Structural identification of a compound preventing calcium oxalate stone disease

描述（由申请人提供）：本项目将在动物实验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。人类70%以上的肾结石主要由草酸钙组成。目前对这部分结石患者（> 70%）没有有效的治疗方法，因此这种化合物具有令人兴奋的可能性，可以对这种广泛而痛苦的疾病的治疗和预防产生重大影响。长期目标是将活性化合物制成药丸形式，可口服以防止结石进一步形成。该化合物还具有重要的潜力，用于去除碎石后的石头碎片和晶体沉积物，从而减少/防止石头复发。我们的初步研究已经清楚地表明，口服植物提取物对高血氧大鼠模型的结石形成和晶体沉积有效。此外，该提取物降低了患有结石疾病的狗的尿钙和草酸盐，将草酸钙的相对过饱和降低到不饱和。在用提取物治疗的任何动物中均未观察到不良副作用。这些发现是一项重大发现。

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.