Oral Administration of Cyclopentane Neuraminidase Inhibitors Protects Ferrets against Influenza Virus Infection

Several cyclopentane inhibitors of influenza virus neuraminidasethat have inhibitory activities in tissue culture similar tothose of zanamivir and oseltamivir have recently been described.These new inhibitors have been examined for efficacy againsta virulent H3N2 influenza virus when administered orally toinfected ferrets. Preliminary studies indicated that oral administrationof BCX-1923, BCX-1827, or BCX-1812 (RWJ-270201) at a dose of5 or 25 mg/kg of body weight was active in ferrets in reducingrespiratory and constitutional signs and symptoms, but theseantivirals affected virus titers in the upper and lower respiratorytracts only marginally. Of the three compounds, BCX-1812 seemedto be the most efficacious and was examined further at higherdoses of 30 and 100 mg/kg. These doses significantly reducedpeak virus titers in nasal washes and total virus shedding asmeasured by areas under the curve. Virus titers in lung homogenateswere also reduced compared to those in controls, but the differencewas not statistically significant. As was observed with BCX-1812at lower doses, the nasal inflammatory cellular response, fever,and nasal signs were reduced while ferret activity was not,with activity remaining similar to uninfected animals.

INTRODUCTION

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Introduction

Influenza, a pandemic viral respiratory disease, results inhigh morbidity and mortality in both humans and domestic animals.Human influenza is predominantly an upper respiratory tractinfection, although influenza pneumonia is a rare complication.Protection can be afforded by annual immunization with a vaccineconsisting of inactivated virus or virion subunits, but limitationson vaccine efficacy, design, uptake, and production have stimulatedthe search for antiviral drugs.

Over the years, a number of compounds with anti-influenza virusactivity have been produced, including nucleoside analoguessuch as ribavirin (23), 2'-deoxy-2'-fluoroguanosine (33), and1,3,4-thiadiazol-2-ylcyanamide (LY217896) (10), but these havenot reached the clinic because of toxicity. Amantadine and itsanalogue rimantadine, inhibitors of virus M2 ion channel activityand hence of uncoating (12), have reached the clinic, but theyare less efficacious (they are active only against influenzaA virus), have adverse side effects, and rapidly generate drug-resistantstrains (6, 11, 13) which are as virulent as amantadine-sensitiveviruses (28).

Novel drugs active against the influenza virus neuraminidasefrom both type A and B strains have recently been described.Some of these have now been approved for use in patients, therebyoffering an alternative antiviral strategy (structures are shownin Fig. 1). On the basis of X-ray crystallographic studies ofthe influenza virus neuraminidase cocrystallized with sialicacid and the unsaturated sialic acid analogue Neu5Ac2en (8,34, 35), several sialic acid analogues have been synthesizedand tested as potential inhibitors of the enzyme. Zanamivir(GG167; 4-guanidino-Neu5Ac2en) is a selective inhibitor of influenzaA and B virus neuraminidases. The efficacy of zanamivir hasbeen demonstrated with animal models of influenza virus infection(21, 35) and in studies with humans (14), but its poor bioavailabilityand rapid elimination from the body necessitate its deliveryas a nasal spray (14, 21). Oseltamivir carboxylate, GS4071,a carbocyclic transition state analogue inhibitor of neuraminidase,has potent in vitro inhibitory activity comparable to that ofzanamivir (15). Furthermore, oseltamivir (GS4104), an ethylester prodrug which is converted to GS4071 in vivo, can be givenorally and has been shown to be active in the mouse and ferretmodels of influenza (16, 18). Based on a rationally designedset of inhibitors, a novel series of cyclopentane derivatives,which are structurally different from sialic acid, has beenfound to exhibit strong and selective inhibitory effects oninfluenza virus neuraminidase (2, 3). These compounds, designatedBCX-1812 (RWJ-270201), BCX-1827 (RWJ-270204), and BCX-1923,have been shown to inhibit neuraminidase activity from variousstrains of influenza A virus, with 50% inhibitory concentrations(IC₅₀s) between 0.09 and 1.4 nM and IC₅₀s ranging from 0.82to 10.8 nM for neuraminidase activity from influenza B virusstrains (4). These compounds had 50% effective (virus-inhibitory)concentrations (EC₅₀) ranging between 0.01 and 1.5 µMfor 4 H1N1, 12 H3N2, and 2 H5N1 influenza A virus strains andbetween 0.02 and 3 µM for 5 influenza B virus strains(24, 25) in tissue culture assays. No cytotoxicity was observedwith concentrations as high as 1 mM (328 µg/ml), and RWJ-270201has been shown to be a potent inhibitor of influenza A and Bvirus infection in mice (24, 25). Here we report the efficaciesof these compounds in comparison to those of oseltamivir inthe ferret influenza virus model (27).

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FIG. 1. Chemical structures of various neuraminidase inhibitors.

MATERIALS AND METHODS

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Materials and Methods

Materials. Oseltamivir, BCX-1827, and BCX-1923 were synthesized at BioCrystPharmaceuticals Inc. (Birmingham, Ala.). BCX-1812 (RWJ-270201)was provided by the R. W. Johnson Pharmaceutical Research Institute(Raritan, N.J.).

Viruses and their assay. The H3N2 clone 7a of the A/Puerto Rico/8/34-A/England/939/69reassortant virus system has been described previously (17).Seed stocks were prepared as previously described (29) and titratedin eggs or allantois-on-shell cultures (egg bits) as describedpreviously (29); titers are expressed as 50% egg infectiousdose (EID₅₀) or 50% egg bit infectious dose (EBID₅₀). The limitof detection of these assays is 1.5 log₁₀ EID₅₀ or EBID₅₀/ml.Some nasal virus samples were titrated by plaque assay in MDCKcells (1), and these titers are expressed in PFU per milliliter;the limit of detection of this assay is 4 PFU/ml.

Intranasal inoculation of ferrets, collection of nasal washings, and counts of inflammatory cells. Adult male ferrets, obtained from Highgate Farm, Normanby-by-Spittle,Market Rasen, Lincolnshire, United Kingdom, and in the weightrange of 1 to 1.5 kg, received intranasal inoculations with10⁶ EID₅₀ of clone 7a under ether anesthesia, and nasal washingswere performed and inflammatory cell counts were obtained asdescribed previously (30, 31). All studies were carried outin accordance with the United Kingdom, Animals (Scientific Procedures)Act 1986, project license number 40/1715.

Ferret temperatures. Microchip temperature transponders (Biomedic Data Inc., Seaford,Del.) were implanted subcutaneously into ferrets between 5 and7 days prior to the experiment, allowing temperatures to berecorded twice daily to determine background temperatures forindividual ferrets as described previously (30, 31). Temperatureswere recorded at 6- to 12-h intervals during the experiment,and rises in temperature above the preinfection mean for eachferret were determined.

Assessment of signs and activity in the ferrets. Signs and activity were monitored and assessed in the ferretsas previously described (20).

Lung collection and maceration. Lung collection and maceration procedures were described byCampbell et al. (9).

Hemagglutination inhibition tests. Hemagglutination inhibition tests were performed on the seraof ferrets prior to infection to establish the lack of priorimmunity of the animals as described by Sweet et al. (29).

Experimental design. Three to four ferrets, shown to have no serum antibody to clone7a, were examined for each treatment group, and an untreatedbut infected group was included in each experiment. Animalswere administered their doses orally by placing a syringe overthe tongue towards the back of the mouth, allowing the ferretsto easily swallow the compound without stress. Compounds wereprepared in the required concentration (e.g., 10 mg/kg bodyweight) in 1 ml of solution (i.e., 10 mg/ml), and thus volumesfor oral doses varied according to the weight of the animalbetween 1 and 1.5 ml. Doses were given 2 h prior to virus inoculationand then at 12, 24, 36, 48, 60, and 72 h postinfection (hpi).Following virus inoculation, nasal washings were taken and temperatureswere recorded every 6 to 12 h at 12, 24, 30, 36, 48, 54, 60,72, 78, 84, and 96 hpi. On day 4 the animals were sacrificedand their lungs were removed for virus titration.

Pharmacokinetics. Three nonfasted male ferrets each received a single oral 10-mg/kggavage dose (1 ml/kg) of BCX-1812 (RWJ-270201) in phosphate-bufferedsaline. Blood samples ( $~$ 0.5 ml) were collected into heparinizedtubes via the jugular vein at predose and 0.5, 1, 2, 4, 8, and24 h postdose. The remaining three nonfasted male ferrets eachreceived a single intravenous (i.v.) 10-mg/kg bolus dose (1ml/kg) of BCX-1812 into the jugular vein, and blood sampleswere collected via the opposing jugular vein at predose and0.1, 0.5, 1, 2, 4, 8, and 24 h postdose. Plasma was separatedby centrifugation and stored frozen. Samples were analyzed forplasma BCX-1812 concentrations using a liquid chromatography-massspectrometry/mass spectrometry assay with a lower limit of quantificationof 1 ng/ml. Noncompartmental pharmacokinetic analysis of thesedata was performed using WinNonlin (version 1.5; Pharsight,Palo Alto, Calif.).

Statistical analysis. Data were collated and analyzed for statistical significanceby the Student's t test.

RESULTS

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Results

Effect of oral administration (5 mg/kg) of oseltamivir and BCX-1827 on influenza infection in ferrets. Influenza in ferrets is largely an upper respiratory tract infectionwith associated respiratory (as monitored by the nasal cellularinflammatory response) and constitutional (as monitored by fever)symptoms, although there is some replication in the lower respiratorytract (26). Thus, in untreated control animals, virus in nasalwashes reached mean peak titers of 10^4.4 to 10^4.7 EBID₅₀/mlbetween 24 and 36 hpi and then declined rapidly; a second minorpeak of replication was observed between 60 and 96 hpi (Fig.2A). Animals that were orally administered oseltamivir at 5mg/kg twice daily also shed virus as early as 12 hpi, with apeak (10^4.3 EBID₅₀/ml) slightly later (30 hpi), but this peakwas less than threefold lower than that observed in untreatedanimals (Fig. 2A). Virus titers declined rapidly, reaching lowlevels (10^1.5 EBID₅₀/ml) by 54 hpi, but again a minor secondpeak of replication was observed. Animals receiving similardoses of BCX-1827 showed greater inhibition of virus replication,being depressed $~$ 4-fold at 24 hpi and $~$ 13-fold at 30 hpi (P <0.004), although at later times virus titers were similar tothose observed in untreated controls (Fig. 2A).

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FIG. 2. Effect of oral administration of compounds on nasal virus titers, nasal inflammatory cell counts, and fever of ferrets infected with influenza virus A (H3N2) virus. Nasal virus titers (A), total numbers of inflammatory cells in nasal washes (B), and changes in temperature relative to baseline temperatures (C) were monitored in ferrets at the indicated times after infection. Ferrets were left untreated ( ${blacksquare}$ ) or were treated twice daily with oral 5-mg/kg doses of GS4104 ( ${blacklozenge}$ ) or BCX-1827 ( ${blacktriangleup}$ ) for 3 days beginning 2 h prior to infection with influenza virus clone 7a. Values are means ± standard errors of the means (error bars) for three to four animals.

While virus titers seemed to be little affected by treatmentwith oseltamivir, nasal inflammatory cell counts were considerablysuppressed; peak cell counts were delayed ( $~$ 24 h) and were significantly(P < 0.0008) lower (approximately sevenfold), and cell countsreturned more quickly to near normal levels (Fig. 2B). In contrast,oral treatment of ferrets with BCX-1827 had little effect onthe inflammatory response, although the onset of this responsewas delayed and declined more rapidly at later times (Fig. 2B).

BCX-1827 had similarly little effect on the febrile response,while oseltamivir delayed the febrile response and its peak(Fig. 2C).

While nasal signs (sneezing, nasal discharge, and mouth breathing)induced by clone 7a in untreated animals were relatively mild,both oseltamivir- and BCX-1827-treated animals exhibited littleor no nasal signs, and their activities were essentially normal(data not shown).

Virus titers in lung 4 days postinfection were not significantly(P > 0.05) reduced in oseltamivir- or BCX-1827-treated ferrets(Fig. 3A).

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FIG. 3. Effect of oral administration of compounds on total virus titers in lung. (A) Ferrets were treated orally with 5-mg/kg doses of GS4014 (bar 1) or BCX-1827 (bar 2) or were untreated (bar 3). (B) Ferrets were treated orally with 25-mg/kg doses of BCX-1827 (bar 1), BCX-1823 (bar 2), or BCX-1812 (bar 3) or were untreated (bar 4). (C) Ferrets were treated orally with BCX-1812 at 10 mg/kg (bar 1), BCX-1812 at 30 mg/kg (bar 2), or BCX-1812 at 100 mg/kg (bar 3) or were untreated (bar 4). All treated animals were given compound for 3 days beginning 2 h prior to infection with influenza virus clone 7a. Values are means + standard errors of the means (error bars) for three to four animals.

Effect of oral administration (25 mg/kg) of BCX-1827, BCX-1923, and BCX-1812 (RWJ-270201) on influenza infection in ferrets. The results obtained with 5-mg/kg doses of oseltamivir in theabove experiment were very similar to those observed previously(18), but inhibition of both respiratory virus and inflammationas well as fever was improved by increasing the dose to 25 mg/kg(18). Thus, a further experiment was carried out in which groupsof four ferrets received oral 25-mg/kg doses of BCX-1827 twicedaily as described above, and the outcome with this compoundwas compared with those with two other cyclopentane derivatives,BCX-1923 and BCX-1812.

None of the compounds showed significant reductions in virusreplication in the upper respiratory tract of ferrets, althoughvirus titers in nasal washes of animals treated with BCX-1812were slightly delayed and significantly lower 24 hpi (P <0.01), and there was little evidence of the second peak of replicationobserved in the untreated control animals (Fig. 4A). However,nasal inflammatory cell counts were delayed in reaching peaklevels, which were reduced (three- to fivefold) by all threecompounds and remained lower until 96 hpi (Fig. 4B). Similarly,febrile responses were delayed and reduced (Fig. 4C). Virustiters in lung were reduced in animals treated with BCX-1812and BCX-1923, but not BCX-1827, although they were not reducedsignificantly (Fig. 3B).

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FIG. 4. Effect of oral administration of compounds on nasal virus titers, nasal inflammatory cell counts, and fever of ferrets infected with influenza virus A (H3N2). Nasal virus titers (A), total numbers of inflammatory cells in nasal washes (B), and changes in temperature relative to baseline temperatures (C) were monitored in ferrets at the indicated times after infection. Ferrets were left untreated ( ${blacksquare}$ ) or were treated twice daily with 25-mg/kg oral doses of BCX-1827 ( ${blacklozenge}$ ), BCX-1823 ( ${blacktriangleup}$ ), or BCX-1812 (•) for 3 days beginning 2 h prior to infection with influenza virus clone 7a. Values are means ± standard errors of the means (error bars) for three to four animals.

Effects of oral administration with different doses of BCX-1812 (RWJ-270201) on influenza infection in ferrets. The above results suggested that oral dosing of ferrets withcompounds had a markedly suppressive effect on sickness in ferretswithout affecting replication to any significant extent in eitherthe upper or lower respiratory tract. To investigate whetherthis was largely an effect of dosage, BCX-1812 treatment wascompared at doses of 10, 30, and 100 mg/kg, the dosing schedulebeing the same as that described above.

While the 10-mg/kg dose had little effect on virus replicationat any one time point in the upper respiratory tract, measurementof the areas under the curve (57.36 ± 10.7 arbitraryunits) showed a reduction, although not statistically significant(P > 0.05), in overall virus shedding compared to that observedin untreated animals (74.11 ± 7.83 arbitrary units) (valuesare means ± standard deviations [SD]). Both the 30- and100-mg/kg doses, however, had a marked effect. Animals treatedwith doses of 30 and 100 mg/kg showed significantly reducedlevels of virus in nasal washes at both 24 (P < 0.002) and30 (P < 0.02) hpi, with a further significant (P < 0.05)reduction (20-fold) in peak titers at 36 hpi (Fig. 5A). Therewas little difference between the responses to these two dosesas indicated by measurement of areas under the curve, whichwere, however, significantly (P < 0.02) smaller (51.8 ±2.90 and 45.03 ± 7.93 arbitrary units for doses of 30and 100 mg/kg, respectively) than in untreated animals and animalstreated (74.11 ± 7.83 arbitrary units) with the 10-mg/kgdose of BCX-1812.

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FIG. 5. Effect of oral administration of compounds on nasal virus titers, nasal inflammatory cell counts, and fever of ferrets infected with influenza virus A (H3N2). Nasal virus titers (A), total numbers of inflammatory cells in nasal washes (B), and changes in temperature relative to baseline temperatures (C) were monitored in ferrets at the indicated times after infection. Ferrets were left untreated ( ${blacksquare}$ ) or were treated twice daily with oral doses of BCX-1812 at 10 mg/kg ( ${blacklozenge}$ ), 30 mg/kg ( ${blacktriangleup}$ ), or 100 mg/kg (•) for 3 days beginning 2 h prior to infection with influenza virus clone 7a. Values are means ± standard errors of the means (error bars) for three to four animals.

The inflammatory response in animals treated with the 10-mg/kgdose of BCX-1812 was similar to that in untreated control animals,but the response in animals treated with the 30-mg/kg dose wasconsiderably decreased (12-fold) and the peak was significantly(P < 0.05) reduced (Fig. 5B). The reduction observed withthe 100-mg/kg dose was even more marked (Fig. 5B). As measuredby areas under the curve, the overall nasal inflammatory responsesin animals treated with the 30-mg/kg (27.92 ± 9.99 arbitraryunits) or the 100-mg/kg (9.33 ± 2.27 arbitrary units)dose were significantly lower (P < 0.03 and P < 0.001,respectively) than that in untreated animals (54.93 ±3.16 arbitrary units).

Animals treated with BCX-1812 at 10 mg/kg showed little fever,although some individuals showed more-prolonged low-level fevers,suggesting that the antiviral was not as effective in all animalsat this dose (Fig. 5C). The fevers in animals given 30 and 100mg/kg were delayed and were of short duration, but results werevariable, as some animals exhibited little or no fever (Fig.5C). Nevertheless, calculations of areas under the curve showedthat the overall level of fever in animals treated with BCX-1812at 100 mg/kg (3.16 ± 0.67 arbitrary units) was significantlyless than that in control untreated animals (7.85 ± 1.89arbitrary units). The areas under the curve for animals treatedwith doses of 10 mg/kg (4.59 ± 1.39) and 30 mg/kg (5.57± 2.10), while reduced, were not statistically differentfrom untreated animals.

All doses of compound produced some reduction in virus titersin lung (Fig. 3C). In animals treated with 10 and 30 mg/kg,mean titers were reduced approximately 10-fold, but largelydue to a significant reduction in only one of the four animalsin each group. In contrast, mean lung titers were reduced approximately60-fold in animals treated with 100-mg/kg doses of compound,but again this was not statistically significant (P > 0.05).

BCX-1812 (RWJ-270201) pharmacokinetics. Profiles of mean concentration in plasma versus time are presentedin Fig. 6. BCX-1812 was rapidly absorbed following an oral doseof 10 mg/kg to male ferrets based on a mean time to maximumconcentration of drug in serum (time to C_max [T_max]) of 1.00h, and was moderately eliminated following both the oral andi.v. doses based on mean half-lives of 3.20 and 3.65 h, respectively.The mean absolute bioavailability following a single oral doseof BCX-1812 was low at 2.5% in male ferrets. The mean volumeof distribution following the i.v. dose (10 mg/kg) was 355 ml/kg,which approximates total body water, indicating that the drugwas uniformly distributed throughout the body. A summary ofthe pharmacokinetic parameters (means and SD) for BCX-1812 followingoral and i.v. administration at 10 mg/kg to male ferrets isshown in Table 1.

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FIG. 6. Profiles of mean (n = 3) concentration of BCX-1812 plasma in male ferrets following single oral and i.v. administration of BCX-1812 (10 mg/kg).

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TABLE 1. Summary of pharmacokinetic parameters for RWJ-270201 in plasma^a

DISCUSSION

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Discussion

A number of antineuraminidase compounds have now reached theclinic, including zanamivir and oseltamivir (36). Zanamiviris a potent antiviral, but it has the disadvantage of beingpoorly bioavailable in both ferrets and humans (35) and hasto be administered intranasally or by aerosol. However, resistantmutants have not been observed in immunocompetent persons treatedclinically with zanamivir (5, 7). In contrast, oseltamivir ismore bioavailable and can be taken orally as its prodrug, butmutants arise with some frequency in the clinic (32).

A novel series of cyclopentane derivatives, which are structurallydifferent from zanamivir and oseltamivir, have been shown tobe inhibitory against influenza virus types A and B in tissueculture and neuraminidase enzyme assays. The 50% effective concentrationsand IC₅₀s of these compounds are comparable to those of zanamivirand oseltamivir (4, 25). Several of these, including BCX-1812(RWJ-270201), were found to be orally active in mice, preventingdeath, inhibiting lung consolidation and reducing virus titersin the lung (24). RWJ-270201 appears to have a similar pharmacokineticprofile to oseltamivir in mice (24).

While the mouse has usually been the animal of choice for studiesof antiviral compounds against influenza virus because of itslow cost, ready availability, and ease of handling (22), mostinfluenza viruses, particularly if they are clinical isolatesnot previously used in mice, cause only a mild subclinical infectionof the upper and lower respiratory tracts (27). Following mousepassage (or adaptation) virus strains cause markedly more lunginvolvement, allowing assessment of disease by time to death,lung weight at death, and histopathology (22). However, influenzain humans is mainly an upper respiratory tract infection withrespiratory (nasal discharge and cough) and constitutional (fever,myalgia, and malaise) symptoms but rare pneumonia, except inthe elderly, where it mostly results from bacterial superinfection(26). Since ferrets can be infected with human clinical isolatesdirectly without passage and produce both respiratory and constitutionalsymptoms, which can be quantified, they are perhaps a more predictiveand objective model than mice for uncomplicated influenza infections(19, 27).

Of the three cyclopentane compounds tested in this study inferrets, RWJ-270201 was the most effective in eliminating allrespiratory and constitutional signs of infection, includingthe febrile response, especially at the highest dose of 100mg/kg. This dose also produced significant reductions in nasalvirus yields and virus titers in lung, although in the lattercase these reductions were not statistically significant. Thebioavailability of RWJ-270201, like that of oseltamivir, appearsrelatively poor in ferrets. While bioavailability studies withRWJ-270201 have not yet been reported for other species, ferretsshowed the lowest oseltamivir bioavailability (11%) in comparisonwith dogs, rats, and mice (30 to 73%) (16). RWJ-270201 was absorbedmore quickly than oseltamivir, with a T_max of 1 h and a C_maxof 1.02 µg/ml, compared to 2 h and 0.20 µg/ml foroseltamivir, although the dose used for oseltamivir was 5 mg/kg,not 10 mg/kg (16). Consequently levels dropped more quicklybut were still above the IC₅₀ for most viruses 24 h postdosing.

These experiments in ferrets have confirmed previous indicationsof oral efficacy in mice (24) and, taken together with the lowtoxicity of this compound and hence a high in vivo margin forsafety (24), the present results support the choice of RWJ-270201for further clinical development for treatment of influenzavirus infections in humans.

FOOTNOTES

* Corresponding author. Mailing address: School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom. Phone: 121-414-6554. Fax: 121-414-5925. E-mail: C.Sweet{at}bham.ac.uk.

REFERENCES

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