항바이러스 치료 중 발생하는 B형 간염 바이러스 polymerase 유전자 변이에 대한 분자생물학적 분석

Abstract

[한글]배경 및 목적: 만성 B형 간염의 장기간 항바이러스 치료에서 약제내성 변이종의 출현은 치료 실패의 주요 원인이다. 이러한 약제내성 변이종의 출현을 방지하기 위한 단기간 두 약제의 병합치료 효과는 임상에서 적용될 수 있는 새로운 치료 전략이나 아직까지 연구된 바가 없다. 따라서 본 연구는 만성 B형 간염 환자에서 혈액 내 B형간염바이러스(HBV)의 HBV DNA 중합효소 유전자 다양성이 존재하는지 확인하고 항바이러스제 투여에 따른 투약 전과 투약 후 매 12주마다의 각 시점 별 바이러스 변이종 분포의 변화를 조사하고, 항바이러스제의 단기병합요법이 바이러스의 감소 정도와 내성변이종 출현의 방지에 긍정적인 영향을 줄 수 있는 지를 확인하고자 하였다. 방법: 대상 환자는 만성 C형간염바이러스(HCV) 또는 HIV 감염이 없고, 암에 걸린 과거력이 없으며, 만성 B형 간염 치료를 위한 항바이러스제 투여력이 없고, HBsAg양성, HBV DNA 20,000 IU/ml(105copies/ml)이상이며, ALT수치가 정상범위 상한치의 2배 이상인 자로 하였다. 2007년 5월부터 2009년 8월까지 전향적으로 총 40명의 환자들이 클레부딘을 매일 30mg 단독으로 투약하는 군(CLV 단독군;n=20)과 매일 클레부딘 30mg과 아데포비어 10mg을 병합하여 12주간 투약 후 아데포비어는 투여 중단하고 매일 클레부딘 30mg을 지속적으로 유지 투약하는 군(CLV+ADV 병합군; n=20)인 두 군으로 나누어 치료받았다. 분자생물학적 분석을 위해서는 항바이러스제 투약 12주째 HBV DNA수치가 103copies/ml이상인 환자들을 대상으로 CLV 단독군에서 3명, CLV+ ADV 병합군에서 4명, 모두 7명을 선택하였다. 7명의 환자에서 약제 복용 전 시점과 약제 복용 시작한 후 12주마다 간기능검사, HBeAg, anti-HBe Ab, HBV DNA농도를 검사하였다. 약제 내성변이종 검출을 위하여 클로닝 및 염기서열분석방법(각 시점별로 20개의 클론) 및 제한효소분절질량다형성(RFMP)법을 사용하였다. 결과: 약제 투약 전의 두 군 사이에 성별, 나이, ALT수치, HBV DNA 수치는 두 군 사이에 통계학적으로 의미 있는 차이를 보이지 않았다. CLV+ADV 병합군에서 투약 12주, 24주까지의 혈청 HBV DNA는 CLV 단독군과 비교하여 통계적으로 의미 있게 감소하였다 [중앙값, CLV 단독군 vs CLV+ADV 병합군; -2.67 vs -4.11, 12주, p =0.001; -4.15 vs -4.97, 24주, p = 0.036]. 하지만 36주와 48주째까지의 HBV DNA 감소 정도는 통계학적으로 의미 있지 않았다. CLV+ADV 병합군 4명의 클로닝 및 염기서열검사결과를 합한 결과와 CLV 단독군 3명의 클로닝 및 염기서열검사를 합한 결과를 비교한 결과 투약 전에 발견되었던 rtV191I변이가 12주에서 관찰되었으나 24주째는 관찰되지 않았다. 두 군 모두에서 투약 12주째와 24주째에 rtR153Q, rtS223A, rtI224V변이가 발견되었다. 24주 째 두 군 모두에서 rtI91L, rtP109S, rtT118N, rtN121I, rtI122L, rtQ130P, rtS135Y, rtK149Q, rtR153Q, rtI163V, rtI187V, rtS223A, rtI224V 변이들이 발견되었다. CLV 단독군의 증례 1 환자에서 rtA181T+rtV191I변이가 치료시작 24주째 클로닝 및 염기서열검사, 및 RFMP 검사에서 발견되었고, rtA181T+rtV191I 변이가 36주째 RFMP 검사로 발견되었다. CLV 단독군의 증례 3 환자에서 rtI163V변이가 치료전, 치료 12주째, 치료 24주째에 클로닝 및 염기서열검사로 발견되었다. CLV 단독군의 증례 3 환자에서 치료 48주째 바이러스돌파현상이 발생하였을 때 시행한 RFMP검사에서 rtM204I변이가 발견되었다. CLV+ADV 병합군의 증례 4 환자는 치료시작 12주째 rtA181T+rtV191I변이가 RFMP 검사로 발견되었고 치료 48주째 RFMP검사로 rtV173L+rtA181T+rtV191I+rtM204I+rtM204V변이가 발견되었다. CLV+ADV 병합군의 증례 5 환자는 치료 24주째와 48주째에 RFMP 방법으로 rtA181T변이가 발견되었다. CLV+ADV 병합군의 증례 6 환자는 치료 24주째 RFMP검사에서 rtA181T변이가 발견되었다. CLV+ADV 병합군의 증례 7 환자는 치료 24주와 치료 36주째 RFMP검사에서 rtV191I+rtM204I변이가 발견되었다. 결론: 만성 B형 간염 치료를 위한 단기간의 항바이러스제 병합요법(CLV+ADV)은 단독요법(CLV)에 비해 효과적인 혈청 HBV DNA 감소를 유도하였다. 하지만 병합요법에도 불구하고 항바이러스 억제 효과가 충분하지 못했던 경우에는 단독요법과 마찬가지로 바이러스 유전자내에 다양한 변이 양상을 보였다. CLV 단독군의 환자에서 바이러스돌파현상이 발생했을 때 L-nucleoside analogue약제들의 내성변이로 알려진 rtM204I변이가 발견되었다. CLV단독군에서 다약제 내성변이인 rtA181T변이도 발견되었다. CLV+ADV 병합군에서는 rtM204I변이, rtA181T변이, rtV173L변이와 동반된 rtM204V변이가 발견되었고, 아데포비어 내성변이인 rtN236T변이는 발견되지 않았다. 향후 이러한 변이들이 임상적으로 의미 있는 항바이러스 내성 발생 및 치료 효과와 직접적인 연관이 있는지에 대한 확인이 필요할 것으로 생각된다.

[영문]Background and aim: The development of drug-resistant mutations during a long-term antiviral therapy is a major cause of the failed treatment of chronic hepatitis B. The short-term combination therapy using two drugs to prevent such drug-resistant mutations is a new treatment strategy, but no research has been conducted about its effect. So, the purpose of this study is: to see what variety of viral quasispecies of HBV exists in HBV DNA polymerase in chronic hepatitis B patients; to examine any changes of the distribution of viral mutations every 12 weeks before and after antiviral drug administration; to check if the short-term combination therapy of antiviral drugs has positive effects on the decrease of virus and the prevention of development of drug-resistant mutations. Methods: The eligible patients had positive HBsAg, with serum HBV DNA levels higher than 20,000 IU/ml(105copies/ml) and serum ALT levels twice higher than the upper limit of normal level. Exclusion criteria included co-infection with hepatitis C or human immunodeficiency virus; evidence of malignancy history; previous exposure to any nucleoside analog that is active against HBV. Between May 2007 and August 2009, a total of 40 chronic hepatitis B Background and aim: The development of drug-resistant mutations during a long-term antiviral therapy is a major cause of the failed treatment of chronic hepatitis B. The short-term combination therapy using two drugs to prevent such drug-resistant mutations is a new treatment strategy, but no research has been conducted about its effect. So, the purpose of this study is: to see what variety of viral quasispecies of HBV exists in HBV DNA polymerase in chronic hepatitis B patients; to examine any changes of the distribution of viral mutations every 12 weeks before and after antiviral drug administration; to check if the short-term combination therapy of antiviral drugs has positive effects on the decrease of virus and the prevention of development of drug-resistant mutations. Methods: The eligible patients had positive HBsAg, with serum HBV DNA levels higher than 20,000 IU/ml(105copies/ml) and serum ALT levels twice higher than the upper limit of normal level. Exclusion criteria included co-infection with hepatitis C or human immunodeficiency virus; evidence of malignancy history; previous exposure to any nucleoside analog that is active against HBV. Between May 2007 and August 2009, a total of 40 chronic hepatitis B Background and aim: The development of drug-resistant mutations during a long-term antiviral therapy is a major cause of the failed treatment of chronic hepatitis B. The short-term combination therapy using two drugs to prevent such drug-resistant mutations is a new treatment strategy, but no research has been conducted about its effect. So, the purpose of this study is: to see what variety of viral quasispecies of HBV exists in HBV DNA polymerase in chronic hepatitis B patients; to examine any changes of the distribution of viral mutations every 12 weeks before and after antiviral drug administration; to check if the short-term combination therapy of antiviral drugs has positive effects on the decrease of virus and the prevention of development of drug-resistant mutations. Methods: The eligible patients had positive HBsAg, with serum HBV DNA levels higher than 20,000 IU/ml(105copies/ml) and serum ALT levels twice higher than the upper limit of normal level. Exclusion criteria included co-infection with hepatitis C or human immunodeficiency virus; evidence of malignancy history; previous exposure to any nucleoside analog that is active against HBV. Between May 2007 and August 2009, a total of 40 chronic hepatitis B patients were prospectively assigned to receive 30mg of clevudine once a day (CLV group; n=20) or 30mg of clevudine and 10mg of adevofir a day for 12 weeks and 30mg of clevudine once a day after 12weeks(CLV + ADV group; n=20). For a molecular analysis, a total of 7 patients who had serum HBV DNA levels on week 12 higher than 1.000 copies/ml (3 patients from the CLV group; 4 patients from the CLV + ADV group) were selected. A liver function test and the HBeAg/anti-HBe Ab/HBV DNA levels of 7 patients were examined every 12 weeks before and after treatment. Cloning (20 clones each time) and the RFMP method were used to find drug-resistant mutations. Results: There were no statistically significant differences between the two groups in sex, age, ALT levels and HBV DNA levels before treatment. The degree of HBV DNA reduction in the CLV+ ADV group was better than that of the CLV group on week 12 and on week 24 statistically, but not on week 36 and on week 48. [median, CLV group vs CLV+ADV group]; -2.67 vs -4.11, week 12, p =0.001; -4.15 vs -4.97, week 24, p = 0.036]. The rtV191I mutation was found before treatment and week 12, but not week 24 when the added results of the cloning and sequencing of 4 patients (CLV+ADV group) were compared with the added results of 3 patients (CLV group). The rtR153Q, rtS223A and rtI224V mutations were found on week 12 and on week 24 in both groups. The rtI91L, rtP109S, rtT118N, rtN121I, rtI122L, rtQ130P, rtS135Y, rtK149Q, rtR153Q, rtI163V, rtI187V, rtS223A and rtI224V mutations were found in both groups on week 24. The rtA181T+rtV191I mutation was found in Case 1 patient of the CLV group on week 24 through cloning and sequencing and the RFMP method. Also, the rtA181T+rtV191I mutation was found in Case 1 patient of the CLV group on week 36 by the RFMP method. The rtI163V mutation was found in Case 3 patient of the CLV group before treatment and on week 12 and week 24 by cloning and sequencing. The rtM204I mutation was found in Case 3 patient of the CLV

group by the RFMP method when a virologic breakthrough developed on

week 48. The rtA181T+rtV191I mutation on week 12 and the

rtV173L+rtA181T+rtV191I+rtM204I+rtM204V mutation on week 48

were found in Case 4 patient of the CLV+ADV group by the RFMP method.

The rtA181T mutation was found in Case 5 patient of the CLV+ADV group

on week 24 and week 48 by the RFMP method. The rtA181T mutation was

found in Case 6 patient of the CLV+ADV group on week 24 by the RFMP

method. The rtV191I+rtM204I mutation was found in Case 7 patient of the

CLV+ADV group on week 24 and week 36 by the RFMP method.

Conclusion: The short-term combination therapy for the treatment of

chronic HBV using clevudine and adefovir reduced HBV DNA levels more

effectively, compared with the clevudine monotherapy. In the case where

antiviral effect was not sufficient, despite the combination therapy, a

variety of mutations were found within a virus gene, same as in the case of the monotherapy. The rtM204I mutation, known as the mutation of

L-nucleoside analogues, was found in the CLV group when a virologic

breakthrough developed. The rtA181T mutation, known as the multi-drug

resistant mutation, was found in the CLV group. The rtM204I mutation, the rtA181T mutation and the rtM204V mutation accompanied by the rtV173L mutation were found in the CLV+ADV group. The rtN236T mutation,

known as a mutation of ADV, was not found in the CLV+ADV group.

Further study to check if these mutations are directly associated with the development of clinically significant antiviral drug resistance and the subsequent treatment outcome is needed.