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Relationship of serum 2.5 oligoadenylate synthetase activity during interferon therapy to pretreatment levels or genotypes of serum HCV-RNA
Internationul
Hepatology International Hepatology Communications 2 (1994) 358-362
ELSEVIER
commLlrlicationS
Relationship of serum 2.5 oligoadenylate synthetase activity during interferon therapy to pretreatment levels or genotypes of serum HCV-RNA Michiari Okuda”*, Keisuke Hino”, Kouzou Kayanoa, Masafumi Kubotaa, Kazuyuki Takenakaa, Kenji Mori”, Aogu Yamashitaa, Isao Sakaida”, Fujio Murakamia, Mitsuru Yasunagaa, Kiwamu Okita”, Tomomi Konishi” “First Department
of Internal Medicine, Yamaguchi University School of Medicine, I144 Kogushi, Ube,
Yamaguchi, 755, Japan bDepartment of Internal Medicine, Shutoh General Hospital, Yamaguchi, Japan
(Received 2 1 February 1994;accepted 28 April 1994)
Abstract 2.5 oligoadenylate synthetase (2SAS) activity is induced during interferon (IFN) therapy of patients with chronic hepatitis C. It is assumed that the genotype and quantitative level of hepatitis C virus RNA (HCV-RNA) in serum preceding IFN therapy are closely associated with the response to IFN. However, it is not clear whether these viral factors may affect 2SAS activity during IFN therapy. We analyzed 2.5AS activity during IFN therapy in patients with different genotypes or levels of serum HCV-RNA. However, there were no statistical differences in 2.5AS activity among them. In the present study, those viral factors were not associated with the induction of 2.5AS activity during IFN therapy, and it is unlikely that differences in response to IFN among patients with different genotypes or quantitative levels of HCV-RNA depend on the 2.5AS pathway. Key words:
Serum 2.5AS activity; Quantitative
serum HCV-RNA level; Genotype
1. Introduction Although the binding of interferon (IFN) to specific liver cell membrane receptors induces the synthesis of multiple antiviral proteins [l], 2.5 oligoadenylate synthetase (2.5AS), the well-known IFN-induced protein, is clinically relevant in establishing the mechanisms of IFN activity and its optimal dosage. Genotypes of hepatitis C virus (HCV)-RNA and quantitative HCV-RNA levels in serum are assumed to be closely *Corresponding author. 0928-4346/94/%07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0928-4346(94)00033-2
M. Okuda et al. IInt. Hepatol. Commun. 2 (1994) 358-362
359
associated with the response to IFN shown by patients with chronic hepatitis C. We studied the changes in 2SAS activity induced by IFN in order to determine whether genotypes and quantitative levels of serum HCV-RNA affect the activity of this protein.
2. Patients and methods Twenty-six patients with chronic hepatitis C who had been treated with IFN were included in this study. There were 12 men and 14 women, mean age 48.0 + 11.2 years. Four had received at least one blood transfusion. The histological diagnosis of patients were; chronic persistent hepatitis in 11, chronic aggressive hepatitis (2A) in 11, and chronic aggressive hepatitis (2B) in four. All patients were positive for serum HCV-RNA before IFN therapy. Of the 22 patients who received natural or recombinant IFN-CX,13 patients received 6 million units of IFN six times a week for 2 weeks, followed by an intramuscular injection given three times a week for 12 or 14 additional weeks. The other nine patients received 6 million units of IFN three times weekly for 14 or 19 weeks. Four patients received 6 million units of IFN-/? six times a week for 7 weeks, or six times a week for 2 weeks, followed by three times a week for an additional 10 weeks. We analyzed the changes in serum 2.5AS activity at several points during treatment and the negativity of serum HCV-RNA at the end of treatment, as related to genotype or to quantitative level of serum HCV-RNA. We also compared those parameters in the two groups of patients who were given IFN six times weekly or three times weekly for the first 2 weeks of therapy. Serum samples for the assay of 2.5AS activity were obtained every 2 weeks for 4 weeks during treatment and at the end of treatment. The activity of 2.5AS in serum was measured with an assay kit from the Eiken Immunochemical Laboratory (Tokyo, Japan). Measurements of enzyme activity were expressed as ratios of the value at each point during treatment to pretreatment value. Serum HCV-RNA was detected by reverse transcription polymerase chain reaction (RT-PCR), using the 5’ non-coding region as a primer [2]. HCV-RNA levels in serum were measured by the method of competitive RT-PCR developed by Kato et al. [3]. Genotypes of HCV-RNA were classified as type I, II, III, or IV by the method of Okamoto et al. [4], with several modifications.
3. Results The quantitative HCV-RNA level in serum just before treatment was more than lo6 copy numbers in 16 patients (group A) and less than lo6 copy numbers in ten patients (group B). The genotype of HCV-RNA was II in 17 patients, III in eight patients, and unclassified in 1 patient. Backgrounds of each group are shown in Table 1, but no remarkable difference was observed in the age, sex, histology, type and administration of IFN. The negativity of serum HCV-RNA was 50% (8116) in group A and 90% (9110) in group B at the end of therapy. Comparison of 2.5AS activity in groups A and
360
M. Okuda et al. /ht. Hepatol. Commun. 2 (1994) 358-362
Table 1 Backgrounds of group A and B, and the patients with HCV genotype II and III
Ageolrs)
A (n = 16)
&O)
&7)
III (n = 8)
51.6 + 10.4
41.8 + 9.6
47.6 + 12.5
48.4 + 8.8
1214
II3
6111
612
Sex
WV Histology CPH* CAH 2A** CAH 2B***
7 6 3
4 5 1
7 7 3
3 4 1
14 2
8 2
14 3
7 1
6 10
3 7
5 12
4 4
IFN type ; Administration Intermittent Continuous
*Chronic persistent hepatitis. **Chronic aggressive hepatitis 2A. ***Chronic aggressive hepatitis 2B.
B after 2 weeks and 4 weeks of treatment showed no statistical difference: 7.1 + 7.0 (group A) cf. 6.1 + 4.0 (group B) after 2 weeks, and 6.5 + 6.1 (group A) cf. 4.8 f 4.4 (group B) after 4 weeks (Table 2). With respect to genotypes of HCV-RNA, the negativity of HCV-RNA was 52.9% (g/17) in type II and 87.5% (7/8) in type III at the end of therapy. There was also no significant difference in 2.5AS activity between types II and III after 2 weeks (7.7 f 6.1 cf. 5.6 + 5.8) and 4 weeks (6.5 f 5.8 cf. 4.2 f 4.6) of treatment (Table 3). When the same analysis related to changes in 2.5AS Table 2 2.5AS activity in relation to quantitative HCV-RNA levels in serum Copy no. (log,,)
2.5AS activity” 2 weeks
4 weeks
7.1 * 7.0
6.5 + 6.1
6.1 + 4.0
4.8 It:4.4
65 (n = 16) 6> (n = 10)
NSb
“2.5AS activity was expressed as the ratio of the value at each point during treatment to pretreatment value. bNot significant.
M. Okuda et al. I ht. Hepatol. Commun. 2 (I 994) 3X-362
361
Table 3 2SAS activity in relation to genotypes of serum HCV-RNA Genotype
2SAS activity” 2 weeks
4 weeks
(n = 17)
7.7 + 6.1
6.5 + 5.8
(n = 8)
5.6 + 5.8
4.2 f 4.6
II
NSb
III
“2.5AS activity was expressed as the ratio of the value at each point during treatment to pretreatment value ‘Not significant.
activity was done using absolute values (pretreatment value subtracted from the value at each point during treatment), results were similar to those given above. A difference in frequency of IFN administration during the first 2 weeks affected 2SAS activity after 4 weeks of therapy, that is, it was significantly higher in patients who received IFN six times weekly than in patients who received IFN three times weekly.
4. Discussion Serum 2SAS activity is useful in evaluating the antiviral efficacy of IFN therapy. Previous studies demonstrated that early induction of 2.5AS activity depended on the dose of IFN [5,6]. In the present study, continuous administration of IFN during the early phase of treatment was preferable for inducing 2.5AS activity. Considering IFN therapy in patients with chronic hepatitis C, the genotypes of HCV-RNA and quantitative HCV-RNA levels just before treatment were probably closely associated with the response to IFN. It is, therefore, important to determine whether or not the genotype or the quantitative level of HCV-RNA may affect the induction of 2.5AS activity. In the present study, those viral factors were not associated with the induction of 2.5AS activity during IFN therapy. Based on these results, it is unlikely that differences in response to IFN among patients with different genotypes or quantitative levels of HCV-RNA depend on the 2.5AS pathway.
References 111Solinas A, Cossu P, Poddighe P, et al. Changes of serum 2’,5’-oligoadenylate synthetase activity during interferon treatment of chronic hepatitis C. Liver 1993;13:253-258.
121Okamoto H, Okada S, Sygiyama Y, et al. Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5’ noncoding region. Jpn J Exp Med 1990;60:215-222. [31Kato N, Yokosuka 0, Omata M, et al. The relationship between the amount of hepatitis C virus
362
M. Okuda et al. Ilnt. Hepatol. Commun. 2 (1994) 358-362
quantified by competitive RT-PCR (CRT-PCR) and the effect of interferon treatment. Acta Hepatol Jpn 1991;32:750-751 (in Japanese). [4] Okamoto H, Sugiyama Y, Okada S, et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. J Gen Virol 1992;73:673-679. [5] Sugawara T, Matsushima T, Karino Y, Miyazaki T, Toyoto J, Okuuti Y. Clinical significance of determination of serum 2’-Soligoadenylate synthetase activities. Acta Hepatol Jpn 1987;28:310-318 (in Japanese). [6] Hino K, Shimoda K, Fujioka S, et al. Clinical significance of serum 2’5’ oligoadenylate synthetase activity in various liver diseases. Igaku to Yakugaku 1989;22:153l-l 542 (in Japanese).
Hepatology International Hepatology Communications 2 (1994) 358-362
ELSEVIER
commLlrlicationS
Relationship of serum 2.5 oligoadenylate synthetase activity during interferon therapy to pretreatment levels or genotypes of serum HCV-RNA Michiari Okuda”*, Keisuke Hino”, Kouzou Kayanoa, Masafumi Kubotaa, Kazuyuki Takenakaa, Kenji Mori”, Aogu Yamashitaa, Isao Sakaida”, Fujio Murakamia, Mitsuru Yasunagaa, Kiwamu Okita”, Tomomi Konishi” “First Department
of Internal Medicine, Yamaguchi University School of Medicine, I144 Kogushi, Ube,
Yamaguchi, 755, Japan bDepartment of Internal Medicine, Shutoh General Hospital, Yamaguchi, Japan
(Received 2 1 February 1994;accepted 28 April 1994)
Abstract 2.5 oligoadenylate synthetase (2SAS) activity is induced during interferon (IFN) therapy of patients with chronic hepatitis C. It is assumed that the genotype and quantitative level of hepatitis C virus RNA (HCV-RNA) in serum preceding IFN therapy are closely associated with the response to IFN. However, it is not clear whether these viral factors may affect 2SAS activity during IFN therapy. We analyzed 2.5AS activity during IFN therapy in patients with different genotypes or levels of serum HCV-RNA. However, there were no statistical differences in 2.5AS activity among them. In the present study, those viral factors were not associated with the induction of 2.5AS activity during IFN therapy, and it is unlikely that differences in response to IFN among patients with different genotypes or quantitative levels of HCV-RNA depend on the 2.5AS pathway. Key words:
Serum 2.5AS activity; Quantitative
serum HCV-RNA level; Genotype
1. Introduction Although the binding of interferon (IFN) to specific liver cell membrane receptors induces the synthesis of multiple antiviral proteins [l], 2.5 oligoadenylate synthetase (2.5AS), the well-known IFN-induced protein, is clinically relevant in establishing the mechanisms of IFN activity and its optimal dosage. Genotypes of hepatitis C virus (HCV)-RNA and quantitative HCV-RNA levels in serum are assumed to be closely *Corresponding author. 0928-4346/94/%07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0928-4346(94)00033-2
M. Okuda et al. IInt. Hepatol. Commun. 2 (1994) 358-362
359
associated with the response to IFN shown by patients with chronic hepatitis C. We studied the changes in 2SAS activity induced by IFN in order to determine whether genotypes and quantitative levels of serum HCV-RNA affect the activity of this protein.
2. Patients and methods Twenty-six patients with chronic hepatitis C who had been treated with IFN were included in this study. There were 12 men and 14 women, mean age 48.0 + 11.2 years. Four had received at least one blood transfusion. The histological diagnosis of patients were; chronic persistent hepatitis in 11, chronic aggressive hepatitis (2A) in 11, and chronic aggressive hepatitis (2B) in four. All patients were positive for serum HCV-RNA before IFN therapy. Of the 22 patients who received natural or recombinant IFN-CX,13 patients received 6 million units of IFN six times a week for 2 weeks, followed by an intramuscular injection given three times a week for 12 or 14 additional weeks. The other nine patients received 6 million units of IFN three times weekly for 14 or 19 weeks. Four patients received 6 million units of IFN-/? six times a week for 7 weeks, or six times a week for 2 weeks, followed by three times a week for an additional 10 weeks. We analyzed the changes in serum 2.5AS activity at several points during treatment and the negativity of serum HCV-RNA at the end of treatment, as related to genotype or to quantitative level of serum HCV-RNA. We also compared those parameters in the two groups of patients who were given IFN six times weekly or three times weekly for the first 2 weeks of therapy. Serum samples for the assay of 2.5AS activity were obtained every 2 weeks for 4 weeks during treatment and at the end of treatment. The activity of 2.5AS in serum was measured with an assay kit from the Eiken Immunochemical Laboratory (Tokyo, Japan). Measurements of enzyme activity were expressed as ratios of the value at each point during treatment to pretreatment value. Serum HCV-RNA was detected by reverse transcription polymerase chain reaction (RT-PCR), using the 5’ non-coding region as a primer [2]. HCV-RNA levels in serum were measured by the method of competitive RT-PCR developed by Kato et al. [3]. Genotypes of HCV-RNA were classified as type I, II, III, or IV by the method of Okamoto et al. [4], with several modifications.
3. Results The quantitative HCV-RNA level in serum just before treatment was more than lo6 copy numbers in 16 patients (group A) and less than lo6 copy numbers in ten patients (group B). The genotype of HCV-RNA was II in 17 patients, III in eight patients, and unclassified in 1 patient. Backgrounds of each group are shown in Table 1, but no remarkable difference was observed in the age, sex, histology, type and administration of IFN. The negativity of serum HCV-RNA was 50% (8116) in group A and 90% (9110) in group B at the end of therapy. Comparison of 2.5AS activity in groups A and
360
M. Okuda et al. /ht. Hepatol. Commun. 2 (1994) 358-362
Table 1 Backgrounds of group A and B, and the patients with HCV genotype II and III
Ageolrs)
A (n = 16)
&O)
&7)
III (n = 8)
51.6 + 10.4
41.8 + 9.6
47.6 + 12.5
48.4 + 8.8
1214
II3
6111
612
Sex
WV Histology CPH* CAH 2A** CAH 2B***
7 6 3
4 5 1
7 7 3
3 4 1
14 2
8 2
14 3
7 1
6 10
3 7
5 12
4 4
IFN type ; Administration Intermittent Continuous
*Chronic persistent hepatitis. **Chronic aggressive hepatitis 2A. ***Chronic aggressive hepatitis 2B.
B after 2 weeks and 4 weeks of treatment showed no statistical difference: 7.1 + 7.0 (group A) cf. 6.1 + 4.0 (group B) after 2 weeks, and 6.5 + 6.1 (group A) cf. 4.8 f 4.4 (group B) after 4 weeks (Table 2). With respect to genotypes of HCV-RNA, the negativity of HCV-RNA was 52.9% (g/17) in type II and 87.5% (7/8) in type III at the end of therapy. There was also no significant difference in 2.5AS activity between types II and III after 2 weeks (7.7 f 6.1 cf. 5.6 + 5.8) and 4 weeks (6.5 f 5.8 cf. 4.2 f 4.6) of treatment (Table 3). When the same analysis related to changes in 2.5AS Table 2 2.5AS activity in relation to quantitative HCV-RNA levels in serum Copy no. (log,,)
2.5AS activity” 2 weeks
4 weeks
7.1 * 7.0
6.5 + 6.1
6.1 + 4.0
4.8 It:4.4
65 (n = 16) 6> (n = 10)
NSb
“2.5AS activity was expressed as the ratio of the value at each point during treatment to pretreatment value. bNot significant.
M. Okuda et al. I ht. Hepatol. Commun. 2 (I 994) 3X-362
361
Table 3 2SAS activity in relation to genotypes of serum HCV-RNA Genotype
2SAS activity” 2 weeks
4 weeks
(n = 17)
7.7 + 6.1
6.5 + 5.8
(n = 8)
5.6 + 5.8
4.2 f 4.6
II
NSb
III
“2.5AS activity was expressed as the ratio of the value at each point during treatment to pretreatment value ‘Not significant.
activity was done using absolute values (pretreatment value subtracted from the value at each point during treatment), results were similar to those given above. A difference in frequency of IFN administration during the first 2 weeks affected 2SAS activity after 4 weeks of therapy, that is, it was significantly higher in patients who received IFN six times weekly than in patients who received IFN three times weekly.
4. Discussion Serum 2SAS activity is useful in evaluating the antiviral efficacy of IFN therapy. Previous studies demonstrated that early induction of 2.5AS activity depended on the dose of IFN [5,6]. In the present study, continuous administration of IFN during the early phase of treatment was preferable for inducing 2.5AS activity. Considering IFN therapy in patients with chronic hepatitis C, the genotypes of HCV-RNA and quantitative HCV-RNA levels just before treatment were probably closely associated with the response to IFN. It is, therefore, important to determine whether or not the genotype or the quantitative level of HCV-RNA may affect the induction of 2.5AS activity. In the present study, those viral factors were not associated with the induction of 2.5AS activity during IFN therapy. Based on these results, it is unlikely that differences in response to IFN among patients with different genotypes or quantitative levels of HCV-RNA depend on the 2.5AS pathway.
References 111Solinas A, Cossu P, Poddighe P, et al. Changes of serum 2’,5’-oligoadenylate synthetase activity during interferon treatment of chronic hepatitis C. Liver 1993;13:253-258.
121Okamoto H, Okada S, Sygiyama Y, et al. Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5’ noncoding region. Jpn J Exp Med 1990;60:215-222. [31Kato N, Yokosuka 0, Omata M, et al. The relationship between the amount of hepatitis C virus
362
M. Okuda et al. Ilnt. Hepatol. Commun. 2 (1994) 358-362
quantified by competitive RT-PCR (CRT-PCR) and the effect of interferon treatment. Acta Hepatol Jpn 1991;32:750-751 (in Japanese). [4] Okamoto H, Sugiyama Y, Okada S, et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. J Gen Virol 1992;73:673-679. [5] Sugawara T, Matsushima T, Karino Y, Miyazaki T, Toyoto J, Okuuti Y. Clinical significance of determination of serum 2’-Soligoadenylate synthetase activities. Acta Hepatol Jpn 1987;28:310-318 (in Japanese). [6] Hino K, Shimoda K, Fujioka S, et al. Clinical significance of serum 2’5’ oligoadenylate synthetase activity in various liver diseases. Igaku to Yakugaku 1989;22:153l-l 542 (in Japanese).