Department of Clinical Pharmacology, University Medical Center, Georg-August-University, Göttingen, Germany.
Biochem Pharmacol. 2012 May 15;83(10):1427-34. doi: 10.1016/j.bcp.2012.01.032. Epub 2012 Feb 10.
Debrisoquine is a probe drug for in vivo phenotyping of human CYP2D6 metabolic activity. However, debrisoquine is positively charged under physiological conditions and it is unclear how it enters the hepatocytes to undergo CYP2D6 metabolism. We analysed whether debrisoquine is a substrate of the hepatic organic cation transporter OCT1 and whether drug-drug interactions at OCT1, or polymorphisms in OCT1 gene, affect debrisoquine uptake. Debrisoquine showed low carrier-independent membrane permeability (P(e) of 0.01×10⁻⁶ cm/s in artificial PAMPA membranes) and strongly inhibited the uptake of the model OCT1 substrate MPP+ (IC₅₀ of 6.2 ± 0.8 μM). Debrisoquine uptake was significantly increased in HEK293 cells overexpressing OCT1 compared to control cells. The OCT1-mediated uptake of debrisoquine followed Michaelis-Menten kinetics (K(M) of 5.9 ± 1.5 μM and V(max) of 41.9 ± 4.5pmol/min/mg protein) and was inhibited by known OCT1 inhibitors and by commonly used drugs. OCT1-mediated debrisoquine uptake was reduced or missing in cells expressing loss-of-function OCT1 isoforms. Deletion of Met420 or substitution of Arg61Cys or Gly401Ser reduced V(max) by 48, 63 and 91%, respectively, but did not affect the K(M). The OCT1 isoforms carrying Cys88Arg or Gly465Arg substitutions completely lacked OCT1-mediated debrisoquine uptake. In conclusion, debrisoquine is a substrate of OCT1 and has the potential to be used as a phenotyping marker for OCT1 activity. Moreover, variations in debrisoquine metabolic phenotypes and their associations with diseases may be due not only to genetic variations CYP2D6, but also in OCT1.
地昔帕明是一种用于体内表型分析人类 CYP2D6 代谢活性的探针药物。然而，地昔帕明在生理条件下带正电荷，目前尚不清楚它如何进入肝细胞进行 CYP2D6 代谢。我们分析了地昔帕明是否是肝有机阳离子转运蛋白 OCT1 的底物，以及 OCT1 上的药物相互作用或 OCT1 基因多态性是否会影响地昔帕明摄取。地昔帕明在人工 PAMPA 膜中显示出低载体非依赖性膜通透性（P(e)为 0.01×10⁻⁶cm/s），并强烈抑制模型 OCT1 底物 MPP+的摄取（IC₅₀为 6.2±0.8μM）。与对照细胞相比，在过表达 OCT1 的 HEK293 细胞中，地昔帕明摄取明显增加。OCT1 介导的地昔帕明摄取遵循米氏动力学（K(M)为 5.9±1.5μM，V(max)为 41.9±4.5pmol/min/mg 蛋白），并被已知的 OCT1 抑制剂和常用药物所抑制。在表达功能丧失的 OCT1 同工型的细胞中，OCT1 介导的地昔帕明摄取减少或缺失。缺失 Met420 或取代 Arg61Cys 或 Gly401Ser 分别使 V(max)降低 48%、63%和 91%，但不影响 K(M)。携带 Cys88Arg 或 Gly465Arg 取代的 OCT1 同工型完全缺乏 OCT1 介导的地昔帕明摄取。总之，地昔帕明是 OCT1 的底物，并有潜力作为 OCT1 活性的表型标志物。此外，地昔帕明代谢表型的变化及其与疾病的关联可能不仅归因于 CYP2D6 的遗传变异，还归因于 OCT1。
