Prevention and treatment of experimental influenza A virus infection in volunteers with a new antiviral ICI 130,685

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1 Journal of Antimicrobial Chemotherapy (186) 18,11-12 Prevention and treatment of experimental influenza A virus infection in volunteers with a new antiviral ICI 13,685 W. Al-Naldb*, P. G. Higgins*, J. WuTman*, D. A. J. TyrreD*, D. L. SwaDowf, B. C Hurstf and A. Rosfatont *MRC Common Cold Unit, Harvard Hospital, Salisbury; timperial Chemical Industries PLC, Pharmaceutical Division, Macclesfield, England The initial prophylactic and therapeutic trials of ICI 13,685 against influenza A virus infection are reported. Prophylaxis with either 2mg/day (38 volunteers received drug and received placebo) or loomg/day (28 volunteers received drug and 28 received placebo) for seven days significantly reduced illness, mean clinical score and nasal secretion weight when volunteers were challenged with 1*' EID 5 of influenza virus A/Eng//83 (H,N a ). Overall, prophylaxis with 2mg/day and loomg/day gave 1% and 72% protection against illness relative to placebo, respectively. In addition, prophylaxis with both regimens for seven days also significantly reduced the number of volunteers who excreted virus. In a therapeutic study, volunteers were inoculated with the same dose of virus and those who developed symptoms which persisted for 6-15 h were treated with 2 mg/day of drug (2 volunteers) or placebo (1 volunteers) for four days. Generally, treatment reduced both the amount of virus excreted and the mean daily clinical score. However, these reductions were only statistically significant (P <-5) on the third day of medication for the amount of virus excreted and on the fourth day of treatment for the mean clinical score. It was concluded that ICI 13,685 is effective in the prevention and treatment of influenza virus infection. An initial tolerance study in 16 volunteers who received either drug (2 mg/day) (8 volunteers) or placebo (8 volunteers) for seven days, indicated that the drug was generally well tolerated. Combining data from all studies, 3% of volunteers who received the drug at the 2 mg/day dosage and 21% who received placebo complained of one or more symptoms. However, symptoms were generally minor and of short duration. At the lower dosage (1 mg/day) the symptoms were qualitatively similar to those reported with placebo. Introduction Amantadine has been licensed for use in the prevention and treatment of influenza A virus infection. In controlled studies it has been shown to be about 7% effective in the prevention of influenza virus infection although a proportion of individuals develop minor central nervous system (CNS) side-effects (Oxford & Galbraith, 18; Bektimirov ei ai, 185). Further, its therapeutic effect has been found to be weak and therefore, it has not been widely prescribed. Rimantadine, a related compound, is widely used in the U.S.S.R. but has not found favour in the West (Oxford & Galbraith, 18). We were, therefore, interested by the discovery of ICI 13,685. This is a cyclo-nonane, containing a primary amine hydrochloride and thus resembles amantadine in this /86/ S2./ C.186 The British Society for Antimicrobial Chemotherapy

2 12 W.Al-Naldbrta/. respect. However, in vitro it has been found to be at least five times as active as amantadine against H 3 N 2 viruses and by mouth, it was more active than amantadine in the prophylaxis of influenza infection in mice (Swallow, 18). Furthermore in ferrets it completely prevented the appearance of symptoms when given prophylactically and significantly reduced symptoms when given therapeutically, whereas amantadine had no effect (Swallow, 18). In this paper we report the first prophylactic and therapeutic evaluation of ICI 13,685 in human volunteers challenged experimentally with influenza virus A/Eng//83 (H 3 N 2 ). This virus was selected because it induces a clear-cut but not a severe response in man and is sensitive to the drug in vitro (MIC=1 mg/1). Materials and Methods Volunteers A total of 23 healthy volunteers of either sex aged 18 to 5 years participated in the four studies designed to determine tolerance, prophylactic and therapeutic effect of the drug. They were housed in isolation in groups of two or three at the MRC Common Cold Unit, Salisbury, in accordance with the Unit routine practice (Beare & Reed, 177). They completed a questionnaire for introversion/extroversion and certain obsessional factors before trials commenced as these have been shown to influence the rate of infection and severity of illness (Totman et al., 18). Study design All trials were conducted double-blind and protocols were approved by the Ethical Committee at Northwick Park Hospital. Written consent was obtained from all volunteers. Volunteers were divided into groups balanced for age and sex and members of each group were randomly allocated to receive drug or placebo. Prior to prophylaxis or treatment they were quarantined for two days and shown to be free of upper respiratory symptoms. The first trial was a tolerance study in which 16 volunteersreceivedeither ICI 13,685 (2mg/day) or placebo for seven days. In the first of the two prophylaxis studies, seventy-eight subjects received either ICI 13,685, two tablets (2 mg) once a day or placebo two tablets once a day for seven days while in the second prophylaxis study 56 volunteers received either ICI 13,685 one tablet (1 mg) once a day or placebo, one tablet once a day for seven days. In both studies, three daily doses were administered prior to virus inoculation (the third dose only 3 h prior to challenge) and the remaining four after challenge. In the therapeutic study volunteers were randomly allocated to receive either ICI 13,685 (2mg/day) or placebo for four days. Treatment was initiated only in the 3 volunteers who had symptoms and/or signs persisting for 6-15 h. The remaining 5 volunteers who did not develop symptoms/signs by h after challenge were then randomly allocated, by code, to receive 2 mg or 1 mg/day of ICI 13,685 or placebo for four days in order to obtain more data on tolerance to the compound. Drug material ICI 13,685 was supplied as 1 mg tablets. Placebo tablets were identical in appearance to active tablets. Active and placebo material were coded and randomized at ICI, Pharma-

3 Prerentkm and treatment of influenza A Thus 121 ccutical Division, Macclesfield, U.K., and the code was not broken until all clinical and laboratory evaluations were completed. Virus challenge The A/Eng//83 (H 3 Nj) (EG26) virus was originally received from Dr M. S. Pereira of the Central Public Health Laboratory, Colindale. It was passed initially three times in embryonated eggs. Diluted allantoicfluidswere inoculated, intranasally, into volunteers and nasal washings collected from those who became infected and symptomatic. Virus was recovered in specific pathogen free eggs and this allantoic fluid was used as the volunteer inoculum. A dose of 1 1 EID J diluted in Hank's saline with -2% bovine plasma albumin was given in nasal drops (OS ml per nostril). Virological investigation Paired sera from volunteers were tested for a four-fold or greater rise in antibody by hacmagglutination inhibition (HAI). Nasal washings were collected before and daily after challenge from all volunteers and the presence of virus was determined by inoculating nasal washings into Madin-Darby canine kidney ((MDCK) cells in the presence of -5 ug of trypsin per ml of maintenance medium. In the therapeutic study the amount of virus in nasal washings was determined by titration in MDCK cells. Methods of assessment All symptoms and signs were graded and combined into a daily clinical score and the weight of nasal secretions was determined on a daily basis as previously described (Bcare & Reed, 177). At the end of each trial volunteers were classified as having had a significant illness or not depending on their respiratory symptoms and/or systemic reaction. Significant illness was classed as mild, moderate or severe cold and/or reaction. Statistical methods As distributions of symptom scores could not be normalized, results were analysed by rank tests including a rank analysis of variance allowing for the effect of antibody. Differences in the frequency of illness and laboratory evidence of infection were tested for significance by x 2 - Results Demography Tables I and II show that the volunteers in the different groups were well matched for age, sex and pre-trial HAI antibody titre. They were also well matched for psychological scores. Tolerance and pharmacokinetics Of the 16 volunteers who participated in the initial tolerance study, eight were given the drug and eight received placebo. Daily analysis of blood pressure and pulse rate showed no significant differences between the treated and placebo group. In the eight volunteers receiving drug, analysis of blood and nasal wash samples for drug concentration at 3 and 2 h after the first dose and again after the last dose indicated that the drug was present at virus inhibitory concentrations in the blood after both the first dose (mean -5 and 1 mg/1 at 3 and 2 h, respectively) and the last dose (mean -8 and -2 mg/1 at 3 and 2 h, respectively) and in nasal washings after both the first dose (mean -7 and

4 Group ICI 13,685 Placebo Pre-trial HAI litre < >2 Total < >2 Total Table I. Prophylaxis with ICI 13,685 (2 mg/day): clinical and virological findings No. of volunteers * 21 1» ' 16 males, 22 females, mean age 3-6± 1-5. * 15 males, 25 femalei, mean age 32-5± 1-6. P--2. '/»-O8. P--5. f 33 paired sera tested. ' 32 paired sera tested. * Illness classed as mild, moderate or severe cold and/or reaction. No. with significant clinical illness* Abrise 1 1 (3%) 13 C (32.5%) (36%/ * (78%/ Virological findings Virus either isolated or both 6 2(63%) ' (85%) (7%) * (2.5%)

5 Either or both (82%) '(3%) Group ICI 13,685 Placebo Pre-trial HAI litre < >2 Total < >2 Total " 1 malen, 1 femnlot mean in ' 1 mala, 1 females, mean age 3O-3±2-O5. ' not significant. /»--2. Illness classed as mik1, moderate or severe cold and/or reaction. Table IL Prophylaxis with ICI 13,685 (1 mg/day): clinical and virological findings No. of volunteers * * No. with significant clinical illness* (1%) '(5%) Abrise 5 18(6%) '(75%) Virological findings Vims isolated (61%) * (8%)

6 12 W.Al-Nakflx/oJ. -3 mg/1 at 3 and 2 h respectively) and the last dose (mean 16 and 7 mg/1 at 3 and 2 h respectively). Since we have estimated that washings contained about % of nasal secretion (unpublished data) drug concentrations present in nasal secretions were four to seven times greater than that present in blood. Haematological and biochemical analysis of blood and urine showed no significant changes following treatment in either those receiving drug or placebo. Forty-three per cent and 21% of volunteers who received 2mg/day of drug or placebo,respectively,for four to ten days (combined data of tolerance, prophylaxis and therapeutic trials) complained of one or more symptoms. Prominent amongst these were gastrointestinal symptoms (mostly mild nausea and anorexia) recorded in 16% of those receiving drug compared with 3-5% in the placebo group, and dry mouth/thirst; (8%) in the drug group compared with nil among placebo recipients. Light headedness occurred in 7% of volunteers who received drug and 2-% given placebo, while mild loss of concentration and disorientation occurred only rarely. Sleep disturbance wasrecordedin 11 % of volunteers in the drug group compared with 6% of those given placebo. In the therapeutic study 5 volunteers did not develop influenzal symptoms following virus challenge. They were given either 2 mg/day, 1 mg/day or placebo for four days. Of the volunteers who had 2 mg, 1 mg or placebo, symptoms were reported by 65%, 5% and 35% respectively. These data, therefore, suggested that the overall incidence of symptoms may be doserelated.medication of four volunteers on 2 mg/day of drug in the therapeutic trial was stopped when they complained of more troublesome symptoms mainly light headedness and dizziness and, in two, palpitations. Medication was also withdrawn for afifthvolunteer, this time in the prophylaxis study with 2 mg/day; he had intermittent vomiting on the sixth day. Prophylaxis Thirty-eight volunteersreceiveddrug (2 mg/day) and receivedplacebo. As seen in Figure 1 and Table I prophylaxis with 2 mg/day significantly reduced illness, mean clinical score on all days of observation, mean nasal secretion weight on day four and virus shedding on days two and three after virus challenge. Reduction in the rate of illness was 1% relative to placebo. Furthermore, there was a lesser but still significant reduction in the number of volunteers who became infected as judged by virus isolation, antibody rise or both combined. Since the incidence of side-effects appeared to be lower with 1 mg/day we conducted another study in order to establish the prophylactic efficacy of this compound when given at the lower dosage. Thus 28 volunteers were given drug (1 mg/day) and another 28 volunteers received placebo and both groups were challenged with virus on the third day of medication as previously described. Figure 2 and Table II show that there was a significant reduction in illness, mean clinical score and nasal secretion weight on days two to five after challenge in the drug group compared with the placebo group. Reduction in the rate of illness was 72% relative to placebo. In addition, prophylaxis with 1 mg/day produced significant reduction in the rates of virus shedding on the third day of medication and in the number of volunteers excreting virus when compared with placebo. Therapeutic efficacy Using the higher dosage of the drug (2 mg/day) we conducted a therapeutic trial as described earlier. As shown in Figure 3 patients who were given drugs had lower clinical

7 Prevention and treatment of tnflnffi A virus 125 Figure 1. Prophylaxis with IC113,685 (2 mg/day): clinical score, nasal secretion and vims excretion., Treated (2mg/day) n»38; D, placebo, n-; *, P <-3;, P <-1; *, P <-1. scores than those who received placebo on all the days of observation and this reached statistical significance on the fourth day of treatment. Similarly, as demonstrated in Figure, those receiving drug generally showed a reduction in the amount of virus present in their nasal wash compared with those who received placebo and this reached statistical significance on the third day of treatment. Discussion Pharmacokinetic studies on ICI 13,685 indicated that drug concentrations of at least -1 mg/1, which is the minimal inhibitory concentration for influenza A virus were achieved in both blood and nasal washings 2 h after first and last medication (2 mg/day). Drug concentrations in nasal secretions were four to seven times higher than those detected in blood indicating that the drug was particularly concentrated therein. This feature may be especially advantageous for compounds directed against viruses such as influenza A virus that primarily multiply in the respiratory tract We found that ICI 13,685 when given to volunteers prophylactically at 2 mg or 1 mg/day dosage effectively prevented clinical illness, and reduced the mean clinical

8 126 W.AJ-Ntkibtfa/. 8 6 I 5 o i 3 * 2 7 ~ 6 If ' eo 6 2 i Medication V I I I Vlrui challenge 1 I I I ( J * Day Figure 2. Prophylaxis with ICI 13,685 (1 mg/day): clinical score, nual secretion and virus excretion. Treated(1mg/day), n-28; D,placebo,n-28; *,P <-5; **,/> < I I 2 Treatmeni day Figure 3. Daily clinical score following treatment with ICI 13,685 (2mg/day). ), Drag, n-2; O, placebo, n-1; *P <OS.

9 Prerentkm and treatment of inflnenza A rirus Day of rrndicotlon Figure. Vims concentration in nasal washings following treatment with ICI 13,685 (2 mg/day),' 13,685,2mg/day, n-2; O, placebo, n-1; *, P <(K)5. score and mean nasal secretion weight following vims challenge compared with placebo. Both regimens also reduced the number of volunteers who excreted virus and at 2 mg/day, the number of volunteers who showed laboratory evidence of infection. Therapeutically, although the drug still reduced the mean daily clinical score and the amount of virus present in nasal washings, these reductions were not statistically significant except on the third day of medication for virus concentration in nasal wash and the fourth day of medication for mean clinical score. It is interesting that the reduction in virus titres preceded that in the clinical score by one day. The reaction to infection with the egg-adapted strain of influenza virus used in this study is mild and short-lived, one to four days, as can be seen from the illness experienced by the placebo treated group. As medication was not begun until between 6 and 15 h after the onset of illness it is not surprising that no significant reduction in virus shedding or clinical score was observed before the third day of illness even if ICI 13,685 is highly effective in treatment. It is possible that in naturally acquired infections, when more severe and protracted illness is known to occur, the difference in the course of the illness between the drug and placebo treated patients will be more dramatic. In this study prophylaxis with 2 mg and 1 mg/day gave protection against illness of 1% and 72% respectively, when volunteers were challenged with influenza A virus. This is higher than has been achieved with amantadine in various artificial challenge studies (Jackson, Muldoon & Akers, 163; Togo, Hornick & Dawkins, 163; Smorodintsev et al., 17; Oxford & Galbraith, 18), in which the average degree of protection was 55% although better protection has been seen in field studies e.g. 78% (Dolin et al., 182). However, a more objective comparison of the efficacy of the two compounds, in our opinion, will have to await controlled comparative trials of both drugs in volunteers challenged with the same virus and assessed under similar experimental conditions. It is of interest to note that in a previous trial we found that lymphoblastoid interferon when given prophylactically gave a protection rate against illness of only 7-5% when volunteers were challenged with this same influenza virus strain (Phillpottse/a/., 18). Since influenza virus infections are generally self-limiting, a pre-requisite for any antiinfluenza compound, is that it should be non-toxic. Amantadine, however, does produce

10 128 W.Al-Naklbrtfli some side-effects, mostly minor neurological symptoms, in about 2% of individuals receiving the drug (Bryson ex al., 18; Oxford & Galbraith, 18). In this study, we found this new compound to be generally well tolerated and minor CNS symptoms at the higher dosage were recorded in only 7% of volunteers receiving drug compared with 2% given placebo. However, the total incidence of symptoms recorded among volunteers who received the drug at 2 mg/day dosage was almost twice as much as that recorded for the placebo group, although most were mild and of short duration. Prominent among the side-effects were gastrointestinal symptoms (mostly mild nausea and anorexia), sleep disturbance and dry mouth/thirst. Medication was withdrawn due to adverse reactions in five volunteers, one in the prophylaxis study and four in the therapeutic study: all received the higher dosage of the drug. It is our opinion, therefore, that the 2 mg/day dosage is not justified in the prophylaxis of influenza virus infection despite its higher protective efficacy but would be acceptable when treating an attack of influenza, particularly since side-effects are rapidly reversed on discontinuing the drug. On the other hand, prophylaxis with 1 mg/day gave 72% protection against illness and produced very few side-effects. We think, therefore, that prophylaxis with this regimen could prove useful in the prevention of influenza A virus infection especially among high risk patients when given for the short periods when virus is known to be in circulation. We advocate that ICI 13,685 should next undergo large but controlled field trials for the prevention and treatment of influenza A virus infection in order that full assessment of its efficacy and side effects be obtained. Acknowledgements The authors wish to thank Mrs N. Bailey, Miss Caroline Dearden, Miss Morag Forsyth and Mrs B. Head for their technical assistance; Mrs K. Callow for help with data analysis, Mrs M. Andrews for her care of the volunteers and the volunteers themselves for their cooperation. References Beare, A. S. & Reed, S. E. (177). The study of antiviral compounds in volunteers. In Chemoprophylaxis and Virus Infection of the Respiratory Tract (Oxford, J. S., Ed.), pp CRC Press, Cleveland. Bektimirov, T. A., Gordon Douglas, Jr. R., Dolin R., Galasso, G. J., Krylov, V. F. & Oxford, J. (185). Current status of amantadine and rimantadine as anti-influenza agents: Memorandum from a WHO meeting. Bulletin of the World Health Organization 63,51-6. Bryson, Y. J., Monahan, C, Pollack, M. & Shields, D. (18). A prospective double-blind study of side effects associated with the administration of amantadine for influenza A virus prophylaxis. Journal of Infectious Diseases 11, Dollin, R., Reichman, R. C, Madore, H. P., Maynard, R., Linton, P. M. & Webber-Jones, J. (182). A controlled trial of amantadine and rimantadine in the prophylaxis of influenza infection. New England Journal of Medicine 37, 58-. Jackson, G. G., Muldoon, R. L. & Akers, L. W. (16). Serological evidence for prevention of inflnmral infection in volunteers by an anti-influenza] drug adamantanamine hydrochloride. In Antimicrobial Agents and Chemotherapy 163 (J. C. Sylvester, Ed.), pp American Society for Microbiology. Michigan. Oxford, J. S. & Galbraith, A. (18). Anti-influenza virus activity of amantadine. A selective review of laboratory and clinical data. In Viral Chemotherapy (Shugar, D., Ed.), pp , Pergamon Press, Oxford.

11 Prerention and treatment of Inflnenra A virus 12 Phillpotts, R. J., Higgins, P. G., Wilhnan, J. S., Tyrrell, D. A. J., Freestone, D. S. & Shepherd, W. M. (18). Intranasal lymphoblastoid interferon ("Wellferon') prophylaxis against rhinovirus and influenza virus in volunteers. Journal of Interferon Research, Smorodintsev, A. A., Karpuchin, G. I., Zlydnikov, D. M., Malysheva, A. M., Shvetsova, E. G., Burov, S. A., Chramtsova, L. M., Romanov, Y. A., Taros, L. Yu., Ivannikov, Y. G. & Novoselov, S. D. (17). The prospect of amantadine for prevention of influenza A 2 in humans. Annals of the New York Academy of Science 173,-61. Swallow, D. L. (18) Antiviral agents In Progress in Drug Research, Vol. 28 (Jucker, E., Ed.), pp , Birkhauser Verlag, Basel. Togo, Y., Hornick, R. B. & Dawlrins, A. T. (168). Studies on induced influenza in man. 1. Double-bund studies designed to assess prophylactic efficacy of amantadine hydrochloride against A2/Rockville/l/65 strain. Journal of the American Medical Association 23,18. Totman, R., Kiff, J., Reed, S. E. & Craig, J. W. (18). Predicting experimental colds in volunteers from different measures of recent life stress. Journal of Psychosomatic Research 2, (Manuscript accepted January 186)