动脉粥样硬化保护性 CD40-TRAF6 抑制剂：临床应用的转化管道,European Heart Journal

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动脉粥样硬化保护性 CD40-TRAF6 抑制剂：临床应用的转化管道
European Heart Journal ( IF 37.6 ) Pub Date : 2023-11-11 , DOI: 10.1093/eurheartj/ehad655.3299
V Bazioti ₁ , K Wichapong ₂ , M Lacy ₃ , K Nitz ₁ , C M Van Tiel ₄ , C Weber ₁ , D Atzler ₁ , E Lutgens ₅

Affiliation

背景共刺激 CD40-CD40 配体轴是动脉粥样硬化形成的核心。激活后，CD40 会招募肿瘤坏死因子受体相关因子 (TRAF) 来诱导下游信号传导。对巨噬细胞中突变的 CD40-TRAF 结合位点的小鼠研究表明，CD40-TRAF6（而不是 CD40-TRAF2/3/5）轴对于动脉粥样硬化至关重要。因此，我们合成了小分子抑制剂 TRAF-STOP 6877002，它选择性阻断 CD40-TRAF6 信号传导。6877002 通过减少 ApoE-/- 小鼠中的单核细胞募集和巨噬细胞活化，成功减少了动脉粥样硬化的发生和发展，且没有免疫抑制副作用。目的评估 6877002 是否是通过转化管道进入临床应用的合适候选者。方法详细的体外和体内试验评估 6877002 的药理特性和潜在毒性。结果与 Cyprotex 一起，我们进行了体外试验，检查 6877002 的 ADME 谱。在肠道通透性试验中，6877002 具有低至中度吸收，没有显着的吸收。外排，表明它不是外排转运蛋白 Pgp/BCRP 或 MRP2 的底物。血浆蛋白结合测定显示未结合的 6877002 分数非常低，表明它与血浆蛋白高度结合。在酶特异性探针底物存在的情况下，使用人肝微粒体评估抑制细胞色素 P450 酶的潜力。虽然 6877002 抑制 CYP1A2 (IC50=4.01 µM)，但没有检测到抑制其他 CYP 亚型的潜力，表明药物间相互作用的可能性较低。为了筛查假定的心血管风险，评估了 hERG 离子通道抑制，但无法确定 IC50。小鼠、大鼠和狗的肝脏清除率很快，而小型猪和人肝细胞的内在清除率较低。我们与 Aptuit 一起开发了适合口服和静脉注射的配方。大鼠体内药代动力学研究显示，与静脉注射 6877002 相比，口服 6877002 的生物利用度较低 (8.43%) 且清除速度快，这可能是由于小鼠肝细胞中快速的首过效应以及巨噬细胞。为了解释 6877002 尽管代谢快速但仍具有高功效，我们评估了 6877002 是否具有结合 CD40-TRAF6 口袋的活性代谢物，并进行了基于 LC-MS/MS 的代谢物分析。我们检测到 17 种代谢物。进一-16.5（6877002）。与 6877002 类似，M4 在西方饮食喂养的 ApoE-/- 小鼠中使动脉粥样硬化减少了 54%，表明 M4 是一种活性代谢物。结论 CD40-TRAF6 抑制剂是一种具有高转化潜力的抗动脉粥样硬化的有吸引力的策略。

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Atheroprotective CD40-TRAF6 inhibitors: translational pipeline towards a clinical application

Background The co-stimulatory CD40–CD40 ligand axis is central in atherogenesis. Upon activation, CD40 recruits tumor necrosis factor receptor-associated factors (TRAFs) to induce downstream signaling. Murine studies with mutated CD40-TRAF binding sites in macrophages have shown that the CD40-TRAF6, rather than CD40-TRAF2/3/5, axis is crucial for atherosclerosis. We thus synthesized the small molecule inhibitor TRAF-STOP 6877002, that selectively blocks CD40-TRAF6 signaling. 6877002 successfully reduced the onset and development of atherosclerosis by reducing monocyte recruitment and macrophage activation in ApoE-/- mice without having immunosuppressive side effects. Purpose To assess whether 6877002 is a suitable candidate to pass the translational pipeline towards a clinical application. Methods Elaborate in vitro and in vivo assays evaluating the pharmacological properties and potential toxicity of 6877002. Results Together with Cyprotex, we performed in vitro assays examining the ADME profile of 6877002. In an intestinal permeability assay, 6877002 had low to moderate resorption with no significant efflux, suggesting it is not a substrate for the efflux transporters Pgp/BCRP or MRP2. A plasma protein binding assay showed very low unbound 6877002 fractions, indicating that it is highly bound to plasma proteins. The potential to inhibit cytochrome P450 enzymes was assessed using human liver microsomes in the presence of enzyme-specific probe substrates. While 6877002 inhibited CYP1A2 (IC50=4.01 µM), no potential for inhibiting other CYP isoforms was detected, suggesting low drug-drug interaction potential. To screen for putative cardiovascular risks, hERG ion channel inhibition was assessed and no IC50 could be determined. Hepatic clearance was rapid in mice, rats and dogs, while minipig and human hepatocytes had lower intrinsic clearance. Together with Aptuit, we developed suitable formulations for oral and i.v. administration. In vivo pharmacokinetics studies in rats showed low bioavailability (8.43%) and rapid clearance of orally administered 6877002 compared to i.v. injected 6877002, likely – and in line with our in vitro data – due to fast first-past effect in mouse hepatocytes and uptake by macrophages. To explain the high efficacy of 6877002 despite its rapid metabolism, we assessed whether 6877002 has active metabolites that bind the CD40-TRAF6 pocket and performed an LC-MS/MS-based metabolite profiling. We detected 17 metabolites. Further MD simulations confirmed that Metabolite (M)4, the most abundant metabolite, binds the CD40-TRAF6 pocket, reflected by the highest binding mode (-19.6; defined as Binding Free Energy derived from 40-50 ns of MD run as compared to -16.5 for 6877002). Similar to 6877002, M4 reduced atherosclerosis by 54% in Western diet-fed ApoE-/- mice, indicative of M4 being an active metabolite. Conclusion CD40-TRAF6 inhibitors are an attractive strategy to combat atherosclerosis with high translational potential.

更新日期：2023-11-11

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