ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2001, p. 743–748 Vol. 45, No.

3
0066-4804/01/$04.00⫹0 DOI: 10.1128/AAC.45.3.743–748.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Cyclopentane Neuraminidase Inhibitors with Potent In Vitro

Anti-Influenza Virus Activities
DONALD F. SMEE,* JOHN H. HUFFMAN, ANN C. MORRISON, DALE L. BARNARD,
AND ROBERT W. SIDWELL

Institute for Antiviral Research, Utah State University, Logan, Utah 84322-5600
Received 12 July 2000/Returned for modification 11 October 2000/Accepted 30 November 2000

A novel series of cyclopentane derivatives have been found to exhibit potent and selective inhibitory effects
on influenza virus neuraminidase. These compounds, designated RWJ-270201, BCX-1827, BCX-1898, and BCX-
1923, were tested in parallel with zanamivir and oseltamivir carboxylate against a spectrum of influenza A
(H1N1, H3N2, and H5N1) and influenza B viruses in MDCK cells. Inhibition of viral cytopathic effect ascer-
tained visually and by neutral red dye uptake was used, with 50% effective (virus-inhibitory) concentrations
(EC50) determined. Against the H1N1 viruses A/Bayern/07/95, A/Beijing/262/95, A/PR/8/34, and A/Texas/36/91,
EC50s (determined by neutral red assay) of the novel compounds were <1.5 ␮M. Twelve strains of H3N2 and
two strains of avian H5N1 viruses were inhibited at <0.3 ␮M. Influenza B/Beijing/184/93 and B/Harbin/07/94
viruses were inhibited at <0.2 ␮M, with three other B virus strains inhibited at 0.8 to 8 ␮M. The novel in-
hibitors were comparable in potency to (or slightly more potent than) zanamivir and oseltamivir carboxylate.

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No cytotoxicity was seen with the compounds at concentrations of <1 mM in cell proliferation assays. The
antiviral activity of RWJ-270201, chosen for clinical development, was studied in greater detail. Its potency and
that of oseltamivir carboxylate decreased with increasing multiplicity of virus infection. Time-of-addition
studies indicated that treatment with either compound needed to begin 0 to 12 h after virus exposure for op-
timal activity. Exposure of cells to RWJ-270201 caused most of the virus to remain cell associated, with ex-
tracellular virus decreasing in a concentration-dependent manner. This is consistent with its effect as a neur-
aminidase inhibitor. RWJ-270201 shows promise in the treatment of human influenza virus infections.

Influenza has continued to be a significant public health
concern, with annual epidemics responsible for serious mor-
bidity and mortality (1, 13). Much attention has conse-
quently been given to the development of antiviral drugs for
the treatment of this disease. Amantadine and rimantadine
both have been approved for prophylaxis of influenza A vi-
rus infection (6). Ribavirin was shown to be effective against
experimental influenza virus infections in mice (9), and was
studied in humans by small-particle aerosol delivery against
severe influenza virus infections (11). However, it was not
effective enough to receive drug approval. As early as 1976
Palese and Compans (19) reported an inhibitor of influenza
virus neuraminidase. This research was largely ignored for
many years, and it was not until recently that the search for
more potent neuraminidase inhibitors has intensified. From
these investigations, zanamivir (GG167) and oseltamivir carbox-
ylate (GS4071) emerged; these compounds were found to be
highly active against both influenza A and B viruses (10, 27).
Zanamivir, a topical agent approved for clinical use, is effective
prophylactically and therapeutically for the treatment of influ-
enza (16, 17). Oseltamivir, the orally active prodrug form of
oseltamivir carboxylate (22), is also clinically approved and has
been found to be effective for both prophylaxis and treatment
of influenza in humans (7, 18).
Structure-activity analyses with the purified influenza virus
neuraminidase enzyme and knowledge of its three-dimensional
* Corresponding author. Mailing address: Institute for Antiviral Re-
search, Department of Animal, Dairy and Veterinary Sciences, Utah
State University, 5600 Old Main Hill, Logan, UT 84322-5600. Phone:
(435) 797-2897. Fax: (435) 797-3959. E-mail: dsmee@cc.usu.edu.
structure (26) have led to the identification of new inhibitors.
A series of cyclopentane derivatives was found to cause potent
and selective inhibition of influenza virus neuraminidase (2).
The chemical structures of the more potent antiviral com-
pounds (Fig. 1) have features in common with both zanamivir
and oseltamivir carboxylate but differ in having a five-mem-
bered-ring structure. In this report the activities of these novel
compounds in vitro against various strains of influenza virus
are presented. Compound RWJ-270201 was evaluated in great-
er detail in secondary assays, since it has been selected for
clinical development.
MATERIALS AND METHODS
Compounds. RWJ-270201, BCX-1827, BCX-1898, BCX-1923, zanamivir, and
oseltamivir carboxylate were synthesized at BioCryst Pharmaceuticals (Birming-
ham, Ala.). Ribavirin was obtained from ICN Pharmaceuticals (Costa Mesa,
Calif).
Viruses. The following viruses were provided by H. Regnery of the Influ-
enza Branch of the Centers for Disease Control and Prevention (Atlanta, Ga.):
A/Texas/36/91 (H1N1), A/Bayern/07/95 (H1N1), A/Beijing/262/95 (H1N1), A/
Washington/05/96 (H3N2), A/Johannesburg/33/94 (H3N2), A/Sydney/05/97
(H3N2), A/Shangdong/09/93 (H3N2), A/Beijing/32/92 (H3N2), B/Beijing/184/93,
B/Panama/45/90, and B/Harbin/07/94. A/NWS/33 (H1N1) was provided by K.
Cochran of the University of Michigan (Ann Arbor). A/PR/8/34 (H1N1) was
obtained from F. Schabel, Jr., Southern Research Institute (Birmingham, Ala.).
A/Victoria/3/75 (H3N2), A/Port Chalmers/1/73 (H3N2), B/Hong Kong/5/72, and
B/Lee/40 were obtained from the American Type Culture Collection (Manassas,
Va.). A/Los Angeles/2/87 (H3N2) and A/Washington/897/80 (H3N2) were from
Program Resources, Inc. (Rockville, Md.). A/X-31 (H3N2), a reassortment virus
containing hemagglutinin and neuraminidase genes from A/Aichi/2/68 (H3N2)
and the remainder of the genes from A/PR/8/34 (H1N1), was obtained from
E. Kilbourne, Mount Sinai School of Medicine, New York Medical College, City
University of New York (New York, N.Y.). A/Port Chalmers/1/73r (H3N2), an
amantadine-resistant virus, was prepared from the wild-type virus by serial pas-
sage in the presence of the drug in this laboratory. A/Virginia/2/88r (H3N2), a

743
744 SMEE ET AL.
FIG. 1. Chemical structures of cyclopentane derivatives, zanamivir, and oseltamivir carboxylate.
clinically isolated amantadine-resistant virus, was provided by F. Hayden, Uni-
versity of Virginia School of Medicine (Charlottesville). A/Duck/MN/1525/81
(H5N1) and A/Gull/PA/4175/83 (H5N1) were obtained from R. Webster of the
St. Jude Children’s Research Hospital (Memphis, Tenn.). All viruses were pas-
saged in cells to prepare pools for use in these experiments.
Cells and media. Madin-Darby canine kidney (MDCK) cells were grown in
antibiotic-free minimum essential medium with nonessential amino acids (Gibco,
Long Island, N.Y.) containing 5% fetal bovine serum (HyClone Laboratories,
Logan, Utah) and 0.1% NaHCO3. Test medium consisted of minimum essential
medium 0.18% NaHCO3, 10 U of trypsin per ml, 1 ␮g of EDTA per ml, and 50
␮g of gentamicin/ml.
Cell culture assays. Three methods were used to assay antiviral activity in
vitro: inhibition of virus-induced cytopathic effect (CPE) determined by visual
(microscopic) examination of the cells, increase in neutral red (NR) dye uptake
into cells, and virus yield reduction. In the CPE inhibition method, which was
reported previously by Sidwell and Huffman (21), seven concentrations of test
drug were evaluated against each virus in 96-well flat-bottomed microplates. The
compounds were added 5 to 10 min prior to virus, which was used at a concen-
tration of approximately 50 cell culture 50% infections doses per well. This virus
challenge dose equated to a multiplicity of infection (MOI) of approximately
0.001 infectious particle per cell. The tests were read after incubation at 37°C for
72 h. In the NR uptake assay, dye (0.34% concentration in medium) was added
to the same set of plates used to obtain the visual scores. After 2 h, the color
intensity of the dye absorbed by and subsequently eluted from the cells was
ANTIMICROB. AGENTS CHEMOTHER.
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determined by the method of Finter (5), using a computerized EL-309 micro-
plate autoreader (Bio-Tek Instruments, Winooski, Vt.). Antiviral activity was
expressed as the 50% effective (virus-inhibitory) concentration (EC50) deter-
mined by plotting compound concentration versus percent inhibition on semi-
logarithmic graph paper. Although the CPE and NR methods were both used for
calculating EC50 against all of the influenza virus strains, for brevity only data
obtained from the NR assays are reported. In general, the EC50 determined by
NR assay were two- to fourfold higher than those obtained by the CPE method.
Cytotoxicity of compounds was assessed in parallel with the antiviral determi-
nations in the same microplates, except in the absence of virus. From these
results, 50% cytotoxic end points (50% cell-inhibitory concentrations [IC50s])
were determined. Later, the compounds were assayed for toxicity in actively
proliferating MDCK cells. This was done by seeding 96-well microplates with 2 ⫻
104 cells per well. Compounds were diluted in medium containing 5% fetal
bovine serum and then were placed into the wells following cell attachment.
After 3 days, the percent inhibition of cell proliferation was assessed by NR assay
as described above.
Virus yield reduction assays were performed by a method which separated and
quantified extracellular (supernatant) from cell-associated virus. These tests,
using A/Texas/36/91 (H1N1), A/Sydney/05/97 (H3N2), and B/Beijing/184/93 vi-
ruses, were initiated in 24-well plates of MDCK cells infected at a virus MOI of
0.001. A visual determination of viral CPE was made after 72 h of incubation,
when cell destruction in untreated cultures was maximum, at which time the
extracellular medium was removed and placed in test tubes. The plates were
VOL. 45, 2001 CYCLOPENTANE INHIBITORS OF INFLUENZA VIRUSES 745

TABLE 1. Activities of cyclopentane derivatives, zanamivir, and oseltamivir carboxylate on influenza virus replication
in MDCK cells as determined by NR assay
Oseltamivir
RWJ-270201 BCX-1827 BCX-1898 BCX-1923 Zanamivir
Virus carboxylate

EC50a(␮M) SIb EC50 SI EC50 SI EC50 SI EC50 SI EC50 SI

H1N1
A/Bayern/07/95 1.0 ⬎1,000 0.64 ⬎1,560 0.36 ⬎2,770 0.72 ⬎1,390 3.4 ⬎290 2.7 ⬎370
A/Beijing/262/95 0.36 ⬎2,770 0.56 ⬎1,780 0.18 ⬎5,550 0.37 ⬎2,700 2.6 ⬎380 2.4 ⬎410
c
A/NWS/33 21 ⬎47 19 ⬎52 21 ⬎47 23 ⬎43 ⬎100 — ⬎100 —
A/PR/8/34 1.5 ⬎660 1.0 ⬎1,000 0.7 ⬎1,420 0.7 ⬎1,420 0.42 ⬎2,380 0.22 ⬎4,540
A/Texas/36/91 0.09 ⬎11,110 0.10 ⬎10,000 0.06 ⬎16,660 0.11 ⬎9,090 0.22 ⬎4,540 0.17 ⬎5,880

H3N2
A/Beijing/32/92 0.07 ⬎14,280 0.14 ⬎7,140 0.05 ⬎20,000 0.05 ⬎20,000 0.65 ⬎1,530 0.23 ⬎4,340
A/Johannesburg/33/94 0.16 ⬎6,200 0.1 ⬎10,000 0.09 ⬎11,110 0.05 ⬎20,000 0.62 ⬎1,610 0.50 ⬎2,000
A/Los Angeles/2/87 0.07 ⬎14,280 0.07 ⬎14,280 0.04 ⬎25,000 0.06 ⬎16,660 0.18 ⬎5,550 0.38 ⬎2,630
A/Port Chalmers/1/73 0.07 ⬎14,280 0.02 ⬎50,000 0.06 ⬎16,660 0.03 ⬎33,330 0.15 ⬎6,660 0.07 ⬎14,280
A/Port Chalmers/1/73r 0.08 ⬎12,500 0.10 ⬎10,000 0.1 ⬎1,800 0.1 ⬎10,000 0.15 ⬎6,660 0.2 ⬎5,000
A/Shangdong/09/93 0.17 ⬎5,880 0.18 ⬎5,550 0.23 ⬎4,340 0.28 ⬎3,570 0.50 ⬎2,000 0.32 ⬎3,120
A/Sydney/05/97 0.19 ⬎5,260 0.13 ⬎7,690 0.10 ⬎1,000 0.22 ⬎4,540 0.45 ⬎2,220 0.31 ⬎3,220
A/Victoria/3/75 0.10 ⬎10,000 0.06 ⬎16,660 0.07 ⬎14,280 0.06 ⬎16,660 0.41 ⬎2,440 0.06 ⬎16,660
A/Virginia/2/88r ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 0.03 ⬎33,330 0.05 ⬎20,000 0.02 ⬎50,000 ⬍0.01 ⬎100,000
A/Washington/897/80 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000
A/Washington/05/96 0.02 ⬎50,000 0.01 ⬎100,000 ⬍0.01 ⬎100,000 0.01 ⬎100,000 0.2 ⬎50,000 0.08 ⬎12,500
A/X-31 0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 ⬍0.01 ⬎100,000 0.4 ⬎2,500 0.12 ⬎8,330

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H5N1
A/Duck/MN/1525/81 0.01 ⬎100,000 0.022 ⬎45,450 0.02 ⬎50,000 0.01 ⬎100,000 0.20 ⬎5,000 0.22 ⬎4,540
A/Gull/PA/4175/83 0.02 ⬎50,000 0.03 ⬎33,330 0.02 ⬎50,000 0.02 ⬎50,000 0.22 ⬎4,540 0.26 ⬎3,840

B
Beijing/184/93 0.06 ⬎16,660 0.09 ⬎11,110 0.02 ⬎50,000 0.02 ⬎50,000 0.03 ⬎33,330 0.11 ⬎9,090
Harbin/07/94 0.12 ⬎8,330 0.16 ⬎6,250 0.06 ⬎16,660 0.06 ⬎16,660 0.20 ⬎5,000 0.26 ⬎3,840
Hong Kong/5/72 2.2 ⬎450 2.4 ⬎410 2.0 ⬎500 1.8 ⬎550 1.0 ⬎1,000 2.5 ⬎400
Lee/40 3.2 ⬎310 8.0 ⬎125 8.0 ⬎125 1.7 ⬎580 0.6 ⬎1,660 3.0 ⬎330
Panama/45/90 2.3 ⬎430 2.8 ⬎350 0.8 ⬎1,250 1.6 ⬎620 1.3 ⬎770 1.5 ⬎660
a
Results are means from two or three independent assays. Assay variability ranged from 20 to 50%.
b
Determined by dividing the IC50 (which was ⬎1,000 ␮M for all of these compounds) by the EC50.
c
—, the SI cannot be calculated, since the highest concentration tested (100 ␮M) was less than the IC50.

refed fresh medium. The tubes containing extracellular virus and cell debris were
centrifuged at 3,200 ⫻ g for 5 min, and most of the supernatant fluid was
removed and transferred to unused tubes. This procedure was carefully done to
avoid collecting any of the cell pellets. The remainder of the medium from each
tube was discarded, and the resulting pellets were recombined with the fresh
medium (and adhered cells) from wells where they originated. Eight wells were
used per concentration of compound. Samples from the eight wells were paired,
yielding a total of four samples for titration. The plates of cells and tubes of
extracellular virus were frozen and thawed, sonicated for 30 s each, and then
assayed for virus titer. Titrations were conducted by adding the serially diluted
samples to four wells each (0.1 ml/well) in 96-well plates of MDCK cells. After
2 h of virus adsorption, the medium was replaced with fresh medium to remove
residual compound present in the original samples. The plates were checked for
virus-induced CPE on days 3 and 6. Quantitation of virus yield titers was by the
end point method of Reed and Muench (20), and the titers were expressed as
log10 50% cell culture infectious doses per 0.1 ml of medium assayed.
Effects of MOI and delay of treatment initiation on antiviral activity. Exper-
iments using CPE inhibition and confirmed by NR uptake were done to ascertain
the effects of various viral challenge doses on antiviral, potency using MOIs of
0.00018, 0.0009, 0.0045, and 0.0225. To examine the influence of delay of treat-
ment initiation on antiviral activity. MOI of approximately 0.001 was used.
Compounds were added to the cells at 24 h pre-virus exposure and then rinsed
off, added at 5 min pre-virus exposure (time zero), or added at 2, 4, 6, 8, 12, or 24 h
post-virus exposure. EC50 were calculated from the NR assay results as described
above. Influenza A/Sydney/05/97 (H3N2) virus was used for these studies.
RESULTS
Antiviral activities against influenza A and B virus strains.
Six neuraminidase inhibitors were evaluated for activity against
several strains of influenza A (H1N1), influenza A (H3N2),
influenza A (H5N1), and influenza B viruses in MDCK cell
culture by the NR method (Table 1). Of the influenza A
(H1N1) viruses, the A/Texas/36/91 strain was the most sensi-
tive to inhibition by the compounds, with EC50s ranging from
0.06 to 0.22 ␮M. The A/Bayern/07/95, A/Beijing/262/95, and
A/PR/8/34 viruses were sensitive to inhibition in the 0.18 to 3.4
␮M range. Activities against the A/NWS/33 virus were up to an
order of magnitude less, at 19 to ⬎100 ␮M. Overall, zanamivir
and oseltamivir carboxylate were slightly less potent (usually
threefold or less) than the cyclopentane derivatives against the
H1N1 viruses.
Twelve influenza A (H3N2) strains were inhibited by the
cyclopentane derivatives at ⬍0.3 ␮M. The activities of zana-
mivir and oseltamivir carboxylate were similar against these
viruses, with 50% inhibition at 0.65 ␮M or less. The A/Wash-
ington/897/80, A/Washington/05/96, and A/X-31 strains were
uniformly more sensitive to inhibition by the cyclopentane
inhibitors than were the other viruses.
Because of the recent emergence of an influenza A (H5N1)
virus from chickens that was transmitted to humans and re-
sulted in lethal consequences (25), the neuraminidase inhibi-
tors were evaluated against two strains of influenza A (H5N1)
virus. Both the A/duck/MN/1525/81 and A/gull/PA/4175/83 vi-
ruses were markedly inhibited by the cyclopentane derivatives
at 0.01 to 0.03 ␮M. Zanamivir and oseltamivir carboxylate
were 10-fold less potent (0.2 to 0.26 ␮M) than the cyclopen-
746 SMEE ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Effect of time of treatment initiation on the concentrations were required to inhibit virus-induced cytopa-
anti-influenza A/Sydney/05/97 (H3N2) virus activities thology. These studies were done with a low input MOI, indi-
of RWJ-270201, oseltamivir carboxylate,
and ribavirin in MDCK cells cating that early treatments were necessary to suppress or
contain the later rounds of virus replication.
Time of treatment EC50a (␮M)
relative to
Effect of virus MOI. Certain compounds which inhibit virus
virus infection
RWJ-270201
Oseltamivir
Ribavirin
in cell cultures infected at low MOI, often are less active (or
(h) carboxylate
even inactive) at higher virus-to-cell ratios. To explore this
⫺24–0b 27 ⫾ 4 100 ⫾ 27 490 ⫾ 102 possibility with these compounds, antiviral activity was deter-
0–72 0.01 ⫾ 0.006 0.02 ⫾ 0.003 8⫾2 mined over a range of infecting MOIs differing five-fold from
2–72 0.03 ⫾ 0.006 0.02 ⫾ 0.004 20 ⫾ 6
each other (Table 3). RWJ-270201 and oseltamivir carboxylate
4–72 0.02 ⫾ 0.004 0.04 ⫾ 0.014 9⫾2
6–72 0.06 ⫾ 0.02 0.02 ⫾ 0.003 22 ⫾ 4 were most potent when virus infections were initiated at low
8–72 0.02 ⫾ 0.004 0.03 ⫾ 0.004 26 ⫾ 6 MOIs, and activities decreased with increasing viral challenge
12–72 0.2 ⫾ 0.03 0.06 ⫾ 0.006 53 ⫾ 8 dose. In contrast, the efficacy of ribavirin was not influenced by
24–72 65 ⫾ 36 ⬎100 530 ⫾ 120
increasing the MOI. This phenomenon has been previously
a
Determined by NR assay. Results are means and standard deviations from reported for ribavirin against other viruses (24).
four replicates.
b
The inhibitor was removed and cells were rinsed twice prior to infection. Virus yield reduction studies. Because neuraminidase is in-
volved in the efficient release of mature viruses from cells, mu-
tant viruses lacking neuraminidase activity aggregate and re-
tane derivatives but were still highly active inhibitors of these main at the cell surface (12, 14). Treatment with a neuraminidase
viruses. inhibitor should produce the same effect. To demonstrate this,

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The influenza B/Beijing/262/95 and B/Harbin 07/94 strain
were sensitive to inhibition by these compounds (EC50s rang-
ing from 0.02 to 0.26 ␮M). However, three other strains (Hong
Kong/5/72, Panama/45/90, and Lee/40) of influenza B virus
were inhibited at 30- to 400-fold-higher concentrations (0.6 to
8 ␮M). Zanamivir and oseltamivir carboxylate were as potent
as the cyclopentane derivatives against the influenza B virus
strains.
Antiviral selectivities. None of the compounds exhibited
cytotoxicity in MDCK cells as determined by visual and NR
assay methods at concentrations of up to 1,000 ␮M. In addi-
tion, actively dividing cells were not inhibited in their growth at
a 1,000 ␮M concentration of each compound. By dividing this
concentration (1,000 ␮M) by the EC50s, selectivity indices (SIs)
were obtained, and these are reported in Table 1. For highly
sensitive viruses such as influenza A/Washington/897/80 (H3N2),
A/Virginia/2/88r (H3N2), and A/duck/MN/1525/81 (H5N1), SIs
were ⬎100,000. SIs against most other viruses were less (⬎125
to ⬎50,000) but were still high compared to those of most
nucleoside-type antiviral agents. Antiviral selectivities were
least against the A/NWS/33 (H1N1) virus (⬎43 to ⬎52 or less)
due to lower potencies.
Time-of-addition studies. Compounds have a certain span of
time when they are most active; this is related to the life cycle
of the virus and the step in the cycle where the compounds act.
The neuraminidase inhibitors RWJ-270201 and oseltamivir
carboxylate were compared with ribavirin (a nucleoside ana-
log) for inhibition of influenza A/Sydney/05/97 (H3N2) virus
when added at a different times relative to virus infection
(Table 2). Treatment of cells starting at the time of infection
required a ⬍0.01 ␮M concentration of the neuraminidase in-
hibitors and 5.7 ␮M ribavirin for inhibition of viral CPE. Treat-
ment of cells prior to infection required much higher concen-
trations of the compounds, indicating a lack of persistence
following their removal from the culture medium. Treatments
with the three compounds could begin at any time from 2 to 12
h post-virus infection and still require about the same concen-
tration to achieve 50% inhibition as was effective for the 0-h
time point of treatment initiation. However, by 24 h, higher
three influenza viruses were exposed to RWJ-270201 for 3
days, followed by assay of extracellular and cell-associated vi-
rus yields (Fig. 2). Ribavirin was evaluated in parallel, it being
an influenza virus inhibitor with different modes of action
(4, 28) unrelated to inhibition of viral neuraminidase. The
A/Texas/36/91 (H1N1), A/Sydney/05/97 (H3N2), and B/Beijing/
184/93 viruses showed a similar pattern, in that more virus was
found extracellularly than cell associated in untreated cultures.
RWJ-270201 blocked the production of extracellular virus in a
dose-dependent manner. As expected, large amounts of cell-
associated virus were present. EC90s for A/Texas, A/Sydney,
and B/Beijing were 0.15, 0.1, and 10 ␮M, respectively. These
values approximated the EC50s obtained against these viruses
from the NR assays (Table 1). In contrast, cell-associated virus
yields were not reduced at ⬎10, ⬎10, and ⬎100 ␮M, respec-
tively, against the three viruses. Ribavirin caused dose-depen-
dent inhibition of both extracellular and cell-associated virus
yields. The EC90s of ribavirin against extracellular A/Texas,
A/Sydney, and B/Beijing viruses were 45, 32, and 5.5 ␮M,
respectively. EC90s of this compound against cell-associated
virus yields were 60, 32, and 12 ␮M, respectively. This is con-
sistent with virus yield reduction results using ribavirin against
other types of viruses (8).
TABLE 3. Effect of virus MOI on the anti-influenza
A/Sydney/05/97 (H3N2) virus activities of RWJ-270201,
oseltamivir carboxylate, and ribavirin in MDCK cells
EC50a (␮M)
MOI
RWJ-270201 Oseltamivir carboxylate Ribavirin
0.00018 0.01 ⫾ 0.006 0.01 ⫾ 0.003 20 ⫾ 2
0.0009 0.1 ⫾ 0.02 0.07 ⫾ 0.05 22 ⫾ 2
0.0045 1.0 ⫾ 0.2 0.9 ⫾ 0.2 24 ⫾ 3
0.0225 ⬎10 ⬎10 24 ⫾ 4
a
Determined by NR assay. Results are means and standard deviations from
four replicates.
VOL. 45, 2001
FIG. 2. Cell-associated (F) and extracellular (■) virus yields produced in the presence of RWJ-270201 and ribavirin. (A and B) Influenza
A/Texas/36/91 (H1N1) virus; (C and D) influenza A/Sydney/05/97 (H3N2) virus; (E and F) influenza B/Beijing/184/93 virus. Data points represent
means from four samples ⫾ standard deviations.
DISCUSSION
The results of this study show that the cyclopentane deriva-
tives were active against a large number of influenza A and B
virus strains in cell culture at nontoxic concentrations. The po-
tencies of these compounds were similar to or slightly greater
than those of zanamivir and oseltamivir carboxylate. Only the
A/NWS/33 (H1N1) virus was clearly less sensitive to inhibition
by these compounds. Viruses with greater resistance to in-
hibition are in the process of being prepared by cell culture
passage in the presence of RWJ-270201. These viruses will
be examined to determine whether the insensitivity is due to
a resistant neuraminidase protein, altered hemagglutinin or
both. Resistance to neuraminidase inhibitors has been the sub-
ject of considerable investigation, as recently reviewed (15).
Some of the viruses resistant in vitro are inhibited in mice and
ferrets by these types of compounds (3), indicating dependence
on the enzyme for spread and disease progression in vivo. Even
CYCLOPENTANE INHIBITORS OF INFLUENZA VIRUSES 747
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the influenza A/NWS/33 (H1N1) virus, which we found to be
relatively insensitive to inhibition in vitro compared to other
viral strains, is highly sensitive to treatment with zanamivir and
oseltamivir in mice (22).
High levels of cell-associated virus were produced in the
presence of RWJ-270201. Yet, considerably less infectious vi-
rus was detected in the extracellular medium relative to virus
yields from inhibitor-free cultures. It was shown with neur-
aminidase-deficient influenza virus mutants that the viruses are
not fully released from cells but remain clumped at the cell
surface (12, 14). An analogous situation should occur with
wild-type virus replication in the presence of a neuraminidase
inhibitor. Clumping is caused because viruses have picked up
cell antigens that attract other viruses, resulting in their aggre-
gation. The function of neuraminidase is to cleave off these
cellular residues so that the virus particles are not attracted to
one another.
748 SMEE ET AL.
In cell culture studies, the potencies of RWJ-270201 and
oseltamivir carboxylate were dependent upon the time of ini-
tiation of treatment and the virus MOI. In order to prevent
neuraminidase activity leading to inefficient virus release (or to
cause virus aggregation as described above), treatments with
the compound needed to be initiated within 12 h of infection.
Increasing the MOI increased the number of cells initially
infected. These cells were not spared by treatment, and high
levels of cell-associated virus were produced. Treatment would
protect neighboring cells during the secondary infection. Thus,
under low-MOI conditions, a larger number of cells were not
initially infected and would have escaped later infection by
antiviral treatment. These results suggest that patients would
most benefit by treatment early in the course of influenza
illness prior to developing high respiratory tract virus titers.
RWJ-270201, which has been selected for clinical develop-
ment, may prove to be effective in humans based upon recent
results of animal studies. Our experiments with this compound,
which are being published separately (23), indicate efficacy in
treating influenza virus infections in mice when administered
orally. RJW-270201 is well tolerated in mice, as are the Food
and Drug Administration-approved neuraminidase inhibitors
zanamivir and oseltamivir (22). RWJ-270201 appears to be a
viable candidate for the treatment of influenza infections in
humans.
ACKNOWLEDGMENTS
This work was supported by contract N01-AI-85348 from the Virol-
ogy Branch, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, and by a grant from The R. W. Johnson
Pharmaceutical Research Institute.
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