L-Cystine Diamides as Inhibitors of L-Cystine Stone Formation in Cystinuria

胱氨酸尿症是一种遗传性的胱氨酸转运障碍，其特征是尿中L-胱氨酸排泄过多，肾脏和膀胱(程度较轻)反复出现胱氨酸结石。在过去的三十年里，目前对胱氨酸尿症的治疗没有改变。建议患者饮用大量的水(每天4升)，以降低尿中游离L-胱氨酸的浓度，并对尿液进行碱化以增加胱氨酸的溶解度，这一建议对患者的坚持提出了挑战。在恶劣条件下的药物治疗仅限于两种与L-胱氨酸反应形成更易溶的混合二硫化物的硫醇药物，但由于不良反应，在某些情况下可能危及生命，这些药物患者的依从性较差。因此，有必要减少或消除与治疗胱氨酸尿症相关的风险。在这一应用中，我们提出了一种新的方法来预防胱氨酸尿症患者复发的胱氨酸结石形成。我们大量的体外研究表明，L-胱氨酸二酰胺是胱氨酸结晶的有效抑制剂，而胱氨酸结晶是结石形成的必要条件。我们的初步体内研究表明，该家族的一种化合物(LH708)可以有效地防止SLC3a1基因敲除小鼠中L-胱氨酸结石的形成，这是我们在实验室建立的胱氨酸尿症的模型。基于我们的体内外数据，我们将设计、合成和评价具有足够代谢稳定性和口服生物利用度的L-胱氨酸二酰胺的新的类似物和前药，从而有效地抑制L-胱氨酸在体内的结晶，从而防止L-胱氨酸结石的形成。这些新化合物抑制L-胱氨酸结晶的有效性将在体外结合使用进行测试 基于荧光的溶解度和实时原位晶体生长抑制分析。然后，将评估最有效的抑制剂在SLC3a1基因敲除小鼠中的体内有效性、安全性和生物利用度，以及它们的临床前ADME-Tox特性。这些研究的综合结果将使我们能够选择一种顶级候选药物和一种备用候选药物进行IND指导的临床前评估，为此我们将寻求NIH NCATS计划的帮助。

Cystinuria, a genetic disorder of cystine transport, is characterized by excessive excretion of L-cystine in the urine and recurrent cystine stones in the kidneys and, to a lesser extent, in the bladder. Current treatment of cystinuria has not changed over the last three decades. Patients are advised to drink substantial amounts of water (>4 liters/day) to reduce the concentration of free L-cystine in urine and to alkalinize the urine to increase cystine solubility, advice that challenges patient adherence. Drug therapy in severe conditions is limited to two thiol drugs that react with L-cystine to form more soluble mixed disulfides, but these drugs have poor patient compliance due to adverse reactions that, in some cases, may be life-threatening. Thus, there is a need to reduce or eliminate the risks associated with therapy for cystinuria. In this application, we propose a new approach to prevent recurrent cystine stone formation in patients with cystinuria. Our substantive in vitro studies indicate that L-cystine diamides are potent inhibitors of cystine crystallization, which is a required condition for stone formation. Our preliminary in vivo studies indicate that one compound (LH708) from this family is effective in preventing L-cystine stone formation in Slc3a1 knockout mice, which we generated in our laboratories as a model for cystinuria. Building on our in vitro and in vivo data, we will design, synthesize, and evaluate new analogs and prodrugs of L-cystine diamides with sufficient metabolic stability and oral bioavailability that can effectively inhibit L-cystine crystallization in vivo and thus prevent L-cystine stone formation. The effectiveness of these new compounds to inhibit L-cystine crystallization will be tested using a combination of in vitro fluorescence-based solubility and real-time in situ crystal growth inhibition assays. The most effective inhibitors then will be evaluated for their in vivo efficacy, safety, and bioavailability in Slc3a1 knockout mice and for their preclinical ADME-Tox properties. The combined results from these studies will enable the selection of one top drug candidate and one backup candidate for IND-directed preclinical evaluations, for which we will seek assistance from the NIH NCATS program.