Drug Transporters in Farm Animals

农场动物的药物转运蛋白 ATP结合盒家族（ABC）中的膜转运蛋白在细胞外排中起关键作用， 内源性代谢物以及包括处方药的外源性物质。一个关键的ABC转运蛋白基因， 存在于哺乳动物和其他物种中的是ABCB 1，其编码P-糖蛋白（P-gp）。高度表达 在环境-生物体屏障组织如肠中，P-gp是药物转运的主要障碍。 吸收限制了其底物的口服生物利用度。因此，筛选候选药物的 通常选择与人P-gp相互作用和与P-gp相互作用有限或没有相互作用的那些， 进一步发展。然而，来自各种哺乳动物物种的P-gp已被证明具有深刻的 其底物和抑制剂特异性的差异。尽管表达人P-gp的细胞系可用于 在药物开发期间进行筛选，目前，表达农场动物P-gps的细胞系不可用。 因此，正在开发治疗动物，特别是农场动物的药物的公司必须依赖于 表达人类P-gp的细胞来预测药物，以推进昂贵的药物开发工作。整体 这项SBIR研究计划的第一阶段目标是建立和表征稳定表达 农场动物P-GPS包括来自鸡、猪、羊和牛的那些。这些细胞系将作为 药物开发人员预测候选药物吸收的资源。第二个目标是创造 并验证表达人P-gp的转化酵母菌株用于测试食品添加剂， 作为人类P-gp的潜在抑制剂。提出了两个目标。在目标1中， 从各种农场动物中建立表达P-gp的稳定细胞系，并表征以下物质的相互作用： 人P-gp与各种动物P-gp的典型底物和抑制剂。在目标2中，我们将 酵母与人P-gp（ABCB 1），并创建用于检测饲料污染物和添加剂的稳健检测方法 因为它们能够抑制人P-gp并对食用动物产品的人产生潜在毒性。 在拟议的研究中使用的方法包括用于创建稳定表达 转运蛋白直系同源物，细胞中的荧光和同位素摄取和通量测定，以及酵母遗传学， 转化方案和细胞毒性测定。总的来说，这些研究将产生一套新的工具， 用于开发和再利用药物治疗农场动物疾病， 评估饲料中的成分或污染物对人P-gp的潜在毒性作用。

Drug Transporters in Farm Animals Membrane transporters in the ATP Binding Cassette Family (ABC) play critical roles in cellular efflux of endogenous metabolites as well as xenobiotics including prescription drugs. A key ABC transporter gene that is present in mammals and other species is ABCB1, which encodes P-glycoprotein (P-gp). Highly expressed in environment-organism barrier tissues such as the intestine, P-gp represents a major impediment to drug absorption limiting the oral bioavailability of its substrates. Accordingly, candidate drugs are screened for their interactions with human P-gp and those with limited or no interactions with P-gp are generally selected for further development. However, P-gp from various mammalian species has been shown to have profound differences in its substrate and inhibitor specificities. Though human P-gp expressing cell lines are available for screening during drug development, currently, cell lines expressing farm animal P-gps are not available. Therefore, companies that are developing drugs to treat animals and in particular farm animals, must rely on cells expressing human P-gp to predict drugs to take forward in costly drug development efforts. The overall goals of this Phase I SBIR research proposal is to create and characterize cell lines stably expressing farm animal P-gps including those from chicken, pig, sheep and cattle. The cell lines will serve as a resource for drug developers to predict the absorption of candidate drugs. A secondary goal is to create and validate a transformed yeast strain expressing human P-gp for use in testing food additives and contaminants in animal feed as potential inhibitors of human P-gp. Two aims are proposed. In aim 1, we will create stable cell lines expressing P-gp from the various farm animals and characterize the interactions of canonical substrates and inhibitors of human P-gp with the various animal P-gps. In aim 2, we will transform yeast with human P-gp (ABCB1) and create a robust assay to use in testing feed contaminants and additives for their ability to inhibit human P-gp and cause potential toxicities to people ingesting animal products. Methods used in the proposed studies include new technologies for creation of cell lines stably expressing the transporter orthologs, fluorescent and isotopic uptake and flux assays in cells, as well as yeast genetics, transformation protocols and cytotoxicity assays. Overall these studies will lead to a set of new tools that can be used in the development and repurposing of drugs for the treatment of diseases of farm animals, and the assessment of ingredients or contaminants in feed for potentially toxic effects on human P-gp.