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Antipyrine clearance and response to interferon treatment in patients with chronic active hepatitis C
Antipyrine Clearance and Response to Interferon Treatment in Patients With Chronic Active Hepatitis C SHIRLEY COVERDALE, K A R E N
BYTH, JACQUELINE FIELD, CHRISTOPHER LIDDLE, RITA LIN, AND GEOFFREY C, FARRELL
0.12 to 0.98; normal range, 0.34 to 1.02 m L / r n i r t / k g body wt), a value that was significantly greater than in 30 n o n r e s p o n d e r s (0~23; 0.08 to 0.67 mlJmin/kg body wt, P < .001). APC was closely associated with response to interferon. The r e s p o n s e rate a m o n g cases with values
months, w h e r e a s a m o n g relapsers (defined b y ALT increase), APC eventually declined to be less than the pretreatment value at 24 months. It is c o n c l u d e d that pretreatment APC is a powerful positive predictor of r e s p o n s i v e n e s s to interferon treatment in patients with chronic hepatitis C, indicating that good hepatic metabolic function m a y be important in determining the effectiveness of interferon treatment. Furthermore, changes in hepatic metabolic function after apparently successful treatment, at least as d e t e r m i n e d by APC, appear to be subtle, delayed in onset, and m a i n t a i n e d o n l y in those w h o remain in biochemical remission. (HEPA-
>0.25 mL/mirt/kg b o d y w e i g h t w a s 79%, the same as in
TOLOGY1995;22:1065-1071.)
To determine w h e t h e r hepatic metabolic function affects the r e s p o n s e to interferon treatment, w e m e a s u r e d
antipyrine clearance (APC) in 85 patients with chronic active hepatitis C and c o m p a r e d the results w i t h treatment outcome. A m o n g 55 patients w h o r e s p o n d e d to interferon b y n o r m a l i z a t i o n o f alAnlne transAminase (ALT), m e d i a n APC before treatment w a s 0.47 (range,
cases without cirrhosis. Cases w i t h o u t cirrhosis and with APC of >0.25 mL/min/kg body w e i g h t h a d an 85% chance of responding to interferon; this w a s unlikely a simple reflection o f histological activity, b e c a u s e the correlation w i t h S c h e u e r score w a s poor in this subgroup (r = -.31, P < .05). A second, independent group of 43 patients w a s u s e d to test the predictive value of APC (using 0.25 mL/min/kg body wt as a cut~ff) for response to interferon treatment. In this group, APC correctly predicted positive r e s p o n s e to interferon in 75% of cases. APC w a s also u s e d to m e a s u r e the effects of treatment o n hepatic metabolic function. Regardless of outcome, there w a s no change in APC at the end o f a 6month course of interferon treatment. Six m o n t h s later, however, i m p r o v e m e n t in APC (14%; P < .05) was evident a m o n g responders but not in those w h o h a d failed to r e s p o n d to interferon. In patients w h o c o n t i n u e d to h a v e normal ALT (18 o f 19 tested were also nonviremic), the i m p r o v e m e n t in APC w a s sustained for at least 24
Abbreviations: ALT, alanine transaminase; APC, antipyrine clearance. From the Storr Liver Unit, Department of Medicine, University of Sydney, and the Department of Gastroenterology and Hepatology, Westmead Hospital, Westmead, NSW, Australia. Received August 15, 1994; accepted May 30, 1995. Supported in part by the Robert W. Storr endowment for liver research. The clinical trials from which these subsets of patients were drawn were supported by Schering-Plough International, Kenilworth, NJ, and its Australian branch, Baulkham Hills, NSW. Presented in part at the Annual Scientific Meeting of the American Association for Study of the Liver, Chicago, IL, November 1993, and has been published in abstract form (HEPATOLOGY1993;18:228A). An earlier pilot study has been published in extended abstract form (reference 1). Address reprint requests to: Professor Geoffrey C. Farrell, Storr Liver Unit, Department of Medicine, Westmead Hospital, Westmead, NSW 2145, Australia. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2204-000753.00/0
A large number of randomized controlled trials have delineated the response rates to interferon t r e a t m e n t of patients with chronic active hepatitis C. 2"9 The outstanding issues concern the suboptimal durability of the interferon response and the health economics of treatment, given t h a t the n a t u r a l history of liver disease caused by hepatitis C virus infection has a prolonged course and a variety of possible outcomes. Efforts to improve the "efficiency" of interferon t r e a t m e n t might follow from more accurate prediction of the responsiveness in various types of cases. Variables t h a t are related to t r e a t m e n t outcome probably include viral determinants, patient characteristics, and the severity of the associated liver disease. In relation to the latter, we and others have identified absence of cirrhosis as a reproducible and informative predictor of response to interferon t r e a t m e n t in hepatitis C as defined by shortterm resolution of serum alanine transaminase (ALT) abnormalities. 4'v'9-11 The response rate is approximately 75% in the absence of cirrhosis and 30% to 40% among cases with cirrhosis. The poorer response to interferon treatment among patients with chronic active hepatitis C and cirrhosis could be due to several reasons. These include longer duration of infection, greater viral load or more virulent genotype, hemodynamic alterations that reduce hepatic availability of interferon, and phenotypic changes of hepatocytes that alter the cellular response to interferon. The present study was designed to examine the proposal that impaired hepatic metabolic function may be the critical change in cirrhosis that determines responsiveness to interferon treatment. Therefore, we measured antipyrine clearance (APC), a sensitive marker of intrinsic hepatic
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TABLE 1. D e m o g r a p h i c Characteristics and P h a r m a c o k i n e t i c Indices in the Two Patient Groups Patient Group
Main group (n = 85) Second group (n = 43)
Age (yrs)
Sex M/F
Smokers
APC (mL/min/kg)
VD (L/kg)
Half-life (hr)
44 (37-53) 37 (33-53)
56/29
31 (36%) 15 (35%)
0.37 (0.22-0.53) 0.39 (0.26-0.50)
0.57 (0.50-0.63) 0.57 (0.51-0.62)
18.5 (12.5-30.5) 16.0 (13.7-23.8)
28/15
NOTE. Data are expressed as median (interquartile range). Abbreviation: VD, volume of distribution.
d r u g clearance, a m o n g patients with chronic active hepatitis C. Studies were initially performed before administration of interferon a n d the results were later correlated with the outcome of treatment, as well as with the quantitative a s s e s s m e n t of liver histology. The a p p a r e n t predictive value of APC as an indicator of t r e a t m e n t outcome was t h e n tested in a second independent group of cases. I n addition, serial studies of APC at the end of t r e a t m e n t and at 6- or 12-month intervals thereafter were performed to determine w h e t h e r a p p a r e n t l y successful interferon t r e a t m e n t improved hepatic metabolic function as determined by this test. PATIENTS AND METHODS Clinical M a t e r i a l . The 85 patients who participated in the major part of this study (i.e., serial &PC testing) were enrolled in two interferon trials, one of which has been reported in full, 7 the other in preliminary form. TM There were 56 men and 29 women, and their median age was 44 years (range, 27 to 73 years). These 85 patients represented 52% of all the patients from this center enrolled in these trials. Selection into the present study was on the basis of logistic considerations, as follows: (1) the patient had been randomized to receive treatment by a protocol that was suitable for the present study (see below, and Table 1), (2) the patient completed treatment and was available to attend for subsequent studies, (3) the patient was not taking substances known to alter &PC, with the exception of cigarette smoke (see below). All patients gave informed consent. The study conformed to current international guidelines for human experimentation and was approved by the Human Ethics Committee of the Western Sydney Area Health Service. The risk factors for acquisition of hepatitis C were similar to those previously reported for Australian patients. 7'12"14Patients taking therapeutic or recreational drugs that are known to influence &PC were excluded, with the exception of cigarette smokers. Thus, 31 subjects smoked cigarettes, and 54 individuals did not (<5 cigarettes per day) (Table 1). There was no change in the number of cigarettes smoked per day during the study. Marijuana smokers were excluded, as were patients enrolled in methadone programs. No patients were known to regularly consume more than 20 g of ethyl alcohol each day. A second group of 43 patients was used to test independently the predictive accuracy of APC as an indicator of response to interferon treatment. These patients included 10 subjects enrolled in the above two clinical trials who did not have subsequent APC studies, and 33 others who were treated by the same protocols outside of these clinical trials. The demographic and clinical characteristics of patients in this second group were similar to those in the first group (Table 1).
All patients (in both groups) had histologically proven chronic active hepatitis, as determined by liver biopsy specimen appearances within 6 months before starting interferon. The presence or absence of cirrhosis was also noted on this biopsy specimen but was not known to the investigators at the time APC studies were performed. In coded biopsy slides, the severity of hepatitis was graded by one of us (GCF) according to the Scheuer scoring system. 15From the main group of 85 patients, 78 pretreatment and 55 posttreatment biopsy specimens were available for scoring. All patients were antiHCV positive by either first-generation (31%) or second-generation (69%) enzyme-linked immunosorbance assay test (Orthodiagnostics Systems; Raritan, NJ). Among 61 cases tested for the presence of HCV-RNA in serum, 36 (59%) were positive, as determined by reverse transcriptase polymerase chain reaction. The reverse transcriptase polymerase chain reaction assay employed primers located in the highly conserved 5'-noncoding region of the HCV genome; the specificity of this assay was confirmed by Southern hybridization with a probe that lies internal to the amplified fragment. TM Other causes of chronic liver disease were systematically excluded, as described elsewhere. 7All patients had persistent elevation of serum ALT levels, which were at least twice the upper limit of the normal laboratory range and had been present for at least 6 months before enrollment. Interferon Treatment. Patients were enrolled into two randomized controlled trials of interferon-a2b (Intron A, Schering-Plough International, Kenilworth, NJ) (interferon). In the first trial, 7 patients were randomized to receive either 3 MU interferon thrice weekly by subcutaneous injection for 6 months, or to receive no treatment; only patients in the treatment arm are considered here (n = 26). In the second trial, TM there were three treatment arms, but only those patients who received 3 MU interferon thrice weekly for 6 months (n = 35) or 5 MU interferon thrice weekly for 6 months (n = 24) are considered here. Patients were assessed clinically on several occasions before randomization into the study and were reviewed every 4 weeks during interferon treatment, at which time blood was taken for determination of liver tests and hematologic parameters. Short-term response to interferon was defined as a return of ALT to normal levels during the first 4 months of interferon treatment, and persistance of normal ALT thereafter for the duration of treatment. Relapse was said to have occurred when ALT abnormality (any extent) was noted after stopping interferon treatment. As expected, 17 biochemical relapse could always be correlated with return to positivity of serum HCV-RNA (Lin R, Liddle C, Farrell G, Unpublished data, 1994). To determine long-term response to interferon treatment, patients were observed at 4-weekly intervals for the first 6 months after completion of interferon treatment. Blood was taken at these times for determination of liver tests. A long-
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COVERDALE ET AL
TABLE 2. R e s p o n s e R a t e s A c c o r d i n g to P r e t r e a t m e n t C h a r a c t e r i s t i c s i n t h e F i r s t G r o u p o f 85 P a t i e n t s APC (mL/min/kg body wt)
APC -< 0.25 APC > 0.25 Total
Liver Histology Cirrhosis
No Cirrhosis
Total
8/22 (36%) 5/10 (50%)
3/7 (43%) 39/46(85%)
11/29 (38%) 44/56(79%)
13/32 (41%)
42/53 (79%)
55/85(65%)
term response to interferon treatment was defined as continued normality of ALT after discontinuation of interferon and until the end of the period of follow-up (at least 6 months). In this study, no relapses were seen later than 4 months after completion of the 6-month course of interferon treatment. Eighteen of 19 tested long-term responders have remained negative for HCV-RNA in serum (Lin R, Liddle C, Farrell G; Unpublished data, 1994). APC Studies. APC was performed in ambulant patients at the following times: (1) within 7 days before starting interferon; (2) at completion of 6 months of interferon treatment, always 7 to 10 days after the last dose of interferon; (3) at 6or 12-monthly intervals thereafter. On each occasion that APC was determined, ALT and other conventional liver tests were measured. In the long-term study, the number of patients examined at each time point is indicated in the figures or figure legends. APC was determined after oral administration of antipyrine (10 mg/kg body weight) given to nonfasted individuals between 8 and 11 AM. Two venous blood samples were collected, the first at 4 hours and the second 24 hours after antipyrine ingestion. Antipyrine was estimated in serum by a sensitive and accurate high-pressure liquid chromatography method, as previously described, ls'19 and clearance was determined from the two concentration/time points. In other studies 1z'2° (and Coverdale S, Farrell G, Unpublished data, 1993), we have noted in healthy subjects and in patients with clinically stable types of liver disease that APC values for any individual do not vary by more than 10% when measured repeatedly during a 2-year interval. Expression of Data and S t a t i s t i c a l Methods. Because of the skewed distribution of the APC results, data are presented in the figures as box and whisker plots, with the median, 25th, 50th, and 75th percentiles indicated (see legends to figures). Logistic regression was used to assess the predictive value of APC and liver histology in relation to a short-term response to interferon. Logistic discriminant analysis was used to determine the cut-off value for APC levels in predicting response to interferon. 21 For this analysis, APC was considered as a continuous dependent variable and response as a dichotomous outcome variable. The performance of the cut-offvalue was independently validated using the second independent series of 43 patients (see Clinical Material). To compare changes in APC over time, Wilcoxon tests were employed. A P value of <.01 was regarded as statistically significant. This is the corrected value after employing the Bonferroni adjustment for five multiple comparisons to the usual P < .05. Spearman's rank correlation was used to test the correlations between liver histology and APC. RESULTS
Response to Interferon Treatment: R e l a t i o n s h i p to Cirrhosis, P r e t r e a t m e n t APC, a n d Conventional L i v e r Tests. T h e s h o r t - t e r m r e s p o n s e r a t e to i n t e r f e r o n t r e a t -
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m e n t for all 85 p a t i e n t s i n c l u d e d in t h e first a n t i p y r i n e s t u d y g r o u p w a s 65%. A m o n g cases c a t e g o r i z e d according to p r e t r e a t m e n t l i v e r biopsy, 79% of t h o s e without cirrhosis r e s p o n d e d to i n t e r f e r o n , c o m p a r e d w i t h 41% of t h o s e w i t h cirrhosis (Table 2). As expected, t h e s e r e s u l t s a r e v e r y s i m i l a r to t h o s e r e p o r t e d for t h e two t r i a l s f r o m w h i c h t h e s u b s e t of p a t i e n t s for a n t i p y r i n e s t u d i e s w e r e d r a w n . 7'12 A m o n g t h e 55 cases w i t h a s h o r t - t e r m r e s p o n s e to i n t e r f e r o n t r e a t m e n t , A P C w a s 0.47 m L / m i n / k g body w e i g h t ( m e d i a n ; range: 0.12 to 0.98), w h i c h w a s h i g h e r t h a n in 30 n o n r e s p o n d e r s (0.23; 0.08 to 0.67; P < .001). I t is a p p a r e n t f r o m i n s p e c t i o n of Fig. 1 t h a t t h r e e - q u a r t e r s of r e s p o n d e r s h a d A P C v a l u e s w i t h i n t h e n o m i n a l n o r m a l r a n g e for t h i s l a b o r a t o r y , w h e r e a s t h r e e - q u a r t e r s of n o n r e s p o n d e r s h a d v a l u e s t h a t w e r e below t h e lower l i m i t of t h e n o r m a l r a n g e . T h e s e d a t a w e r e u s e d to c a l c u l a t e t h e s t r e n g t h of associations between individual characteristics and t r e a t m e n t outcome. F o r t h i s p u r p o s e , l i v e r histology (cirrhosis or not cirrhosis, a n d t o t a l S c h e u e r score) a n d A P C w e r e considered. Logistic d i s c r i m i n a n t a n a l y s i s s u g g e s t e d t h a t A P C > 0.25 w a s a s s o c i a t e d w i t h response. A m u l t i v a r i a t e logistic m o d e l i n c o r p o r a t i n g histology a n d APC (-<0.25 vs. > 0 . 2 5 ) w a s fitted. O n l y A P C a n d cirrhosis w e r e i n d e p e n d e n t l y a s s o c i a t e d w i t h t r e a t m e n t r e s p o n s e . T h u s , 85% of p a t i e n t s w i t h b o t h f a v o r a b l e c h a r a c t e r i s t i c s (no cirrhosis a n d A P C > 0.25) h a d a s h o r t - t e r m r e s p o n s e to i n t e r f e r o n ; 54% of all cases fell into t h i s c a t e g o r y (Table 2). As r e p o r t e d elsew h e r e , 25'7'911 no o t h e r b i o c h e m i c a l indices of liver dise a s e w e r e a s s o c i a t e d w i t h r e s p o n s e to i n t e r f e r o n t r e a t ment. T h e possibility t h a t A P C m a y be m o r e closely r e l a t e d to histologic p r e s e n c e of n e c r o i n f l a m m a t o r y activity t h a n to t h e p r e s e n c e or a b s e n c e of cirrhosis w a s e x a m i n e d u s i n g t h e S c h e u e r score. 15 A l t h o u g h t h e r e w a s a
1.0
°e- °sl "
o
0.41=1
T
Nonresponders n=30
Responders n=S5
FIG. 1. Pretreatment APC in short-term responders and nonresponders to interferon treatment. In these %ox and whisker" plots, the bar within each column represents the median value, the upper and lower borders of the box are the quartiles, and the "whiskers" (error bars) at the extremities indicate the 10th and 90th percentiles. APC in responders is significantly higher than in nonresponders (P < .001).
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HEPATOLOGY October 1995
TABLE 3. R e s p o n s e R a t e s A c c o r d i n g t o P r e t r e a t m e n t C h a r a c t e r i s t i c s i n t h e S e c o n d G r o u p o f 43 P a t i e n t s Liver Histology
APC (mL/min/kg body wt)
Cirrhosis
No Cirrhosis
Total
APC -< 0.25 APC > 0.25
4/9 (44%) 1/5 (20%)
2/2 (100%) 23/27 (85%)
6/11 (55%) 24/32 (75%)
Total
5/14 (36%)
25/29 (86%)
30/43 (70%)
weak correlation between the total Scheuer score and APC (r = -.55, P < .001), total Scheuer score was not an independent predictor of response. Thus, there was minimal difference in median total Scheuer score between responders and nonresponders (respectively, 7 and 8, P = .05). In the 53 patients without cirrhosis, there was no difference in total, portal, or lobular scores or in APC between responders and nonresponders. The correlation between total Scheuer score and APC was much weaker in this group (r = -.31, P < .05). Predictive Value of APC for Response to Interferon Treatment. To test the predictive value of APC as an indicator of response to interferon treatment, a second similar group of 43 patients with chronic active hepatitis C was studied. In this group, the overall short-term response rate to interferon was 70% (Table 3). Among the 32 cases with APC > 0.25 mL/min/kg, 24 had a short-term response to interferon treatment, giving a predictive accuracy for APC of 75%. In this group, the positive predictive accuracy of no cirrhosis was 86% (Table 3). Effects of Cigarette Smoking on APC. As expected from the known effects of cigarette smoking on antipyrine metabolism, 22'23 the pretreatment APC of smokers was higher t h a n t h a t of nonsmokers (median, 0.52; range, 0.13 to 0.98; n = 31; vs. 0.29 [0.08 to 0.92], n = 54, P < .001). The proportion of cigarette smokers appeared to be similar among short-term responders (22 of 55), long-term responders (12 of 27), and nonresponders (9 of 30). There were no significant differences between the percentage changes in APC after treatment between smokers and nonsmokers at any of the times examined (see below). According to smoking status, the "cut-off' APC values t h a t best predicted a response to interferon t r e a t m e n t were respectively 0.31 mL/min/kg for smokers and 0.21 mL/min/kg for nonsmokers. Effects of Interferon Treatment on APC. Previous studies from this laboratory have demonstrated t h a t administration of interferon is itself associated with impairment of A P e . 24 Thus, preliminary studies of APC performed during interferon t r e a t m e n t appeared to be uninformative, as reported elsewhere. 1 Specifically, among 65 patients with &PC performed ->3 but <6 months into a course of interferon treatment, APC increased in 17 (26%), decreased in 23 (35%), and was unchanged in 25 (38%). There was no significant difference in &PC before versus during t r e a t m e n t for the group as a whole nor for the 39 responders or 26 nonre-
sponder subgroups. Studies after completion of interferon t r e a t m e n t were performed 7 to 10 days after the last dose to avoid interferon effects and to precede the onset of relapse. At this time, there was no change in APC compared with the pretreatment value, irrespective of the apparent response during t r e a t m e n t or the subsequent clinical course (Fig. 2). In contrast, the results obtained 6 months after stopping interferon showed a clear improvement in APC in those who had exhibited a short-term response (14%, P < .05) but no improvement in those who did not respond to treatment (Fig. 2). Subsequent Changes of APC. Among patients who had been studied for ->6 months after stopping interferon treatment, the results indicated different patterns of change of APC according to the clinical course. Among short-term responders who experienced relapse, this occurred at a median time of 6 weeks after completion of interferon, t h a t is, 18 weeks before the third APC study (Fig. 3; see 6-month study). Decline of APC values was evident among these cases (Fig. 3A). Thus, by 18 months after the initial study (i.e., 12 months after completion of interferon treatment), values had returned to pretreatment levels. F u r t h e r decline of APC to values t h a t were significantly less t h a n before t r e a t m e n t was evident by 24 months (P < .01) (Fig. 3A). In contrast, long-term response to interferon treatment was associated with an apparent increase in APC
100 U
80
Q.
60 =,,,, • -
~u
40
0
-2o -4o n=29 n=52 End of t r e a t m e n t
n=21 n=32 6 months later
FIG. 2. Change in APC at end of interferon t r e a t m e n t a n d at 6 months after completion of treatment. Graphs show the change of APC, expressed as a percentage of the p r e t r e a t m e n t value, for responders and nonresponders at the end of t r e a t m e n t (i.e., within 7 to 10 days of stopping interferon) and 6 months later (this period corresponds to 12 months after the initial measurement). Short-term responders are indicated by shaded columns, nonresponders by open columns. To improve visual presentation, four outlying values are not shown on the figure; these were for two responders at end of t r e a t m e n t (112%, 144%) a n d two responders 6 months later (129%, 237%). The explanation for the style of plot is indicated in the legend to Fig. 1. The n u m b e r of patients studied in each group and at each time is indicated (see n values). There were no differences between values for nonresponders at either time, or for responders at end of t r e a t m e n t compared with before treatment. The difference between responders at 6 months compared with before t r e a t m e n t is significant (P < .05).
HEPATOLOGY Vol. 22, No. 4, 1995
t 1001 .E
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COVERDALE ET AL
A
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6O 40
-20 -40
F-
1
B ¢j Q. < ¢ .E
120 100 80 60
o
O
40"
20" u 0 ~¢ -zo. -40' "
m
j, O 0
1'2
o
1'8
:;4
Time after stopping interferon (months) Fro. 3. Serial APC among short-term responders to interferon: comparison of relapsers with long-term responders. These data are presented in the same format as Fig. 2. (A) Patients who relapsed after stopping interferon. (B) Long-term responders. The numbers of patients studied at each time (n) are indicated within the boxes. Four outlying values are not shown in (B); these were 0 months, 144%; 6 months, 237%; 12 months, 162%; 18 months, 156%. *Significantly different from p r e t r e a t m e n t value, P < .01. The a p p a r e n t increases in APC among long-term responders (B) are not significant (respectively, 6 months, P = .15; 12 months, P = .11; 18 months, P = .16).
that was sustained throughout the study period (Fig. 3B). Among cases with no response to interferon, there was no consistent change in APC after the initial study (data not shown). It must be borne in mind that these cases exhibited much lower values for APC at the time of entry into the study (Fig. 1). APC studies were discontinued in six patients because of definite or suspected adverse effects of antipyrine. These were all suggestive of allergy and included facial flushing in two, rash in one, conjunctivitis in one, rash plus conjunctivitis in one, and angioedema of lips in one. All adverse effects occurred after multiple (->3) exposures to antipyrine. DISCUSSION
The novel feature of this study is that it reports the relationship between hepatic metabolic function, as determined serially by a quantitative liver function test, and the response to interferon treatment among pa-
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tients with chronic active hepatitis C. The results are interpreted as indicating that hepatic metabolic function is a determinant of interferon responsiveness in this disease. Moreover, changes in liver function occur after treatment with interferon b u t appear to be subtle and delayed. Several viral, demographic, and clinicopathologic determinants of responsiveness to interferon treatment have been identified in hepatitis C, but none correlate as strongly as morphological severity of liver disease. Thus, several studies have confirmed the initial observation 7 that the response among patients without cirrhosis is approximately twice that of those with cirrhosis. 4'9"11 The current findings are consistent with the proposal that response to interferon treatment is suboptimal in cirrhosis because of its associated impairment of hepatic metabolic function. Thus, APC was at least as good at predicting response to interferon as was the presence or absence of cirrhosis. The question arises as to the utility of APC in individual cases. At least half the patients in these two studies had a value for APC (>0.25 mL/kg/min) that allowed prediction of a 75% chance of response, which is as good as that for absence of cirrhosis. Thus, the APC would be particularly useful if information were not available about liver histology, and although our study suggests that it could improve positive predictive value if histology (presence or absence of cirrhosis) is already known, confirmation of this will require study of larger groups of patients. We chose APC as the marker of hepatic metabolic function because of the ease of repeated application of this test during the natural history of a disease that usually continues for decades. There do not appear to be comparative data with other quantitative liver function tests, and it is therefore difficult to consider the relative merits of APC with other t e s t s Y Minor but potentially severe allergy to antipyrine occurred in 6 of the 128 patients in these two studies (5%). This rate is similar to our overall experience of adverse reactions to antipyrine (4%). However, the occurrence of drug allergy clearly restricts the applicability of repeated studies in these cases and underscores the importance of adequate explanation to potential study participants before obtaining informed consent. Further, concomitant intake of drugs and other foreign compounds may confound interpretation of the results of APC. 25 In the current study, a small number of cases were considered unsuitable for APC testing on this basis. It must also be noted that the cut-off values of pretreatment APC to predict response to interferon differed between smokers and nonsmokers. However, the general conclusions from the results did not appear to be altered when cigarette smokers were included in the analysis. An unexpected result of the current study was that beneficial effects of apparently successful interferon treatment on hepatic metabolic function were small, as determined by APC. Moreover, they appeared to be delayed in appearance. Thus, the increase in APC compared with pretreatment levels was 14% (range, - 3 2 %
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to 237%), I n c o n t r a s t , in chronic h e p a t i t i s B (46%) a n d a u t o i m m u n e chronic active h e p a t i t i s (79%), g r e a t e r inc r e a s e s h a v e b e e n e v i d e n t a f t e r r e s p o n s e to t r e a t m e n t w i t h i n t e r f e r o n or corticosteroids, respectively. 19'26 One possible r e a s o n for t h e less i m p r e s s i v e c h a n g e w i t h h e p a t i t i s C is t h a t m o s t r e s p o n d e r s h a v e only m i n i m a l i m p a i r m e n t of h e p a t i c m e t a b o l i c function, t h e r e b y reducing t h e likelihood t h a t c h a n g e s of g r e a t e r m a g n i t u d e c a n be observed. A l t e r n a t i v e l y , t h e r e m a y be less r e v e r s i b i l i t y of liver function a f t e r successful t r e a t m e n t of chronic active h e p a t i t i s C. O t h e r s h a v e n o t e d imp r o v e m e n t in h e p a t i c m e t a b o l i c f u n c t i o n d e t e r m i n e d b y lidocaine m e t a b o l i s m , u s i n g t h e M E G X test, in p a t i e n t s w i t h v a r i o u s t y p e s of chronic h e p a t i t i s (including h e p a titis C) a f t e r successful t r e a t m e n t . 27 I n t h a t study, t h e r e w a s a c o r r e l a t i o n b e t w e e n histologic i m p r o v e m e n t a n d M E G X c h a n g e , w h e r e a s in t h e c u r r e n t s t u d y such correlations between lobular changes and APC were extremely tenuous. Among the several explanations for t h e s e different r e s u l t s is t h e inclusion of a m i x e d d i s e a s e p o p u l a t i o n in t h e M E G X study, b e c a u s e t h e c u r r e n t r e s u l t s w i t h A P C in chronic h e p a t i t i s C a r e q u a l i t a t i v e l y a n d q u a n t i t a t i v e l y different f r o m o u r own o b s e r v a t i o n s in chronic h e p a t i t i s B a n d a u t o i m m u n e chronic active h e p a t i t i s , as e l a b o r a t e d above. T h e d e l a y e d o c c u r r e n c e of a l t e r a t i o n s of A P C in relation to n o r m a l i z a t i o n of A L T levels w a s also u n e x p e c t e d a n d differs f r o m o t h e r t y p e s of chronic active h e p a t i t i s in w h i c h i n c r e a s e s in A P C a p p e a r e d to be c o n c o m i t a n t w i t h c h a n g e s in ALT. 19'26 P e r s i s t e n c e of t h e h e p a t i c i n f l a m m a t o r y r e s p o n s e for s e v e r a l m o n t h s a f t e r resolution of h e p a t i c necrosis m a y p a r t l y a c c o u n t for this, a n d would be in accord w i t h t h e r e s u l t s of histological studies. 2~'7 I t is also i n t r i g u i n g t h a t i m p r o v e m e n t of A P C w a s o b s e r v e d s e v e r a l m o n t h s a f t e r r e l a p s e of h e p atitis C a m o n g t h o s e who h a d only a t e m p o r a r y res p o n s e to i n t e r f e r o n . I f t h i s o b s e r v a t i o n is confirmed, it m a y be feasible a n d a p p r o p r i a t e , in selected cases, to slow t h e clinicopathologic p r o g r e s s i o n of chronic active h e p a t i t i s C b y t r e a t m e n t t h a t fails to p r o d u c e v i r a l cure. T h i s concept differs f r o m t h e c u r r e n t a p p r o a c h t h a t s t r i v e s to produce v i r a l eradication. C o n v e r s e l y , t h e b e n e f i t s to liver function of s u s t a i n e d n o r m a l i z a t i o n of h e p a t i c n e c r o i n f l a m m a t o r y activity, as i n d i c a t e d b y s e r u m ALT, w e r e d e m o n s t r a t e d in t h e c u r r e n t study. T h i s p r o v i d e s f u r t h e r t a n g i b l e evidence for t h e b e n e f i t s of successful i n t e r f e r o n t r e a t m e n t . I n s u m m a r y , serial d e t e r m i n a t i o n of A P C i n d i c a t e d t h a t h e p a t i c m e t a b o l i c function is a m a j o r d e t e r m i n a n t of r e s p o n s i v e n e s s to i n t e r f e r o n t r e a t m e n t a m o n g pat i e n t s w i t h chronic active h e p a t i t i s C. I t is p r o p o s e d t h a t t h i s l a r g e l y e x p l a i n s t h e i m p o r t a n c e of cirrhosis as a p r e d i c t o r of t r e a t m e n t response. A f t e r a s h o r t - t e r m r e s p o n s e to i n t e r f e r o n t r e a t m e n t , APC i n c r e a s e d , e v e n a m o n g i n d i v i d u a l s w h o e x p e r i e n c e d r e l a p s e of t h e i r h e p a t i t i s . T h i s i m p r o v e m e n t in A P C w a s s m a l l a n d d e l a y e d in a p p e a r a n c e , b u t it a p p e a r e d to be s u s t a i n e d a m o n g cases w i t h a l o n g - t e r m r e s p o n s e to interferon. C o n v e r s e l y , d e t e r i o r a t i o n of A P C could be d e t e c t e d
HEPATOLOGYOctober 1995 a f t e r 18 m o n t h s in t h o s e w h o r e l a p s e d a f t e r a n initial r e s p o n s e to i n t e r f e r o n t r e a t m e n t .
Acknowledgment: We g r a t e f u l l y a c k n o w l e d g e t h e a s s i s t a n c e of t h e I n s t i t u t e of Clinical P a t h o l o g y a n d Medical R e s e a r c h , W e s t m e a d H o s p i t a l , for p e r f o r m i n g t h e clinical c h e m i s t r y , v i r a l serology, a n d a n a t o m i c pathology r e q u i r e d for this work, to m a n y colleagues who r e f e r r e d p a t i e n t s for t h e s e studies, to t h e m e d i c a l a n d n u r s i n g s t a f f of W e s t m e a d H o s p i t a l w h o a s s i s t e d w i t h s a m p l e collections, a n d to o u r p a t i e n t s w h o s e i n t e r e s t a n d dedication m a d e t h i s project possible. REFERENCES
1. Farrell GC, Lin R, Coverdale S. Prediction of response to interferon treatment in patients with chronic active hepatitis C, and evidence that this improves hepatic metabolic function. Gastroenterologica Japonica 1991;26(suppl 3):243-246. 2. Davis GL, Balart LA, Schiff EA, Lindsay K, Bodenheimer HC, Perrillo RP, Carey W, et al. Treatment of chronic active hepatitis C with recombinant interferon alfa: a multicenter randomized, controlled trial. N Engl J Med 1989;321:1501-1506. 3. Di Bisceghe AM, Martin P, Kassianides C, Lisker-Melman M, Murray L, Waggoner J, Goodman Z, et al. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, doubleblind, placebo-controlled trial. N Eng] J Med 1989;321:15061510. 4. Causse X, Godinot H, Chevallier M, Chossegros P, Zoulim F, Ouzan D, Heyraud J-P, et al. Comparison of 1 or 3 MU of interferon alfa-2b and placebo in patients with chronic non-A, non-B hepatitis. Gastroenterology 1991; 101:497-502. 5. Davis GL. Recombinant alfa-interferon treatment of non-A, nonB (type C) hepatitis: review of studies and recommendations for treatment. J Hepatol 1990; 11:$72-$77. 6. Farrell GC. Treatment of chronic hepatitis C with alpha-interferon. J Gastroenterol Hepatol 1991;6(suppl 1):36-40. 7. Lin R, Schoeman MN, Bilous M, Grierson J, Craig PI, MacDonald J, Batey RG, et al. Can response to interferon treatment be predicted in patients with chronic active hepatitis C? Aust N Z J Med 1991;21:387-392. 8. Di Bisceglie A, Hoofnagle JH. Therapy of chronic hepatitis C with a-interferon: the answer? or more questions? HEPATOLOGY 1991; 13:601-603. 9. Camps J, Crisdstomo S, Garc~a-Granero M, Riezu-Boj JI, Civiera MP, Prieto J. Prediction of the response of chronic hepatitis C to interferon alfa: a statistical analysis of pretreatment variables. Gut 1993;34:1714-1717. 10. Benelux Multicentre Trial Study Group. Benelux multicentre trial of alpha interferon treatment for chronic hepatitis C: standard v high dose treatment monitored by biochemical and virological markers (interim analysis). Gut 1993;34(suppl):Sl19$120. 11. Camma C, Craxi A, Tine F, Almasio P, Di Marco V, Lo Iacono O, Bruno R, et al. Predictors of response to alpha-interferon (IFN) in chronic hepatitis C: a multivariate analysis on 361 treated patients [Abstract]. HEPATOLOGY1992; 16:131A. 12. Lin R, Zimmerman M, Roach E, Farrell GC. Alpha-interferon 2b in the treatment of chronic active hepatitis C: interim report of the first multicenter Australian trial [Abstract]. HEPATOLOGY 1992; 16:75A. 13. Schoeman MN, Liddle C, Bilous M, Grierson J, Craig PI, Batey RG, Farrell GC. Chronic non-A, non-B hepatitis: a lack of correlation between biochemical and morphological activity and effects of immunosuppressive therapy. Aust N Z J Med 1990;20:56-62. 14. Farrell GC, Weltman M, Dingley J, Lin R. Epidemiology of hepatitis C virus infection in Australia. Gastroenterologica Japonica 1993;28:32-36. 15. Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991;13:372-374. 16. Okamoto H, Okada S, Sugiyama Y, Yotsumoto S, Tanaka T,
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17.
18. 19.
20.
Yoshizawa H, Tsuda F, et al. The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med 1990;60:167-177. Davis GL, Lau JY-N, Urdea MS, Neuwald PD, Wilber JC, Lindsay K, Perrillo RP, et al. Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: definition of optimal conditions for specific collection and clinical application in interferon-treated patients. HEPATOLOGY1994; 19:1337-1341. Farrell GC, Zaluzny L. Accuracy and clinical utility of modified tests of antipyrine metabolism. Br J Clin Pharmacol 1984; 18:559-565. Williams SJ, Farrell GC. Serial antipyrine clearance studies detect altered hepatic metabolic function during spontaneous and interferon-induced changes in chronic hepatitis B disease activity. HEPATOLOGY1989; 10:192-197. Fiatarone JR, Coverdale SA, Batey RG, Farrell GC. Non-alcoholic steatohepatitis: impaired antipyrine metabolism and hypertriglyceridaemia may be clues to its pathogenesis. J Gastroenterol Hepatol 1991;6:585-590.
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21. Armitage P, Berry G. Further analysis of qualitative data. In: Armitage P, Berry G, eds. Statistical methods in medical research. Oxford: Blackwell Scientific Publications, 1987:371-407. 22. Hart P, Farrell GC, Cooksley WGE, Powell LW. Enhanced drug metabolism in cigarette smokers. Br Med J 1976;2:147-149. 23. Vestal RE, Norris AH, Tobin JD, Cohen BH, Shock NW, Andres R. Antipyrine metabolism in man: Influence of age, alcohol, caffeine and smoking. Clin Pharmacol Ther 1975; 18(4)425-432. 24. Williams SJ, Farrell GC. Inhibition of antipyrine metabolism by interferon. Br J Clin Pharmacol 1986;22:610-612. 25. Reichen J. MEGX test in hepatology: the long-sought ultimate quantitative liver function test? J Hepatol 1993; 19:4-7. 26. Coverdale S, Farrell GC. Serial antipyrine clearance in autoimmune chronic active hepatitis: potential to recognize patients with a poor prognosis [Abstract]. HEPATOLOGY1992; 16:525. 27. Shiffman ML, Luketic VA, Sanyal AJ, Duckworth PF, Purdum PP, Contos MJ, Mills AS, et al. Hepatic lidocaine metabolism and liver histology in patients with chronic hepatitis and cirrhosis. HEPATOLOGY1994; 19:933-940.
BYTH, JACQUELINE FIELD, CHRISTOPHER LIDDLE, RITA LIN, AND GEOFFREY C, FARRELL
0.12 to 0.98; normal range, 0.34 to 1.02 m L / r n i r t / k g body wt), a value that was significantly greater than in 30 n o n r e s p o n d e r s (0~23; 0.08 to 0.67 mlJmin/kg body wt, P < .001). APC was closely associated with response to interferon. The r e s p o n s e rate a m o n g cases with values
months, w h e r e a s a m o n g relapsers (defined b y ALT increase), APC eventually declined to be less than the pretreatment value at 24 months. It is c o n c l u d e d that pretreatment APC is a powerful positive predictor of r e s p o n s i v e n e s s to interferon treatment in patients with chronic hepatitis C, indicating that good hepatic metabolic function m a y be important in determining the effectiveness of interferon treatment. Furthermore, changes in hepatic metabolic function after apparently successful treatment, at least as d e t e r m i n e d by APC, appear to be subtle, delayed in onset, and m a i n t a i n e d o n l y in those w h o remain in biochemical remission. (HEPA-
>0.25 mL/mirt/kg b o d y w e i g h t w a s 79%, the same as in
TOLOGY1995;22:1065-1071.)
To determine w h e t h e r hepatic metabolic function affects the r e s p o n s e to interferon treatment, w e m e a s u r e d
antipyrine clearance (APC) in 85 patients with chronic active hepatitis C and c o m p a r e d the results w i t h treatment outcome. A m o n g 55 patients w h o r e s p o n d e d to interferon b y n o r m a l i z a t i o n o f alAnlne transAminase (ALT), m e d i a n APC before treatment w a s 0.47 (range,
cases without cirrhosis. Cases w i t h o u t cirrhosis and with APC of >0.25 mL/min/kg body w e i g h t h a d an 85% chance of responding to interferon; this w a s unlikely a simple reflection o f histological activity, b e c a u s e the correlation w i t h S c h e u e r score w a s poor in this subgroup (r = -.31, P < .05). A second, independent group of 43 patients w a s u s e d to test the predictive value of APC (using 0.25 mL/min/kg body wt as a cut~ff) for response to interferon treatment. In this group, APC correctly predicted positive r e s p o n s e to interferon in 75% of cases. APC w a s also u s e d to m e a s u r e the effects of treatment o n hepatic metabolic function. Regardless of outcome, there w a s no change in APC at the end o f a 6month course of interferon treatment. Six m o n t h s later, however, i m p r o v e m e n t in APC (14%; P < .05) was evident a m o n g responders but not in those w h o h a d failed to r e s p o n d to interferon. In patients w h o c o n t i n u e d to h a v e normal ALT (18 o f 19 tested were also nonviremic), the i m p r o v e m e n t in APC w a s sustained for at least 24
Abbreviations: ALT, alanine transaminase; APC, antipyrine clearance. From the Storr Liver Unit, Department of Medicine, University of Sydney, and the Department of Gastroenterology and Hepatology, Westmead Hospital, Westmead, NSW, Australia. Received August 15, 1994; accepted May 30, 1995. Supported in part by the Robert W. Storr endowment for liver research. The clinical trials from which these subsets of patients were drawn were supported by Schering-Plough International, Kenilworth, NJ, and its Australian branch, Baulkham Hills, NSW. Presented in part at the Annual Scientific Meeting of the American Association for Study of the Liver, Chicago, IL, November 1993, and has been published in abstract form (HEPATOLOGY1993;18:228A). An earlier pilot study has been published in extended abstract form (reference 1). Address reprint requests to: Professor Geoffrey C. Farrell, Storr Liver Unit, Department of Medicine, Westmead Hospital, Westmead, NSW 2145, Australia. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2204-000753.00/0
A large number of randomized controlled trials have delineated the response rates to interferon t r e a t m e n t of patients with chronic active hepatitis C. 2"9 The outstanding issues concern the suboptimal durability of the interferon response and the health economics of treatment, given t h a t the n a t u r a l history of liver disease caused by hepatitis C virus infection has a prolonged course and a variety of possible outcomes. Efforts to improve the "efficiency" of interferon t r e a t m e n t might follow from more accurate prediction of the responsiveness in various types of cases. Variables t h a t are related to t r e a t m e n t outcome probably include viral determinants, patient characteristics, and the severity of the associated liver disease. In relation to the latter, we and others have identified absence of cirrhosis as a reproducible and informative predictor of response to interferon t r e a t m e n t in hepatitis C as defined by shortterm resolution of serum alanine transaminase (ALT) abnormalities. 4'v'9-11 The response rate is approximately 75% in the absence of cirrhosis and 30% to 40% among cases with cirrhosis. The poorer response to interferon treatment among patients with chronic active hepatitis C and cirrhosis could be due to several reasons. These include longer duration of infection, greater viral load or more virulent genotype, hemodynamic alterations that reduce hepatic availability of interferon, and phenotypic changes of hepatocytes that alter the cellular response to interferon. The present study was designed to examine the proposal that impaired hepatic metabolic function may be the critical change in cirrhosis that determines responsiveness to interferon treatment. Therefore, we measured antipyrine clearance (APC), a sensitive marker of intrinsic hepatic
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HEPATOLOGYOctober 1995
TABLE 1. D e m o g r a p h i c Characteristics and P h a r m a c o k i n e t i c Indices in the Two Patient Groups Patient Group
Main group (n = 85) Second group (n = 43)
Age (yrs)
Sex M/F
Smokers
APC (mL/min/kg)
VD (L/kg)
Half-life (hr)
44 (37-53) 37 (33-53)
56/29
31 (36%) 15 (35%)
0.37 (0.22-0.53) 0.39 (0.26-0.50)
0.57 (0.50-0.63) 0.57 (0.51-0.62)
18.5 (12.5-30.5) 16.0 (13.7-23.8)
28/15
NOTE. Data are expressed as median (interquartile range). Abbreviation: VD, volume of distribution.
d r u g clearance, a m o n g patients with chronic active hepatitis C. Studies were initially performed before administration of interferon a n d the results were later correlated with the outcome of treatment, as well as with the quantitative a s s e s s m e n t of liver histology. The a p p a r e n t predictive value of APC as an indicator of t r e a t m e n t outcome was t h e n tested in a second independent group of cases. I n addition, serial studies of APC at the end of t r e a t m e n t and at 6- or 12-month intervals thereafter were performed to determine w h e t h e r a p p a r e n t l y successful interferon t r e a t m e n t improved hepatic metabolic function as determined by this test. PATIENTS AND METHODS Clinical M a t e r i a l . The 85 patients who participated in the major part of this study (i.e., serial &PC testing) were enrolled in two interferon trials, one of which has been reported in full, 7 the other in preliminary form. TM There were 56 men and 29 women, and their median age was 44 years (range, 27 to 73 years). These 85 patients represented 52% of all the patients from this center enrolled in these trials. Selection into the present study was on the basis of logistic considerations, as follows: (1) the patient had been randomized to receive treatment by a protocol that was suitable for the present study (see below, and Table 1), (2) the patient completed treatment and was available to attend for subsequent studies, (3) the patient was not taking substances known to alter &PC, with the exception of cigarette smoke (see below). All patients gave informed consent. The study conformed to current international guidelines for human experimentation and was approved by the Human Ethics Committee of the Western Sydney Area Health Service. The risk factors for acquisition of hepatitis C were similar to those previously reported for Australian patients. 7'12"14Patients taking therapeutic or recreational drugs that are known to influence &PC were excluded, with the exception of cigarette smokers. Thus, 31 subjects smoked cigarettes, and 54 individuals did not (<5 cigarettes per day) (Table 1). There was no change in the number of cigarettes smoked per day during the study. Marijuana smokers were excluded, as were patients enrolled in methadone programs. No patients were known to regularly consume more than 20 g of ethyl alcohol each day. A second group of 43 patients was used to test independently the predictive accuracy of APC as an indicator of response to interferon treatment. These patients included 10 subjects enrolled in the above two clinical trials who did not have subsequent APC studies, and 33 others who were treated by the same protocols outside of these clinical trials. The demographic and clinical characteristics of patients in this second group were similar to those in the first group (Table 1).
All patients (in both groups) had histologically proven chronic active hepatitis, as determined by liver biopsy specimen appearances within 6 months before starting interferon. The presence or absence of cirrhosis was also noted on this biopsy specimen but was not known to the investigators at the time APC studies were performed. In coded biopsy slides, the severity of hepatitis was graded by one of us (GCF) according to the Scheuer scoring system. 15From the main group of 85 patients, 78 pretreatment and 55 posttreatment biopsy specimens were available for scoring. All patients were antiHCV positive by either first-generation (31%) or second-generation (69%) enzyme-linked immunosorbance assay test (Orthodiagnostics Systems; Raritan, NJ). Among 61 cases tested for the presence of HCV-RNA in serum, 36 (59%) were positive, as determined by reverse transcriptase polymerase chain reaction. The reverse transcriptase polymerase chain reaction assay employed primers located in the highly conserved 5'-noncoding region of the HCV genome; the specificity of this assay was confirmed by Southern hybridization with a probe that lies internal to the amplified fragment. TM Other causes of chronic liver disease were systematically excluded, as described elsewhere. 7All patients had persistent elevation of serum ALT levels, which were at least twice the upper limit of the normal laboratory range and had been present for at least 6 months before enrollment. Interferon Treatment. Patients were enrolled into two randomized controlled trials of interferon-a2b (Intron A, Schering-Plough International, Kenilworth, NJ) (interferon). In the first trial, 7 patients were randomized to receive either 3 MU interferon thrice weekly by subcutaneous injection for 6 months, or to receive no treatment; only patients in the treatment arm are considered here (n = 26). In the second trial, TM there were three treatment arms, but only those patients who received 3 MU interferon thrice weekly for 6 months (n = 35) or 5 MU interferon thrice weekly for 6 months (n = 24) are considered here. Patients were assessed clinically on several occasions before randomization into the study and were reviewed every 4 weeks during interferon treatment, at which time blood was taken for determination of liver tests and hematologic parameters. Short-term response to interferon was defined as a return of ALT to normal levels during the first 4 months of interferon treatment, and persistance of normal ALT thereafter for the duration of treatment. Relapse was said to have occurred when ALT abnormality (any extent) was noted after stopping interferon treatment. As expected, 17 biochemical relapse could always be correlated with return to positivity of serum HCV-RNA (Lin R, Liddle C, Farrell G, Unpublished data, 1994). To determine long-term response to interferon treatment, patients were observed at 4-weekly intervals for the first 6 months after completion of interferon treatment. Blood was taken at these times for determination of liver tests. A long-
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TABLE 2. R e s p o n s e R a t e s A c c o r d i n g to P r e t r e a t m e n t C h a r a c t e r i s t i c s i n t h e F i r s t G r o u p o f 85 P a t i e n t s APC (mL/min/kg body wt)
APC -< 0.25 APC > 0.25 Total
Liver Histology Cirrhosis
No Cirrhosis
Total
8/22 (36%) 5/10 (50%)
3/7 (43%) 39/46(85%)
11/29 (38%) 44/56(79%)
13/32 (41%)
42/53 (79%)
55/85(65%)
term response to interferon treatment was defined as continued normality of ALT after discontinuation of interferon and until the end of the period of follow-up (at least 6 months). In this study, no relapses were seen later than 4 months after completion of the 6-month course of interferon treatment. Eighteen of 19 tested long-term responders have remained negative for HCV-RNA in serum (Lin R, Liddle C, Farrell G; Unpublished data, 1994). APC Studies. APC was performed in ambulant patients at the following times: (1) within 7 days before starting interferon; (2) at completion of 6 months of interferon treatment, always 7 to 10 days after the last dose of interferon; (3) at 6or 12-monthly intervals thereafter. On each occasion that APC was determined, ALT and other conventional liver tests were measured. In the long-term study, the number of patients examined at each time point is indicated in the figures or figure legends. APC was determined after oral administration of antipyrine (10 mg/kg body weight) given to nonfasted individuals between 8 and 11 AM. Two venous blood samples were collected, the first at 4 hours and the second 24 hours after antipyrine ingestion. Antipyrine was estimated in serum by a sensitive and accurate high-pressure liquid chromatography method, as previously described, ls'19 and clearance was determined from the two concentration/time points. In other studies 1z'2° (and Coverdale S, Farrell G, Unpublished data, 1993), we have noted in healthy subjects and in patients with clinically stable types of liver disease that APC values for any individual do not vary by more than 10% when measured repeatedly during a 2-year interval. Expression of Data and S t a t i s t i c a l Methods. Because of the skewed distribution of the APC results, data are presented in the figures as box and whisker plots, with the median, 25th, 50th, and 75th percentiles indicated (see legends to figures). Logistic regression was used to assess the predictive value of APC and liver histology in relation to a short-term response to interferon. Logistic discriminant analysis was used to determine the cut-off value for APC levels in predicting response to interferon. 21 For this analysis, APC was considered as a continuous dependent variable and response as a dichotomous outcome variable. The performance of the cut-offvalue was independently validated using the second independent series of 43 patients (see Clinical Material). To compare changes in APC over time, Wilcoxon tests were employed. A P value of <.01 was regarded as statistically significant. This is the corrected value after employing the Bonferroni adjustment for five multiple comparisons to the usual P < .05. Spearman's rank correlation was used to test the correlations between liver histology and APC. RESULTS
Response to Interferon Treatment: R e l a t i o n s h i p to Cirrhosis, P r e t r e a t m e n t APC, a n d Conventional L i v e r Tests. T h e s h o r t - t e r m r e s p o n s e r a t e to i n t e r f e r o n t r e a t -
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m e n t for all 85 p a t i e n t s i n c l u d e d in t h e first a n t i p y r i n e s t u d y g r o u p w a s 65%. A m o n g cases c a t e g o r i z e d according to p r e t r e a t m e n t l i v e r biopsy, 79% of t h o s e without cirrhosis r e s p o n d e d to i n t e r f e r o n , c o m p a r e d w i t h 41% of t h o s e w i t h cirrhosis (Table 2). As expected, t h e s e r e s u l t s a r e v e r y s i m i l a r to t h o s e r e p o r t e d for t h e two t r i a l s f r o m w h i c h t h e s u b s e t of p a t i e n t s for a n t i p y r i n e s t u d i e s w e r e d r a w n . 7'12 A m o n g t h e 55 cases w i t h a s h o r t - t e r m r e s p o n s e to i n t e r f e r o n t r e a t m e n t , A P C w a s 0.47 m L / m i n / k g body w e i g h t ( m e d i a n ; range: 0.12 to 0.98), w h i c h w a s h i g h e r t h a n in 30 n o n r e s p o n d e r s (0.23; 0.08 to 0.67; P < .001). I t is a p p a r e n t f r o m i n s p e c t i o n of Fig. 1 t h a t t h r e e - q u a r t e r s of r e s p o n d e r s h a d A P C v a l u e s w i t h i n t h e n o m i n a l n o r m a l r a n g e for t h i s l a b o r a t o r y , w h e r e a s t h r e e - q u a r t e r s of n o n r e s p o n d e r s h a d v a l u e s t h a t w e r e below t h e lower l i m i t of t h e n o r m a l r a n g e . T h e s e d a t a w e r e u s e d to c a l c u l a t e t h e s t r e n g t h of associations between individual characteristics and t r e a t m e n t outcome. F o r t h i s p u r p o s e , l i v e r histology (cirrhosis or not cirrhosis, a n d t o t a l S c h e u e r score) a n d A P C w e r e considered. Logistic d i s c r i m i n a n t a n a l y s i s s u g g e s t e d t h a t A P C > 0.25 w a s a s s o c i a t e d w i t h response. A m u l t i v a r i a t e logistic m o d e l i n c o r p o r a t i n g histology a n d APC (-<0.25 vs. > 0 . 2 5 ) w a s fitted. O n l y A P C a n d cirrhosis w e r e i n d e p e n d e n t l y a s s o c i a t e d w i t h t r e a t m e n t r e s p o n s e . T h u s , 85% of p a t i e n t s w i t h b o t h f a v o r a b l e c h a r a c t e r i s t i c s (no cirrhosis a n d A P C > 0.25) h a d a s h o r t - t e r m r e s p o n s e to i n t e r f e r o n ; 54% of all cases fell into t h i s c a t e g o r y (Table 2). As r e p o r t e d elsew h e r e , 25'7'911 no o t h e r b i o c h e m i c a l indices of liver dise a s e w e r e a s s o c i a t e d w i t h r e s p o n s e to i n t e r f e r o n t r e a t ment. T h e possibility t h a t A P C m a y be m o r e closely r e l a t e d to histologic p r e s e n c e of n e c r o i n f l a m m a t o r y activity t h a n to t h e p r e s e n c e or a b s e n c e of cirrhosis w a s e x a m i n e d u s i n g t h e S c h e u e r score. 15 A l t h o u g h t h e r e w a s a
1.0
°e- °sl "
o
0.41=1
T
Nonresponders n=30
Responders n=S5
FIG. 1. Pretreatment APC in short-term responders and nonresponders to interferon treatment. In these %ox and whisker" plots, the bar within each column represents the median value, the upper and lower borders of the box are the quartiles, and the "whiskers" (error bars) at the extremities indicate the 10th and 90th percentiles. APC in responders is significantly higher than in nonresponders (P < .001).
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TABLE 3. R e s p o n s e R a t e s A c c o r d i n g t o P r e t r e a t m e n t C h a r a c t e r i s t i c s i n t h e S e c o n d G r o u p o f 43 P a t i e n t s Liver Histology
APC (mL/min/kg body wt)
Cirrhosis
No Cirrhosis
Total
APC -< 0.25 APC > 0.25
4/9 (44%) 1/5 (20%)
2/2 (100%) 23/27 (85%)
6/11 (55%) 24/32 (75%)
Total
5/14 (36%)
25/29 (86%)
30/43 (70%)
weak correlation between the total Scheuer score and APC (r = -.55, P < .001), total Scheuer score was not an independent predictor of response. Thus, there was minimal difference in median total Scheuer score between responders and nonresponders (respectively, 7 and 8, P = .05). In the 53 patients without cirrhosis, there was no difference in total, portal, or lobular scores or in APC between responders and nonresponders. The correlation between total Scheuer score and APC was much weaker in this group (r = -.31, P < .05). Predictive Value of APC for Response to Interferon Treatment. To test the predictive value of APC as an indicator of response to interferon treatment, a second similar group of 43 patients with chronic active hepatitis C was studied. In this group, the overall short-term response rate to interferon was 70% (Table 3). Among the 32 cases with APC > 0.25 mL/min/kg, 24 had a short-term response to interferon treatment, giving a predictive accuracy for APC of 75%. In this group, the positive predictive accuracy of no cirrhosis was 86% (Table 3). Effects of Cigarette Smoking on APC. As expected from the known effects of cigarette smoking on antipyrine metabolism, 22'23 the pretreatment APC of smokers was higher t h a n t h a t of nonsmokers (median, 0.52; range, 0.13 to 0.98; n = 31; vs. 0.29 [0.08 to 0.92], n = 54, P < .001). The proportion of cigarette smokers appeared to be similar among short-term responders (22 of 55), long-term responders (12 of 27), and nonresponders (9 of 30). There were no significant differences between the percentage changes in APC after treatment between smokers and nonsmokers at any of the times examined (see below). According to smoking status, the "cut-off' APC values t h a t best predicted a response to interferon t r e a t m e n t were respectively 0.31 mL/min/kg for smokers and 0.21 mL/min/kg for nonsmokers. Effects of Interferon Treatment on APC. Previous studies from this laboratory have demonstrated t h a t administration of interferon is itself associated with impairment of A P e . 24 Thus, preliminary studies of APC performed during interferon t r e a t m e n t appeared to be uninformative, as reported elsewhere. 1 Specifically, among 65 patients with &PC performed ->3 but <6 months into a course of interferon treatment, APC increased in 17 (26%), decreased in 23 (35%), and was unchanged in 25 (38%). There was no significant difference in &PC before versus during t r e a t m e n t for the group as a whole nor for the 39 responders or 26 nonre-
sponder subgroups. Studies after completion of interferon t r e a t m e n t were performed 7 to 10 days after the last dose to avoid interferon effects and to precede the onset of relapse. At this time, there was no change in APC compared with the pretreatment value, irrespective of the apparent response during t r e a t m e n t or the subsequent clinical course (Fig. 2). In contrast, the results obtained 6 months after stopping interferon showed a clear improvement in APC in those who had exhibited a short-term response (14%, P < .05) but no improvement in those who did not respond to treatment (Fig. 2). Subsequent Changes of APC. Among patients who had been studied for ->6 months after stopping interferon treatment, the results indicated different patterns of change of APC according to the clinical course. Among short-term responders who experienced relapse, this occurred at a median time of 6 weeks after completion of interferon, t h a t is, 18 weeks before the third APC study (Fig. 3; see 6-month study). Decline of APC values was evident among these cases (Fig. 3A). Thus, by 18 months after the initial study (i.e., 12 months after completion of interferon treatment), values had returned to pretreatment levels. F u r t h e r decline of APC to values t h a t were significantly less t h a n before t r e a t m e n t was evident by 24 months (P < .01) (Fig. 3A). In contrast, long-term response to interferon treatment was associated with an apparent increase in APC
100 U
80
Q.
60 =,,,, • -
~u
40
0
-2o -4o n=29 n=52 End of t r e a t m e n t
n=21 n=32 6 months later
FIG. 2. Change in APC at end of interferon t r e a t m e n t a n d at 6 months after completion of treatment. Graphs show the change of APC, expressed as a percentage of the p r e t r e a t m e n t value, for responders and nonresponders at the end of t r e a t m e n t (i.e., within 7 to 10 days of stopping interferon) and 6 months later (this period corresponds to 12 months after the initial measurement). Short-term responders are indicated by shaded columns, nonresponders by open columns. To improve visual presentation, four outlying values are not shown on the figure; these were for two responders at end of t r e a t m e n t (112%, 144%) a n d two responders 6 months later (129%, 237%). The explanation for the style of plot is indicated in the legend to Fig. 1. The n u m b e r of patients studied in each group and at each time is indicated (see n values). There were no differences between values for nonresponders at either time, or for responders at end of t r e a t m e n t compared with before treatment. The difference between responders at 6 months compared with before t r e a t m e n t is significant (P < .05).
HEPATOLOGY Vol. 22, No. 4, 1995
t 1001 .E
"
O
COVERDALE ET AL
A
O
6O 40
-20 -40
F-
1
B ¢j Q. < ¢ .E
120 100 80 60
o
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40"
20" u 0 ~¢ -zo. -40' "
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1'2
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:;4
Time after stopping interferon (months) Fro. 3. Serial APC among short-term responders to interferon: comparison of relapsers with long-term responders. These data are presented in the same format as Fig. 2. (A) Patients who relapsed after stopping interferon. (B) Long-term responders. The numbers of patients studied at each time (n) are indicated within the boxes. Four outlying values are not shown in (B); these were 0 months, 144%; 6 months, 237%; 12 months, 162%; 18 months, 156%. *Significantly different from p r e t r e a t m e n t value, P < .01. The a p p a r e n t increases in APC among long-term responders (B) are not significant (respectively, 6 months, P = .15; 12 months, P = .11; 18 months, P = .16).
that was sustained throughout the study period (Fig. 3B). Among cases with no response to interferon, there was no consistent change in APC after the initial study (data not shown). It must be borne in mind that these cases exhibited much lower values for APC at the time of entry into the study (Fig. 1). APC studies were discontinued in six patients because of definite or suspected adverse effects of antipyrine. These were all suggestive of allergy and included facial flushing in two, rash in one, conjunctivitis in one, rash plus conjunctivitis in one, and angioedema of lips in one. All adverse effects occurred after multiple (->3) exposures to antipyrine. DISCUSSION
The novel feature of this study is that it reports the relationship between hepatic metabolic function, as determined serially by a quantitative liver function test, and the response to interferon treatment among pa-
1069
tients with chronic active hepatitis C. The results are interpreted as indicating that hepatic metabolic function is a determinant of interferon responsiveness in this disease. Moreover, changes in liver function occur after treatment with interferon b u t appear to be subtle and delayed. Several viral, demographic, and clinicopathologic determinants of responsiveness to interferon treatment have been identified in hepatitis C, but none correlate as strongly as morphological severity of liver disease. Thus, several studies have confirmed the initial observation 7 that the response among patients without cirrhosis is approximately twice that of those with cirrhosis. 4'9"11 The current findings are consistent with the proposal that response to interferon treatment is suboptimal in cirrhosis because of its associated impairment of hepatic metabolic function. Thus, APC was at least as good at predicting response to interferon as was the presence or absence of cirrhosis. The question arises as to the utility of APC in individual cases. At least half the patients in these two studies had a value for APC (>0.25 mL/kg/min) that allowed prediction of a 75% chance of response, which is as good as that for absence of cirrhosis. Thus, the APC would be particularly useful if information were not available about liver histology, and although our study suggests that it could improve positive predictive value if histology (presence or absence of cirrhosis) is already known, confirmation of this will require study of larger groups of patients. We chose APC as the marker of hepatic metabolic function because of the ease of repeated application of this test during the natural history of a disease that usually continues for decades. There do not appear to be comparative data with other quantitative liver function tests, and it is therefore difficult to consider the relative merits of APC with other t e s t s Y Minor but potentially severe allergy to antipyrine occurred in 6 of the 128 patients in these two studies (5%). This rate is similar to our overall experience of adverse reactions to antipyrine (4%). However, the occurrence of drug allergy clearly restricts the applicability of repeated studies in these cases and underscores the importance of adequate explanation to potential study participants before obtaining informed consent. Further, concomitant intake of drugs and other foreign compounds may confound interpretation of the results of APC. 25 In the current study, a small number of cases were considered unsuitable for APC testing on this basis. It must also be noted that the cut-off values of pretreatment APC to predict response to interferon differed between smokers and nonsmokers. However, the general conclusions from the results did not appear to be altered when cigarette smokers were included in the analysis. An unexpected result of the current study was that beneficial effects of apparently successful interferon treatment on hepatic metabolic function were small, as determined by APC. Moreover, they appeared to be delayed in appearance. Thus, the increase in APC compared with pretreatment levels was 14% (range, - 3 2 %
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to 237%), I n c o n t r a s t , in chronic h e p a t i t i s B (46%) a n d a u t o i m m u n e chronic active h e p a t i t i s (79%), g r e a t e r inc r e a s e s h a v e b e e n e v i d e n t a f t e r r e s p o n s e to t r e a t m e n t w i t h i n t e r f e r o n or corticosteroids, respectively. 19'26 One possible r e a s o n for t h e less i m p r e s s i v e c h a n g e w i t h h e p a t i t i s C is t h a t m o s t r e s p o n d e r s h a v e only m i n i m a l i m p a i r m e n t of h e p a t i c m e t a b o l i c function, t h e r e b y reducing t h e likelihood t h a t c h a n g e s of g r e a t e r m a g n i t u d e c a n be observed. A l t e r n a t i v e l y , t h e r e m a y be less r e v e r s i b i l i t y of liver function a f t e r successful t r e a t m e n t of chronic active h e p a t i t i s C. O t h e r s h a v e n o t e d imp r o v e m e n t in h e p a t i c m e t a b o l i c f u n c t i o n d e t e r m i n e d b y lidocaine m e t a b o l i s m , u s i n g t h e M E G X test, in p a t i e n t s w i t h v a r i o u s t y p e s of chronic h e p a t i t i s (including h e p a titis C) a f t e r successful t r e a t m e n t . 27 I n t h a t study, t h e r e w a s a c o r r e l a t i o n b e t w e e n histologic i m p r o v e m e n t a n d M E G X c h a n g e , w h e r e a s in t h e c u r r e n t s t u d y such correlations between lobular changes and APC were extremely tenuous. Among the several explanations for t h e s e different r e s u l t s is t h e inclusion of a m i x e d d i s e a s e p o p u l a t i o n in t h e M E G X study, b e c a u s e t h e c u r r e n t r e s u l t s w i t h A P C in chronic h e p a t i t i s C a r e q u a l i t a t i v e l y a n d q u a n t i t a t i v e l y different f r o m o u r own o b s e r v a t i o n s in chronic h e p a t i t i s B a n d a u t o i m m u n e chronic active h e p a t i t i s , as e l a b o r a t e d above. T h e d e l a y e d o c c u r r e n c e of a l t e r a t i o n s of A P C in relation to n o r m a l i z a t i o n of A L T levels w a s also u n e x p e c t e d a n d differs f r o m o t h e r t y p e s of chronic active h e p a t i t i s in w h i c h i n c r e a s e s in A P C a p p e a r e d to be c o n c o m i t a n t w i t h c h a n g e s in ALT. 19'26 P e r s i s t e n c e of t h e h e p a t i c i n f l a m m a t o r y r e s p o n s e for s e v e r a l m o n t h s a f t e r resolution of h e p a t i c necrosis m a y p a r t l y a c c o u n t for this, a n d would be in accord w i t h t h e r e s u l t s of histological studies. 2~'7 I t is also i n t r i g u i n g t h a t i m p r o v e m e n t of A P C w a s o b s e r v e d s e v e r a l m o n t h s a f t e r r e l a p s e of h e p atitis C a m o n g t h o s e who h a d only a t e m p o r a r y res p o n s e to i n t e r f e r o n . I f t h i s o b s e r v a t i o n is confirmed, it m a y be feasible a n d a p p r o p r i a t e , in selected cases, to slow t h e clinicopathologic p r o g r e s s i o n of chronic active h e p a t i t i s C b y t r e a t m e n t t h a t fails to p r o d u c e v i r a l cure. T h i s concept differs f r o m t h e c u r r e n t a p p r o a c h t h a t s t r i v e s to produce v i r a l eradication. C o n v e r s e l y , t h e b e n e f i t s to liver function of s u s t a i n e d n o r m a l i z a t i o n of h e p a t i c n e c r o i n f l a m m a t o r y activity, as i n d i c a t e d b y s e r u m ALT, w e r e d e m o n s t r a t e d in t h e c u r r e n t study. T h i s p r o v i d e s f u r t h e r t a n g i b l e evidence for t h e b e n e f i t s of successful i n t e r f e r o n t r e a t m e n t . I n s u m m a r y , serial d e t e r m i n a t i o n of A P C i n d i c a t e d t h a t h e p a t i c m e t a b o l i c function is a m a j o r d e t e r m i n a n t of r e s p o n s i v e n e s s to i n t e r f e r o n t r e a t m e n t a m o n g pat i e n t s w i t h chronic active h e p a t i t i s C. I t is p r o p o s e d t h a t t h i s l a r g e l y e x p l a i n s t h e i m p o r t a n c e of cirrhosis as a p r e d i c t o r of t r e a t m e n t response. A f t e r a s h o r t - t e r m r e s p o n s e to i n t e r f e r o n t r e a t m e n t , APC i n c r e a s e d , e v e n a m o n g i n d i v i d u a l s w h o e x p e r i e n c e d r e l a p s e of t h e i r h e p a t i t i s . T h i s i m p r o v e m e n t in A P C w a s s m a l l a n d d e l a y e d in a p p e a r a n c e , b u t it a p p e a r e d to be s u s t a i n e d a m o n g cases w i t h a l o n g - t e r m r e s p o n s e to interferon. C o n v e r s e l y , d e t e r i o r a t i o n of A P C could be d e t e c t e d
HEPATOLOGYOctober 1995 a f t e r 18 m o n t h s in t h o s e w h o r e l a p s e d a f t e r a n initial r e s p o n s e to i n t e r f e r o n t r e a t m e n t .
Acknowledgment: We g r a t e f u l l y a c k n o w l e d g e t h e a s s i s t a n c e of t h e I n s t i t u t e of Clinical P a t h o l o g y a n d Medical R e s e a r c h , W e s t m e a d H o s p i t a l , for p e r f o r m i n g t h e clinical c h e m i s t r y , v i r a l serology, a n d a n a t o m i c pathology r e q u i r e d for this work, to m a n y colleagues who r e f e r r e d p a t i e n t s for t h e s e studies, to t h e m e d i c a l a n d n u r s i n g s t a f f of W e s t m e a d H o s p i t a l w h o a s s i s t e d w i t h s a m p l e collections, a n d to o u r p a t i e n t s w h o s e i n t e r e s t a n d dedication m a d e t h i s project possible. REFERENCES
1. Farrell GC, Lin R, Coverdale S. Prediction of response to interferon treatment in patients with chronic active hepatitis C, and evidence that this improves hepatic metabolic function. Gastroenterologica Japonica 1991;26(suppl 3):243-246. 2. Davis GL, Balart LA, Schiff EA, Lindsay K, Bodenheimer HC, Perrillo RP, Carey W, et al. Treatment of chronic active hepatitis C with recombinant interferon alfa: a multicenter randomized, controlled trial. N Engl J Med 1989;321:1501-1506. 3. Di Bisceghe AM, Martin P, Kassianides C, Lisker-Melman M, Murray L, Waggoner J, Goodman Z, et al. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, doubleblind, placebo-controlled trial. N Eng] J Med 1989;321:15061510. 4. Causse X, Godinot H, Chevallier M, Chossegros P, Zoulim F, Ouzan D, Heyraud J-P, et al. Comparison of 1 or 3 MU of interferon alfa-2b and placebo in patients with chronic non-A, non-B hepatitis. Gastroenterology 1991; 101:497-502. 5. Davis GL. Recombinant alfa-interferon treatment of non-A, nonB (type C) hepatitis: review of studies and recommendations for treatment. J Hepatol 1990; 11:$72-$77. 6. Farrell GC. Treatment of chronic hepatitis C with alpha-interferon. J Gastroenterol Hepatol 1991;6(suppl 1):36-40. 7. Lin R, Schoeman MN, Bilous M, Grierson J, Craig PI, MacDonald J, Batey RG, et al. Can response to interferon treatment be predicted in patients with chronic active hepatitis C? Aust N Z J Med 1991;21:387-392. 8. Di Bisceglie A, Hoofnagle JH. Therapy of chronic hepatitis C with a-interferon: the answer? or more questions? HEPATOLOGY 1991; 13:601-603. 9. Camps J, Crisdstomo S, Garc~a-Granero M, Riezu-Boj JI, Civiera MP, Prieto J. Prediction of the response of chronic hepatitis C to interferon alfa: a statistical analysis of pretreatment variables. Gut 1993;34:1714-1717. 10. Benelux Multicentre Trial Study Group. Benelux multicentre trial of alpha interferon treatment for chronic hepatitis C: standard v high dose treatment monitored by biochemical and virological markers (interim analysis). Gut 1993;34(suppl):Sl19$120. 11. Camma C, Craxi A, Tine F, Almasio P, Di Marco V, Lo Iacono O, Bruno R, et al. Predictors of response to alpha-interferon (IFN) in chronic hepatitis C: a multivariate analysis on 361 treated patients [Abstract]. HEPATOLOGY1992; 16:131A. 12. Lin R, Zimmerman M, Roach E, Farrell GC. Alpha-interferon 2b in the treatment of chronic active hepatitis C: interim report of the first multicenter Australian trial [Abstract]. HEPATOLOGY 1992; 16:75A. 13. Schoeman MN, Liddle C, Bilous M, Grierson J, Craig PI, Batey RG, Farrell GC. Chronic non-A, non-B hepatitis: a lack of correlation between biochemical and morphological activity and effects of immunosuppressive therapy. Aust N Z J Med 1990;20:56-62. 14. Farrell GC, Weltman M, Dingley J, Lin R. Epidemiology of hepatitis C virus infection in Australia. Gastroenterologica Japonica 1993;28:32-36. 15. Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991;13:372-374. 16. Okamoto H, Okada S, Sugiyama Y, Yotsumoto S, Tanaka T,
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21. Armitage P, Berry G. Further analysis of qualitative data. In: Armitage P, Berry G, eds. Statistical methods in medical research. Oxford: Blackwell Scientific Publications, 1987:371-407. 22. Hart P, Farrell GC, Cooksley WGE, Powell LW. Enhanced drug metabolism in cigarette smokers. Br Med J 1976;2:147-149. 23. Vestal RE, Norris AH, Tobin JD, Cohen BH, Shock NW, Andres R. Antipyrine metabolism in man: Influence of age, alcohol, caffeine and smoking. Clin Pharmacol Ther 1975; 18(4)425-432. 24. Williams SJ, Farrell GC. Inhibition of antipyrine metabolism by interferon. Br J Clin Pharmacol 1986;22:610-612. 25. Reichen J. MEGX test in hepatology: the long-sought ultimate quantitative liver function test? J Hepatol 1993; 19:4-7. 26. Coverdale S, Farrell GC. Serial antipyrine clearance in autoimmune chronic active hepatitis: potential to recognize patients with a poor prognosis [Abstract]. HEPATOLOGY1992; 16:525. 27. Shiffman ML, Luketic VA, Sanyal AJ, Duckworth PF, Purdum PP, Contos MJ, Mills AS, et al. Hepatic lidocaine metabolism and liver histology in patients with chronic hepatitis and cirrhosis. HEPATOLOGY1994; 19:933-940.