EFFECT OF AN INTERFERON INDUCER,

VIRUS INFECTION IN MICE 9-METHYLSTREPTIMIDONE, ON INFLUENZA

Fujio Suzuki, Noriyuki Saito, and Nakao Ishida

Department of Bacteriology Tohoku University School of Medicine

Sendai, Japan

9-Methylstreptimidone (9-MS), isolated from the culture filtrate of a Strep- tomyces sp. S-885 and having the structure of 3-(5,7-dimethyl-4-0~0-2-hydroxy-6,8- decadieny1)-glutarimide (FIGURE I ) , is a new antiviral antibiotic, which inhibits the growth of poliovirus and Newcastle disease virus (NDV) in virro. Chemical and bio- logical properties of this antibiotic were detailed in our previous report.’ The anti- biotic had no antibacterial or antimycoplasmal activity, but it showed inhibitory activities against the growth of some yeasts such as Saccharomyces sake at the concentration of 4 to 20 pg/ml. The acute LD,?,of the antibiotic by intraperitoneal in- jection was 280 mg/kg in mice, 238 mg/kg in rats, and 138 mg/kg in guinea pigs. The present report describes the protective activity of 9-MS on influenza A2 virus infec- tion in mice. The detection of interferon in mice was correlated with anti-influenza action of the antibiotic.

Materials and Methods

9-MS used in this study was purified from a culture filtrate of Streptomyces sp. S-885.’ The final product, which is pale yellowish viscous oil, was dissolved in sterile phosphate-buffered saline (PBS), pH 7.2, containing 0.1 % ethanol and 0.001 96 Tween 80 and used for animal experiments.

The ddl strain of mice of both sexes weighing 14-16 g were Jsed throughout. They were obtained from the Central Breeding Farm of Tohoku University, where care was taken to prevent infections with Sendai virus and Mycoplasma.

The Kumamoto strain of influenza A2 (H2N2) virus was used for the infection.2 The mouse-adapted virus strain was once propagated in the allantoic cavity of em- bryonated eggs and 48-hr allantoic fluid was used as the inoculum after light centrifu- gation. The egg infectious titer (EID,,) of the fluid was 108.4/ml, which corresponded to LD,,/ml in mice, when it was inoculated intranasally by our standard inhala- tion procedure.

Virus infection was achieved by inhalation of influenza virus. A vaponephrine type nebulizer was connected to the compressor to spray about 10 ml of diluted allantoic fluid within 30 min. This procedure resulted in the inoculation of 1/500 ml of the fluid per mouse.3 The criteria for evaluation of anti-influenza activity were increase in the mean survival time and in the number of survivors 30 days after infection. The former was evaluated statistically by the use of Student’s t-test and the latter, by means of chi-square analysis. In both evaluations, if the p value obtained was below 0.05, the d<yg dose was considered to have a possible antiviral activity.

To determine if 9-MS stimulated interferon, mice were injected intraperitoneally with 9-MS, and at appropriate intervals serum or lung specimens were taken for an- tiviral assay. As a positive control for interferon characteristics, mouse serum in-

667

668 Annals N e w York Academy of Sciences

terferon induced by E. coli endotoxin was tested. Titrations of interferon were made in a system of the thymidine-kinase-less mutant strain of L-ID cells and the Indiana strain of vesicular stomatitis virus (VSV). Interferon units were expressed as the re- ciprocal of the maximal sample dilution giving definite protection against viral cyto- pathic e f f e ~ t . ~

Results

The first part of this presentation will describe experiments showing the effect of 9-MS on influenza infection in mice. Three groups of ten mice and a control group of twenty mice were simultaneously infected intranasally with 5 LD50 of influenza virus. The first two groups received intraperitoneally 20 and 10 mg/kg of 9-MS and the third group, 15 mg/kg of amantadine hydrochloride, three times before infection (-24, -3, and -1 hr), twice after infection ( + i and + 3 hr) and once daily for 4 consecutive days. The 20 control mice received 0.5 ml of PBS according to the same schedule. As shown in TABLE I(A), when 95% of the control mice died within 20 days, 70% of mice treated with 20 mg/kg of 9-MS survived for the observation period of 30

U

FIGURE 1. Chemical structure of 9-methylstreptimidone.

days. Both 10 mg/kg of 9-MS and aman tadine hydrochloride gave 30% survival. The mean survival time of 9-MS-treated mice was also significantly increased.

Even when the challenge dose of influenza virus was increased to 10 LD,,, an a p parent effect of 9-MS on the prolongation of the life span was observed. As shown in TABLE I(B), two groups of ten mice receiving 20 and 10 mg/kg of 9-MS following the above schedule gave survival rates of 50% and 30%, respectively. This was accom- panied by significant increase in mean survival time of the mice treated with both doses of 9-MS [TABLE l(B)]. With such a high challenge dose, all control mice receiv- ing PBS died within 15 days. Further studies revealed that increasing the dose of 9- M S resulted in the proportionate increase in the survival rate, when 1.2 to 22 mg/kg of 9-MS were administered to each group of 10 mice infected with 10 LD,, of influenza virus. Thus, dose-dependent protective activity was observed (FIGURE 2).

The protective effect of 9-MS on influenza virus infection in mice was also de- pendent on the treatment schedule. When the mice were given 30 mg/kg of 9-MS only for three times, 24, 3, and 1 hr before infection of 10 LD,, virus, 50% of the mice survived for more than 30 days (FIGURE 3), whereas all the control mice receiving PBS died within 13 days. When the mice were treated with the same dose of 9-MS for three times, 24, 48, and 72 hr after infection, all the treated mice died within 12 days as did the untreated controls (FIGURE 3). Thus the antiviral effect of 9-MS on

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influenza virus infection in mice was apparent only when the antibiotic was given in a prophylatic manner. In addition, when a single prophylactic injection of 9-MS was given intraperitoneally to mice according to the different time schedules such as 72, 48, 24, or 2 hr before infection, the data shown in TABLE 2 were obtained. In this ex- periment, an almost toxic dose (260 mg/kg) of 9-MS was used. As a result. only one group of mice receiving a single intraperitoneal injection 2 hr before infection gave 40% survival with a statistically significant increase in mean survival time, whereas

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672 Annals New York Academy of Sciences

the mice receiving the same dose of 9-MS at any other time died within 9 days, as did the untreated controls. The effect of a single administration was also examined at a nontoxic dose (5 mg/kg). When this dose was given intraperitoneally 2 hr before virus infection (TABLE 2), all the control mice died within 9 days, and a single administra- tion of 5 mg/kg of 9-MS produced a 20% survival rate (p = 0.027). The mean survival time of those mice also increased significantly.

The second part of this presentation will describe experiments on the antiviral mechanism of the antibiotic. Before everything, in order to test the possibility of direct interaction of the antibiotic with the virus particles, lo7., EID,, of influenza Aa virus was mixed with 1 mg of 9-MS per ml in serum-free Eagle’s MEM and in- cubated for 12 hr a t 37°C. A virus preparation suspended in broth free from the anti- biotic and another preparation mixed with 5% formalin served as negative and posi-

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FIGURE 4. Induction of virus inhibitory factor (=interferon) m mouse serum and lung after intraperitoneally injection of 30 mg/kg of 9-MS. On ordinate the reciprocal of the maximal dilu- tion of serum or 10% lung suspension affording definites protection of L-ID cells from the cyto- pathic effect of 500 TCD,,of VSV was plotted. Lung interferon titer was expressed as a content of the whole organ. 0---0, lung; o--o, serum.

tive controls, respectively. The initial and residual virus contents were determined by titrating infectivity in the chorioallantoic cavity of chick embryos. Though 9-MS did not reveal any virucidal effect on influenza virus, 5% formalin reduced the initial virus titer of 107.5 to less than lo3 EID,,. The effect of 9-MS on in vitro influenza virus growth was examined in chick embryo fibroblasts. For this purpose, after influenza virus was adsorbed to cells a t 37°C for 1 hr, each concentration of 9-MS dissolved in maintenance medium was added, and the total yield of virus was assayed 48 hr after infection by egg infectivity (EID,,). 9-MS showed some inhibitory activity on the growth of influenza virus a t concentrations of more than 12.5 pg/ml, where it also in- hibited the growth of chick embryo fibroblasts.

The results described above, such as, ( I ) that the antiviral effect was demonstrated by prophylactic treatment, (2) that the effect was shown by a single

Suzuki et al.: 9-Methyls t rept imidone 67 3

administration 2 hr before infection, suggested that the antibiotic might be an in- terferon inducer. Therefore, evidence for an interferon activity was sought in serum and lung samples (10% PBS extract) taken from the antibiotic-ti eated mice. As a result, the production of a viral inhibitor in mouse serum and lung after intraperitoneal injection of 30 mg/kg of 9-MS was apparently demonstrated. As shown in FIGURE 4, the inhibitor was found in the serum at the 12th hour postinjec- tion and the titer reached a maximum at the 16th hour. At the 32nd hour the titer was no longer detectable. In contrast, an antiviral substance appeared earlier in lung tissue and reached a peak at the 10th hour. The decrease of titer in lung tissue, however, was almost parallel to that in serum. In addition, when 9-MS was injected at doses of 5 to 260 mg/kg, the same inhibitor was detected in both lung and serum of the mice.

The inhibitor activity was characterized for pH and heat stabilities, trypsin sensitivity, and species specificity, in comparison with mouse serum interferon in- duced 2 hr after intravenous injection of endotoxin. The inhibitory activity in the mouse serum obtained 16 hr after a single 9-MS treatment was abolished after dialysis against glycin-HCI buffer (pH 2.0) for 24 hr a t 4 ° C and after incubation at 56°C for 30 min. Moreover, the inhibitory activity in the serum was completely destroyed by trypsin treatment. Inhibitory activity in mouse serum was not detect- able in rabbit RK-13 cell cultures or in primary cultures of chick embryo fibroblasts when both were challenged with VSV, revealing species specificity of the inhibitor. When the same experiment was conducted with the inhibitor obtained from mouse lung at 10 hr after administration of 9-MS, almost similar results were obtained. Thus, the serum and lung inhibitor induced by 9-MS appears to be an interferon.

Discuss ion

One approach to the control of virus diseases would be to find a nontoxic inducer of interferon. The most studied inducers a re large anionic poIymers,s,6 highly base- paired ribonucleic and polycarboxylates,9.'n all of which were characterized by their polydispersity and toxic side effects.".'* Some low-molecular-weight in- ducers, such as tilorone hydrochloride,I3 BL-20803,I1 CP-2096 I , " and cyclohexi- mide,'O were also examined for their antiviral effect. Neither tilorone hydrochloride, an orally active inducer in mice, nor CP-20961, having a high therapeutic index (300) in the infection with VSV, showed protective activity against influenza virus infection in mice. Cycloheximide caused induction of circulating interferon, but only at the dose where it inhibited protein synthesis in animals."j Other low-molecular-weighr in- terferon inducers have not been examined in the influenza infection in mice.

9-Methylstreptimidone, a unique glutarimide antibiotic, has been shown to be effective in prolonging the life span of the mice infected with influenza virus and to stimulate interferon production in mouse lung and serum. When 9-MS is given in a prophylactic manner, the effect of the antibiotic is reproducible (TABLE I ) . However, when 9-MS is given in a therapeutic manner, it is no longer effective (FIGURE 3). When a high dose (260 mg/kg) of 9-MS was administered once at various times, only one subgroup treated 2 hr before virus infection survived, whereas the other subgroups treated at 24 to 72 hr before infection died at the same time as the nontreated controls (TABLE 2).

On the basis of this result, a completely nontoxic dose of 9-MS ( 5 mg/kg) was administered only once 2 hr before virus infection (TABLE 2). Both survivor numbers and mean survival days of mice increased significantly, indicating that antiviral effect of 9-MS might be displayed not by direct viral inhibitory activity, but rather by host- mediated activity such as interferon induction.

674 Annals New York Academy of Sciences

Although the data are not included in the present article, after a single intraperitoneal administration of an almost toxic dose (260 mg/kg) of 9-MS, the antimicrobial activity against Saccharomyces sake was found in mouse lung at a concentration 0.3 mg/organ 15 min after the injection. However, no 9-MS activity was detected in lung 1 hr after the injection. It is also established that, when mouse- adapted influenza virus is infected by inhalation at a dose of 5 to 20 LDSo, virus growth in mouse lung is initiated 2 days after in fec t i~n .~*~. ' When these observations are taken together, direct interactions between 9-MS and influenza virus in lung are unlikely. In addition, it must be taken into consideration that high-titered in- terferon is detectable in mouse lung from 7 to 25 hr after intraperitoneal injection of 9-MS (FIGURE 4).

However, the direct evidence is still lacking to show that the induced interferon inhibited the virus growth. The antibiotic did not possess any virucidal activity but revealed an inhibitory activity in vilro on the growth of influenza virus when an al- most cytotoxic concentration of the antibiotic was maintained in the cultures from the initial stage of infection. However, it should be emphasized that in vivo protective activity of 9-MS against influenza virus infection was demonstrated at a nontoxic dose.

These data suggest that the protective activity of 9-MS against influenza disease in mice might be due to interferon produced in the lung or, alternatively, to other as yet unestablished effects on host functions. Because of the low toxicity of the anti- biotic,' further detailed evaluation of the antibiotic on various in vivo viral infections are warranted.

Summary

The antiviral effect of 9-MS was examined in mice infected with mouse-adapted influenza Az (HzN,) virus. Either a single or a continuous prophylactic administration of 9-MS protected mice from virus infection, and comparison of the minimal effective dose with the lethal dose gave a therapeutic index of approximately 60. When the treatment was started after infection, however, no antiviral effect was demonstrated. A potent virus-inhibitory factor was detected in the lung and serum after a single intraperitoneal administration of 9-MS in uninfected mice, and on the basis of chem- ical characteristics of this inhibitory factor, it was assumed to be an interferon. These results suggest that the protective activity of the antibiotic in mice is due to in- terferon induction.

Acknowledgments

The authors gratefully acknowledge the staff of the Fermentation Division of Mitsubishi Chemicals Co. Ltd., Tokyo, for the ample supply of 9-MS.

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