Identification of the New Metabolite of Nebivolol Using Liquid Chromatography Coupled with High-Resolution Mass Spectrometry and Chemometrics.

In this study, the phase I hepatic metabolism pathway of a cardiovascular drug nebivolol was proposed on the basis of a human liver microsomes assay with the use of LC-HR-MS coupled with the chemometric method. Six biotransformation products were found with the assistance of chemometric analysis. Five of them were identified as the previously reported products of alicyclic hydroxylation and dihydroxylation, aromatic hydroxylation, as well as alicyclic oxidation of the parent compound. Moreover, one metabolite, not reported so far, was found to be a product of N-dealkylation of nebivolol-2-amino-1-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl)ethan-1-ol. The novel metabolite was submitted to an in silico toxicity analysis to assess its biological properties. The applied computational methods indicated a significantly elevated risk of its mutagenic activity, compared to the parent molecule. Several metabolites of the nebivolol described in the literature were not detected in this study, indicating their non-hepatic origin.

Repurposing old drugs as novel inhibitors of human MIF from structural and functional analysis.

Human macrophage migration inhibitory factor (MIF) is an important pro-inflammatory cytokine that plays multiple pleiotropic functions. It is considered as a promising therapeutic target for the infectious, autoimmune, and cardiovascular diseases and cancers. The development of MIF inhibitors has not been translated into clinical success despite decades of research. Given the time and cost of developing new drugs, existing drugs with clarified safety and pharmacokinetics are explored for their potential as novel MIF inhibitors. This study identified five known drugs that could inhibit MIF's tautomerase activity and MIF-mediated cell chemotaxis in RAW264.7 cells. It was found that compounds D2 (histamine), D5 (metaraminol), and D8 (nebivolol) exhibited micromolar-range inhibition potency close to the positive control ISO-1. Kinetics and the mechanism for inhibition were subsequently determined. Moreover, the detailed inhibitor-binding patterns were investigated by X-ray crystallography, computational molecular docking, and structure-based analysis. Therefore, this study elucidates the molecular mechanism of repurposed drugs acting on MIF and provides a structural foundation for lead optimization to promote the clinical development of MIF-targeted drugs.

Computational study of therapeutic potential of phosphorene as a nano-carrier for drug delivery of nebivolol for the prohibition of cardiovascular diseases: a DFT study.

Density functional theory (DFT) calculations were utilized to assess the drug delivery efficiency of phosphorene carrier for nebivolol drug to treat cardiovascular diseases. The optimized structures, excited state, and electronic properties of nebivolol, phosphorene, and nebivolol-phosphorene (nebivolol-PH) complex were considered to determine the drug delivery ability of phosphorene at the target site. The increased dipole moment (6.08 D) results in the higher solubility of the complex in polar solvents (water). Weak interactive forces between nebivolol and phosphorene were demonstrated by the non-covalent interaction (NCI) plot that facilitated the offloading of nebivolol at the targeted area. The analysis of frontier molecular orbitals (FMOs) revealed that during excitation, the charge was transferred from nebivolol as a higher occupied molecular orbital (HOMO) to phosphorene as a lower unoccupied molecular orbital (LUMO). Thus, the charge-transfer process was further studied by charge decomposition analysis (CDA). The calculated results at the excited state for the nebivolol-PH complex exhibited that the maximum wavelength (λmax) was red-shifted by 6 nm in the gas phase. The electron-hole theory and photoinduced electron transfer (PET) processes were carried out for the exploration of different excited states of the complex. Additionally, phosphorene with + 1 and - 1 charge states indicated the minor structural changes and provide the stable nebivolol-PH complex. This theoretical study also investigated that phosphorene can be exploited as an effective carrier for the delivery of a therapeutic agent as nebivolol to treat cardiovascular diseases. This work will also encourage the researchers to investigate the other 2D nanoparticles as a nano-drug delivery system (NDDS).

Preparative separation of nebivolol isomers by improved throughput reverse phase tandem two column chromatography.

Development of preparative methods for the isolation of chiral molecules has been considered challenging by conventional unit operations due to their identical physical and chemical properties. This has evolved chiral stationary phases for the separation of chiral components using chromatography technique. However, separation method using chiral adsorbents requires high pressure, are expensive, and have low productivity. Generation of bulk quantities purified nebivolols using the available high pressure chiral separation methods is impractical and operating cost-intensive. Thus, there is a need to develop economical methods using nonchiral adsorbents for the purification of nebivolols or similar active ingredients. The present work demonstrates a unique and scalable tandem two-column method for the separation of isomers of nebivolol using inexpensive reverse phase adsorbents. The first column of the scheme causes removal of charged and nonisomeric impurities whereas tandem operation of second column increases resolution of d-nebivolol and l-nebivolol. The maximization of separation due to tandem operation of second column causes enhancement of the throughput of the process. The developed preparative process produces >98% purity of both d-nebivolol and l-nebivolol with overall loading capacity of 56 g (L of adsorbent)-1 and productivity of 20 g L-1 day-1 .

Development and validation of a bio-analytical method for simultaneous quantification of nebivolol and labetalol in aqueous humor and plasma using LC-MS/MS and its application to ocular pharmacokinetic studies.

Beta blockers is the class of choice of drugs in treatment of open angle Glaucoma. However, many of these drugs suffer from systemic side effects due to their absorption into systemic circulation via nasolachrymal duct. To evaluate the safety and efficacy of nebivolol and labetalol for the treatment of open angle glaucoma, it is important to have a bioanalytical method for measuring the drug concentrations both in aqueous humor and plasma. A simple, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with protein precipitation technique was developed for simultaneous quantification of nebivolol and labetalol using nebivolol-d4 and metoprolol, respectively, as internal standards in aqueous humor and plasma. Nebivolol and labetalol were monitored in electrospray positive ionization (ESI) mode at transition 406.2/151.1 and 329.2/162.0, respectively. Mobile phase comprised of mixture of aqueous buffer (solvent A) and organic phase (solvent B) (mixture of A:B in the ratio of 30:70, v/v). The aqueous buffer was 5 mM ammonium acetate buffer adjusted to pH 3.5 ±â€¯0.05 with formic acid while the organic phase was a mixture of methanol and acetonitrile in the ratio of 25:75, v/v. Chromatographic separation was achieved using reverse phase Zorbax SB-C18 column (4.6 × 100 mm, 3.5 µm). Method was linear in both the matrices in the concentration range of 0.43-750 ng/mL for nebivolol and 0.39-668 ng/mL for labetalol with r2 > 0.99. Accuracy values, expressed in terms of bias (%), for nebivolol in aqueous humor and plasma were ≤9.6% and ≤11.4% and for labetalol were ≤8.6% and ≤5.9%, respectively. Inter-day and intra-day precision values, expressed in terms of RSD (%), for both the drugs were within 11.4%. No interference was obtained due to matrix components. Mean recovery (%) values in aqueous humor and plasma were 72.4% and 73.0% for nebivolol and 56.7% and 54.4% for labetalol, respectively. No significant degradation was observed in both the drugs in both the matrices when stored at -20 °C for 1 month. Aqueous humor and plasma samples of nebivolol and labetalol on bench top were stable for 18 h and 8 h, respectively. The developed method was applied for determining pharmacokinetic parameters of both drugs in aqueous humor following single dose ocular administration in rabbits.

Effect of CYP2D6 Poor Metabolizer Phenotype on Stereoselective Nebivolol Pharmacokinetics.

BACKGROUND: Nebivolol is a drug available as a racemate of d-nebivolol (SRRR) and lnebivolol (RSSS). In human liver microsomes, CYP2D6 mainly catalyses the metabolism of lnebivolol, while CYP2C19 catalyses the metabolism of d-nebivolol. Nebivolol stereoselective pharmacokinetics has been described only for extensive metabolizers (EM). OBJECTIVE: To describe the stereoseletive nebivolol pharmacokinetics in CYP2D6 poor metabolizers (PM) and to assess whether the phenotype has an impact on nebivolol pharmacokinetics. METHODS: Three healthy volunteers PM phenotyped (ratios of 20.1, 220 and 244 for the 8 h urinary excretion of metoprolol/alfa-hydroxymetoprolol) received a single oral dose of racemic nebivolol and sequential blood samples were collected between zero (predose) and 48 h. RESULTS: The obtained data were compared to 22 EM subjects with normal kidney function enrolled in our previous study. For both isomers, Cmax, Tmax and AUC0-48 were significantly greater in the PM group compared to the EMs (p = 0.006 - 0.001). For d-nebivolol, Cmax, Tmax and AUC0-48 were, on average, 5.9, 2.7 and 15.0 larger in PMs. The corresponding values for l-nebivolol were 4.4, 2.7 and 17.5. CONCLUSION: The decline in plasma concentration of both nebivolol isomers in PM phenotypes, especially those with MR of 220 and 244, which indicate minimal or absent CYP2D6 activity, points to alternative mechanisms for nebivolol elimination. Collectively, our results are the first to show the significant impact of CYP2D6 PM phenotype on the metabolic disposition and in vivo exposure to both nebivolol isomers.

An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC-MS/MS: Application to clinical pharmacokinetics.

Nebivolol is a racemate of the d-isomer responsible for ß1 adrenergic receptor antagonism and the l-isomer responsible for the release of nitric oxide from endothelial cells. Nebivolol is mainly metabolized to nebivolol glucuronide, which also contribute to the nebivolol ß1 adrenoreceptor antagonism. This study reports the development and validation of an indirect stereoselective method of analysis of nebivolol glucuronides in plasma by LC-MS/MS. The method was applied to the investigation of stereoselectivity in the glucuronidation of nebivolol in elderly hypertensive patients (n=11) CYP2D6 phenotyped as EM and treated with a single oral dose of the racemate. One-milliliter plasma aliquots spiked with internal standard (S)-(-)-metoprolol were incubated with 25µL of ß-glucuronidase (final concentration 2500unit/mL) at pH 5.0 for 16h at 37°C. Linearity for total nebivolol was 0.2-125ng of each isomer per mL plasma and permitted analysis of nebivolol glucuronide isomers up to 48h after administration of a single oral dose of 10mg racemate. Regarding to the nebivolol glucuronide isomers, higher plasma concentrations of the d-isomer were observed compared to the l-isomer (d/l AUC=5.4), explaining at least in part the plasma accumulation of unchanged l-nebivolol (l/d AUC=1.8). This study also showed metabolic glucuronide nebivolol/unchanged nebivolol ratios of approximately 6.5 for the l-isomer (AUC 65.3 vs 10.1ngh/mL) and approximately 62.1 (335.2 vs 5.4ngh/mL) for the d-isomer. Considering that d-nebivolol glucuronide also contributes for ß1 adrenergic receptor antagonism, future studies regarding PK-PD of nebivolol should evaluate not only plasma concentrations of unchanged nebivolol isomers but also glucuronide nebivolol isomers.

Nebivolol-Loaded Microsponge Gel for Healing of Diabetic Wound.

An attempt was made to formulate nebivolol-loaded microsponge gel to access drug at wound area, incorporated into gel that possess optimum moist wound management environment during later stages of wound closure. Nebivolol, antihypertensive drug, exhibits vasodilating effects via nitric oxide pathway, slows diabetic neuropathy, and restores endothelial function in diabetic wounds. Microsponges were prepared by optimizing independent variables; drug to polymer ratio and internal phase volume and their effects on production yield, entrapment efficiency, and particle size. Formulations of microsponges were evaluated for drug content. Differential scanning calorimetry indicated reduction in crystallinity of NB during the formation of microsponges. In vitro study (drug to polymer 1:4 and 10 ml internal phase volume acetone) showed 80% drug released within 8 h. Spherical and porous microsponges confirmed by scanning electron microscopy were incorporated in the carbopol 934 (2%) gel base. Gel was characterized for pH, viscosity, and drug content. Less spreadability determined by texture analyzer demonstrated viscous nature of gel. In vitro diffusion study revealed entrapped drug in porous microsponges with slow release to heal wound. In vivo study performed using streptozotocin-induced diabetic rats and excision wound model showed wound healing and closure activity within day 10. Histology revealed inflammatory cell infiltrations and neovascularization in granulation tissues, ultimately healing wound. Microsponge gel prolonged drug release due to entrapped form in porous structure of microsponges with significant and fast wound healing and closure in diabetic rats. Microsponges with loaded drug fulfilled accessibility at wound area, while gel provided optimum moist wound management environment during later stages of wound closure.

Preparation and characterization of nanofibrous sheets for enhanced oral dissolution of nebivolol hydrochloride.

Nebivolol-loaded electrospun nanofibrous sheets were prepared for the dissolution enhancement of the active with the aim of improving its oral bioavailability. Physicochemical characterization of nanofibers including differential scanning calorimetry, attenuated total reflectance Fourier transform infrared spectroscopy and positron annihilation lifetime spectroscopy were carried out in order to track the physicochemical changes related to the electrospinning process. The obtained results unanimously indicated the amorphous transition of nebivolol as a result of electrospinning, furthermore supramolecular ordering of chains of polyvinyl alcohol matrix could be revealed by positron annihilation lifetime spectroscopy. The crystalline-amorphous conversion of the active, along with the increased specific surface area of the nanofibers enabled rapid and complete dissolution. More than twice amount of active released from the fibrous sheets than from the commercial tablets. In contrast to the control tablets, the dissolution was complete and was not influenced by the pH of the applied media.

Influence of chronic kidney disease and haemodialysis treatment on pharmacokinetics of nebivolol enantiomers.

AIM: The present study evaluated the pharmacodynamics and pharmacokinetics of nebivolol enantiomers in patients with chronic kidney disease (CKD) and in patients undergoing haemodialysis. METHODS: Forty-three adult patients were distributed into three groups: healthy volunteers and hypertensive patients with normal kidney function (n = 22); patients with stage 3 and 4 CKD (n = 11); and patients with stage 5 CKD undergoing haemodialysis (n = 10). The subjects received a single oral dose of 10 mg racemic nebivolol. Serial blood samples were collected up to 48 h after administration of the drug and heart rate variation was measured over the same interval during the isometric handgrip test. The nebivolol enantiomers in plasma were analysed by liquid chromatography-tandem mass spectrometry. RESULTS: The pharmacokinetics of nebivolol is enantioselective, with a greater plasma proportion of l-nebivolol. CKD increased the area under the concentration-time curve (AUC) of l-nebivolol (6.83 ng.h ml(-1) vs. 9.94 ng.h ml(-1) ) and d-nebivolol (4.15 ng.h ml(-1) vs. 7.30 ng.h ml(-1) ) when compared with the control group. However, the AUC values of l-nebivolol (6.41 ng.h ml(-1) ) and d-nebivolol (4.95 ng.h ml(-1) ) did not differ between the haemodialysis and control groups. The administration of a single dose of 10 mg nebivolol did not alter the heart rate variation induced by isometric exercise in the investigated patients. CONCLUSIONS: Stage 3 and 4 CKD increases the plasma concentrations of both nebivolol enantiomers, while haemodialysis restores the pharmacokinetic parameters to values similar to those observed in the control group. No significant difference in heart rate variation induced by isometric exercise was observed between the investigated groups after the administration of a single oral dose of 10 mg nebivolol.

Modification of solid lipid nanoparticles loaded with nebivolol hydrochloride for improvement of oral bioavailability in treatment of hypertension: polyethylene glycol versus chitosan oligosaccharide lactate.

Nebivolol (NB)-loaded solid lipid nanoparticles (SLNs) were prepared and modified with chitosan oligosaccharide lactate (COL) and polyethylene glycol (PEG) stearate for improvement of its oral bioavailability. Compritol, poloxamer and lecithin were used for the preparation of SLNs by homogenisation method. After in vitro characterisation effect of lipase, pepsin, or pancreatin on degradation and release rate were investigated. Cytotoxicity and permeation were studied on Caco-2 cells. As COL concentration increased in SLNs, size and zeta potential increased. PEG concentration was reversely proportional to particle size with no change in zeta potential. Encapsulation efficiencies (EEs) were determined as 84-98%. DSC confirmed solubilisation of NB in lipid matrix. A sustained release with no burst effect was determined. The presence of enzymes affected the release. SLNs did not reveal cytotoxicity and highest permeability was obtained with PEG modification. PEG-modified SLNs could be offered as a promising strategy for oral delivery of NB.

Synthesis of desfluorinated nebivolol isomers.

The syntheses of all possible stereoisomers of desfluorinated side products of the potent antihypertensive ß-blocker nebivolol are reported. A straightforward approach using a common racemic precursor was employed to obtain the desired optically active building blocks. For one series of compounds, a Sharpless asymmetric epoxidation (SAE) route yielded in a direct fashion the required compounds whereas a Mitsunobu reaction was selected to obtain the other series of compounds. This offers a flexible approach to all desfluoronebivolol side-products in order to fully characterize them.