A pilot study of natural interferon γ therapy for chronic hepatitis C

International

ELSEVIER

Hepatology Communications 6 (1997) 264-273

A pilot study of natural interferon y therapy for chronic hepatitis C Michio Sataa,*, Hitoshi Nakano a, Tatsuya Ide a, Taiji Sato b, Norito Matsukuma b, Hiroshi Suzuki a, Kyuichi Tanikawa a a Second

Department b Social

of Medicine, Insurance

Kurume University School of Medicine, 67 Asahi-machi, Kurume 830, Japan Kurume Daiichi Hospital, Kurume, Japan

Received 16 October 1996; received in revised form 24 December 1996; accepted 9 January 1997

Abstract

Background: To assessthe effectiveness and side effects of interferon (1FN)y therapy for chronic hepatitis C. Materials and methods: A single dose of 1 x lo6 IU of natural IFNy was administered intramuscularly daily for 4 weeks to five patients with chronic active hepatitis C. Alanine aminotransferase levels, 2’-5’ oligoadenylate synthetase activity, & microglobulin levels, IFNy activity and HCV RNA levels were measured in sera. Results: ALT level, 2-5’ oligoadenylate synthetase (2-5AS) activity, j& microglobulin (BMG) level, and IFNy activity increased from l-2 weeks after the start of IFNy. However, HCV RNA levels did not decrease during IFNy administration. There were no serious adverse reactions. Conclusions: IFNy, which has attracted attention for its immunoenhancement, is worthy to be investigated as a therapy for chronic hepatitis C. However, the use of IFNy in combination with IFNor, p or other antiviral agents may be more rewarding because of the possibly weak antiviral action of IFNy. 0 1997 Elsevier Science Ireland Ltd. Keywords: Chronic hepatitis C; Interferony; Interferon therapy; Hepatitis C virus

*Corresponding

author. Tel.: + 81 942 317561; fax: + 81 942 342623

0928-4346/97/$17.00 0 1997 Elsevier Science Ireland Ltd. All rights reserved. PII

SO928-4346(97)00356-3

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1. Introduction

Since Hoofnagle et al. reported the effect of interferon (1FN)a in chronic non-A, non-B hepatitis [I], many chronic hepatitis patients have been treated with IFNc( or j’. After completing therapy 30-40% of patients have persistent normalization of serum alanine aminotransferase (ALT) levels and are negative for serum HCV RNA. The efficacy of IFNcl or p depends on the serum HCV RNA level, HCV genotype, extent of hepatic fibrosis and the method of IFN administration [2- 111. Based on the factors predicting such therapeutic effects, IFNc( or j3 has been combined with other drug regimens [12,13] in an attempt to achieve efficacy in patients with chronic hepatitis resistant to IFN therapy. However, the results to date have been unsatisfactory. The importance of cytotoxic T lymphocytes (CTL) and their role in the destruction and elimination of HCV-infected hepatocytes has being studied [14-181. Compared with IFNcl and p, IFNy features strong cellular immunoenhancement such as enhanced HLA class I expression and CTL activity [19-211. Thus, if IFN)’ is used for chronic hepatitis C, a new therapeutic effect might be expected even in patients resistant to IFNcr or ,/3. Therefore we administered natural IFNy (OH-6000) to five patients with chronic hepatitis C and monitored therapeutic effect, blood chemistries, immunological responses and changes in serum HCV RNA levels.

2. Materials

and methods

Informed consent for the study, approved by our institution’s Ethical Guidelines Committee, was obtained from all five patients (three men, two women, mean age 47.0 f 7.1 years). None of the patients had a history of blood transfusion or IFN therapy. Liver biopsies in all five patients, done within 6 months of starting IFNy, were consistent with chronic active hepatitis without cirrhosis (Table 1). The mean serum AST and ALT levels immediately before the start of therapy were 115.6 + 66.4 and 182.7 f 133.3 IUjl, respectively.

Table 1 Backgrounds

of the subjects

Pt No.

Age (sex)

Blood

1 2 3 4 5 Mean

-

k S.D.

36 (M) 49 03 50 (M) 45 (M) 55 (F) 47.0 k 7.1

CAH,

chronic

active

hepatitis

~ ~ ~

transfusion

Histological CAH CAH CAH CAH CAH

findings

ASTiALT

(W/l)

43189 106/171 2241417 93/101 112/157 115.6 k 66.41182.7

& 133.3

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2.1. IFNy udmhistration Natural IFN]’ (OH-6000, Otsuka Pharmaceutical) was obtained by stimulating human myelo-monocytes (HBL-38 cell line) with lipopolysaccharide (LPS). The five patients were given 1 x lo6 IU of natural IFNy intramuscularly daily for 4 weeks. 2.2. Blood examinations The following examinations of the blood were conducted before IFN administration and weekly after the start of administration: AST, ALT, total bilirubin, Al-P, y-GTP, ZTT, LDH, total protein, albumin, BUN, creatinine, uric acid, RBC, Hb, Ht, WBC with differential and platelets. Also urine was tested for protein, sugar and occult blood. The occurrence of adverse reactions and changes in body temperature was also monitored. 2.3. Immunological

examinations

IFNy activity, 2’-5’ oligoadenylate synthe;ase ulin (BMG) levels were measured, using serum 1, 2. and 3 weeks and at completion of IFNy measured by ELISA (Otsuka Pharmaceutical), by RIA (Eiken Chemical).

(2-5AS) activity, and p2 microglobobtained immediately before, after administration. IFNy activity was and both 2-5AS activity and BMG

2.4. Hepatitis virus markers Tests before treatment confirmed that Serum HCV RNA levels were determined method described by Kato et al. [22] obtained immediately before, after 1, 2 administration.

all five patients were negative for HBsAg. by competitive RT-PCR according to the using serum (cryopreserved at - 20°C) and 3 weeks and at completion of IFNy

2.5. Statistical analysis The Kruskal-Wallis test, the X2-test, and the Wilcoxon’s signed rank test were used for ordered data, while analysis of variance, or the paired t-test, was used for measured values. P < 0.05 was considered to be significant.

3. Results 3.1. Serum ALT levels The changes in serum ALT levels of the five patients are shown in Fig. 1. In three of the five, ALT increased after 1 week of IFNy, remained high during administra-

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(IU/llll, 600-

200-

100-

o,, -4

-2

],,,I,, 0 1 2 3 4

[, +2

+4

, +8

[, +12

, +16

,

, +20

,

( +24

(Weeks)

Fig. 1. Time-course changes in ALT 4 week daily dose group.

tion and decreased after treatment completion, but without a return to normal levels. The fourth patient showed an increase after 3 weeks, which peaked at 432 IUjl 4 weeks after treatment completion, but decreased to 178 III/l 8 weeks after completion. The fifth patient, in whom ALT levels were 147 II-J/l immediately before administration, showed a decrease after week 1, with a transient increase after 3 weeks and a subsequent decrease, which continued for 24 weeks after the treatment completion but did not return to normal levels. 3.2. 2-5AS activity, BMG level and IFNy activity 2-5AS activity, BMG level and IFNy activity were measured in four of five patients. 2-5AS activity increased from week 1 to week 2 in three of the four patients, but the peak values were different in each patient (Fig. 2). BMG levels increased in all four patients from week 1 and remained increased until IFNy completion and returned to the pre-treatment levels 2 weeks after completion (Fig. 3). IFNy activity increased in all four patients after week 1 and remained high during administration in three patients. After 2 weeks IFNy completion all values were below the measurement thresholds (Fig, 4). 3.3. HCV RNA levels As shown in Fig. 5, there were no significant decreases in serum HCV RNA levels during IFNy administration in all four patients.

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200180160140izo-

loo80604020O-

I I

I

I

0

1

2

I

3

I

I

4

+2

(Weeks)

Fig. 2. Time-course changes in 2-5A.S activity 4 week daily dose group.

3.4. Blood examinations WBC and neutrophil counts decreased significantly from week 1 of administration, but were at pre-treatment levels 4 weeks after treatment completion (Table 2). There were no significant changes in blood chemistries.

0

'

I 0

I 1

I 2

3

I 4

I

I +2

Fig. 3. Time-course changes in f&MCi 4 level week daily dose group.

(Weeks)

in

16.7+ 8.9

5

5

5

5

* P
Neutrophils (/ mm*) Platelets ( x 1Od/mmJ)

5

+ I 150.2 49.2 i 6.1

5

5

6260

4696f63

5

No.

No.

Mean + S.D.

I week

0 week

15.6+7.4

f 1357.6 30.2f4.1*

4160

466.4 + 43.2

Mean f S.D.

5

5

5

5

No.

177f73

* w4.4* 33.7 f 11.3

4220

480.8 f 50.6

Mean f S.D.

m five patients

2 week

peripheral blood findings due to IFNy admimstratm

RBC ( x 104/ IIld) WBC (/mm’)

Table 2 Changes

5

5

5

5

No.

3 week

15.1 * 7.4

31.8 i 10.5

3280 + 687*

471.2k61.5

MeanfS.D.

5

5

5

5

No.

13.4 f 8.1

i 1073.L?* 29.6 + 13.3

3540

464.0 f 69.7

Mean k SD.

Day after completion of admmistration

4

4

4

4

No

17.1+g3

I+.2174.7 43.2 f 12.8

4925

458.0 f 64.0

Mean f S.D.

2 weeks after completton

4

4

5

5

NO.

17.6k 8.0

39.0* 12.1

6460 f 1546.9

428.8 + 44.

Mean + SD.

4 weeks after completmn

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'

I 0

I 1

I 2

I 4

3

I

I +2

(Weeks)

Fig. 4. Time-course changes in IFN y activity 4 week daily dose group.

3.5. Body temperature and adverse reactions

From the first day, and throughout fevers exceeding 37-39°C.

IFN administration,

all five patients had

4. Discussion Strategies to improve the sustained response of IFNa or /?, such as increasing the dose, prolonging treatment or combining IFNcl with other drugs, have been attempted [9- 131. However, to date there are no reports of an outstanding therapeutic effect against high HCV RNA levels and progression of hepatic fibrosis. We also investigated whether sequential IFNa and p treatment induces a more sustained response. While sequential IFNlr and /3 treatment had a transient beneficial effect in just over half of our patients, we concluded that there was no statistically significant difference in the sustained response rate as compared with previous reports [23]. Bisceglie et al. [24] and Carrend et al. [25] attempted sequential IFNcl and y treatment for chronic hepatitis B, but reported that the addition of IFNy did not increase the antiviral effect or bring the disappearance of hepatitis B e antigen. In a previous study, we administered single daily doses of natural IFNy (0.5 x lo6 or 1 x lo6 IU, IM) to 15 patients with chronic hepatitis B for 4 weeks and investigated the antiviral effect against HBV and changes in liver function [26]. ALT and BMG, the domain of HLA Class I [27], increased simultaneously from week 1 of administration [26] suggesting that IFNy induces enhanced CTL activity and

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enhanced expression of HLA Class I. The action of CTL recognizing HCV antigen epitopes is important in eliminating HCV-infected hepatocytes. If the activity of CTL recognizing this kind of HCV is augmented [14- 181, it may be possible to eradicate persistent HCV infections. From these observation and our hypothesis, we employed IFNy to patients with chronic hepatitis C. We noted an increase in ALT and BMG from week 1 of administration accompanied by an increase in IFNy activity. While this may indicate that CTL activity was enhanced by IFN?, there were no significant decreases of HCV RNA observed during IFNy administration. On the other hand, in our previous study of IFNy therapy for chronic hepatitis B, serum HBV-related DNA polymerase disappeared in five of 13 patients at treatment completion [26]. These findings suggest that the main mechanism of eliminating hepatitis virus-infected hepatocytes due to the host immune reaction is different between chronic hepatitis C and chronic hepatitis B. Since IFNy caused no serious adverse reactions in the present study, it should be considered an alternative to enhancing the therapeutic effect in patients with chronic hepatitis C resistant to IFNa and p therapy. However, since the antiviral effect of IFNy is expected to be weaker than of IFNcr and p, it is important to consider combination therapy with IFNcl or other antiviral agents and to investigate the optimum IFNy dosage. Furthermore, Toyonaga et al. [28] reported that transgenic mice carrying the mouse IFNy gene linked to the promoter of serum amyloid P component gene showed a histological finding of chronic active hepatitis similar to that found in chronic hepatitis patients. This result suggests that administration of IFNy to patients with chronic hepatitis may promote progression or aggravation of the disease. Therefore, further investigation appears to be log,,~HCV-ANA] 81

0

1

2

Fig. 5. Changes in serum HCV RNA levels during IFNy administration: at completion, after 4 weeks.

(Weeks)

pre-dosing, 1 week, 2 weeks,

necessary to select the optimum timing of administration, and subjects for therapy with IFNy.

duration

of treatment,

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[21] Ramadori G, Mitsch A, Pieder H, et al. Alpha-and gamma interferon (IFNr. IFNy) but not interleukin I (IL-l) modulate synthesis and secretion of a,-microglobulin by hepatocytes. Eur J Clin Invest 1988; 18: 3433351. [22] Kato N, Yokosuka 0, Hosoda K, et al. Quantification of hepatitis C virus by competitive reverse transcription-polymerase chain reaction: increase of the virus advanced liver disease. Hepatology 1993: 18: 16-20. [23] Miyajima I, !$ta M, Ide 1, et al. Sequential interferon-r and p treatment in patients with chronic hepatitis C. J Jpn A Infect Dis 1996; 70: 690-695. [24] Bisceglie AMD, Rustgi VK, Kassianides C, et al. Therapy of chronic hepatitis B with recombinant human alpha and gamma interferon, Hepatology 1990; 1 I: 2666270. [25] Carrend V. Moreno A, Galiana F, et al. z- and y-interferon versus a interferon alone in chronic hepatitis B. A randomized controlled study. J Hepatol 1993; 17: 321-325. [26] Sata M, Matsushima T, Kakumu S, et al. Clinical evaluation of intramuscular administration of natural interferon-y in the treatment of chronic hepatitis B. Kurume Med J 1995; 42: 9-20. [27] Peterson PA, Rask L. Lindblom JB: Highly purified papain-solubilized HLA-antigens contain µglobulin. Proc Natl Acad Sci USA 1974; 1: 35-39. [28] Toyonaga T, Hino 0, Sugai S, et al. Chronic active hepatitis in transgenic mice expressing interferon-;. in the liver. Proc Natl Acad Sci USA 1994; 91: 614-618.