以往研究常以结合平衡常数K评价超分子复合物形成难易，而K与药物吸收并不正相关。药物必须解离才能吸收，表征药物解离的动力学速率常数koff，与超分子复合物的吸收密切相关。项目前期建立了koff分子模拟计算和定量亲和色谱（HPAC）测定方法，初步解决了多构型复合物模拟计算与实验测定单一结果对接的瓶颈问题。本项目以系列非甾体抗炎药β-环糊精超分子为对象，进一步完善koff模拟和测定方法的基础上，采用自适应偏置力法计算基元反应自由能，得到各单构型复合物koff,j值并推导koff,j与多构型复合物综合koff的定量关系，使模拟能更准确地反映真实情况；建立基于扩散系数校正的定量亲和色谱koff测定方法，用常规色谱实现koff的低成本快速测定，分子模拟并行计算与色谱质谱联用N-in-one测定相结合，研究不同pH值时的koff及其与Caco-2细胞吸收特征的关系，提高超分子给药系统吸收特征的可预测性。

The equilibrium constant is often used to evaluate the binding strength and the formation of the supramolecular complex, and studies mainly focus on the maximum binding of drug with supermolecules. While binding strongly, the dissociation of the complex and the release of drug from the complex are difficult, which may affect drug absorption. The rate constants (koff), representing the dissociation kinetics of the complex can reflect the dissociation characteristics of drugs. The project has established a novel theory about the calculation of koff with molecular simulation, which link the multiple koff from the molecular simulations of elementary reactions with the single koff from experiments. And quantitative affinity chromatography (HPAC) has been employed to determine koff. For the molecular simulation, only single koff can be obtained from the elementary reaction simulation with the adaptive biasing force (ABF) method. The quantitative relationship between the multiple koff and the integrated koff has been proposed by this project, which enable the koff obtained from the simulation can reflect the true situation more accurately. The modified HPAC method based on the molecular diffusion coefficient has been established to determine koff experimentally, with the advantages of high efficiency, low cost and high throughput. In this study, the non-steroidal anti-inflammatory drugs are chosen as model drugs to validate the theory and methods described above. With the combination of molecular simulation and HPAC, the koff of β-cyclodextrin drug delivery systems under different pH conditions will be calculated and measured by chromatography/mass spectrometry technique and N-in-one cassette dosing method. The correlation between koff and drug absorption on Caco-2 cells will be further investigated, which is important for the predictability of the simultaneous enhancement of drug solubility and drug absorption for the supramolecular drug delivery system.

超分子给药系统可以增加药物溶解度和生物利用度。药物（客分子）与辅料（主分子）结合的动力学常数kon、解离的动力学常数koff，决定药物从给药系统中的解离、吸收程度。现有表面等离子共振（SPR）等方法，不适用于作用较弱、解离较快的以环糊精为代表的超分子体系。项目运用“考虑药物-受体结合动力学行为”的药物发现新理念，发展了表征药物环糊精超分子解离的koff高效液相亲和色谱理论和方法。同时针对超分子存在多种同分异构体，传统模拟只能获得单一构型的koff。基于动力学原理和平衡时各组分的浓度关系，在各单构型复合物的koff与它们综合行为的koff间建立定量关系，使模拟计算得到的koff值能更准确地反映真实情况。对定量亲和色谱理论进行修正，发展了基于常规高效液相色谱的koff测定方法，对常规不保留物质（死时间物质）的塔板高度进行校正，获得理想不保留物质的塔板高度。以对乙酰氨基酚和舍曲林为模型药物，对单流速结果与多流速结果进行了验证和比较，结果与文献报道的毛细管电泳法测得结果数量级一致，表明方法准确、可靠。同时，考察了样品浓度、流速及流动相组成对koff测定结果的影响。采用色谱质谱联用技术建立了N-in-one 方式、同时测定20个药物koff的高通量方法，并与单独测定结果比对，验证了高通量测定具有较高的准确性。分子模拟方面，以氟比洛芬为模型药，建立了单一构型基元反应koff的分子模拟准确计算方法，解决了互为同分异构体、不同电离程度的多构型复合物综合koff的模拟和计算问题，比较了分子型和电离型药物的koff的异同，阐明了koff随pH值的变化规律。最终将药物-环糊精超分子kon/koff相关理论，应用于土荆皮乙酸包合物囊泡的优化给药。项目首次将亲和色谱技术应用于超分子动力学评价的药剂学领域，实现了超分子koff的低成本、快速测定，在低浓度、水溶性差药物相关的koff检测方面较SPR更具优势，并有望成为与SPR互补的koff测定方法，用于其他主客分子相互作用，如药物-功能性超分子、药物-转运蛋白的koff低成本、快速测定。