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Two-year follow-up study after treatment with lamivudine for chronic hepatitis B: seven cases reported
Hepatology Research 17 (2000) 197 – 204 www.elsevier.com/locate/ihepcom
Two-year follow-up study after treatment with lamivudine for chronic hepatitis B: seven cases reported Tatsuya Ide a,*, Ryukichi Kumashiro a, Hiroshi Suzuki a, Kyuichi Tanikawa b, Michio Sata a a
Second Department of Internal Medicine, Kurume Uni6ersity School of Medicine, 67 Asahi-machi, Kurume-shi, Fukuoka-ken 830 -0011, Japan b International Institute for Li6er Reserch, Kurume Reserch Center, 2432 -3 Aikawa-machi, Kurume 839 -0861, Japan Received 29 July 1999; received in revised form 4 October 1999; accepted 13 October 1999
Abstract Recently, lamivudine has been used to treat chronic hepatitis B. The effect of lamivudine has been evaluated in short-term (6 months) follow-up studies after treatment. Here, we report the long-term (2 years) follow-up results of lamivudine therapy for chronic hepatitis B. The subjects were seven patients with chronic hepatitis B, with evidence of hepatitis B virus (HBV) replication. All patients were treated with 100 mg lamivudine daily for 12 months. The patients were monitored for up to 2 years after treatment. Serum HBV DNA by branched DNA assay of all patients became undetectable during the therapy. In two patients, sustained suppression of serum HBV DNA was found after treatment. The remaining five patients exhibited rebound of ALT (\ 2 times baseline) and returned to seropositive for HBV DNA and HBeAg after lamivudine cessation. One of these five patients died of liver failure 3 months after treatment. However, in two of five patients whose alanine aminotransferase (ALT) had rebounded, HBV DNA became undetectable, and the ALT levels markedly decreased 2 years after the end of therapy. Since the disappearance of HBV DNA and stabilization of the ALT level were observed within two patients by 2 years after cessation of treatment, the patients whose ALT had rebounded should be followed up for a long-term period. To confirm the effect of lamivudine, long-term follow-up in many patients is necessary. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lamivudine; Chronic hepatitis B; Long-term follow-up
* Corresponding author. Tel.: +81-942-31-7561; fax: +81-942-34-2623. 1386-6346/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 9 9 ) 0 0 0 7 5 - 3
198
T. Ide et al. / Hepatology Research 17 (2000) 197–204
1. Introduction Recently, several kinds of nucleoside analogues have been used as anti-viral agents for many viral infectious diseases. Lamivudine, a negative enantiomer of 2%3%-dideoxy-3%-thiacytidine, was one of these agents and was initially used to treat human immunodeficiency virus infection. As an inhibitory effect on hepatitis B virus (HBV) replication was noted in vivo and in vitro [1,2], lamivudine has been used to treat HBV-related liver diseases. Several studies have described beneficial effects in chronic hepatitis B patients [3–5] or HBV-related cirrhotic patients after liver transplantation [6 – 8]. Since the suppression of HBV DNA was transient in short-term therapy [3,5], long-term administration was required. Lai et al. [9] examined lamivudine therapy for 1 year. Tanikawa et al. [10] also performed a 1-year study (phase III trial in Japan) in 116 patients. However, the results of long-term follow-up of lamivudine treatment have not been well described. We assess here the outcome of patients with chronic hepatitis B during a 2-year follow-up period after treatment.
2. Patients and methods Lamivudine (100 mg/day) was used for 1 year (52 weeks), according to the protocol of phase III trials in Japan for chronic hepatitis B. The seven patients examined here were included in a phase III trial [10]. Lamivudine treatment was started between July and December 1995 in all patients. All patients were male, and the mean age was 46.69 0.7 years. The diagnosis was based on liver function tests, positive HBsAg and HBV DNA, and liver histology before treatment. Histological findings [11] indicated moderate fibrosis in six patients and severe fibrosis in one patient (patient number 5). The term ‘rebound phenomenon’ was defined as more than twofold elevation of the baseline alanine aminotransferase (ALT) after cessation of therapy. Written informed consent was obtained from all patients. HBeAg and anti-HBe were detected by enzyme immunoassay (AxSYM, HBe, anti-HBe; DAINABOT Co., Ltd., Tokyo, Japan). HBeAg assay results were expressed as + (S/N \ 5.0), 9(2.1 BS/N 5 5.0), − (S/N 5 2.1). HBeAb assay results were expressed as + (% inhibition\70%), 9 (50%B inhibition5 70%), − (% inhibition5 50%). The serum level of HBV DNA was measured by the Quantiplex HBV DNA assay (Chiron Corp., Emeryville, CA, USA), which is based on bDNA technology. Assay results were expressed as the number of copies of HBV genome equivalents per milliliter. Our data were shown in mega106 equivalents per milliliter (Meq/ml). The lower limit of the assay was 0.7 Meq/ml. All patients lacked the hepatitis C virus antibody. The sequence analysis was performed to investigate the amino acid substitutions in the YMDD motif (tyrosine (Y), metionin (M), aspartate (D), aspartate (D)) of RNA-dependent DNA polymerase was investigated. Total DNA isolated from 100 ml serum by the guanidium chloroform method (Isogen LS; Nippon Gene Co. Ltd., Japan) was used for conventional polymerase chain reaction (PCR). The 5% primer for PCR was 5%-AGACCACCAAATGCCCCTAT-3%
T. Ide et al. / Hepatology Research 17 (2000) 197–204
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and the 3% primer was 5%-GGCGTTCACGGTGGTCTCCC-3%. These primers were previously reported by Tipples et al. [12]. Thirty-five reaction cycles were performed and each cycle included denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 45 s. PCR products were inserted into One-Shot TA cloning vector (Invitrogen Corp., San Diego, CA, USA) and sequenced with an Auto Read Sequencing Kit and ALF Red sequencer (Pharmacia LKB Biotechnology, Tokyo, Japan).
3. Result
3.1. During treatment ALT levels which were elevated (mean ALT levels, 1909 95 IU/l) in all patients before treatment decreased to normal range within 24 weeks after the initiation of treatment. Serum HBV DNA became undetectable within 12 weeks in all patients. HBeAg became negative in one of six patients during treatment (one patient was already HbeAg-negative before treatment).
3.2. After treatment (Table 1) In patient numbers 1 and 4, serum ALT rebounded, and serum HBV DNA and HBeAg became positive within 3 months after treatment. Subsequently, the ALT of these patients fluctuated, and HBV DNA and HBeAg were continuously detectable for 2 years. In patient number 5, HBeAg seroconversion (loss of HBeAg and the development of anti-HBe) occurred during the treatment. For 2 years after treatment, HBeAg seroconversion and normalization of ALT were maintained. Patient number 6 was positive for HBV DNA and anti-HBe before treatment. HBV DNA was undetectable during treatment. After treatment, disappearance of HBV DNA and normalization of ALT was maintained. In patient numbers 3 and 7, serum HBV DNA and HBeAg were positive 0.5 years after treatment. However, 2 years after treatment, these HBV markers were negative. Fig. 1 shows the clinical courses of patient number 3, who was a 46-year-old male. In 1991, ALT elevation was first noted, and he was diagnosed as having chronic hepatitis B 2 years later. He was treated with stronger neo-minophagen C (SNMC) and propagermanium. However, the ALT level did not improve, and HBeAg seroconversion did not occur. He then received lamivudine treatment from September 1995 for 1 year. Six months after treatment, the ALT level increased to over 400 IU/l. The patient was treated again with SNMC, which decreased the ALT level gradually. HBV DNA was undetectable in June 1997, and HBeAg was negative in May 1998. The levels of ALT apparently decreased compared with those before treatment. Patient number 7 was a 37-year-old male. In 1994, abnormalities of ALT levels were pointed out, and the patient was diagnosed as having chronic hepatitis B. He received interferon therapy for 1 month in September 1994. However, this therapy was not effective. He received lamivudine from September 1995 for 1 year. Eight months after treatment,
200
Patient number
1 2 3 4 5 6 7
HBV DNA titer (Meq/ml) Before treatment
After 0.5 years
7300 3400 450 270 110 70 1.0
5900 Dead 4.7 750 B0.7 B0.7 3800
HBeAg/anti-HBe After 2.0 years 160 B0.7 180 B07 B0.7 B0.7
Before treatment
After 0.5 years
+/− +/− +/− +/− +/− −/+ +/−
+/− Dead +/− +/− −/+ −/+ +/−
After 2.0 years +/+ −/− +/9 −/+ −/+ − or 9 /+
T. Ide et al. / Hepatology Research 17 (2000) 197–204
Table 1 HBV serological data before and after lamivudine treatment
T. Ide et al. / Hepatology Research 17 (2000) 197–204
201
the ALT levels increased to over 700 IU/l, and HBV DNA markedly increased. He was treated with ursodeoxycholic acid (UDCA) between July and October 1997. Subsequently, the ALT levels and HBV DNA decreased gradually and anti-HBe became positive. In 1998, HBV DNA was undetectable and the ALT level almost returned to normal. Fig. 2 shows the clinical courses of patient number 2, a 41-year-old male. Despite the continued lamivudine administration, serum HBV DNA increased to 640 Meq/ml by the last day of therapy. Two months after cessation of therapy, the patient was admitted because the serum ALT level was 4305 IU/l. The serum level of HBV DNA was 18800 Meq/ml. Intensive care for acute hepatic failure was started. However, the prothrombin index decreased to 18% and the total bilirubin level reached 14.8 mg/dl. As re-activation of HBV occurred despite continuous lamivudine administration, re-administration of lamivudine was not performed. Although the ALT level returned to almost normal range 18 days after admission, there were no signs of hepatic function recovery. The patient died of liver failure 3 months after treatment. Histological findings of the autopsied liver revealed massive necrosis of hepatocytes with marked fibrosis. With regard to YMDD motif mutations, in patient number 2, at 44 weeks of lamivudine treatment, the co-existence of mutant (YMDD YIDD, I representing isoleusin) and wild clones in the serum was confirmed. At 48 and 52 weeks of lamivudine therapy, all clones were defined as mutant. Four weeks after cessation of therapy, co-existence was again observed. Subsequently, all clones became wild type. In patient number 1, the mutation (YMDD YIDD and YVDD, V representing valine) was detected at the end of therapy and 4 weeks after therapy. In patient numbers 3 and 7, a transient mutation in the YMDD locus (YMDD YVDD) was detected during lamivudine therapy (at 36 and 40 weeks, respectively). These mutations disappeared by 2 years after cessation.
Fig. 1. Clinical course of patient number 3. N.D., not determined.
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Fig. 2. Clinical course of patient number 2. T.B., total bilirubin; P.T., prothrombin time; GM, gabexate mesilate; FFP, fresh frozen plasma; GI, glucagon insulin therapy; AT III, anti-thrombin III; PGE1, prostaglandin E1; YMDD mutation, W = YMDD, M =YIDD, M/W=co-existence of YMDD and YIDD.
4. Discussion Lamivudine has potent antiviral activity against HBV. Studies [3–5] on lamivudine therapy for up to 6 months revealed that the levels of HBV DNA decreased to an undetectable level in most patients, but the majority of patients had returned to pretreatment values after treatment. In a 6-month therapy study reported by Nevens et al. [4], four patients (8%) seroconverted to anti-HBe during the study, two during the treatment period, and two patients after treatment. Therefore, long-term administration is required. Lai et al. [9] performed a 1-year lamivudine trial and reported that the highest rate of HBeAg seroconversion, the greatest suppression of HBV DNA, and substantial histologic improvement. However, the course after treatment was not described. Tanikawa et al. [10] also reported a 1-year lamivudine trial, but patients were followed up for only 2 months. Dienstag et al. [13] also reported a 1-year trial, but the patients were followed up for 16 weeks. To date, only the three above mentioned studies on 1-year lamivudine trials have been reported for chronic hepatitis B. Recently, Liaw et al. [14] examined 2-year lamivudine therapy, and the sustained HBe Ag seroconversion rate increased from 17% at year 1 to 27% at year 2. However, the rate after treatment was not described. In this study, within 2 years after cessation of treatment, disappearance of HBV DNA and stabilization of the ALT level were observed in two patients (numbers 3
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and 7). These processes occurred after the rebound phenomenon of ALT over a 1-year period. Although SNMC or UDCA was administered to decrease the ALT elevation due to the rebound phenomenon, these therapies did not likely suppress HBV DNA. Therefore, we considered that elevation of the ALT in these two patients was related to lamivudine therapy and the rebound phenomenon provoked the suppression of HBV DNA. Five of seven patients (71.4%) in this study had ALT elevations after lamivudine cessation. It has been reported that the rate of ALT elevations (rebound phenomenon) was 17.2–22% [10,13,15]. As the difference in the rates is presumably due to the follow-up period, it was speculated that if long-term follow-up was performed, the rate might increase. No study on the seroconversion rate has been reported after treatment in 1-year lamivudine trials. In the study on 6 months of therapy followed by 6 months after cessation, the seroconversion rate after treatment was 4% [4]. In the present study, the incidence of HBeAg disappearance was 28.6% (two of seven patients). Since the treatment and follow-up period were different and the numbers of patients were small, it is unclear whether the rate of this study was high. A long-time follow-up study must be performed using many patients. One patient died of liver failure after cessation of therapy. This was the only fatal case among the 116 patients in the phase III study in Japan. Nevens et al. [4] reported that two patients developed temporary hepatic decompensation after ALT levels increased following treatment, but both patients survived. At 44 weeks of lamivudine therapy, in our case, lamivudine resistant mutation was observed and the treatment was terminated at 52 weeks. HBV reactivation after cessation of lamivudine was mainly due to wild-type virus replication. We considered that replication of mutant clone was increased at the end of therapy and the wild-type virus was very potent in replication. This potent re-activation of wild-type virus might induce the liver failure. The clinical course after lamivudine therapy varies widely. The rebound phenomenon of ALT after treatment may be needed to completely suppress the replication of HBV DNA. However, care must be taken not to develop liver failure. To assess the effectiveness of lamivudine properly, the patients should be followed for a long-term period. Although the efficacy of the long-term lamivudine therapy has been reported [14,16], the optimal duration of therapy remains uncertain. We consider that because the rebound phenomenon after discontinuation of therapy is rarely at risk for liver failure, the treatment should be continued as long as possible. Especially, once the YMDD mutants appear, the therapy must be continued. Further study is required to determine whether combination therapy with other regimens is needed for YMDD mutants. References [1] Doong SL, Tsai CH, Schinazi RF, Liotta DC, Cheng YC. Inhibition of the replication of hepatitis B virus in vitro by 2%,3%-dideoxy-3%-thiacytidine and related analogues. Proc Natl Acad Sci USA 1991;88:8495–9.
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[2] Tyrrell DLJ, Fischer K, Savani K, Ian W, Jewell L. Treatment of chimpanzees and ducks with lamivudine, 2%3%-dideoxy-3%-thiacytidine results in a rapid suppression of hepadnaviral DNA in sera. Clin Invest Med 1993;16(Suppl. 4):B77 (abstract). [3] Dienstag JL, Perrillo RP, Schiff ER, Bartholomew M, Vicary C, Rubin MA. Preliminary trial of lamivudine for chronic hepatitis B infection. N Engl J Med 1995;333:1657– 61. [4] Nevens F, Main J, Honkoop P, et al. Lamivudine therapy for chronic hepatitis B: a six-month randomized dose-ranging study. Gastroenterology 1997;113:1258 – 63. [5] Lai CL, Ching CK, Tung AK, et al. Lamivudine is effective in suppressing hepatitis B virus DNA in Chinese hepatitis B surface antigen carriers: a placebo-controlled trial. Hepatology 1997;25:241 – 4. [6] Andreone P, Caraceni P, Grazi GL, et al. Lamivudine treatment for acute hepatitis B after liver transplantation. J Hepatol 1998;29:985 – 9. [7] Grellier L, Mutimer D, Ahmed M, et al. Lamivudine prophylaxis against reinfection in liver transplantation for hepatitis B cirrhosis. Lancet 1996;348:1212 – 5. [8] Ben AZ, Shmueli D, Mor E, Shapira Z, Tur KR. Beneficial effect of lamivudine in recurrent hepatitis B after liver transplantation. Transplantation 1997;63:393 – 6. [9] Lai CL, Chien RN, Leung NW, et al. A one-year trial of lamivudine for chronic hepatitis B. N Engl J Med 1998;339:61–8. [10] Tanikawa K, Ichida F, Suzuki H, et al. A long-term study of Lamivudine for chronic hepatitis B patients. Kantansui 1998;36:597–611. [11] Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology 1994;19:1513 – 20. [12] Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, Tyrrell DL. Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 1996;24:714–7. [13] Dienstag JL, Schiff ER, Wright T, et al. Lamivudine treatment for one year in previously untreated U.S. hepatitis B patients: histologic improvement and hepatitis Be-antigen (HBeAg) seroconversion (abstract). The American Association for the Study of Liver Diseases, DDW, 1998. p. A-1. [14] Liaw YF, Lai CL, Leung NWY, et al. Two-year lamivudine therapy in chronic hepatitis B infection: results of a placebo controlled multicentre study in asia (abstract). The American Association for the Study of Liver Diseases, DDW, 1998. p. A-1. [15] Honkoop P, Man RA, Heiitjenk RA, Schalm SW. Hepatitis B reactivation after lamivudine. Lancet 1995;346:1156–7. [16] Leung NWY, Lai CL, Chang TT, et al. Three year lamivudine therapy in chronic HBV. J Hepatol 1999;30(suppl 1):59.
.
Two-year follow-up study after treatment with lamivudine for chronic hepatitis B: seven cases reported Tatsuya Ide a,*, Ryukichi Kumashiro a, Hiroshi Suzuki a, Kyuichi Tanikawa b, Michio Sata a a
Second Department of Internal Medicine, Kurume Uni6ersity School of Medicine, 67 Asahi-machi, Kurume-shi, Fukuoka-ken 830 -0011, Japan b International Institute for Li6er Reserch, Kurume Reserch Center, 2432 -3 Aikawa-machi, Kurume 839 -0861, Japan Received 29 July 1999; received in revised form 4 October 1999; accepted 13 October 1999
Abstract Recently, lamivudine has been used to treat chronic hepatitis B. The effect of lamivudine has been evaluated in short-term (6 months) follow-up studies after treatment. Here, we report the long-term (2 years) follow-up results of lamivudine therapy for chronic hepatitis B. The subjects were seven patients with chronic hepatitis B, with evidence of hepatitis B virus (HBV) replication. All patients were treated with 100 mg lamivudine daily for 12 months. The patients were monitored for up to 2 years after treatment. Serum HBV DNA by branched DNA assay of all patients became undetectable during the therapy. In two patients, sustained suppression of serum HBV DNA was found after treatment. The remaining five patients exhibited rebound of ALT (\ 2 times baseline) and returned to seropositive for HBV DNA and HBeAg after lamivudine cessation. One of these five patients died of liver failure 3 months after treatment. However, in two of five patients whose alanine aminotransferase (ALT) had rebounded, HBV DNA became undetectable, and the ALT levels markedly decreased 2 years after the end of therapy. Since the disappearance of HBV DNA and stabilization of the ALT level were observed within two patients by 2 years after cessation of treatment, the patients whose ALT had rebounded should be followed up for a long-term period. To confirm the effect of lamivudine, long-term follow-up in many patients is necessary. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lamivudine; Chronic hepatitis B; Long-term follow-up
* Corresponding author. Tel.: +81-942-31-7561; fax: +81-942-34-2623. 1386-6346/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 9 9 ) 0 0 0 7 5 - 3
198
T. Ide et al. / Hepatology Research 17 (2000) 197–204
1. Introduction Recently, several kinds of nucleoside analogues have been used as anti-viral agents for many viral infectious diseases. Lamivudine, a negative enantiomer of 2%3%-dideoxy-3%-thiacytidine, was one of these agents and was initially used to treat human immunodeficiency virus infection. As an inhibitory effect on hepatitis B virus (HBV) replication was noted in vivo and in vitro [1,2], lamivudine has been used to treat HBV-related liver diseases. Several studies have described beneficial effects in chronic hepatitis B patients [3–5] or HBV-related cirrhotic patients after liver transplantation [6 – 8]. Since the suppression of HBV DNA was transient in short-term therapy [3,5], long-term administration was required. Lai et al. [9] examined lamivudine therapy for 1 year. Tanikawa et al. [10] also performed a 1-year study (phase III trial in Japan) in 116 patients. However, the results of long-term follow-up of lamivudine treatment have not been well described. We assess here the outcome of patients with chronic hepatitis B during a 2-year follow-up period after treatment.
2. Patients and methods Lamivudine (100 mg/day) was used for 1 year (52 weeks), according to the protocol of phase III trials in Japan for chronic hepatitis B. The seven patients examined here were included in a phase III trial [10]. Lamivudine treatment was started between July and December 1995 in all patients. All patients were male, and the mean age was 46.69 0.7 years. The diagnosis was based on liver function tests, positive HBsAg and HBV DNA, and liver histology before treatment. Histological findings [11] indicated moderate fibrosis in six patients and severe fibrosis in one patient (patient number 5). The term ‘rebound phenomenon’ was defined as more than twofold elevation of the baseline alanine aminotransferase (ALT) after cessation of therapy. Written informed consent was obtained from all patients. HBeAg and anti-HBe were detected by enzyme immunoassay (AxSYM, HBe, anti-HBe; DAINABOT Co., Ltd., Tokyo, Japan). HBeAg assay results were expressed as + (S/N \ 5.0), 9(2.1 BS/N 5 5.0), − (S/N 5 2.1). HBeAb assay results were expressed as + (% inhibition\70%), 9 (50%B inhibition5 70%), − (% inhibition5 50%). The serum level of HBV DNA was measured by the Quantiplex HBV DNA assay (Chiron Corp., Emeryville, CA, USA), which is based on bDNA technology. Assay results were expressed as the number of copies of HBV genome equivalents per milliliter. Our data were shown in mega106 equivalents per milliliter (Meq/ml). The lower limit of the assay was 0.7 Meq/ml. All patients lacked the hepatitis C virus antibody. The sequence analysis was performed to investigate the amino acid substitutions in the YMDD motif (tyrosine (Y), metionin (M), aspartate (D), aspartate (D)) of RNA-dependent DNA polymerase was investigated. Total DNA isolated from 100 ml serum by the guanidium chloroform method (Isogen LS; Nippon Gene Co. Ltd., Japan) was used for conventional polymerase chain reaction (PCR). The 5% primer for PCR was 5%-AGACCACCAAATGCCCCTAT-3%
T. Ide et al. / Hepatology Research 17 (2000) 197–204
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and the 3% primer was 5%-GGCGTTCACGGTGGTCTCCC-3%. These primers were previously reported by Tipples et al. [12]. Thirty-five reaction cycles were performed and each cycle included denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 45 s. PCR products were inserted into One-Shot TA cloning vector (Invitrogen Corp., San Diego, CA, USA) and sequenced with an Auto Read Sequencing Kit and ALF Red sequencer (Pharmacia LKB Biotechnology, Tokyo, Japan).
3. Result
3.1. During treatment ALT levels which were elevated (mean ALT levels, 1909 95 IU/l) in all patients before treatment decreased to normal range within 24 weeks after the initiation of treatment. Serum HBV DNA became undetectable within 12 weeks in all patients. HBeAg became negative in one of six patients during treatment (one patient was already HbeAg-negative before treatment).
3.2. After treatment (Table 1) In patient numbers 1 and 4, serum ALT rebounded, and serum HBV DNA and HBeAg became positive within 3 months after treatment. Subsequently, the ALT of these patients fluctuated, and HBV DNA and HBeAg were continuously detectable for 2 years. In patient number 5, HBeAg seroconversion (loss of HBeAg and the development of anti-HBe) occurred during the treatment. For 2 years after treatment, HBeAg seroconversion and normalization of ALT were maintained. Patient number 6 was positive for HBV DNA and anti-HBe before treatment. HBV DNA was undetectable during treatment. After treatment, disappearance of HBV DNA and normalization of ALT was maintained. In patient numbers 3 and 7, serum HBV DNA and HBeAg were positive 0.5 years after treatment. However, 2 years after treatment, these HBV markers were negative. Fig. 1 shows the clinical courses of patient number 3, who was a 46-year-old male. In 1991, ALT elevation was first noted, and he was diagnosed as having chronic hepatitis B 2 years later. He was treated with stronger neo-minophagen C (SNMC) and propagermanium. However, the ALT level did not improve, and HBeAg seroconversion did not occur. He then received lamivudine treatment from September 1995 for 1 year. Six months after treatment, the ALT level increased to over 400 IU/l. The patient was treated again with SNMC, which decreased the ALT level gradually. HBV DNA was undetectable in June 1997, and HBeAg was negative in May 1998. The levels of ALT apparently decreased compared with those before treatment. Patient number 7 was a 37-year-old male. In 1994, abnormalities of ALT levels were pointed out, and the patient was diagnosed as having chronic hepatitis B. He received interferon therapy for 1 month in September 1994. However, this therapy was not effective. He received lamivudine from September 1995 for 1 year. Eight months after treatment,
200
Patient number
1 2 3 4 5 6 7
HBV DNA titer (Meq/ml) Before treatment
After 0.5 years
7300 3400 450 270 110 70 1.0
5900 Dead 4.7 750 B0.7 B0.7 3800
HBeAg/anti-HBe After 2.0 years 160 B0.7 180 B07 B0.7 B0.7
Before treatment
After 0.5 years
+/− +/− +/− +/− +/− −/+ +/−
+/− Dead +/− +/− −/+ −/+ +/−
After 2.0 years +/+ −/− +/9 −/+ −/+ − or 9 /+
T. Ide et al. / Hepatology Research 17 (2000) 197–204
Table 1 HBV serological data before and after lamivudine treatment
T. Ide et al. / Hepatology Research 17 (2000) 197–204
201
the ALT levels increased to over 700 IU/l, and HBV DNA markedly increased. He was treated with ursodeoxycholic acid (UDCA) between July and October 1997. Subsequently, the ALT levels and HBV DNA decreased gradually and anti-HBe became positive. In 1998, HBV DNA was undetectable and the ALT level almost returned to normal. Fig. 2 shows the clinical courses of patient number 2, a 41-year-old male. Despite the continued lamivudine administration, serum HBV DNA increased to 640 Meq/ml by the last day of therapy. Two months after cessation of therapy, the patient was admitted because the serum ALT level was 4305 IU/l. The serum level of HBV DNA was 18800 Meq/ml. Intensive care for acute hepatic failure was started. However, the prothrombin index decreased to 18% and the total bilirubin level reached 14.8 mg/dl. As re-activation of HBV occurred despite continuous lamivudine administration, re-administration of lamivudine was not performed. Although the ALT level returned to almost normal range 18 days after admission, there were no signs of hepatic function recovery. The patient died of liver failure 3 months after treatment. Histological findings of the autopsied liver revealed massive necrosis of hepatocytes with marked fibrosis. With regard to YMDD motif mutations, in patient number 2, at 44 weeks of lamivudine treatment, the co-existence of mutant (YMDD YIDD, I representing isoleusin) and wild clones in the serum was confirmed. At 48 and 52 weeks of lamivudine therapy, all clones were defined as mutant. Four weeks after cessation of therapy, co-existence was again observed. Subsequently, all clones became wild type. In patient number 1, the mutation (YMDD YIDD and YVDD, V representing valine) was detected at the end of therapy and 4 weeks after therapy. In patient numbers 3 and 7, a transient mutation in the YMDD locus (YMDD YVDD) was detected during lamivudine therapy (at 36 and 40 weeks, respectively). These mutations disappeared by 2 years after cessation.
Fig. 1. Clinical course of patient number 3. N.D., not determined.
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Fig. 2. Clinical course of patient number 2. T.B., total bilirubin; P.T., prothrombin time; GM, gabexate mesilate; FFP, fresh frozen plasma; GI, glucagon insulin therapy; AT III, anti-thrombin III; PGE1, prostaglandin E1; YMDD mutation, W = YMDD, M =YIDD, M/W=co-existence of YMDD and YIDD.
4. Discussion Lamivudine has potent antiviral activity against HBV. Studies [3–5] on lamivudine therapy for up to 6 months revealed that the levels of HBV DNA decreased to an undetectable level in most patients, but the majority of patients had returned to pretreatment values after treatment. In a 6-month therapy study reported by Nevens et al. [4], four patients (8%) seroconverted to anti-HBe during the study, two during the treatment period, and two patients after treatment. Therefore, long-term administration is required. Lai et al. [9] performed a 1-year lamivudine trial and reported that the highest rate of HBeAg seroconversion, the greatest suppression of HBV DNA, and substantial histologic improvement. However, the course after treatment was not described. Tanikawa et al. [10] also reported a 1-year lamivudine trial, but patients were followed up for only 2 months. Dienstag et al. [13] also reported a 1-year trial, but the patients were followed up for 16 weeks. To date, only the three above mentioned studies on 1-year lamivudine trials have been reported for chronic hepatitis B. Recently, Liaw et al. [14] examined 2-year lamivudine therapy, and the sustained HBe Ag seroconversion rate increased from 17% at year 1 to 27% at year 2. However, the rate after treatment was not described. In this study, within 2 years after cessation of treatment, disappearance of HBV DNA and stabilization of the ALT level were observed in two patients (numbers 3
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and 7). These processes occurred after the rebound phenomenon of ALT over a 1-year period. Although SNMC or UDCA was administered to decrease the ALT elevation due to the rebound phenomenon, these therapies did not likely suppress HBV DNA. Therefore, we considered that elevation of the ALT in these two patients was related to lamivudine therapy and the rebound phenomenon provoked the suppression of HBV DNA. Five of seven patients (71.4%) in this study had ALT elevations after lamivudine cessation. It has been reported that the rate of ALT elevations (rebound phenomenon) was 17.2–22% [10,13,15]. As the difference in the rates is presumably due to the follow-up period, it was speculated that if long-term follow-up was performed, the rate might increase. No study on the seroconversion rate has been reported after treatment in 1-year lamivudine trials. In the study on 6 months of therapy followed by 6 months after cessation, the seroconversion rate after treatment was 4% [4]. In the present study, the incidence of HBeAg disappearance was 28.6% (two of seven patients). Since the treatment and follow-up period were different and the numbers of patients were small, it is unclear whether the rate of this study was high. A long-time follow-up study must be performed using many patients. One patient died of liver failure after cessation of therapy. This was the only fatal case among the 116 patients in the phase III study in Japan. Nevens et al. [4] reported that two patients developed temporary hepatic decompensation after ALT levels increased following treatment, but both patients survived. At 44 weeks of lamivudine therapy, in our case, lamivudine resistant mutation was observed and the treatment was terminated at 52 weeks. HBV reactivation after cessation of lamivudine was mainly due to wild-type virus replication. We considered that replication of mutant clone was increased at the end of therapy and the wild-type virus was very potent in replication. This potent re-activation of wild-type virus might induce the liver failure. The clinical course after lamivudine therapy varies widely. The rebound phenomenon of ALT after treatment may be needed to completely suppress the replication of HBV DNA. However, care must be taken not to develop liver failure. To assess the effectiveness of lamivudine properly, the patients should be followed for a long-term period. Although the efficacy of the long-term lamivudine therapy has been reported [14,16], the optimal duration of therapy remains uncertain. We consider that because the rebound phenomenon after discontinuation of therapy is rarely at risk for liver failure, the treatment should be continued as long as possible. Especially, once the YMDD mutants appear, the therapy must be continued. Further study is required to determine whether combination therapy with other regimens is needed for YMDD mutants. References [1] Doong SL, Tsai CH, Schinazi RF, Liotta DC, Cheng YC. Inhibition of the replication of hepatitis B virus in vitro by 2%,3%-dideoxy-3%-thiacytidine and related analogues. Proc Natl Acad Sci USA 1991;88:8495–9.
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