Thompson Corbin G, Fallon John K, Mathews Michelle, Charlins Paige, Remling-Mulder Leila, Kovarova Martina, Adamson Lourdes, Srinivas Nithya, Schauer Amanda, Sykes Craig, Luciw Paul, Garcia J Victor, Akkina Ramesh, Smith Philip C, Kashuba Angela D M
aEshelman School of Pharmacy bSchool of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina cSchool of Medicine, Colorado State University, Fort Collins, Colorado dSchool of Medicine, University of California, Davis, California, USA.
AIDS. 2017 Jul 31;31(12):1669-1678. doi: 10.1097/QAD.0000000000001554.
Drug transporters affect antiretroviral therapy (ART) tissue disposition, but quantitative measures of drug transporter protein expression across preclinical species are not available. Our objective was to use proteomics to obtain absolute transporter concentrations and assess agreement with corresponding gene and immunometric protein data.
In order to make interspecies comparisons, two humanized mouse [hu-HSC-Rag (n = 41); bone marrow-liver-thymus (n = 13)] and one primate [rhesus macaque (nonhuman primate, n = 12)] models were dosed to steady state with combination ART. Ileum and rectum were collected at necropsy and snap frozen for analysis.
Tissues were analyzed for gene (quantitative PCR) and protein [liquid chromatography-mass spectrometry (LC-MS) proteomics and western blot] expression and localization (immunohistochemistry) of ART efflux and uptake transporters. Drug concentrations were measured by LC-MS/MS. Multivariable regression was used to determine the ability of transporter data to predict tissue ART penetration.
Analytical methods did not agree, with different trends observed for gene and protein expression. For example, quantitative PCR analysis showed a two-fold increase in permeability glycoprotein expression in nonhuman primates versus mice; however, proteomics showed a 200-fold difference in the opposite direction. Proteomics results were supported by immunohistochemistry staining showing extensive efflux transporter localization on the luminal surface of these tissues. ART tissue concentration was variable between species, and multivariable regression showed poor predictive power of transporter data.
Lack of agreement between analytical techniques suggests that resources should be focused on generating downstream measures of protein expression to predict drug exposure. Taken together, these data inform the use of preclinical models for studying ART distribution and the design of targeted therapies for HIV eradication.
药物转运体影响抗逆转录病毒疗法（ART）的组织分布，但目前尚无临床前各物种药物转运体蛋白表达的定量测定方法。我们的目标是利用蛋白质组学获得转运体的绝对浓度，并评估其与相应基因和免疫测定蛋白数据的一致性。
为了进行种间比较，对两种人源化小鼠[hu-HSC-Rag（n = 41）；骨髓-肝脏-胸腺（n = 13）]和一种灵长类动物[恒河猴（非人灵长类动物，n = 12）]模型给予联合ART至稳态。在尸检时收集回肠和直肠并速冻用于分析。
分析组织中ART外排和摄取转运体的基因（定量PCR）和蛋白[液相色谱-质谱（LC-MS）蛋白质组学和蛋白质印迹]表达及定位（免疫组织化学）。通过LC-MS/MS测定药物浓度。采用多变量回归确定转运体数据预测组织ART渗透的能力。
分析方法不一致，基因和蛋白表达呈现不同趋势。例如，定量PCR分析显示非人灵长类动物与小鼠相比，通透糖蛋白表达增加两倍；然而，蛋白质组学显示方向相反的200倍差异。蛋白质组学结果得到免疫组织化学染色的支持，显示这些组织腔表面有广泛的外排转运体定位。ART组织浓度在不同物种间存在差异，多变量回归显示转运体数据的预测能力较差。
分析技术之间缺乏一致性表明应将资源集中于生成蛋白质表达的下游测量指标以预测药物暴露。综上所述，这些数据为临床前模型用于研究ART分布及设计根除HIV的靶向疗法提供了参考。
