In vitro screening model for compound interactions with human and dairy animal BCRP orthologs

IF 1.5 4区 农林科学 Q3 PHARMACOLOGY & PHARMACY Journal of veterinary pharmacology and therapeutics Pub Date : 2024-06-07 DOI:10.1111/jvp.13460
Lérica Le Roux-Pullen, Jeroen J.M.W. Van den Heuvel, Noraly B. Jonis, Tom Scheer-Weijers, Ilse R. Dubbelboer, Jan B. Koenderink, Frans G.M. Russel, Ronette Gehring
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Abstract

Orthologs of breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) efflux transmembrane transporter, are present in several species. The list of compounds known to interact with BCRP is growing, and many questions remain concerning species-specific variations in substrate specificity and affinity and the potency of inhibitors. As the most abundant efflux transporter known to be present in the blood–milk barrier, BCRP can increase the elimination of certain xenobiotics to milk, posing a risk for suckling offspring and dairy product consumers. Here we developed a model that can be employed to investigate species-specific differences between BCRP substrates and inhibitors. Membrane vesicles were isolated from transiently transduced human embryonic kidney (HEK) 293 cells, overexpressing BCRP, with human, bovine, caprine, and ovine cDNA sequences. To confirm BCRP transport activity in the transduced cells, D-luciferin efflux was measured and to confirm transport activity in the membrane vesicles, [3H] estrone-3-sulfate ([3H]E1S) influx was measured. We also determined the Michaelis–Menten constant (Km) and Vmax of [3H]E1S for each species. We have developed an in vitro transport model to study differences in compound interactions with BCRP orthologs from milk-producing animal species and humans. BCRP transport activity was demonstrated in the species-specific transduced cells by a reduced accumulation of D-luciferin compared with the control cells, indicating BCRP-mediated efflux of D-luciferin. Functionality of the membrane vesicle model was demonstrated by confirming ATP-dependent transport and by quantifying the kinetic parameters, Km and Vmax for the model substrate [3H]E1S. The values were not significantly different between species for the model substrates tested. This model can be insightful for appropriate inter-species extrapolations and risk assessments of xenobiotics in lactating woman and dairy animals.

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化合物与人类和乳用动物 BCRP 同源物相互作用的体外筛选模型。
乳腺癌抗性蛋白(BCRP/ABCG2)是一种 ATP 结合盒(ABC)外排跨膜转运体,其同源物存在于多个物种中。已知能与 BCRP 发生相互作用的化合物越来越多,但关于底物特异性和亲和力以及抑制剂效力的物种特异性差异仍存在许多问题。作为已知存在于血乳屏障中的最丰富的外排转运体,BCRP 可增加某些异种生物在乳汁中的排出量,从而对哺乳后代和乳制品消费者构成风险。在此,我们开发了一种模型,可用于研究 BCRP 底物和抑制剂之间的物种特异性差异。从过量表达 BCRP 的人胚胎肾(HEK)293 细胞中,用人、牛、绵羊和绵羊 cDNA 序列瞬时转导分离出膜囊泡。为了证实转导细胞中 BCRP 的转运活性,我们测量了 D-luciferin 的流出量;为了证实膜泡中的转运活性,我们测量了 [3H] 3-硫酸雌酮([3H]E1S)的流入量。我们还测定了每个物种的[3H]E1S 的迈克尔斯-门顿常数(Km)和 Vmax。我们建立了一个体外转运模型,以研究化合物与产奶动物物种和人类 BCRP 同源物之间相互作用的差异。与对照细胞相比,物种特异性转导细胞中 D-荧光素的积累减少,这表明 BCRP 介导了 D-荧光素的外流,从而证明了 BCRP 的转运活性。通过确认 ATP 依赖性转运以及量化模型底物 [3H]E1S 的动力学参数 Km 和 Vmax,证明了膜囊模型的功能。对于所测试的模型底物,不同物种之间的数值没有明显差异。该模型可用于哺乳期妇女和乳用动物体内异种生物的适当种间推断和风险评估。
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来源期刊
CiteScore
3.10
自引率
15.40%
发文量
69
审稿时长
8-16 weeks
期刊介绍: The Journal of Veterinary Pharmacology and Therapeutics (JVPT) is an international journal devoted to the publication of scientific papers in the basic and clinical aspects of veterinary pharmacology and toxicology, whether the study is in vitro, in vivo, ex vivo or in silico. The Journal is a forum for recent scientific information and developments in the discipline of veterinary pharmacology, including toxicology and therapeutics. Studies that are entirely in vitro will not be considered within the scope of JVPT unless the study has direct relevance to the use of the drug (including toxicants and feed additives) in veterinary species, or that it can be clearly demonstrated that a similar outcome would be expected in vivo. These studies should consider approved or widely used veterinary drugs and/or drugs with broad applicability to veterinary species.
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