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Aliment Pharmacol Ther 1999; 13: 1187±1193.

Effect of ursodeoxycholic acid administration in patients with acute viral hepatitis: a pilot study P. FABRIS*, G. TOSITTI*, G. MAZZELLA , A. R. ZANETTIà, R. NICOLIN*, G. PELLIZZER*, P. BENEDETTI* & F. D E LALLA* *Department of Infectious Diseases, S. Bortolo Hospital, Vicenza;  Department of Gastroenterology, S. Orsola Hospital, Bologna; and àInstitute of Virology, University of Milan, Italy Accepted for publication 4 May 1999

SUMMARY

Background: Ursodeoxycholic acid (UDCA) is able to improve biochemical markers of cholestasis, with a parallel decrease in transaminases, in various cholestatic liver diseases. Aim: To evaluate the effects of UDCA administration on acute viral hepatitis-related cholestasis and the course of acute viral hepatitis. Methods: Seventy-nine consecutive patients with acute viral hepatitis (HBV: 43, HCV: 11, HAV: 15, HEV: 3, Non A-E: 7) were randomized to receive either UDCA for 3 weeks or no treatment. Liver biochemistry and serum bile acid determinations were run at weekly intervals. Results: No signi®cant differences were observed in mean percentage decreases in transaminases between

INTRODUCTION

In the course of cholestasis, intrahepatic accumulation of chenodeoxycholic (CDCA) and deoxycholic acids (DCA) is thought to induce liver damage.1, 2 In fact, the detergent activity of biliary salts seems likely to dissolve phospholipid and cholesterol, which are the main components of cellular membranes.3 Both in animals and in human beings, the ingestion or infusion of CDCA, DCA and lithocholic acids (LCA) induce

Correspondence to: Dr P. Fabris, Department of Infectious Diseases, S. Bortolo Hospital, 36100 Vicenza, Italy. E-mail: [email protected] Ó 1999 Blackwell Science Ltd

treated and untreated patients. By contrast, cholestatic indexes decreased signi®cantly more quickly in patients treated with UDCA than in controls, and this effect was more evident in patients with increasing alanine transaminase levels at admission. After a peak at the end of the ®rst week of therapy, serum levels of conjugated ursodeoxycholic acid (CUDCA) showed a gradual decrease. Conjugated cholic acid (CCA) and chenodeoxycholic acid (CCDCA) showed a progressive decrease with the resolution of viral hepatitis, but no in¯uence of UDCA administration was observed. Conclusions: Our study demonstrates that UDCA significantly improves cholestatic indices in patients with acute viral hepatitis, but this effect does not seem to affect the course of the illness.

hepato-necrosis, although these effects could be prevented by the addition of ursodeoxycholic acid (UDCA).4±8 The protective effect of UDCA has been attributed to the modi®cations of the bile acid pool with the increase of the hydrophilic fraction together with a stabilization of the cell membranes.9±11 Other suggested mechanisms of action are: an immunomodulatory effect; the induction of hypercholeresis; and a competitive inhibition of intestinal absorption of toxic endogenous bile salts.12-14 On this basis, UDCA has become the main treatment for liver diseases characterized by chronic cholestasis, e.g. primary biliary cirrhosis (PBC). Under these conditions liver biochemistry appears to be favourably affected by UDCA treatment.15±17 Moreover, there is some evidence that UDCA, used alone or in 1187

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combination with IFN, is able to improve liver enzymes in patients with chronic hepatitis C. 17±21 More recently, it has been suggested that UDCA could also exert a potential bene®t both in the therapy of cholestatic drugrelated acute hepatitis and in the management of prolonged cholestasis of acute hepatitis B.22, 23 This study was aimed at evaluating the effect of UDCA administration on acute hepatitis-related cholestasis and establishing its potential bene®t in the course of acute viral hepatitis; and also assessing the effect of UDCA therapy on serum conjugated bile acid concentrations in these patients. MATERIALS AND METHODS

Eighty-two consecutive patients with acute viral hepatitis, referred to our Unit from March 1995 to August 1998, were enrolled in the study. In three cases, the disease had a fulminant course that required transfer to the closest liver transplantation centre the day after hospitalization. After informed consent was obtained, 79 patients (M ˆ 62, F ˆ 17, mean age 33 years, range 17±65 years) were randomized to receive either UDCA or no treatment. All patients were randomized by using random number tables the day after hospitalization. Forty patients (M ˆ 33, F ˆ 7) were assigned to be given UDCA (Deursil, Sano®-Winthrop, Milan, Italy) 300 mg twice daily (morning and evening after meals) for 3 weeks, while 39 patients (M ˆ 29, F ˆ 10) were assigned to the control group. At the time of the randomization, alanine transaminase (ALT) serum levels were increasing in 23 patients (12 patients in the UDCA group) and decreasing in 56 patients (28 patients in the UDCA group). Patients were also treated with lactulose for a mean period of 10 days (range 3±21 days) and with 5±10% glucose solutions i.v. (1000±2000 mL/day). Treatments with other drugs, such as steroids (corticosteroids, anabolic steroids, oral contraceptives), enzyme inducers (barbiturates, phenothiazines, sedatives) or drugs interfering with bile acid metabolism (cholestyramine, clo®brate, antacids), were stopped for the entire follow-up period. Of 79 patients recruited, 47 (60%) had a history of intravenous drug use, 13 (16%) had declared homosexual and/or heterosexual intercourse with subjects at risk for blood-borne diseases, 10 (13%) had declared recent exposure to risk for food-borne hepatitis and/or recent travelling abroad, while in the remaining nine (11%) cases no real risk factors could be identi®ed. At admission, aetiology of the

acute illness was known in only ®ve cases; in a further ®ve cases, acute hepatitis occurred in subjects with a well-known history of chronic hepatitis C. The following determinations were run in the same laboratory at baseline, and at weekly intervals for three consecutive weeks: aspartate transaminase (AST), ALT, gammaglutamyl transpeptidase (GGT), alkaline phosphatase (ALP), bilirubin and albumin. At each sampling, two serum specimens for each patient were also collected and immediately stored at ± 80 °C for virological testing and conjugated biliary acid determinations. All patients were tested for HBV, HCV, HAV, HSV, EBV, CMV, T. gondii and HIV, at enrolment and after 3 weeks. In HCV-positive patients, determination of HCV-RNA was carried out. Serum samples obtained from patients with acute hepatitis of unknown aetiology were retrospectively tested for HEV (anti-HEV-IgG, anti-HEV-IgM and HEV-RNA) as well as for HCV-RNA.

Testing for serum bile acids Serum levels of conjugated cholic acid (CCA), chenodeoxycholic acid (CCDCA) and ursodeoxycholic acid (CUDCA) were evaluated by quantitative solidphase competitive enzyme immunoassay.24 Polyclonal antibodies for conjugated bile acids (BA) were raised in rabbits with BA±bovine serum albumin conjugate.25 The antibodies were evaluated for their speci®city titre and af®nity, before puri®cation and immobilization on polystyrene microtitre plates. A suitable BA horseradish peroxidase (HRP) derivative was synthesized from BA using a mixed anhydride method, and used as an enzymatic tracer after puri®cation. The method used for detection is competitive and allows direct analysis of BA in less than 10 lL of serum.

Laboratory determinations Commercial ELISA tests were run for hepatitis A (Abbott IMx, Chicago, IL), hepatitis B (Organon Teknika, Belgium), and HIV (Abbott Laboratories, Chicago, IL). Antibodies against HCV-encoded antigens were assayed using a second-generation HCV EIA system (Ortho Diagnostic Systems, Raritan, NJ) according to the manufacturer's instructions, and con®rmed by RIBA-3. Antibodies against HEV (antiHEV-IgG and anti-HEV-IgM) were tested using commercial ELISA tests (Abbott Laboratories, Chicago, Ó 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13, 1187±1193

UDCA IN ACUTE VIRAL HEPATITIS Table 1. Principal features of the patients studied

Features Sex (M/F) Mean age (years) (range) Duration of symptoms before hospitalization Aetiology HBV* HCV HAV HEV Non A-E Biochemical values at baseline AST (IU/L) ALT (IU/L) GGT (IU/L) ALP (IU/L) Bilirubin (mg/dL) Albumin (g/dL)

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Treated n = 40 (51%)

Untreated n = 39 (49%)

Totals n = 79

33/7 31 (19±58) 8.9 days (range 3±24)

29/10 34 (17±65) 9.0 days (range 4±25)

62/17 33 (17±65) 8.9 days (range 3±25)

21 (52%) 5 (12.5%) 8 (20%) 2 (5%) 4 (10%) Mean ‹ S.D.

22 (56%) 6 (15%) 7 (18%) 1 (2.5%) 3 (7.7%) Mean ‹ S.D.

43 (56%) 11 (14%) 15 (19%) 3 (3.7%) 7 (9%) P-value

1203 ‹ 948 2274 ‹ 1216 215 ‹ 134 187 ‹ 74 8.6 ‹ 7.4 3.9 ‹ 0.3

1248 ‹ 1038 2370 ‹ 1495 195 ‹ 121 168 ‹ 69 8.3 ‹ 4.6 3.8 ‹ 0.4

N.S. N.S. N.S. N.S. N.S. N.S.

Normal ranges: AST £ 40 IU/L; ALT £ 45 IU/L; GGT £ 50 IU/L; ALP £ 130 IU/L; bilirubin 0.2±1.2 mg/dL; albumin 3.2±5.4 g/dL. Mean values were compared using the Mann±Whitney test. S.D., standard deviation; N.S., not signi®cant. * Acute HBV-related hepatitis occurred in ®ve patients (three treated with UDCA and two untreated) with chronic hepatitis C.

IL). HEV-RNA was determined by RT-PCR using nested primers derived from the open reading frame (ORF-1) of the Burnese viral strain.26 Serum HCV-RNA was detected using nested reverse-transcription polymerase chain reaction (RT-PCR) with primers in the 5¢ non-coding region.27 IgG and IgM antibodies against T. gondii, Cytomegalovirus, Herpes viruses (types 1 and 2) and EBV were tested using commercial EIA tests (COBAS, Roche, Switzerland and Medical System, DADE-Behring, Italy, respectively). Statistical analyses To evaluate the success of the randomization, baseline values were compared using the Mann±Whitney nonparametric test. The Mann±Whitney test was also used to compare the variation of liver function tests between UDCA treated and untreated patients. The mean percentage decrease in liver function tests compared with the basal values (T0) was obtained using the following formula: DEC % ˆ Tx ± T0/T0 ´ 100, where Tx represents the interval considered [week 1 (T1), week 2 (T2) and week 3 (T3)].28 A P-value less than 0.05 was considered to be signi®cant. Ó 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13, 1187±1193

RESULTS

Acute hepatitis was HBV-related (HBsAg+, anti-HBc IgM+) in 43 (56%) cases (®ve of which were also affected with chronic C hepatitis); HAV-related (antiHAV-IgM+) in 15 (19%) cases; HCV-related (antiHCV+, HCV-RNA+) in 11 (14%) cases; HEV-related (anti-HEV-IgM+, HEV-RNA+) in three (4%) cases; and Non A-E in seven (9%) cases. At baseline, study and control groups were comparable in sex, mean age, duration of symptoms, aetiology and biochemical features (Table 1). All patients completed the followup and no adverse events related to UDCA were recorded. All cases had a favourable course, and displayed a gradual improvement in biohumoural parameters. Liver enzymes and bilirubin decreased more quickly in patients treated with UDCA than in untreated patients. However, this difference reached statistical signi®cance only for cholestasis indexes (GGT, ALP, bilirubin) (Figure 1). GGT was signi®cantly (P < 0.01) improved by UDCA administration, starting from the end of the ®rst week of treatment () 30.6% (T1), ) 54.5% (T2) and ) 60.8% (T3) in patients treated with UDCA vs. ) 23.1% (T1), ) 30.9% (T2)

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Figure 1. Mean percentage decrease in liver enzymes and bilirubin with respect to basal values at the end of 3 weeks according to the UDCA treatment. *P < 0.05; **P < 0.01; T = Patients treated with UDCA; UN = Untreated.

and ) 29.1% (T3) in untreated patients). Moreover, this effect on GGT behaviour was further con®rmed by strati®cation performed in accordance with the aetiology. In fact, in patients with HBV-related hepatitis, the mean percentage decrease after 3 weeks was ) 53.8% in the treated group and ) 23.5% in controls, P < 0.01; in patients with acute hepatitis of other aetiology the mean decrease was ) 70.2% in patients treated with UDCA and ) 30.8% in untreated patients, P < 0.01. The strati®cation of patients according to the trend of liver enzymes at admission demonstrated that the effect of UDCA administration on cholestasis indexes was mainly evident in patients with increasing ALT levels (Figures 2 and 3). At baseline, albumin was found in the normal range in all patients, and after 3 weeks no signi®cant changes were observed either in patients treated with UDCA or in untreated patients (3.9 ‹ 0.3 g/dL vs. 4.1 ‹ 0.5 g/dL and 3.8 ‹ 0.4 vs. 3.8 ‹ 0.5, respectively, P ˆ N.S.). Follow-up

Figure 2. Mean percentage decrease in liver enzymes and bilirubin at the end of the 3 weeks' follow-up according to treatment in patients with increasing ALT levels at admission. *P < 0.05; **P < 0.01; T = Patients treated with UDCA; UN = Untreated.

With respect to HCV-related acute hepatitis, nine of 11 patients had a chronic course as shown by abnormal liver function tests and HCV-RNA detectability over more than 6 months after the onset of the disease. All patients with acute hepatitis B (treated or untreated with UDCA) recovered, as testi®ed by seroconversion to antiHBs. No signi®cant differences were observed in the timing of seroconversion, because HBsAg clearance occurred within 4 months in all patients. Similarly, all patients with HAV-related acute hepatitis, as well as those with Non A-E acute hepatitis, recovered, with complete normalization of liver transaminases, without any signi®cant difference between patients treated with

Figure 3. Mean percentage decrease in liver enzymes and bilirubin at the end of the 3 weeks' follow-up according to treatment in patients with decreasing ALT levels at admission. *P < 0.05; T = Patients treated with UDCA; UN = Untreated. Ó 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13, 1187±1193

UDCA IN ACUTE VIRAL HEPATITIS

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Figure 6. Mean serum concentrations (‹ s.d.) of CDCA in acute hepatitis according to UDCA treatment. Figure 4. Mean serum concentration (‹ s.d.) of CUDCA in acute hepatitis according to UDCA treatment.

Figure 5. Mean serum concentrations (‹ s.d.) of CCA in acute hepatitis according to UDCA treatment.

UDCA and untreated patients. Seroconversion for HBV, HAV, HCV, EBV, HSV(s), CMV, and for T. gondii acute infection, could be demonstrated in none of the patients with Non A-E hepatitis at the end of the follow-up period. Variation of serum bile acid concentrations At baseline, serum CUDCA could be detected only in traces. After oral UDCA administration, serum concentrations of CUDCA increased in all patients and reached a peak at the end of the ®rst week. Despite the continuation of treatment, a gradual decrease was observed thereafter (Figure 4). In contrast, independently from the aetiology of acute hepatitis, high serum levels of both CCA and CCDCA were detected at baseline; their titres described a slow decrease according to the resolution of acute in¯ammation. No in¯uence of UDCA administration could be demonstrated (Figures 5 and 6). Ó 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13, 1187±1193

DISCUSSION

The potential for UDCA is steadily increasing in the therapy of a variety of chronic liver diseases, particularly when characterized by chronic cholestasis. Several studies have in fact demonstrated that, in patients with PBC,15±17 as well as in patients suffering from other forms of chronic cholestasis29 (i.e. intrahepatic cholestasis of pregnancy, liver disease in cystic ®brosis, primary sclerosing cholangitis, progressive familial intrahepatic cholestasis, Alagylle syndrome, biliary atresia), therapy with UDCA is able to induce a signi®cant improvement in clinical symptoms and/or biochemical markers of cholestasis. Moreover, although the prognostic signi®cance of these ®ndings remain unclear, several randomized double-blind trials in patients with chronic viral hepatitis have shown a favourable effect of UDCA treatment on biochemical parameters, including cytolytic indexes.14, 17±20 The mechanism of action of UDCA has been attributed both to its membrane stabilizing, choleretic, immunomodulatory effect and to the modi®cations of the total pool size by increasing the hydrophilic fraction.14, 29 In the course of acute hepatitis, cholestasis is almost constant,30 probably as a result of massive cytolysis; it could contribute to liver damage through the detergent effect of endogenous hydrophobic biliary salts. Based on these observations, 79 patients with acute viral hepatitis were randomized to receive either UDCA or no treatment. All patients included in the study were followed up at weekly intervals with biochemical (including conjugated serum bile acids) and serological testing. No side-effects were observed during UDCA treatment, con®rming its safety even during acute liver disease. In the UDCA-treated group, transaminases

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displayed a faster reduction rate than in the controls, although statistical signi®cance could not be achieved. Interestingly, among UDCA-treated patients, a signi®cantly higher reduction rate was observed for GGT, ALP and bilirubin serum levels; this effect was most obvious in patients with increasing levels of ALT at admission. The effect on cholestasis indexes was similar to that observed in cholestatic liver diseases, such as PBC, and in patients with chronic hepatitis C.14, 15, 18, 31±33 The mechanism of this effect is dif®cult to explain, although an aspeci®c cytoprotective effect of UDCA can be hypothesized. It has been postulated that UDCA may act either by promoting the excretion of cytotoxic bile acids (CDCA, DCA) or by binding to hepatocyte or biliocyte membranes, thus preventing damage from cytotoxic bile acids.10 Serum bile acid levels derive from a balance between intestinal absorption and hepatic uptake, and because the hepatic ®rst-pass extraction is highly ef®cient, serum bile acid concentrations are low under normal conditions. During cholestasis, both hepatic uptake and biliary secretion of bile acids are impaired. In our study, conjugated serum biliary acids (CCA and CCDCA) were detected at very high levels at baseline, independent of aetiology, con®rming that cholestasis is a constant feature of acute hepatitis, whereas CUDCA could be detected only in traces.14 During follow-up, both CCA and CCDCA serum concentrations displayed a gradual decrease, while serum CUDCA was seen to sharply increase and reach a peak after a week of oral administration. Absorption, probably favoured by the lack of bile salts in the gut, is therefore ef®cient even in patients with acute hepatitis. A similar trend is observed in chronic cholestatic liver diseases, in which serum CUDCA concentrations after oral administration appear to be directly related to the severity of the disease and to the grade of cholestasis.14, 34 In these situations, both hepatic uptake and biliary excretion are severely impaired, and high fractions of bile acids are excreted in the urine. In parallel to the resolution of acute hepatitis, a progressive decrease in CUDCA serum concentration, as well as of CCA and CCDA, was observed. The decrease in serum bile acid concentrations during the resolution phase of acute hepatitis is probably related to the recovery of hepatic uptake and excretion into the biliary tree. UDCA administration appears not to affect the serum concentrations of either CCA or CCDCA, because the titres of these biliary conjugated acids during follow-up were

found to be comparable both in treated and in untreated patients. In patients with chronic cholestatic liver diseases, a signi®cant decrease in serum chenodeoxycholic acid (CDCA), cholic acid (CA) and deoxycholic acid (DCA) concentrations have been reported during UDCA administration, although these modi®cations appear not to signi®cantly affect the total biliary pool size.14, 35±37 It must be emphasized that cholestasis that occurs during acute hepatitis is transient in the majority of cases and therefore the behaviour of serum biliary acid concentrations under UDCA treatment is not comparable to that reported in cholestatic liver diseases. In conclusion, our pilot study demonstrates that UDCA administration in patients with acute hepatitis is able to signi®cantly improve cholestatic indexes but this effect does not seem to affect the course of the disease. REFERENCES 1 Palmer RH. Bile acids, liver injury, and liver disease. Arch Intern Med 1972; 130: 606±17. 2 Greim H, Trulzsch D, Czygan P, et al. Mechanism of cholestasis. Bile acids in human livers with or without biliary obstruction. Gastroenterology 1972; 63: 846±50. 3 Sholmerich J, Becher MS, Schmidt KH, et al. In¯uence of hydroxylation and conjugation of bile salts on their membrane damaging properties. Studies on isolated hepatocytes and lipid membrane vesicles. Hepatology 1984; 4: 661±6. 4 Miyai K, Prince VM, Fisher MM. Bile acid metabolism in mammals. Ultrastructural studies on the intrahepatic cholestasis induced by lithocholic and chenodeoxycholic acids in the rat. Lab Invest 1971; 24: 292±302. 5 King JE, Schoen®eld LJ. Lithocholic acid, cholestasis, and liver disease. Mayo Clin Proc 1972; 47: 725±30. 6 Fisher RL, Anderson DW, Boyer JL, et al. A prospective morphologic evaluation of hepatic toxicity of chenodeoxycholic acid in patients with cholelithiasis: the National Cooperative Gallstone Study. Hepatology 1982; 2: 187±201. 7 Okun R, Van Goldstein LI, Gelder GA, et al. National cooperative gallstone study: non primate toxicology of chenodeoxycholic acid. J Toxicol Environ Health 1982; 9: 727±41. 8 Douglas M, Heuman S, Mills A, et al. Conjugates of ursodeoxycholate protect against cholestasis and hepatocellular necrosis caused by more hydrophobic bile salts. Gastroenterology 1991; 100: 203±11. 9 Armstrong MJ, Carey MC. The hydrophobic±hydrophilic balance of bile salts. Inverse correlation between reversephase high performance liquid chromatographic mobilities and micellar cholesterol-solubilizing capacities. J Lipid Res 1982; 23: 70±80. 10 Attili AF, Angelico M, Cantafora A, et al. Bile acid-induced liver toxicity: relation to the hydrophobic±hydrophilic balance of bile acids. Med Hypotheses 2; 1986(19): 57±68197140: 606±17. Ó 1999 Blackwell Science Ltd, Aliment Pharmacol Ther 13, 1187±1193

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