Randomized, controlled trial of natural interferon-α therapy for e-antigen-positive chronic hepatitis B patients

Hepatology Research 23 (2002) 98 – 104

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Randomized, controlled trial of natural interferon-a therapy for e-antigen-positive chronic hepatitis B patients Yasuji Arase a,*, Akihito Tsubota a, Satoshi Saitoh a, Yoshiyuki Suzuki a, Masahiro Kobayashi a, Fumitaka Suzuki a, Takashi Someya a, Norio Akuta a, Kenji Ikeda a, Mariko Kobayashi b, Hiromitsu Kumada a a

Department of Gastroenterology, Toranomon Hospital, 2 -2 -2 Toranomon, Minato-ku, Tokyo 105, Japan b Hepatic Research Unit, Toranomon Hospital, 2 -2 -2 Toranomon, Minato-ku, Tokyo, Japan Received 11 June 2001; received in revised form 25 September 2001; accepted 5 November 2001

Abstract We evaluated the efficacy of long-term interferon (IFN) therapy in patients with e-antigen-positive chronic hepatitis B. The study design was a prospective, randomized controlled clinical trial. Fifty-three patients were randomly assigned into one of two groups, treated with 3 million units (MU) of IFN (low dose group; n= 27) or 6 MU IFN (high dose group; n=26), administered twice weekly for 52 weeks. Responders were defined as patients whose hepatitis B virus (HBV)-DNA level, determined by branched DNA signal amplification and hepatitis B e-antigen (enzyme immunoassay) were negative and whose serum alanine transaminase (ALT) levels fell to within the normal range (ALT B 50 IU/l) at 6 month after termination of IFN therapy. One patient in high dose group was dropped out because of transfer. Remainder 52 patients were examined by intention-to-treat (ITT) analysis. The response rates by ITT analysis were 40.7% (11/27) in low dose group and 20% (5/25) in high dose group. The difference between low and high dose group was not statistically significant. Univariate analysis of clinical factors that contribute to the response demonstrated that IFN therapy had a significant effect when, (1) the serum HBV-DNA level was B 200 Meq/ml prior to the commencement of IFN therapy (P= 0.0327). (2) Transient acute exacerbation of ALT was present during or after IFN therapy (P =0.0311). Multivariate analysis showed that the risk ratio for the development of response in patients with serum HBV-DNA level of less than 200 Meq/ml was 3.60 compared with patients with that of 200 Meq/ml or more than 200 Meq/ml (95% CI, 1.012– 12.81). In conclusion, the results of this trial show that: (1) long-term twice weekly IFN therapy could be a worthwhile strategy for e-antigen-positive chronic hepatitis B patients with serum HBV-DNA level of less than 200 Meq/ml and (2) patients with transient acute exacerbation of ALT during or after IFN therapy could often respond well after exacerbation of ALT. © 2002 Elsevier Science B.V. All rights reserved. Keywords: e-Antigen-positive chronic hepatitis B; Interferon; Hepatitis B virus-DNA

* Corresponding author. Tel.: + 81-3-3588-1111; fax: + 813-3582-7068. 1386-6346/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 0 1 ) 0 0 1 6 9 - 3

Y. Arase et al. / Hepatology Research 23 (2002) 98–104

1. Introduction Chronic hepatitis B is a serious liver disease with significant mortality and morbidity. However, in patients with seroconversion of hepatitis B e-antigen (HBeAg) to hepatitis B e-antibody (HBeAb) and clearance of hepatitis B virus (HBV) DNA, the prognosis of the disease is generally good [1– 4]. Interferon (IFN) therapy suppresses chronic HBV infection [5– 9] but sustained responses to IFN have proven difficult to attain. Japanese patients treated with 6 million units (MU) IFN daily for 4 weeks exhibited a sustained response rate of only approximately 15– 25%, 1 year after IFN therapy [10– 12]. Other patients remain HBeAg-positive and continue to express abnormal serum alanine transaminase (ALT) levels after completing IFN therapy. Recently, prolonged IFN therapy was reported to increase the response rate of chronic hepatitis B [13]. However, we reported that chronic hepatitis C patients treated with 9 MU of IFN three times weekly for 24 weeks dropped out in high frequent mainly due to adverse effects of IFN [14]. On the other hand, adverse reactions attributed IFN therapy, given 3 MU of IFN twice per week were not severe in previous study [15]. We assessed therefore, the efficacy of intermittent long-term treatment with 3 and 6 MU of IFN for patients with e-antigen-positive chronic active hepatitis and examined the independent factors that might have influenced the response to IFN therapy.

2. Materials and methods

2.1. Patients Fifty-three Japanese patients were enrolled in this trial from April 1995 to March 1997. The patients were randomly assigned to one of two dose groups to receive, natural IFN-a, using randomization sequences (sealed envelope) prepared by Sumitomo Pharmaceutical Co. (Tokyo, Japan), who kindly provided the drug for the study. The selection criteria for patients included in the study were: (1) average ALT increase greater than the upper normal limits (ALTB 50

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IU/l) for longer than 6 months, (2) liver biopsy (taken within 6 months before IFN administration) showing histological features of chronic active hepatitis, (3) positive hepatitis B surface antigen (HBsAg), HBeAg and HBV-DNA (b DNA probe signal amplification technology) for more than 6 months, (4) no corticosteroids, immunosuppressant or antiviral agents use within one year, (5) no serum hepatitis C virus antibodies detectable by enzyme immunoassay (EIA) or radioimmunoassay, and (6) no anitinuclear antibodies or antimitochondrial antibodies detectable in the serum by immunofluorescence on rat liver and kidney.

2.2. Drug assignment IFN-a was administered by intramuscular injection twice week for 52 weeks. The total amount of IFN administered was 312 MU in low dose group (3 MU per dose) and 624 MU in high dose group (6 MU per dose). This study was approved by the institutional review board of our hospital. The physicians in change explained the purpose and method of this clinical trial, as well as potential adverse reactions, to each patient, and informed consent for participation was obtained from all subjects.

2.3. Blood tests Routine biochemical and hematological tests were made at weekly to monthly intervals during treatment with a follow-up more than 6 months after the termination of IFN treatment. The remaining serum samples were divided and stored at − 80 °C until use. HBsAg and HBeAg/HBeAb were determined by EIA (Abbott Diagnostics, Chicago, IL). HBV-DNA was measured by branched DNA signal amplification technology (Chiron Corp, Emeryville, CA), and the results were expressed as 106 genomic equivalents (Meq) per milliliter. The lower limit of the assay was 0.7 Meq/ml. Genotyping of HBV was performed by an ELISA using monoclonal antibodies for the genotype specific epitopes in the pre S2 region product [16]. Anti-hepatitis C virus antibody was detected by third generation enzyme-linked im-

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Y. Arase et al. / Hepatology Research 23 (2002) 98–104

munoassay (Ortho Diagnostics Japan, Tokyo, Japan).

2.4. Definition of response to IFN therapy Responders were defined as patients whose HBV-DNA level by branched DNA signal amplification and HBeAg (EIA) were negative and whose serum ALT levels fell to within the normal range at 6 month after termination of IFN therapy. Response patterns following IFN administration were classified into three types. Type 1 was characterized by transient acute exacerbation during (Ia) or after (Ib) IFN therapy. Type 2 involved in a slight reelevation of ALT after the termination of had ALT normalization during IFN therapy and sustained normalization after the termination of treatment. Type 3 had ALT normalization during IFN therapy and sustained normalization after the termination of treatment.

2.5. Li6er histology The liver histology of chronic hepatitis before IFN therapy was classified according to the extent of fibrosis, into three stages: Stage 1, periportal expansion; Stage 2, portoportal septa; and Stage 3, portocentral linkage or bridging fibrosis [17]. Patients with Stage 0 or Stage 4 were excluded from the diagnosis of chronic hepatitis.

2.6. Statistical analysis The response rates were calculated by the intention-to-treat (ITT) analysis. The  2 test or Fisher’s exact test was used for comparison of group frequencies as appropriate. A P value of B 0.05 by the two-tailed test was considered to indicate a significant difference. Independent factors that might have influenced the response to IFN were studied using multiple logistic regression analysis, and the following variable were evaluated as prognostic factors for the response: sex, age, liver histology, HBV-genotype, serum HBV-DNA level, ALT, the presence or absence of transient acute exacerbation during or after IFN therapy and method of IFN treatment. Transient acute exacerbation was defined as follows: (1) an in-

crease of more than 150 IU/l in serum ALT level in 1 month during or within 3 months after IFN therapy and (2) a decrease of more than 150 IU/l in serum ALT level in 1 month after a peak of ALT. Data analysis was performed using SAS software [18].

3. Results

3.1. Clinical background Fifty-three patients were randomly assigned into two groups to receive 3 or 6 MU of natural IFN-a. One patient in high dose group dropped out 4 months after commencement of IFN therapy because of transfer. This patient was excluded from the analysis because of lack of clinical data. Next, two patients in low dose group and one patient in high dose group dropped out at 3, 4 and 6 months after commencement of IFN therapy, respectively, because of ALT elevation to more than 500 IU/l. One patient in high dose group dropped out 5 months after starting IFN due to depression. In remaining 48 patients, discontinuing of IFN due to adverse reactions was not necessary. Fever (37–40 °C) was observed when IFN was first administered to patients, but this was only mild (up to 38 °C) after the third or fourth dose. Further, IFN therapy did not reduce leukocyte or the platelet count, which were 582092020 and 171 000957 000/ml, respectively, 1 month after initiation of therapy. Other signs and symptoms, such as proteinuria, neurological and psychiatric symptoms, were not observed. Four patients dropped out because of ALT elevation or depression were evaluated by the ITT analysis. In ITT analysis, 52 patients, except one patient who was transferred, were assessed. The background characteristics and baseline measurements for each group are summarized in Table 1. There were no significant differences between the two groups in sex, age, serum ALT, platelet count, HBV-DNA levels, HBV-genotype and the histopathological diagnosis of biopsied liver specimens prior to IFN therapy.

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3.2. Efficacy of IFN therapy

3.3. Representati6e case reports in responders

The response rate was 40.7% (11/27) in low dose group and 20% (5/25) in high dose group by the ITT analysis. Based on per protocol, the response rate was 36% (9/25) in low dose group and 21.7% (5/23) in high dose group. The difference between low dose group and high dose group was not statistically significant. Univariate analysis of clinical factors that contribute to the response was performed (Table 2). This analysis demonstrated that IFN therapy had a significant effect when, (1) the serum HBV-DNA level was B 200 Meq/ml prior to the commencement of IFN therapy (P=0.0327) and (2) transient acute exacerbation of ALT was present during or after IFN therapy (P = 0.0311). The response rate was assessed in relation to the serum HBV-DNA level and transient acute exacerbation of ALT (Fig. 1). Of the cases with serum HBV-DNA level of 200 Meq/ml or more than 200 Meq/ml before IFN therapy, the response rate were 71.4% (5/7) in patients with acute exacerbation of ALT and 8% (2/25) in patients without acute exacerbation of ALT. Multivariate analysis showed that the serum HBV-DNA level was the most important factor for attaining a response outcome. Thus, the risk ratio for the development of response in patients with serum HBV-DNA level of less than 200 Meq/ml was 3.60 compared with patients with that of 200 Meq/ml or more than 200 Meq/ml (95% CI, 1.012–12.81).

Typical examples patients displaying response are shown in Figs. 2 and 3. Type 1a patient was a 40-year-old male. After the administration of IFN, ALT levels displayed transient normalization, but rose again to 742 IU/l 8 month after the initiation of IFN. After stop of IFN therapy, the ALT level reverted to normal again with disappearance of HBeAg (EIA) and HBV-DNA (b DNA probe). Type 1b patient was a 50-year-old male. Following the administration of IFN, ALT levels displayed transient normalization and then rose again to 1500 IU/l one month after the end of treatment. Thereafter, the level of ALT reverted to normal again. HBeAg and HBV-DNA became also negative and remained negative 12 months after completion of the treatment. Type 2 patient was a 36-year-old male. During IFN therapy, ALT levels displayed transient normalization but HBeAg and HBV-DNA were positive. ALT levels rose again to 180 IU/l one month after the termination of IFN. Thereafter, the level of ALT reverted to normal and HBeAg and HBVDNA became negative. Type 3 patient was a 34-year-old male. His ALT level reverted to normal, also HBeAg and HBV-DNA become negative with IFN administration, and subsequently ALT remained normal even after the termination of the administration. HBeAg and HBV-DNA were also found to be negative more than 12 months after the completion of the treatment. The total 16 response patients comprised 5 with type 1a, 2 with type 1b, 6 with type 2 and 3 with type 3.

Table 1 Characteristics of patients before IFN treatment Patient characteristics

Low dose group (3 MU) (n = 27)

high dose group (6 MU) (n =25)

P value

Sex (M/F) Agea ALT (IU/l)a Platelet (×104/ml)a HBV-DNA (b DNA probe Meq/ml)a HBV-genotype (b/c) Liver histology stage (F1/F2/F3)

20/7 34 (20–54) 131 (38–760) 17.3 (9.8–30.2) 680 (1.1 to \3800) 2/25 8/15/4

18/7 38 (21–61) 134(31–685) 18.6(11.6–32.5) 589 (B0.7 to \3800) 3/22 7/15/3

0.686 0.283 0.903 0.779 0.776 0.605 0.828

a

Data are expressed as the median value (range).

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Table 2 Analysis for the predictors of complete response after IFN treatment Factor

Efficacy of IFN treatment

P

CR

NR

16 11/5

36 27/9

Age (year) (B35/]35)

34 (21–50) 9/7

37 (20–61) 16/20

0.551

Liver histology stage (1/2 or 3) Genotype (B/C)

3/13 4/12

12/24 1/35

0.340 0.172

HBV-DNA (Meq/ml)a (B200/]200)

150 (B0.7 to 3300) 9/7

860 (6 to \3800) 11/25

0.0327

ALT (IU/L)a (B150/]150)

125 (38–760) 11/5

143 (1–668) 18/18

0.242

Transient acute exacerbation (presence +/absence −) Method of IFN treatment (low dose/high dose)

7/9 11/5

5/31 16/20

0.0311 0.138

No. of cases Sex (M/F) a

a

0.738

Data are expressed as the median value (range).

4. Discussion Following the report of Hoofnagle and Di Bisceglie [7], many authors have confirmed the therapeutic effect of IFN for HBeAg-positive chronic hepatitis B [4–9]. However, in many series the clearance rate of HBeAg after daily treatment with IFN for 4 weeks was only 20– 30%. Therefore, many patients remain HBeAg-positive or continue to express abnormal serum ALT levels after such treatment regimen. In patients with abnormal ALT levels and high levels of serum HBV-DNA after IFN therapy, the rate of progression of liver cirrhosis may be high [19]. The HBeAg seroconversion rate is 30– 40% with larger IFN doses administered for 4– 6 months. However, patients treated with larger IFN doses often drop out of treatment studies due to IFN-related side effects and a high-dose IFN is also very expensive. Therefore, IFN therapy for HBeAgpositive chronic hepatitis B entails problems with efficacy, cost and side effects. Low-dose IFN therapy with high efficacy and few side effects would thus be a desirable strategy. In a previous study, we examined the efficacy of IFN in a prospective, randomized, double-blind controlled trial in 36 patients with e-antigen-negative chronic hepatitis

B who repeatedly demonstrated abnormal fluctuation in ALT levels [15]. These 36 patients were randomly assigned to three groups, who were treated with 0.3, 1 or 3 MU, administered twice weekly for 24 weeks. IFN therapy did not reduce leukocyte or platelet counts and adverse reactions were very mild in patients treated with 3 MU IFN. Therefore, in this study, we assessed the efficacy of low-dose, long-term IFN therapy in e-antigen-positive chronic hepatitis B infection in a randomized controlled trial. We evaluated the

Fig. 1. Complete response rate based on the serum HBV-DNA level before IFN therapy and transient acute exacerbation after initiation of IFN.

Y. Arase et al. / Hepatology Research 23 (2002) 98–104

Fig. 2. Clinical courses of response cases displayed transient acute exacerbation during or after IFN therapy.

factors that contribute to the efficacy of such an IFN therapy regimen for HBeAg (EIA)-positive, HBV-DNA (bDNA probe)-positive chronic hepatitis patients with repeated abnormal fluctuations in ALT levels. Our results showed that the response after IFN therapy in low dose group (3 MU IFN) were statistically similar to that in high dose group (6 MU). Overall, we did not find that the efficacy of low-dose (3 MU) IFN was not inferior to that of high-dose (6 MU) IFN. In addition, the response rate to IFN was significantly higher in patients with a low level of serum HBV-DNA ( B 200 Meq/ml). Taken together, these results suggest that low-dose, long-term IFN therapy may be a

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worthwhile strategy in patients with levels of HBV-DNA of less than 200 Meq/ml. Next, we found that the response rate was 71% if transient acute exacerbation occurred in patients with levels of HBV-DNA of more than 200 Meq/ml. In contrast, the response rate was 8% if transient acute exacerbation did not occurred in patients with levels of HBV-DNA of more than 200 Meq/ ml. With respect to the HBV-genotype, 80% (4/5) patients with HBV-genotype b showed a response to low-dose IFN. Multivariate regression analysis in our study demonstrated that HBV-genotype was not a determinant of the response to therapy. However, Kao et al. [20] reported that HBV genotype C, compared to genotype B, is associated with a lower response rate to IFN-a therapy. Next, ALT normalization patterns in responders were classified into three patterns. In type 3, inhibition of the HB virus occurs after the initiation of IFN and this kind of viral inhibition continues after the termination of treatment. On the other hand, the serum HBV-DNA level often increase transiently prior to elevation of ALT in type 1 or 2. That is, in type 1 or 2 after HB virus replication and elevation of ALT a response occurs. These findings suggest that intermittent IFN therapy might be related to an immunologic response such as steroid withdrawal therapy. In conclusion, the results of this trial show that: (1) long-term twice weekly IFN therapy could be a worthwhile strategy for e-antigen- positive chronic hepatitis B patients with serum HBVDNA level of less than 200 Meq/ml; and (2) Patients with transient acute exacerbation of ALT during or after IFN therapy could respond well after exacerbation of ALT.

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Fig. 3. Clinical courses of response cases displayed no transient acute exacerbation during or after IFN therapy.

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