HPLC/UV/MS method application for the separation of obeticholic acid and its related compounds in development process and quality control.

An HPLC method with UV and electrospray ionization - mass spectrometry (ESI-MS) detection was developed for the separation and determination of obeticholic acid (OBE) and its related compounds in development process and quality control. OBE and its related compounds were classified into three major group based on the mass spectra profiles: (A) those containing a hydroxyl group at position 3 and 7, (B) those containing a hydroxyl group and/or carbonyl group at position 3, hydrogen, ethyl or ethylidene group at position 6 and a hydroxyl group and/or carbonyl group at position 7, and (C) those containing carbonyl groups at position 3 and 7. ESI-MS ionization of OBE and its related compounds often produced intense adduct ions [M+H+98]+ and/or [M+H+196]+ that were identified as the adduct ions of phosphoric acid ([M+H+H3PO4]+ and [M+H+2H3PO4]+) originating from the mobile phase. The separation on HPLC system was accomplished using stationary phase based on XSelect CSH C18 (3.0×150mm×2.5µm) and a linear gradient elution using acetonitrile and 0.05% of o-phosphoric acid. The condition of chromatographic system was set as follows: flow rate 0.7mL/min, temperature 45°C and UV detection at 192nm. The separation of the 19 compounds was finished in less than 18min (including equilibration time). The HPLC/UV method was partially validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines in terms of repeatability, selectivity, linearity and limit of quantification and detection.

A novel approach for HPLC determination of 2-cynaoacetamide using derivatization procedure with 2-hydroxyacetophenone as a new useful derivatization reagent.

A novel and sensitive derivatization procedure for the determination of 2-cynaoacetamide in pharmaceutical samples using liquid chromatography with the fluorescence detection was discovered. The method is based on derivatization of 2-cynaoacetamide using 2-hydroxyacetophenone as a new derivatization reagent. The product of derivatization reaction was isolated and characterized using spectroscopic techniques namely LC-MS, NMR and IR. The structure of 2-cyanoacetamide derivative was unambiguously assigned as a 2-amino-4-phenylfuran-3-carboxamide. Two derivatization systems were optimized in terms of reaction temperature, reaction time, pH and concentration of 2-hydroxyacetophenone, and a new pre- and post-derivatization HPLC methods were developed. The separations on HPLC with pre-column derivatization were accomplished using stationary phase based on a XBridge C18 column (100×4.6, 3.5µm) and isocratic elution using the mobile phase acetonitrile - 0.1% formic acid (30:70, v/v). The separations on the HPLC with post-column derivatization were performed on stationary phase on a TSKgel Amide-80 column (150×4.6mm, 3µm). The mobile phase was a mixture of acetonitrile, methanol and 10mM sodium formate buffer at pH=4.5 in ratio 3:2:95 (v/v). Both HPLC methods were fully validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy and precision according to ICH guidelines. The pre-column derivatization method was linear in the range 1.1-2000µg/l with method accuracy≥98.2% and method precision RSD≤4.8%. The post-column derivatization method was linear in the range 12-2000µg/l. Method accuracy≥96.3% and method precision RSD≤3.5%. Proposed new methods were proved to be highly sensitive, simple and rapid, and were successfully applied to the determinations of 2-cynaoacetamide in pregabalin.

Synthesis and pharmacological effects of the enantiomers of the N-phenethyl analogues of the ortho and para e- and f-oxide-bridged phenylmorphans.

The N-phenethyl analogues of (1R*,4aR*,9aS*)-2-phenethyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2,3-c]pyridin-6-ol and 8-ol and (1R*,4aR*,9aR*)-2-phenethyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2.3-c]pyridin-6-ol and 8-ol, the ortho- (43) and para-hydroxy e- (20), and f-oxide-bridged 5-phenylmorphans (53 and 26) were prepared in racemic and enantiomerically pure forms from a common precursor, the quaternary salt 12. Optical resolutions were accomplished by salt formation with suitable enantiomerically pure chiral acids or by preparative HPLC on a chiral support. The N-phenethyl (-)- para-e enantiomer (1S,4aS,9aR-(-)-20) was found to be a mu-opioid agonist with morphine-like antinociceptive activity in a mouse assay. In contrast, the N-phenethyl (-)-ortho-f enantiomer (1R,4aR,9aR-(-)-53) had good affinity for the mu-opioid receptor (K(i) = 7 nM) and was found to be a mu-antagonist both in the [(35)S]GTP-gamma-S assay and in vivo. The molecular structures of these rigid enantiomers were energy minimized with density functional theory at the level B3LYP/6-31G* level, and then overlaid on a known potent mu-agonist. This superposition study suggests that the agonist activity of the oxide-bridged 5-phenylmorphans can be attributed to formation of a seven membered ring that is hypothesized to facilitate a proton transfer from the protonated nitrogen to a proton acceptor in the mu-opioid receptor.

Profound inhibition of antigen-specific T-cell effector functions by dasatinib.

PURPOSE: The dual BCR-ABL/SRC kinase inhibitor dasatinib entered the clinic for the treatment of chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia. Because SRC kinases are known to play an important role in physiologic T-cell activation, we analyzed the immunobiological effects of dasatinib on T-cell function. The effect of dasatinib on multiple T-cell effector functions was examined at clinically relevant doses (1-100 nmol/L); the promiscuous tyrosine kinase inhibitor staurosporine was used as a comparator. EXPERIMENTAL DESIGN: Purified human CD3+ cells and virus-specific CD8+ T cells from healthy blood donors were studied directly ex vivo; antigen-specific effects were confirmed in defined T-cell clones. Functional outcomes included cytokine production (interleukin-2, IFN gamma, and tumor necrosis factor alpha), degranulation (CD107a/b mobilization), activation (CD69 up-regulation), proliferation (carboxyfluorescein diacetate succinimidyl ester dilution), apoptosis/necrosis induction, and signal transduction. RESULTS: Both dasatinib and staurosporine inhibited T-cell activation, proliferation, cytokine production, and degranulation in a dose-dependent manner. Mechanistically, this was mediated by the blockade of early signal transduction events and was not due to loss of T-cell viability. Overall, CD4+ T cells seemed to be more sensitive to these effects than CD8+ T cells, and naïve T cells more sensitive than memory T-cell subsets. The inhibitory effects of dasatinib were so profound that all T-cell effector functions were shut down at therapeutically relevant concentrations. CONCLUSION: These findings indicate that caution is warranted with use of this drug in the clinical setting and provide a rationale to explore the potential of dasatinib as an immunosuppressant in the fields of transplantation and T-cell-driven autoimmune diseases.