vx-497: a novel, selective impdh inhibitor and immunosuppressive agent

VX-497: A Novel, Selective IMPDH Inhibitor andImmunosuppressive Agent

JUGNU JAIN, SUSAN J. ALMQUIST, DINA SHLYAKHTER, MATTHEW W. HARDING

Cell Biology and Immunology, Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, Massachusetts 02139

Received 18 July 2000; revised 26 October 2000; accepted 30 October 2000

ABSTRACT: Inosine monophosphate dehydrogenase (IMPDH) is an essential rate-limiting enzyme in the purine metabolic pathway, catalyzing the de novo synthesis ofguanine nucleotides required for lymphocyte proliferation. IMPDH has therefore beenan attractive target for developing immunosuppressive drugs (e.g., CellCept1andmizoribine). Here we describe the immunosuppressive activity of VX-497, a novelnoncompetitive inhibitor of IMPDH. VX-497 (MW 452.5) is orally bioavailable andinhibits the proliferation of primary human, mouse, rat, and dog lymphocytes atconcentrations of �100 nM. The inhibitory effect of VX-497 on lymphocytes is reversedin the presence of exogenous guanosine, but not in the presence of adenosine or uridine,con®rming that the antilymphocytic activity of VX-497 is speci®cally due to inhibition ofIMPDH. The antiproliferative effect of VX-497 in cells is also reversed within 48 h of itsremoval. Based on evaluation of VX-497 in several lymphoid and nonlymphoid cells, theantiproliferative effect of VX-497 is observed to be most pronounced on lymphoid andkeratinocyte cells as compared with ®broblasts. In vivo, oral administration of VX-497inhibits the primary IgM antibody response in a dose-dependent manner, with an ED50

value of �30±35 mg/kg in mice. Single daily dosing of VX-497 is observed to be aseffective as twice-daily dosing in this model of immune activation. These studiesdemonstrate that VX-497 is a potent, speci®c, and reversible IMPDH inhibitor thatselectively inhibits lymphocyte proliferation. ß 2001 Wiley-Liss, Inc. and the American

Pharmaceutical Association J Pharm Sci 90:625±637, 2001

Keywords: IMPDH inhibitor; VX-497; immunosuppression; MPA; lymphocytes; T cells

INTRODUCTION

IMPDH is an essential rate-limiting enzyme thatcatalyzes the NAD� -dependent oxidation of ino-sine 5 0-monophosphate (IMP) to xanthosine 5 0-monophosphate (XMP). XMP in turn is aminatedto form guanosine 5 0-monophosphate (GMP) inthe de novo pathway for biosynthesis of guaninenucleotides. Inhibition of IMPDH leads to thedepletion of the guanylate pools (GMP, GDP,

GTP, dGTP) in cells, with some reduction in theATP and dATP pools as well. Because lympho-cytes depend almost exclusively on the de novopathway for the synthesis of purine nucleotides,inhibition of IMPDH activity markedly limitstheir ability to synthesize DNA and proliferatein response to stimulation.1,2 Cells other thanlymphocytes can compensate for lack of de novosynthesis by the purine salvage pathway. Theimportance of the de novo pathway for nucleotidesynthesis in lymphocytes was suggested byimmunode®ciencies resulting from genetic muta-tions in either adenosine deaminase (ADA) orpurine nucleoside phosphorylase (PNP) enzymes,which cause severe reductions in both the numberand function of lymphocytes. Indeed, inhibitors

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 5, MAY 2001 625

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Journal of Pharmaceutical Sciences, Vol. 90, 625±637 (2001)ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association

of IMPDH enzyme, such as mycophenolic acid(MPA), selectively decrease the proliferation oflymphocytes, but not of other nonlymphoid cellsthat can utilize the salvage nucleotide synthesispathway.3 The addition of exogenous guanine orguanosine is able to overcome the inhibitory effectof MPA on lymphocyte proliferation, con®rmingthat the mode of action of inhibitors such as MPAis IMPDH-dependent.3

IMPDH is a homotetrameric enzyme consistingof 55 kDa subunits of 514 amino acids. There aretwo isoforms of IMPDH, type I and type II, thatare highly homologous both at the nucleotide andat the protein level.4,5 Although earlier reportsimplicated IMPDH type II expression as beingexclusively responsible for the increased proli-feration of leukemic and neoplastic cells, bothIMPDH type I and type II mRNAs have morerecently been shown to be induced on stimulation.Both genes are therefore likely to contribute tothe proliferative responses of lymphocytes.6 MPAinhibits both isoforms of IMPDH with inhibitoryrate constants (Ki) of 6±10 nM against recom-binant human type II enzyme, and 11±37 nMagainst human type I enzyme.7,8 The crystalstructure of Chinese hamster IMPDH type IIprotein complexed with an IMP reaction inter-mediate (XMP�) and MPA has been solved.9 MPAinhibits IMPDH by acting as a replacement forthe nicotinamide portion of the nicotinamideadenine dinucleotide cofactor and a catalytic watermolecule.

IMPDH expression has been observed to behigh in tissues of the immune system (thymus,spleen, bone marrow), in rapidly proliferatingcells (ovary and testis), and in neoplastic cells.2

Mitogenic stimulation of lymphocytes in vitroleads to an increase in IMPDH activity that isblocked by IMPDH inhibitors, including MPA andmizoribine.3,10,11 Mycophenolate mofetil (MMF orCellCept1), the ester prodrug of MPA, is approvedfor the prevention of acute rejection after kidneyand heart transplantation12,13 as part of a com-bination therapy including cyclosporin A andcorticosteroids. MPA or MMF have also beenreported to be effective in the treatment of rheu-matoid arthritis,14 lupus,15 and psoriasis.16±18

Mizoribine (MIZ, Bredinin1) is another immuno-suppressive drug that inhibits IMPDH activityand blocks mitogen-induced T cell proliferationthrough a reduction in intracellular guaninenucleotides.19,20 Mizoribine 5 0-monophosphate isa competitive inhibitor of both IMPDH type I andtype II enzymes, with Ki values of 8 and 4 nM,

respectively.8 Mizoribine is approved for the pre-vention of renal transplant rejection in Japan.Other IMPDH inhibitors include tiazofurin (2-b-D-ribofuranosylthiazole-4-carboxamide), which isconverted to the active moiety TAD (thiazole-4-carboxamide adenine dinucleotide) in cellsand acts as an NAD analogue, and ribavirin(Virazole1), which requires phosphorylation toribavirin 5 0-monophosphate to be active. MPAand tiazofurin have anti-leukemic properties.21

MPA and ribavirin also demonstrate broad-spec-trum antiviral activity.22±24 Ribavirin is currentlyapproved for the treatment of respiratory syncy-tial virus (RSV) infection as an aerosol formu-lation, and for hepatitis C in combination withIFN-a (RebetolTM).25±27

Despite the clinical ef®cacy of MMF andmizoribine, their therapeutic potential has beensomewhat limited by their unfavorable gastro-intestinal tolerability pro®le. Also, MPA is rapidlyconverted to an inactive glucuronide in vivo.20 Toovercome the limitations of current IMPDHinhibitors, new IMPDH inhibitors with bettertolerability were designed at Vertex Pharma-ceuticals based on the IMPDH crystal structure.9

VX-497 is a small, orally bioavailable moleculethat inhibits both isoforms of IMPDH enzyme,with Ki values of 7±10 nM (Cameron Stuver,personal communication). In this study, we pre-sent a detailed in vitro and in vivo evaluation ofthe pharmacological activity of VX-497 in severalcell types from several species.

MATERIALS AND METHODS

Reagents

Most cell culture reagents, unless speci®ed other-wise, were obtained from Gibco-BRL or JRHBiologicals. Complete RPMI was prepared byadding up to a ®nal of 10% fetal bovine serum(FBS), 55 mM b-mercaptoethanol, 50 units/mLpenicillin with 50 mg/mL streptomycin, 300 mg/mLL-glutamine, and 10 mM HEPES pH 7.5 to RPMI1640. Stock solutions of guanosine, adenosine,and uridine (all from Sigma) and phytohemagglu-tinin (PHA-P, Difco) were prepared in FBS-freeRPMI and stored at ÿ 20�C. Staphylococcal pro-tein A immobilized on Sepharose CL-4B (SPAS,Pharmacia) was freshly prepared on the day of theassay in complete RPMI. VX-497 (synthesized inhouse) and MPA (Sigma) were dissolved indimethyl sulfoxide (DMSO) at a concentration of

626 JAIN ET AL.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 5, MAY 2001

20 mM and stored at ÿ 20�C. Tritiated thymidineand leucine were purchased from DuPont NEN.

Isolation of Peripheral Blood MononuclearCells (PBMCs)

Human venous blood was drawn from normalhealthy volunteers using heparin as an anti-coagulant. PBMCs were isolated from blood bycentrifugation over Ficoll-paque gradient orCPT tubes (Becton-Dickinson) using standardconditions. PBMCs were harvested, washed andresuspended in complete RPMI, counted anddiluted to 1� 106 cells/mL. For preparing ratPBMCs, blood was drawn from healthy male ratsby heart puncture using heparin as anticoagu-lant. The isolation and enrichment of rat PBMCswas done as described for human PBMCs. Freshlydrawn, heparinized blood was obtained from twomale mongrel dogs at Tufts University School ofVeterinary Medicine (N. Grafton, MA). The bloodwas diluted 4-fold and the PBMCs isolated asdescribed for human PBMCs.

Isolation of Mouse and Rat Splenocytes

Intact spleens from C57Bl/6 male mice,Sprague-Dawley rats, or Lewis male rats weredissected and placed in cold Hanks balanced saltsolution (HBSS). The splenocytes were releasedfrom the spleens by gentle teasing between thefrosted ends of two microscope slides. The spleno-cyte suspension was centrifuged at 4�C, and thecells resuspended in HBSS. For counting thenumber of mononuclear cells, a hypotonic lysis ofred blood cells (RBCs) was done with an aliquot ofcells. The cell aliquot was brie¯y diluted 1:10 withwater, followed by an isotonic reconstitution with20�phosphate buffered saline (PBS). The rest ofthe cells were centrifuged at 300g for 10 minand resuspended in complete RPMI at 5� 106

cells/mL.

Cell Lines

All cell lines were obtained from American TissueCulture Collection (ATCC), unless noted other-wise, and cultured in the growth medium speci-®ed by ATCC. BJAB, a human EBV-negative Blymphoma cell line, and P3HR1, a humanEBV-positive Burkitt's lymphoma cell line, wereobtained from Dr. S. Sharma (Brown University,Providence, RI), and cultured in complete RPMI.The 8226/S human myeloma cell line (gift of

Dr. W.S. Dalton, University of Florida), wascultured in RPMI 1640 with 2 mM glutamineadjusted to contain a ®nal of 1.5 g/L sodiumbicarbonate, 20% FBS, 4.5 g/L glucose, 10 mMHEPES, 1 mM sodium pyruvate, and 50 units/mLpenicillin with 50 mg/mL streptomycin. Cell plat-ing densities stated are for 96-well plates.

Primary Cells

Normal human epidermal keratinocytes from twodonors were obtained from Clonetics pre-seededin 96-well plates at 10±50,000 cells/well. Normalhuman coronary artery endothelial (CAEC) andsmooth muscle cells (CASMC) were also obtainedfrom Clonetics. All cells were cultured in themedium recommended by Clonetics for thosecells. No mitogen was added in assays performedwith these primary cells.

PBMC and Splenocyte Proliferation Assays

Designated amounts of cells (5� 104 for humanPBMC T cells, 1� 105 for human PBMC B cells,1� 105 for rat PBMC T or B cells, or 1±2� 105 fordog PBMC T or B cells) were added per well to 96-well plates. For mouse and rat splenocytes,5� 105 mononuclear cells were plated. PHA wasadded to a ®nal concentration of 10±20 mg/mL perwell for stimulation of T cells. The mitogens SPAS(®nal concentration of 2 mg/mL per well; forhuman and canine) and LPS (®nal concentrationof 4 mg/mL per well; for murine and rat) were usedto stimulate B cells.

Serial 4-fold dilutions of VX-497 and MPAstocks were made in complete RPMI and addedto cells such that the ®nal concentration of com-pounds ranged from 20 mM to 20 nM, whereas thatof DMSO was maintained at a ®nal concentrationof 0.1%. All samples were tested in triplicate.Tritiated thymidine or leucine (0.4 mCi/well) wasadded for the last 24 h of the 3-day assay. The cellswere harvested onto Betaplate ®lters and countedin a scintillation counter. Concentrations of com-pounds required to inhibit proliferation of cells by50% (IC50 values) were calculated using theSoftMax Pro1 (Molecular Devices) computer soft-ware package.

Proliferation Assays using XTT Reagent

Cells were harvested and resuspended in phenolred-free complete medium. Three days afteraddition of the compounds, 20 mL of a solution

VX-497: A NEW INOSINE MONOPHOSPHATE DEHYDROGENASE INHIBITOR AND IMMUNOSUPPRESSANT 627


of 1 mg/mL XTT reagent (sodium salt of2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetr-azolium-5-carboxyanilide salt) containing 0.01mg/mL PMS (phenazine methosulfate), preparedfresh in phenol red-free complete medium, wasadded per well. The plates were incubated at 37�Cfor 4±5 h. The optical density of samples wasrecorded at 450±630 nM wavelength using amicroplate reader. The IC50 values were deter-mined using the SoftMax Pro1 (MolecularDevices) software.

Reversibility Experiments

PBMCs were cultured for the speci®ed length oftime with VX-497 or MPA and then centrifuged at300g for 5 min at room temperature. Theplates were ¯icked gently to remove medium-containing compounds. Then, 150 mL of freshmedium without compounds was added, and theplates were centrifuged and ¯icked as describedfor a total of three times. Fresh PHA and com-pounds (in the case of addback controls), wereadded after the third wash.

Plaque Forming Cell (PFC) Assay

Adult CD-1 mice (Charles River), sheep red bloodcells (SRBCs, Charles River Pharm Services),guinea pig complement (Gibco-BRL), agar (Difco),DEAE-dextran (Pharmacia), Labrasol1 (Gatte-Fosse, France), Cremophor-EL1 (Sigma), PEG400(Sigma), and propylene glycol (Sigma) wereobtained from speci®ed sources. Five to eightmice were tested in each treatment group. Thevehicle used for VX-497 and MPA was 10%Labrasol1, 10% Cremophor-EL1, 25% PEG400,25% propylene glycol, and 30% water. On dayone, 2.5� 108 SRBCs in saline were injectedintravenously in female CD-1 mice followed bythe oral administration of VX-497, MPA, orvehicle once or twice daily for 4 days. Spleenswere removed on day 5, and the splenocytes wereisolated and normalized to 1� 107 cells/mL. Twodilutions of cells were made in HBSS (1:4 and1:20). Splenocytes (200 mL) from either dilutionwere mixed with SRBCs (50 mL), complement(50 mL, 1:40 ®nal), and agar (700 mL, 0.6% ®nal)and plated as described by Eugui et al.10 Discreteareas of hemolysis or plaques were counted afterincubation of the Petri dishes at 37�C for at least3 h to overnight. Each sample was tested induplicate, and the number of PFC/106 spleen cellswas calculated.

RESULTS

Effect of VX-497 on Human LymphocyteProliferation

The chemical structure of VX-497 is novel andunrelated to other known IMPDH inhibitors(Figure 1). VX-497 inhibited the proliferation ofnormal human PBMC-derived PHA-stimulated Tcells, and SPAS-stimulated B cells in a dose-dependent manner (Figure 2 and Table 1). Theaverage IC50 value of VX-497 calculated from 16independent experiments was comparable inPHA-stimulated T and SPAS-stimulated B cells(100 and 137 nM, respectively), and was verysimilar to the IC50 value obtained with MPA(Table 1). The inhibition of proliferation byVX-497 could be overcome by the addition of50 mM guanosine, demonstrating that VX-497 is aspeci®c inhibitor of IMPDH. The IC50 valuesfor both T and B cells were comparable whetherVX-497 was added immediately or 48 h after theaddition of mitogens in the 3-day proliferationassay (data not shown). These results indicatethat VX-497 is a stable compound, otherwise itsIC50 value would have been lower when added at48 h. The results also indicate that VX-497 canprevent proliferation of cells at the late stage ofnucleotide and DNA synthesis during replication,similar to MPA which has been shown to block cellcycling at the stage of DNA synthesis. Values ofIC50 ranging from 20 to 100 nM have beenreported previously for MPA in human PBMCsstimulated with PHA3 and are similar to ourresults. The T and B cell proliferation assay wasalso performed in leucine-free medium, to whichtritiated leucine instead of tritiated thymidinewas added. The IC50 values obtained using uptakeof tritiated leucine as another marker of cellproliferation were in a similar range as thoseobtained with uptake of tritiated thymidine,con®rming that the inhibition of lymphocyteproliferation by VX-497 was not due to anynonspeci®c block in thymidine uptake (Table 2).

Figure 1. Structural formula of VX-497.

628 JAIN ET AL.


The speci®c inhibition of IMPDH activity byVX-497 was also demonstrated by the ability ofexogenous guanosine, but not of other purinenucleotides such as uridine or adenosine, toreverse the effect of VX-497 (Table 2 and datanot shown).

Reversibility of the AntiproliferativeActivity of VX-497

To examine whether the inhibitory effect ofVX-497 on lymphocytes was reversible over time,normal human PBMCs were stimulated withPHA and incubated with VX-497 for differentperiods of time. After incubation of the cells withVX-497 or MPA for 24 or 48 h, the compoundswere washed out from the medium by replacing itwith fresh medium. As a control for the washingout procedure, VX-497 or MPA was added back toone set of cells after being washed out at 24 h.PHA was added at the beginning of the assay aswell as after removal of the compounds. Tritiatedthymidine was added for the last 24 h, and thecells were harvested at 72 h. Incubation of thecells with VX-497 or MPA for just the ®rst 24 hof mitogenic stimulation did not result in any

signi®cant inhibition of proliferation (IC50 value> 20,000 nM, Table 3). Incubation of cells withVX-497 or MPA for longer periods, 36±48 h in a72-h assay, did inhibit cell proliferation signi®-cantly (Table 3, and data not shown). However,in experiments lasting 96 or 120 h, removal ofVX-497 from cell culture medium, even after38±48 h, allowed cells to proliferate. For example,the IC50 value for VX-497 increased 10-foldfrom 292 to 3000 nM when removed from themedium within 48 h of a 120-h assay (Table 3).

Figure 2. Dose±response curves of VX-497 and MPA for inhibition of T and B cellproliferation. Human PBMCs were stimulated with T (PHA), or B (SPAS) cell mitogensin the presence or absence of VX-497 or MPA for 72 h. Counts per minutes (cpms) oftritiated thymidine were plotted against indicated concentrations of VX-497 or MPA.Error bars represent standard deviation calculated in triplicate samples. The figureshows a representative experiment from > 30 independent experiments.

Table 1. IC50 Values (nM) of VX-497 and MPA inHuman PBMCs Stimulated with T or B Cell Mitogensin the Absence or Presence of Exogenous Guanosinea

Cells VX-497 MPA

PHA-stimulated T cells 104� 18 90� 11� 50 mM guanosine 5955� 1,000 > 20,000Fold reversal 58-fold > 215-foldSPAS-stimulated B cells 132� 21 127� 19� 50 mM guanosine 7126� 1,301 > 20,000Fold reversal 54-fold > 157-fold

aSummary of 16 independent proliteration assays usingthymidine incorporation as a readoct; mean and SEM areshown.



These results indicate that the antiproliferativeeffect of IMPDH inhibitors like VX-497 andMPA is reversed within �48 h of their removalfrom cellular medium.

Effect of VX-497 on Lymphocytes fromOther Species

The ability of VX-497 to inhibit proliferationof lymphocytes obtained from mice, rats, and dogsin response to mitogenic stimulation was alsotested. The IC50 values of �300 nM for VX-497

determined in mouse splenocytes (Table 4)were 2±3-fold higher than those obtained forhuman PBMCs treated with VX-497 or MPA(compare with Table 1). However, the IC50 valuesfor VX-497 in Sprague-Dawley or Lewis ratlymphocytes were similar to the �100 nM humanPBMC IC50 values (Table 4). The IC50 valuesreported for MPA in splenocytes isolated fromC57Bl/6 mice or Hla:(SD)BR rats have beenslightly lower than those observed here.10 Addi-tion of guanosine (50 mM) did not reverse theinhibition by VX-497 and MPA in both mouse andrat lymphocytes. Canine PBMCs were observed tobe very sensitive to both VX-497 and MPA(Table 4). The average IC50 values for VX-497from two independent dog studies were 39 nMfor PHA-stimulated T cells and 83 nM forSPAS-stimulated B cells. The ability of exogenousguanosine to overcome the effect of VX-497 andMPA was variable among individual dogs. Exo-genous guanosine was able to restore PBMCproliferation from one dog but not the other(data not shown). It is not clear whether differ-ences in metabolism between individual dogs, oramong different species, result in the differencesobserved in the ability of guanosine to reverse theantiproliferative effects of VX-497 and MPA.

Effect of VX-497 on Primary Cells and onImmortalized Cell Lines Derived fromLymphoid and Nonlymphoid Origins

To assess the range of speci®c cell types thatare sensitive to VX-497, several immortalizedlymphoid and nonlymphoid cell lines were tested.The IC50 values for VX-497 and MPA are sum-

Table 2. Specificity of the Anti-Proliferative Effect ofVX-497 and MPA

VX-497, MPA,Cells 50 mM 50 mM

T cells (3H-Thy) None 114 94�Guanosine 6460 > 20,000�Adenosine 125 100(3H-Leucine) None 182 196

B cells (3H-Thy) None 111 94�Guanosine 6167 > 20,000�Adenosine 139 101(3H-Leucine) None 229 214

aSpeci®city of the anti-proliferative effect of VX-497 andMPA. IC50 values (nM) were compared for VX-497 and MPA inhuman PBMCs stimulated with mitogens in the presence orabsence of exogenous guanosine versus adenosine. Guanosinebut not adenosine was able to reverse the effect of both IMPDHinhibitors. IC50 values obtained using cpms of 3H-Thy were alsocompared with those using 3H-Leu, and were similar for bothVX-497 and MPA.

Table 3. Reversibility of the Antiproliferative Effect of VX-497 and MPA in HumanPBMCsa

Duration of Length of incubationproliferation assay (h) with compound (h) VX-497 MPA

72 24� 48, add back 103 14472 24 > 20,000 > 20,00072 48 400 40772 72 143 190

120 48� 72, add back 292 448120 48 3000 > 20,000

aIC50 values (nM) were determined after incubation of PHA-stimulated human PBMCs withVX-497 or MPA for the indicated lengths of time using thymidine incorporation as the readout. Thecompounds were removed from the cellular medium in indicated wells at 24 or 48 h in a 72-h or120-h proliferation assay to allow for the resumption of cell proliferation. In some wells, thecompounds were removed but then added back as a control for the washing out procedure (24� 48,add back; 48�72 add back).

630 JAIN ET AL.


marized in Table 5 (established cell lines) andTable 6 (primary untransformed cells). The IC50

values were determined using two methods tomeasure cellular proliferation: uptake of tri-tiated thymidine that is incorporated in theDNA of proliferating cells, and the XTT assaythat measures the activity of the mitochondrialdehydrogenases as a function of viability andnumber of cells.

The L1210 leukemia cell line was observed tobe very sensitive to treatment with VX-497 orMPA (Table 5). The average IC50 value, calculatedfrom 17 independent experiments for VX-497, was449 nM in the XTT assay and 207 nM in thethymidine uptake assay. The IC50 value of 207 nMdetermined by the thymidine uptake method inspontaneously proliferating transformed L1210leukemia cells was therefore very similar to thoseobtained in normal human PBMCs stimulatedwith T or B cell mitogens (104 and 132 nM,respectively, Table 1). Exogenously added guano-sine was unable to restore proliferation of L1210cells (data not shown), which is consistent with

our earlier observations in murine and rodentlymphocytes (see Table 4). However, addition ofhigher concentrations of guanosine alone (up to250 mM) or of 50 mM guanosine in combinationwith 100 mM 8-amino-guanosine was able topartially reverse the antiproliferative effects ofVX-497 and MPA in L1210 cells (data not shown).8-Amino-guanosine is a competitive inhibitor ofPNP that increases the availability of dGTP.28 Inaddition to L1210 cells, VX-497 strongly inhibitedthe proliferation of all the T and B cell linestested, with IC50 values being similar to those inprimary T and B cells in thymidine uptake assaysand submicromolar in the XTT assay (see Table 1for primary cells, and Table 5 for cell lines). Itappears therefore that despite immortalization,lymphocytic cell lines are almost as sensitive toIMPDH inhibitors as normal untransformed peri-pheral blood lymphocytes.

Cell lines derived from the myeloid tissuesvaried in their sensitivity to VX-497. The mono-cytic cell line U937 was observed to be the mostsensitive (IC50 of 591 nM), whereas the 8226/S

Table 4. IC50 Values (nM) of VX-497 and MPA in Lymphocytes or Splenocytes fromDifferent Speciesa

Animal model Cells VX-497 MPA

Mouse splenocytes PHA-T cells 293 256� 50 mM gua. 311 175

LPS- B cells 308 214� 50 mM gua. 222 177

Sprague-Dawley PBMCs PHA- T cells 804 266� 50 mM Gua 236 100

LPS- B cells 126 107� 50mM Gua 108 93

Sprague-Dawley PHA- T cells 138 102Splenocytes � 50 mM Gua 155 128

LPS-B cells 277 176� 50 mM Gua 264 255

Lewis PBMCs PHA- T cells <20 <20� 50 mM Gua <20 23

LPS-B cells 75 54� 50 mM Gua 56 46

Lewis Splenocytes PHA- T cells 85 69� 50 mM Gua 98 80

LPS- cells 66 62� 50 Gua <20 43

Canine PBMCs PHA- T cells 39 32SPAS- B cells 83 80

aC57B1/6 mouse splenocytes (top panel), Sprague- Dawley and Lewis rat splenocytesand PBMCs (middle panel), and dog PBMCs (lower panel) were stimulated with T (PHA) or B(LPS, SPAS) cell mitogens in the presence or absence of VX-497 or MPA. Exogenous guanosine wasadded to determine the reversibility of the anti-proliferative effect of VX-497 or MPA. IC50 valueswere calculated using thymidine incorporation by proliferating cells.



cells showed intermediate sensitivity (1.3 mM)in the XTT assay (Table 5). Using thymidineuptake as the measure of cellular proliferation, allmyeloid cell lines were observed to be quitesensitive to VX-497 as well as MPA. However,inhibition of myeloid cell proliferation required�2-fold higher concentrations of VX-497 thanthose required for inhibiting lymphocyte proli-feration. The erythroleukemia cell line K562 wasobserved to be very sensitive to both VX-497 andMPA, with IC50 values of 277 and 397 nM,respectively, despite being reported to be insen-

sitive to MPA (IC50 > 10 mM).3 Several humancolon carcinoma cell lines were also sensitive toVX-497, but the IC50 values of �1.3±2 mM in XTTand thymidine uptake readouts were again muchhigher than those observed for the leukemia orlymphoma cell lines treated with VX-497. TheC32 melanoma cell line was also strongly in-hibited by VX-497 and MPA.

The antiproliferative effect of VX-497 wasevaluated in two human ®broblast cell lines, WI-38 and Hs-68. Both ®broblast cell lines appearedto be resistant to VX-497 and MPA, with IC50

values being > 20 mM in XTT assays. Theseresults demonstrate that VX-497 is not a cytotoxiccompound because it does not inhibit the pro-liferation of cells that can utilize the salvagepathway to satisfy their requirement for purinenucleotides.

Effect of VX-497 on Primary Cell Types

To examine the effect of VX-497 on keratinocytes,spontaneously proliferating normal human adultkeratinocytes from two donors were incubatedwith VX-497 or MPA for 3 days and assayed forproliferation using the XTT and thymidineuptake assays (Table 6). Keratinocytes from both

Table 5. IC50 Values (nM) of VX-497 and MPA, Measured by the Uptake of Tritiated Thymidine or the XTT Assayin Cell Lines Derived from Different Tissuesa

VX-497 MPA# of cells/well

Cell line cell (x104) 3H-Thy XTT 3H-Thy XTT

T cell lines L1210 1 207 449 77 359Jurkat 1±5 636 636EL-4 1 315 235

B cell lines P3HR1 2 176 401 147 621BJAB 1±5 470 405Raji 2±2.5 180 645 48 298

Monocytic cell lines U937 0.5 353 591 218 606THP-1 4 499 309

Pro-myelocytic HL-60 6 366 2290 252 979Myeloma cell line 8226/S 1 1347 1791Erythro-leukemiacell line K562 1 277 397Colon carcinoma HCT-15 0.25±0.5 2117 2146 1251 814cell lines LoVo 2.5 1318 1647 1062 1474

Caco-2 0.25±1 1312 1796 1303 1261Melanoma cell line C32 1 649 378Fibroblast WI-38 0.5 > 20,000 > 20,000cell lines Hs-68 0.1±0.5 > 20,000 > 20,000

aDifferent cell types were incubated with VX-497 or MPA for 72 hours and evaluated for growth using either the XTT assay (XTT)or thymidine (3H-Thy).

Table 6. IC50 Values (nM) of VX-497 and MPA inUnstimulated Primary Human Keratinocytes,Coronary Artery Smooth Muscle Cells (CASMC), andCoronary Artery Endothelial Cells (CAEC)

VX-497 MPA

Cells 3H-Thy XTT 3H-Thy XTT

Keratinocytes 105 578 96 537� 50 mM Gua 1979 5437 928 > 20,000

CASMC 220 870 279 1235CAEC 2843 12,361 488 14,680

632 JAIN ET AL.


donors were found to be very sensitive to VX-497,with IC50 values approaching those for humanblood lymphocytes (Table 6 and data not shown).The inhibition of proliferation was partiallyreversed with exogenous guanosine, suggestingthat the mechanism of action is due to inhibitionof IMPDH activity by VX-497 and MPA. Normalhuman smooth muscle cells from the coronaryartery (CASMC), and endothelial cells from thecoronary artery (CAEC) were also tested for theirsensitivity to VX-497 and MPA (Table 6). The IC50

values for smooth muscle cells were only 2-foldabove those for normal human lymphocytes,using thymidine uptake as the readout for bothIMPDH inhibitors. Human smooth muscle cellshave been reported to be inhibited by MPA byAllison and Eugui,1 with an IC50 value of�300 nM.Proliferation of normal human arterial endo-thelial cells appears to be relatively less sensitiveto IMPDH inhibitors, in particular to VX-497.Whereas the IC50 value for MPA in endothelialcells was 488 nM, similar to its reported IC50

value of 560 nM,3 the IC50 value for VX-497 inendothelial cells was 2.8 mM as measured bythymidine incorporation (Table 6).

Effect of VX-497 on Antibody Production In Vivo

Based on the good immunosuppressive pro®le ofVX-497 observed against T and B cells in vitro,VX-497 was evaluated in vivo in a murine plaqueforming cell assay (PFC), a model of primary anti-body production in response to an antigenicstimulation. VX-497 inhibited plaque formationin the PFC assay in a dose-dependent manner(Figure 3a). The percents inhibition observed withVX-497 compared with vehicle were 43, 77, and96% for the 25-, 50-, and 100-mg/kg doses, respect-ively. Some differences in response were observedbetween individual animals in the low-dose groupof 25 mg/kg, but the variability was much lower inhigher dose groups. These results demonstratethat VX-497 was pharmacologically active atdoses of 25±100 mg/kg. MPA was also ef®caciousat doses similar to VX-497. Percents inhibition ofplaque formation with MPA were 22, 76, and 99%at the 25-, 50-, and 100-mg/kg doses, respectively.From these results, the ED50 value was calculatedto be �30±35 mg/kg for both VX-497 and MPA.This ED50 value is in concordance with the ED50

value of 25±35 mg/kg published for MPA in asimilar PFC assay.10 The inhibition of plaqueformation was statistically signi®cant at all dosesof VX-497 tested and at the 50- and 100-mg/kg

doses of MPA, with a p value of <0.05 using theStudent t test (Figure 3a). The mice tolerated alldoses of VX-497 very well, and gained weightsimilarly to the vehicle-treated mice. The micehad normal spleen sizes, and splenocyte numbersper spleen were comparable between vehicle-treated and VX-497-treated groups (data notshown).

Given the potent pharmacological activity ofVX-497 observed when dosed twice daily in thePFC assay, a single daily dosing schedule ofVX-497 was compared with the twice-daily dosingschedule. As shown in Figure 3b, single dailydosing of VX-497 was almost as effective as twice-daily dosing in inhibiting the number of plaque-forming cells. In this experiment, the percents

Figure 3. Efficacy of VX-497 in murine plaque form-ing cell assays. (a) Vehicle, VX-497 or MPA were dosedorally twice daily (b.i.d.) at the indicated concentrationsin mice challenged with SRBC antigens. Antibody secret-ing cells were enumerated as plaques as described inMaterials and Methods, and normalized as number ofPFC per million spleen cells. (b) Vehicle or VX-497 weredosed orally once (q.d.) or twice daily (b.i.d.) at theindicated concentrations in the murine PFC model. Thefinal daily dose with q.d. dosing was half that of b.i.d.dosing. Number of plaques in each treatment werecounted as in Figure 3a. The error bars representstandard deviations in both Figures 3a and 3b.



inhibition for VX-497 dosed twice daily werecalculated to be 9, 74, and 86% for 25-, 50-, and100-mg/kg, respectively. For single daily dosing,the percents inhibition of PFC were 34, 70, and84% for 25-, 50-, and 100-mg/kg, respectively.The percent inhibition values were statisticallysigni®cant using the Student t test at the 50- and100-mg/kg doses. Total number of plaques formedin mice dosed with vehicle alone once or twicedaily did not differ signi®cantly. The half-life ofVX-497 in CD-1 mice is rather short, �40±60 min(Caroline Decker, personal communication).Although the half-life of MMF is �2 h, secondarypeaks in plasma levels have been observed due toits glucuronidation followed by enterohepaticrecirculation, which prolongs its presence inblood.20,29 VX-497 does not undergo any suchmodi®cation. Hence, ef®cacy of VX-497 observedin the PFC assay with single daily dosing is mostlikely due to its inhibitory effect on lymphocyteproliferation persisting beyond its actual bloodconcentration.

Given that single daily dosing was almost aseffective as twice-daily dosing in the PFC assay,

additional PFC experiments were performed todetermine the optimal dosing regimen for VX-497.In these experiments, VX-497 was administeredonly during part of the PFC assay. Dosing ofVX-497 was either initiated or terminated atdifferent times (2nd, 3rd, or 4th day) after theSRBC injection on day 1. A dose of 100 mg/kgwas chosen because it reduced PFC numbers by85±95% in previous assays. The results areexpressed as PFC as percent of vehicle control(Figure 4).

As shown on the left-hand side graph in Figure4, dosing twice daily (b.i.d.) or once a day (q.d.)during the entire duration of the PFC assay wasvery effective. Dosing only for the ®rst 3 days ofthe PFC assay was also very effective, but thereduction in plaque numbers was observed to behigher with b.i.d. rather than q.d. dosing (15 versus43% of vehicle respectively). Dosing VX-497 onlyfor the ®rst 2 days after SRBC injection was nodifferent from dosing with vehicle alone. In thesecond PFC assay, administration of 100 mg/kgVX-497 during days 2±4 was very effective, bothfor b.i.d. and q.d. regimens. Similarly, dosing on

Figure 4. Efficacy of 100 mg/kg VX-497 dosed during part of PFC assay only. Dosingof VX-497 was stopped after 2, 3, or 4 days in the PFC assay (left panel) or initiated laterstarting on the 2nd, 3rd, or 4th day of the PFC assay (right panel). Vehicle was dosedb.i.d. for all four days of the PFC assay. The plaques were enumerated as described inMaterials and Methods, and expressed as percent of vehicle. Percent plaques for eachtreatment group are also indicated above the bars in the graphs.

634 JAIN ET AL.


days 3 and 4 alone in the PFC assay was also quiteeffective, with substantial reduction in PFC ascompared with vehicle. VX-497 dosed only on the4th day of the assay was only partly able to reduceplaque numbers; the decrease was only 30±40%that of vehicle. The inhibition of plaque formationwas statistically signi®cant at all dosing regimensof VX-497, with a p value of <0.01 using theStudent t test, except for Days 1±2 b.i.d. or q.d.(no statistical signi®cance) and for Day 4 b.i.d. orq.d. (p < 0.5). These results suggest that the peakof T cell proliferation is �48±72 h after SRBCinjection in the PFC assay, and that an IMPDHinhibitor like VX-497 is very effective in inhibitingthe formation of plaque forming cells if adminis-tered at that time. Dosing of VX-497 only duringthe activation phase of T cells by SRBC antigens(®rst �48 h) does not result in any signi®cantreduction in plaque numbers, and the effect ofdosing only during the ®rst 2 days of the PFCassays is completely washed out by the end of theassay on the 5th day. These results are in agree-ment with our earlier observations in humanPBMCs where effect of VX-497 or MPA is washedout within 48 h. Furthermore, addition of VX-497or MPA 48 h after mitogen is as inhibitory aswhen added immediately with mitogens.

DISCUSSION

The aim of this study was to evaluate the potencyand selectivity of VX-497, a new non-nucleosideIMPDH inhibitor, in several cell types and in ananimal model, and to compare its im-munosuppressive pro®le with that of anotherclinically relevant IMPDH inhibitor, MPA. Theresults presented here demonstrate that VX-497is a potent, speci®c, and reversible IMPDHinhibitor. The cytostatic effect of VX-497 is mostpronounced on lymphocytes, correlating well withthe known dependence of lymphocytes on the denovo pathway for guanosine nucleotide synthesis.VX-497 is equipotent with MPA in its ability tolimit the proliferation of lymphocytes from allspecies tested in our study (human, mouse, rat,and dog). As expected for a speci®c IMPDHinhibitor, the addition of exogenous guanosine tothe growth medium reverses the inhibitory effectof VX-497 on lymphocyte proliferation. Thereversal of inhibition by VX-497 in the presenceof exogenous guanosine, > 50-fold in human bloodlymphocytes, demonstrates the high selectivity ofVX-497 towards its target. Furthermore, VX-497

is not an intrinsically toxic compound as judgedby its lack of toxicity in ®broblast cells that canutilize the salvage purine biosynthesis pathways.Our results suggest that the principal mechanismof action of VX-497 is the speci®c inhibition of theIMPDH enzyme, which exerts an acute anti-proliferative effect on lymphocyte proliferationdue to their almost exclusive dependence on thede novo pathway for synthesis of guanosine.Furthermore, the cystostatic effect of VX-497 isreversible because removal of VX-497 from cellsrestores their ability to proliferate within 48 h ofthe removal of compound.

Among the various cell types evaluated, VX-497 was observed to inhibit the proliferation of allimmortalized lymphoid cell lines. MPA has alsobeen shown to be effective against a number ofleukemia and lymphoma cell lines in vitro but notin vivo, presumably due to the formation of theinactive mycophenolate glucuronide.21, 29 Tiazo-furin however, has demonstrated antitumoractivity in clinical trials for leukemia.21 Hence,VX-497 may also be effective in the treatment ofleukemias and lymphomas. VX-497 also inhibitedthe proliferation of most myeloid, erythroid, andmelanoma cell lines. Treatment of K562 or C32cells with tiazofurin has previously been shown todecrease their proliferation as well as induceterminal differentiation.21 Hence, the ability ofIMPDH inhibitors to not only limit the growth oftransformed, neoplastic cells but to also inducedifferentiation and/or apoptosis may be a distinctadvantage in considering the use of VX-497 in thetreatment of malignancies like melanomas inaddition to hematological cancers.

MPA and its morpholino-ethylester prodrugMMF have both been shown to be effective in thetreatment of moderate to severe psoriasis,16, 18 adisease where abnormal keratinocyte prolifera-tion is mediated by cells of the immune systemthat cause exacerbation of the disease.17 Theef®cacy of MPA and MMF in psoriasis has beenproposed to result from the immunosuppressiveeffects of these drugs blocking an autoimmunestimulation of keratinocytes. We show here thatVX-497 and MPA have direct antiproliferativeeffect on normal human keratinocytes, suggestingthat the therapeutic effect of MPA in psoriasismay in part be due to its ability to directly inhibitkeratinocyte proliferation. These data provide astrong rationale for evaluating VX-497 in thetreatment of psoriasis. Indeed, results from aPhase II psoriasis trial with VX-497 supportthe therapeutic potential of IMPDH inhibitors



for treating psoriasis and other autoimmunediseases. VX-148, a distinct IMPDH inhibitor,has recently been selected by Vertex Pharmaceu-ticals as its lead drug development candidate forthe treatment of autoimmune diseases and iscurrently in preclinical trials.

In the PFC model, VX-497 was shown to beequipotent with MPA and to be highly effective ina single daily dosing regimen. To better under-stand the duration of effect of VX-497, differentdosing schedules of VX-497 were tested. In thisassay, injection of the T cell-dependent SRBCantigens at day 1 probably initiates a synchro-nous activation, followed by the proliferation of Tcells. The proliferative response probably begins�36±48 h after injection of SRBCs, analogous tothe in vitro stimulation of human PBMCs thatpeaks �48 h after the addition of mitogens orantigens. Administration of VX-497 during the®rst 48 h after SRBC injection did not result inany decrease in PFC, suggesting VX-497 does notinhibit the activation of T or B cells by SRBCantigens. In fact, PFC numbers with dosing ofVX-497 on days 2±4 or 1±4 in the two differentPFC assays were very similar (Figure 4). How-ever, dosing of VX-497 on the 3rd day of the PFCassay was crucial, and a difference in b.i.d. versusq.d. dosing was observed at the day 3 time-points.Hence, the ef®cacy of VX-497 dosing regimencorrelated with the time of peak cell proliferation.Twice-daily dosing leading to a higher sustainedblood concentration of the inhibitor was moreeffective than single daily dosing at day 3 in bothPFC assays. These data support the use of a twice-daily dosing regimen for VX-497 in clinical studiesfor achieving its optimal effect in inhibitingrapidly proliferating cells.

In conclusion, the cellular potency and speci®-city of VX-497, combined with its excellent phar-macological activity as an orally dosed compoundin the PFC model, and in rat and dog transplantmodels,30,31 makes it a suitable candidate to beevaluated further as an immunosuppressive agentin autoimmune and transplant indications. Itsantiproliferative effect on leukemia, lymphoma,myeloma, and melanoma cells may be useful intreating speci®c cancers. Furthermore, the in-hibition of keratinocyte and smooth muscle cellproliferation suggests its potential bene®t intreatment of psoriasis, restenosis, and otherdiseases involving vascular arteriopathy. VX-497also demonstrates potent antiviral activity32

and is currently being evaluated in hepatitis Cclinical trials.

ACKNOWLEDGMENTS

We thank Elmar Nimmesgern and Ursula Ger-mann for many helpful suggestions during thecourse of these studies, Cameron Stuver,Olga Futer, and Caroline Decker for sharingunpublished results, Yongping Wang and AngieHeiser for technical help in the PFC assay, andJeff Saunders for Figure 1. We thank IMPDHchemists for providing VX-497 and are grateful tomany IMPDH team members for their help andsupport. We also thank Ursula Germann, ElmarNimmesgern, John Thomson, Vicki Sato, andSteve Lyons for critically reading the manuscript.

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vx-497: a novel, selective impdh inhibitor and immunosuppressive agent

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