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약학석사학위논문 천연물유래합성물질의 shear stress 에의한 혈소판활성화억제작용 Inhibitory Effects of Natural Product Derivatives on Shear Stress-Induced Platelet Aggregation 2017 년 8 월 서울대학교대학원 약학과예방약학전공 노학준
초 록 혈소판은생채내주요방어기전인지혈작용에있어서주요한역할을하지만, 혈소판이과도하게활성화되는경우혈전생성을발생시켜관상동맥질환, 심근경색, 뇌졸중과같은다양한심혈관계질환으로발전할수있다. 그리고심혈관계질환은전세계사망원인의가장큰부분을차지하고있다. 따라서다양한항혈소판제제들이혈전질환및심혈관계질환의치료및예방을위해이용되었다. Aspirin, Clopidogrel 등의기존항혈소판제제의경우심혈관계질환에대한치료및예방효과가입증됐지만, 동시에정상적인지혈작용을저해함으로써출혈부작용을가지는것으로알려져있다. 따라서정상적인지혈작용에는영향을주지않으면서병적인혈전의생성은저해할수있는더욱안전한항혈소판제제를개발하는것이필요하다. 혈소판은생리적인 agonist 들인 thrombin, adenosine diphosphate (ADP), collagen 등과혈류에의한물리적인 shear stress 의해활성화된다. shear stress에의한혈소판응집은다른 agonist 들에의한혈소판응집과는달리정상적인지혈작용에는크게관여하지않지만, 병적인상태에서의혈전생성에주로관여한다. 그래서 shear stress-induced platelet aggregation(sipa) 은새로운항혈소판제의주요개발목표로여겨지고있다. ii
12 종의천연물유래합성물질의 SIPA에대한혈소판응집억제효과를관찰하기위하여다음의연구를수행하였다. 천연물유래합성물질을사람의 platelet rich plasma (PRP) 에가한후 SIPA에대한효과를확인해보았다. 그결과 MG1002, 1009 이가장큰억제효과를보였으며, 그효과는농도의존적이었다. 물리적요인인 SIPA에대한효과와생리적인 agonist (thrombin, ADP, collagen) 에의한혈소판응집에대한효과를비교해보았다. 그결과, agonist 에의한혈소판응집에서 MG1002, 1009는혈소판응집억제효과가나타나지않았지만, SIPA에서 MG1002, 1009에의한억제효과가특이적으로나타났다. 혈소판에대한세포독성지표인 lactate dehydrogenase (LDH) leakage를확인해본결과세포독성은나타나지않았다. 그리고 platelet poor plasma (PPP) 에서의 prothrombin time (PT) 와 activated partial thromboplastin time (aptt) 에영향을주지않는것을확인함으로써지혈시간지연작용에영향을미치지않는다는것을확인하였다. MG1002, 1009의 SIPA 억제효과가실제생체내에서혈전생성에어떤영향을주는지확인하고자 in vivo arterial thrombosis model을이용하였다. MG1002, 1009을경구투여하고 FeCl3를적용한후혈전생성으로인한혈류속도의변화를관찰하였다. 그결과 MG1009는혈류가멈추는데걸리는시간인 occlusion time을지연시켰으며, 이는혈전생성을저해함을의 iii
미한다. 또한항혈소판제제의대표적인부작용인 bleeding time 의문제점을 검증하기위해서 in vivo tail bleeding time model 을이용하였다. MG1002, 1009 모두 bleeding time 을지연시키지않았다. 결론적으로 MG1009는신체내에서중요한혈소판활성화요인중하나인 shear stress에의해유도되는혈소판의활성화와응집에대해서특이적인억제효과를보였으며, 출혈부작용없이혈소판이주요한역할을하는혈전생성을저해하였다. 이는심혈관계, 특히 shear stress와관련된질환들의예방및치료와관련해유용하게활용될수있다고사료된다. 주요어 : 천연물유래합성물질, 혈소판, 혈소판응집, shear stress, 혈전 생성, bleeding time iv
목 차 초 목 록 i 차 v List of Figures and Table vi List of Abbreviations vii 서론 1 실험방법 3 시약및기기 4 혈액시료의준비 5 Shear stress의적용 6 Shear-Induced Platelet Aggregation (SIPA) 측정 6 Agonist-Induced Aggregation 측정 7 Cytotoxicity 측정 8 혈관조직분리 9 혈관의수축력측정 10 In vivo 동맥혈전생성능측정 11 In vivo 혈액응고 Bleeding time 측정 12 통계처리 13 실험결과 14 고찰 26 참고문헌 30 Abstract 33 v
List of Figures and Table Figure 1. Inhibitory effects of MGs on Shear Stress-Induced Platelet Aggregation in human platelet rich plasma. Figure 2. Inhibitory effects of MG1002, 1009 in various concentration on SIPA in human PRP and WP. Figure 3. Effects of MG1002, 1009 on agonist-induced platelet aggregation in human PRP. Figure 4. Effects of MG1002, 1009 on Cytotoxicity. Figure 5. Effects of MG1002, 1009 on coagulation. Figure 6. Effects of MG1009 on Phenylephrine induced contraction in rat aortic ring. Figure 7. Effects of MG1002, 1009 on thrombus formation in rat in vivo arterial thrombosis model. Figure 8. Effects of MG1002, 1009 on thrombus formation in rat in vivo tail bleeding time measurement. Table 1. Comparison of inhibitory effects by MG1002, 1009 on SIPA and agonist-induced platelet aggregation in human PRP. vi
List of Abbreviations ACD : acid citrate dextrose ADP : adenosine diphosphate aptt : activated partial thromboplastin time DMSO : dimethyl sulfoxide EDTA : ethylene diaminetetraacetic acid GP : glycoprotein LDH : lactate dehydrogenase β-nadh : β-nicotinamide adenine dinucleotide, reduced form PE : phenylephrine PGE 1 : prostaglandin E 1 PPP : platelet poor plasma PRP : platelet rich plasma PT : prothrombin time vwf : von Willebrand factor WP : washed platelets vii
서 론 혈액의구성성분으로혈액응고에중요한역할을하는혈소판은골수의거핵세포 (megakaryocyte) 로부터분화되어생성되며혈류를따라전신을순환하는혈액세포로 (Hartwig and Italiano, 2003) 활성화되어지혈작용 (haemostasis) 과혈전생성작용 (thrombosis) 에중요한역할을담당한다 (Ni and Freedman, 2003). 이러한작용은혈소판응집및혈소판과인접조직과의상호작용을통해이루어진다 (Andrews and Berndt, 2004). 지혈작용의경우혈관손상에의한내피하층 (subendothelium) 의노출로인해 collagen이혈류에노출되거나, 여러자극에의해 thrombin 등활성화물질이증가하면, 혈소판활성화가유발된다. 혈소판의활성화는 subendothelium의 collagen과 von Willebreand Factor (vwf) 를매개로한 subendothelium에의부착 (adhension), 분비 (secretion) 및응집 (aggregation) 이라는일련의순서를통해혈소판응집체 (aggregate) 까지형성하는과정을말한다. 여러요인들에의해혈소판이과도한활성화가일어나면, 관상동맥혈전증, 뇌졸중 (stroke), 동맥경화 (atherosclerosis) 등의심혈관계질환을일으킨다 (Michelson, 2004). 혈소판을활성화시키는요인에는생리적 agonist에의한요인과혈류에의해유발되는물리적요인인 shear stress가있다. 이전에심혈관계연구에있어서 thrombin, adenosine diphosphate (ADP), collagen, arachidonic acid 등의생리 1
적 agonist에의한혈소판활성화에관한연구가많이이루어져왔다 (Jackson, 2007). 물리적요인인 shear stress는 agonist에의한혈소판활성화와는다른기전을통해혈소판활성화를유발하며 (Kulkarni et al., 2000), 혈류에의해유발되는환경을반영하기유리하다 (Kroll et al., 1996). 이러한이유로 shear stress에의해유도된혈소판응집을 shear stress-induced platelet aggregation(sipa) 이라정의하고최근많은관심을가지고있다. Shear stress 는혈류에의해작용되는단위면적당힘이라정의하며, 점성이있는혈액의흐름에의해혈과내피세포 (endothelial cell) 나혈소판, 적혈구등의혈액세포에가해지는힘을의미한다 (Hanson and Sakariassen, 1998). 일반적으로정맥에서는 20~200 s -1, 동맥에서는 300~800 s -1, 미세혈관에서는 500~1,600 s -1 정도의 shear rate가나타나며, 동맥경화나뇌경색등의질환과같이병적인상 태의협착성혈관에서는 10,800 s -1 이상까지 shear rate 가증가한다 (Kroll et al., 1996). 따라서협착성혈관에서는 SIPA가증가하면, 혈전의생성이증가하게된다 (Holme et al., 1997). 기존의항혈소판제재로사용되었던약물들에대해서 SIPA에어떤효과가있는지확인된바있다. 대표적인항혈소판제재인 aspirin과 SIPA에효과가없으며 (Nesbitt et al., 2009; Tanigawa et al., 2000), ticlopidine(matsumoto et al., 2005), abciximab, eptifibatide, tirofiban(derhaschnig et al., 2004; Goto et al., 2003; Haga et al., 2002; Wang et al., 2002), cilostazol(minami et al., 1997) 등 2
은 SIPA 또한억제하는것으로보고된바있다. 그리고최근여러천연물과그로부터분리한유효성분들이 SIPA억제효과와혈전생성저해효과가있다고보고되고있다. Protocatechuic acid는전통적으로한약재로사용되어온금은화의유효성분으로혈액순환개선에도움을준다고알려진천연물추출물이다. 여러연구들을통해 Protocatechuic acid가항혈소판, 항혈전작용이있다고보고된바있다 (Kim et al., 2012). 본연구에서는 Protocatechuic acid의유래합성물질 12종을사용하여 SIPA에대한작용을검색하였다. 그리고혈전생성에주는영향에대해연구해보았다. 3
실험방법 시약및기기 Bovine Serum albumin, ADP, thrombin, EDTA, Triton X-100, β-nadh, Ferric chloride 그리고 50% glutaraldehyde등은 Sigma Chemical Co. (St Louis, USA), Collagen은 Chrono-Log Corp에서구입하였고, von Willebrand factor (vwf) 는 Calbiochem (San diego, USA) 에서구입하였으며, MG 유도체화합물은신풍제약으로부터공급받아사용하였다. 그밖의시약은모두특급시약을구입하여사용하였다. 실험동물 Sprague-Dawley 수컷흰쥐를영바이오 (Korea) 로부터공급받아, 서울대학교약학대학실험동물실에서일주일가량적응시킨후실험에사용하였다. 사육실은온도 22±2, 습도 45-55% 를유지하였으며, 오전 7시 30분과오후 7시 30분을기준으로하여밤낮을조절하였다. 사료 ( 애그니브랜드, 퓨리나코리아 ) 와물은제한없이공급하였다. 4
혈액시료의준비 Platelet rich plasma (PRP) 를분리하기위하여, 2주일이상약물을복용하지않은건강한남성의정맥으로부터 3.2% sodium citrate를항응고제로하여혈액을채취한다. 실험전과정에걸쳐서유리용기나유리 pipette의사용은피하였으며. 혈소판분리의전과정은상온에서수행한다. 채혈후 150g에서 15분간원심분리한후상층액 (PRP) 을얻고잔사를 2000g에서 20분간원심분리하여 platelet poor plasma (PPP) 를얻는다. 이와같이얻은 PRP의혈소판수는 hemacytometer를이용하여광학현미경으로세며. PRP를 PPP로희석하여 1 ml당 3ⅹ10 8 개의혈소판이포함되도록한후실험에사용하였다. Washed platelet (WP) 를분리하기위하여, 2주일이상약물을복용하지않은건강한남성의정맥으로부터 acid-citrate-dextrose(acd) 를항응고제로하여혈액을채취한다. 채혈시 prostaglandin E1(PGE 1) 1M을처리하여혈소판의활성화를억제하였다. 채혈한혈액을 150g에서 15분간원심분리하여상층액에서 PRP를얻고, 이를 500g에서 10분간원심분리하여혈소판 pellet을얻는다. 이를 washing buffer (134 mm NaCl, 2.9 mm KCl, 1.0 mm MgCl2.6H2O, 10.0mM HEPES, 5.0 mm dextrose, 12.0 mm NaHCO3,0.34mM Na2HPO4, 10% ACD, 0.3% bovine serum albumin, 1M PGE1, ph7.4) 로현탁하여세척한후, 다시 400g에서 10분간원심분리한다. 여기서얻어진혈소판 pellet을 suspension 5
buffer (134 mm NaCl, 2.9 mm KCl, 1.0 mm MgCl2.6H2O, 10.0 mm HEPES, 5.0 mm dextrose, 12.0 mm NaHCO3, 0.34 mm Na2HPO4, 0.3% BSA, ph7.4) 로현탁한다. WP 중의혈소판수는 hemacytometer를이용하여광학현미경으로세며. suspension buffer로희석하여 1 ml당 3ⅹ10 8 개의혈소판이포함되도록한후실험에사용한다. WP의경우 CaCl 2 를최종농도가 2 mm이되도록가한후실험에사용한다. Shear stress의적용시료 500 μl를 cone-plate viscometer (RotoVisco 1, Haake, Germany) 에올린후 10,800 s -1 의 shear rate로 37 에서 3분동안 shear stress를준다. Shear stress에의한혈소판의응집효과를측정하기위하여, WP의경우 vwf 를최종농도가 10 μg/ml가되도록가하고 shear stress를준다. 각각의실험에서 shear stress를가하지않은대조군은시료를 cone-plate viscometer에올린후, cone을회전시키지않은것으로한다. Shear stress-induced Platelet Aggregation (SIPA) PRP 또는 WP 598.5 μl에 DMSO 또는후보물질를 1.5 μl 가하고 thermomixer를이용하여 37 에서 3 분간배양한다. WP의경우에는배양한 WP 495 μl를 100 μg/ml vwf 55 μl에가하여최종농도가 10 μg/ml가되도록 6
한다. 그리고 PRP 또는 WP 500 μl를 cone-plate viscometer에올린후 10,800 s -1 의 shear rate로 37 에서 3분동안 shear stress를준다. Shear stress를가한후, 20 μl의 PRP를 0.5% glutaraldehyde를포함한 Isoton II buffer 280 μl에넣어고정시킨다. 그후이현탁액중에단독존재하는혈소판수를 hemacytometer를이용하여광학현미경으로세어, aggregation 정도를다음과같이표현한다. Aggregation (%) = (1-A/A0) ⅹ 100 Inhibition (%) = (Aggregation % of sample/aggregation % of control) Maximal Inhibition (%) = Inhibition ⅹ2 A: 후보물질을가한후 shear stress 를준 sample 의혈소판수 A0: Shear stress 를주지않은 sample 의혈소판수 Agonist 에의한후보물질 selectivity 평가 후보물질의 agonist에이한 selectivity를평가하기위하여혈소판의응집정도를 lumi-aggregometer (Chrono-Log Co., USA) 를이용하여 turbidity 변화로써측정한다. PRP의 light transmission을 0%, PPP의 light transmission을 100% 로맞춘후, 37 에서혈소판의 aggregation 정도에따른 light transmission을측 7
정한다. 측정시 silicon으로코팅된 aggregometer cuvette을사용하며, 반응이일어나는동안 1,000 rpm에서지속적으로교반한다. PRP 598.5 μl에 DMSO 또는후보물질을 1.5 μl 가하고 thermomixer를이용하여 37 에서 3분간배양한다. 배양한 PRP 500 μl를 aggregometer cuvette에넣고 1분간 pre-incubation하여 37 가되게한다음혈소판응집유발시료인 thrombin, ADP 그리고 collagen을최대응집을일으키는최소농도를가한다. 이후 thrombin은 6분, ADP 6분, collagen은 6분간반응을관찰한다. PT 및 aptt 혈액응고시간측정사람으로부터분리한혈액을상온에서 2000 g에서 15 분간원심분리하여혈장을얻는다. 혈장응고시간은 BBL Fibrometer를이용하여 aptt, PT를측정하였다. aptt 측정을위하여혈장과 aptt 시약을 fibrometer cup에가한후 3 분간 37 에서배양한다. 배양후 CaCl 2 를가하고, 즉시혈액응고시간을측정한다. PT의측정을위해서가온된혈장에 PT 시약을가하고즉시혈액응고시간을측정한다. Cytotoxicity 측정 Cytotoxicity 측정은혈소판으로부터 lactate dehydrogenase (LDH) 유출은 spectrophotometry 방법을이용한다. PRP 499 μl에 DMSO 또는후보물질을 1 8
μl 가하고 thermomixer를이용하여 37 에서 3분간배양한다. 배양후시료 100 μl를취해 12,000 g에서 2분간원심분리하고, 상층액 80 μl를취하여 assay 전까지냉장보관하며, assay는 24 시간이내에수행한다. Positive control로서 50 μm digitonin을가하여 1시간동안처리한다. 미리가온한 Tris-EDTA NADH 용액 (56 mm Tris(hydroxymethyl) aminomethane, 5.6 mm EDTA, 0.17mM β-nadh ph 7.4) 1 ml에시료 25 μl를가한다음, 37 에서 5 분동안배양한다. 여기에 37 에서미리가온해놓았던 100 μl의 14 mm pyruvate 용액을가하고, 즉시 UV-Vis spectrophotometer (UV-2201, Shidmadzu, Japan) 를이용하여 339 nm의파장에서 1분간흡광도감소를측정한다. 흡광도감소속도는 NADH의산화속도를의미하며, 이는혈소판으로부터유리된 LDH의활성도를나타낸다. 0.3 % Triton X-100으로혈소판의 lysis를유발하여 LDH의총활성도를측정한다. 각시료의활성도는 LDH 의총활성도에대한백분율로측정한다. 혈관조직분리 혈관의반응은 organ bath system을이용하여측정하였다. 흰쥐를실혈치사시킨후개흉하여신속하게흉부대동맥 (thoracic aorta) 을적출하고, 즉시 95% O2/5% CO2의혼합가스로포화된 ice-cold Krebs-Ringer 완충액 (NaCl 115.5 mm, KCl 4.6 mm, KH2PO4 1.2 mm, MgSO4 1.2 mm, CaCl2 2.5 mm, NaHCO3 9
25.0 mm, disodium. Ca2+-EDTA 0.026 mm, glucose 11.1 mm, ph 7.4) 으로옮 겼다. 혈관내의혈액과주변의결합조직및지방등을제거하고길이 3~4 mm 의혈관 ring 을만들었다. 내피세포가없는혈관은안과용핀셋끝을혈관내 에넣고 KR 완충액으로적신거름종이위에서 3 초씩 10 회정도회전시켜내피 를제거하였다. 매 3 초마다 KR 완충액에다시담그고혈관이건조해지지않도 록하였다. 혈관의수축력측정만들어진혈관 ring을혈관에자극이가해지지않도록주의하면서 KR 완충액 10 ml가채워진 organ bath에현수하였다. Organ bath내의 KR 완충액은 circulator bath (PolyScience, USA) 를이용하여 37 를유지하고, 95% O2/5% CO2 의혼합가스를지속적으로공급하여 ph 7.4 를유지시켰다. 혈관 ring 은 두개의텅스텐사를이용하여고정하였는데하부의텅스텐사는 organ bath 내 의 holder 에부착되어있고, 하부의철사와평행되게장치한상부의텅스텐사 는 Grass FT03 force transducer (Grass Instrument Co., Quincy, USA) 에연결 되어혈관의장력을감지하도록하였다. 감지된장력의변화는 BIOPAC's MP100 data acquisition system (BIOPAC Systems Inc. Goleta, USA) 에의해 computer로전달되고, Acknowledge III computer program으로결과를기록, 분석하였다. 10
혈관 ring은초기 30분간점증적으로장력을주어 resting tension인 2g이된후 30분이상방치하여평형상태에도달하도록하였다. 혈관을 phenylephrine (PE) 10-6 M으로수축시킨후, acetylcholine (Ach) 10-6 M 을가하여이완시켰을때이완율이 80% 이상인것을표본으로사용하였다. 그후 30분간혈관장력을 2g의평형상태로유지시킨다음, NaCl과의이온평형을고려하여제조한 90 mm KCl을함유한 KR 완충액 (37 ) 으로 Organ bath내의완충액을교환하여혈관의수축을유도하였다. 이것을최대수축으로삼아혈관수축률을구하였다. 그후 3번 wash 한후혈관장력이 2g의평형상태로유지되도록 30분간 KR 완충액상에두었다. MG1009의용량의존성효과를관찰하기위해서 2g의평형상태에서, 혈관에용매 (DMSO) 혹은 MG1009를 30분전처리한다음, Organ bath 상에서수축유발 agonist인 PE를점증적으로가하여혈관장력의변화를관찰하였다. In vivo 동맥혈전생성능측정 실험에는 Sprague-Dawley 수컷흰쥐중체중이 250 ~ 300 g이되는것을실험에사용하며, 실험동물은실험에사용하기전에하룻밤절식한다. 실험동물에 Control 시료 (DMSO : DW : Tween 80 = 1 : 2 : 17) 또는후보물질을농도별로경구투여한후 30 분뒤에 urethane (1.25 g/kg) 을복강투여하여실험동물을마취시킨다. 마취가되면수술대위해실험동물을고정시키고, 전수 11
술과정동안 heating pad를이용하여실험동물의체온을유지시킨다. 실험동물의목부근을절개하여 right carotid artery를조심스럽게노출시키고, 혈관에붙은지방조직을조심스럽게제거한다. Doppler flow probe (0.5 mm-diameter, DBF-20A, Crystal Biotech, USA) 를노출된 carotid artery에고정하고, Doppler flowmeter (PD-20, Crystal Biotech, USA) 에연결시킨다. 경구투여후 60분이되는시점에서 50% FeCl 3 에적신 whatman No. 1 여과지 (4 mm ⅹ 2 mm) 조각을 probe가고정된바로밑혈관위에부착한다. 여과지는 10분후제거하며, 제거한시점부터 60분동안 carotid artery 내의혈전생성에의한혈류의변화를측정한다. Occlusion time은최초로혈류가 0 이되어 30초이상지속되는시점으로한다. In vivo 혈액응고 Bleeding time 측정 실험에는 Sprague-Dawley 수컷흰쥐중체중이 250 ~ 300 g이되는것을실험에사용하며, 실험동물은실험에사용하기전에하룻밤절식한다. 실험동물에 Control 시료 (DMSO : DW : Tween 80 = 1 : 2 : 17) 또는후보물질을농도별로경구투여한후 30 분뒤에 urethane (1.25 g/kg) 을복강투여하여실험동물을마취시킨다. 마취가되면수술대위해실험동물을고정시키고, 전수술과정동안 heating pad를이용하여실험동물의체온을유지시킨다. 경구투여 60분후 rat의꼬리끝 3 mm 부분을자르고매 30초마다 whatman No. 1 12
여과지를이용하여흘러나온혈액을조심스럽게닦아준다. 출혈시간은더이상 혈액이묻어나오지않을때까지시간으로측정하며, 출혈이 30 분이상지속될 경우출혈시간은 30 분으로기록한다. 통계처리실험결과는 mean ± S.E.M으로표시하였다. 두군사이의차이는 t-test로서검증하였으며, 세군이상의비교에서는 one-way ANOVA test를수행한후 Duncan's multiple range test를실시하여유의성을검증하였다. 유의적인차이는 p value가 0.05 이하인경우에인정한다. 13
실험결과 심혈관계질환에효과가알려진천연물 MG의유도체 12종을 25 μm의농도로인체의혈액으로부터분리된 PRP에 3분간배양후 SIPA에대한억제효과를검색하였다. 검색한결과를토대로 Aggrgation (%) 과 Maximal Inhibition (%) 를계산하여도출했다 (Fig. 1). 그결과 MG1002, MG1009에서높은 SIPA 억제효과가나타났다. MG1002와 MG1009를선택하여 10, 25, 50 μm 농도에따른 SIPA 억제효과를확인해본결과농도의존적인 SIPA 억제경향성이나타났다 (Fig. 2A, B). 혈소판을활성화시키는요인들중 SIPA와생리적인 agonist에의한혈소판응집에 MG1002와 MG1009의억제효과를비교하기위해서 thrombin, ADP, collagen을 agonist로하여 PRP에서확인하였다. 그결과 MG1002와 MG1009 는 250 μm까지모든 agonist에서혈소판응집억제효과가거의관찰되지않았다 (Fig. 3A, B, C). WP에서 SIPA에대한 MG1002, MG1009의혈소판에대한독성을확인해보기위해 LDH의유출정도를확인해본결과 (Fig. 4A) positive control로사용한 50 μm의 digitonin의경우세포독성이뚜렷하게보인반면 MG1002, MG1009 에서는세포독성이확인되지않았다. 또한간세포 HepG2 cell-line에대한 14
MG1002, MG1009의독성을확인해보기위해서 MTT assay로 cell viability를측정한결과 (Fig. 4B) positive control로사용한 40 mm의 acetaminophen에서는 cell viability가현저하게감소하였지만 250 μm의 MG1002, MG1009에서는세포독성이확인되지않았다. 혈액응고부작용을평가하기위해서 MG1002, MG1009을혈장에 3분간처리한후 PT, aptt assay를수행하였다 (Fig. 5A, B). positive control로서 direct thrombin inhibitor (DTI) 2 μm을 3분간처리하였다. 그결과, DTI를처리한실험군에서는 PT, aptt 모두현저히증가한반면, MG1002, MG1009은 PT, aptt이증가하지않았다. 즉 MG1002, MG1009은혈장내에서의혈액응고에직접적인영향을주지않았다. 혈관의수축에미치는영향을실험하기위해서 organ bath system에서 MG1009 50, 100, 250 μm씩 30분간혈관에전처리한후혈관수축물질인 phenylephrine (PE) 을점증적으로가하여혈관수축의변화를관찰하였다 (Fig. 6). 혈전생성에있어서혈소판은중요한역할을하므로, MG1002, MG1009에의한 SIPA 억제효과가혈전생성에어떠한영향을주는지확인하고자 in vivo arterial thrombosis model을이용하였다. MG1002, MG1009을 5, 10, 25 mg/kg 으로경구투여하고 1시간후에 50% FeCl 3 를 10분간적용하여혈전생성을유발하고이후혈류의변화를관찰하였다 (Fig. 7A, B). 그결과 positive control인 15
8 mg/kg clopidogrel을투여한그룹의 Occlusion time을현저히증가시켰지만, MG1002는 Control값대비유의미한차이가없었다. 반면, MG1009는농도의존적으로혈류가멈추는데걸리는시간인 Occlusion time을지연시켰다. MG1009의 10, 25 mg/kg의경우유의적인효과가나타났다. 출혈부작용에대한효과를검증하기위해 in vivo tail bleeding time model을도입했다. MG1002, MG1009을 25, 100 mg/kg으로경구투여하고 1시간후에꼬리끝 3 mm 부분을절단한후출혈이멈출때까지의시간을측정하였다 (Fig. 8A, B). Positive control인 8 mg/kg clopidogrel을투여한그룹의 bleeding time 을현저히증가한반면, MG1002, 1009을 25, 100 mg/kg 에서는 control 그룹과큰차이가없었다. 16
100 Maximal Inhibition (%) 80 60 40 20 0 MG1001 MG1002 MG1003 MG1004 MG1005 MG1006 MG1007 MG1008 MG1009 MG1010 MG1011 MG1012 Figure 1. Inhibitory effects of MGs on Shear Stress-Induced Platelet Aggregation(SIPA) in human platelet rich plasma(prp). Human PRP was incubated with 12 MGs (25 μm) for 3 min at 37 and then sheared at 10,800 s -1 for 3 min. Values are mean ± SEM of three independent experiments. 17
A B Maximal Inhibition (%) 100 80 60 40 20 0 MG1002 MG1009 10 25 50 10 25 50 Concentration ( M) Maximal Inhibition (%) 100 80 60 40 20 0 MG1002 MG1009 10 25 50 10 25 50 Concentration ( M) Figure 2. Inhibitory effects of MG1002, 1009 in various concentration on SIPA in human PRP and WP. A. Human PRP or B. Human WP were incubated with various concentration of MG1002, 1009 for 3 min at 37 and then sheared at 10,800 s -1 for 3 min. Values are mean ± SEM of three independent experiments. 18
A B C Aggregation (%) 120 100 80 60 40 20 MG1002 MG1009 Aggregation (%) 120 100 80 60 40 20 MG1002 MG1009 Aggregation (%) 120 100 80 60 40 20 MG1002 MG1009 0 0 25 100 250 25 100 250 0 0 25 100 250 25 100 250 0 0 25 100 250 25 100 250 Concentration ( Concentration ( Concentration ( Figure 3. Effects of MG1002, 1009 on agonist-induced platelet aggregation in human PRP. Human PRP was incubated with various concentration of MG1002, 1009 for 3 min at 37, and then aggregation was initiated by a sub-threshold of A. thrombin, B. ADP, C. collagen. Values are mean ± SEM of three independent experiments. 19
Table 1. Comparison of inhibitory effects by MG1002, 1009 on SIPA and agonist-induced platelet aggregation in human PRP. Human PRP was incubated with various concentration of MG1002, 1009 for 3 min at 37 and then various platelet activators were added. Values are mean ± SEM of three independent experiments. 20
LDH leakage (% of total) A 80 60 40 20 0 0 MG1002 MG1009 25 100 250 25 100 250 Concentration ( Digitonin Cell viability (% of control) B 100 80 60 40 20 0 MG1002 MG1009 250 um AAP Figure 4. Effects of MG1002, 1009 on Cytotoxicity. A. Human WP was incubated with various concentration of MG1002, 1009 for 3 min at 37, and then release of LDH (lactate dehydrogenase) was determined. Digitonin(50 μm) was used as a positive control. B. HepG2 cells were treated with MG1002, 1009(250 μm) or Acetaminophen(40 mm) and incubated for 3 min at 37. Values are mean ± SEM of two to three independent experiments. 21
60 A 100 B 50 80 PT (sec) 40 30 20 10 MG1002 MG1009 aptt (sec) 60 40 20 MG1002 MG1009 0 0 25 100 250 25 100 250 Concentration ( DTI 0 0 25 100 250 25 100 250 Concentration ( DTI Figure 5. Effects of MG1002, 1009 on coagulation. Human plasma was isolated and B. activated partial thromboplastin time (aptt) or A. prothrombin time (PT) was determined. Values are mean ± SEM of three independent experiments. 22
Contraction ( % of 90 mm K + ) 100 50 0 9 8 Control MG1009 50uM MG1009 100uM MG1009 250uM 7 6 5 -log [PE(M)] Figure 6. Effects of MG1009 on Phenylephrine (PE) induced contraction in rat aortic ring. After MG1009 was treated to aortic ring with endothelium for 30 min, dose-dependent curve of phenylephrine induced contraction. Values are mean ± SEM of three independent experiments. 23
A B Occlusion time (min) 60 40 20 Occlusion time (min) 60 40 20 * * * 0 0 5 10 25 MG1002 Dose (mg/kg) 8 Clopidogrel 0 0 5 10 25 MG1009 Dose (mg/kg) 8 Clopidogrel Figure 7. Effects of MG1002, 1009 on thrombus formation in rat in vivo arterial thrombosis model. 1 h after oral administration of 5, 10, 25 mg/kg of A. MG1002, B. MG1009, 50% of FeCl3 was applicated to carotid artery for 10 min. The time needed for occlusion was measured for up to 60 min. Values are mean ± SEM of three to four independent experiments. * represents significant difference from corresponding control (p<0.05) 24
A B Bleeding time (min) 30 20 10 * Bleeding time (min) 30 20 10 * 0 0 25 100 MG1002 Dose (mg/kg) 8 Clopidogrel 0 0 25 100 MG1009 Dose (mg/kg) 8 Clopidogrel Figure 8. Effects of MG1002, 1009 on thrombus formation in rat in vivo tail bleeding time measurement. 1h after oral administration of 25, 100 mg/kg of MG1002, 1009 and clopidogrel(8 mg/kg), the bleeding time was determined in the rat tail transection model. Values are mean ± SEM of three to four independent experiments. * represents significant difference from corresponding control (p<0.05) 25
고 찰 본연구에서는심혈관계질환에효과를보인다고알려진기존의천연물중에서천연물유래합성물질 12종을합성하여 SIPA에대한억제효과를검색하였다. 그결과 12종의천연물유래합성물질중 MG1002, MG1009 2종이 SIPA에대한억제효과가가장컸다. MG1002, MG1009는 PRP와 WP 모두에서농도의존적으로 SIPA에의한억제효과를보여주었으며, PRP와 WP에서의 SIPA에대한억제효과가크게차이가없음을알수있다. 그결과 MG1002, MG1009는혈장단백과결합이크지않음을의미한다. 사람의 PRP에서 MG1002, MG1009의 SIPA에대한효과와 agonist에대한억제효과를비교해보았다. MG1002, MG1009의 agonist에대한억제효과는 SIPA에서효과를보인농도 25 µm의 10배인 250 µm까지유의미한억제효과는보이지않았다. SIPA에대한억제효과의 IC 50 는 MG1002는 18.6 ± 1.3 µm, MG1009는 20.0 ± 4.3 µm 이었다반면, 생리적 agonist에서는 250 µm 이상의 IC 50 값을보여주었다. 이는 MG1002, MG1009가 shear stress 에대한혈소판활성화억제에선택적억제효과를보이는것을의미한다. MG1002, MG1009의세포독성을확인하기위해서혈소판에서 LDH assay, 간세포 cell-line에서 MTT assay를실행하였다. 그결과세포독성은관찰 26
되지않았으며, 이는 MG1002, MG1009의 SIPA 억제효과가혈소판의세포독성으로인한것이아님을의미한다. MG1002, MG1009에대한 coagulation 부작용을확인하기위해 PT / appt assay를실행한결과고농도에서도 PT와 appt는증가하지않았다. 그결과 MG1002, MG1009는혈소판응집에관여하는혈장내의 factor에직접적인영향을주지않아혈장내에서의혈액응고에영향을주지않았다. 혈관의수축에미치는영향을실험하기위해서 organ bath system에서 MG1009의혈관수축의변화를관찰하였다 SIPA실험에서사용한최고농도인 50 μm에서는유의미한혈관수축억제작용은보이지않았지만 100, 250 μm에서는혈관수축억제작용이관찰되었다. 고농도에서의혈관수축억제작용이독성에의한비가역적수축인지, 약리적효과에의한가역적수축억제작용인지실험으로확인해봐야할것이다. SIPA를억제한다고보고되어있는기존의항혈소판제 cilostazol (Goto, 2005; Goto et al., 2003) 은혈소판의 c-amp phosphodiesterase type III를선택적으로억제하여혈소판활성화억제및혈관수축억제등의다양한 작용을한다. 혈소판내의 c-amp 농도상승으로혈소판내의 Ca 2+ 이온을 Ca으로저장과립화시킴으로써 ADP, Serotonin 등의방출억제작용을통한혈소판활성화억제작용을하며, 동시에혈관평활근세포내의 phosphodiesterase 활성저해에의한혈관수축억제작용을한다 (Tanaka 27
et al., 1988). MG1009는 cilostazol과유사하게 SIPA에대한억제효과를가지고있으면서혈관수축억제작용을가지고있다그래서작용기전연구에있어서대조연구가필요하다. In vitro 실험을통해확인한항혈소판제후보물질들의효과를 In vivo 상태에서확인하는데여러 thrombosis model이이용되고있다. Shear stress 는혈류에의해작용되는힘이기때문에혈류속도가중요한역할을한다. 그래서정맥보다는동맥, 그리고협착성혈관에서주로발생하므로동맥에손상을주어혈전생성을유도하고혈관폐색을유발하는 In vivo arterial thrombosis model을이용하였다. SD rat에 MG1002 또는 MG1009를 5, 10, 25 mg/kg으로경구투여후 FeCl 3 를이용하여인위적으로혈전생성을유발하고그로인해생성된혈전이혈관을폐색하여혈류가 0이되는시점인 occlusion time을측정하였다. 그결과양성대조군인 clopidogrel 투여그룹에서는 occlusion time이현저하게증가시켰지만 MG1002 투여그룹에서는 control 그룹대비유의미한차이가없었다. 반면 MG1009 투여그룹에서는농도의존적으로 occlusion time을증가시켰는데이는농도의존적으로동맥에서의혈전생성을억제시켰음을의미한다. MG1002는 In vitro 실험에서는효과가있었지만 In vivo 실험에서는효과를보이지않았다. 약리적효과가보이지않는원인을찾기위해서 pharmacokinetics 실험으로확 28
인해봐야할것이다. 항혈소판제의대표적인부작용인출혈부작용을검증하기위해서 In vivo tail bleeding time measurement 실험을이용하였다. 에 MG1002 또는 MG1009를 25, 100 mg/kg 용량으로경구투여 1시간뒤꼬리끝을절단한후출혈이멈출때까지의시간을측정한결과, 양성대조군인 clopidogrel은 bleeding time이현저히증가한반면 MG1002, MG1009 투여그룹에서는 control 그룹대비유의미한차이가없었으며고농도인 100 mg/kg에서도 bleeding time에영향을주지않았다. 반복투여하였을때도 bleeding time을연장시키지않는지확인해봐야할것이다. 결론적으로혈소판활성화및응집은심근경색, 뇌졸중, 동맥경화와같은다양한심혈관계질환에서주요원인이다. 그러므로혈소판활성화및응집억제는이러한심혈관계질환에있어서효과적인예방및치료의방법으로사용되고있다. 본연구자는심혈관계질환에효과가있다고알려진천연물유례합성물질을이용하여 SIPA에대한효과를확인해본결과 MG1009가 Shear Stress에의해유발되는혈소판활성화억제에가장효과가있었다. 그리고혈소판을활성화시키는요인들중생리적요인인 agonist에의한혈소판응집은억제하지않고 SIPA에특이적으로작용하였다. 약리적효과를보이는농도에서세포독성은나타나지않았으며 MG1009는혈전생성을억제하지만출혈부작용이없다. 따라서 MG1009는심혈관계질환의예방및치료와관련해유용하게활용할수있다고사료된다. 29
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Abstract HAKJUN NOH Preventive Pharmacy The Graduate School Seoul National University The platelet of the blood has a huge role functioning as a vital defense mechanism of the body. However, excessive activation of the platelet can cause thrombosis, which can lead to several cardiovascular diseases such as coronary artery disease, myocardial infarction, and cerebral infarction. Because cardiovascular disease is one the most prevalent cause of death worldwide, a variety of antiplatelet drugs were created to treat and prevent thrombosis and cardiovascular diseases. While anti-platelet drugs such as Aspirin and Clopidogrel are known to be effective treatment for cardiovascular diseases, they tend to cause bleeding, a side effect caused by inhibition to the hemostasis. Therefore, it is necessary to develop anti-platelet drugs that effectively reduces the 33
abnormal formation of blood clots while still not affecting normal hemostasis. Platelets are activated by both physiological agonists such as thrombin, adenosine diphosphate (ADP), and collagen and physical shear stress of the blood stream. Platelet aggregation from shear stress, different from other platelet aggregation due to agonists, generally do not participate in the production of normal hemostasis. However, it is involved in the pathological formation of thrombosis. As a result, an important goal for the production of a new anti-platelet drug is developing a Shear Stressinduced Platelet Aggregation (SIPA). We conducted a research examining the inhibition of platelet aggregation of the 12 kinds of natural product derivatives of SIPA. After adding the natural product derivatives to the platelet rich plasma (PRP) of a person, we examined the effect of SIPA. The results that showed the largest inhibitory effect was MG1002 and MG1009, which were dependent on the concentration of PRP. We compared the effect of SIPA, the physical factor, and the effect of platelet aggregation caused by physiological agonists (thrombin, ADP, collagen). According to the results, when looking at the effect of platelet aggregation caused by agonists, MG1002 34
and MG1009 did not show inhibitory effect of platelet aggregation. On the other hand, when examining the effect of SIPA, inhibitory effects from MG1002, and MG1009 did specifically show up. We found that there was no sign of cytotoxicity after examining the lactate dehydrogenase leakage(ldh), which is a cytotoxicity indicator of the platelet. In addition, after we checked that the prothrombin time (PT) and the activated partial thromboplastin time (aptt) of the platelet poor plasma (PPP) was left unaffected we drew a conclusion that MG1002 and MG1009 did not have an effect on the coagulation. In order to see how the SIPA inhibitory effect of MG1002 and MG1009 affects the production of thrombosis in our bodies, we used the in vivo arterial thrombosis model. After orally administrating MG1002 and MG1009 and applying FeCl 3, we examined the change in the velocity of blood flow after the formation of thrombosis. The results show that MG1009 delayed the occlusion time, which shows the time it take for the blood flow to stop. This means that it inhibits the production of thrombosis. Furthermore, in order to verify the issues regarding the bleeding time side effect of antiplatelet drugs, we used the in vivo tail bleeding time model. Both MG1002 and MG1009 did not delay the bleeding time. 35
Because MG1009 has an inhibitory effect on platelet activation and aggregation, which is induced by an important platelet activation factor, shear stress, it inhibits the formation of thrombosis of the platelet without bleeding side effects in our bodies. Therefore, MG1009 is especially useful in preventing and curing cardiovascular diseases, especially those related to shear stress. Key words: Natural product derivatives, Platelet, Platelet aggregation, Shear stress, Thrombus formation, bleeding time Student number: 2015-23180 36