Ineffectiveness of Adenine Arabinoside and Adenine Arabinoside 5'-Monophosphate in Simian Varicella Infectiont

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1980, p /80/ /06$02.00/0 Vol. 18, No. 1 Ineffectiveness of Adenine Arabinoside and Adenine Arabinoside 5'-Monophosphate in Simian Varicella Infectiont KENNETH F. SOIKE,* AMBHAN D. FELSENFELD, SUSAN GIBSON, AND PETER J. GERONE Delta Regional Primate Research Center, Tulane University, Covington, Louisiana Adenine arabinoside and adenine arabinoside 5'-monophosphate (ara-amp) have been evaluated for antiviral activity against simian varicella virus infection in monkeys. In a preliminary study for toxicity, intramuscular injection of ara- AMP at 15 mg/kg per day as a single injection for 5 days to two normal patas monkeys caused no detectable local reaction, no weight loss or changes in serum transaminase levels, and no hematological abnormalities. When this dose was given in the treatment of four simian varicella virus-infected patas monkeys, no effect was observed on the clinical course of infection, as compared with four infected monkeys which received phosphate-buffered saline. Treatment was begun 43 h after virus inoculation and was continued for 16 days. Toxicity of intravenously administered ara-amp at 100 or 50 mg/kg per day for 5 days to pairs of uninfected patas monkeys was evident by hematological and hepatic histological alterations, as well as the death of one monkey in each pair. No gross evidence of toxicity occurred in two monkeys which received 20 mg of ara-amp per kg per day. The antiviral efficacy of intravenous treatment was studied in groups of four African green monkeys which received adenine arabinoside at 15 mg/kg per day or ara-amp at 18.4 mg/kg per day. Drug administration began 48 h after inoculation with simian varicella virus and continued for 10 days. The monkeys that received treatment did not respond to infection differently from four infected control monkeys similarly treated with phosphate-buffered saline. Antiviral activity of adenine arabinoside (ara- A) against varicella-zoster virus (VZV) has been demonstrated in vitro (11). The minimum inhibitory concentration of ara-a for VZV was shown to be slightly greater than that required for herpes simplex virus (7,11). Upon further examination of a number of fresh clinical isolates of VZV, it was found that sensitivity varied among the isolates over a range of inhibitory concentrations of ara-a from 0.5 to 20.0,g/ml (9) İn vivo studies may provide a more relevant test for the ultimate application of an antiviral compound. Herpes simplex virus has been used in a variety of animal models ranging from genital infection, cutaneous lesions, and keratitis to encephalitis. Response to ara-a treatment in these models has been variable (1,3,8,10) but sufficiently encouraging to result in the therapeutic use of ara-a in human herpes encephalitis with reported beneficial results (13). Evaluation of antiviral compounds against VZV in vivo has been hampered by the unavailability of a suitable laboratory model for investigation. An animal model for VZV infection is desirable, recognizing that human VZV infection t. Publication no. 43 from the Cooperative Antiviral Testing Group of the Antiviral Substances Program. can produce such conditions as pneumonia, keratitis, central nervous system involvement, and severe congenital infection. A simian varicella virus isolated from a natural outbreak at this Center produces varicella-like disease in some species of Old World nonhuman primates, antigenically resembles human VZV, and clinically resembles severe generalized humapn varicella infection (5,6). This model has been used to examine the efficacy of ara-a and adenine arabinoside 5'-monophosphate (ara-amp) in the treatment of this forn of varicella infection. Results obtained in these studies are presented in this report. MATERIALS AND METHODS Monkeys. Patas monkeys (Erythrocebuspatas) or African green monkeys ( Cercopithecus aethiops) were purchased as wild-caught monkeys from a commercial importer and maintained in quarantine for 90 days after arrival at this Center. During quarantine, sera from each monkey were tested and determined to be free of antibody to simian varicella virus. Upon the completion of quarantine, the monkeys were moved to the infection control unit, where they were housed in individual cages. Base-line clinical chemistry and hematology values were determined, and sera were again examined for neutralizing antibody to the simian varicella virus before use. The monkeys were sedated with Ketamine for all medication and sampling. 142

2 VOL. 18, 1980 In vitro studies. Concentrations of ara-amp of 1, 10, 25, 50, 100, 500, and 1,000,ug/ml were prepared in Eagle minimum essential medium with 10% fetal calf serum at a ph of 7.6. Vero cells at 105 cells per ml were suspended in growth medium containing the ara- AMP concentrations and seeded in duplicate in 5-ml amounts into 60-mm culture dishes. The simian varicella virus was inoculated at the time of seeding in a 0.2-ml volume containing 200 to 300 plaque-forming units. The cultures were observed microscopically for the development of plaques and for drug-related cytotoxicity. After incubation at 37 C in 5% CO2 for 5 days, the plates were washed in phosphate-buffered saline (PBS), fixed in methanol, and stained with a mixture of methylene blue and basic fuchsin. Plaques were counted to determine the inhibition of virus growth by the concentrations of ara-amp. Ara-A and ara-amp solutions. The drugs were supplied in powder form by Warner-Lambert/Parke Davis Laboratories. In experiments to investigate the intramuscular and intravenous toxicities and in an experiment evaluating the effect of intramuscular treatment, ara-amp was dissolved in PBS (ph 7.6) and sterilized by filtration through a 0.45-,gm pore size membrane filter (Millipore). In an experiment to compare the intravenous efficacy of ara-a and ara-amp treatment, the drugs were weighed into 250-ml Falcon tissue culture flasks and sterilized by ethylene oxide. This was necessary because of the insolubility of ara- A. Sterile PBS was added to the sterilized drugs to give concentrations that would provide the desired doses when administered at 5 ml/kg of body weight. In vivo toxicity studies. Two patas monkeys received intramuscular injections of ara-amp at 15 mg/ kg per day once daily for 5 days. The monkeys were weighed before the initial injection and weekly for approximately 3 weeks. Hematology and clinical chemistry evaluations were made on the same schedule Ȧn additional eight patas monkeys were used for investigation of intravenous toxicity of ara-amp. Three groups of two monkeys were given ara-amp at 100, 50, or 20 mg/kg per day, administered as a daily intravenous drip over a 6-h period between 9 a.m. and 3 p.m. on 5 consecutive days. Two monkeys received PBS in the same manner as controls. All monkeys were restrained without sedation during the period of daily intravenous treatment. Hematology and clinical chemistry were evaluated before treatment and after 5 days of drug administration. Liver biopsies were also performed at these times. In vivo studies for antiviral activity. After collection of base-line hematology and clinical chemistry data and confirmation of the nonimmune status, the monkeys were weighed and arranged in groups of four. Each group was comparable with respect to the weight ranges of the monkeys. Infection was accomplished by inoculation of 1.5 mi of simian varicella virus transtracheally by puncture of the crycothyroid cartilage, and 1.5 ml of the same virus by the subcutaneous route. The virus inoculum was titrated at the time of inoculation for plaque-forming units. In the experiment to evaluate ara-amp treatment by the intramuscular route, 1.35 x 105 plaque-forming units of simian varicella virus were administered. Ara-AMP was administered as a single daily injection at 15 mg/kg per day ARA-A AND ARA-AMP IN SIMIAN VARICELLA 143 for 16 days, and treatment was initiated 43 h after virus inoculation. Infected control monkeys received a similar PBS injection on the same schedule. The monkeys used to determine the effect of intravenous ara-a or ara-amp treatment were infected with a total dose of 7.5 x 104 plaque-forming units of simian varicella virus. Treatment was begun 48 h after virus inoculation and, in an attempt to achieve a more sustained dose, was given in divided doses twice daily at 9 a.m. and 3 p.m. for 10 days. Ara-A was given at a daily dose of 15 mg/kg, and ara-amp was given at 18.4 mg/kg per day. PBS was administered intravenously on the same schedule to the infection control monkeys. For evaluation of antiviral activity, blood was collected on days 0, 3, 5, 7, and 9 for hematological and clinical chemistry monitoring and for virological assessment. A complete blood count and differential was performed, and clinical chemistry parameters evaluated were blood urea nitrogen, creatinine, total protein, albumin, globulin, transaminases, and bilirubin. To determine virus infection, 3.0 ml of blood from patas monkeys or 1.5 ml of blood from African green monkeys was collected in Panheparin at 10 U/ml, and the lymphocytes were separated over a Ficoll-Hypaque gradient. The lymphocytes were washed twice in RPMI medium with 20% fetal calf serum and inoculated into three tubes of Vero cells. Throat swabs were collected and expressed into 1.0 ml of tissue culture medium which was used as the inoculum for three additional Vero cell cultures. A maintenance medium of minimum essential medium with 2% fetal calf serum was used, and the cultures were observed for cytopathic changes for 14 days. Tissue from animals dying during the course of evaluation was homogenized by mortars and pestles to an approximate 10% homogenate and used as the inoculum for Vero cell cultures. RESULTS Comparison of patas and vervet monkey susceptibility to simian varicella virus. Experiments were performed to compare the relative susceptibilities and course of clinical disease in patas and vervet (African green) monkeys infected with the simian varicella virus. Inoculation of the virus by the combined intratracheal and subcutaneous routes led to similar clinical disease in both species. Virus isolation from the buffy coat of blood was possible between days 3 and 10 postinfection from both species, and the viremia continued for 5 to 7 days. Vesicular rash, characteristic of the disease, occurred in both the patas and vervet monkeys at 8 to 9 days after inoculation and cleared by day 14. The vesicles observed in the vervet monkeys were somewhat larger than those seen in the patas monkeys. Serum transaminase levels increased in both species between 3 and 10 days after inoculation and returned to normal between 14 and 21 days after inoculation. A greater fluctuation in liver function tests occurred in the vervet monkeys, although both species responded with

3 144 SOIKE ET AL. abnormal values in serial serum specimens collected during the course of disease. In vitro studies. Ara-AMP at concentrations of 10 to 100 jg/ml effectively inhibited the development of 80 to 100% of the plaques of simian varicella virus when 200 to 300 plaque-forming units were seeded into 60-mm petri plates seeded with Vero cells at 105 cells per ml. Inhibition of the virus was not observed at a concentration of 1,ug/ml, and concentrations of 500 to 1,000,g/ ml appeared to be toxic for the Vero cells as evidenced by granularity, rounding of cells, and subsequent sloughing. Intramuscular toxicity. Toxicity of daily intramuscular injections of ara-amp was examined in two patas monkeys which received single daily injections of 15 mg/kg for 5 days. No local reactions occurred at the injection sites, and no loss of weight occurred during the 5-day period. Also, no changes in serum transaminases or alterations in complete blood counts were observed during a 19-day period of observation. A decline in blood platelets in one monkey may suggest a possible depression of bone marrow. Intravenous toxicity. The intravenous toxicity of ara-amp was investigated by administration of an intravenous drip daily for 6 h on 5 consecutive days. Pairs of patas monkeys received doses of 100, 50, or 20 mg/kg per day, and one pair of control monkeys received normal PBS in the same manner. Serum transaminase values were observed to increase in all eight monkeys, including the controls. The elevations may have been the consequence of the necessary restraint of the monkeys in a prone position during treatment. Considerable straining of the monkeys occurred during this period of confinement while unsedated. Erythrocyte counts, hematocrit, and hemoglobin declined in monkeys receiving ara-amp at 100 and at 50 mg/kg per day. One monkey at 100 mg/kg per day and one at 50 mg/kg per day died on days 15 and 17, respectively, after the initiation of treatment. Both monkeys showed weakness and anorexia before death. The monkey receiving the 100-mg/kg per day dose was found dead 15 days after initiation of ara-amp treatment. The second monkey, which received ara-amp at 50 mg/kg per day, was euthanized 17 days after beginning treatment. Both monkeys were observed to have extensive degeneration and necrosis of skeletal muscle, presumably related to the straining during restraint. The lungs of both animals revealed pneumonitis, with the monkey receiving 100 mg/ kg per day exhibiting perivascular and intraalveolar hemorrhage. The monkey at 50 mg/kg per day showed severe but diffuse edema. The kidneys of both monkeys showed a mild lymphocyte infiltrate and hemorrhage. The adrenal ANTIMICROB. AGENTS CHEMOTHER. gland of the monkey at 100 mg/kg per day revealed hyaline droplet degeneration, cytoplasmic vacuolization, and some necrosis. Testicular tissue of the monkey receiving 50 mg/kg per day showed severe, diffuse aspermatogenesis. The monkey at the 100 mg/kg per day dose was a female, and the ovaries were not examined. No significant findings were observed in the brain of the monkey at 50 mg/kg per day, and only minimal multifocal areas of hemorrhage were seen in the animal receiving 100 mg of ara-amp per kg per dy. Liver biopsies taken from all of the monkeys after the 5 days of treatment showed areas of cloudy swelling and granulation in hepatocytes of those receiving ara-amp at 100 and at 50 mg/ kg per day. Liver changes and hematological changes were not seen in those receiving 20 mg of ara-amp per kg per day. Intramuscular treatment of simian vancelia virus infection with ara-amp. Treatment of four patas monkeys with a singular daily intramuscular injection of ara-amp did not appreciably alter the course of clinical disease. Nine days after virus inoculation, a vesicular rash, typical of simian varicella infection, appeared on the face, chest, and abdomen and on the extremities of two control and one ara-amptreated monkey. By day 11, all eight monkeys were clinically ill with varicella-like lesions. One control monkey was found dead on the morning of day 14. Gross and histopathological examinati'on indicated a disseminated varicella infection. The remaining seven animals showed complete resolution of the rash within 8 days of onset. Virus was recovered from lymphocytes of each of the control and ara-amp-treated monkeys (Table 1). In addition, simian varicella virus was recovered from throat swabs of control monkey TABLE 1. Simian varicella virus viremia in control and ara-amp-treatedpatas monkeys Virus isolation from Monkey Treatment buffy coat on days no. group, postinfection: Control Control Control Control Ara-AMP Ara-AMP Ara-AMP Ara-AMP a Control, normal saline administered intramuscularly. Ara-AMP, Ara-AMP administered intramuscularly at 15 mg/kg per day as a single daily injection for 16 days beginning 43 h after virus inoculation.

4 VOL. 18, 1980 ARA-A AND ARA-AMP IN SIMIAN VARICELLA 5780 on day 9 and from throat swabs of control AMP. Twelve African green monkeys were infected with simian varicella virus and divided monkey 5786 on days 7 and 9. Virus was also isolated from the vesicles of monkey 5786 on day into three groups of four monkeys each to compare the antiviral activity of ara-a and ara-amp 11. Monkey 5781, which received ara-amp, had virus in its throat on day 9 in addition to the administered intravenously. Three days after demonstrated viremia. No virus was recovered the virus was administered, viremia was detected in each of the four untreated control from lung, liver, spleen, kidneys, lymph nodes, or spinal, trigeminal, or facial nerve ganglia from monkeys, in one of four ara-a-treated monkeys, the monkey that died from the infection. Failure and in two of four monkeys receiving ara-amp to recover virus was believed to be due to the (Table 3). During the succeeding 4 days, each of time elapsed between death and necropsy. the treated monkeys developed viremia. Similarly, each of the monkeys in the control and Serum transaminase values were observed to be higher in the control monkeys than in the two treatment groups developed the typical ara-amp-treated monkeys (Table 2). Both the rash. serum glutamic oxalacetic and pyruvic transaminase values were generally lower in the monkeys occurred on day 3 and remained ele- Elevations in serum transaminase in all 12 treated monkeys. The lower serum transaminase vated through day 9 (Table 4). The moderation values observed in the ara-amp-treated monkeys suggest a possible moderation in the liver periment with ara-amp was not seen. By day of transaminase values seen in the previous ex- disease resulting from this infection, although 15, the transaminase values declined to normal these differences were not statistically significant by means of Student's t test. was found when the means at each observation or near-normal levels. No statistical significance Intravenous treatment of simian varicella virus infection with ara-a or ara- in hematological values or clinical chemistry period were compared. No appreciable changes TABLE 2. Mean transaminase values in untreated control and ara-amp-treatedpatas monkeys infected with simian varicella virus SGOT SGPTb Days post- (Mean ± SEc) (Mean ± SE) infection Control Ara-AMP Control Ara-AMP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.4 a SGOT, Serum glutamic oxalacetic transaminase. b SGPT, Serum glutamic pyruvic transaminase. c SE, Standard error. TABLE 3. Viremia and rash in African green monkeys infected with simian varicella virus and treated with ara-a and ara-amp Monkey Treatment Virus isolation from buffy Appearance of rash (days no. groupa coat (days postinfection) postinfection) Control Control Control Control Ara-A Ara-A Ara-A Ara-A Ara-AMP Ara-AMP Ara-AMP Ara-AMP a Control, PBS administered intravenously. Ara-A, Ara-A administered intravenously at 15 mg/kg per day twice daily for 10 days beginning 48 h after virus inoculation. Ara-AMP, Ara-AMP administered intravenously at 18.4 mg/kg per day, twice daily for 10 days beginning 48 h after virus inoculation. 145

5 146 SOIKE ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 4. Mean transaminase values in untreated control and ara-a- or ara-amp-treated African green monkeys infected with simian varicella virus SGOTa (Mean ± SE') SGPTb (Mean ± SE) Days postinfection Control Ara-A Ara-AMP Control Ara-A Ara-AMP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.7 a SGOT, Serum glutamic oxalacetic transaminase. b SGPT, Serum glutamic pyruvic transaminase. SE, Standard error. Xresults were observed during the course of dis- -ease. Fifteen days after virus inoculation, monkey 5803, from the group receiving ara-amp, was found dead. Necropsy revealed a generalized varicella infection with involvement of the lungs, liver, gastrointestinal tract, and lymph nodes. Simian varicella virus was isolated from the lung and liver. DISCUSSION Simian varicella virus was shown to be susceptible to inhibition by ara-amp in vitro. A concentration of ara-amp at 10,ig/ml inhibited the development of greater than 80% of the inoculated plaque-forming units of the virus. Intramuscular administration of ara-amp to patas monkeys once daily at 15 mg/kg had no effect on the appearance and duration of viremia nor upon the development of the rash. As serum levels of ara-amp were not determined, it is not known whether adequate antiviral levels of the drug were attained nor how long serum concentrations of ara-amp persisted. A moderation of liver disease may have occurred, based on the observed differences of serum transaminase levels between control and ara-amp-treated monkeys. Statistical analysis, however, failed to establish the significance of these differences. The ineffectiveness of ara-amp when given as a single daily intramuscular injection may have been the result of deamination in vivo, preventing a single daily dose from achieving adequate or persistent levels in the circulation. Therefore, a subsequent experiment was performed in which ara-a and ara-amp were administered, twice daily, at a comparable dose. This experiment also failed to demonstrate antiviral activity against the simian varicella virus. Viremia occurred in all control and treated monkeys, and rash was observed in all monkeys. No effect was seen on the liver disease associated with this infection, as transaminase values rose in all groups and no statistically significant differences occurred between groups. It is evident that the transaminase values obtained in the two experiments are appreciably different. These differences were not due to differences between the species of monkeys employed but occurred due to the use of two different methods of assay. In one experiment, the values were obtained by a method employing a Calbiochem ultraviolet assay, whereas, in the second experiment, the values were obtained with a Hycel HMA Chemical Analyzer ultraviolet assay. The range of values for normal human control sera by these two methods shows an approximate twofold difference, with the HMA assay giving higher values. Presumably enzymatic degradation of ara-a and its 5'-monophosphate could still account for differences in in vitro and in vivo activity. As serum levels of the drugs were not determined, we cannot substantiate this possibility. It is strongly suggested that treatment of simian varicella infection with these drugs in combination with a deaminase inhibitor be attempted based on reported findings of potentiation of activity (14). Investigation of the intramuscular and intravenous toxicity of ara-amp suggested no adverse effects of treatment at 15 to 20 mg/kg per day. However, it was observed that one monkey in each pair of monkeys receiving 50 or 100 mg/ kg per day intravenously died. The significance of the findings upon necropsy of these monkeys is not known at this time. Some of the histological observations could be associated with drug toxicity. The significance of the pulmonary edema, aspermatogenesis, and adrenal changes requires clarification by further studies. Because of this potential toxicity, experiments in which ara-a and ara-amp are used in combination with a deaminase inhibitor should be preceded by appropriate studies for toxicity. The simian varicella virus serves as a suitable

6 VOL. 18, 1980 laboratory model for human VZV infection. Clinically it produces a severe generalized varicella-like illness in patas monkeys (2), can infect a number of Old World species of nonhuman primates (6,12), and is immunologically related to human VZV (5). Like human varicella virus, it is cell associated, and recovery from tissues and vesicle fluids is difficult. Isolation from lymphocytes during the period of viremia has been found to be reproducible during early infection and persists over several days. This detectable viremia has served to monitor infection by the virus and has been shown to be inhibited by treatment with an effective antiviral agent (4). ACKNOWLEDGMENTS This work was supported under Public Health Service contract no. N01-AI with the National Institute of Allergy and Infectious Diseases. The technical assistance of Stephen Williams is gratefully acknowledged. ARA-A AND ARA-AMP IN SIMIAN VARICELLA 147 LITERATURE CITED 1. Alenius, S., and B. Oberg Comparison of the therapeutic effects of five antiviral agents on cutaneous herpesvirus infection in guinea pigs. Arch. Virol. 58: Allen, W. P., A. D. Felsenfeld, R. H. Wolf, and H. F. Smetana Recent studies on the isolation and characterization of Delta herpesvirus. Lab. Anim. Sci. 24: Collins, P., and D. J. Bauer Comparison of activity of herpes virus inhibitors. J. Antimicrob. Chemother. 3(Suppl. A): Felsenfeld, A. D., C. R. Abee, P. J. Gerone, K. F. Soike, and S. R. Williams Phosphonoacetic acid in the treatment of simian varicella. Antimicrob. Agents Chemother. 14: Felsenfeld, A. D., and N. J. Schmidt Inununological relationship between Delta herpesvirus of patas monkeys and varicella-zoster virus of humans. Infect. Inunun. 12: Felsenfeld, A. D., and N. J. Schmidt Antigenic relationships among several simian varicella-like viruses and varicella-zoster virus. Infect. Immun. 15: Fiala, M., A. W. Chow, K. Mizaschi, and L. B. Yuze Susceptibility of herpesviruses to three nucleoside analogues and their combinations and enhancement of the antiviral effect at acid ph. J. Infect. Dis. 129: Kern, E. R., J. T. Richards, J. C. Overall, Jr., and L. A. Glasgow Genital Herpesvirus hominis infection in mice. II. Treatment with phosphonoacetic acid, adenine arabinoside, and adenine arabinoside 5'-monophosphate. J. Infect. Dis. 136: Luby, J. P., S. R. Jones, M. T. Johnson, and D. Mikulec Sensitivities of herpes simplex types 1 and 2 and varicella-zoster virus to adenine arabinoside and hypoxanthine arabinoside, p In D. Pavan- Langston, R. A. Buchanan, and C. A. Alford, Jr. (ed.), Adenine arabinoside: an antiviral agent. Raven Press, New York. 10. Marks, M. I Evaluation of four antiviral agents in the treatment of herpes simplex encephalitis in a rat model. J. Infect. Dis. 131: Shannon, W. M Adenine arabinoside: antiviral activity in vitro, p In D. Pavan-Langston, R. A. Buchanan, and C. A. Alford, Jr. (ed.), Adenine arabinoside: an antiviral agent. Raven Press, New York. 12. Wenner, H. A., S. Barrick, D. Abel, and P. Seshumurty The pathogenesis of simian varicella virus in cynomolgus monkeys. Proc. Soc. Exp. Biol. Med. 150: Whitley, R. J., S. Soong, R. Dolin, G. J. Galasso, L. T. Chien, C. A. Alford, Jr., and the Collaborative Study Group Adenine arabinoside therapy of biopsy-provide herpes simplex encephalitis. National Institute of Allergy and Infectious Diseases Collaborators Antiviral Study. N. Engl. J. Med. 297: Willlams, B. B., and A. M. Lerner Antiviral activity of an adenosine deaminase inhibitor: decreased replication of herpes simplex virus. J. Infect. Dis. 131: