A study of the relative bioavailability of cysteamine hydrochloride, cysteamine bitartrate and phosphocysteamine in healthy adult male volunteers.
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AIMS: Cysteamine, the only drug available for the treatment of cystinosis in paediatric patients, is available as the hydrochloride, the bitartrate and as sodium phosphocysteamine salts. It has been suggested that cysteamine bitartrate and phosphocysteamine are better tolerated and may have a better bioavailability than cysteamine hydrochloride. This has, however, never been demonstrated. METHODS: We compared the pharmacokinetics and tolerance of these three formulations of cysteamine in 18 healthy adult male volunteers in a double-blind, latin-square, three-period, single oral dose cross-over relative bioavailability study. RESULTS: No statistical difference was found between relative bioavailabilities, AUC (0, infinity) (geometric mean and s.d. in micromol l(-1) h: 169+/-51, 158+/-46, 173+/-49 with cysteamine hydrochloride, phosphocysteamine and cysteamine bitartrate respectively), Cmax (geometric mean and s.d. in micromol l(-1); 66+/-25.5, 59+/-12, 63+/-20) and tmax (median and range in h: 0.88 (0.25-2), 1.25 (0.25-2), 0.88 (0.25-2)) with each of the three forms of cysteamine tested. Bioequivalence statistics (90% confidence intervals) showed non equivalence of Cmax of cysteamine base as the only non equivalence of pharmacokinetics between the three formulations: 90% CI for Cmax relative ratios to cysteamine hydrochloride were [75.6-105.81 for phosphocysteamine and [74.2-124.2] for cysteamine bitartrate. The only significant adverse event was vomiting whose frequency was inversely correlated with body weight (Spearman's r=-0.76, P<0.001). The nature of the salt tested did not influence vomiting. CONCLUSIONS: While none of the three forms of cysteamine tested has a clear advantage over the others in terms of pharmacokinetics and tolerance profile, this should now however be addressed in patients treated for cystinosis during repeat administrations. (+info)
Exogenous cysteamine increases basal pancreatic exocrine secretion in the rat.
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To determine whether exocrine pancreatic secretion is regulated by endogenous somatostatin, somatostatin deficiency was induced by cysteamine. Rats were subcutaneously administered a single dose of cysteamine (30 mg/100 g body weight) 12 hr before experiment. Anesthetized rats were prepared with cannulation into bile duct, pancreatic duct, duodenum, and jugular vein and pancreatic juice was collected. For in vitro study, isolated pancreata of rats, pretreated with cysteamine, were perfused with an intraarterial infusion of Krebs-Henseleit solution (37 degrees C) at 1.2 mL/min, and pancreatic juice was collected in 15-min samples. In vivo experiment of the rat, the mean basal pancreatic secretions, including volume, bicarbonate, and protein output were significantly increased from 18.4+/-0.5 microL/30 min, 0.58+/-0.05 microEq/30 min, and 214.0+/-26.1 microg/30 min to 51.6+/-3.7 microL/30 min, 1.52+/-0.11 microEq/30 min, and 569.8+/-128.9 microg/30 min, respectively (p<0.05). In the isolated perfused pancreas, cysteamine also resulted in a significant increase in basal pancreatic secretion (p<0.05). Simultaneous intraarterial infusion of octreotide (10 pmol/hr) to isolated pancreata partially reversed the effect of cysteamine on basal pancreatic secretion. These findings suggest that endogenous somatostatin play an important role on the regulation of basal pancreatic exocrine secretion. (+info)
Male pronuclear formation in denuded porcine oocytes after in vitro maturation in the presence of cysteamine.
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The present study was conducted to examine effects of cysteamine in culture medium on progression of meiosis, glutathione (GSH) content, kinase activities (histone H1 kinase and mitogen-activated protein kinase), and male pronuclear formation after in vitro insemination of cumulus-denuded oocytes (DOs) in the pig. DOs, obtained by mechanically removing cells from cumulus-oocyte complexes (COCs) with a small-bore pipette, were cultured for 45 h in TCM199 supplemented with sodium pyruvate, gonadotropins, estradiol, and 10% porcine follicular fluid, with or without cysteamine (150 microM). Maturation rates of DOs cultured with and without cysteamine were not different (60-70%) but were significantly lower than those of COCs (90-100%) (p < 0.05). GSH content of matured DOs cultured with cysteamine was significantly higher than that of DOs cultured without cysteamine (p < 0.05). Values for both types of kinase activity in matured DOs cultured with and without cysteamine were not different (p > 0.05). After in vitro insemination, DOs cultured with cysteamine showed significantly higher rates of male pronuclear formation (80.3 +/- 3.0%) than DOs cultured without cysteamine (16.4 +/- 0.5%) (p < 0.05). These results indicate that the addition of cysteamine to culture medium increased oocyte GSH content and promoted male pronuclear formation after sperm penetration of porcine DOs but had no effects on their maturation rates or kinase activities. (+info)
New method for determining cystine in leukocytes and fibroblasts.
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BACKGROUND: Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required. METHODS: Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection. RESULTS: The assay was linear to 200 micromol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 micromol/L. Added cysteine, up to 200 micromol/L, was completely removed, and recovery of added cystine was 69-86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 degrees C CONCLUSIONS: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine. (+info)
Selective incorporation and specific cytocidal effect as the cellular basis for the antimelanoma action of sulphur containing tyrosine analogs.
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Tyrosine analogs are good candidates for developing melanoma chemotherapy because melanogenesis is inherently toxic and uniquely expressed in melanocytic cells. Sulphur containing substrate (tyrosine) analogs, N-acetyl-4-S-cysteaminylphenol (NAcCAP) and N-propionyl-4-S-cysteaminylphenol (NPrCAP), have been shown to have potent antimelanoma activity in mice bearing melanoma. Both NAcCAP and NPrCAP show selective cytotoxicity towards melanoma cell lines. But the mechanism leading to selectivity is not clear as these drugs are also toxic to other cell lines to a lesser extent. Here we show that these drugs have both cytostatic and cytocidal effects, which could account for this. Cytostatic effect is suggested by DNA flow cytometry. The drug causes cell cycle changes in four human cell lines (normal skin fibroblasts, HeLa cells, and melanoma cell lines, C32 and SK-MEL-23) in a dose-dependent manner blocking cells in S phase with concomitant decrease in the number of cells in G1 phase. There is also a gradual decrease in cells in G2 + M phases. The dose-concentration curves give IC50 values in the range of 50-400 microM and the melanotic melanoma cell line SK-MEL-23 has the lowest IC50 value consistent with our hypothesis that these drugs are selective towards melanoma cells. The concentration-dependent accumulation of cells in S phase suggest a cytostatic effect as a consequence of inhibition of DNA synthesis in agreement with [3H] thymidine incorporation assay. There is a highly specific uptake of [14C]NAcCAP and irreversible damage to DNA synthesis machinery in SK-MEL-23 cells, indicating a melanotic-specific cytocidal effect as well. Trypan blue exclusion study and competitive inhibition assay indicated that visible cytocidal effect occurs slowly and oxidative stress resulting from tyrosinase mediated oxidation of the drug appears to be the underlying mechanism. The primary antimelanoma effect of cysteaminylphenols derives from a selective cytostatic effect, but is followed by a specific cytocidal action rendering the drugs useful for targeted melanoma chemotherapy. (+info)
Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling.
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Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component. (+info)
Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases.
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BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation. The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate specificity is important for the engineered biosynthesis of new compounds. RESULTS: We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC. Comparing L- and D-Phe-SNACs as upstream (donor) substrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity. CONCLUSIONS: Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate specificity of C domain-catalyzed peptide bond formation. (+info)
Contribution of dithiol ligands to in vitro and in vivo trypanocidal activities of dithiaarsanes and investigation of ligand exchange in an aqueous solution.
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Twelve new dithiaarsanes were evaluated for their in vitro and in vivo trypanocidal properties in regard to their three parent molecules, 4-amino-phenylarsenoxide, melarsenoxide, and 4-dansylamino-phenylarsenoxide. The most potent dithiaarsane, compound 2b, had a minimum effective concentration of 1.5 nM after 48 h of incubation and at a dose of 0.39 micromol/kg of body weight (0.2 mg/kg) administered subcutaneously cured 100% of mice acutely infected with Trypanosoma brucei brucei CMP. With this model, the chemotherapeutic index of compound 2b was 512, compared to 256 for melarsamine dihydrochloride (Cymelarsan) under the same conditions. With a chronic infection produced by T. brucei brucei GVR, compound 2b cured 100% of mice after treatment at a dose of 25 micromol/kg (12.5 mg/kg) for 4 consecutive days, whereas melarsamine dihydrochloride and potassium melarsonyl (Trimelarsan) cured less than 50% mice at this dose. For both acute and late-stage infections, dithiaarsanes having a melaminophenyl ring exhibited the most-potent trypanocidal activity. Compound 2b is thus one of the most active organoarsenicals described in a mouse trypanosomiasis model. Considering that the main intracellular targets of organoarsenicals are thiol groups, we studied the possibility of ligand exchange between Cymelarsan and several dithiols. In aqueous solution, we observed a rapid exchange of cysteamine from melarsamine with free cysteamine and also with various dithiols always in favor of more stable cyclic derivatives. These ligand exchanges suggest the ability of trivalent organoarsenicals to react with targets such as trypanothione and dihydrolipoic acid. Among several ligands, a 1,3-dimercaptopropane moiety appeared the most suitable for trypanocidal activity. (+info)