hofstee plot(x-axis: V/S, y-axis: V). Eadie-Hofstee plots were used to visually detectdeviation from linearity. The formation of the thiolactone metabolites fromprasugrel by the recombinant hCE2 indicated a non–straight line in theEadie-Hofstee plot, suggesting involvement of the substrate inhibitionkinetic properties. Therefore, the data were fitted to eq. 3 using WinNonlinProfessional (version 4.0.1; Pharsight Corporation, St. Louis, MO) tocalculate the Km and Vmax values. S (mM) is the substrate concentration,Km (mM) is the Michaelis-Menten constant, Vmax (pmol/min per micro-gram of protein) is the maximal reaction rate and, Ki is the inhibitionconstant for the substrate:

V ¼ Vmax�1þ ðKm=SÞ þ ðS=KiÞ� ð3Þ

On the other hand, Km and Vmax for the recombinant RKIP were calculated byusing WinNonlin professional based on a pharmacodynamic compiled model(model no.101) since the best model was a Michaelis-Menten kinetics model.

Fig. 7. Kinetic analysis of the thiolactone produced from prasugrelusing recombinant RKIP and hCE2 by Eadie-Hofstee plots (A) anddirect plots (B).

TABLE 1

Enzymatic kinetic parameters of the thiolactone metabolite produced from prasugrelusing recombinant RKIP and hCE2

Enzymatic kinetic parameters are expressed as mean 6 S.E. of parameter estimate. Theformation of thiolactone metabolite from prasugrel in the recombinant hCE2 indicated a non–straight line in the Eadie-Hofstee plot, suggesting involvement of the substrate inhibition kineticproperties. Therefore, the data were fitted to eq. 3 to calculate the Km and Vmax values. S (mM) isthe substrate concentration, Km (mM) is the Michaelis-Menten constant, Vmax (pmol/min/mgprotein) is the maximal reaction rate, and Ki is the inhibition constant for the substrate. Km andVmax for the recombinant RKIP were calculated by using WinNonlin professional based on apharmacodynamic compiled model (model no.101).

Km Vmax Vmax/Km Ki

mM pmol/min/mg protein ml/min/mg protein

RKIP 49.9 6 7.96 14,114 6 647 283 N.D.hCE2 49.8 6 2.54 54,839 6 1510 1101 1380 6 498

N.D., no data.

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The contribution ratio (%) of RKIP and hCE2 that was responsible for theformation of the thiolactone metabolite in human small intestinal S9 wasdetermined by using eq. 4 and eq. 5:

CLintðmL=min=ng  enzyme  concentrationÞ

¼ Vmax � each  enzyme  concentrationðng=mg  human  small  intestinal  S9ÞKm

ð4Þ

The  contribution  ratioð%Þ ¼ CLint  for  each  enzyme � 100

+CLint  for  each  enzymeð5Þ

Additionally, the remaining hydrolysis activity (%) in the inhibition study wasdetermined by eq. 6 and eq. 7.

The  remaining  hydrolysis  activityð%Þ

¼ RKIP� depleted  S9 with  or without  BNPPðnmol=mg=minÞ � 100Control  antibody  treated  S9 without  BNPP  ðnmol=mg=minÞ

ð6Þ

The  remaining  hydrolysis  activityð%Þ

¼ Depleted  S9 with  BNPPðnmol=mg=minÞ � 100Control  antibody  treated  S9 without  BNPPðnmol=mg=minÞ ð7Þ

The calculated V and Vmax are expressed to an integral number. The calculatedKm is expressed to three significant figures. The mean values, S.D. of thecontribution ratio, and the remaining hydrolysis activity are also expressed tothree significant figures. If the calculated value exceeded three digits, however, itwas expressed to an integral number.

Results

Identification of Several Hydrolases in Human and MonkeySmall Intestine. To define subcellular localization of the hydrolaseinvolved in the prasugrel hydrolysis reaction, we determined theprasugrel hydrolysis activity using each subcellular fraction(i.e., homogenate, S9, microsome, cytosol) of monkey small intestine.The hydrolysis activity of prasugrel was localized mainly in thecytosolic fraction of the monkey small intestine (Fig. 2). Additionally,the gel filtration of human small intestinal S9 and monkey smallintestinal cytosolic fraction was performed using a Superdex 75 columnto confirm the characterization of expressing hydrolysis enzymes ofprasugrel in humans and monkeys (Fig. 3). Two hydrolysis activitypeaks of prasugrel were detected in the human small intestinal S9fractions. The first peak was observed at about 60-kDa molecularweight and the prasugrel hydrolase activity in this peak was inhibited by70% with BNPP, a carboxylesterase (CES)-specific chemical inhibitor.On the other hand, prasugrel hydrolase activity in the second peak wasnot inhibited with BNPP, and its molecular weight was estimated to beabout 20 kDa (Fig. 3A). These results suggested that the first peakactivity consists of CES2, and there was an unidentified hydrolase in thesecond peak. In the case of monkey small intestinal cytosolic fraction,a roughly similar result was obtained as human small intestinal S9fraction (Fig. 3B).Purification of Hydrolase. The monkey intestinal cytosolic fraction

was primarily subjected to the HiPrep heparin column, and twohydrolysis activity peaks of prasugrel were detected (Fig. 4A). Eachfraction of heparin chromatography was analyzed by Western blotting

A B

Fig. 8. Expression of RKIP and hCE2 in humansmall intestinal S9 by Western blot analysis. (A)Expression of RKIP in human small intestinal S9.Lanes 1, 2, 3, 4, and 5 are recombinant RKIP atthe concentrations of 12.5, 25, 50, 75, and100 ng/10 ml, respectively. Lane 6, 7, 8, and 9are individual human small intestinal S9. (B)Expression of hCE2 in human small intestinal S9.Lanes 1, 2, 3, 4, and 5 are recombinant hCE2at the concentrations of 10, 25, 50, 75, and100 ng/10 ml, respectively. Lanes 6, 7, 8, and9 are individual human small intestinal S9.

TABLE 2

Estimation of the contribution ratio of RKIP and hCE2 involved in the formation of the thiolactone metabolite from prasugrel in human small intestinal S9 by the enzymatickinetic parameters

The contents of RKIP and hCE2 in individual human small intestinal S9 (n = 4) were determined using recombinant RKIP and PNGase F-treated hCE2 by Western blotting method. The contributionratio of RKIP and hCE2 involved in the hydrolysis reaction of prasugrel in human small intestinal S9 was estimated by using the obtained contents data and enzyme kinetic parameters.

Vmax/Km Individual Human Small Intestinal S9 Enzyme Contents CLint Contribution Ratio (%)

ml/min/mg protein ng/human small intestinal S9 mg) ml/min/mg enzyme contents

RKIP

283 HIS-063-S3 14.9 4217 52.7 42.9 9.82HIS-067-S3 12.8 3622 29.4HIS-084-S3 15.6 4415 43.4HIS-111-S3 7.48 2117 46.2

hCE2

1101 HIS-063-S3 3.44 3787 47.3 57.1 9.82HIS-067-S3 7.91 8709 70.6HIS-084-S3 5.24 5769 56.6HIS-111-S3 2.24 2466 53.8

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using anti-CES2 antibody (Fig. 4B). It was shown that CES2 wasincluded in the bound fractions (Fig. 4B). Taken together, it was shownthat the unknown prasugrel hydrolase activity was separated from theCES2 catalyzed activity and eluted in flow-through fraction in theHiPrep heparin step. In the next step of mono-S fractionation of HiPrepflow-through sample, the unknown prasugrel hydrolysis activities wereobserved both in the flow-through and bound fraction (data not shown).The physical basis of this heterogeneity was not determined. The boundfraction, containing most of the activity, was used for the next step ofmono Q chromatography. SDS-PAGE and Flamingo staining of themono Q fractions revealed a band of 21 kDa associated with prasugrel

hydrolase activity (Fig. 5A). Compared with other fractions, fractions6 and 7 contained higher prasugrel hydrolyzing activity and only21 kDa of protein content was higher in these two fractions whereasother protein band contents were not. Because the mono Q activefraction contained several bands (Fig. 5A), we applied the mono Qactive fraction (bound fraction) next to the mini S column. As a result ofsuccessive four-step purification, our target unknown prasugrel hydro-lyzing protein was purified. SDS-PAGE and Flamingo staining of themini S active fractions revealed a single band of 21 kDa associated withprasugrel hydrolysis activity (Fig. 5B). Faint 60-kDa bands wereobserved but did not correlate with the activity. Those bands are wellknown to be human keratins, which were often contaminated in smallamounts during experiments and visualized by high-sensitivity stainingsuch as fluorescent dye and silver staining.Protein Identification by LC-MS/MS. To identify the protein

associated with prasugrel hydrolysis activity, the mini S active fractionswere subjected to SDS-PAGE, and the 21-kDa band was excised andsubjected to in-gel trypsin digestion. The fragmented peptides wereanalyzed by LC-MS/MS, identified by Mascot. Twenty-one peptidesmatched the amino acid coverage of 96% for monkey Raf-1 kinaseinhibitory protein (RKIP) (Fig. 6).Estimation of the Enzymatic Kinetic Parameters for Prasugrel

Hydrolysis by hRKIP and hCE2. The V and V/S values of prasugrelhydrolysis for recombinant hRKIP and hCE2 were determined accord-ing to eq. 1 and eq. 2; the Eadie-Hofstee plots of these data are shown inFig. 7. The enzyme kinetic parameters, Km and Vmax for recombinanthCE2, were estimated according to eq. 3 since the formation ofthiolactone from prasugrel indicated a substrate inhibition pattern in theEadie-Hofstee plots. On the other hand, Km and Vmax values forrecombinant RKIP were calculated byMichaelis-Menten model since itfit the data best, although we tried other models such as a substrateinhibition model. The CV (%) for the parameter estimates in theMichaelis-Menten model was the smallest of the models tested.In the formation of thiolactone metabolite from prasugrel using

recombinant RKIP, Km and Vmax were 49.9 6 7.96 mM and 14,114 6647 pmol/min per microgram of protein (Table 1). Similarly, in the caseof using recombinant hCE2, Km and Vmax were 49.8 6 2.54 mM and54,839 6 1510 pmol/min per microgram of protein (Table 1).Thiolactone formation from prasugrel in GFP was almost neverdetected (data not shown).Estimation of the Contribution of hRKIP and hCE2 Involved in

the Prasugrel Hydrolysis in Human Small Intestinal S9. Wedetermined the contents of hRKIP and hCE2 in individual human smallintestinal S9 by Western blotting method (Fig. 8). As results, RKIP andhCE2 in individual human small intestinal S9 were 7.48 (ng/mg S9)–15.6 (ng/mg S9) and 2.24 (ng/mg S9)–7.91(ng/mg S9), respectively. Fromthese data and enzyme kinetic parameters, it was estimated that thecontribution ratio of RKIP and hCE2 involved in the hydrolysis reactionof prasugrel in human small intestinal S9 was 42.9%6 9.82% (mean6S.D.) and 57.1% 6 9.82%, respectively (Table 2).

Fig. 9. Immunodepletion of RKIP from human small intestine S9 fraction. (A)Immunoblot analysis of the human small intestine S9 fractions depleted of RKIP. Theanti-RKIP antibodies or nonspecific rabbit IgGs were used to immunodeplete theRKIP. The RKIP-depleted S9 fraction was subjected to SDS-PAGE, followed byimmunoblotting analysis with the indicated antibodies. b-actin was used as an internalcontrol. (B) The relative prasugrel hydrolysis activity of the RKIP-depleted S9 fraction.The depleted S9 fractions were incubated with 40 mM prasugrel for 20 minutes at37�C in the presence or absence of 0.1 mM BNPP. The data are mean (n = 2).

TABLE 3

Immunodepletion of RKIP and effect of BNPP

The relative prasugrel hydrolase activity of RKIP depleted human small intestine S9. The depleted S9 fractions were incubated with 40 mM prasugrel for 20 minutesat 37�C in the presence of or absence of 0.1 mM BNPP.

Individual Human Small Intestinal S9Production of Thiolactone Form (nmol/mg protein/min) Remaining Hydrolysis Activity (%)

Control Anti-RKIP Treatment Control Anti-RKIP Treatment

BNPP (-) BNPP (+) BNPP (-) BNPP (+) BNPP (-) BNPP (+) BNPP (-) BNPP (+)

HIS-063-S3 54.5 33.4 34.8 11.2 100 61.4 63.8 20.6HIS-111-S3 50.5 18.8 34.2 8.09 100 37.3. 67.9 16.0

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Inhibition Study of Prasugrel Hydrolysis in Human SmallIntestinal S9. RKIP was immunodepleted from human small intestinalS9 fraction using the anti-RKIP antibody (Fig. 9A). This immunode-pletion of RKIP clearly inhibited prasugrel hydrolysis activity by about34.7%; inhibition by BNPP was about 50.7% (Fig. 9B; Table 3).

Discussion

Prasugrel and clopidogrel are both thienopyridine-type antiplateletprodrugs and both need to be bioactivated via a thiolactone interme-diate to their pharmacologically active metabolites (Farid et al., 2010).Prasugrel was converted more rapidly and more efficiently to thethiolactone metabolite compared with clopidogrel since for prasugrel,the thiolactone formation is via rapid ester group hydrolysis duringgastrointestinal absorption. Prasugrel itself is not detected in theblood circulation, but there is rapid appearance of the thiolactonemetabolite. On the other hand, conversion of clopidogrel to itsthiolactone metabolite is via hepatic cytochrome P450 oxidation,including CYP2C19, which is known to have large interindividualvariability. Furthermore, most of the clopidogrel undergoes hydrolysisof its ester group to form a carboxylic acid metabolite, which cannot beconverted to the active metabolite (Tang et al., 2006; Hagihara et al.,2009; Kazui et al., 2010). We previously reported that prasugrel wasconverted to the thiolactone metabolite primarily by hCE2, with a lessercontribution by hCE1, and that at high prasugrel concentrations inexcess of 109 mM, substrate inhibition was observed for hCE2(Williams et al., 2008). The prasugrel concentrations in the gastroin-testinal tract at the doses of 2.5, 10, and 75 mg are calculated to be 26.8,107, and 803 mM, respectively, assuming that these doses of prasugrelare dissolved in a standard glass of water (250 ml). If the metabolicenzyme involved in the formation of thiolactone in the small intestine isonly hCE2, the human exposure to thiolactone metabolite mightsaturate at more than 10-mg doses of prasugrel; however, the humanplasma exposure to the thiolactone metabolite after oral administrationof prasugrel at the doses of 2.5, 10, and 75 mg were dose dependent(Asai et al., 2006), meaning that the observed inhibition in vitro doesnot translate to in vivo relevance. Thus, we hypothesized anotherenzyme other than hCE2 might contribute to the prasugrel hydrolysis inthe intestine. We also tried to determine the contributions of hCE2 andunknown enzyme to the prasugrel hydrolysis process.As first step, we found that the prasugrel hydrolysis activity was

localized in the cytosolic fraction of monkey small intestine, in addition tomicrosome fraction, which contains most of CESs, suggesting that anotherprasugrel hydrolase exists in the cytosol fraction (Fig. 2). Accordingly, wecompared prasugrel hydrolysis activity profiles of human intestinal S9fraction and monkey intestinal cytosol fraction in size-exclusion columnchromatography, which resulted in almost the same profiles. In bothmatrices, first CES activity peak and second unknown activity peak wereobserved (Fig. 3, A and B). Therefore, further purification of the unknownenzyme was performed using monkey small intestinal cytosolic fractionsince human source availability was limited. As a result of successivefour-step column chromatography purification, target enzyme protein wassuccessfully purified. SDS-PAGE and Flamingo staining of the mini Sactive fractions revealed a single band of 21 kDa associated with prasugrelhydrolysis activity (Fig. 5, A and B). The fragmented peptides of the21-kDa band were analyzed by LC-MS/MS, identified by Mascot. As aresult, 21 peptides were found to match with the amino acid coverage of96% for RKIP (Fig. 6). RKIP is a widely known protein as a member ofthe phosphatidylethanolamine-binding protein family. It is a small,evolutionarily conserved cytosolic protein that plays a pivotal modula-tory role in several protein kinase signaling cascades (Bazzi et al., 1992;Yeung et al., 2000; Yeung et al., 2001; Corbit et al., 2003; Lorenz et al.,

2003). Additionally, has been reported that RKIP exerts a significantimpact on controlling the cell cycle and is also associated withcentrosomes and kinetochores in cultured mammalian cells (Al-Mullaet al., 2013). Taken together, RKIP play several roles in regulating theprocess of cell growth. It is already known that a lot of hydrolysisenzymes such as carboxylesterase, paraoxonase, butyrylcholinesterase,acetylcholinesterase, carboxymethylenebutenolidase, and albumin areinvolved in the bioconversion of ester-based prodrugs (Liedere andBorchardt, 2006; Ishizuka et al., 2013); however, it is not known thatRKIP plays a role of the drug-metabolizing enzyme (i.e., hydrolase).This study is, to the best of our knowledge, the first report describinghydrolase activity of RKIP.We determined the contributions of hRKIP and hCE2 for prasugrel

hydrolysis by estimating the enzymatic kinetic parameters of recombi-nant enzymes and by the inhibition study using human small intestinalS9. The experiment for estimating the enzymatic kinetic parametersshowed that both RKIP and hCE2 have similar Km values for thehydrolysis of prasugrel, suggesting that they bind prasugrel with similaraffinity (Table 1), but the Vmax value for hCE2 appears to be four timeshigher than that for RKIP (Table 1).From these enzyme kinetic parameters with the enzyme contents

determined by Western method, the contribution ratio of RKIP andhCE2 involved in prasugrel hydrolysis in the human small intestinal S9was estimated to be 42.9%6 9.82% and 57.1%6 9.82%, respectively(Table 2). Additionally, the prasugrel hydrolysis in human smallintestinal S9 was inhibited about 30%–40% by anti-RKIP antibodyand a further 40%–50% by BNPP (Table 3, Fig. 9B). The data from theinhibition study were consistent with the estimated contribution ratiofrom the enzyme kinetic parameters obtained using the recombinantenzymes. Therefore, we judged that the contribution of RKIP and hCE2to prasugrel hydrolysis in human intestine was about 30%–40% and60%, respectively.In conclusion, hRKIP was identified as capable of hydrolyzing

prasugrel to its thiolactone metabolite and may play a significant rolewith hCE2 in prasugrel bioactivation in the human intestine. RKIP isknown to have diverse functions as a master modulator of manyintracellular signaling cascades. This is the first report describing a newfunction of RKIP as hydrolase involved in drug metabolism.

Acknowledgments

The authors thank Dr. Mary Pat Knadler (Eli Lilly and Company) for helpfulcomments and discussion on this manuscript.

Authorship ContributionsParticipated in research design: Kazui, Kurihara.Conducted experiments: Kazui, Ogura, Kubota, Hagihara.Performed data analysis: Kazui, Ogura, Kubota.Wrote or contributed to the writing of the manuscript: Kazui, Kurihara.

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Address correspondence to: Miho Kazui, Drug Metabolism & PharmacokineticsResearch Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-Ku, Tokyo, 140-8710, Japan. E-mail: kazui.miho.yk@daiichisankyo.co.jp

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