In-vitro metabolic profiling study of potential topoisomerase inhibitors 'pyrazolines' in RLMs by mass spectrometry.

Taking into consideration of the cytotoxicity and topo-IIα inhibitory activity of pyrazoline derivatives (1-3) against HCT15 cells, and known topo-IIα inhibitor, etoposide, respectively, the compounds were biotransformed in rat liver microsomes. LC-MS/MS and MALDI mass spectrometric techniques has been used for analysis. All three compounds were biotransformed into demethylated metabolites. Among three compounds, compounds 1 and 2 were biotransformed into mono-hydroxylated metabolites and compound 3 biotransformed into reduced and epoxidized metabolites. Reduced and reduced along with demethylation metabolites were identified from MALDI Orbitrap spectrometric analysis. Without NADPH or microsomes no compounds (1-3) were generated metabolites, it shows CYP450 enzymes involvement in the presence of NADPH in the metabolisms.

Gemifloxacin mesylate (GFM): dissolution test based on in vivo data.

Gemifloxacin mesylate (GFM) is a synthetic, broad-spectrum, fluoroquinolone antibacterial agent. It is different from other class members because it achieves adequate plasma concentrations to inhibit both topoisomerase IV and gyrase. The aim of this study was to develop and validate a dissolution test for GFM in coated tablets, using a simulated absorption profile based on in vivo data obtained from the literature. The fraction and percentage of the dose absorbed were calculated using model-dependent Loo-Riegelman approach for two compartments. The best in vitro dissolution profile was obtained using 900 mL of pH 6.0 phosphate buffer as a dissolution medium at 37 °C ± 0.5 °C and paddles at 50 rpm. The in vitro dissolution samples were analyzed using a liquid chromatography method, and the validation was performed according to USP 34 (2011). The method showed specificity, precision, accuracy, robustness and linearity. Under these conditions, a level-A in vitro-in vivo correlation was suggested (r = 0.9926). The prediction errors were calculated to determine the validity and accuracy of the suggested correlation. The dissolution test can be used to evaluate the dissolution profile of GFM-coated tablets and minimize the number of bioavailability studies as part of new formulation development.

Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons.

Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A). In neurons, the paternal allele of UBE3A is intact but epigenetically silenced, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.