细胞内伊拉曲韦的药效在体外模型中存在种属差异：对临床前研究设计的影响。
Intracellular islatravir pharmacology differs between species in an in vitro model: implications for preclinical study design.
机构信息
UNC Eshelman School of Pharmacy, Chapel Hill, North Carolina, USA.
RTI International, Research Triangle Park, North Carolina, USA.
出版信息
J Antimicrob Chemother. 2022 Mar 31;77(4):1000-1004. doi: 10.1093/jac/dkac015.
BACKGROUND
Islatravir (4'-ethynyl-2-fluoro-2'-deoxyadenosine; EFdA) is a first-in-class nucleoside reverse transcriptase translocation inhibitor (NRTTI) being investigated for HIV treatment and prevention. EFdA is intracellularly phosphorylated to EFdA-triphosphate (EFdA-tp), a competitive substrate of deoxyadenosine-triphosphate (dATP). Thus, translating safety and efficacy findings from preclinical studies relies on the assumption that EFdA's intracellular pharmacology can be extrapolated across species.
OBJECTIVES
We investigated how EFdA is phosphorylated across animal species commonly used for preclinical models in drug development to identify those that most closely matched humans.
METHODS
PBMCs were isolated from whole blood of six species (human, rhesus macaque non-human primate (rmNHP), rat, minipig, dog, and rabbit) using Ficoll separation and counted on a haemocytometer by Trypan blue staining. One million live cells were cultured in media supplemented with 10 U/mL human IL-2, 10% FBS and 1% antibiotics and treated with 0, 17, 170, and 1700 nM EFdA (n = 3 replicates per concentration). After 24 h, representative cell counts were derived from untreated control wells (as above), cells were washed in PBS, and lysed with 70:30 methanol:water. EFdA-tp and dATP concentrations were quantified by HPLC-MS/MS and normalized to the representative live cell counts for each species.
RESULTS
When compared to human values, EFdA-tp concentrations for each EFdA treatment concentration were lower in all species (rmNHP 1.5-2.1-fold, rat 4.5-15-fold, minipig 37-71-fold, dog and rabbit >100-fold). Additionally, rmNHP and dog PBMCs exhibited significantly higher (7-10-fold; P < 0.001) dATP when compared with human PBMCs.
CONCLUSIONS
Given intracellular pharmacology differences, these preclinical models may be a conservative estimate of EFdA's intracellular pharmacokinetics and efficacy in humans.
背景
伊伐布雷定（4'-乙炔基-2-氟-2'-脱氧腺苷；EFdA）是一种首创的核苷逆转录酶易位抑制剂（NRTTI），正在被研究用于 HIV 的治疗和预防。EFdA 在细胞内被磷酸化为 EFdA-三磷酸（EFdA-tp），这是脱氧腺苷三磷酸（dATP）的竞争性底物。因此，将临床前研究的安全性和疗效发现转化为临床应用，依赖于假设 EFdA 的细胞内药理学可以在物种间推断。
目的
我们研究了 EFdA 在常用于药物开发临床前模型的动物物种中的磷酸化情况，以确定最接近人类的物种。
方法
使用 Ficoll 分离法从全血中分离出六种物种（人、恒河猴非人类灵长类动物（rmNHP）、大鼠、迷你猪、狗和兔）的 PBMC，并通过台盼蓝染色在血细胞计数器上计数。将 100 万个活细胞培养在含有 10 U/mL 人白细胞介素 2（IL-2）、10%胎牛血清（FBS）和 1%抗生素的培养基中，并以 0、17、170 和 1700 nM 的 EFdA 处理（每个浓度重复 3 个样本）。24 小时后，从未经处理的对照孔中提取代表性细胞计数（如上所述），用 PBS 洗涤细胞，并用 70:30 甲醇：水裂解。通过 HPLC-MS/MS 定量 EFdA-tp 和 dATP 的浓度，并将其归一化为每个物种的代表性活细胞计数。
结果
与人类值相比，每种 EFdA 处理浓度的 EFdA-tp 浓度在所有物种中均较低（rmNHP 为 1.5-2.1 倍，大鼠为 4.5-15 倍，迷你猪为 37-71 倍，狗和兔 >100 倍）。此外，与人类 PBMC 相比，rmNHP 和狗的 PBMC 表现出明显更高的（7-10 倍；P < 0.001）dATP。
结论
鉴于细胞内药理学的差异，这些临床前模型可能是 EFdA 在人类细胞内药代动力学和疗效的保守估计。
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