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Circulation. 1996;94:2254-2259

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(Circulation. 1996;94:2254-2259.)
© 1996 American Heart Association, Inc.

Articles

Antiviral Treatment With WIN 54 954 Reduces Mortality in Murine Coxsackievirus B3 Myocarditis

J. Fohlman, MD, PhD; K. Pauksen, MD, PhD; T. Hyypia, MD, PhD; G. Eggertsen, MD, PhD; A. Ehrnst, MD, PhD; N.G. Ilback, PhD; G. Friman, MD, PhD

the Department of Infectious Diseases, University Hospital, Uppsala, Sweden (J.F., K.P., N.G.I., G.F.); the Department of Virology, University of Turku, Finland (T.H.); Toxicology and Safety Assessment, Kabi Pharmacia AB, Helsingborg, Sweden (N.G.I.); the Division of Clinical Virology, Huddinge University Hospital, Karolinska Institute, Huddinge, Sweden (A.E.); and the Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Huddinge Hospital, Huddinge, Sweden (G.E.).

Correspondence to Dr J. Fohlman, Department of Infectious Diseases, University Hospital, 751 85 Uppsala, Sweden.


*    Abstract
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 *Abstract
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Background Coxsackieviruses B (CBVs) are dominant causative agents in myocarditis and are associated with pathogenesis in some cases of dilated cardiomyopathy, a clinical entity with a poor survival without heart transplantation.

Methods and Results In vitro, the antiviral agent WIN 54 954 was shown to inhibit replication of CBV3 at a minimal inhibitory concentration value of 0.02 mg/L. Administration of WIN 54 954, 100 mg/kg BID PO, beginning on the day of infection resulted in complete protection from enteroviral mortality (P<.01). WIN 54 954 treatment did not abrogate the inflammatory reaction in the myocardium. No difference was found in the expression of surface lymphocyte subset markers. At 3 weeks, macrophages seemed to dominate the inflammatory reaction, regardless of treatment. There was no difference in CBV3 antibody titers, indicating that WIN 54 954 does not interfere with the development of protective immunity. Complement factors C3 and B were synthesized at a higher level during infection and correlated well with the degree of inflammatory reaction.

Conclusions The results show that WIN 54 954 is a potent antiviral agent with a highly significant effect on survival in CBV-induced myocarditis in the A/J mouse if treatment is started early. It is suggested that the reduction in mortality seen with WIN 54 954 administration is due to an inhibitory effect on virus replication in affected organs that does not interfere with cellular or humoral immunity.


Key Words: isoxazole • viruses • myocarditis • immunohistochemistry • cardiomyopathy


*    Introduction
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Previous studies have shown that the coxsackieviruses B (CBVs) may cause myocarditis in humans as well as in mice1 2 3 4 and that they may be the principal causative agents in the clinical disease setting.5 Although the majority of patients suffering from acute CBV myopericarditis recover uneventfully, a few develop persistent symptoms, sometimes cardiac insufficiency or fatal arrhythmias. Several studies suggest that not only the virus but also the antiviral host defense mechanisms contribute to the tissue damage.6 7 8 Previous attempts using immune suppressants to eliminate or diminish the tissue inflammatory reaction have been successful, but no reduction in experimental animal mortality was described.2 9 However, the broad antiviral effect of the interferon inducer poly I:C10 reduces mortality as well as myocardial inflammation if given within 2 days after inoculation with the virus. The immune stimulator linomide and selenium, although they have different modes of action in experimental CBV myocarditis, also lower mortality significantly.10 11 These results may be due to increased natural killer cell activity and scavenging selenium-containing enzymes (superoxide dismutase and glutathione peroxidase), respectively. Immunocompromised nude mice that develop sparse inflammation have been shown to be more resistant to CBV-mediated myocarditis, with lower mortality, than immunocompetent adult mice.5 There is also evidence that mice with high natural killer cell activity might be more resistant to infection with this virus.12

Recently, a new class of antiviral compounds (isoxazoles) has been synthesized.13 14 15 These analogues have been shown to interact with picornaviruses. The drugs inhibit viral uncoating and virus-receptor interaction of the major receptor group of rhinoviruses.16 By x-ray crystallography, they have been shown to bind to a hydrophobic pocket in VP1 at the bottom of the "canyon."17 This cleftlike structure has been suggested to act as a receptor recognition unit in the enterovirus and rhinovirus groups of Picornavirus. The cleft is too narrow for antibodies to penetrate, thereby protecting the fundamental element of the virus from host immune reconnaissance. In fact, hypervariable regions seem to exist around the canyon, which correlates with the high number of serotypes of enteroviruses and rhinoviruses (roughly 70 and 103, respectively). Binding of drugs to the bottom of the canyon will thus inhibit cell penetration, viral uncoating, and thereby viral replication.17 18

2,6-Disubstituted analogues of disoxaril show a broad-spectrum antipicornavirus activity in vitro and have also been shown to be effective in vivo, as demonstrated in echovirus- and poliovirus-infected mouse models.14 15 A newly synthesized drug of this family, WIN 54 954, showed a broad-spectrum antipicornaviral action in vitro as well as in vivo19 and appeared especially interesting with regard to myocarditic enteroviral variants.

Here, we report the effects of the disoxaril analogue WIN 54 954 in CBV3-infected inbred strain of A/J mice.


*    Methods
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Animals
Male A/J mice (Bomholtsgaard, Denmark) and CBA mice,20 4 to 8 weeks old, were used for the experiments. CBA mice were used as an additional control to show the inherent tendency for myocarditis and mortality in enteroviral infection. Mice were kept under strict hygienic barriers at 23±1°C and were controlled for humidity. Food (Ewos) and water were given ad libitum. Different animal groups used in the experiments were randomized with similar initial mean body weights and sized to allow for losses due to mortality as determined from preexperiments. Infected, treated, and control mice were studied simultaneously. Experiments had been reviewed by an ethics committee.

Virus
CBV3 (Woodruff strain) obtained from Dr S. Huber, Burlington, Vt, was used for infection of the animals. The virus was stored at -20°C in aliquots from the same stock solution and before infection was suspended in the proper concentration to inject 5x103 PFU in 0.2 mL physiological saline IP. For in vitro testing, other virus strains were obtained from different sources.

Experimental Design and Antiviral Therapy
WIN 54 954 is not water soluble, but it was easily solubilized in corn oil at a concentration of 10 mg/mL after ultrasonication and was then administered intragastrically by means of feeding cannulas. The drug (0.2 mL, corresponding to 2 mg, roughly 100 mg/kg mouse) was given twice daily (at about 8 AM and 6 PM) for 5 days starting roughly 1 hour after virus inoculation. A noninoculated control group was given the same drug preparation. Animals were observed daily for at least 3 weeks and were killed on days 7 and 21, respectively, after viral infection. Blood was withdrawn under ether anesthesia through cervical vessels. The heart was excised in toto, divided transversely, and placed into formalin or frozen in a pentane/dry ice mixture.

In Vitro Plaque Reduction Assay
M-199 medium was aspirated from confluent monolayers of LLC-MK2D cells, and the cells were infected with 1.0 mL of virus ({approx}80 PFU per well) in M-199 medium. The cultures were incubated for 1 hour at 37°C. The virus inoculum was removed, and the cells were overlaid with M-199 containing 5% FCS, 30 mmol/L MgCl2, 0.5% agarose, and WIN 54 954 at various concentrations. Virus was allowed to replicate (forming plaques) for 2 days at 37°C in a 2% CO2 atmosphere. The cells were fixed with 5% glutaraldehyde and stained with 0.25% crystal violet. The minimal concentration of WIN 54 954 that inhibited plaque formation by 50% was determined and recorded as the MIC. The MIC determined in this manner had an SD of <±50%.

In Vitro Assay of Virus Drug Sensitivity
The in vitro sensitivity against different enterovirus strains was tested by incubation of various dilutions of the virus in the presence of WIN 54 954 at a concentration of 0.3 µg/mL. The results were expressed as end-point titers of virus growth and were compared with the titers of the untreated stocks.

Neutralizing Antibody Titers
Sera for analysis of virus antibodies were collected individually when the animals were killed. They were assayed for CBV3 neutralizing activity in GMK cells. For details, see Reference 21.

Histology
Formalin-fixed tissue was processed for hematoxylin-eosin staining, and immune histology was performed as presented elsewhere.2

Image Analysis
Automatic image analysis was performed with an Olympus BH2 light microscope, a Dage CCD black-and-white video camera, and a Macintosh II cx computer equipped with Optilab software and a Neotech card. The analysis permits evaluation of a gray scale. Image analysis was used for surface area measurement.

Quantification of mRNA
The livers were cut into small pieces and frozen on blocks of dry ice in cryotubes. Total RNA was prepared from the mouse livers according to a previously described method.22 Separation of total RNA in formaldehyde gels and blotting onto nylon membranes (Hybond N) were performed according to established techniques.23 Hybridization of the immobilized RNA with 32P-labeled cDNA probes was carried out as described.24 Suitable cDNA probes were labeled by use of the Pharmacia oligolabeling system. The following cDNA probes were used: mouse factor B,25 human C3,26 and GAPDH.27 Radioactive signals were quantified by densitometry or by scintillation counting of excised areas from the filters.

Statistics
{chi}2 analysis was performed on a Macintosh Plus computer with Statview SE+ graphics software.


*    Results
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In Vitro Assays
The antiviral activity of WIN 54 954 was tested against different enteroviruses (TableDown). It showed high antiviral activity at a concentration of 0.3 µg/mL against two tested myocarditic virus variants, CBV3 and CBV4, poliovirus 3, and coxsackievirus A9, but less activity was noted for the other strains tested (TableDown). In general, WIN 54 954 was more potent against the viruses tested than was WIN 51 711. The MIC of WIN 54 954 against the myocarditic CBV3 (which was used in the in vivo model) was determined to be 0.02 µg/mL. Two other myocarditic CBV3 variants (Gauntt and Frisk) had MIC values of 0.03 and 0.05 µg/mL, respectively. A nonmyocarditic variant (RD), however, had an MIC 100 times higher, 2.00 µg/mL.


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  		Table 1. In Vitro Antiviral Effects of WIN Compounds

Survival of the Animals
In numerous previous studies of the present experimental CBV3 infection in BALB/c and A/J mice, we have consistently observed pancreatitis, myocarditis, and hind leg paresis. Animals lose weight, appear apathetic, and most likely restrict food and water intake to a minimum. No signs of infection were apparent in CBA mice, which thus appear naturally resistant to "clinical" disease. After treatment with the compound WIN 54 954, a clear therapeutic effect was apparent that was very consistent in all the experiments performed (more than 500 animals observed). There was a clear dose dependence, and at doses >200 mg·kg-1·d-1 for 5 consecutive days, all animals survived for 10 days (Fig 1Down). There was a slight loss of animals with further time lapse, but even so, at 3 weeks, >80% were alive compared with the infected control group with about 10% survival. This beneficial effect of drug treatment at 200 mg·kg-1·d-1 versus placebo treatment was statistically significant at P<.01, Fig 2Down.



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  		Figure 1. Dose dependence of WIN 54 954 in CBV3-infected BALB/c mice at day 10 after viral inoculation.



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  		Figure 2. Survival at 7 and 21 days after CBV3 inoculation in WIN 54 954 (200 mg·kg-1·d-1 BID) treated and untreated (CTRL) BALB/c mice.

Neutralizing Antibody
Infected WIN-treated animals developed antibody titer levels equivalent to those observed in the infected control animals. A mean reciprocal titer of 2560 was obtained for the WIN-treated group versus 2296 for the infected control group (not significant). This indicated active infection in the treated animals as well, which is consistent with the microscopic findings of myocarditis (see below). It also means that the immunological response and protective immunity are not impaired by treatment.

Histopathology
Noninfected animals and noninfected WIN 54 954–treated animals showed no inflammatory reaction. CBV-inoculated animals treated with WIN 54 954 (200 mg·kg-1·d-1) showed infiltration of inflammatory cells to a slightly (but nonsignificantly) higher degree than untreated mice. Estimation of the mean inflammatory ratio (value from 10 animals in each group) is depicted in Fig 3Down. It appears that treated animals show more inflammatory reaction, which may be explained by a higher survival in animals with more severe disease. CBA mice are known to have unusually high levels of natural killer cells.20 They showed little inflammatory reaction (Fig 3Down) and had a much higher survival rate (almost no mortality at all). Noninfected animals, with or without treatment with WIN 54 954, showed negligible amounts of inflammation. Immune histological staining (Fig 4Down) showed the typical appearance of lymphocyte subsets.2 Mainly macrophage-positive (MAC-staining) cells were seen together with T helper cells (not shown). T killer cells were in the minority. There was no difference among the groups in this respect.



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  		Figure 3. Inflammatory area observed in the different groups (mean of 8 animals) at 7 (left) and 21 (right) days after CBV3 infection in CBA and A/J mouse strains. Control (CTRL) animals were untreated as well as drug treated but not inoculated with virus (WIN indicates WIN 54 954).



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  		Figure 4. Number of surface marker–expressing cells per defined area at day 7. Markers were (left) Lyt 2 for T killer cells and (right) MAC for macrophages. CB3 indicates CBV3 infection.

Correlation Between Complement Factor Synthesis and Inflammatory Reaction
The results of RNA blotting for complement factors C3 and B are shown in Figs 5Down and 6.Down The banding pattern was analyzed with a densitometer. It is clear that the activation of complement factor synthesis is greatest in the infected WIN 54 954–treated groups. A good correlation existed between the degree of inflammation (Fig 3Up) and the level of complement factor synthesis. The correlation coefficient between inflammatory area and C3 mRNA level was 0.97. A slightly lower figure was obtained for factor B (0.81). The correlation between factors B and C3 was 0.92.



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  		Figure 5. Northern blot analysis of total RNA hybridized with a 32P-labeled cDNA probe against human C3 mRNA (arrow) with 28-hour exposure time. A lanes are control animals; B lanes, animals infected with CBV3; and C lanes, animals infected with CBV3 and treated with WIN 54 954. 28S and 18S denote the positions of the ribosomal RNA.



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  		Figure 6. Same as Fig 5Up, but hybridized with a cDNA probe against murine factor B (f.B) mRNA and human GAPDH (GAP) mRNA (arrows). Exposure time was 15 hours. Lane designation as in Fig 5Up.


*    Discussion
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 up arrowAbstract
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 up arrowResults
 *Discussion
down arrowReferences
 
Myocarditis is a well-known clinical entity with a favorable prognosis in most cases.1 28 29 Several lines of evidence support the hypothesis that CBV can persist after a clinically silent myocarditis, finally causing dilated cardiomyopathy and end-stage heart failure. This is indicated by high persisting CBV IgM titers30 as well as polymerase chain reaction and in situ hybridization studies showing enteroviral RNA in the heart.31 32 33 At present, no established, causal medical treatment exists for these conditions.

In the present article, the viral capsid–binding drug WIN 54 954 was shown to be effective in preventing death in CBV3-induced murine myocarditis. The lethal mechanisms have not been explored in the present infection model; concomitant encephalitis, pancreatitis, and hepatitis are possibilities. WIN 54 954 has been shown to eradicate enterovirus from the central nervous system in other experimental situations.15 34 The earlier it was given during the course of the infection, the more effective it was.35 CBV also has affinity for the pancreas. Thus, exocrine pancreatitis with electrolyte-fluid imbalance or massive coagulative hepatocellular necrosis36 cannot be ruled out as perhaps more severe manifestations than the myocarditis. Despite the clear effect on survival, WIN 54 954 did not abrogate myocardial inflammation in CBV3-infected mice, thus indicating that organ infection does take place and initiates an immune response, even with WIN 54 954 treatment. With WIN 54 954, antibody production and complement activation occurred as well. This is in contrast to a previous study with ribavirin in murine CBV3 myocarditis, in which the myocardial inflammation was virtually abrogated.37 It has not been shown that ribavirin has an anti-inflammatory, immune-suppressive effect per se; the most prominent side effect was reversible anemia.38 Perhaps the inhibition of viral metabolism and replication by ribavirin is more effective in inhibiting virus-induced cytolysis and ensuing inflammation than the capsid-binding, receptor-blocking action of WIN 54 954. The question of virus-induced cytopathology rather than immune-pathological mechanisms in the cardiac lesions has been hard to resolve. Since WIN 54 954 does not inhibit heart inflammation, whereas ribavirin does, this may be taken as an indication that viral synthesis is important in the pathogenic process.

The mRNA expression of complement factor C3 and factor B in the liver was found to increase moderately after CBV3 infection. We also observed a correlation between the size of the inflammatory lesion in the myocardium and the increase in C3 and factor B mRNA expression. C3 and factor B belong to the group of acute-phase reactants whose transcription can be significantly increased by cytokines.39 40 41 42 43 Generation of inflammatory mediators may be expected in the infected mice, as reflected in the increase of mRNA for C3 and factor B. However, variations in the patterns exist between individual mouse strains, and it is difficult to evaluate the amplitude of the response.44 Generally, it is well known to the clinician that acute-phase reactants, like C-reactive protein, are less notably affected by virus than by bacterial infections. Whether increased complement synthesis is a measure of the inflammatory reaction, a response to tissue destruction, or a parallel phenomenon was not addressed here. Complement synthesis is partially under the control of interleukin-1 and tumor necrosis factor,44 and interleukins are activated during enterovirus infection.45

In the pharmaceutical industry, long-term projects have been devoted to the search for antipicornaviral drugs. The most promising so far appear to be disoxaril (WIN 51 711), Ro 09-0881, Ro 09-0535, and Ro 09-0696 (Roche Laboratories). R-61837 was effective against rhinovirus infection in a prophylactic, double-blind, placebo-controlled clinical trial in human volunteers.46 Antiviral drugs are presently available clinically for several viral infections, including herpes simplex, herpes zoster, cytomegalovirus, HIV, hepatitis B and C, respiratory syncytial virus, and influenza A. With regard to the picornavirus family, including the coxsackieviruses, much work remains to be done to find the best drugs and evaluate them in clinical trials for different indications. WIN 54 954 represents a promising candidate toward this goal. One clinical application might be for prophylaxis (and even early treatment) in the setting of serious enteroviral epidemics, which rarely occur in neonatal wards.


*    Acknowledgments
 
The project was funded by the National Heart and Lung Association, Uppsala University Funds, and the Swedish Diabetes Association. The skillful assistance of Gertrud Bertilsson Pfrehl, Maria Israelsson, Marita Maaronen, and Ninni Rosen is gratefully acknowledged. The expert know-how, assistance, and supply of the drug by Maureen Woods and Mark McKinlay at Sterling Drug Co, Rensselaer, NY, were indispensable for the completion of this study.

Received November 2, 1995; revision received May 22, 1996; accepted June 7, 1996.


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 *References
 

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