PG1 PG Course 2015 Viral Hepatitis B 형간염의새로운치료제 고려대학교구로병원 김지훈 Ji Hoon Kim Korea University Guro Hospital, Korea 서 론 만성 B형간염의치료는 1990년대초반부터인터페론이사용되면서본격적인치료가이루어졌다. 그후 1998년에라미부딘이라는경구용항바이러스제가임상에서사용되기시작하면서만성 B형간염치료는급격한발전을이루기시작했다. 현재 B형간염치료제는경구용 6가지약제와주사제인 peg-ifn이임상에서사용되고있다. 인터페론제제의몇몇장점에도불구하고주사제라는불편과비대상성환자에서는사용이제한된다는문제로현재만성 B형간염치료에서는경구용항바이러스제가주류를이루고있다. 특히초기의항바이러스제가장기사용에서내성이많이발생하는문제로인해임상에서많은어려움을야기하였으나최근의엔테카비어나테노포비어는장기사용에도불구하고거의내성이없어만성 B형간염치료를보다용이하게하고있다. 하지만이들약제모두간내의 cccdna 까지제거하여만성 B형간염의완치를가능케하지는못하고있어완치를기대할수있는새로운치료가여전히요구되고있는것이사실이다. 2012년에그동안베일에가려졌던 B형간염의간내유입의수용체가 sodium taurocholate cotransporting polypeptide (NTCP) 라는사실이밝혀졌고 1 이를통한 B형간염유입을차단하는 B형간염치료제가연구되어기대를모으고있으며또한 B형간염의여러생활사의각단계를차단하는치료제개발도전에없이상당한진전을이루고있다. 새롭게개발되는약제들은크게새로운 polymerase inhibitor, 바이러스침입억제제 (entry inhibitor), cccdna inhibitor, nucleocapsid assembly inhibitor, HBsAg secretion inhibitor, small molecules, gene silencing, immune modulator/ therapeutic vaccine 으로나눌수있다. 본고에서는현재사용중인 B형간염치료제외에새로이연구되고있는다양한치료제에대해알아보고자한다. B형간염바이러스의생활사와치료의표적 B형간염바이러스가 1967년 Blumberg 에의해발견된후지난 50여년간 B형간염의생활사에대한다양한연구로많은것이밝혀졌다 (Figure 1). 하지만 2012년에야 B형간염바이러스의간세포내침입에관여하는수용체가확인되었을만큼아직많은부분알려지지않은것이있다. 현재의대표적인두치료제인 peg-ifn은주로면역증강을통하여 B형간염을치료하며경구용항바이러스제들은 polymerase inhibitor로 HBV 증식을 3
Postgraduate Course 2015 Figure 1. HBV life cycle and suggested therapeutic target. 2 억제하는역할만을수행한다. 따라서 ccc (covalently closed circular) DNA를포함한 B형간염바이러스의박멸을위해서는좀더근원적인치료방법이모색되어야하는데새로운치료법의표적은바이러스의생활사에서찾아볼수있다. 완전한형태의 B형간염바이러스인 Dane 입자는 3.2kb의이중가닥 DNA의바이러스와그를둘러싸는캡시드단백질 (capsid protein) 그리고그외부를둘러싸는표피단백질의형태를갖는다. B형간염바이러스가간세포표면에서 heparin sulfate에부착되면표피단백질 (HBsAg) 의 pre-s1 와 B형간염수용체인 NTCP가결합, 상호작용하여 NTCP를통해바이러스가세포내로침투한다 ( 침투과정 ). 간세포내로유입된후에는표피단백질이벗겨지고핵내로침입하여부분이중가닥이던 HBV DNA는원형의 cccdna 로변형되어핵내에존재하며전사의증식을시작한다 ( 증식과정 ). cccdna 로부터전사된 pregenomic RNA 등은 HBsAg, HBeAg, polymerase 등을만들고이들은다시재조립과정을거쳐완전한바이러스형태를갖추어다시핵내로침입하거나간세포밖으로빠져나간다 ( 배출과정 ). 2 따라서침투, 증식, 재조립, 배출과정이모두새로운치료의표적이될수있다. 새로운치료제 1. 새로운 polymerase inhibitor 1) Besifovir (LB80380) 베시포비어는아데포비어나테노포비어와유사한 acyclic nucleotide phosphonate 이다. 베시포비어는간에서빠르게활성대사물인 guanosine monophosphate의형태로바뀌며엔테카비어와유사한방식으로 polymerase의 4
Ji Hoon Kim 특정 tyrosine residue에결합하여엔테카비어와같은강력한바이러스증식억제작용을나타낸다. 1상연구에서일일 60 mg부터 240 mg까지적정한 HBV 증식억제반응을보였다. 3 2상연구에서엔테카비어와비교되었는데 48주치료에 HBV DNA가검출한계이하로감소하는정도는베시포비어에서 63%, 엔테카비어에서 58% 로유사하였으며내성은확인되지않았다. 4,5 베시포비어를사용한환자의 94% 에서혈청 L-carnitine이감소하는부작용이발견되었으나 L-carnitine을복용함으로써정상화되었다. 베시포비어의이런특이한부작용은베시포비어의 pivalic acid 구조때문으로알려져있으며 pivalic acid 구조를가지는아데포비어에서도이와같은부작용이보고되었으나임상적으로유의한문제는없었다. 6 현재베시포비어는베시포비어와함께 L-carnitine을복용하는 3상연구가진행중이다. 2) Prodrug of tenofovir 강력한항바이러스제인테노포비어보다생체활성도를높이고항바이러스작용을강화하며신기능의부작용을줄이고자 prodrug들이개발되고있다. 가장대표적인약제는 tenofovir alafenamide (TAF, GS 7340) 이다. TAF 는체내에서 tenofovir-alanine conjugate (TFV-Ala) 로변환된후다시 tenofovir 를거쳐활성대사물인 tenofovir diphosphate (TFV-DP) 로인산화된후작용한다. 최근 HIV환자에서테노포비어와비교한 3상연구가발표되었으며유사한항바이러스효과를보이면서혈청크레아틴의상승, 단백뇨의발생, 골밀도의감소에서유의하게좋은반응을보였다. 7 B형간염환자에서용량적정을위한 28일간의 TAF 사용연구에서 8 mg에서 120 mg 까지특이한부작용없이유사한항바이러스반응을보여 25 mg을사용한 2상과 3상연구가진행되고있다. 8 그외에도테노포비어의 hexadecyloxypropyl conjugate인 CMX157와또다른구조의 AGX-1009 등이일상또는이상연구가진행되고있다. 2. 바이러스침입억제제바이러스침입을억제하면 B형간염의발생자체를차단할수있고새로운간세포에더이상의바이러스가침입할수없도록할수있으므로매우매력적인표적이라할수있으나 2012년에야 NTCP가발견되어앞으로이에대한연구가활발해질것으로보인다. 현재까지 NTCP를통해 B형간염바이러스가침입하지못하도록직접개발된약제는없으나이전부터알려진약제들은상당수존재한다. 1) Myrcludex NTCP가발견되기전에 B형간염바이러스의 Pre-S1 단백질이세포내침입과정의 ligand이라는데착안하여 pre-s1 단백질의 47개아미노산을합성하여만들어진약제이다. Myrcludex는 in-vitro 9-12 와 in-vivo 13-15 실험에서 B형간염의바이러스의침입을효과적으로억제할수있다는사실이확인되었으며 1상연구에서경정맥또는경피로투여하였을때대체적으로별다른부작용은없었다고알려져있다. 16 현재러시아에서 2상연구가진행중이다. 2) Cyclosporin cylcosporin A는면역억제제로보편적으로사용되고있는약제로 NTCP를억제하여담즙정체의부작용을나타낼수있는것으로알려져있었다. 따라서실제적으로부작용을이용하는것이긴하지만 NTCP를억제하는첫합성약물이라하겠다. 또한 cyclosporine A는 HIV, HCV, influenza, HPV 등다양한종류의바이러스를억제할수있다는연구도있어왔다. 최근 cyclosporin 이 HepG2-hNTCP 를이용한세포실험에서용량의존적으로 B형간염바이러스감염을억제시킬수있다는연구가있었다. 17-19 또한 Cyclosporin B도 NTCP 억제기능이 5
Postgraduate Course 2015 뛰어나며 18 cyclosporin의유도체중하나인 SCYX1454139은 cyclosporine A보다 10배이상의저해효과를보인다는보고가있다. 17 따라서이들이가지는면역억제기능을조절할수있다면앞으로 B형간염치료제로유용하게사용될가능성을기대할수있다. 3) 기타기존의 heparin과 dextran sulfate는 B형간염바이러스의부착을저해한다고알려져있고 NTCP의기질인 UDCA, cholate, progesterone 등도바이러스의침입을억제할수있다고알려져있다. 최근이미승인되어사용되고있는약제들을실험실적으로스크리닝하여 NTCP의저해능을확인했을때 statin계열과 angiotensin inhibitor 들이효과적일수있다는보고도있었다. 20 3. cccdna inhibitor cccdna는핵내로침입함 HBV 유전자가안정된형태로존재하는상태로지속적인 HBV 복제의주형이되는데현재의항바이러스제는이를제거하지못한다. 따라서 cccdna를직접제거할수있는약제는새로운 B형간염치료의지평을열게될것이다. cccdna 를표적으로하는약제는형성억제제, 절단제, 제거제정도로구분될수있고, 최근 cccdna가 histone에감겨 minichromosome을형성하고있고 histone 단백질의아세틸화나메틸화와같은후성적 (epigenetic) 변화에의해 HBV의복제정도가결정된다고알려지면서 21 이들을제어하는 cccdna 기능억제제가연구되고있다. 1) cccdna 형성억제제실험실적 cell-based cccdna assay를통해 85,000개의후보물질을검증하여 sulfonamide의유사체인 disubstituted sulfonamides 두가지가 (CCC-0975, CCC-0346) cccdna 의형성을억제한다는보고가있다. 22 이연구에서그기전으로 cccdna의전구물질인 relaxed circular DNA의 deproteinization 을방해하는것을제시하였다. 2) cccdna 절단제 Zinc-finger nucleases (ZFNs) 와같은 DNA cleavage 효소를이용하여 cccdna의염기를효과적으로절단할수있다는연구가최근발표되고있다. 23-25 하지만이들효소를감염된간세포로전달하고 cccdna 만을표적으로하기위해서는아직많은연구가필요하다. 3) cccdna 제거제 HBV에감염된간세포에서 HBV를제거하는데는세포면역을통해간세포를감염간세포를제거하는것이주가되고이에따라특히인터페론등의항바이러스치료를하는중 ALT 상승과같은부작용이초래되기도한다. 또다른방법은 cytokine을이용한 noncytotoxic한 HBV 제거방법이있겠으나현재까지 cccdna 를선택적으로제거하는 noncytotoxic한증거는확인되지않았다. 최근 Lucifora 등은 IFN-α와 lymphotoxin β receptor를활성화시키는항체 (BS1, CBE11) 를투여함으로써 APOBEC3A와 APOBEC3B cytidine deaminases를유도하여이들이 cccdna 를 deamination 시킴으로써선택적으로 cccdna를제거할수있으며 genomic DNA 에는영향이없었다는보고를하였다. 26 4) cccdna 기능억제제 cccdna의후성적변화가전사등의기능을조절한다고알려지면서후성적조절을할수있는 Histone 6
Ji Hoon Kim deacetylase inhibitor (HDACi), JMJD3 histone demethylase inhibitor, p300/pcaf histone acetyltransferase (HAT) inhibitor 등의물질들이관심을끌고있다. 전통적인 B형간염치료제인 IFN-α 에있어서도세포배양과마우스 (chimeric upa/scid) 실험에서 IFN-α 가 histone의아세틸화를저해함으로써 cccdna로부터 RNA를전사하는과정을억제하는후성적기능이최근추가적으로알려졌다. 27 4. Nucleocapsid assembly inhibitor HBV의캡시드는게놈의 packing, 역전사, 세포내수송 (trafficking), 안정적감염의유지에전반적으로작용하며 HBV의자연사에서필수적인역할을수행한다. Heteroaryldihydropyrimidines (HAPs) 은 assembly inhibitor 종류로비뉴클레오사이드계열의 B형간염바이러스억제효과가밝혀지고있다. 28-31 HAPs 은이성체적 (allosteric) 반응기 (effector) 로조립과정에서다양한변형된바이러스입자를만들게하여극적으로올바른모양으로결합하지못하도록방해한다. 32,33 개발되고있는 HAPs중하나인 BAY 41-4109 는 humanized upa/scid 마우스실험에서 5일치료에1log 10 정도의 HBV DNA 감소를보였고끊고 5일이후에재반등이있었다. 28 GLS4도 HAPs의일종으로 HBV를억제하는소입자로 small molecule 야생형과아데포비어 HBV DNA를억제한다고보고되었다. 34 5. HBsAg secretion inhibitor 바이러스입자인 HBsAg은 HBV에대한체내면역을억제한다고알려져있다. 따라서감염된간세포에서분비되는 HBsAg을줄여서면역능을회복시키고 HBV 치료효과를증강시킬수있다. REP 9AC은이런목적으로개발되고있는약재로양친매성 (amphipathic) 뉴클레오티드폴리시티딘 DNA이다 (nucleotide polycytidine DNA). REP 9AC를주 1회 500 mg 정맥주사하여치료 1-32주안에 8명의환자중 7명에서 HBsAg이사라지고 anti-hbs 가나타났다. 이 7명중 3명에서 HBV DNA가감소하였다. 35 향후추가적인연구가기대된다. 6. Small molecule Cryptosporidium과 Giardia lamblia 의치료에주로쓰이는항생제의일종인 nitazoxanide는 HIV와 HBV 또는 HCV가감염된환자의설사병을치료하다가우연히 HBV와 HCV에효과에있음을발견한약제이다. Nitazoxanide 계열의약물은 protein kinase의활성화를통한면역유도작용으로항바이러스효과를나타내는 small molecule로구분되는약제이다. Nitazoxanide은라미부딘아데포비어와함께사용할때효과의증강을보였으며라미부딘또는아데포비어의내성바이러스에도효과적이었다. 36 4명의 HBeAg 양성환자에서 nitazoxanide 500 mg을 1일 2회 1년이상사용하였을때 2명에서 HBV DNA가음전되고 3명이 HBeAg 소실이있었고 1명에서 HBsAg 소실이있었다. 37 7. Gene silencing microrna 와 sirna 는간섭현상을통해 sequence specific한방법으로유전자의발현을억제하는것으로알려져있으며 HBV는불필요한중복이적은조밀한유전체를가지고있어이들의좋은표적이될수있다. 38 세의환자에서 NUC B100 5 mg의 DNA 용량을투여한 1상연구에서약제투여후 IFBN-α 와 γ IL-10 등의상승이확인되었으며모두근육통, 발열이있었으나해열제 1회투여로안정화되었다. 39 7
Postgraduate Course 2015 최근전달효과가뛰어난 dynamic polyconjugate 약물전달방법을이용하여개발된 sirna인 ARC-520 은형질전환마우스모델과 40 HBV 감염된침팬지모델에서 41 HBV DNA, HBsAg, HBeAg의감소효과를보였다. 최근의 36명을대상으로한 1상연구에서 42 24명의 ARC-520 을투여받은환자와위약을받은 12명에서유의한이상작용의차이는없어현재 2상이진행중이다. 8. Immune modulator / therapeutic vaccine 만성 B형간염환자는자연살해세포에의한 cytokine 생성이원활하지않고세포면역기능을조절하는조절 T세포에의해면역이억제되어있는상태이므로 43-45 면역을향상시키는약제로 HBV를치료하려는시도도있다. 1) TLR-7 작용제 (agonist) TLR는내인성면역체계를구성하는인자로외부에서침입하는병원체에제일먼저반응하는작용체중의하나이며 HBV는 TLR 관련내인성면역을억제한다. 따라서 TLR 작용제로내인성면역을강화시키는것은좋은 HBV 치료의표적일수있다. 경구용 TLR-7의작용제로개발된 GS-9620 은침팬지를이용한 in-vivo 실험에서상당한 HBsAg, HBeAg, HBV DNA 감소효과를보였으며간세포의세포자살을유도하였고 46 75명을대상으로하여 1회 12 mg까지증량하는 1상연구에서도안전성이입증되었다. 47 2) GS-4774 (GI-13020, Tarmogen) 효모를근간으로재조합하여만들어진치료백신으로 HBx, S, core 항원을포함하고있다. 1상연구에서안정성을확인하였고 48 현재 2상연구중이다. 결 론 최근 20여년간만성 B형간염의치료는비약적인발전을하였다. 이에따라현재의만성 B형간염치료는간염의악화, 간경변으로의진행, 비대상성악화뿐아니라간암의발생률도감소시킬수있다. 49, 50 또한최근의경구용항바이러스제는장기사용에도내성이발현이거의없어장기사용에따른간경변의조직학적개선도이루어지는것으로알려졌다. 51 하지만, 현재의약제는만성 B형간염의 cccdna를포함하는완전한퇴치를이룰수없어약제를종료하지못하고평생투약을해야하며지속적인간암감시검사를받아야하는상황이다. 전술한많은새로운항바이러스제가연구되고있으나이들이만성 B형간염의완전한퇴치를이루어낼수있을지는아직은불분명하다. 다만최근의만성 C형간염에서강력한경구용약제로거의모든환자에서치료후바이러스가완치될수있는것과같이이러한많은연구와약제의개발이궁극적으로는만성 B형간염의퇴치를이룰수있을것으로기대해본다. 참고문헌 1. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 2012;1:e00049. 8
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Ji Hoon Kim 48. Gaggar A, Coeshott C, Apelian D, Rodell T, Shen G, Subramanian M. Safety, Tolerability, and Immunogenicity of GS-4774, an HBV-Specific Therapeutic Vaccine, in Healthy Volunteers. Hepatology 2013;58:656 [Abstract]. 49. Hosaka T, Suzuki F, Kobayashi M, Seko Y, Kawamura Y, Sezaki H, et al. Long-term entecavir treatment reduces hepatocellular carcinoma incidence in patients with hepatitis B virus infection. Hepatology 2013;58:98-107. 50. Liaw YF, Sung JJ, Chow WC, Farrell G, Lee CZ, Yuen H, et al. Lamivudine for patients with chronic hepatitis B and advanced liver disease. N Engl J Med 2004;351:1521-1531. 51. Marcellin P, Gane E, Buti M, Afdhal N, Sievert W, Jacobson IM, et al. Regression of cirrhosis during treatment with tenofovir disoproxil fumarate for chronic hepatitis B: a 5-year open-label follow-up study. Lancet 2013;381:468-475. 11