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Item does not contain fulltext OBJECTIVES: To assess whether copper deficiency plays a role in the recently described cysteamine toxicity in patients with cystinosis, and to examine whether polymorphisms in copper transporters, lysyl oxidase, and/or type I procollagen genes could be responsible for the occurrence of cysteamine toxicity in a small subset of patients with cystinosis. STUDY DESIGN: Thirty-six patients with cystinosis were included: 22 with Fanconi syndrome (including 7 with cysteamine toxicity), 12 after renal transplantation, 1 receiving hemodialysis, and 1 with ocular cystinosis. Serum copper and ceruloplasmin levels and urinary copper/creatinine ratio were measured. Genes ATP7A and CTR1 (encoding copper transporters), LOX (encoding lysyl oxidase), and COL1A1 and COL1A2 (encoding type I procollagen) were analyzed in patients with (n = 6) and without (n = 5) toxicity. Fibroblast (pro)collagen synthesis was compared in patients with (n = 3) and those without (n = 2) cysteamine toxicity. RESULTS: All 22 patients with Fanconi syndrome had increased urinary copper excretion. Serum copper and ceruloplasmin levels were decreased in 9 patients, including all 7 patients with cysteamine toxicity. No specific sequence variations were associated with toxicity. All fibroblasts exhibited normal (pro)collagen synthesis. CONCLUSION: Patients with cystinosis with cysteamine toxicity demonstrate copper deficiency. This can cause decreased activity of lysyl oxidase, the enzyme that generates the aldehydes required for collagen cross-linking. Thus, copper supplementation might prevent cysteamine toxicity.

Item does not contain fulltext Nephropathic cystinosis is an autosomal recessive metabolic, lifelong disease characterized by lysosomal cystine accumulation throughout the body that commonly presents in infancy with a renal Fanconi syndrome and, if untreated, leads to end-stage kidney disease (ESKD) in the later childhood years. The molecular basis is due to mutations in CTNS, the gene encoding for the lysosomal cystine-proton cotransporter, cystinosin. During adolescence and adulthood, extrarenal manifestations of cystinosis develop and require multidisciplinary care. Despite substantial improvement in prognosis due to cystine-depleting therapy with cysteamine, no cure of the disease is currently available. Kidney Disease: Improving Global Outcomes (KDIGO) convened a Controversies Conference on cystinosis to review the state-of-the-art knowledge and to address areas of controversies in pathophysiology, diagnostics, monitoring, and treatment in different age groups. More importantly, promising areas of investigation that may lead to optimal outcomes for patients afflicted with this lifelong, systemic disease were discussed with a research agenda proposed for the future.

Objective To report new adverse effects of cysteamine.Study design Detailed clinical information was obtained from the patients' physicians.Results New adverse events were reported in 8 of 550 patients with cystinosis treated with cysteamine in Europe during the last 5 years. Detailed clinical information was not available for 2 of these patients, 1 of whom died from cerebral ischemia. The 6 evaluable patients developed vascular elbow lesions (6/6), neurologic symptoms (1/6), bone and muscle pain (2/6), and/or skin striae (2/6). Analysis of biopsy specimens from the elbow lesions demonstrated angioendotheliomatosis with irregular collagen fibers. In 3 of the 6 patients, the daily cysteamine dose exceeded the recommended maximum of 1.95 g/m(2)/day. Dose reduction led to improvement of signs and symptoms in all 6 patients, suggesting a causal relationship with cysteamine administration.Conclusion Cysteamine administration can be complicated by the development of skin, vascular, neurologic, muscular, and bone lesions. These lesions improve after cysteamine dose reduction. Doses >1.95 g/m(2)/day should be prescribed with great caution, but underdosing is not advocated. (J Pediatr 2011;159:1004-11).

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Cysteamine bitartrate is taken lifelong, every 6 h and for the treatment of cystinosis. Recent studies using cysteamine for for other diseases such as neurodegenerative disorders adopt the same dosing regimen for cysteamine. Regular cysteamine bitartrate (Cystagon) may cause upper gastrointestinal symptoms in some patients. WHAT THIS STUDY ADDS • This is the only study that provides pharmacokinetic data for cysteamine delivered in an enteric-release preparation in normal subjects. EC-cysteamine is very well tolerated and does not cause increased gastrin concentrations, even at relatively high doses. • EC-cysteamine at the higher dose results in better drug uptake as measured by Cmax and AUC and is more likely to be effective. AIMS Cysteamine bitartrate (Cystagon®) is the approved treatment for cystinosis. Poor compliance and patient outcome may occur because the drug needs to be taken every 6 h and in some patients causes gastrointestinal symptoms due to hypergastrinaemia. A formulation of cysteamine requiring twice daily ingestion would improve the quality of life for these patients. This study compares the pharmacokinetics and gastrin production following cysteamine bitartrate non-enteric-coated and cysteamine bitartrate enteric-coated in normal healthy subjects. METHODS Enteric-coated cysteamine was prepared. Following single doses of cysteamine bitartrate non-enteric-coated 450 mg and cysteamine bitartrate enteric-coated 450 mg and 900 mg, serial plasma cysteamine and gastrin concentrations were measured. Two subjects also received cysteamine bitartrate non-enteric-coated 900 mg. Gastrointestinal (GI) symptoms were recorded. RESULTS Six healthy adults (mean age 20.7 years, range 18–24 years; mean weight 59.3 kg) received drug. All post-dose gastrin concentrations were within the normal range (

Objective Cystinosis causes renal and other organ failure. Treatment with 6-hourly cysteamine bitartrate (Cystagon, Mylan, Morgantown, West Virginia) reduces intracellular cystine and the rate of organ deterioration. A recent study showed that an enteric-release cysteamine required less frequent daily dosing. This report describes the long-term use of enteric-coated (EC) cysteamine bitartrate (Cystagon) in children with cystinosis. Study design After a pharmacokinetic and pharmacodynamic study of EC-cysteamine in children with cystinosis, 5 patients remained on twice-daily treatment. White blood cell cystine levels were measured 12 hours after ingestion every 4 to 8 weeks. These levels were then compared with the patient's previous 6-h post-dose levels taken while on regular cysteamine bitartrate before entering the study. Blood chemistry was also measured. Results Five children with cystinosis (mean age, 9 years; range, 8 to 17 years) who previously took cysteamine bitartrate (mean dose, 47 mg/kg body wt), received EC-cysteamine for 10 to 27 months (mean dose, 25 mg/kg body wt) and had mean white blood cell cystine levels of 0.77 and 0.71 nmol half-cystine/mg protein, respectively. During the study period, patients maintained adequate growth and there was no significant deterioration in renal or thyroid function. Two children were required to restart acid suppression after 6 months on EC-cysteamine therapy. Conclusions Long-term, twice-daily EC-cysteamine, given at approximately 60% of the previous daily dose of cysteamine bitartrate, was effective at maintaining white blood cell cystine levels within a satisfactory range. There was no significant deterioration in renal or thyroid function.

To test the hypothesis that a controlled-release preparation of cysteamine, with fewer daily administrations, would improve the quality of life for patients with cystinosis.A specifically designed nasoenteric tube was used to administer cysteamine directly into the stomach, small intestine (SI) and colon and serial plasma cysteamine, serum gastrin and leukocyte cystine levels were measured.Eight control subjects (mean age 23.2 years) and 6 subjects with cystinosis (mean age 15.2 years) were studied. Cysteamine absorption (maximum concentration and area under the curve of the concentration-time gradient) was greater from the SI than stomach or cecum (P.01). Leukocyte cystine depletion was greater after delivery of cysteamine into the SI than stomach or cecum; this effect was associated with the plasma cysteamine maximum concentration and area under the curve (P.001 and.02, respectively). Gastrin levels were not affected by site of drug delivery and were elevated only in patients with cystinosis with gastrointestinal symptoms.The absorption of cysteamine and the effect of this agent on leukocyte cystine depletion are more profound after SI administration. Enteric-coated cysteamine, targeted for SI release, may require fewer daily dosages. Not all patients with cystinosis require acid-suppression therapy.

Aims Cysteamine is used to reduce tissue cystine content in patients suffering from nephropathic cystinosis. The objectives of the current study were to investigate pharmacokinetics and pharmacodynamics of cysteamine bitartrate in children and young adults with nephropathic cystinosis. Methods Cysteamine bitartrate was administered to 11 cystinosis patients at their regular dose level in a single-dose, open-label, steady-state study. Blood samples were collected and analysed for plasma cysteamine and white blood cell cystine content and pharmacokinetic and pharmacodynamic parameters estimated by NONMEM analysis using a linked pharmacokinetic–pharmacodynamic model. Results Cysteamine was rapidly cleared from the plasma (mean CL/F = 32.3 ml min−1 kg−1, range = 17.3–52.2), appeared to be extensively distributed (mean Vss/F = 15.1 l, range 2.7–32.3) and exhibited a mean Tmax of 1.4 h. White blood cell cystine content post-dosing was significantly decreased compared with pre- and post-dose values (average decrement approximately 47%). A counter-clockwise hysteresis was noted in all patients, suggestive of a lag time (mean Tlag = 0.44 h, range 0.22–0.92) between drug concentration and effect. Conclusions The results of this study establish that cysteamine is rapidly cleared from the plasma but that an every 6 h dosing interval adequately maintains white blood cell cystine content below the target of 1 nmol cystine per mg protein.

In this technical paper, the authors apply a cumulative genetic algorithm and a neural network to the transportation network design problem. Constraints of the problem must be addressed during the design process to determine the best feasible solution. After introducing the algorithm, it is demonstrated in examples of restrictions encountered in practice. Then, an analysis is made of alternative constraint processing methods and the optimization process is modified within the cumulative genetic algorithm. Two groups of constraints are defined for a test network to verify the applicability of the method. Results indicate that the search process yields the best feasible solutions, confirming the use of the cumulative genetic algorithm and neural network in processing constraints.

Fourteen families of children with infantile nephropathic cystinosis were evaluated using the Stanford-Binet Intelligence Scale, Fourth Edition [Thorndike et al., 1986: Stanford-Binet Intelligence Scale, Fourth Ed.]. The IQs of 15 children with cystinosis, their 23 sibs and 24 parents were compared in order to evaluate a potential effect of cystinosis on intelligence. Children with cystinosis had a significantly lower mean IQ than their sibs and their parents (P = .001). Thus, even though the mean IQ of the children with cystinosis (94.4 +/- 10) was within the average range, there is evidence that these children have a mild global intellectual deficit relative to their expected IQ based upon the IQs of other relatives. In addition, to a subset of the subjects we administered a measure of scholastic ability, the Wide Range Achievement Test-Revised [Jastak and Wilkinson, 1984: The Wide Range Achievement Test-Revised], which consists of spelling, reading, and arithmetic subtests. The 11 cystinosis subjects scored significantly lower (P = .01) than their 16 sibs and their 14 parents in the area of spelling, whereas they did not significantly differ in their performance in the areas of reading and arithmetic.

Cysteamine bitartrate is taken lifelong, every 6 h and for the treatment of cystinosis. Recent studies using cysteamine for for other diseases such as neurodegenerative disorders adopt the same dosing regimen for cysteamine. Regular cysteamine bitartrate (Cystagon) may cause upper gastrointestinal symptoms in some patients.This is the only study that provides pharmacokinetic data for cysteamine delivered in an enteric-release preparation in normal subjects. EC-cysteamine is very well tolerated and does not cause increased gastrin concentrations, even at relatively high doses. EC-cysteamine at the higher dose results in better drug uptake as measured by Cmax and AUC and is more likely to be effective.Cysteamine bitartrate (Cystagon) is the approved treatment for cystinosis. Poor compliance and patient outcome may occur because the drug needs to be taken every 6 h and in some patients causes gastrointestinal symptoms due to hypergastrinaemia. A formulation of cysteamine requiring twice daily ingestion would improve the quality of life for these patients. This study compares the pharmacokinetics and gastrin production following cysteamine bitartrate non-enteric-coated and cysteamine bitartrate enteric-coated in normal healthy subjects.Enteric-coated cysteamine was prepared. Following single doses of cysteamine bitartrate non-enteric-coated 450 mg and cysteamine bitartrate enteric-coated 450 mg and 900 mg, serial plasma cysteamine and gastrin concentrations were measured. Two subjects also received cysteamine bitartrate non-enteric-coated 900 mg. Gastrointestinal (GI) symptoms were recorded.Six healthy adults (mean age 20.7 years, range 18-24 years; mean weight 59.3 kg) received drug. All post-dose gastrin concentrations were within the normal range (100 pg ml(-1)). The tmax following cysteamine bitartrate non-enteric-coated (mean and SD is 75+/-19 min) was shorter than cysteamine bitartrate enteric-coated (220+/-74 min) (P=0.001), but only the Cmax and AUC estimates following 900 mg cysteamine bitartrate enteric-coated were significantly greater than any of the other preparations or doses (P0.05). One patient had GI symptoms following both 900 mg cysteamine bitartrate non-enteric-coated and cysteamine bitartrate enteric-coated.Although patient numbers were low, single high doses of cysteamine bitartrate enteric-coated were better tolerated than similar doses of cysteamine bitartrate non-enteric-coated in the healthy subjects and all had normal gastrin concentrations. The delayed tmax following cysteamine bitartrate enteric-coated suggested that the cysteamine was released enterically.

In many urban-planning, geographic analysis, and engineering studies, a classical problem is finding the shortest paths within a bounded area, or the best path that goes around or through special regions, called barriers. In this paper we present two algorithms for finding the shortest or best paths. The first one is designed to find the shortest Euclidean path in an area represented as a polygon. In this algorithm, we first select an arbitrary path between the two points, then modify this path until it is the shortest. Its computational complexity is proven to be superior to that of previous ones. The other algorithm is for finding the best path between two locations, using a rectilinear metric in the presence of penetrable barriers. To minimize both total travel time and cost, the route can either go around or through each barrier. When selecting the best path, multiple-weighted criteria are used in the evaluation. Examples are presented to illustrate how these two algorithms work.

Three patients with nephropathic cystinosis and chronic renal disease, treated since early childhood with orally administered cysteamine, had an accelerated rate of rise of serum creatinine values after receiving recombinant human growth hormone. During 18 to 24 months of growth hormone treatment, each patient had a twofold to fourfold increase in growth velocity. The slope of the plot of reciprocal serum creatinine values versus age for each patient after growth hormone treatment was significantly steeper than the pretreatment slope. Growth hormone treatment had no effect on the rate of change of the uncorrected 24-hour renal creatinine clearance. We conclude that these patients gained body size but failed to compensate for their increased creatinine production with an increase in glomerular filtration rate. The result was an accelerated rate of rise of their serum creatinine values, hastening renal transplantation in one patient and the anticipated need for transplantation in another.

Sirs, We are concerned that the manner in which leukocytes are prepared for the determination of cystine levels may cause imprecise results which could adversely affect the treatment of patients with cystinosis. Leukocyte cystine levels are used to diagnose cystinosis, monitor Cystagon therapy, and for multiple purposes in research studies. The determination of cystine in leukocytes is performed only in a small number of laboratories worldwide, one of which is the Cystine Determination Laboratory at the University of California San Diego (UCSD). In the late 1970s, when the first cysteamine treatment trial was started, we evaluated ways to have blood shipped to the laboratory for the separation of leukocytes and the determination of intracellular cystine. It was determined that the leukocyte cystine level could only reliably be measured from samples that had been separated immediately after the blood was drawn. To ensure uniform preparation, our practice has been to ship reagents to point-of-care clinical laboratories, with precise instructions for sample preparation (http://biochemgen.ucsd.edu/cystinosis/), as in the published method [1]. Separated and lysed leukocyte samples are acidified, frozen and transported to our laboratory on dry ice for protein and cystine determinations. Other laboratories accept heparinized whole blood shipped on wet ice for the determination of leukocyte cystine levels. The leukocytes are separated from the sample upon arrival at the laboratory, typically 24–48 h after the blood was drawn. This frees the point-of-care laboratory from the task of preparing the leukocytes, a procedure which is not complicated but which is time consuming and may be seen as an inconvenience. Our laboratory therefore would naturally be interested in changing the procedure to accept whole blood. However, based on our experiences in the 1970s, we felt it was important to evaluate the stability of intracellular cystine in blood samples during storage and shipping. Until recently, the demanding cystine binding assay method used for the cystine assay made such an evaluation impractical, but a newer method using tandem mass spectrometry [3] has reduced sample analysis time drastically. The aim of our small study was to evaluate whether intracellular cystine levels would be reliable if leukocyte separation from heparinized whole blood were delayed for up to 24–48 h, as would be expected with express mail shipment. Three cystinotic patients were enrolled into the study. Patients on Cystagon therapy were asked to discontinue therapy 24 h before participating. Blood was drawn from each subject, with aliquots anticoagulated in Li-heparin or Na-heparin. Blood was divided into 3-mL samples, and leukocytes were prepared in triplicate by the ACD-dextran method immediately after collection (0 h, baseline) or after being stored for 24 ± 1 or 48 ± 1 h at either room temperature or 4°C. All reagents for the separation were prepared by the UCSD Cystine Determination Laboratory. Protein content was determined using the Lowry method [3], and cystine content was measured using tandem mass spectrometry [2, 4]. Leukocyte cystine content was calculated as nanomoles half-cystine per milligram protein and is presented in Table 1 as the percentage of baseline value. Samples stored for 48 h at room temperature and collected in Li- and Na-heparin, respectively, did not separate when mixed with ACD-dextran, and leukocytes could not be isolated from these samples. Leukocytes could be prepared from all other samples. Table 1 Leukocyte cystine levelsa after the storage of whole blood for 24 or 48 h in Li- and Na-heparin (results averaged together) at room temperature or 4°C before separation of leukocytes No differences were observed between Li- and Na-heparin, and the results for both anticoagulants were therefore combined. The levels determined at baseline and after storage for 24 and 48 h at 4°C and for 24 h at room temperature are shown in Table 1. Cystine levels in samples stored for 24 h at room temperature varied between 52 and 117% of baseline values. Even greater variations were measured when the samples were stored at 4°C for 24 h (37–214%). After 48 h, only samples stored at 4°C could be separated, and the cystine levels determined were between 52 and 240% of baseline values. Although we tested only a small number of samples, we believe these results clearly show that results will be imprecise if leukocytes are not prepared immediately after heparinized blood is drawn. Accordingly, we encourage nephrologists treating cystinosis patients to take into account the time involved in storing and shipping samples prior to separating leukocytes for cystine determination. We are hoping to expand this study and evaluate different agents which might stabilize leukocytes and obviate the need for immediate cell preparation, but we believe it important to share these results immediately with all pediatric nephrologists treating cystinosis patients.

Objective Cystinosis causes renal and other organ failure. Regular 6-hourly cysteamine bitartrate (Cystagon; Mylan, Morgantown, West Virginia) reduces intracellular cystine and the rate of organ deterioration. A formulation of cysteamine requiring less frequent dosing may improve compliance and possibly patient outcome. Methods Enteric-release cysteamine was prepared. For a period of 1 month, patients received their regular cysteamine dose every 6 hours (stage I). The patients then underwent pharmacokinetic and pharmacodynamic studies following washout periods using single-doses of cysteamine and enteric-release cysteamine (stage II). Finally, the patients commenced regular enteric-release cysteamine therapy (stage III). Weekly trough white blood cell (WBC) cystine levels were recorded. Results Seven children with cystinosis (mean age, 11.8 years; range, 8-17 years) who received cysteamine and enteric-release cysteamine (mean dose, 45 and 28.8 mg/kg body weight/day, respectively) had mean WBC cystine levels of 0.7 ± 0.3 and 0.41 ± 0.22 nmol half-cystine/mg protein in study stages I and III, respectively. Study stage II showed that the mean time (Tmax) to reach the maximum plasma cysteamine level (Cmax) was longer for enteric-release cysteamine than for cysteamine (176 minutes vs 60 minutes; P = .001), but the mean Cmax at the same dose was similar. Mean serum gastrin levels were similar after ingestion of cysteamine and enteric-release cysteamine. Conclusions Twelve-hour enteric-release cysteamine, given at approximately 60% of the previous daily dose of cysteamine, was effective in maintaining trough WBC cystine levels within a satisfactory range.

We have studied a child with cystic fibrosis (CF), nephropathic cystinosis, and manifestations of Bartter syndrome, a finding reported previously in both of these diseases (CF and cystinosis). The chance of an individual inheriting a mutant allele for both CF and cystinosis from each of his parents by independent segregation is very small. Therefore, other mechanisms of inheritance were investigated, including whether his diseases were caused by a chromosome deletion or rearrangement that caused defects in both genes, whether his phenotype was caused by a new mutation or variant of either disease, or whether both diseases were inherited together due to inheritance of 2 copies of the same chromosome from one of the parents (uniparental disomy). An investigation was made of whether having mutations for both CF and cystinosis resulted in a different phenotype for either disease and whether the child was a heterozygote rather than a homozygote for one of the mutations. The results suggest that neither disease influenced the expression of the defect in the other and that this child inherited a mutant allele for both diseases independently from each parent.

We characterize here a lysosomal cystine transporter in mouse L-929 fibroblasts. Granular fractions from cells preloaded with cystine demonstrated countertransport that showed no dependence on Na+ or K+. The Michaelis constant for infinite-trans influx was 0.27 +/- 0.06 mM (n = 3), and a nonsaturable component of cystine entry was observed with Kd = 0.8-1.8 nmol of cystine.min-1.unit of hexosaminidase-1.mM-1. We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport. The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine. Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required. Some di- as well as tetrapolar amino acids were recognized. We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur.

The cystine that accumulates within cystinotic lysosomes comes primarily from proteins which have been degraded within this organelle. The individual amino acids have specific transport mechanisms to exit the lysosome. The lysosomal cystine transporter is defective in all types of cystinosis. When cells from patients with nephropathic and benign cystinosis were fused, the defect was not corrected and the cystine level remained elevated. This strongly indicates that the genetic defects are allelic (i.e., on the same chromosome). Cysteamine is a weak base which enters the cystinotic lysosome and reacts with cysteamine. forming a mixed disulfide of half-cystine and cysteamine. This mixed disulfide rapidly exits the lysosome via the transport system for cationic amino acids which is normal in cystinosis. Because of the success of renal transplantation, many cystinosis patients are alive in their twenties and even early thirties. Unfortunately, these patients have developed damage to other organs including thyroid, eye, central nervous system, pancreas, and muscle. Cysteamine and its analog, phosphocysteamine, are very beneficial to cystinosis patients, especially when started early in life. These drugs may prevent the need for transplantation. It is too early to know if they will prevent damage to other organs.

Aims Although cysteamine was first used in the treatment of cystinosis in 1976 and approved by the FDA as cysteamine bitartrate (Cystagon™) in 1994, surprisingly little pharmacological data are available for this compound. Cysteamine and its related drugs are currently being evaluated for the treatment of Huntington’s and Parkinson’s disease. The aim of te study was to understand the pharmacokinetics of cysteamine bitartrate following gastrointestinal infusion. Method Cysteamine bitartrate was delivered through a naso-enteric catheter into the stomach (n = 8), small intestine (n = 8) and caecum (n = 4) of normal subjects. Plasma cysteamine concentrations were determined using LC-MS/MS. Results The rate and extent of drug absorption were assessed by comparing AUC(0, ∞), Cmax and tmax, among the gastrointestinal infusion sites. Total cysteamine exposure, expressed as area under the curve (AUC(0, ∞)) was greatest when the drug was infused into the small intestine (4331.3 ± 1907.6 min × µm) followed by stomach (3901.9 ± 1591.9 min × µm) and caecum (3141.4 ± 1627.6 min × µm). Cysteamine infusion into the small intestine resulted in the most rapid rise to maximal plasma concentrations (tmax = 21 ± 0.56 min); tmax was delayed to 50 ± 26 min and 64 ± 26 min after gastric and caecal infusion, respectively. The maximum cysteamine plasma concentration (Cmax) was reached after infusion of the drug into the small intestine (51 ± 21 µm), which was higher than plasma Cmax concentrations after gastric (39 ± 16 µm) and caecal infusion (23 ± 15 µm). Conclusions The pharmacokinetic data generated help extend our understanding of cysteamine.

Consensus Statement First NIH/Office of Rare Diseases Conference on Cystinosis: past, present, and future Robert Kleta, Frederick Kaskel, Ranjan Dohil, Paul Goodyer, Lisa M. GuayWoodford, Erik Harms, Julie R. Ingelfinger, Vera H. Koch, Craig B. Langman, Mary B. Leonard, Roslyn B. Mannon, Minnie Sarwal, Jerry A. Schneider, Flemming Skovby, Barbara C. Sonies, Jess G. Thoene, Doris A. Trauner and William A. Gahl 19

Nephropathic cystinosis (MIM 21980) is an autosomal recessive disorder due to defective transport of the amino acid cystine out of lysosomes1. Cystine storage leads to acidosis, dehydration, rickets and growth retardation in the first year of life, followed by renal glomerular failure at approximately ten years of age2. Renal transplantation is highly successful, but cystine continues to accumulate in other tissues, resulting in complications such as corneal ulcerations and retinal blindness, a distal vacuolar myopathy, delayed puberty, swallowing difficulties, pancreatic deficiency and central nervous system involvement2,3. Treatment with the cystine-depleting agent, cysteamine (CystagonR), or phosphocysteamine, has proven successful in retarding glomerular deterioration and enhancing growth4,5. Although cystinosis represents the prototypic disorder of lysosomal membrane transport, neither the cystinosis gene nor the lysosomal cystine carrier has been isolated. We now report linkage of the cystinosis gene to markers on the short arm of chromosome 17 (Zmax=10.89, θ=0.03) for marker D17S1584. Multipoint analysis and haplotypes in recombinant families suggest that the gene is located between markers D17S1583 and D17S796.

Cysteamine prevents organ damage in children with cystinosis, but may cause gastrointestinal (GI) symptoms. In this study we evaluated the nature of GI disease, and the value of omeprazole in controlling GI symptoms in these children.Upper GI disease was evaluated with endoscopy, gastrin levels, and acid secretion studies after oral administration of cysteamine, before and after 16 weeks of therapy with omeprazole. A symptom score was devised.Eleven children (mean age, 5.7 years) were studied. After cysteamine ingestion, before and after omeprazole therapy, the mean maximum acid output was significantly higher than the mean basal acid output. The maximum acid output was measured within 60 minutes of cysteamine ingestion and was reduced by omeprazole therapy (P.01). The mean peak gastrin level was 30 minutes postcysteamine and was higher than baseline (P.01). The initial mean symptom score (maximum score, 14) was 6.9 and fell to 0.7 (P.0001) after 16 weeks of omeprazole therapy. At endoscopy, two children had diffuse gastric nodularity, and nearly all had cystine crystal deposits.GI symptoms in children with cystinosis receiving cysteamine are often acid-mediated and improve with omeprazole. Cystine crystals were detected in the GI tract and may signify inadequate treatment with cysteamine.

Aim: To evaluate the safety and efficacy of a new topical cysteamine formulation, stable at room temperature, for the treatment of corneal cystine crystals in cystinosis. Methods: 20 study subjects were enrolled in the safety study and 16 in the efficacy study. Both studies were randomised and double blind. The primary outcome for the safety study was the occurrence of predefined serious adverse reactions over 6 months and for the efficacy study the reduction of corneal cystine crystal score (CCCS) by 1.00 or more units on photographs graded by a reading centre using a standardised protocol. Results: No study subject developed any serious adverse reactions. In the efficacy study, 47% of eyes receiving the standard formulation experienced a reduction in the CCCS of ≥1.00 after 1 year, while 7% of eyes on the new formulation experienced such a decrease (p=0.04). Conclusion: Although no serious adverse reactions were observed with either formulation, the new formulation was not as effective as the standard formulation.

Cystinosis, an autosomal recessive lysosomal storage disorder, is rarely diagnosed in African Americans. The disease results from mutations in the gene CTNS ; at least 55 such mutations have been reported. By far the most common is a 57,257-bp deletion of Northern European origin encompassing most of the CTNS gene. We performed mutation analysis on DNA from four African American patients with cystinosis. In one individual with classical, nephropathic cystinosis, we identified a new molecular defect, i.e., a homozygous GT→CC substitution at the +5 position of IVS 5 of CTNS (IVS 5+5 GT→CC). The out-of-frame splicing of exon 5 creates a null allele consistent with the patient's severe phenotype. Two patients were heterozygous and one homozygous for the common 57-kb deletion allele, reflecting the admixture of African and Northern European gene pools in North America. The two African Americans heterozygous for the 57-kb deletion were also hemizygous for a 928G→A change, associated with ocular or nonnephropathic cystinosis. These two individuals are the only known African Americans with ocular cystinosis. We conclude that the diagnosis of cystinosis should be entertained in African Americans with symptoms of the disease, and that mutation analysis for the 57-kb deletion should be considered in this group of patients.

Ocular nonnephropathic cystinosis, a variant of the classic nephropathic type of cystinosis, is an autosomal recessive lysosomal storage disorder characterized by photophobia due to corneal cystine crystals but absence of renal disease. We determined the molecular basis for ocular cystinosis in four individuals. All had mutations in the cystinosis gene CTNS, indicating that ocular cystinosis is allelic with classic nephropathic cystinosis. The ocular cystinosis patients each had one severe mutation and one mild mutation, the latter consisting of either a 928 G-->A (G197R) mutation or an IVS10-3 C-->G splicing mutation resulting in the insertion of 182 bp of IVS10 into the CTNS mRNA. The mild mutations appear to allow for residual CTNS mRNA production, significant amounts of lysosomal cystine transport, and lower levels of cellular cystine compared with those in nephropathic cystinosis. The lack of kidney involvement in ocular cystinosis may be explained by two different mechanisms. On the one hand (e.g. the G197R mutation), significant residual cystinosin activity may be present in every tissue. On the other hand (e.g. the IVS 10-3 C-->G mutation), substantial cystinosin activity may exist in the kidney because of that tissue's specific expression of factors that promote splicing of a normal CTNS transcript. Each of these mechanisms could result in minimally reduced lysosomal cystine transport in the kidneys.

Six patients with the intermediate form of cystinosis are described. Two have new mutations not previously described. The disease occurs due either to the combination of one mild mutation and one which is known to cause nephropathic cystinosis or to homozygosity for a predicted mild mutation. Partial phenotypic correction of cystinotic fibroblasts by transfection with normal cDNA or a cDNA derived from a mutation causing intermediate cystinosis is demonstrated. © 1999 Academic Press

A group of children and adolescents with infantile nephropathic cystinosis underwent cognitive testing and were examined for cortical atrophy using magnetic resonance imaging or computed tomography. Ten of 11 patients demonstrated cortical atrophy. A consistent pattern of lower cognitive performance was found in patients with greater atrophy; however, only the relationship between atrophy and short-term memory approached statistical significance. In addition, evidence for greater impairment of visual memory than of other cognitive functions was observed. This latter observation did not appear to be related to the degree of atrophy.

Objective. Cysteamine is a recently licensed orphan drug used to treat the inherited metabolic disease cystinosis. The drug delays the onset of renal failure in cystinotic patients and may provide many other significant health benefits. This study examined the cost-effectiveness of the administration of cysteamine to cystinotic patients prior to end-stage renal disease (ESRD). Method. Decision-tree analysis and cost-effectiveness analysis. Cost data were estimated from current clinical charges and Medicare public-access reports. Life expectancy outcomes were derived from both published and unpublished clinical studies and from the U.S. Renal Data System. Results. Cysteamine therapy can extend the life of kidneys and delay renal transplantation, thereby increasing life expectancy for patients with cystinosis. Patients receiving cysteamine therapy prior to renal failure have lifetime-treatment drug costs of $234,000, in comparison with $238,000 for those who are not medicated. Costs of cysteamine therapy are offset by savings associated with delaying transplantation and costs of dialysis. Conclusions. Use of the orphan drug cysteamine both improves health outcomes and reduces health care costs for patients with cystinosis. Key words: cystinosis; cysteamine ; decision analysis; cost-effectiveness. (Med Decis Making 1997;17:193-198)

Cysteamine bitartrate capsules (Cystagon) have been approved by the US Food and Drug Administration for use in patients with nephropathic cystinosis. Plasma cysteamine concentrations were virtually identical at various times following ingestion of either cysteamine hydrochloride or Cystagon capsules in 24 normal control subjects. A transfer study was done with eight cystinosis patients who had been receiving either cysteamine hydrochloride or phosphocysteamine for many years. The plasma cysteamine concentration was significantly higher 2h after Cystagon and the leukocyte cystine content was significantly lower at all times after Cystagon compared to older forms of the drug. These differences are probably the result of greater patient compliance in taking the capsules compared to the older, liquid forms of the drug. A new method for following the course of renal glomerular deterioration in diseases such as cystinosis has been published recently. This method was used to re-analyse data on the efficacy of cysteamine treatment and to re-analyse new data on treating cystinosis patients with either of two doses of cysteamine (1.30 g/m2 per day and 1.95 g/m2 per day). This new method agrees well with other methods and shows that both doses of drug are equally effective in maintaining glomerular function.

We report the case of a patient with infantile nephropathic cystinosis who required renal transplantation at age 30 months. Exhaustive evaluation did not identify a cause of progressive renal failure other than cystinosis. The patient's genetic lesion was allelic with those of other patients with cystinosis; fusion of this patient's fibroblasts with fibroblasts from another patient with infantile nephropathic cystinosis did not demonstrate complementation of the biochemical defect.

Lysosomes are a major intracellular site for the degradation of a large variety of macromolecules including proteins, nucleic acids, complex carbohydrates, and lipids. These macromolecules are delivered to lysosomes by the processes of pinocytosis, receptor-mediated endocytosis, phagocytosis, or autophagy. In the lysosome these large molecules are degraded by hydrolases, many of which function optimally at the acidic pH of the intralysosomal environment. The resulting degradation products often require specific transport systems in order to exit the lysosomal compartment. This review focuses on the amino acid transport systems of mammalian lysosomes which allow the release of amino acids generated within the lysosome following proteolysis. As in the plasma membrane, the specificity of lysosomal amino acid transport systems is based upon the net charge and dimensions of the amino acid. Thus, there are specific transport routes for the cationic amino acids, for the anionic amino acids, and multiple routes for the neutral amino acids. In addition, three highly specific lysosomal transport routes have been characterized for cystine, cysteine, and proline. All of the lysosomal transport systems described thus far are Na+-independent.

Cystinosis is an autosomal recessive metabolic disorder in which nonprotein cystine accumulates within most body tissues due to a defect in lysosomal cystine transport. Neurologic declarations are only recently being recognized. We studied 13 cystinotic subjects (aged 5 to 21 years old), determining median motor and sensory nerve conduction velocities, F waves, peroneal motor nerve conduction velocities, sural sensory nerve conduction velocities, median sympathetic skin response, electrocardiogram R-to-R variability, and blink reflex analysis. The results were normal. We conclude that neurophysiologic testing suggests relative sparing of the peripheral nervous system in nephropathic cystinosis.

The predicted reciprocal creatinine at age 10 years (PRC10), a parameter of renal function based upon the linear relationship between reciprocal serum creatinine and age, incorporates age, serum creatinine, and rate of renal deterioration into a single term. PRC10 measurements were employed to assess renal function in children with nephropathic cystinosis treated with oral cysteamine, a cystine-depleting agent. In 71 children receiving oral cysteamine for at least 1 year, PRC10 decreased linearly with initial serum creatinine concentration. This indicated that, although established renal damage in cystinosis was irreversible, early intervention with cysteamine therapy could favorably alter the rate of glomerular deterioration. In other analyses, mean PRC10 was shown to increase with duration of cysteamine therapy and extent of leukocyte cystine depletion. The predicted reciprocal creatinine value at a certain age can be useful in analyzing the effects of therapeutic intervention in a disease with a relatively uniform rate of renal deterioration.

Three patients with nephropathic cystinosis and chronic renal disease, treated since early childhood with orally administered cysteamine, had an accelerated rate of rise of serum creatinine values after receiving recombinant human growth hormone. During 18 to 24 months of growth hormone treatment, each patient had a twofold to fourfold increase in growth velocity. The slope of the plot of reciprocal serum creatinine values versus age for each patient after growth hormone treatment was significantly steeper than the pretreatment slope. Growth hormone treatment had no effect on the rate of change of the uncorrected 24-hour renal creatinine clearance. We conclude that these patients gained body size but failed to compensate for their increased creatinine production with an increase in glomerular filtration rate. The result was an accelerated rate of rise of their serum creatinine values, hastening renal transplantation in one patient and the anticipated need for transplantation in another.

Methodology for the preparation of cultured fibroblasts for measurement of their free (nonprotein) amino acids was studied in two ways. First, the effect of storage of fibroblasts at −20°C, −70°C, and −196°C was investigated. The recovery of free-cystine declined about 50% after 24 hr of storage and another 25% following six more days of storage at these temperatures. Second, three methods of removing fibroblasts from the surface of tissue culture vessels were studied. The recovery of amino acids was significantly lower when the cells were removed with a silicone rubber scraper than when trypsin or trypsin plus tetrasodium ethylenediaminetetracetate was used.

Cystine binding protein isolated from Escherichia coli W3092 in high yield was used to develop a binding assay for the disulfide amino acid cystine. Filtration of a mixture of [14C]cystine and cystine binding protein through nitrocellulose filters separated the [14C]cystine-cystine binding protein complex from the unbound cystine. The binding of [14C]cystine-cystine binding protein complexes to the filter was optimal (100%) at pH 4 to 5, and was insensitive to Mg2+ (10 mm), EDTA (10 mm), or concentrations of NaCl to 0.5 m. Bovine serum albumin in excess of 30 µg decreased binding of the 14C complex to the filter. The binding of [14C]cystine to the cystine binding protein was inhibited by lanthionine, much less by diaminopimelic acid, cystathionine, and d-cystine, and was not inhibited by reduced or oxidized glutathione, penicillamine disulfide, cysteic acid, methionine, taurine, homocystine, carboxymethyl cysteine, or the N-ethylmaleimide adduct of cysteine. Nonradioactive cystine competed with [14C]cystine resulting in the expected diminution of the bound radioactivity, permitting the direct calculation of nonradioactive cystine concentrations of unknown samples. With the cystine binding protein assay, the cystine concentrations of human plasma were in the range of 87 to 100% of the values obtained by ion exchange chromatography, and the cystine contents of cultured skin fibroblasts from patients with nephropathic cystinosis were in the range of 89 to 121% of the values obtained by ion exchange chromatography. As little as 10 pmoles of cystine were measured.

Human diploid fibroblasts underwent lysis when resuspended in isotonic sucrose. After preparation of a granular fraction by differential centrifugation, lysosomes were almost completely purified by free-flow electrophoresis. Electron micrographs of the final fraction of fibroblast lysosomes (about 25-fold purified) showed mostly secondary lysosomes having an appearance identical to those in intact cells. There was no indication that the purification steps selectd for any lysosomal subpopulation. The lysomal nature of the isolated organelles was confirmed by cytochemical staining for acid beta-glycerophosphatase. As judged by a 92% structure-linked latency of beta-N-acetylglucosaminidase in the final fraction, the structural integrity of the isolated lysosomes was unaffected by the purification procedure.

Urine glyceraldehyde excretion is elevated in the renal Fanconi syndrome. We analyzed urinary constituents using GC/MS in 16 children with the renal Fanconi syndrome and 13 normal individuals. Urine glyceraldehyde levels were strikingly elevated in the renal Fanconi syndrome group (mean 5.1 ± 4.8 mg/mg creatinine) compared to levels in the normal group (mean 0.04 ± 0.04 mg/mg creatinine, P < 0.001). Urine lactate levels were also elevated in the renal Fanconi syndrome group (mean 2.3 ± 2.6 mg/mg creatinine) compared to normals (mean 0.01 ± 0.01 mg/mg creatinine, P < 0.003). Only small elevations of glyceraldehyde and lactate were found in urine from children with other renal disorders. Serum levels of glyceraldehyde and lactate were no greater in individuals with the Fanconi syndrome than in the normals. The fractional reabsorption of both glyceraldehyde and lactate was virtually complete in the normals, but was markedly impaired in the Fanconi syndrome patients where, in some cases, glyceraldehyde excretion greatly exceeded the excretion of creatinine. We conclude that marked glyceraldehyde excretion is a previously unrecognized feature of the renal Fanconi syndrome which may result from disordered proximal tubular glycolytic metabolism. Further studies will be required to determine the role of glyceraldehyde loss in the pathogenesis of this generalized disturbance of proximal tubular function.

Cystinosis is a recessively inherited metabolic disorder characterized biochemically by a high intracellular content of free (nonprotein) cystine which appears to be compartmentalized within lysosomes. This results in crystal deposition in the cornea, conjunctiva, bone marrow, lymph nodes, leukocytes, and internal organs. The primary metabolic defect which leads to cystine accumulation is unknown.

Cystinotic fibroblasts contain approximately 100 times more cystine than do normal control fibroblasts. When cystinotic fibroblasts were placed in the presence of 30 mM cysteine-glutathione mixed disulfide (CSSG) for 24 hr, their cystine content increased about 3-fold. Similar treatment of normal fibroblasts and fibroblasts from patients heterozygous for cystinosis resulted in a 6- to 7-fold increase in cystine content. In all three cell types, the intracellular free cystine is located within lysosomes. When placed in cystine-free medium after 24 hr in CSSG-containing medium, the normal and heterozygous fibroblasts rapidly lost their lysosomal cystine (t 1/2 = 20 min), but the cystine content of the cystinotic cells remained stable for over 90 min. In contrast to the findings in intact fibroblasts, cystine loss could not be demonstrated from isolated, cystine-loaded lysosomes from any of the three cell types.

Boman, H. and Schneider, J. A. (Institute of Medical Genetics, University of Oslo, Oslo, Norway, and Department of Pediatrics, School of Medicine, University of California San Diego, La Jolla, California, USA). Prenatal diagnosis of nephropathic cystinosis: Pregnancy at risk ascertained through heterozygote diagnosis of parents. Acta Paediatr Scand, 70:389, 1981.–The biochemical diagnosis of cystinosis in a deceased girl was made indirectly through the demonstration of heterozygote values of free-cystine contents in leukocytes from both parents. Amniocentesis was performed on the mother in the 16th week of two subsequent pregnancies. Amniotic fluid cell lysate from the first fetus had 6–8 times normal levels of radioactivity in the cystine band determined by a 35S-cystine pulse labeling method followed by high voltage electrophoresis on paper. Values for the cystine contents in various organs of the affected fetus are given. The second fetus was found to be not affected, and this was subsequently confirmed by the birth of a healthy child. The results of the laboratory analyses in these two cases were available to the parents 21 and 23 days following amniocentesis, respectively. The interruption of the first pregnancy was performed in the 19th week. This was 4 and 6 weeks earlier, respectively, than in the two previously reported induced abortions of affected fetuses, and should increase the acceptability of the present method for prenatal diagnosis of nephrophatic cystinosis.