臨床藥理學會誌第 21 卷第 2 號 2013 J Korean Soc Clin Pharmacol Ther 2013;21(2): 사람혈장에서비발리루딘농도측정을위한분석방법의유효성

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1 臨床藥理學會誌第 21 卷第 2 號 2013 J Korean Soc Clin Pharmacol Ther 2013;21(2): 사람혈장에서비발리루딘농도측정을위한분석방법의유효성검증및약동학시험에의적용 1 서울아산병원임상약리학과, 2 울산대학교의과대학, 3 서울아산병원임상시험센터임상약리실험실 김요한 1,2, 박현정 3, 최희연 1,2, 임형석 1,2, 배균섭 1,2 =Abstract= Validation of LC-MS/MS Method for Determination of Bivalirudin in Human Plasma: Application to a Pharmacokinetic Study Yo Han Kim MD 1,2, Hyun Jeong Park 3, Hee Youn Choi MD 1,2, Hyeong-Seok Lim MD, PhD 1,2, Kyun-Seop Bae, MD, PhD 1,2 1 Department of Clinical Pharmacology and Therapeutics, Asan Medical Center, Seoul, Korea, 2 University of Ulsan College of Medicine, 3 Clinical Trial Center, Asan Medical Center, Seoul, Korea Background: Bivalirudin is a direct thrombin inhibitor for patients with unstable angina undergoing percutaneous coronary intervention. Methods: A sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of bivalirudin, in human plasma using nafarelin as internal standard (IS). Chromatographic separation was performed using a Shiseido MG3 mm column ( mm) with a gradient mobile phase consisting of water and acetonitrile containing 0.1 % formic acid at a flow rate of 0.4 ml/min, and total run time was within 5 min. Detection and quantification was performed by the mass spectrometer using a multiple reaction-monitoring mode at m/z for bivalirudin, and m/z for IS. Results: The assay was linear over a concentration range of ng/ml with a lower limit of quantification of 10 ng/ml in human plasma. Conclusion: This method was successfully applied for pharmacokinetics study after intravenous administration of bivalirudin to healthy Korean male volunteers. Key words: Bivalirudin, Liquid chromatography, Pharmacokinetics 본연구는씨트리 의지원에의하여이루어졌음. 교신저자 : 배균섭소속 : 서울아산병원임상약리학과 / 울산대학교의과대학주소 : 서울시송파구올림픽로 43길서울아산병원임상약리학과전화번호 : , 팩스 : , ksbae@amc.seoul.kr 접수일자 : 수정일 : 게재확정일 :

2 YH Kim et al: LC-MS/MS Method for Determination of Bivalirudin 서론비발리루딘은가역적으로작용하는직접트롬빈억제제 (direct thrombin inhibitor) 로심장동맥중재술시혈액이굳는것을막기위하여투여하는항응고제로사용되고있다. 1,2) FDA 및 EMEA 로부터각각, 2000년및 2004년도에승인을받은후, 제품명안지오멕스 (ANGIOMAX ) 및안지옥스 (ANGIOX ) 로서시판중인제품이나, 국내에서는동일성분및동일제제로서허가받은제품은아직없다. 3,4) 다양한문헌을통해비발리루딘의약동학및약력학특성이알려져있다. 비발리루딘의소실반감기는 1시간이내이며, 투여한양의 80 % 는세포내에서단백질가수분해되고, 남은 20 % 는신장으로제거된다. 5) 또한, 활성화부분트롬보플라스틴시간 (activated partial thromboplastin time, aptt) 이나활성화응고시간 (activated clotting time, ACT) 을약력학파라미터로하여, 투여용량과약력학적효과의상관관계를관찰한임상시험에서투여용량과항응고효과사이에밀접한상관관계가있음이보고된바있다.6,7) 기존에사용중인헤파린의경우, 간접트롬빈억제제로응고된혈액에붙어있는트롬빈 (clot-bound thrombin) 을억제하지못하는점, 비선형적인약동학 / 약력학특성을보인다는점이단점으로지적되고있다. 이에반해, 비발리루딘의경우반감기가짧고, 주된제거과정이특정장기에의존하지않는점, 약동학 / 약력학효과가예측이용이한점, 트롬빈의결합부위뿐아니라트롬빈의항응고효과를나타내는활성화부위에모두결합하여항응고효과를나타낸다는점으로인해헤파린을대체하는역할을할것으로기대된다. 8-10) 비발리루딘제제의약동학및약력학특성이널리 알려진것에반해비발리루딘제제의분석법과관련하여서는소수의논문에서만보고된바있다. 11,12) 이에본논문에서는혈장에서 LC/MS/MS 를이용하여비발리루딘의농도를측정하기위한분석법을확립하고, 이를가지고 씨트리에서제조한비발리루딘제제의약동학특성을확인하는데적용하였다. 연구대상및방법 1. 시험약물및내부표준물질표준물질인비발리루딘 (88.84 %) 은의뢰사인 시트리에서공급받았으며, 내부표준물질로사용한나파렐린 (Nafarelin) 은 Sigma-Aldrich 사 (MA, USA) 에서구입하였다. 용매로사용된메탄올 (methanol) 과아세토니트릴 (Acetonitrile) 은 J.T.Baker (HPLC grade, USA) 에서구입하였다. 2. 분석조건시료의분석에는 Agilent 1200 series liquid chromatograph/api 4000 mass spectrometer (Applied Biosystems, USA) 를사용하였고, 질량분석기의이온화방식에는전기분무이온화 (electrospray ionization, ESI) 의양이온모드를사용하였다. HPLC 분석조건의경우, 컬럼은 Shiseido MG3mm, 50 mm (length) 2.0 mm (i.d.) 를사용하였으며, 이동상으로물과아세토니트릴을그래디언트 (gradient) 방법을이용하여분리하였고, 유속은 0.4 ml/min 로하였다. Mass Spectrometer 분석조건의경우, multiple reaction monitoring (MRM) 모드에서비발리루딘은 , 나파렐린은 의조건에서검출하였다. 167

3 김요한외 : 비발리루딘의농도측정방법의유효성검증 (A) (B) (C) Figure 1. Chromatograms of (A) a human blank plasma sample; (B) a human plasma sample spiked with 10 ng/ml of bivalirudin and IS (nafarelin); (C) a subject s plasma sample at 5 min after intravenous bolus administration of bivalirudin mg/kg. 3. 시료전처리및검량선작성비발리루딘표준품을메탄올에녹여농도가비발리루딘으로서 1 mg/ml 이되도록만들고, 이용액을순차적으로희석하여물과메탄올 10:90(v/v) 의비율에 0.1 % formic acid를첨가하여표준액을조제하였다. 표준검량선을작성하기위하여공혈장 200 ml에비발리루딘의혈장중농도가 10, 50, 100, 500, 1000, 2000, 5000 및 ng/ml의농도가되도록첨가하였다. 각시 168

4 YH Kim et al: LC-MS/MS Method for Determination of Bivalirudin 험관에비발리루딘의내부표준물질인나파렐린 (1000 ng/ml) 20 ml와아세토니트릴을 400 ml 가한후 2분간진탕한후원심분리하고유기용매상층을 500 ml 취해 1.5 ml 튜브에옮긴후 45, 120분간건조시켰다. 건조된각튜브에물과메탄올 90:10 (v/v) 의비율에 0.1 % formic acid 를 100 ml 가한후 2분간혼합하여재용해하였다. 원심분리하고유기용매상층액 5 ml를 LC/MS/MS에주입하였다. 여기에서얻은내부표준물질의피크면적에대한비발리루딘피크면적비를가지고검량선을작성하였다. 결과 1. 특이성시료전처리과정에기술한대로검체를처리하여 LC/MS/MS 로분석하였을때얻어진크로마토그램은 Figure 1과같았으며비발리루딘의피크의머무름시간은약 2.20분이었고내부표준물질인나파렐린피크의머무름시간은약 2.22분이었다. 분석조건에서비발리루딘과내부표준물질인나파렐린의분리상태는양호하였다. 4. 분석법검증 2. 직선성, 정확성, 정밀성 검량선작성용표준시료이외에별도로조제한정도관리시료를저농도 (20 ng/ml), 중농도 (5000 ng/ml), 고농도 (8000 ng/ml) 를만들어정확성및정밀성을확인하였다. 하루에실험을 5번시행하여일내재현성을구하고 5일간실험을반복시행하여일간재현성을구하였다. 5. 약동학시험총 3개의용량군 (0.375 mg/kg, 0.75 mg/kg, 1.5 mg/kg) 으로나누어각용량군별 6명의건강자원자를대상으로비발리루딘을정맥내일시주사하였다. 채혈시점은기존문헌을참고로하여정맥내주사전 (pre-dose), 주사후 (post-dose) 5, 10, 20, 40, 60, 90, 120, 180, 240, 300분에채혈을시행하였다. 5) 각시점마다헤파린튜브에 7 ml의혈액을채취하였고, 4, 1800 g, 조건에서 8분동안원심분리하여상층혈장을분리하였다. 해당검체는 20 이하에냉동보관후 7일이내에 70 이하냉동고로옮긴후농도분석시까지보관하였다. 비발리루딘분석시 1/x 2 에대한가중치로단순회귀분석하였을때 ng/ml 범위에서회귀계수는 에서 사이의양호한직선성을나타내었다. 정도관리시료의일내분석에서 bivalirudin 의정밀성은 10 ng/ml의농도에서 7.03 %, 20 ng/ml 에서 %, 5000 ng/ml에서 5.03 % 및 8000 ng/ml 에서 4.76 % 로나타났다. 일내분석에서정확성은각농도에서 %, %, 4.02 % 및 2.28 % 로나타났다 (Table 1). 정도관리시료의일간분석에서 bivalirudin 의정밀성는 10 ng/ml의농도에서 8.02 %, 20 ng/ml 에서 5.78 %, 5000 ng/ml 에서 5.13 % 및 8000 ng/ml 에서 6.34 % 로나타났다. 일간분석에서정확성은각농도에서 9.46 %, 6.73 %, 5.90 % 및 4.69 % 로나타났다 (Table 1). 3. 안정성저농도 (20 ng/ml), 고농도 (8000 ng/ml) 에해당 169

5 김요한외 : 비발리루딘의농도측정방법의유효성검증 Table 1. Intra- and inter-day precision and accuracy data for this assay of bivalirudin in human plasma (n=5) QC 10 ng/ml QC 20 ng/ml QC 5000 ng/ml QC 8000 ng/ml Intra-day Mean (ng/ml) Precision (%) Accuracy (RE*, %) Inter-day Mean (ng/ml) Precision (%) Accuracy (RE*, %) RE = 100% (spiked concentration mean calculated concentration) / spiked concentration. Table 2. Stability of bivalirudin in human plasma Storage conditions QC 20 ng/ml QC 8000 ng/ml Short-term (room temperature for 24 h) % % Long-term (-70 for 40 days) -0.89% -7.68% Three Freeze/thaw cycles 3.66% -9.07% After extraction (stored in HPLC autosampler at 4 for 24 h) -7.66% 0.37% Data are presented as percent difference from baseline (%). 하는시료를모든전처리과정을거친다음, 상온에서의안정성, 장기안정성, 냉해동안정성, 추출후안정성을확인하였다. 그결과, Table 2에서알수있듯이실험을수행한모든상황에서안정함을알수있었다. 4. 최저정량한계최저정량한계는정확성및정밀성이 ± 20 % 이내인 LLoQ의농도로결정하되크로마토그램상에서신호대잡음비 (S/N ratio) 가 10 이상이거나최저정량한계에서분석물질의반응이공시료의반응과비교할때최소 5배이상이어야하는조건을만족하는 10 ng/ml 를최저정량한계로결정하였다. 5. 약동학시험에의적용 Figure 2. Mean plasma concentration time curves of bivalirudin after single intravenous bolus injection. Data are expressed as mean ± SD (each group, n=6). 본시험에서개발된분석법을이용하여측정한시간에따른혈장농도양상은 Figure 2와같다. 채혈시점의적절성을확인하기위해 WinNonlin Version 6.1 (Pharsight Co., Mountain View, CA, USA) 을이용하여 AUC % Extrap = (AUC inf AUC last ) / AUC last 100을확인한결과, mg/kg, 0.75 mg/kg, 1.5 mg/kg 군에서각각 1.48 ± 0.31, 0.72 ± 0.17, 0.44 ± 0.24였다. 이상을고려 170

6 YH Kim et al: LC-MS/MS Method for Determination of Bivalirudin 해볼때, 본농도측정법의최저정량한계 (10 ng/ml) 및채혈시점은약동학시험에적용하기에적절한것으로생각된다. 고찰비발리루딘은히루딘구조를토대로한단일체인의 20개아미노산폴리펩타이드로서합성펩타이드분자량은 2180 Da로알려져있다. 13) 최근들어펩타이드제제가치료제로개발되는경우가늘어남에따라, 펩타이드제제에대한분석법개발의중요성이대두된바있다 ) 비발리루딘의농도분석법과관련하여서는몇몇문헌에서보고된바가있다. Farthing 등은고체상추출법 (solid-phase extraction, SPE) 과 OPA (o-phthalaldehyde) 유도체화를이용하여 ng/ml 의검량선범위에서분석법을확립한바있다. 12) Robson 등은고체상추출법을적용한분석법을가지고임상시험에적용하였고, 당시의검량선범위는 ng/ml 였다. 5) 그러나, 고체상추출법을사용한분석법의경우비용소모가큰점, 분석시간이오래걸리는단점이있다. 이런점을보완하고자, Pan 등은고체 상추출법대신메탄올을이용한단백질침전 (protein precipitation) 을이용하여 ng/ml 의검량선범위에서분석법을확립하였고, 분석실행시간 (run time) 은 7분이었다. 11) 본시험에서는아세토니트릴을이용한단백질침전을이용하여 ng/ml의검량선범위에서분석법을확립하였고, 분석실행시간 (run time) 은 5분이었다. 이는 Pan이보고한분석법과비교하였을때, 검체당분석실행시간도단축했을뿐아니라, 더넓은검량선범위를확보했다는데의의를둘수있다. 넓은검량선범위를확보함으로써검체의희석을최소화하여분석을진행할수있었다. 실제임상시험용의약품을투여한결과, 최고용량군인 1.5 mg/kg 군에서는검량선범위의상한인 ng/ml 를넘어, 희석이필요한채혈포인트가일부있었다. 희석이필요한채혈포인트의분석에앞서 20 (QC1), 5000 (QC2), (Dilution 1/10) (QC3), 8000 (QC4) ng/ml 과같이희석 QC (quality control) 시료를포함하여적합성을확인하였고앞서기술된방법으로시료전처리후분석하였다. 그결과, QC시료중 67 % 가예상되는이론값의 15 % 이내여야하는조건을모두 Table 3. Suitability of bivalirudin in human plasma Analyte concentration (ng/ml) Calculated concentration (ng/ml) Accuracy (%) (Dilution 1/10)* QC samples of ng/ml were diluted 1:10 to prepare QC sample of 5000 ng/ml. Those diluted QC samples were used to analyze diluted samples of bivalirudin in case of the unexpected high concentration over ng/ml. 171

7 김요한외 : 비발리루딘의농도측정방법의유효성검증 만족하였다 (Table 3). 17) 이를종합하여볼때, 이미분석법이어느정도알려져있다하더라도분석법의개선을통해충분한검량선범위를확보후약동학시험에적용하는것이중요함을확인할수있다. 참고문헌 1. Gladwell TD. Bivalirudin: a direct thrombin inhibitor. Clin Ther, 2002;24(1): Lincoff AM, Bittl JA, Harrington RA, Feit F, Kleiman NS, Jackman JD, Sarembock IJ, Cohen DJ, Spriggs D, Ebrahimi R, Keren G, Carr J, Cohen EA, Betriu A, Desmet W, Kereiakes DJ, Rutsch W, Wilcox RG, de Feyter PJ, Vahanian A, Topol EJ, Investigators R-. Bivalirudin and provisional glycoprotein IIb/IIIa blockade compared with heparin and planned glycoprotein IIb/IIIa blockade during percutaneous coronary intervention: REPLACE-2 randomized trial. JAMA, 2003;289(7): EMEA. Scientific discussion for the approval of Bivalirudin [cited Oct]. Available from: library/epar_-_scientific_discussion/human/ /WC pdf. 4. FDA. NDA Angiomax (Bivalirudin): Clinical Pharmacology Biopharmaceutics Review. Rockville, MD: Department of Health and Human Services, US Food and Drug Administration; Robson R, White H, Aylward P, Frampton C. Bivalirudin pharmacokinetics and pharmacodynamics: effect of renal function, dose, and gender. Clin Pharmacol Ther, 2002;71(6): Lidon RM, Theroux P, Juneau M, Adelman B, Maraganore J. Initial experience with a direct antithrombin, Hirulog, in unstable angina. Anticoagulant, antithrombotic, and clinical effects. Circulation, 1993;88(4 Pt 1): Topol EJ, Bonan R, Jewitt D, Sigwart U, Kakkar VV, Rothman M, de Bono D, Ferguson J, Willerson JT, Strony J, et al. Use of a direct antithrombin, hirulog, in place of heparin during coronary angioplasty. Circulation, 1993;87(5): Reed MD, Bell D. Clinical pharmacology of bivalirudin. Pharmacotherapy, 2002;22(6 Pt 2): 105S-111S. 9. Anand SX, Viles-Gonzalez JF, Mahboobi SK, Heerdt PM. Bivalirudin utilization in cardiac surgery: shifting anticoagulation from indirect to direct thrombin inhibition. Can J Anaesth, 2011;58(3): Shammas NW. Bivalirudin: pharmacology and clinical applications. Cardiovasc Drug Rev, 2005;23(4): Pan G, Wang X, Huang Y, Gao X, Wang Y. Development and validation of a LC-MS/MS method for determination of bivalirudin in human plasma: Application to a clinical pharmacokinetic study. J Pharm Biomed Anal, 2010;52(1): Farthing D, Larus T, Fakhry I, Gehr T, Prats J, Sica D. Liquid chromatography method for determination of bivalirudin in human plasma and urine using automated ortho-phthalaldehyde derivatization and fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci, 2004;802(2): Maraganore JM, Bourdon P, Jablonski J, Ramachandran KL, Fenton JW, 2nd. Design and characterization of hirulogs: a novel class of bivalent peptide inhibitors of thrombin. Biochemistry, 1990;29(30): Zhang X, Nieforth K, Lang JM, Rouzier-Panis R, Reynes J, Dorr A, Kolis S, Stiles MR, Kinchelow T, Patel IH. Pharmacokinetics of plasma enfuvirtide after subcutaneous administration to patients with human immunodeficiency virus: Inverse Gaussian density absorption and 2-compartment disposition. Clin 172

8 YH Kim et al: LC-MS/MS Method for Determination of Bivalirudin Pharmacol Ther, 2002;72(1): Douglas SA. Human urotensin-ii as a novel cardiovascular target: heart of the matter or simply a fishy tail? Curr Opin Pharmacol, 2003;3(2): John H, Walden M, Schafer S, Genz S, Forssmann WG. Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography-mass spectrometry. Anal Bioanal Chem, 2004;378(4): FDA. Bioanalytical Method Validation (Draft Guidance). Rockville, MD: Department of Health and Human Services, US Food and Drug Administration;

한약재품질표준화연구사업단 강활 ( 羌活 ) Osterici seu Notopterygii Radix et Rhizoma 생약연구과

한약재품질표준화연구사업단 강활 ( 羌活 ) Osterici seu Notopterygii Radix et Rhizoma 생약연구과 - 2 - - 3 - KP 11 Osterici seu Notopterygii Radix et Rhizoma Ostericum koreanum Maximowicz, Notopterygium incisum Ting, Notopterygium