Vol. 33, No. 12ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1989, p. 2069-20740066-4804/89/122069-06$02.00/0Copyright C 1989, American Society for Microbiology

In Vitro and In Vivo Activities of WIN 54954,a New Broad-Spectrum Antipicornavirus Drug

M. G. WOODS,'* G. D. DIANA,2 M. C. ROGGE,3 M. J. OTTO,1t F. J. DUTKO,1 AND M. A. McKINLAY1Departments of Virology and Oncopharmacology,l Medicinal Chemistry,2 and Drug Metabolism and Deposition,3

Sterling Research Group, Rensselaer, New York 12144

Received 20 June 1989/Accepted 20 September 1989

WIN 54954 (5-[5-[2,6-dichloro-4-(4,5-dihydro-2-oxazolyl)phenoxy]pentyl]-3-methylisoxazole) is a new mem-ber of the class of broad-spectrum antipicornavirus compounds known to bind in a hydrophobic pocket withinvirion capsid protein VP1. In plaque reduction assays, WIN 54954 reduced plaque formation of 50 of 52rhinovirus serotypes (MICs ranged from 0.007 to 2.2 ,ug/ml). A concentration of 0.28 ,Lg/ml was effective ininhibiting 80% of the 52 serotypes tested (EC..). WIN 54954 was also effective in inhibiting 15 commonlyisolated enteroviruses, with an EC80 of 0.06 ,Lg/ml. Furthermore, WIN 54954 was effective in reducing the yieldof two selected enteroviruses in cell culture by 90% at concentrations approximately equal to their MICs. Thetherapeutic efficacy of intragastrically administered WIN 54954 was assessed in suckling mice infected withcoxsackievirus A-9 or echovirus type 9 (Barty) 2.5 days prior to initiation of therapy. Single daily doses of 2 and100 mg/kg protected 50% of the mice from developing paralysis (PD..) following infection with coxsackievirusA-9 and echovirus-9, respectively. At the PD50 doses for these two viruses, levels of WIN 54954 in serum weremaintained above the in vitro MICs for a significant portion of the dosing interval. The dose-dependentreduction in viral titers observed in coxsackievirus A-9-infected mice correlated well with the therapeutic doseresponse. The potency and spectrum of WIN 54954 make it a potentially useful compound for the treatment ofhuman enterovirus and rhinovirus infections.

Picornaviruses are a family of small, single-stranded RNAviruses, which includes a number of significant humanpathogens. The rhinoviruses and enteroviruses are twogroups within the Picornaviridae family which cause a rangeof human viral syndromes. Rhinoviruses are a major causeof mild upper respiratory infection in humans (8). Theenteroviruses, which include the polioviruses, echoviruses,and coxsackieviruses, are associated with many syndromes,including poliomyelitis, myocarditis, aseptic meningitis, en-cephalitis, and upper respiratory tract infections (16). Atpresent, no effective antiviral agents are commercially avail-able for the treatment of the diseases caused by theseviruses.

Previous reports have shown that the antipicornaviralagent disoxaril (WIN 51711) is systematically active intreating enterovirus infections in mice (9, 10, 12). This classof compounds has been shown to bind in a specific hydro-phobic pocket within the capsid protein VP1 (4) and preventviral attachment or uncoating, depending on the picornavi-rus serotype involved (13). Based on these findings, furtherchemical synthesis was undertaken in an effort to increaseboth the spectrum and potency of this class of antiviralcompounds. WIN 54954 (Fig. 1) was synthesized (6) and, asdescribed in this report, found to be a potent inhibitor ofrhinovirus replication in vitro and of selected enterovirusesin vitro and in vivo.

MATERIALS AND METHODSMedia and solutions. The following media and solutions

were used: minimal essential medium, medium 199 (M-199),and 2x M-199 (Flow Laboratories, Inc., McLean, Va.),newborn calf serum and fetal calf serum (FCS) from GIBCO

* Corresponding author.t Present address: E.I. du Pont de Nemours & Co., Wilmington,

DE 19898.

Laboratories (Grand Island, N.Y.), 1% Seakem agarose inwater (FMC Corp., Marine Colloids Div., Rockland,Maine), 3 mg of DEAE-dextran per ml (Sigma Chemical Co.,St. Louis, Mo.; Mr 500,000), 3 M MgCl2, 5% gluteraldehydefor fixing, and 0.25% crystal violet for staining. For plaquereduction assays, a set of 200x stock solutions ofWIN 54954were prepared in dimethyl sulfoxide (DMSO) and diluted inM-199 to achieve final concentrations of 0.001 to 6.2 ,ug/ml.

Cells and viruses. Coxsackievirus A-9 was originally ob-tained from C. Wilfert, Duke University, N.C., and culturedin LLC-MK2 cells. Coxsackievirus B-3 was originally ob-tained from C. Guantt, University of Texas, San Antonio,and cultured in Vero cells. Echovirus types 11 and 9 wereobtained from the Wadsworth Center for Laboratories andResearch, Albany, N.Y., and cultured in RD-2 cells. Echo-virus type 9 (Barty) was originally obtained from RockefellerUniversity, New York, N.Y. A 10% suspension of tissuefrom suckling mice infected with echovirus type 9 (Barty)was made in M-199 medium. All other viruses were obtainedfrom the American Type Culture Collection, Rockville, Md.Human rhinoviruses were cultured in HeLa (Ohio) cells.Echoviruses and coxsackieviruses were cultured in RD-2cells. All cell cultures used in plaque reduction and virusyield reduction assays were grown in six-well 35-mm2 tissueculture plates (Costar, Cambridge, Mass.) in a 2% CO2atmosphere.

Plaque reduction assay. Medium was aspirated from con-fluent monolayers of cells and cells infected with 1.0 ml ofthe appropriate virus (approximately 80 PFU per well) inM-199 medium. The cultures were incubated for 1 h at 37°Cfor enteroviruses or at 33°C for rhinoviruses. The virusinoculum was removed, and the cells were overlaid withM199 containing 5% newborn calf serum or 5% FCS, 30 mMMgCl2, 0.5% agarose, and WIN 54954 at various concentra-tions. The overlay for human-rhinovirus-infected cells alsocontained 15 ,ug of DEAE-dextran per ml. Enteroviruses

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C1 ~~~~CH3

c N~~~~

ci

FIG. 1. Chemical structure of WIN 54954 (5-[5-[2,6-dichloro-4-(4,5-dihydro-2-oxazolyl)phenoxy]pentyl]-3-methylisoxazole).

were allowed to replicate (forming plaques) for 1 to 3 days at370C and rhinoviruses for 3 to 4 days at 33°C in a 2% CO2atmosphere. Cells were fixed with 5% gluteraldehyde andstained with 0.25% crystal violet. The concentrations ofWIN 54954 which inhibited plaque formation by 50% were

determined for each virus and recorded as the MIC. TheMICs determined in this manner had a standard deviation ofless than ±50%.

Virus yield reduction assay. Confluent 1-day-old monolay-ers of RD-2 cells were infected with coxsackievirus A-9 or

echovirus type 9 (Barty) at a multiplicity of 0.01 PFU per

cell. WIN 54954 (solubilized at 100x in DMSO) was diluted100-fold in the virus inoculum at the indicated concentra-tions. After 1 h at 37°C and 2% C02, the incoculum was

removed and the monolayers were washed once with me-

dium containing the appropriate concentration of WIN54954. Fresh medium (3 ml of M199 plus 5% FCS) containingWIN 54954 was added to each well, and the plates wereincubated at 37°C for 16 h for coxsackievirus A-9 or 24 h forechovirus type 9 (100% destruction of cell sheet by the viruscontrol) and then frozen at -70°C. The virus yield in eachwell was quantitated by plaque assay on monolayers of RD-2cells. No effort was made to remove drug before progenyvirus was plaque assayed. Other experiments (data notshown) have demonstrated reversible binding of drug tovirus. Thus, serial dilution into drug-free medium duringquantitation by plaque assay dissociates drug from virus. Inthe plaque assay, the number of plaques followed a dilutionresponse, indicating that drug carryover is not a problem.

Evaluation of cytotoxicity by measurement of cell growth.RD-2 cells were seeded in six-well tissue culture plates(Costar) with M-199 plus 5% FCS and grown for 24 h at 37°Cand 2% CO2 to result in 5 x 105 viable cells per well. WIN54954 dissolved in DMSO at 10Ox the final concentrationwas diluted 1:100 into M-199 plus 5% FCS. A 3-ml sample ofmedium containing WIN 54954 at the desired concentration,ranging from 0.3 to 66 ,ug/ml, was added to each well at day0. Control wells contained medium with 1% DMSO alone.On days 1 through 3, triplicate wells for each drug concen-tration and' controls were examined microscopically forobvious toxic effects. The growth medium was then removedand the cell layer was rinsed with 0.5 ml of a 1:1 mixture of4 mM disodium EDTA and 0.25% trypsin, followed byincubation with 2 ml of this mixture for 10 min at 37°C. Afterthis time, the cells were detached and separated by gentleagitation. Cell suspensions were counted in duplicate bytrypan blue extension and a hemacytometer. By day 3, theuntreated control cell number (1% DMSO) increased byapproximately 10-fold above the day 0 level.

Mice. Swiss female albino ICR mice (Blue Spruce Farms,Altamont, N.Y.) that had delivered babies within the last 24h were placed in plastic mouse boxes and provided withnesting material. The dams and pups arrived on the day theexperiment was initiated. The suckling mice were pooled,weighed, and distributed to nursing dams in groups of 10 to15. Each pup weighed between 1.2 and 1.9 g.

Infection of mice. Each mouse was infected subcutane-ously with 0.03 ml of diluted virus over the right shoulder

with a 0.5-ml Glaspak syringe and a 27-gauge, 0.5-in. (ca.1.25-cm) needle. All suckling mice were infected within 24 hof birth. Coxsackievirus A-9 was diluted in phosphate-buffered saline to give 10 50% lethal doses (LD50s; 1 LD50equals 1 PFU per mouse). Echovirus type 9 (Barty) wasdiluted in phosphate-buffered saline to give 10 LD50s (1 LD50equals 22 to 84 PFU per mouse).

Preparation of WIN compound, medication route, andregimen. WIN 54954 was prepared as a suspension in sterile1% gum tragacanth (Fisher Scientific). Suckling mice weremedicated intragastrically with the use of a 1-in. 24-guagePerfectum animal feeding needle (Popper and Sons Biomed-ical Instrument Division, New Hyde Park, N.Y.) on a 0.5-mlGlaspak syringe. The WIN 54954 suspension (0.03 ml) orplacebo (1% gum tragacanth) was administered once a day(q.d.) beginning 2.5 days postinfection and continuing for 5days. Five days of medication have been shown to besufficient (9). Longer medication regimens result in similarefficacy (data not shown). WIN 54954 is efficacious prophy-lactically (data not shown). The regimen used represents atherapeutic regimen, which is a stringent test of antiviralefficacy.

Observation. Mice were checked twice a day (b.i.d.) forevidence of flaccid limb paralysis. Paralyzed animals werenoted, and the percentage of nonparalyzed animals wascalculated for each dose level for each day of the test. The50% protective dose (PD50) values were calculated by probitanalysis (Proc. Probit Subroutine in SAS User's Guide;Statistics, 1982 ed., SAS Inc., Cary, N.C.).

Quantitation of virus titers in suckling mice. Titration ofcoxackievirus A-9 was performed on homogenized 'tissuefrom mice killed on day 5 after infection, when peak titerswere reached in control animals (data not shown). Mice weremedicated b.i.d. for 2.5 days beginning' 2.5 days postinfec-tion. Five mouse pups from groups medicated with 1.2, 3.7,11, and 33 mg of WIN 54954 per kg per day or placebo werekilled, and the carcasses were washed with sterile 0.85%NaCl. Ten percent suspensions of individual carcasses weremade in M-199 plus penicillin (100 U/ml) and streptomycin(100 mg/ml) with a Duall tissue grinder (Kontes, Vineland,N.J.). Homogenates were frozen and thawed and thencentrifuged to remove debris. The supernatants were placedin ampules and stored at -70°C for use in a standard tissueculture plaque assay with RD-2 cells (9).

Quantitation ofWIN 54954 levels in serum in suckling mice.Three-day-old suckling mice were medicated with a singleintragastric dose of WIN 54954 in 1% gum tragacanth at adose of 2, 14, or 100 mg/kg. At 1, 2, 4, 8, and 24 h afteradministration, groups of 20 mice per time interval wereexsanguinated. The blood was pooled and allowed to clot,and the serum was obtained by centrifugation. Serum levelsof WIN 54954 were measured by gas chromatography with aHewlett-Packard 5710A gas chromatograph equipped with anickel-63 electron capture and linked to a computerized dataacquisition system (Hewlett-Packard model 3357 LAS) forcollection and processing of data.

RESULTS

Inhibition of human rhinovirus and human enterovirusplaque formation. WIN 54954 was a potent inhibitor ofplaque production of 50 of the 52 human rhinoviruses tested(Table 1). The MICs ranged from 0.007 to 2.2 ,ug/ml. Onlyrhinovirus 5 and rhinovirus 8 were not susceptible to thecompound. A concentration of 0.28 ,ug/ml was effective ininhibiting 80% of the 52 serotypes tested (EC80). Plaque

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TABLE 1. Anti-human rhinovirus activity of WIN 54954

Human MIC Human MICrhinovirus rhinovirus (Rg/ml)serotype (gm)aserotype (L/I

36 0.007 67 0.112 0.009 28 0.1321 0.010 15 0.1378 0.012 6 0.1322 0.013 86 0.1489 0.015 32 0.1511 0.017 12 0.177 0.017 17 0.1838 0.018 25 0.1850 0.032 10 0.1833 0.040 39 (ATCC) 0.2030 0.040 66 0.2019 0.044 13 0.2316 0.048 27 0.2449 0.048 18 0.2647 0.048 31 0.2823 0.052 41 0.4144 0.055 72 0.6220 0.056 54 0.6524 0.056 14 0.8261 0.067 1A 0.9829 0.068 3 1.175 0.070 4 1.59 0.075 87 2.21B 0.09 8 NDb (>6.2)39 (strain T-39) 0.10 5 ND (>6.2)

a Defined as the concentration required to reduce plaque number by 50%.Values are the mean of at least two separate assays and a standard deviationof less than ±50%.

b ND, Not determined.

formation of 15 commonly isolated enteroviruses was inhib-ited by WIN 54954 (Table 2). The MICs ranged from 0.004 to0.43 ,ug/ml. The EC80 for the enterovirus serotypes was 0.06,ug/ml.

Effect of WIN 54954 on virus yield. The effect of WIN54954 on multiple rounds of replication of coxsackievirus

TABLE 2. Activity of WIN 54954 against 15 commonlyisolated enterovirusesa

Virus Serotype MICb (qg/ml)Echovirus 3 0.01

4 0.065 0.436 0.027 0.0049 0.0611 0.3624 0.0230 0.25

Coxsackievirus B-1 0.03B-2 0.007B-3 0.009B-4 0.02B-S 0.03

A-9 0.004a Most commonly isolated enteroviruses from 1970 to 1983 (15).b Defined as the concentration required to reduce plaque number by 50%

compared with the untreated control. Values are the mean of at least twoseparate assays and had a standard deviation of less than ±50%.

a0I-z0

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\ ECHOVIRUS TYPE 9

SACKIEVIRUS A-9

0.001 0.010 0.100 1.000WIN 54954 UG/ML

FIG. 2. Effect of WIN 54954 on the yield of two enteroviruses invitro. RD-2 cells were infected with the indicated virus in thepresence of the appropriate concentration of WIN 54954. Afterincubation of the cells at 37°C for 16 h (coxsackievirus A-9) or 24 h(echovirus type 9 [Barty]), the cells were frozen and thawed, and theyield of infectious virus was determined by plaque assay. Each pointis the average of two separate experiments.

A-9 and echovirus type 9 (Barty) is shown in Fig. 2. A doseof 0.003 p.g/ml resulted in a 90% reduction in coxsackievirusA-9 yield. A dose 10 times this concentration reduced thevirus yield by 106-fold and completely inhibited viral multi-plication. WIN 54954 at 0.14 pug/ml reduced echovirus type 9(Barty) yield by 90%. The maximum yield reductionachieved was 103-fold (99.6%) at 0.5 p,g/ml.RD-2 cell growth in the presence of WIN 54954. The in vitro

cytotoxicity of WIN 54954 was determined by assessing theeffects of the drug on RD-2 cell growth over a 3-day periodin two separate experiments (data not shown). In each case,the cell number in the untreated control (1% DMSO) in-creased approximately 10-fold above the day 0 level. Atconcentrations of WIN 54954 of 2.5 ,ug/ml or lower, therewas no effect ('11% inhibition) on cell growth at any timepoint (day 1, 2, or 3 after addition of WIN 54954). At 7 ,ug/ml,cell growth was inhibited by 50%, and at a concentration of22 ,ug/ml, cell growth was essentially arrested.

Oral efficacy against coxsackievirus A-9 infection in sucklingmice. Dose-dependent therapeutic efficacy of WIN 54954 inmice infected with coxsackievirus A-9 is shown in Fig. 3.The' combined results of three dose-response studies arepresented. The results are shown as the mean percentnonparalyzed animals on each day. Q.d. intragastric doses ofWIN 54954 beginning 2.5 days after infection prevented thedevelopment of paralysis in mice in a dose-dependent man-ner. On day 13 postinfection, only 10% of the placebo-medicated animals were symptom free, compared with 36,66, 87, and 95% of the mice medicated with WIN 54954 atdoses of 1.2, 3.7, 11, and 33 mg/kg per day, respectively.From the data in Fig. 3, the PD50 for WIN 54954 wasdetermined to be 2.0 mg/kg per day q.d.

Effect of WIN 54954 on coxsackievirus replication in suck-ling mice. WIN 54954 prevented the replication of coxsack-ievirus A-9 in suckling mice in a dose-dependent manner

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100

90

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FIG. 3. Oral therapeutic efficacy ofWIN 54954 in coxsackievirusA-9-infected suckling mice. Mice were medicated q.d. for 5 daysbeginning 2.5 days after infection with WIN 54954 at 1.2 (90 mice),3.7 (89 mice), 11 (90 mice), and 33 (60 mice) mg/kg per day or withplacebo (88 mice).

(Fig. 4). Suckling mice were medicated intragastrically b.i.d.(b.i.d. and q.d. dosing were equally efficacious) for 2.5 daysbeginning 2.5 days postinfection. The titers of coxsackievi-rus A-9 isolated from these mice were 7.9 x 106, 1.3 x 107,

8.0

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MG/KG/DAY OF WN 54954FIG. 4. Virus titers in coxsackievirus A-9-infected suckling mice

medicated orally with WIN 54954 b.i.d. for 2.5 days beginning 2.5days postinfection. Error bars indicate standard deviation.

70 100 MG/KG/DAY

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DAY POST-INFECTIONFIG. 5. Oral therapeutic efficacy ofWIN 54954 in echovirus type

9 (Barty)-infected suckling mice. Mice were medicated q.d. for 5days beginning 2.5 days after infection with WIN 54954 at 3.7 (85mice), 11 (88 mice), 33 (88 mice), and 100 (74 mice) mg/kg per day orwith placebo (87 mice).

1 x 105, 3.9 x 103, and 1.6 x 102 PFU per mouse at WIN54954 doses of 0, 1.2, 3.7, 11, and 33 mg/kg per day,respectively. To determine whether drug carryover in mousetissue was responsible for the reduction in virus titers,homogenized samples from WIN 54954-medicated mice (33mg/kg per day) were spiked with a known quantity of virus.All of the inoculated virus was recovered from these sam-ples.

In vitro and in vivo activities of WIN 54954 against echovi-rus type 9 (Barty). WIN 54954 inhibited plaque formation ofechovirus type 9 (Barty) by 50% at a concentration of 0.31,ug/ml [the MIC for echovirus type 9 (Barty), a mouse-adapted virus, is different from the MIC for echovirus type 9(ATCC) shown in Table 2]. The dose-dependent oral thera-peutic efficacy of WIN 54954 in suckling mice infected withechovirus type 9 (Barty) is shown in Fig. 5. The combinedresults of three dose-response studies are presented. Rever-sal of paralysis was seen in all dose groups, includingplacebo. Survival data on the day that the dose groupshowed the fewest number of symptom-free mice were usedto assess efficacy (worst-case analysis). Under these condi-tions, 7% of the placebo-treated mice were asymptomatic,compared with 10, 10, 33, and 55% of the mice medicatedwith WIN 54954 at doses of 3.7, 11, 33, and 100 mg/kg perday q.d., respectively. The PD50 for WIN 54954 againstechovirus type 9 (Barty) was determined to be 100 mg/kg perday q.d.Serum WIN 54954 concentrations in suckling mice. Serum

WIN 54954 levels in suckling mice medicated orally with 2,14, or 100 mg/kg are shown in Fig. 6. Maximum concentra-

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N.Ecm

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FIG. 6. WIN 54954 concentrations in serum following a singleintragastric dose of WIN 54954 in suckling mice. The minimalquantifiable level (MQL) was 0.020 jig/ml. Each point represents thevalue for pooled serum from 20 mice.

tions in serum were 0.12, 0.79, and 3.41 jig/ml for the 2-, 14-,and 100-mg/kg dose groups, respectively. The apparenthalf-life (t,12) values ranged from 2.4 h at 2 mg/kg to 4.9 h at14 mg/kg and to 8.8 h at 100 mg/kg. The areas under theserum concentration-time curve (AUC) were 0.48, 8.08, and36.09 jig. h/ml for the 2-, 14-, and 100-mg/kg doses, respec-

tively.

DISCUSSION

The data presented here demonstrate the in vitro and invivo efficacy of WIN 54954, a new broad-spectrum antipi-cornavirus drug. Compared with disoxaril, the previouscompound in this chemical class, a greater than 20-foldimprovement in spectrum and potency was observed against52 human rhinovirus serotypes, as evidenced by the drop inEC80 from >6.2 ,ug/ml for disoxaril to 0.28 ,ug/ml for WIN54954 (data not shown). WIN 54954 was also effective ininhibiting 15 commonly isolated enteroviruses, with an EC80of 0.06 ,ug/ml. Against two selected enteroviruses, WIN54954 was effective in reducing the yield of virus in RD-2cells by 90% at concentrations equal to or below the MICs.Replication of coxsackievirus A-9, a susceptible virus withan MIC of 0.004 ,ug/ml, was completely inhibited at 10 timesthe MIC. This antiviral activity is specific and not due to

cytotoxicity, since a dose of 2.5 ,ug ofWIN 54954 per ml hadno significant effect on RD-2 cell growth. The specificity wasexpected because this class of compounds bind in a specificsite within virion capsid protein VP1 (4). The hydrophobiccompounds may also associate with cell membranes, but thisassociation does not result in observable toxicity at antiviralconcentrations. Mode of action studies indicate that WIN54954 inhibits replication of human rhinovirus 14 by blockingadsorption to cellular receptors (13). Disoxaril, the first-generation compound, was shown to have no effect on theadsorption of human rhinovirus type 2 and poliovirus type 2

to host cells but has been shown to inhibit rhinovirus type 2and poliovirus type 2 uncoating events (7, 17). Additionalstudies are in progress to determine whether WIN 54954 caninhibit uncoating as well as adsorption of human rhinovirus14.The therapeutic efficacy of WIN 54954 demonstrated in

suckling mice infected with coxsackievirus A-9 and echovi-rus type 9 (Barty) indicates that the compound is sufficientlybioavailable when administered orally to prevent the devel-opment of flaccid limb paralysis due to severe polymyositisand striated muscle necrosis (5, 11). Q.d. intragastric dosesof WIN 54954, initiated 2.5 days after coxsackievirus A-9infection, prevented the onset of paralysis in .90% of themouse pups at doses as low as 11 mg/kg per day, and dosesof only 2 mg/kg per day protected 50% of the animals. Virustiters in carcasses of coxsackievirus A-9-infected sucklingmice medicated with WIN 54954 were reduced in a dose-dependent manner and correlated well with the survivalcurves, indicating that the protective effects of WIN 54954were related to the ability of the drug to inhibit virusreplication in mouse tissue. At 3.7 mg/kg per day, the dose atwhich -50% of the animals were protected (PD50), a 99%reduction in virus titers was observed. At higher doses ofWIN 54954, 103- to 105-fold reductions in virus titers re-sulted in .90% protection. These findings suggest that thereis a threshold titer for coxsackievirus A-9 beyond whichsymptoms become evident. WIN 54954 administered afterthe establishment of infection is able to maintain virus titersbelow this threshold.WIN 54954, administered to echovirus type 9 (Barty)-

infected suckling mice with a regimen identical to that usedagainst coxsackievirus A-9, resulted in a significantly higherPD50 of 100 mg/kg per day. These results are consistent withthe higher MIC of WIN 54954 for echovirus type 9 (0.31pug/ml) than for coxsackievirus A-9 (0.004 ,ug/ml). Also, invirus yield reduction assays, WIN 54954 completely re-pressed coxsackievirus A-9 viral multiplication at a concen-tration of 0.03 jxg/ml. Maximum inhibition of 103-fold wasobserved with echovirus type 9 (Barty) at 0.5 ,ug/ml.

Determination of WIN 54954 levels in serum at the PD50doses of 2 and 100 mg/kg indicated that levels in serum weremaintained above the in vitro MICs for a portion of, andpossibly over the whole, dosing interval. At a dose of 100mg/kg, levels in serum at 24 h postmedication were 0.4,ug/ml, while the MIC for echovirus type 9 (Barty) was 0.31,ug/ml. A dose of 2 mg/kg resulted in levels in serum fallingbelow the minimum quantifiable level of 0.02 ,ug/ml by 8 hpostmedication. It is possible, however, that levels in serumremained above the MIC of 0.004 ,ug/ml for coxsackievirusA-9 for most of the dosing interval. These results suggestthat to demonstrate efficacy in these models, levels of WIN54954 in serum must be above the in vitro MIC for at least aportion of the dosing interval.WIN 54954 has been shown to be nonmutagenic in the

Ames test, the human lymphocyte assay, and the Chinesehamster ovary HG phosphoribosyltransferase forward mu-tation assays. As expected, this virus-specific inhibitor haslow acute toxicity, with oral 50% acute lethal doses of 4 g/kgin rats and mice. One-month chronic toxicology studies inmonkeys and rats have shown that daily doses of 100 and 160mg/kg, respectively, were generally well tolerated (R. Fa-bian, personal communication).A number of compounds have in vitro activity against

rhinovirus mediated through direct binding to the viruscapsid. Dichloroflavan, Ro 09-0410/Ro 09-0415, RMI 15731,and 44,081 R.P. have all been shown to have in vitro

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antirhinovirus activity but have lacked clinical efficacy (14).New compounds Ro 09-0881, Ro 09-0535, and Ro 09-0696(Roche Laboratories) have been shown to be more activethan the earlier chalcone Ro 09-0410 (2). Another newantirhinovirus compound, R-61837, has potent in vitro activ-ity and was effective in a prophylactic, double-blind, place-bo-controlled clinical trial in human volunteers (1, 3). Thefinding of these new more potent antirhinovirus compounds,including WIN 54954, may lead to future clinical candidatesfor the control of rhinovirus infection in humans.The increased potency and broader spectrum of activity of

WIN 54954 represent a significant advance over disoxaril,the first compound in this series. WIN 54954 has been shownto be absorbed when given orally and efficacious againstsystemic enteroviral diseases in mice and represents a can-didate for clinical evaluation in the treatment of humanpicornaviral infections.

ACKNOWLEDGMENTS

We thank V. Csontos, A. Erenberg, M. Fancher, P. Felock, R.Rowland, W. Shave, D. Vigars, and A. Visosky for their technicalcontributions and M. Celentano for manuscript preparation.

LITERATURE CITED1. Al-Nakib, W., P. G. Higgins, G. I. Barrow, D. A. J. Tyrreli, K.

Andries, G. Vanden-Bussche, N. Taylor, and P. A. J. Janssen.1989. Suppression of colds in human volunteers challenged withrhinovirus by a new synthetic drug, R-61837. Antimicrob.Agents Chemother. 33:522-525.

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