IntroductionNebivolol, a third-generation β-blocker, is subject to extensive first-pass metabolism and produces active β-blocking hydroxylated metabolites, like 4-OH-nebivolol. It is primarily a substrate of CYP2D6, a metabolic pathway that is under polymorphic genetic regulation. The objective of this study was to assess the metabolizer phenotype and to evaluate the interphenotype bioavailability and metabolism of nebivolol.Material and methodsForty-three healthy volunteers were included in this open-label, non-randomized clinical trial and each volunteer received a single dose of 5 mg nebivolol. Non-compartmental pharmacokinetic analysis was performed to determine the pharmacokinetic parameters of nebivolol and its active metabolite. The phenotypic distribution was assessed based on the AUC (aria under the curve) metabolic ratio of nebivolol/4-OH-nebivolol and statistical analysis. An interphenotype comparison of nebivolol metabolism and bioavailability was performed based on the pharmacokinetic parameters of nebivolol and its active metabolite.ResultsNebivolol/4-OH-nebivolol AUC metabolic ratios were not characterized by a standard normal distribution. The unique distribution emphasized the existence of two groups and the 43 healthy volunteers were classified as follows: poor metabolizers (PMs)=3, extensive metabolizers (EMs)=40. The phenotype had a marked impact on nebivolol metabolism. The exposure to nebivolol was 15-fold greater for PMs in comparison to EMs.Conclusion40 EMs and 3 PMs were differentiated by using the pharmacokinetic parameters of nebivolol and its active metabolite. The study highlighted the existence of interphenotype differences regarding nebivolol metabolism and bioavailability.

Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have here established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify a peptide with 9-fold higher affinity than the starting peptide.

Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in superresolution microscopy. In this work, we have established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 complementing peptide variants using high-density peptide microarrays and examined them for binding and functional complementation of the LOO-FP fragment into Green Fluorescent Protein. We have studied the effect of peptide length and the effect of different linkers to the solid support. The peptide microarray platform allowed us to map the effect of all possible amino acid substitutions on each position of the complementing peptides as well as in the context of some single and double amino acid substitutions. As all peptides in different formats were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing determination of robustness and precision of the method.Importantly, we showed that the microarray fluorescence correlated with the affinity in solution between the LOO-FP and peptides. A double substitution yielded a peptide with 9-fold higher affinity than the starting peptide.