Shinjuku, Tokyo 160, Japan. histamine were used as were the standard reference. drugs of anti-inflammatory substances, aspirin and

Transcription

1 ANTIMICROBAL AGENTS AND CHEMOTHERAPY, Jan. 1973, p Copyright i 1973 American Society for Microbiology Vol. 3, No. 1 Printed in U.SA. Dual Effect in the Mechanism of Action of Anti-Influenza Virus Amino Acid Analogues HARUHISA FUJITA Division of Chemotherapy, Pharmaceutical Institute, School of Medicine, Keio University, Shinanomachi Shinjuku, Tokyo 160, Japan Received for publication 17 April 1972 In comparative research studies to clarify the mechanism governing the effects of anti-influenza amino acid analogues, it was established that amino acid analogues exhibit definite chemotherapeutic properties. They possess dual activity capable of inhibiting multiplication and pulmonary consolidation induced by influenza virus infection. Amantadine possesses a relatively narrow antiviral spectrum and exhibited an inhibitory effect only when administered in a relatively early stage of infection (1, 4, 7, 11). However, benzamidine derivatives (2) and amino acid analogues (H. Fujita and S. Toyoshima, Int. Congr. Chemother. Proc., 7th, p. A-5/53, 1971; S. Toyoshima, H. Fujita, and S. Kaneo, Int. Congr. Chemother. Proc., 7th, p. A-5/20, 1971; Y. Seto, Y. Nakamura, and S. Toyoshima, Int. Congr. Chemother. Proc., 7th, p. A-5/38, 1971) had a broad anti-influenza virus spectrum. One distinctive characteristic is the fact that they present an inhibitory effect even when treatment is started rather late after viral inoculation. Experiments utilizing amino acid analogues originally discovered by S. Toyoshima and his researchers provided much new and valuable information regarding the behavior of anti-influenza virus drugs. In comparative research studies to clarify the mechanism of the characteristics that govern the effects of anti-influenza virus amino acid analogues, it was established that the inhibiting properties of antiinfluenza amino acid analogues on viral-induced inflammation definitely play an important role in the clarification of the action mechanism of these drugs. This paper presents a resume of the results of these studies. MATERIALS AND METHODS Materials. Viral strains were influenza virus strains A2/Adachi and B/Lee. Cells were fibroblast cells derived from 10-day-old chicken embryos. Male DD strain mice weighing 10 to 12 g and male Wister strain rats weighing 150 to 200 g were used. Inflammatogenic substances, carrageenan, yeast extract, serotinin, kaolin, Formalin, hyaluronidase, bradikinin, dextran sulfate, egg white, mustard, and histamine were used as were the standard reference drugs of anti-inflammatory substances, aspirin and hydrocortisone. To inactivate infectivity of virus, merzonine was used. Amino acid analogues were used throughout the experiments as shown in Fig. 1. Method of evaluating the activity of an antiinfluenza drug on mice. After being intranasally inoculated with 5 to 10 mean lethal doses (LD,0) of either the AJ/Adachi strain or the B/Lee strain, the mice were injected intraperitoneally with an appropriate dose of the experimental compounds. Tests were conducted on the inoculated mice at varying intervals during the 14-day observation periods. Calculations were made on the mean survival rate, the degree of development of pulmonary consolidation, and the amount of virus in mouse lungs. Pulmonary consolidation was evaluated according to Ledinko's evaluation (6). Method of estimation of the anti-inflammatory action. By the method of Winter et al. (12) the measurement of the volume of unilateral hind paw of rats was followed by an intraperitoneal administration of test compounds in various doses. After a 30-min interval, an inflammatogenic substance was injected into the hind paws, and they were then examined in order to ascertain the extent of edema. Precise measurements of hind paw volume were made 30, 60, 120, and 180 min after the injection of the causative substance. Method of cultivation of chicken embryo fibroblast cells. Chicken embryos were harvested aseptically into petri dishes from 10-day-old eggs. After removal of heads and legs, embryos were finely cut up with scissors and then were placed in Hanks solution, supplemented with 0.25% trypsin, and left at room temperature for 2 hr. The resulting supernatant fluid was discarded, and the process was repeated. The culture was held at 4 C for 16 to 20 hr and strained through an 80-mesh net. 57

2 58 FUJITA A281:N-,3-naphthylaminomethyl-L-a-alanine FIG. 1. NH-CH2-NH-C H-COOH A286:N-2-fluorensulfonyl-3-alanine 82 SO-NH-CH-CH2-COOH Chemical structure of amino acid analogues The resulting filtrate was centrifuged at 600 to 1,000 rev/min 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 10" cells/ml. Method of counting plaques of influenza virus. The monolayers of chicken embryo fibroblast cells were washed repeatedly with a phosphate-buffered saline solution and then were infected with virus, which was allowed to be absorbed by the cells at 4 C for 30 min. Finally, Lacto-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 z- to 3-day incubation period at 37 C, the plaques were counted. Method of inactivation of influenza virus. The lung homogenates of mice infected with influenza virus were centrifuged at 3,000 rev/min for 30 min. The resulting supernatant fluid was centrifuged (Beckman ultracentrifuge, model L-2) at 20,000 rev/min for 30 min. The precipitated virus was suspended with saline in one-tenth of the original volume and centrifuged again at 3,000 rev/min 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 hr and allowed to stand at 4 C for 24 hr in order to inactivate viral infectivity. RESULTS Effects of amino acid analogues on mice infected with influenza. Three days after intranasal inoculation of 5 to 10 LD,0 mouseadapted A2/Adachi strain, the mice were intraperitoneally injected with a 0.2 LD50 of a testing compound. Both the control and the experimental groups of mice were subjected to examinations for the determination of mortality and pulmonary consolidation. The control group of mice were injected with physiological saline only. The results of the experiments are shown in Table 1. As can be seen in Table 1, the distinctive chemotherapeutic properties of amino acid analogues inhibit the development of pulmonary consolidation. This characteristic property definitely contributes to the prolongation of the mean survival rate and a substantial decrease in the mortality rate of in- ANTIMICROB. AG. CHEMOTHER fected mice. These properties were not observed in amantadine. Therefore, it is evident that the characteristics of the antiviral effect of amino acid analogues are superior to that of amantadine in that they display curative effects even under restricted conditions such as delaying initial injection for 3 days after viral infection. On this basis, the following experiment was carried out to determine the differences in the action mechanism of amino acid analogues and amantadine. Antiviral effects of amino acid analogues on influenza virus in tissue culture. Primarily, experiments were conducted to examine the effects of these compounds on the multiplication of virus at cellular level. Chicken embryo fibroblast cells were infected with A2/Adachi strain 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 hr. The total amount of virus was estimated by plaque-forming technique. The results obtained are shown in Table 2. As shown in Table 2, when the maximum nontoxic dose of each respective compound was used the multiplication of AJ/Adachi strain was inhibited completely by amantadine, but less markedly so by amino acid analogues. 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 after viral inoculation. Amino acid analogues, however, displayed a curative effect even when administered at a later stage. These results suggest that there may be a difference in the action mechanism between amantadine and any one of the amino acid analogues. It is presumed that amino acid analogues may have an inhibitory effect in arresting pulmonary inflammation induced by the influenza virus infection. The following investigations were carried out to resolve this problem. Anti-inflammatory effects of compounds on pharmacological inflammatogenic substances. Additional research was conducted to determine the inhibitory effect of amino acid analogues on inflammation induced by a pharmacological inflammatogenic substance. Rats were injected intraperitoneally with standard reference drugs and amino acid analogues. Thirty minutes later, the hind paw was injected with 0.1 ml of inflammatogenic substance. To determine inhibitory effect of the analogues, edema intensity measurements

3 VOL. 3, 1973 ANTI-INFLUENZA VIRUS AMINO ACID ANALOGUES 59 TABiE 1. Effects of amino acid analogues on mice infected with A2/Adachi virus straina Compound Mortality Lung consolidation Dose Prolongation of e survival time (mg/kg/day) Reduction Sor Reduction (days) Saline Amantadine O ± 0.56 A b b b A k k b a Each group was comprised of 20 mice. p = <0.01 TABLE 2. Antiviral effects of amino acid analogues on influenza virus in tissue culture Inhibition (%) Compound Maximum administerec; nontoxic dose _giml AJAdachi B/Lee Amantadine A A were taken at 30, 60, 120, and 180 min after the injection. The effective dose (ED50) was calculated from the dose response of A281 and A286 in relation to the inflammatogenic substance by the method of Reed and Muench (10). Table 3 shows the specific dose response rate of the test compounds against edema induced with the inflammatogenic substances. ED,0 values of A281 were less than 10 mg/kg against bradikinin and hyaluronidase; 15 mg/ kg against kaolin; 45 mg/kg against egg white; 80 mg/kg against mustard; and ineffective against carrageenan, yeast extract, serotonin, Formalin, dextran sulfate, and histamine. Values of A286 were less than 10 mg/kg against serotonin, kaolin, and hyaluronidase; 20 mg/kg against carrageenan; 25 mg/kg against bradikinin; 20 mg/kg against egg white; 50 mg/kg against mustard; 55 mg/kg against histamine; and 110 mg/kg against dextran sulfate; however, A286 was ineffective against Formalin. These facts indicate that these compounds display satisfactory anti-inflammatory action. Inhibitory effect on the consolidation development in mice infected with merzonine-inactivated virus. It has been reported (8) 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 then the administration of a compound was started immediately after infection and continued once daily for 4 consecutive days. On the 5th day the mice were examined in order to determine the degree of pulmonary consolidation. The results obtained are shown in Table 4. As shown in Table 4, the rate of inhibition on the development of pulmonary consolidation was A281, 47.9%; A286, 30.3%; hydrocortisone, 43.3%; aspirin, 26%; and amantadine, 0%. These findings indicate that amino acid analogues had a slightly higher rate of inhibition than aspirin. To study these results further, an attempt was made to isolate inflammatogenic factors from the lesions of pulmonary consolidation. The effect of amino acid analogues on the inflammatogenic factors induced by viral infection was also examined. Isolation of an inflammatogenic factor from virus-infected mouse lungs. Mice, 10 to 12 g in weight, were inoculated with the A2/Adachi strain of influenza virus. Lungs were removed 5 to 7 days after infection. A 10% homogenate was prepared by adding physiological saline to the infected lungs. Preliminary centrifugation of the homogenate was at 3,000 rev/min for 20 min. The resulting supernatant fluid was subjected to two 1-hr ultracentrifugation periods at 4,000 rev/min. The supernatant fluid obtained was sterilized by use of a Millipore type HA filter. This crude preparation was inoculated into 10-day-old embryonated eggs. After incubation at 36 C for 40 hr, chorioallantoic fluids were collected from the eggs and examined

4 i60 FUJITA ANTIMICROB. AG. CHEMOTHER. TABLE 3. Anti-inflammatory effects of compounds on edema induced with several chemical mediators in rats Anti-inflammatory activity (edema inhibition percentage) Compound Dose Hya- BE Histaadministered admiistred (mg/ Carra- Yeast Sero- Kali Forma- uroni- Brai Dextran gg Mustard mine kg) geenan extract tonin Kaln ln luoi kinin Deta hite Msadmn (1%) (10%) (0.04%) (20%) (3) dase g wa 05) (25)(0 ) (15%) 2.5%) (500 Hydrocortisone a b a 43.3a 26.7 NDc Oa a b 44.8a 45.2a 73.1 a 69.3b ND Oa Oa 44.9a 0 ND ND ND ND ND ND Aspirin ga a 68.9b a ND b O 77.6b 43.5a O ND a a b 79.3b 62.6a ND A a a 86.2b b a 81.0b a 44.0a b ND 58.2a 53.3a ND ND ND 34.9 ND 91.4b ND ND ND 37.5 A k 58.3b a 40.0a b b 62.1a a 49.3a 44.4a b 50.0a b 45.2a 68.6b 60.Ob 61.6a k 63. Ob 86.2i 80.3k b ND 68.7b 82.1k 84. Ob 68.1b ap = <0.05. P = <0.01. cnd = not done. TABLE 4. Inhibitory effects on development of lung consolidation in mice infected with merzonine-inactivated virus Lung consolidation Compound Dosea administered (mg/kg) Score Inhibition Saline Amantadine ± Hydrocortisone ± Aspirin A k 47.9 A Merzonine-inactivated virus was administered to mice and was immediately followed by doses of the test compounds which were continued for 4 consecutive days. Each group was comprised of 10 mice. " p = <0.05. for hemagglutinating activity. A non-hemagglutinin was used as crude inflammatogenic material for the following experiments. Occurrence of inflammatory symptoms by pulmonary homogenate prepared from the lesion of pulmonary consolidation induced by influenza virus and from lungs of healthy mice. Rats were inoculated into their hind paws with 0.45 mg of protein contained in 0.1 ml of the crude material. Foot measurements were taken 30, 60, and 120 min after inoculation to determine the intensity of edema. Control rats were injected with an equal amount of physiological saline. The results obtained are shown in Table 5. As is clear from Table 5, a distinct inflammation occurred in the rats of the experimental group inoculated with the crude material prepared from viral consolidation lesions. By contrast, the degree of inflammation was low in the rats of the experimental group inoculated with the crude material prepared from the homogenate from healthy mouse lungs. These results suggest that lungs infected with influenza virus may contain elements causing viral inflammation. Rise and fail of factors causing inflammation induced with influenza virs. In experiments carried out to determine the relationship between the production of the factors causing viral inflammation and the occurrence of consolidation in lungs, seven groups of five mice each were all infected with the A JAdachi strain. All groups were examined for the degree of pulmonary consolidation 1, 2, 3, 4, 5, 7, and 8 days after infection, respectively. Crude material was then prepared from the mouse lungs by the method mentioned above and inoculated into their hind paws. Later the intensity of edema was measured. The results obtained are shown in Table 6.

5 VOL. 3, 1973 ANTI-INFLUENZA VIRUS AMINO ACID ANALOGUES 61 TABLE 5. Occurrence of inflammatory symptoms by pulmonary homogenates prepared from the lesion of pulmonary consolidation induced by influenza virus and from lungs of healthy mice Material Edema volume at time after injection (ml)a 0.5hr lhr 2hr 3hr 4hr Saline 0.09 ± ± ± ± ± 0.05 Healthy lung homogenate ± ± ± ± ± 0.08 Influenza-infected lung ho io0. 09c 0.54 ± 0. 09' 0.51 ± 0.08c 0.41 ± 0.07b 0.28 ± mogenate a Each group was comprised of five rats. P = <0.01. cp= <0.05. As shown in Table 6, the development of consolidation in lungs began about 3 days after infection and continued to increase linearly in intensity up to about 8 days after infection. On the other hand, the factor causing viral inflammation began to increase about 2 days after viral infection, reaching a peak about 5 days after infection and remained at this level indefinitely. These results suggest that the development of consolidation in lungs is closely related to the formation of the inflammatogenic factor induced by virus. Inhibitory effects of amino acid analogues on the development of inflammation induced by the factor causing viral inflammation. Experiments were conducted to clarify the effects of compounds on the development of inflammation induced by the factor causing viral inflammation. Rats were first injected intraperitoneally with a 0.2 LD,0 of the test compound. Thirty minutes later, their hind paws were inoculated with 0.45 mg of protein contained in the crude inflammatogenic preparation. The intensity of edema was measured 2 hr later. In this manner, a comparision of the inhibitory effects were made of various compounds. Hydrocortisone and aspirin were used as standard reference drugs in these experiments. The results obtained are shown in Table 7. As shown in Table 7, A281 and A286 revealed an outstanding inhibitory effect upon viral inflammation. 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 A281 and A286. Amantadine possessed no inhibitory effect at all. DISCUSSION In the tissue culture system, amantadine was shown to exhibit significant inhibition of viral replication. It has been reported that amantadine inhibits the process of uncoating TABLE 6. Rise and fall of the factor causing inflammation to be induced with influenza virus Date harvested Lung Inflammatory (days after consolidation activity viral infection) (score) (ml)a NDb ± c d d d d Healthy control a Edema volume at 60 min after injection; each group was comprised of five rats. b ND = not done. cp= <0.05. d p = <0.01. TABLE 7. Inhibitory effects of various compounds on the development of inflammation induced by the factor causing viral consolidationa Anti-inflammatory Compound Dose activity administered (mg/kg) Edema volume Inhibition (ml) (%) Saline Hydrocortisone ± t Aspirin ± Amantadine A k A c a Each group was comprised of five rats. b p = <0.01. cp= <0.05. in the course of intracellular viral replication (5). However, these experiments showed that the effect of amino acid analogues on viral replication was definitely inferior to amantadine. These results suggest that amino acid analogues may have a second action mechanism

6 62 FUJITA by which they exhibit chemotherapeutic effects different from those of amantadine. This dual-action mechanism has an anti-inflammatory effect on inflammation induced by virus. Therefore, we may assume that the whole action mechanism of anti-influenza amino acid analogues may be considered to result from the following two effects: (i) inhibition of viral replication and (ii) an inhibitory effect on the appearance of the factor 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. On the other hand, amino acid analogues clearly display intensified dual chemotherapeutic effects which inhibit both viral replication and the occurrence of inflammation induced by influenza virus. Ginsberg pointed out (3) that xerosine had no effect against viral replication, but inhibited the development of consolidation. He presumed that this factor might have been produced by the action of a harmful substance released from cells injured primarily by the virus. Ogasawara and Aida (9) conducted studies on myxoviruses including inactivated influenza virus, hemagglutinating virus of Japan, and Newcastle disease virus. They found that the administration of autonomic nerve-blocking drugs had the effect of inhibiting the formation of consolidation. They assumed that specific factor to 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. In the present studies on the development of consolidation observed in mice infected with influenza virus, only crude materials were used. ANTIMICROB. AG. CHEMOTHER. ACKNOWLEDGMENTS This work was performed under the direction of Shigeshi Toyoshima, Head, Division of Chemotherapy, Pharmaceutical Institute, School of Medicine, Keio University, Tokyo, Japan. I express my gratitude to him for his warm support and guidance. 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: Fujita, H., H. Tonegi, S. Toyoshima, J. Abe, T. Wantabe, and K. Fujimoto Effect of amidine derivatives on influenza virus, p In H. Umezawa (ed.), Progress in antimicrobial and anticancer chemotherapy: proceedings, vol. 2. University Park Press, Baltimore. 3. Ginsberg, H. S Suppression of influenza viral pneumonia in mice by the nonspecific action of xerosine. J. Immunol. 75: Grunert, R. P The in vivo antiviral activity 1-adamantanamine hydrochloride I. Prophylacted 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. W. Mcgaken, and W. L. Davis Antiviral activity of amantadine hydrochloride in tissue culture and in ovo. Proc. Soc. Exp. Biol. and Med., 119: Ogasawara, K., and A. Aida. 1958, Lung consolidation by nasal inoculation with inactivated influenza virus or HVJ in mice. (1). Virus 8: Ogasawara, K., and A. Aida Lung consolidation by nasal inoculation with inactivated influenza virus or HVJ in mice. (2). Virus 8: Reed, L. J., and H. Muench A simple method of estimating fifty percent endpoints. Amer J. Hyg. 27: Solovgov, V. N., R. P. Grunert, and C. F. Hoffaman Therapeutic effect of amantadine aerosol in experimental influenza infection of white mice. Acta Virol. 11: Winter, C. A., E. A. Risley, and G. W. Nuss, Carrageenin-induced edema in hind paw of the rat as an assay for antiinflammatory drugs. Proc. Soc. Exp. Biol. Med. 111:544.