Mechanism of Action of Anti-Influenza Benzamidine Derivatives

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1975, p Copyright American Society for Microbiology Vol. 7, No. 4 Printed in U.SA. Mechanism of Action of Anti-Influenza Benzamidine Derivatives HARUHISA FUJITA,* YOSHIKO SETO, AND SHIGESHI TOYOSHIMA Pharmaceutical Institute, School of Medicine, Keio University, Shinano-cho, Shinjuku-ku, Tokyo 160 Japan Received for publication 19 August 1974 Benzamidine derivatives exhibited a high antiviral effect in vivo against influenza virus strains A2/Adachi and B/Lee. The inhibiting properties of anti-influenza benzamidine derivatives on the virus-induced inflammation undoubtedly plays an important role in the clarification of the mechanism of action of these drugs. Amantadine possesses two defects: (i) it has a narrow antiviral spectrum and (ii) its effects are only apparent when the drug is administered in the early phase of viral infection (1, 6, 9, 13). We have tried to discover a highly effective antiinfluenza virus agent which would exert a curative effect rather than a prophylactic effect against influenza virus infection. Fujita et al. and Toyoshima et al. (3, 15) reported that amidine derivatives and amino acid analogues have a broad anti-influenza virus spectrum and exhibit an inhibitory effect even when treatment is started late after viral inoculation (2, 4, 12, 14). The present report concerns the results obtained from studies to clarify the mechanism of the,se drugs in governing the effects of antiinfluenza benzamidine derivatives on viral replication and on virus-induced pulmonary inflammation. MATERIALS AND METHODS Materials. Viral strains used were influenza virus A2/Adachi and B/Lee. Cells used were fibroblast cells derived from 10-day-old chicken embryo. Male dd strain mice weighing 10 to 12 g and male Wistar strain rats weighing 150 to 200 g were used. As an inflammatory substance, hyaluronidase (Sanko Pure Chemical Co., Tokyo) was used. Hydrocortisone, fulfenamic acid, aspirin (Fujisawa Pharmaceutical Co., Tokyo) and amantadine (DuPont Co.) were used as standard reference drugs. Merzonine (Wako Pure Chemical Co., Tokyo) was used to inactivate infectivity of the virus. The benzamidine derivatives used are shown in Fig. 1. Evaluator of the activity of anti-influenza drugs in mice. After being intranasally inoculated with 5 to 10 mean lethal doses of either A2/Adachi strain or B/Lee strain in ether anesthesia, mice were injected intraperitoneally with an appropriate dose of an experimental compound. In all studies, during and/or after the 14-day observation period, evaluation was made on the parameters, including mean survival rate, degree of development of pulmonary consolidation, and amount of virus in mouse lungs. Pulmonary consolidation was evaluated by the method of Ledinko (8). Estimation of anti-inflammatory action. Antiinflammatory action of compounds was measured by the method of Winter et al. (16). Inflammatory agent (virus) was injected into the hind footpads, and 30 min later test compounds were injected intraperitoneally. The animals were then examined to determine the amount of edema, which was estimated by measuring the hind footpad volumes at 0.5, 1, 2, and 3 h after injection of the inflammatory agent. Cultivation of chicken embryo fibroblast celis. Chicken embryos were harvested aseptically into petri dishes from 10-day-old eggs. After removal of head and legs, embryos were finely cut up with scissors and placed in Hanks solution supplemented with 0.25% trypsin and left at room temperature for 2 h. The resulting supernatant fluid was discarded, and the process was repeated. The culture was held at 4 C for 16 to 20 h and strained through an 80-mesh net. The resulting filtrate was centrifuged at 600 to 1,000 rpm for 10 min to allow for collection of cells. This procedure was repeated several times. Cells were incubated at 37 C. At the start of an experiment each tube contained 2 x 106 cells/ml. Counting plaques of influenza virus. The monolayers of chicken embryo fibroblast cells were washed repeatedly with a phosphate-buffered saline solution and then infected with virus, which was allowed to be absorbed by the cells at 4 C for 30 min. Finally, Lact-Hanks solution, containing 0.003% neutral red and a 1% final concentration of agar, was laid on the surface of the cell monolayer. After a 2- to 3-day incubation period at 37 C the plaques were counted. Inactivation of influenza virus. The lung homogenates of mice infected with influenza virus were centrifuged at 3,000 rpm for 30 min. The resulting supernatant fluid was centrifuged at 20,000 rpm for 30 min. The precipitated virus was suspended with saline in one-tenth of the original volume and centrifuged again at 3,000 rpm for 30 min. The resulting supernatant fluid, to which a 1:10,000 concentration of merzonine was added, was incubated at 37 C for 4 h 426

2 VOL. 7, 1975 ANTI-INFLUENZA BENZAMIDINE DERIVATIVES T - 40 N, N - DI - N - HEXYL - PHENYLACETOAMIDINE HYDROCHLORIDE NHC6HI3 C6HISCHj2 X HCL NC6H13 T N - CYCLOHEXYL - N - N - BUTYLBENZAMIDINE HYDROCHLORIDE 427 NH / CH3 \ C HCL N(CH2)4CH3 T N - CYCLOHEXYL - N - BENZYL - P - BENZAMIDINE HYDROCHLORIDE NH/\ C < O *HCL NCH2CH:CH T N - CYCLOHEXYL - N - N - PROPYL - BENZAMIDINE HYDROCHLORIDE NH/\ / CX-H HCL N(Ca2)2CH3 FIG. 1. Chemical structure of benzamidine derivatives. and allowed to stand at 4 C for 24 h to inactivated viral infectivity. RESULTS Effects of benzamidine derivatives in mice infected with influenza virus. Three days after intranasal inoculation of five mean lethal doses of mouse-adapted A2/Adachi strain, the mice were intraperitoneally injected with one injection of one-fifth mean lethal dose of each compound. The control group of mice were injected with physiological saline only. The results are shown in Table 1. N,N',di-n-hexyl-phenylacetoamidine hydrochloride (T-40) and N-cyclo-hexyl-N-n-butylbenzamidine hydrochloride (T-217) exhibited distinctive chemotherapeutic effects; however, these effects were not observed in amantadine. Therefore, it is evident that the characteristics of the antiviral effect of amidine derivatives are superior to that of amantadine in that they display curative effects even under restricted conditions, such as delaying initial injection for up to 3 days after viral infection. On this basis, the following experiment was carried out to determine the differences in the mechanism of action of amidine derivatives and amantadine. Antiviral effects of amidine derivatives on influenza virus in tissue culture. Experiments were conducted primarily to examine the effects of amidine derivatives on the multiplication of virus at the cellular level. Chicken embryo fibroblast cells were infected with A2/Adachi or B/Lee strains and allowed to stand at 4 C for 30 min. A maximum nontoxic dose of the testing compound was added to the culture, which was then incubated at 37 C for 22 h. The total amount of virus was estimated by plaque-forming technique. The results obtained are shown in Table 2. The multiplication of A2/Adachi strain was virtually inhibited by benzamidine derivatives and amantadine when using a maximum nontoxic dose of each respective compound (Table 2). When one-tenth of the maximum nontoxic dose of each respective compound was used, the multiplication of A2/Adachi strain was inhibited completely by amantadine but less markedly so by benzamidine derivatives. With regard to the inhibitory effect on viral multiplication in tissue culture, amantadine displayed the most outstanding values; however, the curative effect in vivo showed that amantadine was effective only when it was administered directly before or after viral inoculation. Benzamidine derivatives, however, displayed a curative effect in vivo even when administered on the third day after viral inoculation (Table 1). These results suggest that there may be a differences in the mechanism of action between amantadine and any one of the benzamidine derivatives. It is presumed that the benzamidine derivatives may have an inhibitory effect in arresting pulmonary inflammation induced by the influenza virus infection. The following investigation was carried out to resolve this problem.

3 428 FUJITA, SETO, AND TOYOSHIMA ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Effects of benzamidine derivatives on mice infected with strain A2/Adachia Mortality" Lung consolidation" Viral amounts in lungsd Compound Dose Drug administered (mg/kg) Reduction Score Reduction toxicityc EID,/ml Inhibition (% (% (log,0) (% Viral control Amantadine T e e T ' e T ' a Each group was comprised of 20 mice. b The data indicates the mean values of results of three experiments. c Expressed as the number of mice which died. d Viral amounts in lungs was estimated on day 4 after viral inoculation. e p = <0.01. tp = <0.05. Anti-inflammatory effects of compounds on edema induced with pharmacological inflammatory agents. Additional research was conducted to determine the inhibitory effect of benzamidine derivatives on edema induced by a pharmacological inflammatogenic agent. Rats were injected intraperitoneally with standard reference drugs and benzamidine derivatives. Thirty minutes later, the hind paw was injected with 0.1 ml of an inflammatory agent. To determine the inhibitory effect of the compounds, edema intensity measurements were taken at 1, 2, and 3 h after the injection. The results obtained against edema induced with hyaluronidase are shown in Table 3. The edema was moderately inhibited by benzamidine derivatives, and the inhibition rate was equivalent to that of flufenamic acid or aspirin (Table 3). However, amantadine did not show any anti-edema effect. These facts indicate that these amidine derivatives displayed satisfactory anti-inflammatory action. Inhibitory effect on the consolidation development in mice infected with merzonineinactivated virus. It has been reported (10) that when influenza virus is treated with merzonine its infectivity is inactivated, but it retains its ability to induce hemagglutination. However, our studies showed that when mice were infected intranasally with this inactivated virus, pulmonary consolidation developed. Mice were infected with 3,200 hemagglutination units of merzonine-inactivated virus, and the administration of a compound was started immediately after infection and continued once daily for 4 consecutive days. On day 5 the mice were examined to determine the degree of pulmonary consolidation (Table 4). The findings indicate that benzamidine derivatives inhibit the development of pulmonary consolidation induced with inactivated-virus inoculation. To study TABLE 2. Antiviral effects of benzamidine derivatives on influenza virus in tissue culture Compound Drug Mean plaque numbers administered concentration (,Ug/ml) A2/Adachi B/Lee Saline control Amantadine (98.2)a 76.5 (0) T (93.3) 45.4 (40) T (95.5) 2.9 (96.2) T (86.8) 31.3 (58.9) Amantadine (96.2) 81 (0) T (14) 77 (0) T (17.6) 79.3 (0) T (20) 82.2 (0) a Parentheses indicate the percentage of inhibition of plaque formation. these results further, the effect of benzamidine derivatives on the inflammatory factors induced by viral infection was also examined. Inhibitory effects of various compounds on the development of edema induced by the factors causing viral consolidation. In the studies to clarify the mechanism of action of anti-influenza amino acid analogues, an attempt was made to isolate inflammatory factors from the lesion of pulmonary consolidation (2). Experiments were conducted to clarify the effects of amidine derivatives on the development of edema induced by the inflammatory factors induced by virus. Rats were first injected intraperitoneally with a 0.2 mean lethal dose of the tested compounds. Thirty minutes later, their hind paws were inoculated (by the method of Folin-Ciocalteu) with 0.45 mg of protein contained in the crude inflammatory factor prepared as previously described (2). In this manner a comparison was made of the inhibitory effects of various

4 VOL. 7, 1975 ANTI-INFLUENZA BENZAMIDINE DERIVATIVES 429 TABLE 3. Anti-inflammatory effects of benzamidine derivatives on edema induced with hyaluronidase in rats Anti-inflammatory activity" Compound Dose administered (mg/kg) 1 h 3 h Edema vol (ml) Inhibition (%) Edema vol (ml) Inhibition (%) Saline 1.6 ± Amantadine ± Flufenamic acid ± k ± Aspirin ± ± k Hydrocortizone c 0.5 ± b ± b 0.7 ± T b O b T ± b 0.7 ± k ± b 0.9 ± a Time after injection of hyaluronidase. Each group was comprised of five rats. P = <0.05. c p = <0.01. TABLE 4. Inhibitory effects on development of lung consolidation in mice infected with merzonine-inactivated A2/Adachi virus straina Lung consolidation Compound Dose" administered (mg/kg) Score Inhibition (Mean ± SDc (%) Saline 2.3 ±0.7 Amantadine ± Hydrocortizone ± d Aspirin ± T le T ± e a Each group was comprised of 10 mice. b Merzonine-inactivated virus was administered to mice and immediately followed by doses of the testing compounds which were continued for 4 consecutive days. c SD, Standard deviation. P p = <0.05. e p = <0.01. compounds. Hydrocortisone and aspirin were used as standard reference drugs in these experiments. The results are shown in Table 5. T-40 and T-217 revealed an outstanding inhibitory effect upon viral inflammation (Table 5). The effect of these compounds was of the same degree as that of hydrocortisone. However, aspirin possessed a somewhat lower inhibitory effect on viral inflammation than did T-40 and T-217. Amantadine possessed no inhibitory effect at all. TABLE 5. Inhibitory effects of various compounds on the development of edema induced by the factors causing viral consolidationa Edema-inducible activity Compound Dose administered (mg/kg) Edema vol Inhibition (ml) (%) Saline 1.14 ± 0.10 Hydrocortizone ± Ob Aspirin ± Amantadine ± T ± b T ± Ob a Each group was comprised of five rats. b p = <0.01. DISCUSSION In the tissue culture system, amantadine was shown to exhibit significant inhibition of viral replication of A2/Adachi strain. It has been reported that amantadine exhibits the process of uncoating in the course of intracellular viral replication (7). However, these experiments showed that the effect of amidine derivatives on viral replication was definitely inferior to amantadine. These results suggest that amidine derivatives may have a second mechanism of action by which they exhibit chemotherapeutic effects different from those of amantadine. Ginsberg (5) pointed out that xerosine had no effect on viral replication but inhibited the

5 430 FUJITA, SETO, AND TOYOSHIMA development of lung consolidation. He presumed that this lung consolidation-producing factor might have been produced by the action of a harmful substance released from cells injured primarily by the virus. Ogasawara and Aida (11) conducted studies on myxoviruses, including inactivated influenza virus, hemagglutinating virus of Japan, and Newcastle disease virus, and they found that the administration of autonomic nerve-blocking drugs had the effect of inhibiting the formation of consolidation. They assumed that a specific factor inherent in virus might stimulate the nervous system, particularly the terminal portion of the,autonomic nervous system, and that the resulting excessive excitement might induce consolidation in the lungs. They called this factor the consolidation factor. I isolated inflammatory factor as crude materials from the mouse lungs infected with influenza virus (2). We do not know at the present whether this factor is the same as the consolidation factor of Ogasawara and Aida (11). However, the development of consolidation in lungs may be closely related to the formation of the inflammatory factor induced by virus. Benzamidine derivatives were found to reveal a highly inhibitory effect upon both pharmacological and viral inflammatory factors. Therefore, we may assume that the whole mechanism of action of anti-influenza benzamidine derivatives results from the following two effects: (i) inhibition of viral replication and (ii) an inhibitory effect on the appearance of the factors causing viral inflammation. Amantadine and almost all other antiviral drugs that have been reported up to this time have inhibitory effects on the process of viral replication. LITERATURE CITED 1. Davis, W. L., R. F. Haff, and C. E. Hoffman Influenza virus growth and antibody response in amantadine treated mice. J. Immunol. 95: ANTIMICROB. AGENTS CHEMOTHER. 2. Fujita, H Dual effect in the mechanism of action of anti-influenza virus amino acid analogues. Antimicrob. Agents Chemother. 3: Fujita, H., H. Tonegi, S. Toyoshima, J. Abe, T. Watanabe, and K. Fujimoto Effect of amidine derivatives on influenza virus. Prog. Antimicrob. Anticancer Chemother. II: Fujita, H., and S. Toyoshima Inhibitory effect of antiviral drugs on viral specific inflammatory factors produced with influenza virus. Adv. Antimicrob. Antineoplast. Chemother. 1: Ginsberg, H. S Suppression of influenza viral pneumonia in mice by the nonspecific action of zerosine. J. Immunol. 75: Grunert, R. P The in vivo antiviral activity 1-adamantanamine hydrochloride. 1. Prophylactic and therapeutic activity against influenza virus. Virology 26: Kato, N., and H. J. Eggers Inhibition of uncoating of fowl plaque virus by 1-adamantanamine hydrochloride. Virology 37: Ledinko, N., and B. Perry Studies with influenza virus B of recent human origin. 1. Adaptation to the mouse lung. J. Immunol. 74: Newmayer, E. M., J. M. Mcgaken, and W. L. Davis Antiviral activity of 1-adamantanamine hydrochloride in tissue culture and in ovo. Proc. Soc. Exp. Biol. Med. 119: Ogasawara, K. and A. Aida Lung consolidation by nasal inoculation with inactivated influenza virus or HVJ in mice. 1. Virus (in Japanese) 8: Ogasawara, K., and A. Aida Lung consolidation by nasal inoculation with inactivated influenza virus or HVJ in mice. 2. Virus (in Japanese) 8: Seto, Y., Y. Nakamura, and S. Toyoshima Site of action of new amino acid analogs on influenza virus. Adv. Antimicrob. Antineoplast. Chemother. I: Solovgov, V. N., R. P. Grunert, and C. F. Hoffman Therapeutic effect of amantadine aerozol in experimental influenza infection of white mice. Acta Virol. 2: Toyoshima, S., H. Fujita, and S. Kanao Chemotherapeutic effect of new amino acid analogs on influenza. Adv. Antimicrob. Antineoplast. Chemother. I: Toyoshima, S., Y. Seto, and K. Saito Antiviral effect of noformicine. Chemotherapy (in Japanese) 14: Winter, C. A., E. A. Risely, and G. W. Nuss Carrageenine-induced edema in hind paw of the rat as an assey for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med. 111: