Reliable Assays for Pentosan Polysulfate Sodium

镰状细胞病(SCD)仍然是一种治疗不善的疾病。基于内皮细胞P-选择素是 作为SCD中异常微血管血流的核心，我们的治疗针对的是这种分子。我们的 体外和初步临床数据表明，多聚戊聚糖硫酸钠(PPS)可改善微血管。 SCD的血流情况。然而，商业化的PPS并不是一种可行的治疗方法，因为它的口腔边缘 生物利用度和有限的作用时间。我们计划开发改进的第二代PPS-2 要克服这些限制，目前由于缺乏有用的PPS分析而被排除在外。创建可靠的 化验将使PPS-2的开发和商业化成为可能。SCD是一种遗传性疾病，困扰着 全球有数百万患者，美国约有10万名患者。大多数SCD的发病是由于血流异常， 值得注意的是，由于微血管血流停止而导致的急性疼痛危机。我们的首要目标是 将口服P-选择素阻滞剂PPS-2推向市场改善SCD患者的生活质量 长期服药防止镰刀状红细胞粘连血管内壁，改善 血液流动，并避免急性疼痛发作。在本应用程序中，我们建议创建可靠的检测方法 对血浆中PPS-2候选化合物进行定量，并测定其在血液中的活性。化验结果将是 对临床前和临床试验是必要的，对临床监测是有用的。标准的不足 检测涉及广泛的PPS分子异质性，缺乏特异性PPS抗体，以及血浆 干扰化验的物质。首先，我们将利用较低分子量的更大均一性 PPS组分作为物理定量分析方法的目标。我们将采用亲水相互作用 层析(HILIC)-MS分析我们的PPS组分。我们将在血浆中测试这种检测的可靠性。 来自实验动物的样本给予PPS组分。接下来，我们将利用精致的专一性 由人类组合PPS单抗提供，克服了干扰血浆物质的挑战 用于杂交瘤产生的单抗和多克隆抗体。我们将从Abd Serotec那里获得 用噬菌体展示选择产生的重组PPS单抗对用于夹心ELISA。我们将测试 人血浆中高、低分子量PPS-2候选蛋白的定量测定 实验动物对PPS测试项目进行测试。最后，拉霍亚过敏研究所的克劳斯·莱伊 免疫学将设计一种微流控设备，用于P-选择素阻断的护理点功能评估 一滴血中的活动。测定生物活性而不是物理浓度克服了 挑战PPS的生物变异，并允许监测患者的功能活动。该设备将 通过评估添加了PPS或PPS组分的全血中的P-选择素阻断活性和 每种剂量的小鼠的血液样本。这一阶段的SBIR将提供可靠的定量和 PPS-2开发所必需的PPS和PPS组分的功能分析。

Sickle cell disease (SCD) remains a poorly treated disease. Based on evidence that endothelial P-selectin is central to the abnormal microvascular blood flow in SCD, we are targeting this molecule with our therapy. Our in vitro and preliminary clinical data show that pentosan polysulfate sodium (PPS) improves microvascular blood flow in SCD. However, commercially available PPS is not a viable therapy because of its marginal oral bioavailability and limited duration of action. Our plan to develop an improved second generation PPS-2 that will overcome these limitations is precluded at this time by the lack of useful PPS assays. Creation of reliable assays will enable development and commercialization of PPS-2. SCD is an inherited disorder that afflicts millions of patients worldwide, ~100,000 in the US. Most SCD morbidity is due to abnormal blood flow, conspicuously the acute pain crises that are caused by stoppage of microvascular flow. Our overarching goal is to improve the quality of life of patients with SCD by bringing to market PPS-2 as an oral P-selectin blocking drug for long-term administration to prevent sickle red blood cell sticking to the lining of blood vessels, improve blood flow, and avert acute painful episodes. In this application we propose to create reliable assays for quantifying PPS-2 candidate compounds in plasma and determining its activity in blood. The assays will be necessary for preclinical and clinical testing and useful for clinical monitoring. The inadequacy of standard assays relates to extensive molecular heterogeneity of PPS, lack of specific PPS antibodies, and plasma substances that interfere with assays. First, we will exploit the greater homogeneity of a lower molecular weight fraction of PPS as a target for a physical quantification assay method. We will adapt hydrophilic interaction chromatography (HILIC)-MS for assaying our PPS fraction. We will test the reliability of this assay in plasma samples from experimental animals administered PPS fraction. Next we will utilize the exquisite specificity provided by human combinatorial PPS MAb, which overcomes challenges that interfering plasma substances posed for hybridoma-generated MAb and polyclonal antibodies. We will obtain from AbD Serotec a recombinant PPS MAb pair generated using phage display selection for use in a sandwich ELISA. We will test the assay for quantification of high and lower molecular weight PPS-2 candidates in plasma samples from experimental animals administered the PPS test items. Lastly, Klaus Ley of the La Jolla Institute of Allergy and Immunology will devise a microfluidics device for point-of-care functional assessment of P-selectin blocking activity in a drop of blood. Determining biological activity rather than physical concentration overcomes challenges with biological variation of PPS and allows monitoring functional activity in patients. The device will be validated by assessing P-selectin blocking activity in whole blood spiked with PPS or PPS fraction and in blood samples from mice dosed with each. This Phase I SBIR will provide the reliable quantitative and functional assays for PPS and PPS fraction necessary for PPS-2 development.