Supplementary Material for: Phytochemical Comparison of Medicinal Cannabis Extracts and Study of Their CYP-Mediated Interactions with Coumarinic Oral Anticoagulants

<b><i>Introduction:</i></b> Treatment with cannabis extracts for a variety of diseases has gained popularity. However, differences in herb-drug interaction potential of extracts from different plant sources are poorly understood. In this study, we provide a characterization of cannabis extracts prepared from four cannabis chemotypes and an in vitro assessment of their Cytochrome P450 (CYP)-mediated herb-drug interaction profiles. <b><i>Methods:</i></b> Plant extracts were either commercially obtained or prepared using ethanol as solvent, followed by overnight decarboxylation in a reflux condenser system. The extracts were characterized for their cannabinoid content using NMR and HPLC-PDA-ELSD-ESIMS. CYP inhibition studies with the cannabis extracts and pure cannabinoids (tetrahydrocannabinol [THC] and cannabidiol [CBD]) were performed using pooled, mixed gender human liver microsomes. Tolbutamide and testosterone were used as specific substrates to assess the inhibitory potential of the extracts on CYP2C9 and CYP3A4, and the coumarinic oral anticoagulants warfarin, phenprocoumon, and acenocoumarol were studied as model compounds since in vivo herb-drug interactions have previously been reported for this compound class. <b><i>Results:</i></b> In accordance with the plant chemotypes, two extracts were rich in THC and CBD (at different proportions); one extract contained mostly CBD and the other mostly cannabigerol (CBG). Residual amounts of the corresponding acids were found in all extracts. The extracts with a single major cannabinoid (CBD or CBG) inhibited CYP2C9- and CYP3A4-mediated metabolism stronger than the extracts containing both major cannabinoids (THC and CBD). The inhibition of CYP3A4 and CYP2C9 by the extract containing mostly CBD was comparable to their inhibition by pure CBD. In contrast, the inhibitory potency of extracts containing both THC and CBD did not correspond to the combined inhibitory potency of pure THC and CBD. Although being structural analogs, the three coumarin derivatives displayed major differences in their herb-drug interaction profiles with the cannabis extracts and the pure cannabinoids. <b><i>Conclusion:</i></b> Despite the fact that cannabinoids are the major components in ethanolic, decarboxylated cannabis extracts, it is difficult to foresee their herb-drug interaction profiles. Our in vitro data and the literature-based evidence on in vivo interactions indicate that cannabis extracts should be used cautiously when co-administered with drugs exhibiting a narrow therapeutic window, such as coumarinic anticoagulants, regardless of the cannabis chemotype used for extract preparation.

**引言：** 使用大麻提取物治疗多种疾病的做法日益普及。然而，不同植物来源提取物的草药-药物相互作用潜力差异却鲜为人知。本研究对四种大麻化学型（chemotype）制备的大麻提取物进行了表征，并对其细胞色素P450（Cytochrome P450，CYP）介导的草药-药物相互作用特征开展体外评估。**方法：** 本研究中的植物提取物或购自商业渠道，或以乙醇为溶剂制备，随后在回流冷凝系统中进行过夜脱羧反应。采用核磁共振波谱（Nuclear Magnetic Resonance, NMR）与高效液相色谱-光电二极管阵列检测器-蒸发光散射检测器-电喷雾电离质谱（High Performance Liquid Chromatography-Photodiode Array Detector-Evaporative Light Scattering Detector-Electrospray Ionization Mass Spectrometry, HPLC-PDA-ELSD-ESIMS）对提取物的大麻素类成分含量进行表征。采用混合性别混合供体人肝微粒体，开展大麻提取物与纯大麻素类化合物（四氢大麻酚（tetrahydrocannabinol, THC）和大麻二酚（cannabidiol, CBD））的CYP抑制实验。以甲苯磺丁脲（tolbutamide）和睾酮（testosterone）作为特异性底物，评估提取物对CYP2C9与CYP3A4的抑制活性；同时选用香豆素类口服抗凝药华法林（warfarin）、苯丙香豆素（phenprocoumon）与醋硝香豆素（acenocoumarol）作为模型化合物，因已有文献报道该类化合物存在体内草药-药物相互作用。**结果：** 与大麻化学型一致，两种提取物富含THC与CBD（比例各异）；一种提取物主要含有CBD，另一种则主要含有大麻萜酚（cannabigerol, CBG）。所有提取物中均检测到对应羧酸类成分的残留量。仅含单一主要大麻素（CBD或CBG）的提取物，对CYP2C9与CYP3A4介导的代谢的抑制作用强于同时含有两种主要大麻素（THC与CBD）的提取物。主要含CBD的提取物对CYP3A4与CYP2C9的抑制活性，与纯CBD的抑制活性相当。与之相反，同时含有THC与CBD的提取物的抑制效力，并不等同于纯THC与纯CBD的联合抑制效力。尽管三种香豆素类衍生物为结构类似物，但它们与大麻提取物及纯大麻素的草药-药物相互作用特征存在显著差异。**结论：** 尽管经脱羧处理的乙醇提取物中，大麻素类为主要成分，但仍难以预判其草药-药物相互作用特征。本研究的体外数据以及已发表的体内相互作用文献证据均表明，当与治疗窗较窄的药物（如香豆素类抗凝药）联用时，无论用于制备提取物的大麻化学型为何，均应谨慎使用大麻提取物。