A New and Rapid LC-MS/MS Method for the Determination of Cysteamine Plasma Levels in Cystinosis Patients.

Cystinosis is a rare lysosomal storage disorder caused by autosomal recessive mutations in the CTNS gene that encodes for the cystine transporter cystinosin, which is expressed on the lysosomal membrane mediating the efflux of cystine. Cysteamine bitartrate is a cystine-depleting aminothiol agent approved for the treatment of cystinosis in children and adults. In this study, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of cysteamine levels in plasma samples. This LC-MS/MS method was validated according to the European Medicines Agency (EMA)'s guidelines for bioanalytical method validation. An ultra-performance liquid chromatograph (UPLC) coupled with a 6470 mass spectrometry system was used for cysteamine determination. Our validated method was applied to plasma samples from n = 8 cystinosis patients (median, interquartile range (IQR) = 20.5, 8.5-26.0 years). The samples were collected before cysteamine oral administration (pre-dose) and 1 h after (post-dose). Our bioanalytical method fulfilled the regulatory guidelines for method validation. The cysteamine plasma levels in pre-dose samples were 2.57 and 1.50-3.31 µM (median and IQR, respectively), whereas the post-dose samples reported a cysteamine median concentration of 28.00 µM (IQR: 17.60-36.61). Our method allows the rapid determination of cysteamine plasma levels. This method was successfully used in cystinosis patients and, therefore, could be a useful tool for the evaluation of therapy adherence and for future pharmacokinetic (PK) studies involving a higher number of subjects.

MFSD12 depletion reduces cystine accumulation without improvement in proximal tubular function in experimental models for cystinosis.

Cystinosis is an autosomal recessive lysosomal storage disorder, caused by mutations in the CTNS gene, resulting in an absent or altered cystinosin (CTNS) protein. Cystinosin exports cystine out of the lysosome, with a malfunction resulting in cystine accumulation and a defect in other cystinosin-mediated pathways. Cystinosis is a systemic disease, but the kidneys are the first and most severely affected organs. In the kidney, the disease initially manifests as a generalized dysfunction in the proximal tubules (also called renal Fanconi syndrome). MFSD12 is a lysosomal cysteine importer that directly affects the cystine levels in melanoma cells, HEK293T cells, and cystinosis patient-derived fibroblasts. In this study, we aimed to evaluate MFSD12 mRNA levels in cystinosis patient-derived proximal tubular epithelial cells (ciPTECs) and to study the effect of MFSD12 knockout on cystine levels. We showed similar MFSD12 mRNA expression in patient-derived ciPTECs in comparison with the control cells. CRISPR MFSD12 knockout in a patient-derived ciPTEC (CTNSΔ57kb) resulted in significantly reduced cystine levels. Furthermore, we evaluated proximal tubular reabsorption after injection of mfsd12a translation-blocking morpholino (TB MO) in a ctns-/- zebrafish model. This resulted in decreased cystine levels but caused a concentration-dependent increase in embryo dysmorphism. Furthermore, the mfsd12a TB MO injection did not improve proximal tubular reabsorption or megalin expression. In conclusion, MFSD12 mRNA depletion reduced cystine levels in both tested models without improvement of the proximal tubular function in the ctns-/- zebrafish embryo. In addition, the apparent toxicity of higher mfsd12a TB MO concentrations on the zebrafish development warrants further evaluation.NEW & NOTEWORTHY In this study, we show that MFSD12 depletion with either CRISPR/Cas9-mediated gene editing or a translation-blocking morpholino significantly reduced cystine levels in cystinosis ciPTECs and ctns-/- zebrafish embryos, respectively. However, we observed no improvement in the proximal tubular reabsorption of dextran in the ctns-/- zebrafish embryos injected with mfsd12a translation-blocking morpholino. Furthermore, a negative effect of the mfsd12a morpholino on the zebrafish development warrants further investigation.

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis.

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns-/- zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS-/- kidney cells and injection into ctns-/- zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns-/- larvae, and restoration of the zebrafish pronephros function.

Genistein improves renal disease in a mouse model of nephropathic cystinosis: a comparison study with cysteamine.

Cysteamine is currently the only therapy for nephropathic cystinosis. It significantly improves life expectancy and delays progression to end-stage kidney disease; however, it cannot prevent it. Unfortunately, compliance to therapy is often weak, particularly during adolescence. Therefore, finding better treatments is a priority in the field of cystinosis. Previously, we found that genistein, an isoflavone particularly enriched in soy, can revert part of the cystinotic cellular phenotype that is not sensitive to cysteamine in vitro. To test the effects of genistein in vivo, we fed 2-month-old wild-type and Ctns-/- female mice with either a control diet, a genistein-containing diet or a cysteamine-containing diet for 14 months. Genistein (160 mg/kg/day) did not affect the growth of the mice or hepatic functionality. Compared with untreated mice at 16 months, Ctns-/- mice fed with genistein had lower cystine concentrations in their kidneys, reduced formation of cystine crystals, a smaller number of LAMP1-positive structures and an overall better-preserved parenchymal architecture. Cysteamine (400 mg/kg/day) was efficient in reverting the lysosomal phenotype and in preventing the development of renal lesions. These preclinical data indicate that genistein ameliorates kidney injury resulting from cystinosis with no side effects. Genistein therapy represents a potential treatment to improve the outcome for patients with cystinosis.

Benefits and Toxicity of Disulfiram in Preclinical Models of Nephropathic Cystinosis.

Nephropathic cystinosis is a rare disease caused by mutations of the CTNS gene that encodes for cystinosin, a lysosomal cystine/H+ symporter. The disease is characterized by early-onset chronic kidney failure and progressive development of extra-renal complications related to cystine accumulation in all tissues. At the cellular level, several alterations have been demonstrated, including enhanced apoptosis, altered autophagy, defective intracellular trafficking, and cell oxidation, among others. Current therapy with cysteamine only partially reverts some of these changes, highlighting the need to develop additional treatments. Among compounds that were identified in a previous drug-repositioning study, disulfiram (DSF) was selected for in vivo studies. The cystine depleting and anti-apoptotic properties of DSF were confirmed by secondary in vitro assays and after treating Ctns-/- mice with 200 mg/kg/day of DSF for 3 months. However, at this dosage, growth impairment was observed. Long-term treatment with a lower dose (100 mg/kg/day) did not inhibit growth, but failed to reduce cystine accumulation, caused premature death, and did not prevent the development of renal lesions. In addition, DSF also caused adverse effects in cystinotic zebrafish larvae. DSF toxicity was significantly more pronounced in Ctns-/- mice and zebrafish compared to wild-type animals, suggesting higher cell toxicity of DSF in cystinotic cells.

Renal and Extra Renal Manifestations in Adult Zebrafish Model of Cystinosis.

Cystinosis is a rare, incurable, autosomal recessive disease caused by mutations in the CTNS gene. This gene encodes the lysosomal cystine transporter cystinosin, leading to lysosomal cystine accumulation in all cells of the body, with kidneys being the first affected organs. The current treatment with cysteamine decreases cystine accumulation, but does not reverse the proximal tubular dysfunction, glomerular injury or loss of renal function. In our previous study, we have developed a zebrafish model of cystinosis through a nonsense mutation in the CTNS gene and have shown that zebrafish larvae recapitulate the kidney phenotype described in humans. In the current study, we characterized the adult cystinosis zebrafish model and evaluated the long-term effects of the disease on kidney and extra renal organs through biochemical, histological, fertility and locomotor activity studies. We found that the adult cystinosis zebrafish presents cystine accumulation in various organs, altered kidney morphology, impaired skin pigmentation, decreased fertility, altered locomotor activity and ocular anomalies. Overall, our data indicate that the adult cystinosis zebrafish model reproduces several human phenotypes of cystinosis and may be useful for studying pathophysiology and long-term effects of novel therapies.

Therapeutic Drug Monitoring Is a Feasible Tool to Personalize Drug Administration in Neonates Using New Techniques: An Overview on the Pharmacokinetics and Pharmacodynamics in Neonatal Age.

Therapeutic drug monitoring (TDM) should be adopted in all neonatal intensive care units (NICUs), where the most preterm and fragile babies are hospitalized and treated with many drugs, considering that organs and metabolic pathways undergo deep and progressive maturation processes after birth. Different developmental changes are involved in interindividual variability in response to drugs. A crucial point of TDM is the choice of the bioanalytical method and of the sample to use. TDM in neonates is primarily used for antibiotics, antifungals, and antiepileptic drugs in clinical practice. TDM appears to be particularly promising in specific populations: neonates who undergo therapeutic hypothermia or extracorporeal life support, preterm infants, infants who need a tailored dose of anticancer drugs. This review provides an overview of the latest advances in this field, showing options for a personalized therapy in newborns and infants.

Cell-Based Phenotypic Drug Screening Identifies Luteolin as Candidate Therapeutic for Nephropathic Cystinosis.

BACKGROUND: Mutations in the gene that encodes the lysosomal cystine transporter cystinosin cause the lysosomal storage disease cystinosis. Defective cystine transport leads to intralysosomal accumulation and crystallization of cystine. The most severe phenotype, nephropathic cystinosis, manifests during the first months of life, as renal Fanconi syndrome. The cystine-depleting agent cysteamine significantly delays symptoms, but it cannot prevent progression to ESKD and does not treat Fanconi syndrome. This suggests the involvement of pathways in nephropathic cystinosis that are unrelated to lysosomal cystine accumulation. Recent data indicate that one such potential pathway, lysosome-mediated degradation of autophagy cargoes, is compromised in cystinosis. METHODS: To identify drugs that reduce levels of the autophagy-related protein p62/SQSTM1 in cystinotic proximal tubular epithelial cells, we performed a high-throughput screening on the basis of an in-cell ELISA assay. We then tested a promising candidate in cells derived from patients with, and mouse models of, cystinosis, and in preclinical studies in cystinotic zebrafish. RESULTS: Of 46 compounds identified as reducing p62/SQSTM1 levels in cystinotic cells, we selected luteolin on the basis of its efficacy, safety profile, and similarity to genistein, which we previously showed to ameliorate other lysosomal abnormalities of cystinotic cells. Our data show that luteolin improves the autophagy-lysosome degradative pathway, is a powerful antioxidant, and has antiapoptotic properties. Moreover, luteolin stimulates endocytosis and improves the expression of the endocytic receptor megalin. CONCLUSIONS: Our data show that luteolin improves defective pathways of cystinosis and has a good safety profile, and thus has potential as a treatment for nephropathic cystinosis and other renal lysosomal storage diseases.

Phenformin Inhibits Hedgehog-Dependent Tumor Growth through a Complex I-Independent Redox/Corepressor Module.

The antidiabetic drug phenformin displays potent anticancer activity in different tumors, but its mechanism of action remains elusive. Using Shh medulloblastoma as model, we show here that at clinically relevant concentrations, phenformin elicits a significant therapeutic effect through a redox-dependent but complex I-independent mechanism. Phenformin inhibits mitochondrial glycerophosphate dehydrogenase (mGPD), a component of the glycerophosphate shuttle, and causes elevations of intracellular NADH content. Inhibition of mGPD mimics phenformin action and promotes an association between corepressor CtBP2 and Gli1, thereby inhibiting Hh transcriptional output and tumor growth. Because ablation of CtBP2 abrogates the therapeutic effect of phenformin in mice, these data illustrate a biguanide-mediated redox/corepressor interplay, which may represent a relevant target for tumor therapy.

Cefoxitin Prophylaxis During Pediatric Cardiac Surgery: Retrospective Exploration of Postoperative Trough Levels.

BACKGROUND: This study aimed to explore inter-individual variability of cefoxitin trough levels, predictors of serum cefoxitin concentration and the probability of target attainment of drug levels above 4 mg/L after pediatric cardiac surgery. METHODS: Retrospective study on children scheduled for elective cardiac surgery and having cefoxitin trough levels available up to 24 hours postsurgery. RESULTS: Overall, 68 children (9 neonates, 34 infants, 15 children below or equal to 10 years old and 10 patients above this age) were included. Of these, 16 surgeries were performed off cardiopulmonary bypass and 52 were performed on cardiopulmonary bypass. The free cefoxitin concentrations showed a median (interquartile range) concentration of 1.7 (0.6-4.2) mg/L. The range of cefoxitin concentrations showed a 150-fold and 340-fold variability at cardiac intensive care unit admission and after 24 hours, respectively. The pharmacodynamics (PD) targets of free cefoxitin at 100% of the dosing interval, considering Eucast breakpoints for Methicillin Sensitive Staphylococcus Aureus (4 mg/L) and E.Coli (8 mg/L), were obtained in 28% and 16% of patients, respectively. Patient weight (odds ratio, 0.7; 95% confidence interval, 0.62-0.92; P = 0.006) and serum creatinine concentrations (odds ratio, 25; 95% confidence interval, 18-36; P = 0.004) showed a significant relationship with the PD targets. CONCLUSIONS: Cefoxitin trough concentrations vary significantly in the first 24 hours after pediatric cardiac surgery. Both serum creatinine and body weight showed independent associations with cefoxitin concentration. The PD target was not obtained in the vast majority of the explored population, regardless of the target bacteria.

Eltrombopag-Induced Acute Liver Failure in a Pediatric Patient: A Pharmacokinetic and Pharmacogenetic Analysis.

Eltrombopag is an oral thrombopoietin receptor agonist approved for the treatment of patients with chronic idiopathic thrombocytopenic purpura (ITP), who are more than 1 year old, and show poor response to first-line therapy. ITP is a hematological disorder characterized by isolated thrombocytopenia in the absence of secondary causes or disorders. Eltrombopag is generally well tolerated in the pediatric population; therefore, therapeutic drug monitoring (TDM) is not usually performed in clinical practice.We presented the case study of a 3-year-old girl with chronic ITP. She arrived in the pediatric intensive care unit with acute liver failure due to eltrombopag toxicity despite taking the standard drug dosage. A very high eltrombopag plasma concentration, indicating drug toxicity, was found through TDM. The patient also carried the allelic variations that are involved in drug metabolism [CYP2C8 and UDP glucuronosyltransferase (UGT) 1A1 (UGT1A1)] and drug cellular transportation [ABCG2 (ATP-binding cassette G2)]. This observation highlights the importance of using TDM and pharmacogenetic approaches to manage patients' unusual complications associated with standard pharmacological treatment regimens.