Structural identification of a compound preventing calcium oxalate stone disease

描述（由申请人提供）：本项目将在动物研究中分离和鉴定一种有效治疗和预防草酸钙肾结石疾病的植物化合物的化学和结构组成。据估计，美国约有2000万结石患者。在人类受试者中，超过70%的肾结石主要由草酸钙组成。目前对这部分结石患者（> 70%）没有有效的治疗，因此该化合物具有对治疗和预防这种广泛和痛苦的疾病产生实质性影响的令人兴奋的可能性。长期目标是将活性化合物制成可口服的药丸形式，以防止进一步的结石形成。该化合物还具有显著的潜力用于在碎石术后去除结石碎片和晶体沉积物，从而减少/预防结石复发。我们的初步研究已经清楚地表明，口服给予植物提取物在预防高尿酸大鼠模型中的结石形成和晶体沉积方面是有效的。此外，该提取物降低了结石病犬的尿钙和草酸盐，将草酸钙的相对过饱和度降低至饱和度以下。在接受浸提液处理的任何动物中均未观察到不良副作用。这些发现是一个重大发现。 本申请的主要目的是进一步纯化活性化合物并阐明其结构。一个高活性的植物提取物，在体外抑制草酸钙晶体生长的有效部分，已被确定，并通过一个既定的程序，使96%的纯度。将从该馏分中纯化活性化合物并阐明其结构。将使用各种色谱技术以及适用于该化合物的提取和沉淀程序进一步纯化该级分。通过聚丙烯酰胺凝胶电泳、高效液相色谱法和核磁共振（NMR）确认活性化合物的纯度。 纯化的活性化合物将进行化学和结构程序以鉴定，1）通过气相色谱-质谱法（GC-MS）鉴定其化学组成; 2）通过甲基化分析-GC-MS鉴定化合物的单个单体的键合位置; 3）使用基质辅助激光解吸/电离飞行时间质谱仪的分子量测定（MALDI-TOF MS）和液相色谱电喷雾离子化质谱（LC-ESI MS）;和4）通过许多标准NMR技术确定的化合物的异头构型的类型（，）、其它取代基和序列。我们预计，这种新型化合物将证明在治疗人类受试者中是有效的，这将代表结石病的临床治疗和管理的重大进展。

DESCRIPTION (provided by applicant): This project will isolate and identify the chemical and structural composition of a plant compound effective in treating and preventing calcium oxalate kidney stone disease in animal studies. It is estimated that there are about 20 million stone patients in the United States. More than 70% of kidney stones in human subjects are composed mainly of calcium oxalate. There is at present no effective treatment for this segment of stone patients (> 70%) and consequently this compound holds the exciting possibility of making a substantial impact on the treatment and prevention of this widespread and painful disease. The long-term objective is to have the active compound in a pill form that can be taken orally to prevent further stone formation. The compound also has significant potential for use in removing stone fragments and crystal deposits following lithotripsy thereby reducing/preventing stone recurrence. Our initial studies have clearly demonstrated that oral administration of the plant extract was effective in preventing stone formation and crystal deposition in a hyperoxaluric rat model. Furthermore, the extract lowered urinary calcium and oxalate in dogs with stone disease, reducing the relative supersaturation of calcium oxalate to under saturation. No adverse side effects were observed in any of the animals treated with the extract. These findings represent a major discovery. The major aims of this application are to further purify the active compound and elucidate the structure. A highly active fraction of the plant extract, potent in inhibiting crystal growth of calcium oxalate in vitro, has been identified and brought to 96% purity by an established procedure. The active compound will be purified from this fraction and its structure elucidated. This fraction will be further purified using a variety of chromatography techniques as well as extraction and precipitation procedures appropriate to this compound. Purity of the active compound will be confirmed by polyacrylamide gel electrophoresis, high performance liquid chromatography and by nuclear magnetic resonance (NMR). The purified active compound will be submitted to chemical and structural procedures to identify, 1) it's chemical composition by gas chromatography-mass spectrometry (GC-MS); 2) linkage positions of the individual monomers of the compound by methylation analysis-GC MS; 3) molecular weight determination using matrix assisted laser desorption/ionization time-of-flight mass spec (MALDI-TOF MS) and liquid chromatography electrospray ionization mass spec (LC-ESI MS); and 4) the type of anomeric configuration (,), other substituents and sequence of the compound, determined by a number of standard NMR techniques. We anticipate that this novel compound will prove effective in the treatment of human subjects which would represent a major advance in the clinical treatment and management of stone disease.