An artificial neural network for membrane-bound catechol-O-methyltransferase biosynthesis with Pichia pastoris methanol-induced cultures.

BACKGROUND: Membrane proteins are important drug targets in many human diseases and gathering structural information regarding these proteins encourages the pharmaceutical industry to develop new molecules using structure-based drug design studies. Specifically, membrane-bound catechol-O-methyltransferase (MBCOMT) is an integral membrane protein that catalyzes the methylation of catechol substrates and has been linked to several diseases such as Parkinson's disease and Schizophrenia. Thereby, improvements in the clinical outcome of the therapy to these diseases may come from structure-based drug design where reaching MBCOMT samples in milligram quantities are crucial for acquiring structural information regarding this target protein. Therefore, the main aim of this work was to optimize the temperature, dimethylsulfoxide (DMSO) concentration and the methanol flow-rate for the biosynthesis of recombinant MBCOMT by Pichia pastoris bioreactor methanol-induced cultures using artificial neural networks (ANN). RESULTS: The optimization trials intended to evaluate MBCOMT expression by P. pastoris bioreactor cultures led to the development of a first standard strategy for MBCOMT bioreactor biosynthesis with a batch growth on glycerol until the dissolved oxygen spike, 3 h of glycerol feeding and 12 h of methanol induction. The ANN modeling of the aforementioned fermentation parameters predicted a maximum MBCOMT specific activity of 384.8 nmol/h/mg of protein at 30°C, 2.9 mL/L/H methanol constant flow-rate and with the addition of 6% (v/v) DMSO with almost 90% of healthy cells at the end of the induction phase. These results allowed an improvement of MBCOMT specific activity of 6.4-fold in comparison to that from the small-scale biosynthesis in baffled shake-flasks. CONCLUSIONS: The ANN model was able to describe the effects of temperature, DMSO concentration and methanol flow-rate on MBCOMT specific activity, as shown by the good fitness between predicted and observed values. This experimental procedure highlights the potential role of chemical chaperones such as DMSO in improving yields of recombinant membrane proteins with a different topology than G-coupled receptors. Finally, the proposed ANN shows that the manipulation of classic fermentation parameters coupled with the addition of specific molecules can open and reinforce new perspectives in the optimization of P. pastoris bioprocesses for membrane proteins biosynthesis.

Hepatic UDP-glucuronosyltransferase is responsible for eslicarbazepine glucuronidation.

Eslicarbazepine acetate (ESL) is a once-daily novel antiepileptic drug approved in Europe for use as adjunctive therapy for refractory partial-onset seizures with or without secondary generalization. Metabolism of ESL consists primarily of hydrolysis to eslicarbazepine, which is then subject to glucuronidation followed by renal excretion. In this study, we have identified that human liver microsomes (HLM) enriched with uridine 5'-diphosphoglucuronic acid give origin to a single Escherichia coli ß-glucuronidase-sensitive eslicarbazepine glucuronide (most likely the O-glucuronide). The kinetics of eslicarbazepine glucuronidation in HLM was investigated in the presence and absence of bovine serum albumin (BSA). The apparent K(m) were 412.2 ± 63.8 and 349.7 ± 74.3 µM in the presence and absence of BSA, respectively. Incubations with recombinant human UDP glucuronosyltransferases (UGTs) indicated that UGT1A4, UGT1A9, UGT2B4, UGT2B7, and UGT2B17 appear to be involved in eslicarbazepine conjugation. The UGT with the highest affinity for conjugation was UGT2B4 (K(m) = 157.0 ± 31.2 and 28.7 ± 10.1 µM, in the absence and presence of BSA, respectively). There was a significant correlation between eslicarbazepine glucuronidation and trifluoperazine glucuronidation, a typical UGT1A4 substrate; however, no correlation was found with typical substrates for UGT1A1 and UGT1A9. Diclofenac inhibited eslicarbazepine glucuronidation in HLM with an IC(50) value of 17 µM. In conclusion, glucuronidation of eslicarbazepine results from the contribution of UGT1A4, UGT1A9, UGT2B4, UGT2B7, and UGT2B17, but the high-affinity component of the UGT2B4 isozyme may play a major role at therapeutic plasma concentrations of unbound eslicarbazepine.

In-vivo evaluation of prolonged release bilayer tablets of anti-Parkinson drugs in Göttingen minipigs.

OBJECTIVES: Patients with Parkinson's disease can benefit from controlled released levodopa dosage forms since there is a clear clinical advantage in obtaining sustained plasma concentrations. The purpose of this study was to obtain a tablet that prolonged the release of levodopa. METHODS: A novel bilayer tablet, consisting of an immediate release layer containing nebicapone (100 mg) and an erosion-matrix type prolonged release layer containing levodopa (100 mg) and carbidopa (25 mg) was developed (LCN PR). A pharmacokinetic study in Göttingen minipigs was performed to evaluate this formulation. KEY FINDINGS: LCN PR tablets prolonged the in-vitro release of levodopa in HCl 0.1 m for more than 3 h. In-vivo plasma levodopa levels peaked at a later time point with LCN PR tablets as compared with that obtained with Sinemet 100/25 (2.7 vs 0.5 h). Nebicapone increased the maximum plasma concentration and area under the plasma concentration-time curve values for levodopa. CONCLUSIONS: The results obtained suggested that LCN PR tablets may have decreased the number of tablets and daily intake in the treatment of patients with Parkinson's disease.