Development of N-tert-(Butyl)hydroxylamine (NtBuHA) as a therapeutic agent for treating Infantile Neuronal Ceroid Lipofuscinosis (INCL)

项目摘要/摘要 治疗婴儿神经元性脑炎的治疗方法在医学上有很大的需求，但尚未得到满足。 脂褐素沉着症(CLN1型巴顿病)，估计有(1/10万)患者 美国目前没有治疗疾病的选择。我们将在以下时间开始临床前项目 一种被NICHD的一名高级研究员选为先导化合物的小分子， 阿尼尔·慕克吉博士。这种小分子N-(叔丁基)-羟胺(NtBuHA)已被选中 在一个由12个羟胺衍生物组成的小组中，其体外和体内毒性最低，其 它在水溶液中的溶解性及其取代缺失棕榈酰基蛋白质的优异化学活性 硫代酯酶1(PPT1)活性，这是INCL患者病理生理的基础。为…做准备 作为一个支持IND的计划，我们将通过完成物理化学分析DMPK来降低NtBuHA的风险 研究、生物分布研究和先导毒理学研究。溶解度、稳定性和亲脂性研究 将在体外进行，以确认NtBuHA在进一步体内试验之前具有足够的化学性质 工作。来自四个物种(小鼠、大鼠、狗和人类)的血浆蛋白结合研究将确定 NtBuHA的热力学结合参数将用于PK分析，这将有助于提供 从动物数据估计人类全身暴露。代谢稳定性将在 四个物种(小鼠、大鼠、狗、人)的微粒体和肝细胞以估计跨物种到 人的第一相代谢和第二相代谢。四种沙门氏菌肝细胞代谢物的分析 将进行物种(小鼠、大鼠、狗、人)，以帮助选择毒理学物种，并 确定任何人类特定的代谢物。INCL患者通常采用联合抗肿瘤药物治疗。 癫痫、疼痛和抗痉挛药物(如丙戊酸盐、芬太尼、巴氯芬、替扎尼定)，因此 有必要进行药物-药物相互作用(CYP抑制和诱导)研究 人体肝脏样本。我们将在大鼠身上完成PK研究，以确定剂量依赖关系 药物动力学参数既依赖于吸附(通过口服途径)又依赖于吸附 独立(通过静脉途径)，以及估计人体PK参数和剂量 药理反应所需的养生法。剂量递增的大鼠生物分布研究将是 重点研究NtBuHA在大脑中的积累，以确定最低限度 达到脑组织治疗相关浓度所需的剂量。最多7天 将完成耐受量(MTD)研究，以建立NtBuHA的治疗窗口，以及 确保可以安全地达到与治疗相关的大脑浓度。《赫尔格》、《艾姆斯》和 微核试验将作为体外毒理学门控研究完成，这将有助于确定 是否值得提交第二阶段SBIR。在完成拟议的工作后，我们将有一个 具有更全面的配方、安全性和有效性的铅化合物 体内评价。在成功完成这些研究后，我们打算进入第二阶段SBIR 将使用GLP设施和GMP材料的研究，更长的重复剂量毒性方案 大型动物和安全性研究(心脏、呼吸等)，将更准确地预测NtBuha 这将支持后续的IND应用程序。最终，我们希望NtBuha 可以成为一种对包括患者在内的患者具有临床意义的上市产品。

PROJECT SUMMARY/ABSTRACT There is a significant unmet medical need for a therapy to treat patients with Infantile Neuronal Ceroid Lipofuscinosis (type CLN1 Batten Disease), for which there are (<1 in 100,000) estimated patients in the United States that have no current disease-treating options. We will begin a pre-clinical program on one small molecule that has been selected as a lead compound by a senior investigator at the NICHD, Dr. Anil Mukherjee. This small molecule, N-(tert-butyl)-hydroxylamine (NtBuHA), has been selected amongst a panel of 12 hydroxylamine derivatives for its minimal in-vitro and in-vivo toxicity profile, its solubility in aqueous solution, and its superior chemical activity in replacing deficient Palmitoyl-Protein Thioesterase 1 (PPT1) activity, that underlies the pathophysiology of INCL patients. In preparation for an IND-enabling program, we will de-risk NtBuHA by completing physiochemical analysis, DMPK studies, biodistribution studies, and pilot toxicology studies. Solubility, stability, and lipophilicity studies will be conducted in-vitro to confirm NtBuHA has adequate chemical properties prior to further in-vivo work. Plasma protein binding studies from four species (mouse, rat, dog, human) will determine thermodynamic binding parameters of NtBuHA to be used in PK analyses and that will help provide an estimation of human systemic exposure from animal data. Metabolic stability will be performed in microsomes and hepatocytes of four species (mouse, rat, dog, human) to estimate cross-species-to- human phase I and phase II metabolism, respectively. Metabolite profiling in hepatocytes of four species (mouse, rat, dog, human) will be performed to aid in the selection of toxicology species, and to identify any human specific metabolites. INCL patients are often treated with a combination of anti- epileptic, pain, and anti-spasticity medications (e.g. valproate, fentanyl, baclofen, tizanidine), thus necessitating drug-drug interaction (CYP inhibition and induction) studies that will be performed in human liver samples. We will complete PK studies in rats to determine the dose dependent pharmacokinetic parameters that are both adsorption dependent (via oral gavage route) and adsorption independent (via intravenous route), as well as to estimate both human PK parameters and dose regimen needed for pharmacological response. Dose-escalating biodistribution studies in rats will be conducted with focus on accumulation of NtBuHA within the brain, in order to determine the minimum dose required to achieve therapeutically relevant concentrations in brain tissues. A 7-day maximum tolerated dose (MTD) study will be completed to establish the therapeutic window for NtBuHA, and ensure therapeutically relevant brain concentrations can be safely achieved. The hERG, AMES and micronucleus tests will be completed as in-vitro toxicology gating studies that will aid in determining whether a phase II SBIR submission is merited. Upon the conclusion the proposed work, we will have a lead compound that is characterized for more comprehensive formulation, safety and efficacy evaluation in vivo. Upon successful completion of these studies, we intend to enter into Phase II SBIR studies that will employ GLP facilities and GMP materials, longer repeat dosing toxicity protocols in large animals, and safety studies (cardiac, respiratory, etc.), that will more precisely predict NtBuHA behavior in humans and that will support a subsequent IND application. Ultimately, we hope NtBuHA can become a marketed product that has clinically meaningful impact on INCL patients.