Recombinant Leukocyte A Interferon Treatment in Patients With Chronic Hepatitis B Virus Infection

GASTROENTEROLOGY 1985;88:870-80

Recombinant Leukocyte A Interferon Treatment in Patients With Chronic Hepatitis B Virus Infection Pharmacokinetics, Tolerance, and Biologic Effects MASAO OMATA, FUMIO IMAZEKI, OSAMU YOKOSUKA, YOSHIMI ITO, KATSUO UCHIUMI, JUNKO MORI, and KUNIO OKUDA

First Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan

Large doses of recombinant leukocyte A interferon were administered to 20 patients with deoxyribonucleic acid polymerase- and hepatitis B e antigenpositive chronic hepatitis B to study the maximum tolerated dose, its pharmacokinetics, and its antiviral activity. The first group of 5 patients received a constant dose of 36 x 10 6 Ulday for 28 consecutive days. When it was well tolerated, the second, third, and fourth groups (5 patients each) received 50, 72, and 100 x 106 Ulday, respectively . All 20 patients completed the 28-day treatment. Hourly and daily profile of serum interferon level showed a dosedependent effect with an increasing dosage, and cumulative effects during the treatment. The mean peak serum interferon concentration ranged from 93 Ulml on day 1 in the first group to 1271 Ulml on day 28 in the fourth group. Inhibition of serum deoxyribonucleic acid polymerase activity and hepatitis B virus-deoxyribonucleic acid during the. treatment was compared between the groups with low doses (36 and 50 x 10 6 U) and high doses (72 and 100 x Received May 10, 1984. Accepted October 18, 1984. Address requests for reprints to: Masao Ornata, M.D., First Department of Medicine, Chiba University School of Medicine, Chiba, Japan 280. This work was supported in part by Japanese Ministry of Education Grant (B) 58480215. a grant from the Viral Hepatitis Research Foundation of Japan, and a grant from the Japan Medical Research Foundation. The authors thank Dr. Leon L. Bernhardt, Dr. Zofia Dziewanowska (Hoffmann-La Roche, Nutley, N.J.)' and Dr. Kouichi Takano (Nippon Roche, Japan) for support of this study. They also thank Dr. ShudoYamazaki (National Institute of Health, Japan) for .helpful suggestions and Mr. Hiroaki Ishiko for excellent technical assistance. © 1985 by the American Gastroenterological Association 0016-5085/85/$3 .30

10 6 U). Low doses of interferon suppressed deoxyribonucleic acid polymerase activity to the same extent as did the high doses. Prednisolone withdrawal was combined with interferon in 5 patients. Three patients treated with such combination became seronegative for hepatitis B e antigen during the treatment, whereas all 15 with interferon alone remained seropositive. These results suggest that a maximum antiviral effect of recombinant leukocyte A interferon is below the maximum tolerated doses . Interferon, a naturally occurring protein discovered in 1957, has potent antiviral, anti proliferative , and immunomodulating properties. Progress in defining the clinical role of interferon has been hampered by limited availability of species-specific human interferon. Leukocyte interferon of - 1% purity produced from human buffy-coat cells has shown certain clinical activity against human viral diseases. Recently, biologic synthesis of several species of leukocyte interferon in Escherichia coli by recombinant deoxyribonucleic acid (DNA) techniques has been achieved (1,2). Recombinant leukocyte A interferon (rIFN-aA), the first of a series of biosynthetic interferons, has been shown to have biologic activities in preclinical (2) and clinical trials in cancer patients (3) .

Hepatitis B virus (HBV) is a partially double stranded DNA virus, and its persistent infection in the liver is associated with a variety of liver diseases Abbreviations used in this paper: DNA-P, deoxyribonucl eic acid polymerase; m-IgG, mouse · immunoglobulin G; rIFN-aA, recombinant leukocyte A interferon; SDS, sodium dodecyl sulfate.

April 1985

including hepatocellular carcinoma. Conventional interferon from human buffy-coat cells reduced HBV-associated DNA polymerase (DNA-P) in blood during treatment (4). Chronic hepatitis B is a particularly good model to study the antiviral effect of interferon, as infected persons normally have stable levels of various viral marker~ in both blood and liver, and any measurable decline would indicate reduced infectivity and virus replication. We present the results of a clinical study in patients positive for hepatitis B "e" antigen (HBeAg) and DNA-P activity who were treated with a recombinant interferon and report on the pharmacokinetics, tolerance, biologic activity of rIFN-aA, and its antiviral effects.

Materials and Methods Recombinant Leukocyte Interferon Isolation, expression, and purification cif rIFN-aA were performed at Hoffmaim-La Roche (Nutley, N.J.) and Genetech, as have been described (2,5). Purification _of rIFN-aA was done using a specific monoclonal antibody column to leukocyte A interferon (6). This purified protein was homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel. The material was >95% pure, arid the specific activity was 2-4 x 10 8 U/mg of protein when tested on human fibroblasts or bovine kidney cell lines (6). Patients having chronic hepatitis B who had been positive for hepatitis B surface antigen for 1 yr and in whom DNA-P activity had been stable for 3 mo were treated. Criteria Jor entrance into the study included preserved functions of the kidney (serum creatinine :51.4 mg/dl), liver (normal albumin levels and prothrombin time :51.3 x control value), and blood (leukocyte count ~4000/mm3, granulocytes 2: 1500/mm3 , and platelets ~50,000/mm3). The types of patients and diseases excluded from this study were symptomatic ischemic heart diseases, chronic obstructive lung diseases, kidney diseases, blood diseases, active neurologic diseases, and pregnant or lactating women. Five patients were entered stepwise in each of the four dosage groups. The first group received a constant dose of 36 x 10 6 Ulday of rIFN-aA administered intramuscularly in two divided doses for 28 consecutive days, ~nd was evaluated for tolerance before increasing the dosage to 50 x 10 6 Ulday for 28 days in the second group of 5 patients. The third group was treated with 72 x 10 6 Ulday, and the fourth with 100 x 10 6 Ulday. Patients receiving the following drugs for the indicated number of days before study were excluded: adenine arabinoside (30 days), barbiturates (7 days), cardioactive agents (2 days), theophylline (2 days), aspirin (2 days), aminoglycoside aritibiotics (2 days), and nonsteroidal antiinflammatory agents (2 days). In 5 patients, 40 mg of prednisolone was administered orally for 4 wk from 1 to 4 mo before interferon administration. One of the objectives of the study was to determine the maximum tolerated dose in the dose escalation schedule.-

rIFN-aA AND HEPATITIS B VIRUS INFECTION

871

If any adverse effect occurred in blood (leukocyte count :51000/mm 3, granulocytes :5500/mm3 , and platelets :520,000/mm3 ), liver function (albumin level ~75% reduction and prothrombin time ~2_5 x control), and renal function (serum creatinine ~3.5 mg/dl), interferon administration was stopped and the patient was observed until the abnormalities in question returned to baseline. In addition, if any other adverse reactions occurred that were judged clinically significant, interferon administration could be stopped. The maximum tolerated dose was defined as the dose in which 3 of 5 patients experienced aforementioned toxicities. If 2 patients in one dose group experienced these toxicities, as many as 5 additional patients could be entered at that dose until a third patient experienced these toxicities. For safety, vital signs such as heart rate, body temperature,blood pressure, and respiration were measured four times per day ori days 1 through 14, and before each injection on days 15 through 28. A complete physical examination, inchiding weight measurement, was performed before the first interferon injection on day 1, again on day 14, and within 1 wk after the last injection. An electrocardiogram and chest x-ray were taken before the study and after the final dose.

Laboratory Studies For safety, a panel of tests which encompassed complete blood count including differential, reticulocyte, and platelet counts, serum alkaline phosphatase, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, total serUm bilirubin, blood glucose, blood urea nitrogen, total protein, uric acid, serum albumin, lactate dehydrogemise, creatinine, urinalysis, and prothrombin time were rUn at baseline (just before the first injection) and on days 7,14, and 28, and at the end of the study. In the event that significant changes occurred iIi laboratory parameters, the investigators should drop that patient from the study with replacement.

Viral Markers _The following HBV-associated markers were measured at baseline, on days 3, 7, 11, 14, 21, 28,42, and 56. Endogenous deoxyribonucleic acid polymerase activity. Serum DNA polymerase activity was measured by the method of Robinson (7). Serum (200 ~l) was diluted to 1.0 ml in TN buffer (0.01 M Tris-HCl, pH 7.4, and 0.15 M NaCl) and centrifuged at 10,000 g for 10 min. The supernatant was laid over 2.5 ml of 30% (wt/vol) sucrose containing TN, b.OOl M ethylenediaminetetraacetic acid (EDTA), O.llljo 2-mercaptoethanol, and 1 mg/ml bovine serUm albumin and centrifuged for 4 h at 50,000 rpm and 4°C in a Spineo SW 5b rotor. The pellet was resuspended in 50 ~l of TN containing i % Nonidet P-40 and 0; 1 % 2-inercaptoethanol,and then 25 ~l of assay mixture (0.2.M Tris-HCI, pH 7.4, 0.08 M MgCI 2 , 0.24-M NH4 Cl, 1.0 mM dATP, 1.0 mM TTP, 0.025 nM of 3H-dCTP and 3H-dGTP each, both 21 Ci/mM) was added. The mixture was incubated at 37°C for 3 hand 50 ~l was then spotted on a Whatman 3 MM paper disk (Whatman Laboratory Products, Clifton, N.J.),

872

GASTROENTEROLOGY Vol. 88 , No.4

OMATA ET AL.

washed, and assayed for acid-precipitable 3H by liquid scintillation counter. In addition to the aforementioned method of DNA-P activity measuremept, we analyzed the reaction product by slab gel electrophoresis and autoradiography as described by Mason et al. (8), to ascertain the specificity of HBV-speCific DNA-P reaction . In short, 100 ILl of serum was layered onto 5 ml of 10%-20% (wt/vol) suc~ose gradient, and centrihiged for 3 h at 48,000 rpm. The pellet was suspended iI1 10 pI of assay mixture, cbnsisting bf 0.15 M NaCl , 0.05 M Tris-HCl (pH 8.0), 0.01 M MgCI 2, 1 mM dithiothreitol, 0.1% (vol/vol) Triton XI0G; 0.1 mM TTP, 0.1 mM dGTP, 0.1 mM dATP, and 1 ILM 32p_dCTP (-800 Ci/mM). AfteJ; a 2-h incubation at 37°C, the reaction was terminated by the addition of 15 ILl of a mixture Of 10 mM EDTA, 10 mM Tris-HCl (pH 7.4), 0.5 mg/ml of pronase, 0.1% (wtlvol) SDS. The mixture was then analyzed by electrophoresis on a horizontal i.5% slab gel. The presence of HBV-specific DNA-P activity in a serum sample was indicated by the radiolabeling of a DNA species of -3200 base pairs. Serum, hepatitis B virus deoxyribonucl~ic acid. Senirri (10 pI) was spotted directly on nitrocellulose filter paper (0.45 ILm pore) and air-dried. The nitrocellulose was submerged first in 0.1 M NaOH and 1.5 M NaCl for:3 min, then in 0.1 M Tris-HCl (pH 7.4) and 1.5 M NaCI for 3 min, and baked at 80°C under vacuum for 2 h. The filter was hybridized with [a- 32 PJ-dCTP labeled HBV-DNA isolated from an EcoRi digestion of the viral genome cloned in the plasmid vector, pBR 322. The filter was washed three times at room temperature with 0.1% SDS in 2 x SSC, for a totai of 20 min, and washed three flme~ at 56°C with 0.1 % SDS in 0.1 x SSC (20 min per w~sh), dried, and autographed. Serum hepatitis B surface antigen and hepatitis B e antigen. Serum HBeAg and antibody to HBeAg (anti-HBe) were assayed by a solid-phase enzyme-linked immunoassay (Abbott Laboratories, North Chicago, Ill.). Hepatitis B surface antigen was measured by reversed passive hemagglutination and enzyme immunoassay (Abbott Laboratories) .

Pharmacokinetics of Recombinant Leukocyte A InteIjeron Serum samples were collected at 0,1,2,3,4,6,8, 12, and 24 h after the first injection in the morning on days 1, 14, and 28 and once on day 42. Serum interferon titer was measured by a bioassay using FL cells as targets and Sindbis virus (.9). The development of antibodies to rlFNCiA was tested using a modification of the bioassay in which neutralitation of the antiviral effect of interferoncontaining samples was measured (10).

Table 1. Clinical Features of 20 Patients With Chronic Hepatitis B Treated With Recombinant Leukocyte A Interferon Administered Twice a Day for 28 Days Daily dose

Histologic diagnosis

Patient No.

Age (yr)

Sex

1 :1

41 24 30 31 24

M M M M M

CAH CPH CPH CPH CAH

No No Yes No No

9 10

36 28 37 47 24

M M M M M

CAH CPH Cirrhosis CAH CPH

No No No No No

11 12 13 14 15

33 22 33 26 26

M M M M

F

CAH CAH CRH CPH CPH

No No No Yes No

16 17 18 19 20

32 44 26 25 Hi

M M M M M

Cirrhosis CPH CPH CAH CPH

Yes No No Yes Yes

Group 1 36 X 10 6 U

3

4 5

Group 2 50 x 10" U

6 7 8

Group 3 72 x iO" U

Group 4 iOox10" U

Prior prednisolone

CAH, chronic active hepatitis; CPH, chronic persistent hepatitis.

Results Table 1 shows the clinical features of the 20 patients in this study, including their dosage of rIFNerA, age, sex, and diagnosis. The patients, 19 men and 1 woman, ranged in age from 19 to 47 yr with a mean of 30.4 yr. Histologically, 11 patients had chronic persistent hepatitis, 7 patients had chronic active hepatitis, and 2 patierits had cirrhosis. Maximum Tolerated Dose In this dose-escalation schedule, the first 5 patiehts were started on 36 miliion Ulday for 28 consecutive days, and the fourth group received the highest dose of 100 million Ulday. None of the 20 patients developed a severe adverse reaction and, thus, none were dropped from this study. The maximum tolerated dose appeared to be above the highest dose administered to our patients. Body Temperature

Liver Biopsy Specimens . For histologic examination of the liver, blind biopsy was performed with Tru-cut needles before and after the treatment in all but 1 patient.

Fever developed virtually in all patients . There was, however, no correlation between the febrile response and increasing interferon dose. High grade fever of 38°e to 39°e occurred in all four

April 1985

rIFN-aA AND HEPATITIS B VIRUS INFECTION

Liver Function Tests

Table 2. Number of Patients Having Adverse Clinical Reactions Associated With Recombinant Leukocyte A Interferon Treatment Daily dose (x 10 6 U) Aqverse reactions

36

50

72

100

Total

Fever Hair lQss Anorexia Fatigue Headache Chills/shivering Myalgia Arthralgia Mucosal bleeding (nasal, oral, and/or anal)

5 4 5

5 5 4 3 3 3 2 2 0

5 4 4 4

5 4

20 17 15

1 2 0 1

2 2 2 2

2

4

2 4

2 2 1 0

2 5

873

14 13

9 6 6 6

dosage groups, and lasted from 1 to 2 wk. The febrile response gradually became less during the treatment, and most of the patients were afebrile in the fourth week. .

Elevations of serum glutamic oxaloacetic transaminase and gl\ltam~c pyruvic transaminase, more than twice the baseline values, were observed during the treatment in '1 1 and in 7 ca1?es, respectively. Peak elevations of the enzymes wflre noted in the fourth week in 16 of these 18 patients. The enzyme increase patterns are shown in Figure 2. Elevation of glutamic oxaloiicetic transaminase was more marked, and the ratio of glutamic pyruvic transaminase to glutamic oxaloacetic trans,,!-minase was reversed during the treatment. No significant effect of interferon was observed in other biochemIcal parameters including total bilirubin, alkaline phosphatase, total protein, serum albumin, and prothrombjn time (Table 3). Liver Histology Pretreatment biopsy specimens were obtained within 2 wk of start of interferon administration, and

. )

Body Weight

Mean body weight of the 20 patients at the beginning of the study was 62 .6 kg, decreased to 59.9 kg on day 14, and was 60.0 kg on day 42. No correlation was noted between the interferon dose and weight loss.

'"Z

"'<

%

100

S. O

:z: u

...z

'"U 0: ...'"

...'">-

'.'t

%

.. '"' . 1---':""-- ..................

50

-

u

Adverse Experiences

The clinical adver~e experiences associated with rIFN-aA are shown in Table 2. Fever wa~ observed in all patients. Anorexia, fatigue, and headache occurred in >50% of the patients. All adverse reactions but fatigue and mucosal bleeding appeared unrelated to the dose of injected interferon (Table 2). Fatigue and nasal bleeding were mOre often encountered in patients receiving higher doses (Table 2). Hair loss was ob~erved in 17 patients. This was usually noted 1-3 mo after the drug had been discontinued. All adverse effects disappeared in due time. Leukocyte and Platelet Count

0

'"'1

---1'/ .

36 X 10 6 &50:.:10 6

u

(n~

:::>

10 )

: 72:.:10 6 &100 )(10 6 (n ~ 10 )

'"....

Day 1

Day 7

D~y

Day 42

14

b)

%

100

'"

"z

I

C :I:

I

U

... Z

'"

u

0:

...'" ... .... '" '"... .......

)-----r---~(

% 50

I

00:

Leukocytopenia was most marked in the second week, and the mean white blood cell count in 20 patients dropped to 44% of the pretreatment level. The higher-dosage groups (72 and 100 million Ulday) showed a greater ' 1?uppression of leukocyte count~ than the lower-dosage groups (36 · and 50 million U/day) on day 14 (p < 0.05) (Figure 10). Similarly, platelet counts tended to decrease slightly more in the higher-dosage groups (Figure lb).

Day 1

Day 7

Day 14

Day 28

Day 42

Figure 1. a. Leukocyte count is shown as percent changes from the baseline count (100%) in the Jow- and high-dose groups. A statistically significant difference in the count reductiop between the two gr'oups wall noted on day 14 tA) (p' < 0.05). b. The percent change in pJatfllet coul1t was compared b,etween the two groups. 1\ statistically significqnt differencfl in changes was seen on day 213 (6) (p < o.in).

874

GASTROENTEROLOGY Vol. 88, No . 4

OMATA ET AL.

30

/1\

;' 1"\

J/ \

,.j

.

vi

+1

C

e

200

... ,,-

L-----:Jt-=-=..:--_-______ ,

II.

~

... ::. of"

0


:::I III:

100

••

(

,/

\

,/

GP T

GOT

... •• III

groups (Table 4). When the lowest-dose group was compared with the highest, a twofold to threefold increase of serum interferon concentration was observed (Table 4). Development of antibodies to interferon was studied before and after the treatment by the neutralizing method. No evidence of antibody development was obtained (Table 5). To exclude the possibility of contamination by E. coli protein and mouse immunoglobulin G (m-IgG) in the recombinant interferon preparation, aIiti-E. coli protein and anti-m-IgG antibodies were studied. Development of antibodies to these proteins was not demonstrated in any of the patient sera:

OL Day 1

DaY 7

Day 14

Day 28

Serum Deoxyribonucleic Acid Polymerase

Day 42

Figure 2. Mean serum glutamic ox~loacetic trans
posttreatment biopsy specimens were performed in the second week after tr!,)atment. Histologic changes, illcluding portal and parenchymal inflammation, disruption of limiting plates, hepatocellular degeneration, and fibrosis, were semi quantitatively graded and compared in the paired biopsy specimens from 19 patients. Inflammatory infiltrates became less severe in 8 cases, worsened in 2, and remained unchanged in 9 after treatment. Disruption of limiting plates became less prominent in 6 cases, more seve're in 3, imd . unchanged in 10. Hepatocellular degeneration seemed unchang(3d before and after the treatment (degeneration was lessened in 3, worsened in 4, and unchanged in 12). Thus, it seemed that inflammatory infiltrates became less prominent, but the severity of hepatocellular degeneration did not change after treatment. Serum Interferon Concentration At the baseline, no illterferon activity in serum was detl3cted by bioassay in any of "the 20 patients. Hourly and daily profiles 6f serum interferon level by bioassay are shown in Figllres 3 and 4. We administered rIFN-aA twice a day, at 9:00 AM and 9:00 PM, A maximum serum interferon activity was reached 2-6 h after the intrarimscular administration (Figures 3 and 4). Although fixed interferon doses were administered, there was a daily incre~ ment of serum interferon activity. The mean peak serupl interferoQ activities on day 1 were 93 Dlml with 3~ million U, 157 with 50 million U, 205 with 72 million U, and 323 with 100 million U. These values increased to 526, 525, 739, and 1271 Dlml on day 28 in the respective dosage

A marked reduction of serum DNA-P by the treatment was noted in all four groups (Figure 5). The mean DNA-P activity in these patients before treatment was 10,057 cpm/ml. 'The activity was decreased to 1319 cpm (87% reduction) on day 3, 1197 cpm (88% reduction) on day 7, 1234 cpm (88% reduction) on day :i1, 1056 cpm (89% reduction) pn day 14, and 938 cpm (91 %reduction) on day 21. The activity, however, started to increase while the patient was still under treatment, and rose to 1327 cpm (87% reduction) on day 28. It further increased to 3841 cpm (62% reduction) on day 42 and 5169 cpm (49% reduction) on day 56. To assess the interferon dose-related response of DNA-P activity, perce~t reduction of DNA-p during treatment was calculated both for lower doses (36-million and 50-million groups) and higher doses (72-million and 100-mil. lion groups). There was no difference in the suppressive effect (m DNA-P activity between the two groups (Figure 6). Deoxyribonucleic acid polymerase activity became negative at least once in 13 cases; 6 in the lower-dose group, and 7 in the higher-dose group. Of Table 3. Serum Biochemical Parameters Before and After Recombinant Leukocyte A Interferon Treatment Parameters

n

Baseline

Total bilirubin (mg/d/) Tqtal protein (g/d/) Serum albumin (g/d/) Alkaline phosphatase (UlL) Lactate dehydrogenase (lUlL) Prothrombin time (s) Blood glucose (g/d/) Blood urea nitrogen (!TIg/d/) Uric acid (mg/d/) Creatinine (mg/d/)

19 19 19 19 19 18 17 . 19 19 18

1.0 :t 0.5 7.2 ± 0.4 4.4 ± 0.4 123 :t 54 181 ± 66 12 .8 ± 0.7 83 .2 ± 8.8 13 £ 3 5.4 ± 0.9 1.1 ::t 0.2

a

Mean ± SD.

Day 42 0

1.1 6.7 4.0 128 200 12.3 80.8 11 5.4 1.0

± :t :t ± ± ± ± :t ± :t

1.2 0.5 0.5 73 89 0.8 7.0 3 1.1 0.2

rIFN-aA AND HEPATITIS B VIRUS INFECTION

April 1985

875

Serum IFN Level/36X 106 U b.Ld.

a Day 1 U/ol rlFN-QA

2.000

rlF'N · A- A

laxll,.u

18 x, lt'u

I

I

,

Day 14

,

rlFN-"'A

rIFN- ..... 18xlO"U

18xIO"U

Day 28 to Day 42 rlFN · a A

18 xl 0"U

I

1.000

100

10

D1 234

6

8

12 Time (hour)

Serum IFN Level/50X 106 U b.i.d.

b

Day 1

U/ ol

2 . 000

rIFN -a A

25 x lO-O

I

,

r! FN-" A

2S x lO"U

Day14

,

Day 28 to Day 42 rl FN -u A

rIFN -a'"

25x lItV

25 x IO''V

I

1.000

100

10

6

o12 34

12

24012346

Time (hour)

Figure 3. Hourly and daily profile of serum interferon activity (mean ± SD) measured by bioassay in patients receiving 36 x 10 6 U/day (a), and in patients receiving 50 x 10" U/day (b).

these 13 cases, 4 remained negative up to 4 wk after discontinuation of the drug. Hepatitis B Virus Deoxyribonucleic Acid and Deoxyribonucleic Acid Polymerase Reaction Product In addition to DNA-P activity measurement by the aforementioned method, we analyzed the poly-

merase reaction product by slab gel electrophoresis and autoradiography using 32p_dCTP. In this method, HBV-DNA was demonstrated by the presence of radioactive signals of 3.2-4 .0 kilo base pairs on the autoradiogram. In 13 patients, DNA-P. reaction product became undetectable during this treatment. Furthermore, HBV-DNA was studied directly by spot hybridization using a 32P-Iabeled HBV-DNA probe. This highly specific method for detection of

876

OMATA ET AL.

GASTROENTEROLOGY Vol. 88, No.4

Serum IFN Level/l00X 106 U b.i.d.

b

Day 1

,

,

rIFN-crA

U/."

SOX!O"u

SQX11T'U

2.000

Day 14

,

rIFN-aA

,rFN-aA

5OXIO"U

,

Day 28 to Day 42

,

rlFN-aA

rlFN·aA

rIFN-aA

SOX10"U

SOXID"U

SOXIO"U

1.000

100

10

Time (hour)

Serum IFN Level/72X 106 U b.i.d.

a Day 1 U/""

2.000 1.000

,

,

rIFN-,tA 36X10"U

rIFN-IlA 36x10"U

Day 14

,

Day 28 to Day 42

,

rlFN'"A

l6xl0'U

,

,

rIFN-nA

rtFN-,,"

36XIO"U

36Xl0"U

rlFN-"A

36xl0"U

100

10

012:i 4 6

8

)2

24 0 1 234

6

8

12

24

0 1 234

6

8

12

Time (hour) Figure 4. Hourly and daily profile of serum interferon activity (mean ± SD) in patients receiving 72 x 10 6 U/day (a). and in patients receiving 100 x 10 6 U/day (b).

viral DNA confirmed these results of the measurement of DNA-P activity using 3H-dCTP and 3H_ dGTP, and analysis of its reaction product using 32p_ dCTP (Figure 7). Prednisolone Withdrawal Effect In' 5 patients, 40 mg of prednisolone was administered daily for 4 wk from 1 to 4 mo before

interferon. When DNA-P activity was compared between the interferon-treated patients with prior prednisolone treatment and those without, mean serum DNA-P activity became nearly negative in the prednisolone-treated group, and this difference became statistically significant on days 4-2 through 56 (Figure 8). Serum HBeAg was measured by a solid-phase enzyme-linked immunoassay in both groups. Mean

rIFN-aA AND HEPATITIS B VIRUS INFECTION

April 1985

rIFN-I1'A

Table 4. Dosage of Interferon Administered and Mean Peak Serum Interferon Activity in Four Groups n Group 1 36 x 10 6 U Group 2 50 X 10 6 U Group 3 72 x 10 6 U Group 4 100 x 10 6 U a

Day 1

Day 14

~ 16000

Day 28

5

93 ± 20 0

449 ± 131

526 ± 320

5

157 ± 44

571 ± 343

525 ± 367

5

205 ± 74

552 ± 110

739 ± 466

5

323 ± 93

847 ± 419

1,271 ± 453

.---- 36X10·u/day

-

14000

. - - - 5OX10·u/day ,---<> 72 X10·u/day

12000

D--
E E

c.

., .,'"~ 0

~

10000

E

8000

~ 0

D.. 1

Interferon activity, measured by bioassay (mean ± SD).

c(

cut-off indices for HBeAg were significantly lower in patients receiving combined therapy than in patients receiving interferon alone. The difference was statistically significant on days 14 through 56 (Figure 9). None of the 15 patients with interferon treatment alone became seronegative for HBeAg, whereas 3 of 5 with combined therapy became seronegative during the treatment. One patient (No. 19) developed antiHBeAg antibody during the treatment.

Discussion Owing to successful molecular analysis and the recombinant technique, large quantities of biologically active interferon have become available. In this study, we used large doses of recombinant leukocyte interferon in patients with chronic hepatitis B, and examined its pharmacologic profile, dose tolerance, and biologic effects. In previous studies of Table 5. Serum Antibody Against Interferon Before and After Treatment

Daily dose

877

Patient number

Anti-interferon antibody Before

ND

1 2

3

After

Z 0

6000 4000 2000

1~ 3

7

11

21

14

28

56

42

Day of Treatment

Figure 5. Endogenous deoxyribonucleic acid polymerase activities serially measured in the four groups. Mean values of 5 patients in each group are shown.

interferon treatment for chronic hepatitis B (4,11,12), partially purified leukocyte interferon of ~1 % purity from human buffy-coat cells (Cantell type) (13) had been used. Compared with these studies, the doses of recombinant interferon used in our study were much larger. Using this larger dose was an attempt to determine the maximum tolerated dose. Although we increased the dosage up to 100 million U/day, all 20 patients completed 28 days of treatment, and the maximum tolerated dose seemed to be above 100 million U/day. This result is somewhat at variance with that observed in cancer patients treated with the same recombinant interferon. It was reported that fatigue, frequently progressive, was potentially dose limiting at 72 million U in cancer patients (14). Increased incidence of fatigue was similarly ob-

ND ND

4 5 6

%

ND

7 8

>0

100

:~

36 x 10& .. 50 x 10 6 U

u C

72 x 10' & 100x 10' U

9

10 ND

11

12 13 14 15 16 17 18 19 20 ND , not done.

.~.

----,: 1

3

11

21

14

Day

ND

ND

28

"

56

Figure 6. Deoxyribonucleic acid polymerase changes are expressed as percent changes in the low- and high-dose groups. No appreciable difference in the suppressive effect of interferon was noted between the two groups.

878

GASTROENTEROLOGY Vol. 88, No.4

OMATA ET AL.

2

N.18

N.19

3

4

5

••••• •

•

6

7

•• 8

9

•

Figure 7. Hepatitis B virus deoxyribonucleic acid was serially analyzed by spot hybridization technique. Two representative cases are shown (No. 18 and No. 19). Hepatitis B virus deoxyribonucleic acid of patient No. 18 was detectable before (lane 1), during (lanes 2 through 7). and after (lanes 8 and 9) the interferon treatment. In patient No. 19, 40 mg of prednisolone was administered for 4 wk before interferon treatment. Hepatitis B virus deoxyribonucleic acid was markedly increased during (lanes 2 and 3) the steroid, and interferon treatment was started on the day of lane 4. During interferon treatment, hepatitis B virus deoxyribonucleic acid became undetectable (lanes 5 through 7), and remained negative after the combination therapy (lanes 8 and 9). Hepatitis B e antigen became undetectable, and anti-HBeAg appeared during the interferon treatment in this patient.

served in our high-dose groups (Table 2). As most of our patients were young men (mean age 30 yr) with a much better physical condition than cancer patients, perhaps our patients tolerated the dose-limiting fatigue better, hence high interferon dosages. Adverse reactions to recombinant interferon were similar to those previously reported with Cantelltype leukocyte interferon (15), and included fever, headache, muscle pain, and anorexia. All side effects abated in the latter half of the treatment period, and rapidly disappeared upon discontinuation. A somewhat unexpected adverse reaction was the marked loss of hair in 17 of 20 patients 2-3 mo after cessation of the drug. Clinically, obvious hair loss occurred in only 8% of patients treated with Cantelltype interferon (15). The difference in incidence could well be due to larger doses of rIFN-aA in our study. Elevation of serum transaminase level was noted during the treatment. Serum glutamic oxaloacetic transaminase was elevated more markedly than serum glutamic pyruvic transaminase. In a previous study the same recombinant interferon was used in cancer patients, but no serum transaminase elevation was noted (3). Similar elevation of these enzyme activities by Cantell-type interferon was previously reported in patients with chronic hepatitis B (15). Thus, interferon, regardless of the purity, may have some ejIect on liver enzymes in individuals with chronic liver diseases. Our study demonstrated the hourly and daily profile of serum interferon activity in patients treat-

-: 10.000 .:

cpm

c

Interferon Alone

i.

: Prednisolone & Interferon

.. >

u

C

5.000 cpm

i

... 0

~----T"--.

Z

0

1

1.-~-r-

11

3

-._ 21

14

28

42

56

Day

Figure 8. Deoxyribonucleic acid polymerase activity was compared between interferon treatment alone (n = 15) and in combination with prednisolone (n = 5). Deoxyribo· nucleic acid polymerase activity was more suppressed in patients with the combined therapy, and the differ· ence was statistically significant on days 42 and 56 (iI) (p < 0.05).

ed with 2-400 times greater cumulative doses than those in the previous buffy-coat interferon treatment (11,12,16). Rapid absorption of rIFN-aA administered intramuscularly and dose-dependent increase of serum interferon activity were shown in our study. In addition, a cumulative increase in serum interferon level, not reported previously, was demonstrated during this consecutive 28-day treatment period. The serum interferon levels on day 28 were usually three to five times greater than those on day 1 in these different dosage groups (Table 4). The

i •

T"10

iTT,)"

c

: ~ ! -

'n'ed,,,,, (36 ("=9)

x

1O'& 50XlO')

Interferon (72 )( 1 0 6 & 100x 10 6 )

1

1'-'- -1---1- --1

("=6)

: Prednisolone and

Interferon

("=5)

1

3

II

14

21

28

.,

56

Day

Figure 9. Serum hepatitis B e antigen was determined by enzyme-liked immunoassay. Cut-off indices were expressed as the ratio of absorbancy values at 492 nm between the specimen and negative control. Although no difference of cut-off indices WilS noted in patients treated with low and high doses of-interferon alone, there was a significant decrease of hepatitis B e antigen in 5 patients with combined prednisolone and interferon. The difference between interferon alone and interferon in combination with prednisolone was statistically significant on days 14 through 56.

rIFN-aA AND HEPATITIS B VIRUS INFECfION

April 1985

highest m!3an peak serum interferon activity was obtained on day 28 in group 4, and was 1271 Ulml. This was higher thanthe reported interferon concentration during treatmept for chronic hepatitis B (4,15,17) . The enzyme-linked immunoassay, a newly developed method (18), was used in our study, and it confirmed the dose-dependent and cumulative effects of rIFN-aA shown by bioassay (data not shown). Furthermore, we examined the development of antibodies to rIFN-aA by the neutralization assay. Neither the pretreatment nor the posttreatment sera showed the antibody. This was in contrast to tlle results of rIFN-aA treatment for cancer patients in which 3 of i6 patients developed low-titer antibodies (3). The difference could be due to a longer duration of interferon treatment in cancer patients. Ip the previous studies in patients with chronic hepatitis B (4,11,12,15-17,19-21), neutralizing antibody wa~ not checked. Because intermittent longterm treatment has been advocated, development of neutralizing antibody against interferon that interferes with the biologic activity of interferon and may result in immune complex disorders should be carefully checked in the future study. Our data indicate that leukocyte interferon produced by recombinant technique will markedly suppress DNA-P and HBV-DNA ip serum. A significant antiviral effect of rIFN-aA was similarly shown by Smith et al. (22) during sequential 8-day courses of treatment in 9 patients. Although we chose young male patients with very high DNA-P activity for treatment, DNA-P became undetectable at least once in 13 patients during the treatment. No difference in antiviral effect, however, was noted between the low doses (36 and 50 million U) and high doses (72 and 100 million U) . The reduGtion of DNA-P activity by our lowest-dose regimen appeared even more marked than that obtained in the previous studies using buffy-coat interferon. Thus, it is likely that a mflximum antiviral effect of interferon was reached even at a dose of 36 million Ulday. In 5 patients, prednisolone was administered before interferon in order to combine the withdrawal effect of steroid with antiviral treatment. Corticosteroid and cancer chemotherapy are known to increase DNA-P activity (23-25) and decrease the activity upon discontinuation (24-26). When DNA-P activity and serum HBeAg were compared between the patients receiving interferon alone and those receiving interferon in combination with prednisolone withdrilwal showed a clear difference in efficacy; combination therapy was more effective in reducing these viral markers. Although we used massive doses of interferon arone in 15 patients, none became seronegative for HBeAg during treatment. In contrast, 3 of 5 who received the combination therapy became

879

seronegative for HBeAg, and 1 patient developed antibody to HBeAg during the treatment. These 3 · patients remained seronegative for HBeAg at least for 6 mo after treatment. In a separate study, ;3 of 14 patients (21 %) treated with prednisolone alone with a similar dose schedule became seronegative in the averl;ige follow-up period of 6 mo. To ' corroborate efficacy of the combination therapy, a randomized, controlled tdal is required. In summary, our study revealed that leukocyte interferon produced by the recombinant technique had a significant antiviral effect against HBV, and that a maximum biologic effect could be obtained below the maximum tolerated doses. Our trial of combination therapy suggests that synergistic effects of immunomodulating steroid withdrawal and antiviral therapy may offer Illore promise for the treatment of chronic hepatitis B than antiviral treatment alone. However, this has yet to be proven in a controlled study.

. References 1.

2. 3.

4. 5.

6.

7. 8. 9.

10. 11. 17.

S, Taira H, Hall A, et al. Synthesis in E. coli of a polypeptide with human leucocyte interferon activity. Nature 1980;284:316-20. Goeddel D, Yelverton E. Ullrich A. et al. Human leucocyte interferon produced by E. coli is biologically active. Nature 1980;287:411-6. Gutterman JU. Fine S. Quesada J, et al. Recombinant leucocyte A interferon: pharmacokinetics. single-dose tolerance. and biologic effect in can,:;er patients . Ann Intern Med 1982; 96:549-56. Greenberg HB . Pollard RB . Lutwick LI. et al. Effect of human leucocyte interferon on hepatitis B virus infection in patients with chronic active hepatitis. N Engl J Med 1976;295:517-22. Maeda S. McCandliss R. Gross M. et al. Construction and identification of bacterial plasmids containing nucleotide sequence for human leucocyte interferon. Proc Natl Acad Sci USA 1980;77:7010-3 . Staehelin T. Hobbs DS. Kung HF. et .al. Purification and characterization of recombinant human leucocyte interferon (IFLrA) with monoclonal antibodies. J Bioi Chern 1981 ;256: 9750-4. Robinson WS. DNA and DNA polymerase in the core of the Dane particles of hepatitis B. Am J Med Sci 1975 ;270:151-9. Mason WS. Seal G. Summers J. Virus of pekin ducks with structural and biological relatedness 'to human hepatitis B virus. J Virol 1980;36:829-36. Kohase M. Kohno S. Yamazaki S. et al. Potency standardization of human interferon preparations for clinical trials. In : Kono R. Vilcek J, eds . The clinical potential of interferons. Tokyo : University of Tokyo Press. 1982:299_309. Trown PW. Kramer MJ, Dennin RA. et al. Antibodies to human leucocyte interferon in cancer patients. Lancet 1983; i:81-4. Weimar W. Heijtink RA. Tenkate FJP. et al. Double-blind study of leucocyte interferon administration in chronic HBsAg-positive hepatitis. Lancet 1980;i:336-8. Scullard GH. Pollard RB . Smith JL. et al. Antiviral treatment of chronic hepatitis B virus infection. 1. Changes in viral markers with interferon combined with adenine arabinoside. J Infect Dis 1981;143 :772-83. Nagat~

880

OMAT A ET AL.

t3. Cantell K, Hirvonen S. Large scale production of human leucocyte interferon containing 10 units per ml. J Gen Virol 1978;39:541-3. 14. Leavitt RD, Duffey P, Wiernik PH, et al. A phase 1 trial of twice dajly recombinant human leucocyte A interferon (IFLrA) in cancer patients (abstr). Am Soc Clin One 1982; C-162 . . . 15. Sacks SL, Scullard GH, Pollard RB, et al. Antiviral treatment of chronic hepatitis B virus infection. 4. Pharmacokinetics and side effects of interferon and adenine arabinoside alone and in combination. Antimicrob Agents Chemother 1982;21: 95-100. 16. Scullard GH, Alberti A, Wansbrough-Jones MH, et al. Effects of human leucocyte interferon on hepatitis B virus replication and immune response in patients with chronic hepatitis B infection. J Clin Lab Immunol 1979;1:277-82. 17. Kingham JGC, Ganguly NK: Shaari ZD, et al. Treatment of HBsAg-positive chronic active hepatitis with human fibroblast interferon. Gut 1978;19:91-4. 18. Staehelin T, Durrer R, Schmidt J, et al. Production of hybridomas secreting monoclonal antibodies to the human leucocyte interferons. Proc Natl Acad Sci USA 1981;78:1848-52. 19. Desmyter J, Ray MB, Groote JD, et al. Administration of human fibroblast interferon in chronic hepatitis B infection. Lancet 1976;ii:645-7.

GASTROENTEROLOGY Vol. 88, No.4

20. Iwarson S, Norkrans G, Nordenfelt E, et al. Interferon treatment in acute hepatitis B infection with prolonged course. Scand J Infect Dis 1980;12 :233-4. 21. Dolen JG , Carter WA, Horoszewicz JS, et al. Fibroblast interferon treatment of a patient with chronic active hepatitis. Am J Med 1979;67:127-31 . 22. Smith CI, Weissberg J, Bernhardt L, et al. Acute Dane particle suppression with recombinant leucocyte A interferon in chronic hepatitis B virus infection. J Infect Dis 1983;146:90713. 23. Sagnelli E, Manzillo G, Maio G, et al. Serum levels of hepatitis surface and core antigens during immunosuppressive treatment of HBsAg-positive chronic active hepatitis. Lancet 1980; ii:395-7. 24. Scullard GH, Smith CL, Merigan TC, et al. Effects of immunosuppressive therapy on viral markers in chronic active hepatitis B. Gastroenterology 1981;81:987-91. 25. Hoofnagle JH, Dushe'iko GM, Schaffer DE, et al. Reactivation of chronic hepatitis B virus infection by cancer chemotherapy. Ann Intern Med 1982;96:447-9 . 26. Rakela J, Redeker AG , Weliky B. Effect of short-term prednisone therapy on aminotransferase levels and hepatitis B virus markers in chronic type B hepatitis. Gastroenterology 1983; 84:956-60.