Basic and Translational Science Novel Cystine Ester Mimics for the Treatment of Cystinuria-induced Urolithiasis in a Knockout Mouse Model Amrik Sahota, Jaspreet S. Parihar, Kathleen M. Capaccione, Min Yang, Kelsey Noll, Derek Gordon, David Reimer, Ill Yang, Brian T. Buckley, Marianne Polunas, Kenneth R. Reuhl, Matthew R. Lewis, Michael D. Ward, David S. Goldfarb, and Jay A. Tischfield OBJECTIVE To assess the effectiveness of L-cystine dimethyl ester (CDME), an inhibitor of cystine crystal growth, for the treatment of cystine urolithiasis in an Slc3a1 knockout mouse model of cystinuria. MATERIALS AND METHODS CDME (200 mg per mouse) or water was delivered by gavage daily for 4 weeks. Higher doses by gavage or in the water supply were administered to assess organ toxicity. Urinary amino acids and cystine stones were analyzed to assess drug efficacy using several analytical methods. RESULTS Treatment with CDME led to a significant decrease in stone size compared with that of the water group (P ¼ .0002), but the number of stones was greater (P ¼ .005). The change in stone size distribution between the 2 groups was evident by micro computed tomography. Overall, cystine excretion in urine was the same between the 2 groups (P ¼ .23), indicating that CDME did not interfere with cystine metabolism. Scanning electron microscopy analysis of cystine stones from the CDME group demonstrated a change in crystal habit, with numerous small crystals. L-cysteine methyl ester was detected by ultra-performance liquid chromatographyemass spectrometer in stones from the CDME group only, indicating that a CDME metabolite was incorporated into the crystal structure. No pathologic changes were observed at the doses tested. CONCLUSION These data demonstrate that CDME promotes formation of small stones but does not prevent stone formation, consistent with the hypothesis that CDME inhibits cystine crystal growth. Combined with the lack of observed adverse effects, our findings support the use of CDME as a viable treatment for cystine urolithiasis. UROLOGY 84: 1249.e9e1249.e15, 2014. Ó 2014 Elsevier Inc. C ystinuria is a rare cause of kidney stones, ac- counting for only 1% of urolithiasis cases in adults, but it accounts for 6%-8% of pediatric cases. 1-4 Stone formation secondary to cystinuria often presents in the first decade of life, and the majority of patients have their first stone by the end of their teenage years. 4,5 Although rare, cystine stones can lead to serious consequences for patients, because they are large and tend to recur, often resulting in multiple treatments and pro- gressive decline in renal function in pediatric and adult patients. 3-5 Patients with cystine stones have a greater incidence of chronic kidney disease than patients suffering from the more common calcium oxalate stones. 6 Despite the morbidity associated with cystine urolithiasis, treatments for cystinuria have not substantially changed in the past 30 years. 7,8 At the molecular level, cystinuria is characterized by defective transport of cystine and dibasic amino acids in the kidney and small intestine. 7-9 Cystinuria is an auto- somal recessive disorder caused by mutations in either SLC3A1 or SLC7A9. These genes encode the rBAT and b 0,þ AT subunits, respectively, of the heterodimeric cystine transporter. 10,11 The inability to reabsorb cystine and dibasic amino acids from the glomerular filtrate results in the hyperexcretion of these amino acids in the urine (cystine excretion >400 mg/day compared with <30 mg/ day in normal subjects). This can lead to the formation of cystine stones in the kidney and, to a lesser extent, in the Financial Disclosure: The authors declare that they have no relevant financial interests. New York University is the assignee of patent number US 8,450,089 B2 entitled “Compounds as L-cystine crystallization inhibitors and uses thereof”. Funding Support: This work was supported in part by a pilot project grant (no. 434056) from the Rare Kidney Stone Consortium (U54KD083908), which is a part of the NIH Rare Diseases Clinical Research Network, supported through collaboration between the NIH Office of Rare Diseases Research at the National Center for Advancing Translational Sciences and National Institute of Diabetes and Digestive and Kidney Disease. This work was also supported in part by National Institute of Environmental Health Sciences Centre grant ES05022. From the Department of Genetics, Rutgers University, Piscataway, NJ; Division of Urology, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ; Rutgers Cancer Institute of New Jersey, New Brunswick, NJ; Laboratory Animal Services, Rutgers University, Piscataway, NJ; Environmental and Occupational Health Sciences Institute, Rutgers University, Piscataway, NJ; Department of Surgery and Cancer, Imperial College London, London, United Kingdom; Department of Chemistry, New York University, New York, NY; and Nephrology Division, New York University Langone Medical Center, New York, NY Address correspondence to: Amrik Sahota, Ph.D., Department of Genetics, Life Sciences Building, Rutgers University, 145 Bevier Road, Piscataway, NJ 08854. E-mail: sahota@biology.rutgers.edu Submitted: December 15, 2013, accepted (with revisions): July 24, 2014 ª 2014 Elsevier Inc. All Rights Reserved http://dx.doi.org/10.1016/j.urology.2014.07.043 0090-4295/14 1249.e9