Protein covalent binding of maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in rats.

(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one) (MaxiPost, BMS-204352) is a potent and specific opener for maxi-K channels and has potential to prevent and treat ischemic stroke. Following single intravenous doses of [14C]BMS-204352 to rats, only 10 to 12% of radioactivity was extractable from plasma with organic solvents. The unextractable radioactivity remained associated with the proteins (mostly albumin) after SDS-polyacrylamide gel electrophoresis or dialysis. Following acid hydrolysis in 6 M HCl for 24 h at 110 degrees C from plasma proteins collected from nine rats dosed with [14C]BMS-204352, one major radioactive product was isolated and identified as a lysine-adduct of des-fluoro des-O-methyl BMS-204352 by liquid chromatography/mass spectrometry and NMR analyses as well as by comparison with the synthetic analog, lysine-adduct of des-fluoro BMS-204352 (BMS-349821). The covalent binding of BMS-204352 results from the displacement of the ring-fluorine atom of des-O-methyl BMS-204352 with the epsilon-amino group of a lysine residue. Microsomal incubations of [14C]BMS-204352 resulted in low levels of covalent binding of radioactivity to proteins. This in vitro covalent binding required cytochrome P450-reductase cofactor NADPH and was attenuated by glutathione. P4503A inhibitors ketoconazole and troleadomycin selectively prevented the covalent binding in vitro. Based on these observations, a two-step bioactivation process for the protein covalent binding of BMS-204352 was postulated: 1) P4503A-mediated O-demethylation leading to spontaneous release of HF and the formation of an ortho-quinone methide reactive metabolite and 2) nucleophilic addition of the epsilon-amino group of protein lysine residue(s) in protein to form des-fluoro des-O-methyl BMS-204352 lysine adduct.     

Two flares of Still’s disease after two doses of the ChAdOx1 vaccine

Clinical Rheumatology - We report the case of an 18-year-old male with Still’s disease for the last 3 years, in remission, who developed two flares of his disease after receiving two...     

Evaluation of Biocompatibility and Osteogenic Potential of Tricalcium Silicate–based Cements Using Human Bone Marrow–derived Mesenchymal Stem Cells

Introduction

The success of endodontic regeneration lies in the appropriate combination of stem cells and bioactive materials. Several novel dental materials are available on the market in this regard. Hence, the current study aimed to evaluate the proliferation, differentiation, and osteogenic potential of human bone marrow–derived mesenchymal stem cells (hBMSCs) onto biomaterials like ProRoot MTA (MTA; Dentsply Tulsa Dental, Tulsa, OK), Biodentine (BD; Septodont, Saint Maur de Fosses, France), and EndoSequence Root Repair Material (ERRM; Brasseler USA, Savannah, GA).

A congenital myasthenic syndrome refractory to acetylcholinesterase inhibitors.

We studied 4 siblings (3 men and 1 woman), ages 22 to 43 years, with congenital ptosis, external ophthalmoplegia, proximal muscle weakness and fatigability unresponsive to acetylcholinesterase (AChE) inhibitors. Repetitive nerve stimulation showed a significant compound muscle action potential (CMAP) area decrement at 2 or 3 Hz. Nerve conduction studies and concentric needle electromyography were normal, and repetitive CMAPs to single nerve stimulation were not observed. Voluntary single fiber electromyography (SFEMG) showed increased jitter and blocking. Assessment of individual end-plates using SFEMG with intramuscular axonal microstimulation showed no uniform relationship between jitter and the rate of stimulation, consistent with a postsynaptic defect of neuromuscular transmission. Edrophonium eliminated the decremental response to repetitive nerve stimulation, but caused no significant clinical improvement, suggesting an additional mechanism for weakness in these patients.     

In vitro and in vivo metabolism of a gamma-secretase inhibitor BMS-299897 and generation of active metabolites in milligram quantities with a microbial bioreactor

BMS-299897 is a gamma-secretase inhibitor that has the potential for treatment of Alzheimer's disease. The metabolism of [(14)C]BMS-299897 was investigated in human liver microsomes, in rat, dog, monkey and human hepatocytes and in bile duct cannulated rats. Seven metabolites (M1-M7) were identified from in vitro and in vivo studies. LC-MS/MS analysis showed that M1 and M2 were regioisomeric acylglucuronide conjugates of BMS-299897. Metabolites M3, M4 and M6 were identified as monohydroxylated metabolites of BMS-299897 and M5 was identified as the dehydrogenated product of monooxygenated BMS-299897. In vivo, 52% of the radioactive dose was excreted in bile within 0-6 h from bile duct cannulated rats following a single oral dose of 15 mg/kg of [(14)C]BMS-299897. Glucuronide conjugates, M1 and M2 accounted for 80% of the total radioactivity in rat bile. In addition to M1 and M2, M7 was observed in rat bile which was identified as a glucuronide conjugate of an oxidative metabolite M5. For structure elucidation and pharmacological activity testing of the metabolites, ten microbial cultures were screened for their ability to metabolize BMS-299897 to form these metabolites. Among them, the fungus Cunninghamella elegans produced two major oxidative metabolites M3 and M4 that had the same HPLC retention time and mass spectral properties as those found in in vitro incubations. NMR analysis indicated that M3 and M4 were stereoisomers, with the hydroxyl group on the benzylic position. However, M3 and M4 were unstable and converted to their corresponding lactones readily. Based on x-ray analysis of the synthetically prepared lactone of M3, the stereochemistry of benzylic hydroxyl group was assigned as the R configuration. Both the hydroxy metabolites (M3 and M4) and the lactone of M3 showed gamma-secretase inhibition with IC(50) values similar to that of the parent compound. This study demonstrates the usefulness of microbial systems as bioreactors to generate metabolites of BMS-299897 in large quantities for structure elucidation and activity testing. This study also demonstrates the biotransformation profile of BMS-299897 is qualitatively similar across the species including rat, dog, monkey and human which provides a basis to support rat, dog and monkey as preclinical models for toxicological testing.     

Managing rheumatoid arthritis during COVID-19

Clinical Rheumatology - The outbreak of coronavirus in the world has led to an uncertainty about treatment of patients with autoimmune disorders because of their weakened immune system coupled with...     

Management of Dens Invaginatus Type II Associated with Immature Apex and Large Periradicular Lesion Using Platelet-rich Fibrin and Biodentine

Dens invaginatus (DI) poses peculiar challenges in endodontic treatment of teeth because of distortion of pulpal space. A case of Oehlers type II DI with open apex and large periapical lesion is reported. The case was managed using cone-beam computed tomography (CBCT), operating microscope, platelet-rich fibrin (PRF), and Biodentine. A 15-year-old male patient presented with palatal swelling. Pulp sensibility testing of right maxillary lateral incisor was negative. Intraoral periapical digital radiograph revealed an Oehlers type II DI with open apex and periapical radiolucency. A CBCT scan was performed to study the anatomy, determine the true extent of the periapical lesion, and form a treatment plan. A diagnosis of Oehlers type II DI with pulp necrosis and acute periapical abscess was made. Two-visit endodontic treatment was performed. In the first visit, the invaginated central mass was removed under operating microscope, chemo-mechanical preparation was done, and double antibiotic paste dressing was placed. In the second visit, the canal was sealed with apical matrices of PRF and Biodentine as filling material. The patient was asymptomatic and radiographs revealed continued healing of the osseous defect at follow-up visits. A CBCT scan at 30 months showed complete continuity of periodontal ligament space, healing of labial and palatal cortical plates, and formation of intercortical bone. The advances in endodontic armamentarium and technology, like CBCT and operating microscope, have made successful treatment of challenging cases possible. PRF and Biodentine as apical matrices and filling material, respectively, proved to be effective in the present case.