Dendrimer-conjugated nSMase2 inhibitor as a novel therapeutic approach for Alzheimer's Disease

患有阿尔茨海默病（AD）的人数正在稳步上升，目前的治疗方法只有 提供轻微的症状改善。最近针对淀粉样蛋白-β（Aβ）产生或 清除率令人失望，促使人们重新审视AD的治疗方法。AD的大脑 患者表现出神经酰胺的积累，神经酰胺是一种信号分子，也是外泌体的组成部分。 膜。神经酰胺的一个主要来源是通过中性蛋白酶催化鞘磷脂的水解， 鞘磷脂酶2（nSmase 2）。即使通过nSMase 2上调神经酰胺一过性增加， 是正常大脑功能的一部分，实验证据表明慢性nSMase 2上调导致 包括神经炎症和氧化应激的负面影响。最近的研究表明，nsmase 2与 通过神经胶质细胞外泌体分泌的Aβ聚集和tau蛋白增殖。此外，抑制 通过遗传或药理学抑制nSM酶2合成外泌体，可阻断Aβ聚集 以及tau在体外和体内AD模型中的传播，从而为AD治疗开辟了新的途径。 不幸的是，没有临床上有用的nSMase 2抑制剂。电流抑制剂较弱（µM-mM）， 物理化学性质和/或有限的脑渗透。我们与NCATS合作， nSMase 2高通量筛选（HTS）> 350，000种化合物。HTS命中的过滤和分析导致 发现2，6-二甲氧基-4-（5-苯基-4-（噻吩-2-基）-1H-咪唑-2-基）苯酚（DPTIP）第一个nM抑制剂 (IC50= 30 nM）。发现DPTIP是选择性的并且能够剂量依赖性地抑制外泌体释放 在神经胶质细胞培养中。不幸的是，在体内DPTIP表现出快速清除，导致半衰期短（t1/2< 0.5h）。 口服生物利用度差（F<5%）。结构修饰（我们小组合成的约200种类似物） 并没有带来实质性的改善。鉴于其显著的临床潜力，我们建议解决 利用树枝状聚合物纳米颗粒将DPTIP选择性递送至活化的神经胶质细胞的药代动力学限制 脑细胞。我们的团队发现，系统性给药羟基封端的聚（酰胺胺） （PAMAM）树枝状聚合物（尺寸约4 nm）靶向受损大脑中的激活神经胶质细胞，而无需靶向 配体，在健康大脑中显示出最小的摄取。虽然树枝状聚合物被内吞并被保留， 大脑中的活化神经胶质细胞维持暴露>2周，它们迅速从外周清除 （血浆t1/2 ~ 6-24 h）。我们已经在多个小型和大型研究中验证了脑靶向、安全性和有效性。 动物模型，并与我们的第一个树枝状聚合物产品（D-NAC在儿童期的1期临床试验 脑肾上腺脑白质营养不良）。在此，我们建议在体内合成和评估 与DPTIP偶联的两种不同大小的树枝状聚合物（D-DPTIP）的药代动力学和靶向结合 口服给药后。通过脑成像、LC/MS生物分析和 将在两个建立的小鼠模型中测试神经胶质nSM酶2活性的功能性抑制的功效和安全性 AD模型我们已经组建了一支经验丰富的团队，拥有树枝状纳米颗粒的专业知识 （Rangaramanujam）、药代动力学、生物标志物和靶点结合研究（Rais），以及 药理学、药物发现和临床翻译（Slusher）。

The number of people suffering from Alzheimer's disease (AD) is steadily rising and current treatments only provide minor symptom amelioration. Results from recent clinical trials targeting amyloid-β (Aβ) production or clearance were disappointing, prompting a reexamination of approaches to AD treatment. Brains from AD patients exhibit accumulation of ceramide, a signaling molecule and an integral component of exosomal membranes. One major source of ceramide is through the hydrolysis of sphingomyelin catalyzed by neutral sphingomyelinase 2 (nSmase2). Even though transient increases in ceramide through nSMase2 upregulation are part of normal brain functioning, experimental evidence indicates that chronic nSMase2 upregulation results in negative effects including neuroinflammation and oxidative stress. Recent studies implicate nSMase2 in both Aβ aggregation and tau protein propagation through exosome secretion from glial cells. Moreover, inhibition of exosome synthesis by genetic or pharmacological inhibition of nSMase2 was shown to block Aβ aggregation and tau propagation in both in vitro and in vivo AD models, thus opening a new avenue for AD therapeutics. Unfortunately, there are no clinically useful nSMase2 inhibitors. Current inhibitors are weak (µM-mM) with poor physicochemical properties and/or limited brain penetration. In collaboration with NCATS we carried out a human nSMase2 high throughput screen (HTS) of >350,000 compounds. Filtering and analysis of HTS hits led to discovery of 2,6-dimethoxy-4-(5-phenyl-4-(thiophen-2-yl)-1H-imidazol -2-yl) phenol (DPTIP) the first nM inhibitor (IC50 = 30 nM). DPTIP was found to be selective and capable of dose-dependently inhibiting exosome release in glial cultures. Unfortunately, in vivo DPTIP exhibited rapid clearance resulting in a short half-life (t1/2< 0.5h) and had poor oral bioavailability (F<5%). Structural modifications (~200 analogs synthesized by our group) have not led to substantial improvements. Given its significant clinical potential, we propose to address the pharmacokinetic limitations by utilizing dendrimer nanoparticles to deliver DPTIP selectively to activated glial cells in the brain. Our team discovered that systemically-administered hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimers (~4 nm in size) target activated glia in the injured brain, without the need for targeting ligands, showing minimal uptake in healthy brains. While the dendrimers are endocytosed and retained by activated glial cells in the brain maintaining exposure for >2 weeks, they are rapidly cleared from the periphery (plasma t1/2 ~ 6-24 h). We have validated the brain targeting, safety, and efficacy in multiple small and large animal models, and are in Phase 1 clinical trials with our first dendrimer product (D-NAC in childhood cerebral adrenoleukodystrophy). Herein, we propose to synthesize and evaluate the in vivo pharmacokinetics and target engagement of two differently sized dendrimers conjugated to DPTIP (D-DPTIP) following peroral administration. The optimal conjugate assessed by brain imaging, LC/MS bioanalysis, and functional inhibition of glial nSMase2 activity will be tested for efficacy and safety in two established mouse models of AD. We have assembled a highly experienced team with expertise in dendrimer nanoparticles (Rangaramanujam), pharmacokinetics, biomarkers and target engagement studies (Rais) and pharmacology, drug discovery and clinical translation (Slusher).