A novel sustained-release cysteamine bitartrate formulation for the treatment of cystinosis: Pharmacokinetics and safety in healthy male volunteers.

The strict intake regimen of cysteamine bitartrate formulations, associated with side effects, is a concern for the treatment compliance in cystinosis therapy. Therefore, there is a need for a cysteamine formulation with an improved pharmacokinetic profile. This study investigated the pharmacokinetics, safety and tolerability of a new sustained-release cysteamine dosage form, PO-001, in healthy volunteers. This was a randomized, investigator-blinded, three-way cross-over study to compare single doses (600 mg) of PO-001 with Cystagon® (immediate-release) and Procysbi® (delayed-release). Collected blood samples were analyzed for plasma cysteamine concentrations and pharmacokinetic parameters were estimated by noncompartmental analysis. In addition, plasma cysteamine concentrations were analyzed using a population pharmacokinetic approach using NONMEM® . Pharmacokinetics showed clear sustained-release characteristics of PO-001 over time with a lower Cmax and longer Tmax compared to Cystagon® and Procysbi® . All treatment-emergent adverse events were of mild severity, with the exception of two subjects who reported moderate severity gastrointestinal problems including vomiting and diarrhea, which were related to Cystagon® intake. Population PK simulations showed a favourable PK profile based on Cmax and Ctrough concentrations at steady state. In conclusion, a single dose of 600 mg PO-001 was well tolerated with no findings of clinical concern. This new cysteamine bitartrate formulation showed pharmacokinetics of a sustained-release formulation, which may be beneficial for the treatment of cystinosis patients. This study supports advancing this type of sustained-release formulation into a subsequent study to confirm reduced dosing frequency with efficient control of white blood cells (WBCs) cystine levels. Netherlands Trial Registry (NTR) (NL67638.056.18).

Carbon Black Tinted Contact Lenses for Reduction of Photophobia in Cystinosis Patients.

PURPOSE: To examine if current development on using contact lenses for drug delivery of cysteamine to treat ocular symptoms of cystinosis can be tinted to mitigate photophobia common in patients by reducing transmittance Methods: Commercial contact lenses were placed in a carbon black solution to examine loading after lens synthesis. Silicone hydrogel contact lenses were also synthesized with carbon black added prior to UV curing. Transmittance was measured using UV-vis spectrophotometry over the range of 190-1190 nm and compared to unmodified contact lenses. Lens parameters of refractive index, ion permeability, and Young's modulus were measured using a refractometer, release of sodium chloride, and the cantilever method. Cysteamine release was measured by loading lenses into 5% cysteamine solution and then monitoring the release of the drug using UV-vis spectrophotometry. Vitamin E diffusion barriers were also added to lenses via ethanol solution, and the release of cysteamine from these modified lenses was also examined. RESULTS: No leeching of carbon black was detected during experiments. Loading of pre-made contact lenses led to uneven distribution of carbon black throughout lens. Adding 0.3% carbon black to lens monomer solution prior to UV-curing led to even distribution and a transmittance reduction of approximately 50%. Ion permeability was reduced from 6.19 ± 0.90 x 10-3 to 1.28 ± 0.06 x 10-3 mm2 min-1, and Young's modulus was decreased from 1.58 ± 0.08 to 1.29 ± 0.06 MPa. Cysteamine releases from carbon black lenses with and without vitamin E were comparable to controls, although the loading solution of vitamin E/ethanol had to be tripled to achieve a similar mass loading to control. CONCLUSIONS: Carbon black increases the softness of contact lenses, but a loading of 0.3% maintains lens parameters required for wear. The release of cysteamine is also possible with carbon black lenses, albeit requiring a higher loading concentration of vitamin E to achieve similar release times.

Cystamine and cysteamine as inhibitors of transglutaminase activity in vivo.

Cystamine is commonly used as a transglutaminase inhibitor. This disulphide undergoes reduction in vivo to the aminothiol compound, cysteamine. Thus, the mechanism by which cystamine inhibits transglutaminase activity in vivo could be due to either cystamine or cysteamine, which depends on the local redox environment. Cystamine inactivates transglutaminases by promoting the oxidation of two vicinal cysteine residues on the enzyme to an allosteric disulphide, whereas cysteamine acts as a competitive inhibitor for transamidation reactions catalyzed by this enzyme. The latter mechanism is likely to result in the formation of a unique biomarker, N-(γ-glutamyl)cysteamine that could serve to indicate how cyst(e)amine acts to inhibit transglutaminases inside cells and the body.

A Phase 1 Pharmacokinetic Study of Cysteamine Bitartrate Delayed-Release Capsules Following Oral Administration with Orange Juice, Water, or Omeprazole in Cystinosis.

INTRODUCTION: Cystinosis is a rare, metabolic, autosomal recessive, genetic lysosomal storage disorder characterized by an accumulation of cystine in various organs and tissues. Cysteamine bitartrate (CB) is a cystine-depleting aminothiol agent approved in the United States and Europe in immediate-release and delayed-release (DR) formulations for the treatment of nephropathic cystinosis in children and adults. It is recommended that CBDR be administered with fruit juice (except grapefruit juice) for maximum absorption. Omeprazole is a proton pump inhibitor that inhibits gastric acid secretion and, theoretically, may cause the premature release of cysteamine by increasing intragastric pH, thereby affecting the PK of CBDR. METHODS: This open-label, three-period, randomized study in healthy adult subjects was designed primarily to compare the pharmacokinetics of CBDR capsules after a single oral dose administered with orange juice, water, or multiple oral doses of omeprazole with water at steady state. A total of 32 subjects were randomly assigned to receive study agents in one of two treatment sequences. RESULTS: All subjects completed the study and baseline characteristics of the overall population and the two treatment sequence populations were similar. Peak mean plasma cysteamine concentrations following co-administration of CBDR capsules with orange juice (1892 ng/mL) were higher compared with co-administration with water (1663 ng/mL) or omeprazole 20 mg and water (1712 ng/mL). Mean time to peak plasma concentration was shorter with omeprazole co-administration (2.5 h) compared with orange juice (3.5 h) or water (3.0 h). Statistical comparisons between treatment groups indicated that exposure as assessed by AUC0-t, AUC0-∞, and Cmax were all within the 80-125% bioequivalence ranges for all comparisons. All treatments were generally well tolerated. CONCLUSION: Overall, the pharmacokinetics of cysteamine bitartrate DR capsules are not significantly impacted by co-administration with orange juice, water only, or omeprazole (with water). FUNDING: Horizon Pharma, Inc.

PEGylated Polyamidoamine dendrimer conjugated with tumor homing peptide as a potential targeted delivery system for glioma.

Glioblastoma multiforme (GBM) is the most common and aggressive primary central nervous system (CNS) tumor with a short survival time. The failure of chemotherapy is ascribed to the low transport of chemotherapeutics across the Blood Brain Tumor Barrier (BBTB) and poor penetration into tumor tissue. In order to overcome the two barriers, small nanoparticles with active targeted capability are urgently needed for GBM drug delivery. In this study, we proposed PEGylated Polyamidoamine (PAMAM) dendrimer nanoparticles conjugated with glioma homing peptides (Pep-1) as potential glioma targeting delivery system (Pep-PEG-PAMAM), where PEGylated PAMAM dendrimer nanoparticle was utilized as carrier due to its small size and perfect penetration into tumor and Pep-1 was used to overcome BBTB via interleukin 13 receptor α2 (IL-13Rα2) mediated endocytosis. The preliminary availability and safety of Pep-PEG-PAMAM as a nanocarrier for glioma was evaluated. In vitro results indicated that a significantly higher amount of Pep-PEG-PAMAM was endocytosed by U87 MG cells. In vivo fluorescence imaging of U87MG tumor-bearing mice confirmed that the fluorescence intensity at glioma site of targeted group was 2.02 folds higher than that of untargeted group (**p<0.01), and glioma distribution experiment further revealed that Pep-PEG-PAMAM exhibited a significantly enhanced accumulation and improved penetration at tumor site. In conclusion, Pep-1 modified PAMAM was a promising nanocarrier for targeted delivery of brain glioma.

Effect of pH and penetration enhancers on cysteamine stability and trans-corneal transport.

Ocular cystinosis is a rare metabolic disorder characterized by the presence of insoluble cystine crystals inside the corneal stroma, with consequent photophobia, keratopathies and frequent corneal erosions. The current therapy consists in the lifetime ophthalmic administration of cysteamine, drug characterized by extremely high hydrophilicity, low molecular weight (77g/mol), and easy oxidization to disulfide. Ocular delivery of cysteamine is very challenging, for its poor permeability and stability in solution. The purpose of the present paper was to study the impact of formulation pH and composition on (1) the trans-corneal delivery and (2) the stability in solution of cysteamine, with particular focus on the use of alpha-cyclodextrin (α-CD), benzalkonium chloride (BAC) and disodium edetate (EDTA). Permeation experiments were performed ex vivo through freshly excised porcine cornea; stability was evaluated for six months at -20°, +4° and +25°C; irritation potential was evaluated using HET-CAM assay. The results showed that cysteamine trans-corneal diffusion is strictly dependent on both pH (7.4 preferred to 4.2) and buffering capacity, that negatively impact on the permeation; EDTA did not enhance the trans-corneal diffusion of cysteamine neither at pH 7.4 nor at pH 4.2, while benzalkonium chloride (BAC), antimicrobial agent present within commercial eye-drops, significantly enhanced it. Notably, α-CD was able to promote the trans-corneal diffusion of cysteamine and, at a 5.5%, a 4-fold higher penetration compared to the BAC-containing formulation was obtained. EDTA and acidic pH demonstrated to be essential for cysteamine stability. The formulation obtained by combining α-CD and EDTA was characterized by significant permeation, good stability profile, and no irritation potential, even if the tolerability should be further confirmed by in vivo test.

An Open-Label Investigation of the Pharmacokinetics and Tolerability of Oral Cysteamine in Adults with Cystic Fibrosis.

BACKGROUND AND OBJECTIVE: Cysteamine is licensed for use in nephropathic cystinosis but preclinical data suggest a role in managing cystic fibrosis (CF). This study aimed to determine whether oral cysteamine is absorbed in adult CF patients and enters the bronchial secretions. Tolerability outcomes were also explored. METHODS: Patients ≥18 years of age, weighing >50 kg with stable CF lung disease were commenced on oral cysteamine bitartrate (Cystagon(®)) 450 mg once daily, increased weekly to 450 mg four times daily. Serial plasma cysteamine concentrations were measured for 24 h after the first dose. Participants were reviewed every week for 6 weeks, except at 4 weeks. Plasma cysteamine concentrations were measured 8 h after dosing when reviewed at 1, 2 and 3 weeks and 6 h after dosing when reviewed at 5 weeks. Sputum cysteamine concentration was also quantified at the 5-week assessment. RESULTS: Seven of the ten participants reported adverse reactions typical of cysteamine, two participants discontinued intervention. Following the first 450-mg dose, mean (SD) maximum concentration (C max) was 2.86 (1.96) mg/l, the time corresponding to C max (T max) was 1.2 (0.7) h, the half-life (t ½) was 3.7 (1.7) h, clearance (CL/F) 89.9 (30.5) L/h and volume of distribution (V d/F) 427 (129) L. Cysteamine appeared to accumulate in sputum with a median (interquartile range) sputum:plasma cysteamine concentration ratio of 4.2 (0.98-8.84). CONCLUSION: Oral cysteamine is absorbed and enters the bronchial secretions in patients with CF. Although adverse reactions were common, the majority of patients continued with cysteamine. Further trials are required to establish the risk benefit ratio of cysteamine therapy in CF.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts 'on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment.

Synthesis of diacylated γ-glutamyl-cysteamine prodrugs, and in vitro evaluation of their cytotoxicity and intracellular delivery of cysteamine.

To overcome the major disadvantages of cysteamine, the only registered treatment for the rare genetic disease cystinosis, nine prodrugs of γ-glutamyl-cysteamine (4) were synthesized for evaluation. Esterification of the thiol conferred oxidative stability, while sufficient lipophilicity for oral bioavailability was achieved by acylation of the α-carboxyl group of γ-glutamyl-cysteamine (4). Low cytotoxicity was observed in cultured HaCaT keratinocytes using the MTT assay, with EC50 values higher than or similar to that of cysteamine. Successful uptake of the esterified prodrugs and the subsequent release of cysteamine into cultured human proximal tubule epithelial cells were demonstrated using CMQT derivatisation and HPLC with UV detection. These prodrugs show potential as novel delivery vehicles of cysteamine to improve the treatment of the genetic disorder nephropathic cystinosis.

Development of 4-hexadecyl-4,7-diaza-1,10-decanedithiol (HDD) kit for the preparation of the liver cancer therapeutic agent Re-188-HDD/lipiodol.

INTRODUCTION: A lipiodol solution of (188)Re-4-hexadecyl-2,2,9,9-tetramethyl-4,7-diaza-1,10-decanedithiol (HTDD) has been successfully developed for liver cancer therapy; however, its preparation requires a multi-step synthesis and it is characterized by a low labeling yield. METHODS: We synthesized a new compound, 4-hexadecyl-4,7-diaza-1,10-decanedithioacetate (AHDD), without gem dimethyl groups to address these issues. AHDD was formulated into a kit and was labeled with (188)Re. Biodistribution study was performed using normal BALB/c mice. RESULTS: The kit was labeled with (188)Re with a high efficiency (98.8±0.2%). After extraction with lipiodol, the overall yield of (188)Re-HDD/lipiodol was as high as 90.2±2.6%. A comparative biodistribution study of (188)Re-HTDD and (188)Re-HDD was performed in normal mice after intravenous injection. The lungs were identified as the main uptake site due to capillary-blockage. (188)Re-HDD/lipiodol showed a significantly higher lung uptake than that of (188)Re-HTDD/lipiodol (p<0.05). CONCLUSION: The newly synthesized (188)Re-HDD/lipiodol showed improved radiolabeling yield and biodistribution results compared to (188)Re-HTDD/lipiodol, and may therefore be more suitable for liver cancer therapy.

Pharmacokinetics of cysteamine bitartrate following intraduodenal delivery.

Cysteamine is approved for the treatment of cystinosis and is being evaluated for Huntington's disease and non-alcoholic fatty liver disease. Little is known about the bioavailability and biodistribution of the drug. The aim was to determine plasma, cerebrospinal fluid (CSF), and tissue (liver, kidney, muscle) cysteamine levels following intraduodenal delivery of the drug in rats pretreated and naïve to cysteamine and to estimate the hepatic first-pass effect on cysteamine. Healthy male rats (n = 66) underwent intraduodenal and portal (PV) or jugular (JVC) venous catheterization. Half were pretreated with cysteamine, and half were naïve. Following intraduodenal cysteamine (20 mg/kg), serial blood samples were collected from the PV or the JVC. Animals were sacrificed at specific time points, and CSF and tissue were collected. Cysteamine levels were determined in plasma, CSF, and tissue. The Cmax was achieved in 5-10 min from PV and 5-22.5 min from JVC. The PV-Cmax (P = 0.08), PV-AUC0-t (P = 0.16), JVC-Cmax (P = 0.02) and JVC-AUC0-t (P = 0.03) were higher in naive than in pretreated animals. Plasma cysteamine levels returned to baseline in ≤120 min. The hepatic first-pass effect was estimated at 40%. Peak tissue and CSF cysteamine levels occurred ≤22.5 min, but returned to baseline levels ≤180 min. There was no difference in CSF and tissue cysteamine levels between naïve and pretreated groups, although cysteamine was more rapidly cleared in the pretreated group. Cysteamine is rapidly absorbed from the small intestine, undergoes significant hepatic first-pass metabolism, crosses the blood brain barrier, and is almost undetectable in plasma, CSF, and body tissues 2 h after ingestion. Sustained-release cysteamine may provide prolonged tissue exposure.

Feasibility of corneal drug delivery of cysteamine using vitamin E modified silicone hydrogel contact lenses.

Cystinosis is an inherited genetic disease characterized by the accumulation of cystine crystals in several tissues including the cornea. The corneal manifestations of cystinosis are treated by hourly instillation of cysteamine eye drops each day while awake. The high frequency of eye drop instillation along with the long duration of treatment leads to poor compliance in many patients. We have combined in vitro experiments with mathematical modeling to investigate the feasibility of daily use of cysteamine loaded contact lenses to replace the hourly instillation of drops. Our approach was based on incorporation of vitamin E diffusion barriers into commercially available contact lenses to increase the duration of drug release. Contact lenses were first soaked in a solution of vitamin E in ethanol. Subsequently, the lenses were soaked in an aqueous solution of cysteamine to load the drug. The drug release profiles from vitamin E treated lenses were measured under sink conditions. In addition, drug oxidation rates were measured after exposing drug loaded contact lenses to humidified room air. To study further the feasibility of using contact lenses for the delivery of cysteamine, a mass transfer model was used to determine the rates at which the drug loaded in the lens is delivered to the cornea. The results show that vitamin E loading increases the release duration from 10 min to about 3 h in solution, thus allowing the possibility of extended drug delivery. In addition to improving the release profiles, vitamin E loading also improved the drug stability by reducing the oxidation rates. The mathematical modeling of drug transport in the eye suggested that the vitamin E loaded contact lens can provide the daily therapeutic dose without causing toxicity, while significantly increasing the bioavailability compared to eye drops. Based on the in vitro experimental results and the mathematical modeling, it is likely that a single contact lens worn for about 2h could achieve the same therapeutic effects as hourly instillation of eye drops.

Folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation.

BACKGROUND: A novel dual ligand-modified liposome, folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier (FP-Lipo), was designed to overcome the nonselectivity of conventional penetrating peptide-tagged nanoparticulates and to provide the advantage of selective targeting of the folic acid receptor, which is frequently overexpressed on epithelial cancer cells. METHODS: FP-Lipo was prepared by a sequential process of formation of a maleimide-derivatized small unilamellar vesicle, postinsertion of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-folate to the vesicle, and Pep-1 peptide conjugation via thiol-maleimide linkage. Conformational and physical characteristics of the FP-Lipo nanocarriers were investigated for the extent of Pep-1 peptide and folic acid on the surface, vesicle size, and zeta potential. In vitro cellular uptake behaviors of the novel carrier containing a fluorescein dextran isothiocyanate probe were examined by spectrophotometry or by confocal laser scanning microscopy. RESULTS: A novel nanocarrier bearing approximately 750 folate ligands and 100 penetrating peptides per vesicle was successfully prepared. The physical properties were as follows: 140 nm in size; 5 mV in zeta potential; less than 0.3 in polydispersity index. An in vitro cellular uptake study revealed that the FP-Lipo nanocarrier system exhibited more than twofold enhanced translocation into the folic acid receptor-positive HeLa cells compared with the single Pep-1 peptide-modified liposome. Meanwhile, its cellular association and internalization into the folic acid receptor-negative normal HaCaT cells was comparable with that of Pep-1 peptide-modified liposome. CONCLUSION: An advanced dual ligand-modified liposome is potentially useful for the treatment of folic acid receptor-positive tumors with high translocation capability of the penetrating peptide-modified liposome.

Halitosis in cystinosis patients after administration of immediate-release cysteamine bitartrate compared to delayed-release cysteamine bitartrate.

Halitosis due to dimethylsulfide (DMS) generation is a major side effect of cysteamine in the treatment of cystinosis. Recently, an enteric coated formulation of cysteamine bitartrate (RP103) administered twice daily was demonstrated to be non-inferior for lowering WBC cystine levels compared to the non-enteric coated formulation (Cystagon®), administered 4 times per day. Since both formulations had different pharmacokinetic profiles, we compared DMS breath levels after administration of either RP103 or Cystagon® in four cystinosis patients. Although cysteamine areas under the curve (AUCs) were comparable, AUC of DMS was lower after the administration of RP103 compared to Cystagon®. This observation is of importance in cystinosis patients, since halitosis hampers compliance with cysteamine therapy.

Population pharmacokinetics and pharmacodynamics of cysteamine in nephropathic cystinosis patients.

BACKGROUND: Nephropathic cystinosis is an autosomal recessive disorder resulting in an impaired transport of cystine trough the lysosomal membrane causing an accumulation of free cystine in lysosomes. The only specific treatment for nephropathic cystinosis is cysteamine bitartrate. This study was aimed to describe the relationship between cysteamine plasma concentrations and white blood cell cystine levels, and to simulate an optimized administration scheme to improve the management of patients with cystinosis. METHODS: Cysteamine and cystine concentrations were measured in 69 nephropathic cystinosis patients. A total of 250 cysteamine plasma concentrations and 243 intracellular cystine concentrations were used to perform a population pharmacokinetic and pharmacodynamic analysis. An optimized administration scheme was simulated in order to maintain cystine levels below 1 nmol half-cystine/mg of protein and to investigate the possibility of administrating the treatment less than 4 times a day (QID, recommended). The current dosing recommendations are 1.3 g/m2/day for less than 50 kg BW and 2 g/day thereafter; the maximum dose should not exceed 1.95 g/m2/day. RESULTS: Cysteamine concentrations were satisfactorily described by a one-compartment model. Parameter estimates were standardized for a mean standard bodyweight using an allometric model. WBC cystine levels were adequately described by an indirect response model where the first-order removal rate constant is stimulated by the cysteamine concentrations. CONCLUSIONS: According to simulations, in order to increase the percentage of patient with cystine levels below 1 nmol half-cystine/mg of protein, the current dosages could be changed as follows: 80 mg/kg/day (QID) from 10 to 17 kg, 70 mg/kg/day (QID) from 17 to 25 kg, 60 mg/kg/day (QID) from 25 to 40 kg and 50 mg/kg/day (QID) from 40 to 70 kg (these dosages remain under the maximum recommended dose). However an 8-hourly daily treatment (TID) did not provide acceptable cystine levels and should not be proposed.

Pharmacokinetics of enteric-coated cysteamine bitartrate in healthy adults: a pilot study.

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 (<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 (P<0.05). One patient had GI symptoms following both 900 mg cysteamine bitartrate non-enteric-coated and cysteamine bitartrate enteric-coated. CONCLUSION: 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.

Cysteamine, the molecule used to treat cystinosis, potentiates the antimalarial efficacy of artemisinin.

Malaria continues to be a major threat to global health. Artemisinin combination therapy (ACT) is the recommended treatment for clinical malaria; however, recent reports of parasite resistance to artemisinin in certain areas where malaria is endemic have stressed the need for developing more efficacious ACT. We report that cysteamine (Cys), the aminothiol used to treat nephropathic cystinosis in humans, strongly potentiates the efficacy of artemisinin against the Plasmodium parasite in vivo. Using a mouse model of infection with Plasmodium chabaudi AS, we observe that Cys dosing used to treat cystinosis in humans can strongly potentiate (by 3- to 4-fold) the antimalarial properties of the artemisinin derivatives artesunate and dihydroartemisinin. Addition of Cys to suboptimal doses of artemisinin delays the appearance of blood parasitemia, strongly reduces the extent of parasite replication, and significantly improves survival in a model of lethal P. chabaudi infection. Cys, the natural product of the enzyme pantetheinase, has a history of safe use for the clinical management of cystinosis. Our findings suggest that Cys could be included in novel ACTs to improve efficacy against Plasmodium parasite replication, including artemisinin-resistant isolates. Future work will include clinical evaluation of novel Cys-containing ACTs and elucidation of the mechanism underlying the potentiation effect of Cys.

Intracellular delivery of proteins into mouse Müller glia cells in vitro and in vivo using Pep-1 transfection reagent.

Direct protein transfection is a potentially valuable tool for studying protein function in basic and clinical research. A major challenge is to enable a sufficiently large amount of protein to penetrate the plasma membrane of the transfected cells. Pep-1, a protein transfection reagent, was evaluated for its ability and efficiency in delivering proteins and antibodies into mouse Müller cells in vitro and in vivo. Pep-1 delivered active beta-galactosidase enzyme and antibodies (non-specific IgG and Cy3-conjugated anti-vimentin) into cultured Müller cells with high efficiency. Transfection efficiency increased with increasing concentration of the protein in the complex and with incubation time. Following intravitreal injection of Pep-1/IgG complexes in vivo, retinal histology was preserved and immunostaining showed that the antibodies were distributed widely across the retinal surface, with the most intense staining located near the retino-vitreal border. For complexes using non-specific IgG, double staining with anti-glutamine synthetase identified many IgG-positive cells as Müller glia. IgG immunoreactivity was also detected in the cytoplasm and occasionally in the nuclei of inner retinal neurons. Dark-adapted flash electroretinogram (ERG) recordings from injected eyes were nearly identical to ERG recordings from control eyes, suggesting that injection of Pep-1/IgG complex has minimal effects on retinal function. Therefore, Pep-1 is a useful tool for intracellular delivery of antibodies to study the role of proteins in living cells.