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Antimicrobial Agents and Chemotherapy, March 2001, p. 743-748, Vol. 45, No. 3
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 ascertained 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 Influenza has continued to be a significant
public health concern, with annual epidemics responsible for
serious morbidity and mortality (1, 13). Much attention
has consequently 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 virus 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
carboxylate (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 influenza (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 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 compounds (Fig.
1) have features in common with both
zanamivir and oseltamivir carboxylate but differ in having a
five-membered-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 greater detail in
secondary assays, since it has been selected for clinical development.
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
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
1.5 µM. Twelve strains of H3N2 and two strains of
avian H5N1 viruses were inhibited at <0.3 µM. Influenza
B/Beijing/184/93 and B/Harbin/07/94 viruses were inhibited at <0.2
µM, with three other B virus strains inhibited at 0.8 to 8 µM. The
novel inhibitors were comparable in potency to (or slightly more
potent than) zanamivir and oseltamivir carboxylate. 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 optimal activity. Exposure of cells to
RWJ-270201 caused most of the virus to remain cell associated,
with extracellular virus decreasing in a concentration-dependent manner. This is consistent with its effect as a neuraminidase inhibitor. RWJ-270201 shows promise in the treatment of human influenza
virus infections.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (18K):
[in a new window]
FIG. 1.
Chemical structures of cyclopentane derivatives,
zanamivir, and oseltamivir carboxylate.
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MATERIALS AND METHODS |
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Compounds. RWJ-270201, BCX-1827, BCX-1898, BCX-1923, zanamivir, and oseltamivir carboxylate were synthesized at BioCryst Pharmaceuticals (Birmingham, Ala.). Ribavirin was obtained from ICN Pharmaceuticals (Costa Mesa, Calif).
Viruses. The following viruses were provided by H. Regnery of the Influenza 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 passage in the presence of the drug in this laboratory. A/Virginia/2/88r (H3N2), a clinically isolated amantadine-resistant virus, was provided by F. Hayden, University 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 passaged 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 µg of EDTA per ml, and 50 µg 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 concentration 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 determined by the method of Finter (5), using a computerized EL-309 microplate autoreader (Bio-Tek Instruments, Winooski, Vt.). Antiviral activity was expressed as the 50% effective (virus-inhibitory) concentration (EC50) determined by plotting compound concentration versus percent inhibition on semilogarithmic 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 determinations 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 viruses, 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 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. Experiments 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 treatment 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.
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RESULTS |
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Materials and Methods
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Discussion
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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 sensitive to inhibition by the compounds, with EC50s ranging from 0.06 to 0.22 µM. 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 µM range. Activities
against the A/NWS/33 virus were up to an order of magnitude less, at 19 to >100 µM. Overall, zanamivir and oseltamivir carboxylate were slightly less potent (usually threefold or less) than the cyclopentane derivatives against the H1N1 viruses.
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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 µM. In addition, actively dividing cells were not inhibited in their growth at a 1,000 µM concentration of each compound. By dividing this concentration (1,000 µM) 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 analog) 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
µM concentration of the neuraminidase inhibitors and 5.7 µM
ribavirin for inhibition of viral CPE. Treatment of cells prior to
infection required much higher concentrations 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 concentration to achieve 50% inhibition as was effective for the
0-h time point of treatment initiation. However, by 24 h, higher
concentrations were required to inhibit virus-induced cytopathology.
These studies were done with a low input MOI, indicating that early
treatments were necessary to suppress or contain the later rounds of
virus replication.
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Effect of virus MOI.
Certain compounds which inhibit virus in
cell cultures infected at low MOI, often are less active (or even
inactive) at higher virus-to-cell ratios. To explore this possibility
with these compounds, antiviral activity was determined over a range of
infecting MOIs differing five-fold from each other (Table
3). RWJ-270201 and oseltamivir
carboxylate were most potent when virus infections were initiated at
low MOIs, and activities decreased with increasing viral challenge
dose. In contrast, the efficacy of ribavirin was not influenced by
increasing the MOI. This phenomenon has been previously reported for
ribavirin against other viruses (24).
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Virus yield reduction studies.
Because neuraminidase is
involved in the efficient release of mature viruses from cells,
mutant viruses lacking neuraminidase activity aggregate and
remain at the cell surface (12, 14). Treatment with
a neuraminidase inhibitor should produce the same effect. To
demonstrate this, three influenza viruses were exposed to RWJ-270201
for 3 days, followed by assay of extracellular and cell-associated
virus 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 µM, 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 µM, respectively, against the three viruses. Ribavirin caused dose-dependent 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 µM, respectively. EC90s of this compound
against cell-associated virus yields were 60, 32, and 12 µM,
respectively. This is consistent with virus yield reduction results
using ribavirin against other types of viruses (8).
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) 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.
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DISCUSSION |
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The results of this study show that the cyclopentane derivatives were active against a large number of influenza A and B virus strains in cell culture at nontoxic concentrations. The potencies 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 inhibition 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 subject 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 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 virus was detected in the extracellular medium relative to virus yields from inhibitor-free cultures. It was shown with neuraminidase-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 aggregation. The function of neuraminidase is to cleave off these cellular residues so that the virus particles are not attracted to one another.
In cell culture studies, the potencies of RWJ-270201 and oseltamivir carboxylate were dependent upon the time of initiation 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 development, 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.
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ACKNOWLEDGMENTS |
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This work was supported by contract N01-AI-85348 from the Virology 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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FOOTNOTES |
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* Corresponding author. Mailing address: Institute for Antiviral Research, 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{at}cc.usu.edu.
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Antimicrobial Agents and Chemotherapy, March 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.
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