학술연구용역사업최종결과보고서 항결핵제의혈중약물농도측정법개발및임상적적용 차년도 Development of analytical method for the determination of plasma concentration of anti-tuberculosis agents and its clinical application 주관연구기관 : 인제대학교의과대학 질병관리본부 - 1 -
주 의 1. 이보고서는질병관리본부에서시행한학술연구용역사업의최종결 과보고서입니다. 2. 이보고서내용을발표할때에는반드시질병관리본부에서시행한 학술연구용역사업의연구결과임을밝혀야합니다. 3. 국가과학기술기밀유지에필요한내용은대외적으로발표또는공개 하여서는아니됩니다. - 2 -
질병관리본부학술연구용역사업최종결과보고서 과제명 수행기관 / 연구책임자 항결핵제의혈중약물농도측정법개발및임상적적용 (1 차년도 ) (Development of analytical method for the determination of plasma concentration of anti-tuberculosis agents and its clinical application) 인제대학교의과대학 발주부서 질병관리본부에이즈결핵과 - 1 -
목 차 Ⅰ. 연구개발결과요약문 요약문 ---------------------------------------------------------------- 3 Summary -------------------------------------------------------------- 4 Ⅱ. 학술연구용역사업연구결과 제1장최종연구개발목표 -------------------------------------------------- 5 제2장최종연구개발내용및방법 ------------------------------------------ 15 제3장최종연구개발결과 ------------------------------------------------- 20 제4장연구결과고찰및결론 ---------------------------------------------- 53 제5장연구성과및활용계획 ---------------------------------------------- 55 제6장기타중요변경사항 -------------------------------------------------- 56 제7장연구비사용내역 -------------------------------------------------- 56 제8장첨부서류 --------------------------------------------------------- 58-2 -
μ μ μ μ μ μ μ μ μ μ - 3 -
μ μ μ μ μ μ μ μ μ μ - 4 -
학술연구용역사업연구결과 1. 연구배경 : 2. 연구목적 : 3. 당해연도 (1차년도) 사업내용 - 5 -
1.2 목표달성도및관련분야에대한기여도 1.3 국내 외기술개발현황 - 6 -
Table 1. 2010 년도결핵발병률과사망률추정치 구분 한국 프랑스 독일 캐나다 미국 호주 일본 영국 발병률 ( 천명 ) 47 5.9 4 1.6 13 1.4 27 7.9 사망률 ( 천명 ) 2.6 0.45 0.2 0.078 0.55 0.45 2 0.4 < 자료원 > WH Global Tuberculosis Control 2011 Table 2. 2010년도다제내성발생추정치 구분 한국 프랑스 독일 캐나다 미국 호주 일본 영국 다제내성발병률 1,780 49.9 57 12 100 32 290 53 < 자료원 > WH Global Tuberculosis Control 2011-7 -
Table 3. 임상약리학서비스 (CKPS) 유뮤에따른입원환자들의입원기간및사망률 - 8 -
- 9 -
bserved Concentration Peak Concentration Concentration Trough Concentration Time Figure 1. Type of target concentration - 10 -
Ward, utpatient Unit Required Information (Drug Administration History, Sampling Time, Demographics, Some Clinical Information) TDM Report Sample from Patient Drug Concentration Measurement Unit Concentration Information TDM Service Unit Figure 2. Work flow-chart of TDM - 11 -
라 항결핵제 - 12 -
Table 4. 항결핵제 TDM 을실시하는국가및기관 미국 독일 국가별 TDM을실시하는기관 Virginia Department of Health, Virginia, USA New York City Department of Health and Mental Hygiene, New York, USA Wake County Human Services and Duke University Medical Center, North Carolina, USA Veterans Affairs Medical Center, Tennessee, USA National Jewish Medical and Research Center, Colorado, USA National Jewish Medical and Research Center, Denver, Colorado, USA Beth Israel Medical Center, New York, USA Chest Hospital Heckeshorn and Universitatsmedizin Berlin, Berlin, 약제명 Isoniazid, Rifampicin, Pyrazinamide, ethambutol Cycloserine, floxacin, Ethionamide, Ciprofloxacin, Ethambutol, Pyrazinamide, PAS, Streptomycin Isoniazid, Rifampin, Rifabutin Rifampin Ethambutol Isoniazid, Rifampin, Ethambutol, Pyrazinamide Rifampin, Ethambutol Ethambutol - 13 -
네덜란드호주캐나다영국 Germany Universitatsmedizin Berlin, Berlin, Germany Radboud University Nijmegen Medical Center, Netherlands University center for chronic Dekkerswald St. Vincent's Hospital and Westmead Hospital, Sydney, Australia Montreal Chest Institute and Respiratory Epidemiology & Clinical Research Unit of McGill University, Montreal, Canada Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Malawi and University of Liverpool, United Kingdom Rifampin, Ethionamide Isoniazide, Rifampicin, Pyrazinamide, Ethambutol Rifampicin, Isoniazid Isoniazid, Rifabutin, Rifampin, Pyrazinamide Pyrazinamide, Ethambutol - 14 -
제 2 장최종연구개발내용및방법 - 15 -
μ μ μ μ μ μ μ μ μ μ - 16 -
- 17 -
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Figure 3. NNMEM code - 19 -
제 3 장최종연구개발결과 제 절 분석대상약물의선정 Table 5. WH guideline의약물권고사항 단계 요령 항결핵제 1 일차약제중사용가능한 Pyrazinamide, Ethambutol 약제를포함한다. 2 주사약제중하나를선택한다. Kanamycin, amikacin, streptomycin, capreomycin 3 퀴놀론중하나를선택한다. Levofloxacin, moxifloxacin, ofloxacin 4 4군의약제중필요한개수 p-aminosalicylic acid, cycloserine, 만큼선택한다. ethionamide (or prothionamide) 5 4단계까지거쳐도 4가지약제가 Amoxicillin/clavulanate, clarithromycin, 구성되지않으면 5군에속한 high-dose isoniazid, clofazimine, 약제를사용해볼수있다. linezolid H 2 N H H H H H H N H H 2 N Amikacin Streptomycin H N H NH 2 NH 2 H H H H H H H H H H 2 N H H H H H H 2 N NH 2 NH 2 Kanamycin H N NH 2 H NH 2 N NH 2 N F NH 2 H C 2 H 5 H N Linezolid H NH 2 H H 3 C N Amoxicillin CH NH H N CH 3 H S N CH 3 H Clarithromycin HN Cycloserine H NH 2 NH H Rifabutin N H F N N NH Moxifloxacin F H N N N F floxacin H N N H HN NH 2 Ciprofloxacin p-aminosalicylic acid (PAS) H 2 N N NH H 2 N N S Prothionamide Isoniazid NH N H H H H H NH H Rifampin Figure 4. Structure of TB drugs included in this study C 2 H 5 N N H N H H H Roxithromycin N S N NH 2 CH 3 Ethionamide CH 3 N CH 3 H H H H N N H Ethambutol H H 2 N N N Pyrazinamide Cl N N Clofazimine N NH Cl - 20 -
제 절 분석대상약물의정량농도범위설정 Table 6. Doses and pharmacokinetic parameters of TB drugs Drug Dose Cmax AUC Peak conc Trough conc (mg/ml) (mg*h/ml) (mg/ml) (mg/ml) Reference Amikacin 15 mg/kg i.v 48 138 S.Ehrmann, 2007 Kanamycin Streptomycin Arnold, 2007 Amoxicillin 500mg, p.o 10.63 28.56 A.Ullah, 2008 Clarithromycin 7.5 mg/kg I.v 1.0 2.9 JWC.Alffenaar, 2010 Cycloserine 250mg p.o 12.56 183.49 DS.Patel, 2011 Gatifloxacin 400mg i.v qd 4.93 0.004 DN.Fish, 2007 Linezolid 600mg i.v 14.4 98.1 AM.Lovering, 2009 Moxifloxacin 400mg p.o 3.8 39.4 M.Weiner, 2007 floxacin 800mg p.o 10.5 103 M.Zhu, 2002 600mg p.o 8.52 68.7 p-aminosalicylic acid(pas) Prothionamide 6.2 mg/kg p.o 2.2 11 HW.Lee, 2009 Rifabutin 300mg p.o twice weekly 0.3 2.71 C.Boulanger, 2009 Roxithromycin 150mg p.o 7.51 86.7 T-hang, 2007 Ciprofloxacin 400mg i.v bid 6.92 1.22 Arthur, 2008 Isoniazid 200mg p.o 6.5 25 Helen, 2006 Rifampin 600mg p.o, qd 5.49 29.21 David, 2005 Ethambutol 400mg p.o 5 19.9 Helen, 2006 20mg/kg i.v, qd 1.54 7.68 David, 2005 Pyrazinamide 500mg p.o 52.7 288.4 Helen, 2006 Table 7. Maximum therapeutic concentration of TB drugs proposed by Peloquin - 21 -
μ μ μ Table 8. Target analytical concentrations of TB drugs. 대상약물 정량범위 (μg/ml) Group 1 Amikacin 1.0-50.0 Kanamycin 1.0-50.0 Streptomycin 1.0-50.0 Group 2 Amoxicillin 0.4-20.0 Clarithromycin 0.2-10.0 Cycloserine 0.8-40.0 Linezolid 0.4-20.0 Moxifloxacin 0.2-10.0 floxacin 0.2-10.0 PAS 1.0-50.0 Prothionamide 0.2-10.0 Rifabutin 0.04-2.0 Roxithromycin 0.4-20.0 Clofazimine 0.1-5.0 Ethionamide 0.1-5.0 Ciprofloxacin 0.1-5.0 Ethambutol 0.2-10.0 Isoniazid 0.2-10.0 Pyrazinamide 2.0-100 Rifampin 0.2-10.0-22 -
Figure 5(a). Full scan product ion mass spectra and the proposed fragmentation pattern of amikacin (A), kanamycin (B), and streptomycin (C). - 23 -
Figure 5(b). Full scan product ion mass spectra and the proposed fragmentation pattern of amoxicillin (D), clarithromycin (E) and cycloserine (F). - 24 -
Figure 5(c). Full scan product ion mass spectra and the proposed fragmentation pattern of linezolide (G), moxifloxacin (H), ofloxacin (I) and PAS (p-aminosalycilic acid) (J). - 25 -
Figure 5(d). Full scan product ion mass spectra and the proposed fragmentation pattern of prothionamide (K), rifabutin (L), roxithromycin (M) and ciprofloxacin (N). - 26 -
Figure 5(e). Full scan product ion mass spectra and the proposed fragmentation pattern of ethambutol (), isoniazid (P), pyrazinamide (Q) and rifampin (R). - 27 -
Figure 5(f). Full scan product ion mass spectra and the proposed fragmentation pattern of clofazimine (S) and Ethionamide (T). - 28 -
Table 9. Retention times, SRM transitions, MS/MS parameter and calibration range for the tested 20 anti-tuberculosis drugs. Compound Retention SRM (m/z) Collision Calibration Polarity Energy range Time (min) Q1 Q3 (ev) (μg/ml) Group 1 Amikacin 0.63 586.2 425.3 ES + 30.0 1.0-50.0 Kanamycin 0.65 485.5 163.3 ES + 30.0 1.0-50.0 Streptomycin 0.71 582.2 263.3 ES + 42.5 1.0-50.0 Group 2 Amoxicillin 2.83 366.0 114.0 ES + 30.0 0.4-20.0 Clarithromycin 2.49 748.6 590.7 ES + 30.0 0.2-10.0 Cycloserine 2.92 103.0 75.0 ES + 10.0 0.8-40.0 Linezolid 2.07 338.0 296.0 ES + 30.0 0.4-20.0 Moxifloxacin 2.93 402.0 384.0 ES + 30.0 0.2-10.0 floxacin 3.22 362.0 318.0 ES + 30.0 0.2-10.0 PAS 1.91 154.0 119.0 ES + 30.0 1.0-50.0 Prothionamide 2.46 181.0 154.3 ES + 30.0 0.2-10.0 Rifabutin 2.48 847.6 815.5 ES + 30.0 0.04-2.0 Roxithromycin 2.71 837.6 679.6 ES + 30.0 0.4-20.0 Clofazimine 2.56 474.2 432.4 ES + 50.0 0.1-5.0 Ethionamide 2.84 167.1 107.0 ES + 30.0 0.1-5.0 Ciprofloxacin 2.68 332.0 231.2 ES + 20.0 0.1-5.0 Ethambutol 5.65 205.0 116.0 ES + 20.0 0.2-10.0 Isoniazid 3.03 138.0 121.0 ES + 20.0 0.2-10.0 Pyrazinamide 2.24 124.0 107.0 ES + 20.0 2.0-100 Rifampin 2.44 823.8 791.3 ES + 20.0 0.2-10.0 SRM: selected reaction monitoring μ μ μ μ - 29 -
Figure 6(a). Representative SRM chromatograms of amikacin (A), kanamycin (B), streptomycin (C). Left panel, blank plasma (dotted line) and LLQ (lower limit of quantification, solid line). Right panel: ULQ (upper limit of quantification, solid line) - 30 -
Figure 6(b). Representative SRM chromatograms of amoxicillin (D), clarithromycin (E) and cycloserine (F). Left panel, blank plasma (dotted line) and LLQ (lower limit of quantification, solid line). Right panel: ULQ (upper limit of quantification, solid line) - 31 -
Figure 6(c). Representative SRM chromatograms of linezolid (G), moxifloxacin (H), ofloxacin (I) and p-aminosalicylic acid (J). Left panel, blank plasma (dotted line) and LLQ (lower limit of quantification, solid line). Right panel: ULQ (upper limit of quantification, solid line) - 32 -
Figure 6(d). Representative SRM chromatograms of prothionamide (K), rifabutin(l), Roxithromycin (M) and ciprofloxacin (N). Left panel, blank plasma (dotted line) and LLQ (lower limit of quantification, solid line). Right panel: ULQ (upper limit of quantification, solid line) - 33 -
Figure 6(e). Representative SRM chromatograms of ethambutol (), isoniazid (P), pyrazinamide (Q), and rifampin (R). Left panel, blank plasma (dotted line) and LLQ (lower limit of quantification, solid line). Right panel: ULQ (upper limit of quantification, solid line) - 34 -
Figure 6(f). Representative SRM chromatograms of clofazimine (S) and ethionamide (T). Left panel, blank plasma (dotted line) and LLQ (lower limit of quantification, solid line). Right panel: ULQ (upper limit of quantification, solid line) - 35 -
Table 10. Back-calculated concentrations of spiked moxifloxacin in human plasma batch Back-calculated calibration standards (μg/ml) Curve parameters 0.2 0.4 1 2 5 10 Slope Intercept Correlation #1 0.183 0.42 0.997 2.09 5 9.91 0.111-0.00393 0.9997 #2 0.198 0.401 0.963 2.07 5.13 9.84 0.122-0.0000987 0.9997 #3 0.22 0.344 1.03 1.9 5.49 9.62 0.0931 0.00744 0.9974 #4 0.21 0.411 0.935 2 4.85 10.2 0.115-0.000478 0.9996 #5 0.174 0.414 1.04 2.11 5.16 9.7 0.114 0.00552 0.9991 Mean 0.20 0.40 0.99 2.03 5.13 9.85 weighting SD 0.02 0.03 0.04 0.09 0.24 0.22 7. Moxifloxacin 1/x Table 11. Day-to-day linear regression equation for 20 anti- tuberculosis drugs (n=5) Compounds Slope* intercept* Correlation coefficient* (R) Amikacin 0.0014 ± 0.00006 0.00003 ± 0.000198 0.9947 ± 0.00150 Kanamycin 0.0052 ± 0.00229 0.00036 ± 0.000480 0.9934 ± 0.00144 Streptomycin 0.0012 ± 0.00022 0.00046 ± 0.000168 0.9959 ± 0.00188 Amoxicillin 0.0706 ± 0.00632-0.01682 ± 0.004901 0.9988 ± 0.00077 Clarithromycin 0.0155 ± 0.00153-0.00033 ± 0.000445 0.9990 ± 0.00065 Cycloserine 0.0155 ± 0.00582-0.00071 ± 0.001358 0.9985 ± 0.00069 Linezolid 0.1057 ± 0.00787 0.00010 ± 0.005720 0.9995 ± 0.00022 Moxifloxacin 0.1110 ± 0.01080 0.00169 ± 0.004669 0.9991 ± 0.00098 floxacin 0.6168 ± 0.03849-0.00997 ± 0.009828 0.9991 ± 0.00063 PAS 0.1322 ± 0.01639-0.00561 ± 0.015299 0.9994 ± 0.00040 Prothionamide 0.4138 ± 0.07225-0.02029 ± 0.011524 0.9988 ± 0.00107 Rifabutin 1.2720 ± 0.05310-0.00192 ± 0.004193 0.9996 ± 0.00032 Roxithromycin 1.1480 ± 0.09680-0.11258 ± 0.100104 0.9987 ± 0.00050 Ciprofloxacin 0.0085 ± 0.00068 0.00001 ± 0.000091 0.9993 ± 0.00046 Ethambutol 0.0832 ± 0.01428-0.00189 ± 0.001114 0.9995 ± 0.00071 Isoniazid 0.1928 ± 0.03229 0.00745 ± 0.005676 0.9994 ± 0.00035 Pyrazinamide 0.0018 ± 0.00018 0.00062 ± 0.000891 0.9989 ± 0.00088 Rifampin 0.0843 ± 0.00360 0.00308 ± 0.001115 0.9997 ± 0.00013 Clofazimine 0.1031 ± 0.00419 0.00194 ± 0.000777 0.9988 ± 0.00142 Ethionamide 0.4434 ± 0.02907 0.00247 ± 0.010244 0.9987 ± 0.00172 * Values are mean ± standard deviation. - 36 -
μ μ μ μ μ μ μ Table 12(a). Inter- and intra-day precision and accuracy data for assays of anti-tb drugs in human plasma Compounds Amikacin Kanamycin Streptomycin Amoxicillin INTER-DAY (n=10) INTRA-DAY (n=5) Conc. Precision Precision Accuracy (μg/ml) Accuracy (%) (CV %) (CV %) (%) 1 7.0 105.6 8.4 106.8 3 7.1 95.3 2.3 92.9 20 5.5. 93.2 2.4 89.5 40 6.4 100.1 6.3 99.5 1 7.2 105.9 3.4 113.3 2 4.8 92.5 3.7 90.0 20 4.8 95.9 1.2 87.5 40 5.1 100.1 7.5 97.5 1 8.5 98.1 7.3 109.7 3 8.6 103.6 8.3 101.1 20 6.4 95.2 3.9 91.8 40 9.1 97.7 6.3 94.7 0.4 3.5 115.0 5.8 102.5 1.2 6.8 99.8 3.2 97.5 8 6.8 104.4 3.2 102.2 16 8.8 101.5 5.1 107.1-37 -
Clarithromycin Cycloserine 0.2 7.2 107.1 10.4 96.2 0.6 7.7 101.2 7.9 101.2 4 3.5 108.2 3.4 104.0 8 7.0 101.9 4.0 106.9 0.8 10.7 108.8 6.3 91.7 2.4 7.1 97.7 11.8 92.0 16 4.5 95.2 2.6 87.0 32 5.8 95.1 1.0 86.1 Table 12(b). Inter- and intra-day precision and accuracy data for assays of anti-tb drugs in human plasma Compounds Linezolid Moxifloxacin floxacin PAS Prothionamide Rifabutin Roxithromycin INTER-DAY (n=10) INTRA-DAY (n=5) Conc. Precision Precision Accuracy (μg/ml) Accuracy (%) (CV %) (CV %) (%) 0.2 11.9 101.8 7.7 90.7 0.6 6.1 100.0 5.1 95.9 4 4.9 107.5 1.9 101.9 8 7.5 105.1 5.5 107.3 0.2 9.0 101.0 5.1 88.7 0.6 6.3 98.2 7.5 97.0 4 3.5 108.1 4.7 105.9 8 7.5 103.9 3.7 107.7 0.2 6.1 112.0 4.3 102.3 0.6 6.0 97.1 5.7 95.4 4 4.5 103.8 2.4 102.5 8 7.9 103.7 5.0 106.7 1 7.0 108.8 5.5 93.7 3 5.2 99.5 5.1 97.3 20 4.8 105.8 3.6 102.7 40 8.6 102.6 5.1 107.2 0.2 7.0 111.6 6..8 106.0 0.6 2.8 95.1 0.8 96.7 4 10.0 101.0 3.2 101.5 8 9.8 102.3 4.7 108.7 0.04 6.8 108.8 5.1 93.4 0.12 3.6 99.4 6.0 93.9 0.8 3.5 106.5 2.9 101.6 1.6 7.6 103.7 7.0 104.5 0.4 4.0 114.9 8.4 90.2 1.2 7.9 98.8 5.0 91.8 8 6.9 106.6 2.9 105.8 16 10.7 104.4 6.4 108.5-38 -
Table 12(c). Inter- and intra-day precision and accuracy data for assays of anti-tb drugs in human plasma Compounds Ciprofloxacin Ethambutol Isoniazid Pyrazinamide Rifampin Clofazimine Ethionamide INTER-DAY (n=10) INTRA-DAY (n=5) Conc. Precision Accuracy Precision Accuracy (μg/ml) (CV %) (%) (CV %) (%) 0.1 14.8 105.2 4.3 107.5 0.3 7.8 103.2 9.2 103.3 2 5.6 104.4 5.4 103.9 4 5.4 103.2 4.3 99.3 0.2 4.8 113.6 3.8 114.0 0.6 7.9 99.3 5.0 97.9 4 5.1 102.6 0.9 99.7 8 6.0 101.3 3.2 103.1 0.2 9.9 104.7 3.0 87.3 0.6 7.1 100.1 5.3 95.3 4 6.9 103.7 3.8 101.6 8 5.1 104.7 3.5 102.6 2 16.9 102.2 14.3 98.3 6 8.5 98.1 6.0 98.9 40 4.8 108.4 4.7 99.3 80 6.6 104.4 3.8 98.2 0.2 4.1 104.7 4.9 98.9 0.6 4.6 101.5 2.7 101.7 4 4.0 105.3 2.8 101.9 8 6.0 102.6 2.6 102.0 0.1 10.9 90.7 8.5 94.3 0.3 6.8 103.6 5.4 103.8 2 5.7 99.8 8.2 99.4 4 7.1 103.4 4.7 102.1 0.1 11.6 96.2 23.7 106.4 0.3 9.1 102.3 8.4 96.1 2 5.0 103.3 7.7 103.2 4 6.1 103.7 5.7 109.2-39 -
Table 13(a). Matrix effect of anti-tb drugs from six different sources of human plasma. Compounds Conc. (μg/ml) RSD (%) Accuracy (%) Amikacin Kanamycin Streptomycin Amoxicillin Clarithromycin Cycloserine Linezolid Moxifloxacin floxacin PAS Prothionamide Rifabutin Roxithromycin Ciprofloxacin Ethambutol Isoniazid Pyrazinamide Rifampin Clofazimine Ethionamide 3 9.7 96.5 40 14.0 109.7 3 10.4 94.6 40 12.3 114.5 3 5.0 107.7 40 11.9 111.4 1.2 4.3 91.6 16 9.4 100.3 0.6 6.7 101.0 8 6.4 101.2 2.4 43.9 141.6 32 54.9 131.8 1.2 4.5 102.0 16 5.9 100.6 0.6 4.0 99.1 8 6.1 101.1 0.6 4.4 96.2 8 5.8 100.1 3 3.0 99.3 40 6.3 99.9 0.6 7.2 102.8 8 9.4 107.3 0.12 3.7 98.9 1.6 6.6 97.7 1.2 8.2 115.5 16 3.7 105.7 0.3 12.8 94.3 4 8.1 94.3 0.6 6.0 101.3 8 6.0 104.8 0.6 4.2 97.0 8 4.5 99.1 6 6.8 100.1 80 3.7 97.0 0.6 4.0 99.1 8 3.0 97.6 0.3 4.4 62.3 4 2.7 67.5 0.3 5.0 113.1 4 3.3 84.3-40 -
Table 14(a). Total recovery and extraction efficiency (%) Compounds QC Extraction efficiency (recovery) * (%) Amikacin Kanamycin Streptomycin Amoxicillin Clarithromycin Cycloserine Linezolid Moxifloxacin QC1 65.1 ± 16.1 QC2 69.7 ± 0.5 QC3 50.6 ± 5.0 QC1 68.9 ± 14.9 QC2 70.4 ± 4.6 QC3 53.7 ± 2.0 QC1 76.2 ± 24.2 QC2 72.2 ± 0.3 QC3 59.0 ± 0.8 QC1 103.6 ± 2.0 QC2 69.1 ± 1.9 QC3 75.2 ± 4.8 QC1 120.7 ± 25.9 QC2 84.3 ± 1.0 QC3 90.1 ± 3.5 QC1 36.3 ± 0.7 QC2 31.3 ± 0.8 QC3 26.6 ± 0.4 QC1 114.5 ± 0.1 QC2 82.2 ± 1.5 QC3 89.1 ± 2.4 QC1 97.4 ± 3.7 QC2 75.5 ± 4.7 QC3 82.3 ± 2.4 *Values are mean ± standard deviation - 41 -
Table 14(b). Total recovery and extraction efficiency (%). Compounds QC Extraction efficiency (recovery) * (%) QC1 98.7 ± 10.0 Ciprofloxacin QC2 68.1 ± 3.2 QC3 74.2 ± 3.3 QC1 99.5 ± 1.6 Ethambutol QC2 79.3 ± 0.7 QC3 82.5 ± 4.3 QC1 99.1 ± 2.1 floxacin QC2 73.0 ± 1.1 QC3 79.3 ± 3.5 QC1 96.1 ± 5.9 PAS QC2 71.2 ± 0.3 QC3 76.5 ± 2.9 QC1 93.6 ± 1.8 Prothionamide QC2 70.3 ± 2.3 QC3 78.7 ± 2.6 QC1 104.7 ± 8.7 Rifabutin QC2 77.9 ± 5.7 QC3 84.3 ± 0.8 QC1 80.3 ± 7.0 Roxithromycin QC2 70.8 ± 6.0 QC3 81.3 ± 8.6 QC1 102.9 ± 0.6 Isoniazid QC2 75.4 ± 0.5 QC3 79.8 ± 0.1 QC1 110.4 ± 10.5 Pyrazinamide QC2 91.5 ± 8.5 QC3 85.7 ± 0.3 QC1 105.2 ± 20.6 Rifampin QC2 81.6 ± 2.1 QC3 87.0 ± 3.1 QC1 80.4 ± 3.2 Clofazimine QC2 84.8 ± 3.5 QC3 77.0 ± 0.6 QC1 91.5 ± 5.6 Ethionamide QC2 104.1 ± 6.0 QC3 106.9 ± 4.0 *Values are mean ± standard deviation - 42 -
Table 15. Stability of prothionamide after 1, 2, 3 and 6 hours at room temperature in human plasma (n=2) Tested time Added (μg/ml) Measured (μg/ml) * RSD (%) Accuracy (%) 1hr 0.6 0.69 ± 0.01 2.0 114.6 8 8.37 ± 0.35 4.2 104.6 2hrs 0.6 0.44 ± 0.02 3.6 73.0 8 6.33 ± 0.32 5.1 79.1 3hrs 0.6 0.37 ± 0.02 4.8 61.2 8 5.46 ± 0.28 5.2 68.3 6hrs 0.6 0.18 ± 0.01 3.3 30.1 8 3.16 ± 0.18 5.6 39.6 RSD (%): relative standard deviation * Values are mean ± standard deviation - 43 -
Table 16. Stability of 20 anti-tb drugs after 24 hours at room temperature in human plasma Compounds Conc. (μg/ml) Measured (μg/ml) RSD (%) Accuracy (%) Amikacin 3 2.9 ± 0.2 8.5 96.5 40 41.3 ± 2.2 5.4 103.1 Kanamycin 3 2.7 ± 0.1 5.0 90.9 40 42.5 ± 0.9 2.1 106.0 Streptomycin 3 3.0 ± 0.2 5.8 100.9 40 42.1 ± 1.3 3.2 105.0 1.2 1.06 ± 0.03 3.0 88.1 Amoxicillin 16 14.43 ± 0.23 1.6 90.2 Clarithromycin 0.6 0.63 ± 0.02 3.6 105.0 8 8.65 ± 0.41 4.8 108.3 Cycloserine 2.4 2.28 ± 0.00 0.0 94.9 32 32.30 ± 1.83 5.7 101.0 Linezolid 1.2 1.29 ± 0.04 2.7 107.7 16 17.03 ± 0.15 0.9 106.7 Moxifloxacin 0.6 0.66 ± 0.03 5.0 110.7 8 8.89 ± 0.15 1.7 111.3 floxacin 0.6 0.61 ± 0.02 2.7 102.4 8 8.52 ± 0.15 1.8 106.7 PAS 3 3.2 ± 0.10 3.0 107.7 40 44.4 ± 0.10 0.2 111.0 0.6 0.69 ± 0.01 2.0 114.6 Prothionamide 8 8.37 ± 0.35 4.2 104.6 0.12 0.123 ± 0.005 4.1 102.2 Rifabutin 1.6 1.720 ± 0.055 3.2 107.7 Roxithromycin 1.2 1.25 ± 0.03 2.4 104.0 16 18.47 ± 0.40 2.2 115.3 Ciprofloxacin 0.3 0.34 ± 0.02 6.6 113.7 4 4.58 ± 0.14 3.0 114.7 Ethambutol 0.6 0.75 ± 0.01 0.8 125.0 8 9.55 ± 0.10 1.0 119.3 Isoniazid 0.6 0.54 ± 0.03 5.2 89.9 8 7.63 ± 0.13 1.7 95.3 Pyrazinamide 6 6.9 ± 0.3 4.0 116.3 80 92.4 ± 2.7 2.9 115.7 Rifampin 0.6 0.72 ± 0.03 4.0 119.7 8 8.99 ± 0.16 1.7 112.0 Clofazimine 0.3 0.35 ± 0.03 9.8 115.0 4 4.23 ± 0.18 4.2 106.0 Ethionamide 0.3 0.20 ± 0.01 4.1 68.0 4 3.29 ± 0.07 2.1 82.3 RSD (%): relative standard deviation, PAS: p-aminosalycylic acid *Values are mean ± standard deviation stability of amoxicillin after 12 hours at room temperature in human plasma - 44 -
Table 17. Stability of 20 anti-tb drugs after three Freeze-Thaw cycles in human plasma Compounds Conc. (μg/ml) Measured (μg/ml) RSD (%) Accuracy (%) Amikacin 3 3.5 ± 0.2 4.3 116.0 40 47.1 ± 2.7 5.6 117.7 Kanamycin 3 3.6 ± 0.0 1.1 119.7 40 50.0 ± 3.0 6.0 125.0 Streptomycin 3 3.5 ± 0.0 1.3 118.3 40 46.6 ± 1.3 2.7 116.3 Amoxicillin 1.2 1.26 ± 0.05 3.8 104.3 16 18.53 ± 0.78 4.2 115.7 Clarithromycin 0.6 0.66 ± 0.06 8.8 109.9 8 8.70 ± 0.18 2.0 108.7 Cycloserine 2.4 2.31 ± 0.05 2.1 95.9 32 34.45 ± 0.21 0.6 107.5 Linezolid 1.2 1.24 ± 0.06 4.4 103.7 16 16.60 ± 0.66 4.0 103.7 Moxifloxacin 0.6 0.63 ± 0.02 3.9 104.7 8 8.52 ± 0.16 1.9 106.3 floxacin 0.6 0.59 ± 0.03 5.1 98.5 8 8.30 ± 0.17 2.0 104.0 PAS 3 3.3 ± 0.1 3.4 109.3 40 44.5 ± 1.1 2.4 111.3 0.6 0.65 ± 0.02 3.2 108.0 Prothionamide 8 8.87 ± 0.20 2.3 111.0 0.12 0.126 ± 0.007 5.2 105.2 Rifabutin 1.6 1.700 ± 0.056 3.3 106.3 Roxithromycin 1.2 1.51 ± 0.06 4.0 125.7 16 19.37 ± 0.85 4.4 121.0 Ciprofloxacin 0.3 0.32 ± 0.02 5.1 106.0 4 4.28 ± 0.22 5.1 107.0 Ethambutol 0.6 0.67 ± 0.02 2.3 111.3 8 8.98 ± 0.10 1.1 112.3 Isoniazid 0.6 0.65 ± 0.07 11.2 108.2 8 9.11 ± 0.13 1.4 114.3 Pyrazinamide 6 6.7 ± 0.1 2.2 112.0 80 90.2 ± 2.0 2.2 112.7 Rifampin 0.6 0.68 ± 0.03 3.8 114.0 8 8.96 ± 0.04 0.4 112.3 Clofazimine 0.3 0.32 ± 0.04 13.8 107.6 4 3.72 ± 0.33 8.7 93.0 Ethionamide 0.3 0.29 ± 0.02 6.8 97.4 4 3.59 ± 0.10 2.8 89.7 RSD (%): relative standard deviation, PAS: p-aminosalycylic acid *Values are mean ± standard deviation - 45 -
Table 18. Stability of 20 anti-tb drugs after 12 hours in extracts at room temperature in human plasma Compounds Conc. (μg/ml) Measured (μg/ml) RSD (%) Accuracy (%) Amikacin 3 2.7 ± 0.2 6.4 88.2 40 45.1 ± 5.8 12.9 112.7 Kanamycin 3 2.8 ± 0.3 9.7 91.8 40 44.2 ± 5.9 13.4 111.0 Streptomycin 3 3.0 ± 0.4 11.8 98.5 40 42.7 ± 3.1 7.3 107.0 Amoxicillin 1.2 1.19 ± 0.02 1.3 98.9 16 15.13 ± 0.51 3.4 94.6 Clarithromycin 0.6 0.61 ± 0.03 4.6 101.8 8 8.18 ± 0.28 3.4 102.1 Cycloserine 2.4 2.28 ± 0.01 0.3 94.8 32 32.20 ± 0.99 3.1 100.8 Linezolid 1.2 1.25 ± 0.01 0.5 104.3 16 16.43 ± 0.32 2.0 102.7 Moxifloxacin 0.6 0.62 ± 0.02 2.5 103.0 8 8.06 ± 0.18 2.2 100.7 floxacin 0.6 0.58 ± 0.01 1.1 97.4 8 8.15 ± 0.07 0.8 102.0 PAS 3 3.0 ± 0.1 3.5 98.5 40 41.5 ± 0.2 0.4 103.7 0.6 0.67 ± 0.01 0.9 110.7 Prothionamide 8 9.28 ± 0.08 0.9 116.0 0.12 0.125 ± 0.005 3.8 104.3 Rifabutin 1.6 1.670 ± 0.017 1.0 104.7 Roxithromycin 1.2 1.20 ± 0.04 3.0 99.5 16 17.17 ± 0.15 0.9 107.3 Ciprofloxacin 0.3 0.33 ± 0.02 5.1 110.7 4 4.01 ± 0.12 3.1 100.3 Ethambutol 0.6 0.62 ± 0.01 0.9 103.3 8 8.32 ± 0.19 2.3 104.0 Isoniazid 0.6 0.50 ± 0.01 2.8 82.6 8 7.50 ± 0.60 7.9 93.6 Pyrazinamide 6 6.4 ± 0.6 9.8 106.0 80 87.5 ± 0.5 0.5 109.3 Rifampin 0.6 0.67 ± 0.02 2.8 110.7 8 8.59 ± 0.23 2.7 107.3 Clofazimine 0.3 0.36 ± 0.03 7.4 121.5 4 4.43 ± 0.13 2.9 110.5 Ethionamide 0.3 0.35 ± 0.12 5.0 117.5 4 4.58 ± 0.09 2.0 114.5 RSD (%): relative standard deviation, PAS: p-aminosalycylic acid *Values are mean ± standard deviation - 46 -
Fig. 7(a). Peoduct ion mass chromatograms of 20 anti-tb drugs in plasma from patient A. - 47 -
Fig. 7(b). SRM chromatograms of 20 anti-tb drugs in plasma from patient A. - 48 -
A. 환자 B 에서의검출약물과농도추이 B. 환자 C 에서의검출약물과농도추이 Figure 8. 다제내성환자 B 와 C 의내원시수집된혈액에서의검출된약물과농도 - 49 -
μ μ μ Fig. 9. 다제내성결핵환자에서의 4 가지항결핵제의약물농도 - 50 -
Figure 10. cycloserine 을복용한 4 명의혈중농도 - 시간프로파일. 점선은집단예측치, 실선은개인 예측치, 점선은관찰치임. - 51 -
Table 19. Cycloserine 의집단약동학파라미터 Parameters Point estimate Standard Error RSE(%) CL/F (L/hr) 1.38 0.21 15.2 Vd/F (L) 88.0 25.3 28.8 Ka (1/hr) 5.13 1.58 30.8 IIV of CL (%) 37.0 7.5 20.2 IIV of Vd (%) NE NE NE IIV of Ka (%) NE NE NE RSE, relative standard error; CL, total clearance of cycloserinel; V/F, apparent volume of distribution of cycloserine; IIV, inter-individual variability;ne, not estimated (fixed to zero) - 52 -
제 4 장연구결과고찰및결론 - 53 -
- 54 -
제 5 장연구성과및활용계획 5.1 활용성과 과제명항결핵제의혈중약물농도측정법개발및임상적적용 차년도 과제책임자 김동현 / 인제대학교의과대학 / 대사독성 번호논문제목저자명저널명집 ( 권 ) 페이지 Impact factor 1 2 국내 / 국외 SCI여부 번호발표제목발표형태발표자학회명연월일발표지 1 2 국내 / 국제 번호출원 / 등록 1 2 특허명출원 ( 등록 ) 인출원 ( 등록 ) 국출원 ( 등록 ) 번호 IPC 분류 기타관련정책에활용예를구체적으로기술함. 한국인다제내성결핵환자에서의항결핵제의약동학적특성을규명하고임상적 outcome 결과를종합하여적정투여용량을설정하고 TDM을통한최적의치료가이드라인제시연구에활용할계획 언론홍보및대국민교육내용, 일자등을간략히기술함. 임상시험, 관련 DB 구축, 워크샾또는심포지움개최등의경우구체적으로기술함. - 55 -
5.2 활용계획본연구결과는차년도다제내성환자에서항결핵제의약동학적특성연구와더불어혈중약물농도와임상적 outcome 또는부작용과의연관성규명연구에활용하고자한다. 연구결과는 Therapeutic Drug Monitoring 잡지에투고하고자함. 제 6 장기타중요변경사항 중요변경사항없음 제 7 장연구비사용내역 단위 원 구 분 비 목 금액구성비비고 인건비소계 책 임 연 구 원 연 구 원 연 구 보 조 원 보 조 원 경비소계 여 비 유 인 물 비 전 산 처 리 비 시약및연구용재료비 회 의 비 임 차 료 교 통 통 신 비 감 가 상 각 비 위 탁 정 산 수 수 료 일반관리비 이윤 계 - 56 -
제 8 장참고문헌 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. - 57 -
18. 19. 20. 21. 22. 23. 24. 25. 제 9 장첨부서류 없음 - 58 -