Abstract Gaucher disease is an autosomal recessive disease, caused by a lack or functional deficiency of the lysosomal enzyme, glucocerebrosidase (GCase). Recently, mutations in the ...glucocerebrosidase gene (GBA) have been associated with Parkinson's disease (PD) and GBA mutations are now considered the most important genetic vulnerability factor for PD. In this study, we have investigated (i) in vivo whether inhibition of the enzyme glucosylceramide synthase by miglustat may protect C57Bl/6 mice against subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and (ii) in vitro whether a decrease of GCase activity may render dopaminergic neurons susceptible to MPP+ (1-methyl-4-phenylpyridinium) or alpha-synuclein (α-Syn) toxicity and amenable to miglustat treatment. We could demonstrate that reduction of glucocerebroside by inhibition of glucosylceramide synthase partially protects mice against MPTP-induced toxicity. Conversely, we could show that inhibition of GCase activity with conduritol-B-epoxide (CBE) enhances both α-Syn and MPP+ induced toxicity in vitro . However, only CBE-induced enhancement of MPP+ toxicity could be reversed by miglustat. Moreover, we were unable to reveal any alterations of complex I activity or cell respiration upon treatment with either CBE or miglustat. Our findings suggest that the reduction of GCase activity rather than an accumulation of glucocerebroside increases aSyn toxicity.
We describe the discovery and optimization of new, brain-penetrant T-type calcium channel blockers. We present optimized compounds with excellent efficacy in a rodent model of generalized ...absence-like epilepsy. Along the fine optimization of a chemical series with a pharmacological target located in the CNS (target potency, brain penetration, and solubility), we successfully identified an Ames negative aminopyrazole as putative metabolite of this compound series. Our efforts culminated in the selection of compound 20, which was elected as a preclinical candidate.
Biomarkers have shown to improve success rates in the development of novel drugs, providing essential information in the early phases of clinical development for decision-making. Chemoattractant ...receptor-homologous molecule expressed on Th2 cells (CRTH2) is pursued as a drug target for a number of inflammatory diseases. CRTH2 antagonists block the activation and migration of key inflammatory cells such as eosinophils, basophils, and Th2 cells. The mechanism of action of CRTH2 antagonists was established in cells isolated from human blood. Biomarkers derived from these experiments were included in clinical studies to investigate the mechanism of action and potency of CRTH2 antagonists in human. For clinical phase I studies with the CRTH2 antagonist ACT-453859, a follow-up molecule of setipiprant, inclusion of the most precise and robust pharmacodynamic (PD) biomarker with a clinically relevant target effect was desired to aid phase II dose selection.
Candidate biomarkers such as IL-13 secretion from Th2 cells and CRTH2, CD11b and CD203 modulation on basophils and eosinophils in whole blood were compared in terms of signal intensity and variability. Blockade of CRTH2 receptor internalization was finally chosen as PD biomarker and rigorously tested in a feasibility study. The assay showed excellent robustness, an intra-assay precision of 5% and inter-subject variability smaller than 15%. Based on phase II clinical study results with setipiprant, 90% CRTH2 receptor blockade was defined as clinically relevant PD effect. This target PD effect provides the means to take decisions based on the data generated in the phase I clinical studies with ACT-453859.
•BedsideBiomarkers offer a great potential to influence decisions taken during early clinical development. For clinical phase I studies with the CRTH2 antagonist ACT-453859, a follow-up molecule of setipiprant, inclusion of a biomarker was desired to aid phase II dose selection. In order to facilitate decision-making, we developed a biomarker that delivers high quality data under clinical circumstance and defined a relevant target biomarker effect.•BenchsideIn-vitro experiments with human whole blood identified CRTH2 receptor internalization on basophils and eosinophils as the most precise and robust biomarker. Clinical results obtained with setipiprant in a seasonal allergic rhinitis study were used to define the clinically relevant target biomarker effect of 90% CRTH2 receptor blockade. Proof for the chosen target biomarker effect remains to be demonstrated in phase II clinical studies with ACT-453859.
The transformation of S-methyl-14Cadenosyl-l-methionine (14C-SAM) and 2-(or 5-)methyl-14C-2,5-dimethyl-4-hydroxy-3(2H)-furanone (14C-1) was investigated in detached ripening strawberry fruits over a ...three-day period. Radiochemical analysis of the different fruit parts revealed that 46% and 51% of the applied 14C remained in the stems for 14C-SAM and 14C-1, respectively. In samples obtained by solid phase extraction, 1, 2,5-dimethyl-4-methoxy-3(2H)-furanone (2), and glycosidically bound 1 were analyzed by reversed-phase high-performance liquid chromatography. Incorporation of 14C-SAM and 14C-1 into 2 was observed with transformation rates of 0.4% and 0.3% of the applied 14C, respectively. In addition, 0.7% of the applied 14C-1 was recovered as glycosidically bound 1. The data support the conclusions that 14C-SAM is the source of the methyl group in the 4-methoxy compound 2 and that 1 is the precursor of 2 and the glycosides studied. Keywords: 2,5-Dimethyl-4-hydroxy-3(2H)-furanone; 2,5-dimethyl-4-methoxy-3(2H)-furanone; Furaneol; methoxyfuraneol; S-adenosyl-l-methionine; metabolism; strawberry
The transformation of 12 radioactively labeled compounds into 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF), glycosidically bound DMHF, and 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF) was ...investigated in detached ripening strawberry fruits (Fragaria × ananassa) over a 3-day period. Radiochemical analysis of the different fruit parts revealed that major portions of the applied radioactivity (up to 66%) remained in the stems and calyx. Incorporation levels of 2-14Cdihydroxyacetone, d-1-3Hglucose, d-U-14C-glucose, d-U-14Cglucose 6-phosphate, d-U-14Cfructose, and d-U-14Cfructose 1,6-bisphosphate into the total amount of furanone derivatives were 0.022, 0.032, 0.035, 0.147, 0.202, and 0.289% of the radioactivity entering the fruits, respectively. Minor amounts of radioactivity (<0.001%) were detected in the furanone structures after the administration of 1-14Cacetate and 3-14Cpyruvate. l-1-14CFucose, l-6-3Hfucose, l-1-3Hrhamnose, l-U-14Cthreonine, l-U-14Clactaldehyde, and 2-14Cmalonic acid were not transformed into DMHF or a derivative thereof. Keywords: Biosynthesis; carbohydrate metabolism; 2,5-dimethyl-4-hydroxy-3(2H)-furanone; Fragaria × ananassa; Rosaceae
Using synthesized reference compounds a screening for benzyl, 2-phenylethyl, geranyl, citronellyl, and 2,5-dimethyl-4-hydroxy-3(2H)-furanone 6‘-O-malonyl β-d-glucopyranosides in various plant tissues ...was performed by high-performance liquid chromatography−electrospray ionization−tandem mass spectrometry (HPLC−ESI−MS/MS). The results obtained with fruits (guava; raspberry; strawberry), leaves (green tea; vine), and mountain papaya (Carica pubescens) peel indicate that malonylation of glycoconjugates is a common pathway in plant secondary metabolism. Keywords: Malonylated glycosides; benzyl 6‘-O-malonyl β-d-glucopyranoside; 2-phenylethyl 6‘-O-malonyl β-d-glucopyranoside; geranyl 6‘-O-malonyl β-d-glucopyranoside; citronellyl 6‘-O-malonyl β-d-glucopyranoside; 2,5-dimethyl-4-hydroxy-3(2H)-furanone 6‘-O-malonyl β-d-glucopyranoside; HPLC−MS/MS, electrospray ionization (ESI)
The glycosidic fraction isolated from Vitis vinifera cv. Riesling leaves by XAD-2 adsorption and methanol elution was separated by multilayer coil countercurrent chromatography (MLCCC) to identify ...genuine precursors of the off-flavor-causing compound 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN). A twostep procedure utilizing preparative MLCCC in combination with analytical MLCCC yielded two major vine leaf glycosides in pure form, i.e., the beta-D-glucopyranosides of 3-oxo-7,8-dihydro-alpha-ionol (Blumenol C) and 3-oxo-4,5-dihydro-alpha-ionol, respectively. The determination of minor vine leaf constituents in separated MLCCC fractions was achieved after acetylation and subsequent HPLC purification of the per-O-acetylated derivatives. This procedure allowed the identification of 3,4-dihydroxy-7,8-dihydro-beta-ionone 3-O-beta-D-glucopyranoside as a major TDN precursor in Riesling leaves. In addition, the beta-D-glucopyranosides of 3,4-dihydroxy-beta-ionone and zingerone were identified
Regioselective acylation of glycoconjugates with malonic acid was achieved by using phenyl β-d-glucopyranoside as a model glycoside, malonic acid, and tert-butyl isocyanide in aprotic solvents. ...Structural elucidation of phenyl 6‘-O-malonyl-β-d-glucopyranoside was performed by 1H and 13C NMR spectroscopy and high-performance liquid chromatography−atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC−APCI-MS/MS). This one-step reaction opens the way to the preparation of reference substances which are required for the spectroscopic identification of malonylated glycosides in complex natural matrices. Keywords: Phenyl 6‘-O-malonyl-β-d-glucopyranoside; malonylated glycosides; HPLC−MS/MS; atmospheric pressure chemical ionization (APCI)
2,5-Dimethyl-4-hydroxy-32H-furanone 6'-O-malonyl-beta-D-glucopyranoside was isolated from a glycosidic extract of strawberry fruit (Fragaria x ananassa, cv. Senga Sengana) by means of countercurrent ...chromatography and reverse-phase HPLC. Identification was achieved by comparison of chromatographic and 1H, 13C and 2D-NMR, as well as mass spectral data with those of the synthesized reference compound. In ripe strawberry fruit, a 1:1 ratio of the malonylated glucoside to its deacylated glucoconjugate was determined by on-line liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.
PDF
