基于靶细胞PK/PD模型研究伏立康唑预防侵袭性曲霉菌病的机制及给药方案优化

Pulmonary epithelial cells invaded by Aspergillus fumigatus conidia in human body is the first step of causing invasive aspergillosis (IA), and IA is still related to high rates of morbidity and mortality after the utilization of antifungal agents. Therefore, how to design the optimum prophylactic dosage regimen to prevent IA is an intractable problem in clinical practice. Voriconazole has a strong activity against Aspergillus fumigatus but a low concentration in pulmonary epithelial cells. It is vital to explore the concentration of voriconazole in epithelial lining fluid (ELF) and the intracellular activity of voriconazole against Aspergillus fumigatus in pulmonary epithelial cells. Since it’s difficult to detect the concentration of voriconazole in ELF in clinical practice, this project aims to study the relationship between the concentration of voriconazole in ELF and that in blood firstly applying physiologically based pharmacokinetic model, and then build a cell model infected by Aspergillus fumigatus to investigate the dose-effect relationship of intracellular activity of voriconazole and acquire the pharmacokinetic/pharmacodynamic (PK/PD) breakpoint of intracellular activity of voriconazole against Aspergillus fumigatus based on the "ELF - pulmonary epithelial cells - Aspergillus fumigatus" environment in human body, and finally, use the result of physiologic based pharmacokinetics model and PK/PD breakpoint to calculate the probability of target attainment to obtain the optimum prophylactic dosage regimen, and apply immunocompromised rats to validate the dosage regimen. The completion of this project will clarify the mechanism of voriconazole preventing IA on a cellular level, and provide novel theoretical and experimental basis for the optimization of voriconazole prophylactic dosage regimen and the reduction of IA morbidity.

烟曲霉菌侵袭肺上皮细胞（PEC）是引起侵袭性曲霉菌病（IA）的首要环节。尽管临床已使用抗真菌药物进行预防，IA发病率和死亡率仍较高，如何制定最佳预防方案是临床面临的难点。伏立康唑（VOR）抗烟曲霉菌活性强，但PEC内浓度低，研究其在PEC表面衬液（ELF）中的浓度和PEC内抗菌作用可为优化预防给药方案提供依据。鉴于此，本项目拟采用生理药动学研究VOR的ELF浓度与血药浓度的关系，以克服临床难以获得其ELF浓度的问题；模拟人体“ELF-PEC-烟曲霉菌”环境，构建烟曲霉菌感染的细胞模型，探讨VOR在细胞内抗菌作用的量效关系，获得抗菌作用的药动学/药效学（PK/PD）折点；利用PK/PD折点和生理药动学结果获取达标概率，以遴选最佳预防方案，并用免疫缺陷大鼠对给药方案进行验证。本项目将从细胞水平阐明VOR预防IA的机制，可为临床优化VOR预防给药方案和降低IA的发病率提供新的理论依据和实验基础。

侵袭性曲霉菌病（IA）在免疫缺陷患者中具有较高的发病率和死亡率，烟曲霉菌侵袭肺上皮细胞（A549）是引起IA的首要环节。伏立康唑是预防和治疗IA的一线药物，如何制定最佳预防方案是临床面临的难点，而研究伏立康唑在肺上皮细胞内的药动学/药效学特征可为抗真菌预防方案制定提供依据。本项目构建了伏立康唑-肺上皮细胞-烟曲霉菌三者之间相互作用的体外研究体系。建立了LC-MS/MS法测定细胞内伏立康唑浓度，明确了伏立康唑在A549细胞中的药动学特征与参数。结果表明伏立康唑在细胞内吸收呈线性动力学特征，在细胞内消除快，消除半衰期约为10.2 min；构建了烟曲霉菌感染的A549细胞模型，研究了伏立康唑细胞内抗烟曲霉菌作用的药效学特征和PK/PD折点。结果表明伏立康唑在细胞内抗菌后效应短，烟曲霉菌MIC值对伏立康唑细胞内抗菌作用有影响。伏立康唑可使细胞内烟曲霉菌AF293和AF26最多下降0.79和0.84 lg cfu。抑制性S型最大效应模型结果显示伏立康唑可抑制细胞内84.01%烟曲霉菌的生长，当伏立康唑孵育浓度与MIC的比值（Ce/MIC）大于35.53时，可抑制细胞内50%烟曲霉菌的生长。利用蒙特卡洛模拟法结合PPK模型与PK/PD 折点结合制定了伏立康唑预防给药方案，结果显示口服200 mg/12 h的给药方案可以有效预防侵袭性曲霉菌病，其累积反应分数为91.48%。伏立康唑在肺上皮细胞衬液和血液中的浓度大于17.77 mg/L 和1.55 mg/L时可保证PK/PD参数达标。进一步构建了基于CYP3A4酶时间依赖性抑制的伏立康唑生理药动学模型，该模型可准确预测伏立康唑在患者体内的血药浓度。利用生理药动学进行模拟表明伏立康唑在CYP2C19快代谢者需采用250 mg/12 h的维持剂量，在CYP2C19中间代谢者中可采用200 mg/12 h的维持剂量，在CYP2C19慢代谢者可采用150 mg/12 h的维持剂量，依据CYP2C19基因多态性制定的给药方案可保证伏立康唑血药浓度维持在预防有效浓度范围内。本研究为抗真菌药物预防给药方案的制定和降低烟曲霉菌在免疫缺陷患者中的发病率提供了理论依据。