Application of a dual mechanistic approach to support bilastine dose selection for older adults.

The objective of this study was to evaluate bilastine dosing recommendations in older adults and overcome the limitation of insufficient data from phase I studies in this underrepresented population. This was achieved by integrating bilastine physicochemical, in vitro and in vivo data in young adults and the effect of aging in the pharmacology by means of two alternative approaches: a physiologically-based pharmacokinetic (PBPK) model and a semi-mechanistic population pharmacokinetic (Senescence) model. Intestinal apical efflux and basolateral influx transporters were needed in the PBPK model to capture the observations from young adults after single i.v. (10 mg) and p.o. (20 mg) doses, supporting the hypothesis of involvement of gut transporters on secretion. The model was then used to extrapolate the pharmacokinetics (PKs) to elderly subjects considering their specific physiology. Additionally, the Senescence model was develop starting from a published population PK) model, previously applied for pediatrics, and incorporating declining functions on different physiological systems and changes in body composition with aging. Both models were qualified using observed data in a small group of young elderlies (N = 16, mean age = 68.69 years). The PBPK model was further used to evaluate the dose in older subjects (mean age = 80 years) via simulation. The PBPK model supported the hypothesis that basolateral influx and apical efflux transporters are involved in bilastine PK. Both, PBPK and Senescence models indicated that a 20 mg q.d. dose is safe and effective for geriatrics of any age. This approach provides an alternative to generate supplementary data to inform dosing recommendations in under-represented groups in clinical trials.

Opposite alterations of 5-HT2A receptor brain density in subjects with schizophrenia: relevance of radiotracers pharmacological profile.

The status of serotonin 5-HT2A receptors (5-HT2ARs) in schizophrenia has been controversial. In vivo positron emission tomography neuroimaging and in vitro post-mortem binding studies have reported conflicting results about 5-HT2AR density. Radiotracers bind different receptor conformations depending on their agonist, antagonist or inverse agonist properties. This study investigates 5-HT2AR density in the post-mortem prefrontal cortex from subjects with schizophrenia and controls using three radiotracers with a different pharmacological profile. The specific binding parameters of the inverse agonist [18F]altanserin, the agonist [3H]lysergic acid diethylamide (LSD) and the antagonist [3H]MDL100907 to brain cortex membranes from 20 subjects with schizophrenia and 20 individually matched controls were evaluated under similar methodological conditions. Ten schizophrenia subjects were antipsychotic-free at death. Saturation curve analyses were performed by non-linear regression to obtain a maximal density of binding sites (Bmax) and the affinity of the respective radiotracers (Kd). In schizophrenia subjects, 5-HT2AR density was decreased when quantified by [18F]altanserin binding, whereas increased when evaluated by [3H]LSD binding. However, [3H]MDL100907 binding was unaltered. A slight loss of affinity (higher Kd) was observed exclusively in [3H]LSD binding. The findings were more evident in antipsychotic-free subjects than in antipsychotic-treated subjects. In conclusion, a higher proportion of the 5-HT2AR-active functional conformation, which is rather identified by agonist radiotracers, was observed in schizophrenia patients. A consequent reduction of the inactive 5-HT2AR conformation, which is preferentially identified by inverse agonist radiotracers, was also obtained. Antagonist radiotracers do not distinguish between molecular conformations of the receptor, and accordingly, the absence of changes was shown. These results are compatible with the proposed increased functional activity of brain cortical 5-HT2ARs in schizophrenia.

Di-aryl guanidinium derivatives: Towards improved α2-Adrenergic affinity and antagonist activity.

Compounds with excellent receptor engagement displaying α2-AR antagonist activity are useful not only for therapeutic purposes (e.g. antidepressants), but also to help in the crystallization of this particular GPCR. Therefore, based on our broad experience in the topic, we have prepared eighteen di-aryl (phenyl and/or pyridin-2-yl) mono- or di-substituted guanidines and 2-aminoimidazolines. The in vitro α2-AR binding affinity experiments in human brain tissue showed the advantage of a 2-aminoimidazolinium cation, a di-arylmethylene core, a conformationally locked pyridin-2-yl-guanidine and a di-substituted guanidinium to achieve good α2-AR engagement. After different in vitro [35S]GTPγS binding experiments in human prefrontal cortex tissue, it was possible to identify that compounds 7a, 7b and 7c were α2-AR partial agonist, whereas 8h was a potent α2-AR antagonist. Docking and MD studies with a model of α2A-AR and two crystal structures suggest that antagonism is achieved by compounds carrying a di-substituted guanidine which substituent occupy a pocket adjacent to TM5 without engaging S2005.42 or S2045.46, and a mono-substituted cationic group, which favorably interacts with E942.65.

Pharmacokinetics and safety of bilastine in children aged 6 to 11 years with allergic rhinoconjunctivitis or chronic urticaria.

Bilastine, a second-generation antihistamine, is approved in Europe for the treatment of allergic rhinoconjunctivitis and urticaria in adults and children aged ≥ 6 years. Pharmacokinetic data for children aged 6-11 years were extracted post hoc from a study in which children (2-11 years) with allergic rhinoconjunctivitis or urticaria received oral bilastine (10 mg/day). Maximum plasma concentration (Cmax) and area under the plasma concentration curve (AUC) data were compared with adult pharmacokinetic data from seven clinical studies (bilastine 20 mg/day). Safety data for children aged 6-11 years were extracted post hoc from a phase III randomized controlled trial of children (2-11 years) with allergic rhinoconjunctivitis or chronic urticaria receiving once-daily bilastine 10 mg or placebo for 12 weeks. Exposure and Cmax values were similar for children (6-11 years) and adults: median pediatric/adult ratios for AUC0-24 and Cmax were 0.93 and 0.91, respectively. There was no significant difference in the incidence of treatment-emergent adverse in children (6-11 years) receiving bilastine 10 mg or placebo.Conclusion: Pharmacokinetic and safety analyses in children aged 6-11 years support the suitability of the pediatric dose of bilastine 10 mg and confirm that the safety profiles of bilastine and placebo are similar.What is Known:• Bilastine, a second-generation antihistamine, is approved in Europe for the treatment of allergic rhinoconjunctivitis and urticaria in adults (20 mg/day) and children aged ≥ 6 years (10 mg/day).• An ontogenic model based on adult data and pharmacokinetic/pharmacodynamic simulations supported the selection of a bilastine dose of 10 mg/day in children aged 2-11 years. Bilastine 10 mg/day was shown to have a safety profile similar to that of placebo in a large phase III randomized clinical trial in children aged 2-11 years.What is New:• As bilastine is approved in Europe for children aged ≥6 years, the current study reports the results of two post hoc analyses of pharmacokinetic and safety data in children aged 6-11 years.• Analysis of pharmacokinetic and safety data in children aged 6-11 years supports the suitability of the pediatric dose of bilastine 10 mg and confirms that its safety profile is similar to that of placebo.

Bioequivalence Evaluation of Three Pediatric Oral Formulations of Bilastine in Healthy Subjects: Results from a Randomized, Open Label, Crossover Study.

BACKGROUND AND OBJECTIVE: Bilastine is a non-sedating H1 antihistamine indicated for the treatment of allergic rhinoconjunctivitis and urticaria. The aim of this trial was to assess the bioequivalence of three novel pediatric oral formulations of bilastine. METHODS: An open label, randomized, four-treatment-period, four-sequence, crossover, single-center study was conducted in 23 healthy volunteers. Each subject received four single doses of bilastine under fasting conditions: a 10-mg orodispersible tablet (DT1), a 10-mg oral solution (SOL), a 10-mg orodispersible tablet without water (DT2dry), and a 10-mg orodispersible tablet with water (DT2water, reference formulation). Blood samples were collected during 72 h with a washout period of at least 7 days. Bilastine maximum plasma concentration (Cmax) and area under the plasma concentration-time curve between 0 to t time (AUC0-t) were calculated to assess bioequivalence. Tolerability was evaluated throughout the study. RESULTS: The three oral pediatric formulations tested were bioequivalent to the reference formulation as determined by the ratio test/reference of the geometric mean and their 90% confidence intervals (between 0.80 and 1.25) for the Cmax, AUC0-t and AUC0-∞. Bilastine was well tolerated when administered indistinctly as an orodispersible tablet or as an oral solution. CONCLUSION: The three oral pediatric formulations tested were found to be bioequivalent to the reference formulation. All formulations were well tolerated. TRIAL REGISTRATION: Spanish Clinical Studies Registry (REEC) number 2014-000786-41.

Efficacy and safety of bilastine in reducing pruritus in patients with chronic spontaneous urticaria and other skin diseases: an exploratory study.

Purpose: To evaluate the efficacy/safety of bilastine in pruritus relief in patients with chronic spontaneous urticaria (CSU) or other pruritic skin diseases.Methods: In this multicenter, open-label, exploratory study (EudraCT No.: 2016-001505-17), 115 adults with CSU (n = 34), eczema/dermatitis (n = 30), prurigo (n = 25) or cutaneous pruritus (n = 26), received bilastine 20 mg once daily for 8 weeks, or in non-responder patients (<30% improvement in pruritus score at week 2), 40 mg/day from week 2.Results: The mean change in weekly pruritus severity score from baseline to week 8 (primary endpoint) was reduced with bilastine (overall and by disease group); overall, percentage and absolute reductions were 71.16% and 1.63 points, respectively (p < .001). Updosed non-responders (n = 31) had improved weekly pruritus severity scores from baseline to week 8; percentage and absolute reductions were 49.08% and 1.13 points, respectively (p < .001). Bilastine improved the Dermatology Life Quality Index at weeks 4 and 8 (p < .001) in all disease groups, and the 7-day Urticaria Activity Score in CSU patients (p < .001). Bilastine was well tolerated.Conclusions: Bilastine relieved pruritus associated with urticaria and other skin diseases, with a very good safety profile.

Serotonin 5-HT2A receptor expression and functionality in postmortem frontal cortex of subjects with schizophrenia: Selective biased agonism via Gαi1-proteins.

Serotonin 5-HT2A receptors (5-HT2ARs) have been implicated in schizophrenia. However, postmortem studies on 5-HT2ARs expression and functionality in schizophrenia are scarce. The 5-HT2AR mRNA and immunoreactive protein expression were evaluated in postmortem tissue from dorsolateral prefrontal cortex (DLPFC) of antipsychotic-free (n = 18) and antipsychotic-treated (n = 9) subjects with schizophrenia, and matched controls (n = 27). Functional coupling of 5-HT2AR to G-proteins was tested by measuring the activation induced by the agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride ((±)DOI) in antibody-capture [35S]GTPγS scintillation proximity assays (SPA). In antipsychotic-free schizophrenia subjects, 5-HT2AR mRNA expression and protein immunoreactivity in total homogenates was similar to controls. In contrast, in antipsychotic-treated schizophrenia subjects, lower mRNA expression (60±9% vs controls) and a trend to reduced protein immunoreactivity (86±5% vs antipsychotic-free subjects) just in membrane-enriched fractions was observed. [35S]GTPγS SPA revealed a significant ~6% higher stimulation of Gαi1-protein by (±)DOI in schizophrenia, whereas activation of the canonical Gαq/11-protein pathway by (±)DOI remained unchanged. Expression of Gαi1- and Gαq/11-proteins did not differ between groups. Accordingly, in rats chronically treated with clozapine, but not with haloperidol, a 30-40% reduction was observed in 5-HT2AR mRNA expression, 5-HT2AR protein immunoreactivity and [3H]ketanserin binding in brain cortical membranes. Overall, the data suggest a supersensitive 5-HT2AR signaling through inhibitory Gαi1-proteins in schizophrenia. Together with previous results, a dysfunctional pro-hallucinogenic agonist-sensitive 5-HT2AR conformation in postmortem DLPFC of subjects with schizophrenia is proposed. Atypical antipsychotic treatment would contribute to counterbalance this 5-HT2AR supersensitivity by reducing receptor expression.

Antipsychotic-induced Hdac2 transcription via NF-κB leads to synaptic and cognitive side effects.

Antipsychotic drugs remain the standard for schizophrenia treatment. Despite their effectiveness in treating hallucinations and delusions, prolonged exposure to antipsychotic medications leads to cognitive deficits in both schizophrenia patients and animal models. The molecular mechanisms underlying these negative effects on cognition remain to be elucidated. Here we demonstrate that chronic antipsychotic drug exposure increases nuclear translocation of NF-κB in both mouse and human frontal cortex, a trafficking event triggered via 5-HT2A-receptor-dependent downregulation of the NF-κB repressor IκBα. This upregulation of NF-κB activity led to its increased binding at the Hdac2 promoter, thereby augmenting Hdac2 transcription. Deletion of HDAC2 in forebrain pyramidal neurons prevented the negative effects of antipsychotic treatment on synaptic remodeling and cognition. Conversely, virally mediated activation of NF-κB signaling decreased cortical synaptic plasticity via HDAC2. Together, these observations may aid in developing therapeutic strategies to improve the outcome of schizophrenia treatment.

A group II metabotropic glutamate receptor 3 (mGlu3, GRM3) isoform implicated in schizophrenia interacts with canonical mGlu3 and reduces ligand binding.

As well as being expressed as a full-length transcript, the group II metabotropic glutamate receptor 3 (GRM3, mGlu3) gene is expressed as an mRNA isoform which lacks exon 4 (GRM3Δ4) and which is predicted to encode a protein with a novel C terminus (called mGlu3Δ4). This variant may contribute to the mechanism by which GRM3 acts as a schizophrenia risk gene. However, little is known about the properties or function of mGlu3Δ4. Here, using transiently transfected HEK293T/17 cells, we confirm that GRM3Δ4 cDNA is translated, with mGlu3Δ4 existing as a homodimer as well as a monomer, and localizing primarily to cell membranes including the plasma membrane. Co-immunoprecipitation shows that mGlu3Δ4 interacts with canonical mGlu3. mGlu3Δ4 does not bind the mGlu2/3 antagonist [3H]LY341495, but the presence of mGlu3Δ4 reduces binding of [3H]LY341495 to mGlu3, paralleled by a decrease in the abundance of membrane-associated mGlu3. These experiments indicate that mGlu3Δ4 may negatively modulate mGlu3, and thereby impact on the roles of GRM3/mGlu3 in schizophrenia and as a therapeutic target.

Substituted conformationally restricted guanidine derivatives: Probing the α2-adrenoceptors' binding pocket.

In this paper we report the design, synthesis and pharmacological evaluation of new N-substituted 2-amino-1,4-dihydroquinazolines, 2-amino-1,4-dihydropyridopyrimidines and 2-amino-4,5-dihydro-1,3-benzodiazepines as α2-adrenoceptors ligands. Computational studies show that the proposed substitutions and guanidine-containing ring size will probe an extensive area of the active site. Preparation of these molecules involved novel routes than those previously utilised in our laboratory for the preparation of the acyclic aryl-guanidine counterparts. Compounds 8b and 18c showed the highest affinity and antagonistic activity, within their series, towards the α2-adrenoceptor in human brain tissue in vitro experiments. Structure-activity relationships have been established for the design and biological evaluation of novel α2-adrenoceptor ligands.

Metabotropic glutamate receptor 3 (mGlu3; mGluR3; GRM3) in schizophrenia: Antibody characterisation and a semi-quantitative western blot study.

BACKGROUND: Metabotropic glutamate receptor 3 (mGlu3, mGluR3), encoded by GRM3, is a risk gene for schizophrenia and a therapeutic target. It is unclear whether expression of the receptor is altered in the disorder or related to GRM3 risk genotype. Antibodies used to date to assess mGlu3 in schizophrenia have not been well validated. OBJECTIVE: To characterise six commercially available anti-mGlu3 antibodies for use in human brain, and then conduct a semi-quantitative study of mGlu3 immunoreactivity in schizophrenia. METHODS: Antibodies tested using Grm3-/- and Grm2-/-/3-/- mice and transfected HEK293T/17 cells. Western blotting on membrane protein isolated from superior temporal cortex of 70 patients with schizophrenia and 87 healthy comparison subjects, genotyped for GRM3 SNP rs10234440. RESULTS: One (out of six) anti-mGlu3 antibodies was fully validated, a C-terminal antibody which detected monomeric (~100kDa) and dimeric (~200kDa) mGlu3. A second, N-terminal, antibody detected the 200kDa band but also produced non-specific bands. Using the C-terminal antibody for western blotting in human brain, mGlu3 immunoreactivity was found to decline with age, and was affected by pH and post mortem interval. There were no differences in monomeric or dimeric mGlu3 immunoreactivity in schizophrenia or in relation to GRM3 genotype. The antibody was not suitable for immunohistochemistry. INTERPRETATION: These data highlight the value of knockout mouse tissue for antibody validation, and the need for careful antibody characterisation. The schizophrenia data show that involvement of GRM3 in the disorder and its genetic risk architecture is not reflected in total membrane mGlu3 immunoreactivity in superior temporal cortex.

Allosteric signaling through an mGlu2 and 5-HT2A heteromeric receptor complex and its potential contribution to schizophrenia.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Previous findings demonstrated that the Gq/11-coupled serotonin 5-HT2A receptor and the Gi/o-coupled metabotropic glutamate 2 (mGlu2) receptor-GPCRs that are involved in signaling alterations associated with psychosis-assemble into a heteromeric complex in the mammalian brain. In single-cell experiments with various mutant versions of the mGlu2 receptor, we showed that stimulation of cells expressing mGlu2-5-HT2A heteromers with an mGlu2 agonist led to activation of Gq/11 proteins by the 5-HT2A receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to Gi/o proteins, and we determined the relative location of the Gi/o-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of Gq/11, but not Gi/o, was reduced in the frontal cortex of 5-HT2A knockout mice and was reduced in the frontal cortex of postmortem brains from schizophrenic patients. These findings offer structural insights into this important target in molecular psychiatry.

Repressive epigenetic changes at the mGlu2 promoter in frontal cortex of 5-HT2A knockout mice.

Serotonin 5-HT(2A) and metabotropic glutamate 2 (mGlu2) are G protein-coupled receptors suspected in the pathophysiology of psychiatric disorders, such as schizophrenia, depression, and suicide. Previous findings demonstrate that mGlu2 mRNA expression is down-regulated in brain cortical regions of 5-HT2A knockout (KO) mice. However, the molecular mechanism responsible for this alteration remains unknown. We show here repressive epigenetic changes at the promoter region of the mGlu2 gene in frontal cortex of 5-HT(2A)-KO mice. Disruption of 5-HT(2A) receptor-dependent signaling in mice was associated with decreased acetylation of histone H3 (H3ac) and H4 (H4ac) and increased tri-methylation of histone H3 at lysine 27 (H3K27me3) at the mGlu2 promoter, epigenetic changes that correlate with transcriptional repression. Neither methylation of histone H3 at lysine 4 (H3K4me1/2/3) nor tri-methylation of histone H3 at lysine 9 (H3K9me3) was affected. We found that Egr1, a transcription factor in which promoter activity was positively regulated by the 5-HT(2A) receptor agonist 4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide, binds less to the mGlu2 promoter in frontal cortex of 5-HT(2A)-KO, compared with wild-type mice. Furthermore, expression of mGlu2 was increased by viral-mediated gene transfer of FLAG-tagged Egr1 in mouse frontal cortex. Together, these observations suggest that 5-HT(2A) receptor-dependent signaling epigenetically affects mGlu2 transcription in mouse frontal cortex.

HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity.

Histone deacetylases (HDACs) compact chromatin structure and repress gene transcription. In schizophrenia, clinical studies demonstrate that HDAC inhibitors are efficacious when given in combination with atypical antipsychotics. However, the molecular mechanism that integrates a better response to antipsychotics with changes in chromatin structure remains unknown. Here we found that chronic atypical antipsychotics downregulated the transcription of metabotropic glutamate 2 receptor (mGlu2, also known as Grm2), an effect that was associated with decreased histone acetylation at its promoter in mouse and human frontal cortex. This epigenetic change occurred in concert with a serotonin 5-HT(2A) receptor-dependent upregulation and increased binding of HDAC2 to the mGlu2 promoter. Virally mediated overexpression of HDAC2 in frontal cortex decreased mGlu2 transcription and its electrophysiological properties, thereby increasing psychosis-like behavior. Conversely, HDAC inhibitors prevented the repressive histone modifications induced at the mGlu2 promoter by atypical antipsychotics, and augmented their therapeutic-like effects. These observations support the view of HDAC2 as a promising new target for schizophrenia treatment.