Prediction of CYP3A mediated drug-drug interaction using the endogenous metabolic markers and physiologically based pharmacokinetic model = 내인성 대사체 마커 및 생리학 기반 약물동태 모델을 이용한 CYP3A 매개 약물-약물 상호 작용 예측

다국어 초록 (Multilingual Abstract)

Introduction: Cytochrome P450 3A (CYP3A) is a phase I drug-metabolizing enzyme that accounts for the metabolism of drugs and endogenous substances in the gut and liver. By developing biomarkers for CYP3A activity, it is possible to predict new drug entities’ potential of modulating CYP3A activity in the early clinical stage. Because, previously identified markers were only evaluated for hepatic CYP3A activity with midazolam probe, identifying whether it reflects intestinal CYP3A activity and assessing in other CYP3A-mediated drug-drug interaction (DDI) will enhance the utility of the CYP3A markers. Physiologically based pharmacokinetic (PBPK) model is one of the emerging tools to predict pharmacokinetic (PK) changes considering the mechanism underlying physiology and drug interaction quantitatively. It allows not only to predict potential PK differences in various CYP3A activities and disease states, but also to estimate CYP3A inhibition on various DDI scenarios. Therefore, the study aimed to evaluate whether endogenous CYP3A activity reflects intestinal CYP3A activity, and assess whether those markers predict DDI between other CYP3A substrate (sildenafil) and two inhibitors with a different mechanism of CYP3A suppression. We further developed a PBPK model to simulate clinical trials to interpret the DDI results comprehensively with multidirectional approaches involving PK, endogenous metabolic marker, PBPK modeling.
Methods: To evaluate endogenous metabolites for assessing intestinal CYP3A activity, an open-label, three-period, six-treatment, one-sequence clinical trial was performed in 16 healthy Korean males. In the control phase, all subjects received a single dose of intravenous (IV) and oral midazolam (1 mg and 5 mg, respectively). Clarithromycin (500 mg) was administered twice daily for 4 days to inhibit hepatic and intestinal CYP3A, and 500 mL of grapefruit juice was given to inhibit intestinal CYP3A. To assess utility of markers in other CYP3A-mediated DDI, an open-label, one-sequence, one-period, two-treatment parallel study was conducted in 32 healthy Korean subjects. Each of 16 subjects were randomly assigned to the clarithromycin and itraconazole groups. Both groups received a single dose of sildenafil 25 mg as a control, and either clarithromycin 250 mg or itraconazole 100 mg was administered four times to inhibit CYP3A activity. Full PBPK models of CYP3A substrate (sildenafil, N-desmethyl sildenafil) and CYP3A inhibitor (clarithromycin, itraconazole) were constructed using physiological parameters including in-vitro, in-vivo study results from literature and several parameters were estimated using clinical studies results. The virtual clinical trial was simulated with the same observed DDI schedule using the final model.
Results: In the midazolam study, clarithromycin significantly inhibited total CYP3A activity, and the clearance of IV and apparent clearance of oral midazolam decreased by 0.15- and 0.32-fold, respectively. Grapefruit juice only reduced the apparent clearance of oral midazolam by 0.84-fold, which indicates a slight inhibition of intestinal CYP3A activity. Urinary markers, including 6β-OH-cortisol/cortisol and 6β-OH-cortisone/cortisone, were significantly decreased 0.5-fold after clarithromycin administration but not after grapefruit juice. The fold changes in 6β-OH-cortisol/cortisol and 6β-OH-cortisone/cortisone did not correlate to changes in intestinal availability but did correlate to hepatic availability. In the DDI study, pharmacokinetics of sildenafil showed the similar magnitude of inhibitory effects of the two inhibitors (clarithromycin and itraconazole) on total CYP3A activity; both inhibitors similarly increased systemic exposure of sildenafil by 2-fold. Urinary 6β-OH-cortisone/cortisone and plasma 4β-OH-cholesterol were significantly decreased after clarithromycin administration but not after itraconazole. A significant correlation between sildenafil CL/F and metabolic markers of CYP3A activity was observed after clarithromycin administration. The constructed PBPK model of sildenafil, N-desmethyl sildenafil, clarithromycin, itraconazole was well predict the observed DDI scenario. Although predicted maximum hepatic intrinsic clearance (CLint) of sildenafil was more reduced by itraconazole, CLint was highly fluctuated compared to clarithromycin. Moreover, the median fraction metabolized by liver CYP3A4 in relation to systemic clearance of sildenafil was decreased approximately 16% after clarithromycin, while only 6% after itraconazole. The p-gp mediated CLint of sildenafil and Ki of p-gp for clarithromycin and itraconazole have no impact on AUC of sildenafil after concomitant administration of either clarithromycin or itraconazole.
Conclusions: Our results suggest the limitation of steroid-based endogenous markers because these markers are not feasible to predict intestinal CYP3A activities. The DDI study between sildenafil with clarithromycin and itraconazole, suggested that those endogenous metabolic markers well reflected the mechanism-based CYP3A inhibition of clarithromycin but not for competitive CYP3A inhibition of itraconazole. The PBPK model confirmed that the observed pharmacokinetic interactions between sildenafil and CYP3A inhibitors were not due to transporter-mediated interaction. The endogenous metabolic markers may be utilized to predict potential hepatic CYP3A inhibition with moderate to strong alteration, but there are limitations in predicting intestinal CYP3A mediated DDI. The PBPK model is useful to predict and interpret observed DDI results with underlying mechanistic understanding.

국문 초록 (Abstract)

서론: Cytochrome P450 3A (CYP3A)는 간과 장에서 약물 및 내인성 물질 대사에 관여하는 phase I 약물 대사 효소이다. CYP3A 활성에 대한 바이오마커를 개발한다면, 초기 임상 단계에서 개발하는 약물이 잠재적으로 CYP3A 활성을 변화시키는지 예측할 수 있다. 이전에 확인된 마커들은 midazolam을 이용하여 간의 CYP3A 활성에 대해서만 평가되었기 때문에, 본 연구에서는 이들 마커가 장의 CYP3A 활성도 반영하는지 확인하고, 다른 CYP3A 매개 약물-약물상호작용을 평가하는데 유용한지 확인하고자 하였다. 생리학 기반 약물동태 모델링(PBPK)은 기전적으로 생리학 및 약물의 상호작용을 고려하여 약물의 약동학 변화를 정량적으로 예측할 수 있는 도구 중 하나이다. 이 방법을 이용하면 다양한 CYP3A 활성 및 질환 상태 뿐만 아니라 다양한 약물-약물 상호작용 시나리오에서 잠재적인 약동학 차이를 예측할 수 있다. 따라서 본 연구에서는 내인성 대사체 마커가 장의 CYP3A 활성을 반영하는지 평가하고, 이들 마커가 CYP3A의 기질인 sildenafil과 서로 다른 CYP3A 억제 기전을 갖는 두 개의 CYP3A 저해제를 병용한 약물-약물 상호작용을 예측하는지 확인하고자 한다. 또한 PBPK 모델을 이용하여 약물-약물 상호작용 임상시험 결과를 복합적으로 해석하고자 하였다.
방법: 내인성 대사체 마커가 장 CYP3A 활성을 반영하는지 평가하기 위해, 공개, 3군, 6치료군, 1 순서군 임상시험을 16명의 건강한 한국인 남성에서 수행하였다. 대조군에 모든 대상자는 정맥주사 (IV) 및 경구 midazolam (각각 1 mg 및 5 mg)을 단회 투여 받았다. Clarithromycin 500 mg은 1일 2회 용법으로 4일간 투여하여 간 및 장 CYP3A 활성을 억제하였고, 500 mL의 자몽주스를 투여하여 장 CYP3A 활성만 억제하였다. CYP3A 매개 약물-약물 상호작용을 예측하는데 마커들이 유용한지 확인하기 위하여, 공개, 2치료군, 1 순서군, 평행 설계 임상시험을 32명의 건강한 한국인 남성에서 수행하였다. 각 16명 씩 무작위로 clarithromycin 그룹과 itraconazole 그룹에 배정되었다. 두 그룹은 모두 대조군으로 sildenafil 25 mg을 단회 투여 받았고, CYP3A 활성을 억제하기 위해 그룹에 따라 clarithromycin 250 mg 또는 itraconazole 100 mg 을 4회 투여 받았다. CYP3A 기질인 sildenafil과 CYP3A 저해제인 clarithromycin, itraconazole에 대하여 문헌의 in-vitro, in-vivo 연구 결과를 포함한 생리학적 파라미터를 이용하여 PBPK 모델을 구축하였고, 일부 파라미터들은 임상시험 결과들을 이용하여 추정하였다. 최종 모델을 이용하여 관찰된 약물-약물상호작용 연구와 동일한 일정의 가상의 임상시험을 시뮬레이션 하였다.
결과: midazolam을 이용한 연구에서 clarithromycin은 전체 CYP3A 활성을 유의하게 억제하였고, IV 및 경구 투여 시 midazolam의 청소율은 각각 0. 15배, 0.32배 감소하였다. 자몽주스는 midazolam의 겉보기 청소율만 0.84 배 감소시켜, 장 CYP3A 활성을 약하게 감소시킨 것을 확인하였다. 소변 마커인 6β-OH-cortisol/cortisol 및 6β-OH-cortisone/cortisone은 clarithromycin 투여 후 유의하게 0.5 배 감소하였으나, 자몽주스 투여 후 변화하지 않았다. 6β-OH-cortisol/cortisol 및 6β-OH-cortisone/cortisone의 변화 정도는 장 생체이용률과 상관관계를 보이지 않았으나, 간 생체이용률과는 상관관계를 보였다. 약물-약물 상호작용 연구에서 sildenafil의 약동학은 두개의 CYP3A 저해제 (clarithromycin, itraconazole) 병용 투여 후 비슷하게 변화하여 두 CYP3A 저해제는 유사한 정도로 CYP3A를 억제하였다. 두 저해제는 sildenafil의 전신 노출을 유사하게 2배 가량 증가하였고, 소변 6β-OH-cortisone/cortisone 및 혈장 4β-OH-cholesterol 은 clarithromycin 투여 후에만 유의하게 감소하였다. Sildenafil의 겉보기 청소율과 CYP3A 활성에 대한 대사체 마커 간에는 clarithromycin 그룹에서만 유의한 상관관계를 확인하였다. 예측된 sildenafil의 최대 간 고유 청소율(CLint)은 itraconazole 투여 후 더 감소하였지만, clarithromycin에 비해 CLint의 변화율이 매우 컸다. 또한, sildenafil의 systemic clearance와 관련하여 간 CYP3A4에 의해 대사되는 비율은 clarithromycin 투여 후 약 16% 감소한 반면, itraconazole 투여 후 에는 약 6%만 감소하였다. Sildenafil의 p-gp 매개 CLint 및 clarithromycin 및 itraconazole의 p-gp에 대한 Ki 값은 clarithromycin 또는 itraconazole 투여 후 sildenafil의 AUC에 영향을 미치지 않았다.
결론: 본 연구에서는 스테로이드 기반 내인성 대사체 마커가 장 CYP3A 활성을 예측하는 것에 한계점이 있음을 확인하였다. Sildenafil과 clarithromycin 및 itraconazole 간의 약물-약물 상호작용 결과를 통해, 내인성 대사체 마커가 clarithromycin과 같은 mechanism 기반 CYP3A 억제에는 잘 예측하지만 itraconazole 과 같은 경쟁적 CYP3A 억제시에는 잘 예측하지 못하는 것을 확인하였다. PBPK 모델을 이용하여 관찰된 sildenafil과 CYP3A 억제제 간의 약동학적 상호작용이 수송체 매개 상호작용에 의한 것은 아님을 확인하였다. 본 내인성 대사 마커들을 이용하여 잠재적으로 간 CYP3A 활성에 중등도에서 강하게 영향을 주는 약물에 대해서 예측하도록 활용할 수 있으나, 장 CYP3A 매개 약물-약물 상호작용에 대한 예측력에는 한계를 가진다. PBPK 모델은 기전적인 이해를 통해 관찰된 DDI 결과를 해석하고 예측하는데 활용 할 수 있다.

목차 (Table of Contents)

• INTRODUCTION 1
• METHODS 7
• Study design and subjects 7
• Sample collection 11
• Pharmacokinetic analysis 12
• Endogenous metabolic marker analysis 13
• Pharmacogenomic analysis 15
• Statistical analysis 15
• Development of PBPK model 17
• Verification of PBPK model 19
• Simulation with PBPK model 20
• RESULTS 22
• PART I. Evaluation of endogenous CYP3A markers whether reflect on hepatic and intestinal CYP3A activity in healthy subjects 22
• Subjects demographics 22
• Changes in pharmacokinetics 23
• Changes in metabolic markers of CYP3A activity 32
• PART II. Utility of endogenous CYP3A marker in drug-drug interaction between sildenafil and CYP3A inhibitors, clarithromycin and itraconazole 40
• Subjects demographics 40
• Changes in pharmacokinetics 42
• Changes in metabolic markers of CYP3A activity 49
• PART III. PBPK model to evaluate CYP3A mediated drug-drug interaction 53
• Development and Verification of PBPK model 53
• Simulation with PBPK model 66
• DISCUSSION 79
• PART I. Evaluation of endogenous CYP3A markers whether reflect on hepatic and intestinal CYP3A activity in healthy subjects 79
• PART II. Utility of endogenous CYP3A marker in drug-drug interaction between sildenafil and CYP3A inhibitors, clarithromycin and itraconazole 85
• PART III. PBPK model to evaluate CYP3A mediated drug-drug interaction 90
• CONCLUSION 97
• REFERENCES 98
• 국문 초록 104

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