Vol. 10, No. 3
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1976, p. 571-572
Printed in U.S.A.
Copyright © 1976 American Society for Microbiology
Antiviral Effects of Virazole in Chronic Hepatitis B Surface Antigen-Seropositive Chimpanzees ALEXANDER E. DENES,* JAMES W. EBERT, KENNETH R. BERQUIST, BERT L. MURPHY, AND JAMES E. MAYNARD Phoenix Laboratories Division, Center for Disease Control, Phoenix, Arizona 85014
Received for publication 17 May 1976
Virazole (Ribavirin, ICN 1229), a broad-spectrum, antiviral chemotherapeutic agent was used to treat two adult chronically hepatitis B surface antigen (HBS Ag)-seropositive chimpanzees. No significant change in serum hepatitis B surface antigen was noted and no adverse reactions were observed. The role of viral replication in the chronic carrier state of hepatitis B is discussed. Virazole (Ribavirin, ICN 1229, 1-/3-u-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a synthetic nucleoside with demonstrated activity against several ribonucleic acid and deoxyribonucleic acid viruses (8). The active metabolite, Virazole 5'-phosphate, is thought to inhibit guanosine 5'-phosphate biosynthesis in infected cells at the step involving the conversion of inosine 5'-phosphate to xanthosine 5'-phosphate and thus inhibit viral replication (9). In South America, preliminary clinical studies with the drug have been reported with encouraging results in acute hepatitis B as well as in chronic carriers of hepatitis B surface antigen (HB, Ag) (5, 10, 11). We treated two adult chronically HB, Agseropositive chimpanzees with Virazole. Both animals were in good health and had no evidence of active liver disease either clinically or by liver biopsy. Virazole was given orally, as a liquid suspension, for 21 days. It was administered at 7 a.m. and 4 p.m. daily in 600-mg doses, with a total daily dose of 1,200 mg (ca. 20 mg/ kg). Prior to initiation of the study, each animal had a complete blood count and automated chemistry profile (SMA-12), which includes the following serum chemistry determinations: calcium, phosphorus, urea nitrogen, glucose, uric acid, cholesterol, total proteins, albumin, total bilirubin, alkaline phosphatase, lactic dehydrogenase, and glutamic-oxaloacetic transaminase. These determinations, as well as a serum HBS Ag determination, were performed twice a week during the period of drug administration and for 4 weeks thereafter. Liver biopsies were obtained weekly and examined after staining with hematoxylin and eosin and also by immunofluorescence for HB, Ag and hepatitis B core antigen (HBC Ag) (3). All serological determinations for HB, Ag were by radioimmunoassay (Ausria II, Abbott Laboratories) (7).
No adverse reactions were noted. There were no significant changes in hematocrit, hemoglobin, or leukocyte counts. The only significant change in blood chemistry was a decrease in serum alkaline phosphatase in both animals, which returned to pretreatment values after the study was completed. Both animals remained highly seropositive for HB, Ag during the investigation (Table 1). Serial dilutions of serum also did not demonstrate any significant decrease in the titer of HB, Ag. We were not able to show any beneficial effect of Virazole in our chronically HB, Ag-seropositive animals. Although this study involved only two animals, our results suggest that Virazole may not be as effective in terminating the chronic HB, Ag carrier state as has been reported (5). Although the results can be interpreted as demonstrating a lack of antiviral activity of this agent against the hepatitis B virus, they also raise an intriguing question about the role of viral replication in some chronically healthy carriers of HB, Ag. Serial liver biopsies from both animals demonstrated
cytoplasmic HB, Ag by immunofluorescence prior to and after treament with Virazole but failed to show HB, Ag in hepatocyte nuclei. TABLE 1. HBS Ag content of Virazole-treated chimpanzees HB, Ag content (SR units a) on day: Animal no.
0
7
14
21
42
50
37
36
33
37
28 37
35
109
39
33
33
120
30
34
30
32
31
30
31
30
Sample ratio (SR) units counts per minute of test sample/counts per minute of control sample. Ratios above 2.6 were recorded as HBS Ag positive. For titration purposes all specimens were assayed with one radioimmunoassay kit. a
571
=
572
NOTES
Early studies using immunofluorescence and immune electron microscopy demonstrated hepatitis B antigens in both cytoplasm and nuclei of hepatocytes (4, 6). The identification of the two antigens HB, Ag and HB, Ag resolved this problem (1). Subsequently, several investigators showed the exclusive localization of HB, Ag in hepatocyte nuclei and its association with viral replication (2, 3). During acute hepatitis B virus infection, it is possible that a segment of the viral genome (a component of HB, Ag) is integrated into the host's genome in much the same way that bacteriophages interact with lysogenic bacteria. Under certain circumstances, this interaction could result in the continuous cytoplasmic production of HB5 Ag without concurrent viral replication. In this situation, an agent such as Virazole would be without effect.
ANTIMICROB. AGENTS CHEMOTHER.
4.
5. 6.
7.
8.
9.
Virazole was supplied by ICN Pharmaceuticals, Irvine, Calif. LITERATURE CITED 1. Almeida, J. D., M. Rubenstein, and E. J. Scott. 1971. New antigen-antibody system in Australia-antigen positive hepatitis. Lancet 2:1225-1227. 2. Barker, L. F., F. U. Chisari, P. P. McGrath, D. W. Dalgart, R. L. Kirschstein, J. D. Almeida, T. S. Edgington, D. G. Sharp, and M. R. Peterson. 1973. Transmission of type B viral hepatitis to Chimpanzees. J. Infect. Dis. 127:648-662. 3. Berquist, K. R., J. M. Peterson, B. L. Murphy, J. W.
10.
11.
Ebert, J. E. Maynard, and R. H. Purcell. 1975. Hepatitis B antigens in serum and liver of chimpanzees acutely infected with hepatitis B virus. Infect. Immun. 12:602-605. Brzosko, W. D., K. Madalinski, K. Krawczynski, and A. Nowoslawski. 1973. Duality ofhepatitis B antigen and its antibody. I. Immunofluorescence studies. J. Infect. Dis. 127:424-428. Galvao, P. A. A., and I. 0. Castro. 1974. Treatment of acute viral hepatitis with a new antiviral compound. Rev. Bras. Clin. Ter. 3:221-228. Huang, S.-N., I. Millman, A. O'Connell, A. Aronoff, H. Gault, and B. S. Blumberg. 1972. Virus-like particles in Australia antigen-associated hepatitis. Am. J. Pathol. 67:453-462. Ling, C., and L. Overby. 1972. Prevalence of hepatitis B surface antigen as revealed by direct radioimmunoassay with '25I-antibody. J. immunol. 109:834-841. Sidwell, R. W., J. H. Huffman, G. P. Khare, L. B. Allen, J. T. Witkowski, and R. K. Robins. 1972. Broad spectrum antiviral activity of Virazole: 1-/3-Dribofuranosyl-1,2,4-triazole-3-carboxamide. Science 177:705-706. Streeter, D. G., J. T. Witkowski, P. K. Gyaneshwar, R. W. Sidwell, R. J. Bauer, R. K. Robins, and L. N. Simon. 1973. Mechanism of action of 1-f8-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole), a new broad-spectrum antiviral agent. Proc. Natl. Acad. U.S.A. 70:1174-1178. Vilelea, M. P., W. A. Siqueira, S. H. S. Domingues, and F. S. Rodrigues. 1974. Behaviour of Australia Antigen in cases of hepatitis treated with 1-beta-DRev. ribofuranosyl-1,2,4-triazole-3-carboxamide. Bras. Clin. Ter. 3:27-28. Zuniga, C. B., C. Almeida, A. C. S. Iervolino, I. 0. Castro, and P. A. A. Galvao. 1974. Action of 1-beta-D ribofuranosyl-1,2,4,-triazole-3-carboxamide (viramid, ICN 1229) in the treatment of acute viral hepatitis. Rev. Assn. Med. Brazil. 20:386-390.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1976, p. 571-572
Printed in U.S.A.
Copyright © 1976 American Society for Microbiology
Antiviral Effects of Virazole in Chronic Hepatitis B Surface Antigen-Seropositive Chimpanzees ALEXANDER E. DENES,* JAMES W. EBERT, KENNETH R. BERQUIST, BERT L. MURPHY, AND JAMES E. MAYNARD Phoenix Laboratories Division, Center for Disease Control, Phoenix, Arizona 85014
Received for publication 17 May 1976
Virazole (Ribavirin, ICN 1229), a broad-spectrum, antiviral chemotherapeutic agent was used to treat two adult chronically hepatitis B surface antigen (HBS Ag)-seropositive chimpanzees. No significant change in serum hepatitis B surface antigen was noted and no adverse reactions were observed. The role of viral replication in the chronic carrier state of hepatitis B is discussed. Virazole (Ribavirin, ICN 1229, 1-/3-u-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a synthetic nucleoside with demonstrated activity against several ribonucleic acid and deoxyribonucleic acid viruses (8). The active metabolite, Virazole 5'-phosphate, is thought to inhibit guanosine 5'-phosphate biosynthesis in infected cells at the step involving the conversion of inosine 5'-phosphate to xanthosine 5'-phosphate and thus inhibit viral replication (9). In South America, preliminary clinical studies with the drug have been reported with encouraging results in acute hepatitis B as well as in chronic carriers of hepatitis B surface antigen (HB, Ag) (5, 10, 11). We treated two adult chronically HB, Agseropositive chimpanzees with Virazole. Both animals were in good health and had no evidence of active liver disease either clinically or by liver biopsy. Virazole was given orally, as a liquid suspension, for 21 days. It was administered at 7 a.m. and 4 p.m. daily in 600-mg doses, with a total daily dose of 1,200 mg (ca. 20 mg/ kg). Prior to initiation of the study, each animal had a complete blood count and automated chemistry profile (SMA-12), which includes the following serum chemistry determinations: calcium, phosphorus, urea nitrogen, glucose, uric acid, cholesterol, total proteins, albumin, total bilirubin, alkaline phosphatase, lactic dehydrogenase, and glutamic-oxaloacetic transaminase. These determinations, as well as a serum HBS Ag determination, were performed twice a week during the period of drug administration and for 4 weeks thereafter. Liver biopsies were obtained weekly and examined after staining with hematoxylin and eosin and also by immunofluorescence for HB, Ag and hepatitis B core antigen (HBC Ag) (3). All serological determinations for HB, Ag were by radioimmunoassay (Ausria II, Abbott Laboratories) (7).
No adverse reactions were noted. There were no significant changes in hematocrit, hemoglobin, or leukocyte counts. The only significant change in blood chemistry was a decrease in serum alkaline phosphatase in both animals, which returned to pretreatment values after the study was completed. Both animals remained highly seropositive for HB, Ag during the investigation (Table 1). Serial dilutions of serum also did not demonstrate any significant decrease in the titer of HB, Ag. We were not able to show any beneficial effect of Virazole in our chronically HB, Ag-seropositive animals. Although this study involved only two animals, our results suggest that Virazole may not be as effective in terminating the chronic HB, Ag carrier state as has been reported (5). Although the results can be interpreted as demonstrating a lack of antiviral activity of this agent against the hepatitis B virus, they also raise an intriguing question about the role of viral replication in some chronically healthy carriers of HB, Ag. Serial liver biopsies from both animals demonstrated
cytoplasmic HB, Ag by immunofluorescence prior to and after treament with Virazole but failed to show HB, Ag in hepatocyte nuclei. TABLE 1. HBS Ag content of Virazole-treated chimpanzees HB, Ag content (SR units a) on day: Animal no.
0
7
14
21
42
50
37
36
33
37
28 37
35
109
39
33
33
120
30
34
30
32
31
30
31
30
Sample ratio (SR) units counts per minute of test sample/counts per minute of control sample. Ratios above 2.6 were recorded as HBS Ag positive. For titration purposes all specimens were assayed with one radioimmunoassay kit. a
571
=
572
NOTES
Early studies using immunofluorescence and immune electron microscopy demonstrated hepatitis B antigens in both cytoplasm and nuclei of hepatocytes (4, 6). The identification of the two antigens HB, Ag and HB, Ag resolved this problem (1). Subsequently, several investigators showed the exclusive localization of HB, Ag in hepatocyte nuclei and its association with viral replication (2, 3). During acute hepatitis B virus infection, it is possible that a segment of the viral genome (a component of HB, Ag) is integrated into the host's genome in much the same way that bacteriophages interact with lysogenic bacteria. Under certain circumstances, this interaction could result in the continuous cytoplasmic production of HB5 Ag without concurrent viral replication. In this situation, an agent such as Virazole would be without effect.
ANTIMICROB. AGENTS CHEMOTHER.
4.
5. 6.
7.
8.
9.
Virazole was supplied by ICN Pharmaceuticals, Irvine, Calif. LITERATURE CITED 1. Almeida, J. D., M. Rubenstein, and E. J. Scott. 1971. New antigen-antibody system in Australia-antigen positive hepatitis. Lancet 2:1225-1227. 2. Barker, L. F., F. U. Chisari, P. P. McGrath, D. W. Dalgart, R. L. Kirschstein, J. D. Almeida, T. S. Edgington, D. G. Sharp, and M. R. Peterson. 1973. Transmission of type B viral hepatitis to Chimpanzees. J. Infect. Dis. 127:648-662. 3. Berquist, K. R., J. M. Peterson, B. L. Murphy, J. W.
10.
11.
Ebert, J. E. Maynard, and R. H. Purcell. 1975. Hepatitis B antigens in serum and liver of chimpanzees acutely infected with hepatitis B virus. Infect. Immun. 12:602-605. Brzosko, W. D., K. Madalinski, K. Krawczynski, and A. Nowoslawski. 1973. Duality ofhepatitis B antigen and its antibody. I. Immunofluorescence studies. J. Infect. Dis. 127:424-428. Galvao, P. A. A., and I. 0. Castro. 1974. Treatment of acute viral hepatitis with a new antiviral compound. Rev. Bras. Clin. Ter. 3:221-228. Huang, S.-N., I. Millman, A. O'Connell, A. Aronoff, H. Gault, and B. S. Blumberg. 1972. Virus-like particles in Australia antigen-associated hepatitis. Am. J. Pathol. 67:453-462. Ling, C., and L. Overby. 1972. Prevalence of hepatitis B surface antigen as revealed by direct radioimmunoassay with '25I-antibody. J. immunol. 109:834-841. Sidwell, R. W., J. H. Huffman, G. P. Khare, L. B. Allen, J. T. Witkowski, and R. K. Robins. 1972. Broad spectrum antiviral activity of Virazole: 1-/3-Dribofuranosyl-1,2,4-triazole-3-carboxamide. Science 177:705-706. Streeter, D. G., J. T. Witkowski, P. K. Gyaneshwar, R. W. Sidwell, R. J. Bauer, R. K. Robins, and L. N. Simon. 1973. Mechanism of action of 1-f8-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole), a new broad-spectrum antiviral agent. Proc. Natl. Acad. U.S.A. 70:1174-1178. Vilelea, M. P., W. A. Siqueira, S. H. S. Domingues, and F. S. Rodrigues. 1974. Behaviour of Australia Antigen in cases of hepatitis treated with 1-beta-DRev. ribofuranosyl-1,2,4-triazole-3-carboxamide. Bras. Clin. Ter. 3:27-28. Zuniga, C. B., C. Almeida, A. C. S. Iervolino, I. 0. Castro, and P. A. A. Galvao. 1974. Action of 1-beta-D ribofuranosyl-1,2,4,-triazole-3-carboxamide (viramid, ICN 1229) in the treatment of acute viral hepatitis. Rev. Assn. Med. Brazil. 20:386-390.
