Antifungal Effects and Potential Mechanisms of Benserazide Hydrochloride Alone and in Combination with Fluconazole Against Candida albicans.

PURPOSE: The resistance of C. albicans to traditional antifungal drugs brings a great challenge to clinical treatment. To overcome the resistance, developing antifungal agent sensitizers has attracted considerable attention. This study aimed to determine the anti-Candida activity of BEH alone or BEH-FLC combination and to explore the underlying mechanisms. MATERIALS AND METHODS: In vitro antifungal effects were performed by broth microdilution assay and XTT reduction assay. Infected Galleria mellonella larvae model was used to determine the antifungal effects in vivo. Probes Fluo-3/AM, FITC-VAD-FMK and rhodamine 6G were used to study the influence of BEH and FLC on intracellular calcium concentration, metacaspase activity and drug efflux of C. albicans. RESULTS: BEH alone exhibited obvious antifungal activities against C. albicans. BEH plus FLC not only showed synergistic effects against planktonic cells and preformed biofilms within 8 h but also enhanced the antifungal activity in infected G. mellonella larvae. Mechanistic studies indicated that antifungal effects of drugs might be associated with the increasement of calcium concentration, activation of metacaspase activity to reduce virulence and anti-biofilms, but were not related to drug efflux. CONCLUSION: BEH alone or combined with FLC displayed potent antifungal activity both in vitro and in vivo, and the underlying mechanisms were related to reduced virulence factors.

Benserazide Perturbs Kif15-kinesin Binding Protein Interaction with Prolonged Metaphase and Defects in Chromosomal Congression: A Study Based on in silico Modeling and Cell Culture.

The interaction of Kif15 with kinesin binding protein (KBP) is critical for its microtubule localization, bundling of kinetochore microtubules and proper alignment of chromosomes at the metaphase plate. The Kif15-KBP structure was prepared from the crystal structure of Kif15 and nonhomologous model of KBP through docking. Benserazide was retrieved when we did a screening of the ZINC Drug Database using the pharmacophore model generated from the potential binding site on Kif15 in an effort to identify molecules for repurposing as Kif15 inhibitors. Live cell imaging of HeLa cells revealed that benserazide delayed metaphase to anaphase-onset by 47±10 min compared to control cells. Benserazide treatment perturbed the kinetochore and microtubule interaction and inhibited the proliferation of HeLa cells with an IC50 of 101 µM with a mitotic block of 12 %. It did not bind to tubulin in the in vitro assays suggesting that the observed effects could be due to its perturbation of Kif15-KBP interaction.

Development, Optimization, and Evaluation In Vitro/In Vivo of Oral Liquid System for Synchronized Sustained Release of Levodopa/Benserazide.

To enhance efficiency, convenience, and safety of Parkinson's disease (PD) treatment for geriatric patients, an advanced suspension of Levodopa/Benserazide hydrochloride (LD/BH) has been prepared by cation-exchange resin and used to synchronize sustained release of LD and BH by optimizing coating parameters and prescription. For the purpose, LD and BH were immobilized on the surface of cation-exchange resin, respectively. Based on HPLC results, the cation-exchange resin showed high loading capacity. The studies on drug loading mechanism indicated that both drugs were immobilized by electrostatic interaction rather than physical adsorption. After PEG modification, pretreated drug-resin complexes were coated by emulsion-solvent evaporation method. In order to control drug release in a sustained manner, coating parameters of drug-resin microcapsules were optimized respectively by single-factor analysis. Further, coating prescription of the microcapsules was optimized to synchronize sustained release of LD and BH in vitro by orthogonal design. Utilizing optimal LD-resin microcapsules and BH-resin microcapsules, LD/BH suspension, containing both of them, was prepared by an optimal formulation and characterized by accelerated test and pharmacokinetic study in vivo. The accelerated test confirmed high stability of LD/BH suspension. According to pharmacokinetic results in vivo, in contrast with LD/BH commercial tablets, LD/BH suspensions did not only synchronize sustained release of both drugs but also show good bioequivalence. As LD/BH sustained release suspension can synchronize sustained release of multiple active ingredients by oral administration, the suspension presents promising oral dosage forms for geriatric patients with PD. An advanced Levodopa/Benserazide hydrochloride (LD/BH) suspension, prepared by cation-exchange resin and optimized microencapsulation, synchronizes sustained releases of LD and BH in vivo to benefit Parkinson's disease treatment for geriatric patients.

Nanocarrier for levodopa Parkinson therapeutic drug; comprehensive benserazide analysis.

Loss of dopamine-secreting neurons in the midbrain causes Parkinson's disease. L-DOPA, the precursor to the neurotransmitters dopamine, crosses vast majority of physiological and biochemical barriers that dopamine cannot. But most levodopa is decarboxylated to dopamine before it reaches the brain. This causes to little therapeutic gain with strong peripheral side effects. Benserazide is an irreversible inhibitor of peripheral aromatic L-amino acid decarboxylase that prevents the breakdown of levodopa in the bloodstream. The challenges are to increase the therapeutic efficiency, the bioavailability and decreasing the unfavourable side effects of Levodopa drug. Biocompatible nano-sized drug carriers could address these challenges at molecular level. Thus calculations of drug loading ability of acid-functionalized CNT for the benserazide as a nanodug carrier complex for L-DOPA were performed. In this regard, evaluation of all adsorption features of the most stable conformer of benserazide molecule onto carboxylated carbon nanotube is critical. To determine the minimum energy conformer of benserazide, the molecular structure and conformational analysis of 512 possible conformers have been subjected to first principle quantum mechanical calculations. Our work established a novel and easy-to-make formulation of benserazide/carboxylated CNT conjugate with extremely high drug loading efficiency of Levodopa for Parkinson disease treatment.

Application of silver nanoparticles and principal component-artificial neural network models for simultaneous determination of levodopa and benserazide hydrochloride by a kinetic spectrophotometric method.

A multicomponent analysis method based on principal component analysis-artificial neural network model (PC-ANN) is proposed for the simultaneous determination of levodopa (LD) and benserazide hydrochloride (BH). The method is based on the reaction of levodopa and benserazide hydrochloride with silver nitrate as an oxidizing agent in the presence of PVP and formation of silver nanoparticles. The reaction monitored at analytical wavelength 440 nm related to surface plasmon resonance band of silver nanoparticles. Differences in the kinetic behavior of the levodopa and benserazide hydrochloride were exploited by using principal component analysis, an artificial neural network (PC-ANN) to resolve concentration of analytes in their mixture. After reducing the number of kinetic data using principal component analysis, an artificial neural network consisting of three layers of nodes was trained by applying a back-propagation learning rule. The optimized ANN allows the simultaneous determination of analytes in mixtures with relative standard errors of prediction in the region of 4.5 and 6.3 for levodopa and benserazide hydrochloride respectively. The results show that this method is an efficient method for prediction of these analytes.

A feasibility study of differential delivery of levodopa ester and benserazide using site-specific intestinal loops in rats.

To study the effect of varied intestinal delivery of levodopa ester, levodopa and its butyl ester were administered as bolus or continuous infusions into site-specific, in situ ligated intestinal loops of rats. Benserazide, a carboxylase inhibitor, was not administered, coadministered with ester, or infused into the duodenal loop prior to ester dosing. While the proximal colon minimally absorbed levodopa itself, it substantially absorbed the ester. Coadministration of benserazide and ester at the colon did not increase absorption; however, prior infusion of benserazide into the duodenum enhanced the colonic absorption of ester. Compared to bolus infusion, continuous delivery of the ester resulted in a more sustained levodopa concentration in plasma, and less metabolism into dopamine. The results were repeated for methyl ester, and the relative differences between the results of methyl and butyl esters versus levodopa were similar. The overall results at the duodenum, jejunum, and ileum were also comparable, likewise were those for the proximal, middle, and distal colons. The results of the study are encouraging: a combination of the continuous delivery of levodopa ester with an immediate-release of benserazide optimizes levodopa bioavailability, potentially leading to a much more effective use of levodopa in the treatment of Parkinson's patients.

Continuous wavelet and derivative transforms for the simultaneous quantitative analysis and dissolution test of levodopa-benserazide tablets.

Simultaneous analyses and dissolution tests of levodopa-benserazide tablets were carried out by continuous wavelet transform (CWT) and classic derivative spectrophotometry (DS) without using any chemical separation step. The developed two spectrophotometric resolutions are based on the transformation of the original UV spectra. The original absorption spectra of levodopa and benserazide in the concentration range of 1-80 microg/mL and 5-240 microg/mL in USP simulated gastric juice were registered in the spectral range of 250-310 nm, respectively. Various wavelet families and different spectrophotometric derivative orders were tested to find the optimal signal processing for obtaining desirable calibration graphs and reliable determinations of the investigated drugs. Under the optimized conditions of the methods, symlets wavelet family using a=128 with sixth order (SYM6-CWT) and the first derivative transform with Deltalambda=10nm were identified as optimal signal processing methods for the determinations and dissolution tests. The calibration functions for each drug were obtained by measuring the values of the CWT and derivative amplitudes. The validation of the developed methods was confirmed by analyzing various synthetic mixtures of the investigated drugs. Mean recovery values were found between 99.1% and 104.7% for DS and 100% and 102.9% for CWT, respectively for determination of BEN and LEV in synthetic mixtures. Each developed approaches were successfully applied to the simultaneous determination and dissolution test of levodopa and benserazide in their commercial tablets and a good agreement was observed.

Structural insight into Parkinson's disease treatment from drug-inhibited DOPA decarboxylase.

DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinson's disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.

Quality control of benserazide-levodopa and carbidopa-levodopa tablets by capillary zone electrophoresis.

In modern practice, the treatment of Parkinson's disease and syndrome is carried out using pharmaceutical formulations containing a combination of levodopa and a decarboxylation inhibitor (carbidopa or benserazide). Two pharmaceutical formulations were quantified by capillary zone electrophoresis using two procedures which differed only in the kind of background electrolyte used. One procedure used a 25 mM phosphate buffer, pH 2.5, while the second one used a 25 mM borate buffer, pH 8.5. The electrophoretic analysis was carried out using an uncoated fused- silica capillary, a separation voltage of 20 kV with currents typically less than 60 microA, and spectrophotometric detection at 205 nm. Calibration curves were performed for levodopa (concentration range 1-100 microg/mL), for carbidopa and benserazide (1-50 microg/mL), and the plots of the peak area versus concentration were found to be linear with a correlation coefficient better than 0.9990. Satisfactory results were obtained when commercial tablets were analyzed in terms of accuracy (98-102%), repeatability (0.6-2.0%), and intermediate precision (1.1-2.6%).

Effectiveness of Madopar HBS plus Madopar standard in patients with fluctuating Parkinson's disease: two years of follow-up.

Clinical response to a new galenic formulation of levodopa plus benserazide, Madopar HBS, was studied in 25 fluctuating parkinsonians. This open study was planned in two phases. In the first phase, the administering of HBS alone resulted in a surprisingly high number of dropouts. In the second phase, Madopar standard in association with Madopar HBS, the follow-up period was 24 months. A stable long-lasting improvement in predictable fluctuations and their severity was maintained for the whole period without any change in drug dose. Nocturnal and early morning akinesia improved too. The study shows that Madopar HBS plus Madopar standard is effective in producing a prolonged and stable response in parkinsonian fluctuating patients.