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theses

Cystinuria is a lifelong and recurrent stone disease characterized by renal L-cystine calculi due to defective renal and intestinal reabsorption of L-cystine and dibasic amino acids. Cystinuria is caused by defects in one or both SLC3A1 and SLC7A9 genes located on chromosomes 2p and 19q, respectively. L-Cystine crystallizes in a zwitterionic form with stable crystals and low solubility due to the hydrogen-bond networks within the solid. The presence of four terminal groups, two amino and two carboxylic groups, with the ability to form hydrogen-bond networks makes L-cystine more unique than other amino acids or standard dipeptides. The medical management of the disease focuses mainly on lowering the concentration of L-cystine in urine or increasing L-cystine solubility in urine. Hyperdiuresis to decrease the concentration of L-cystine in urine and alkalinization are used to improve L-cystine solubility and if these techniques fail the next step in the treatment algorithm is introduction of thiol drugs. Drugs like D-penicillamine and tiopronin react with L-cystine to form more soluble mixed disulfides, but they are poorly tolerated due to their numerous adverse side effects. A new alternative approach to the prevention of L-cystine kidney stones through molecular mimicry was suggested recently by Ward and colleagues. L-CDME was able to reduce the mass yield of crystallization and maintain a metastable supersaturated L-cystine concentration which is sufficient for preventing stone formation. However, the in vivo stability of L-CDME is a real concern for it to be used as a therapeutic since esters are liable to esterase enzyme cleavage and its metabolite is L-cystine which would worsen the disease. Our group converted the ester to the amide which is more stable in vivo that lead to better inhibition with enhanced stability profile. The diamide analogues were more active and more stable according to our crystallization inhibition assay, real-time in situ atomic force microscopy (AFM) and in vitro chemical stability study. Research in this dissertation focused on the structure-activity relationship (SAR) of the most active analogues. Our results showed the importance of keeping the core structure of L-cystine intact in order to maintain the strong binding affinity to the L-cystine crystal and inhibit the crystallization of L-cystine. Our research led to compound 8-L-cystinyl bis(1,8-diazaspiro[4.5]decane) (56) with an EC50 of 25.1 nM which is 1.7 fold more potent than LH708 (9), L-cystine bis(N′-methylpiperazide), in our crystallization inhibition assay. The bioavailability of LH708 (9) in SLC3A1 knockout mice was found to be 18%. To improve the oral bioavailability of LH708, several prodrugs with better lipophilicity were designed, synthesized, and evaluated for their stability and activation.

ETD_9414

Ph.D.

Includes bibliographical references

by Haifa Albanyan

doi:10.7282/t3-mmez-hv87

ETD doctoral

The author owns the copyright to this work.