Reliable Assays for Pentosan Polysulfate Sodium

戊聚糖多硫酸钠的可靠测定

• 批准号: 8648586
• 负责人: Stephen H. Embury
• 金额: $ 22.64万
• 依托单位: VANGUARD THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648586
• 关键词: Acute; Acute Pain; Adhesions; Affect; Animals; Antibodies; Biological; Biological Assay; Biological Availability; Blood; Blood Vessels; Blood flow; Blood specimen; Boston; Chromatography; Clinic; Clinical; Clinical Data; Clinical Research; Clinical Trials; Consultations; Detection; Development; Devices; Disease; Dose; Drops; Drug Formulations; Drug Kinetics; Drug Monitoring; Enzyme-Linked Immunosorbent Assay; Event; Failure; Generations; Goals; Hemoglobin; Heterogeneity; Human; Hybridomas; Hypersensitivity; Immunoassay; Immunology; In Vitro; Inborn Genetic Diseases; Individual; Inherited; Inorganic Sulfates; Institutes; Legal patent; Leukocytes; Marketing; Mass Spectrum Analysis; Measurement; Measures; Methods; Microfluidic Microchips; Molecular; Molecular Weight; Monitor; Monkeys; Monosaccharides; Morbidity - disease rate; Mus; Oligosaccharides; Oral; P-Selectin; Pain; Patients; Pentosan Polysulfate; Phage Display; Pharmaceutical Preparations; Pharmacodynamics; Phase; Plasma; Polysaccharides; Preclinical Testing; Property; Prophylactic treatment; Publishing; Quality of life; Rattus; Recombinants; Sampling; Sickle Cell; Sickle Cell Anemia; Small Business Innovation Research Grant; Sodium; Specificity; Symptoms; Technology; Testing; Therapeutic; Time; United States; Universities; Unspecified or Sulfate Ion Sulfates; Variant; Whole Blood; base; combinatorial; commercialization; improved; neutrophil; pill; point of care; polyclonal antibody; prevent; public health relevance; research clinical testing; response; scale up; theories

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.

镰状细胞病（SCD）仍然是一种治疗效果不佳的疾病。基于内皮P-选择素是 SCD中异常微血管血流的中心，我们的治疗靶向这种分子。我们 体外和初步临床数据显示，戊聚糖多硫酸钠（PPS）可改善微血管 SCD中的血流。然而，市售PPS不是一种可行的治疗方法，因为其边缘口服给药。 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， 将克服这些限制是排除在这个时候缺乏有用的PPS测定。建立可靠的 这将使PPS-2的开发和商业化成为可能。SCD是一种遗传性疾病， 全球数百万患者，美国约10万。大多数SCD发病率是由于异常血流， 明显的是由微血管流动停止引起的急性疼痛危象。我们的首要目标是 通过将PPS-2作为口服P-选择素阻断剂推向市场，改善SCD患者的生活质量 长期给药的药物，以防止镰状红细胞粘附在血管内壁，改善 血液流动，避免急性疼痛发作。在本申请中，我们提出建立可靠的测定， 定量血浆中PPS-2候选化合物并测定其在血液中的活性。试验将 这是临床前和临床测试所必需的，并且对临床监测有用。标准的不足 分析涉及PPS的广泛分子异质性，缺乏特异性PPS抗体，以及血浆 干扰测定的物质。首先，我们将利用较低分子量的更大均匀性， PPS部分作为物理定量测定方法的目标。我们将调整亲水相互作用 色谱（HILIC）-MS用于测定我们的PPS级分。我们将在血浆中测试该测定的可靠性 来自给予PPS组分的实验动物的样品。接下来我们将利用 由人组合PPS MAb提供，其克服了干扰血浆物质 杂交瘤产生的单克隆抗体和多克隆抗体。我们将从AbD Serotec获得 使用噬菌体展示选择产生的重组PPS MAb对用于夹心ELISA。我们将测试 用于定量血浆样品中高分子量和低分子量PPS-2候选物的测定法 实验动物给予PPS测试项目。最后，拉霍亚过敏症研究所的克劳斯·莱伊和 免疫学将设计一种微流体装置，用于P-选择素阻断的即时功能评估 一滴血中的活性确定生物活性而不是物理浓度克服了 挑战PPS的生物学变异，并允许监测患者的功能活动。该设备将 通过评估加标PPS或PPS组分的全血中的P-选择素阻断活性以及 来自给予每种药物的小鼠的血液样品。第一阶段SBIR将提供可靠的定量和 PPS-2开发所需PPS和PPS组分的功能测定。