ADME properties of CHF6366, a novel bi-functional M3 muscarinic receptor antagonist and ß2 adrenoceptor agonist (MABA) radiolabelled at both functional moieties.

CHF6366, a dual action ß2-receptor agonist and M3-muscarinic receptor antagonist developed for chronic obstructive pulmonary disease (COPD) was [14C]-radiolabelled on the two different functional moieties of the molecule (either aminobutanolic or carbamate) to characterise its ADME profile following intravenous (IV), intratracheal (IT) and oral (PO) administration.A very low oral bioavailability and a good balance between absorption and lung retention after IT administration were observed, together with a rapid distribution throughout the body and a complete metabolic transformation of the parent drug without relevant gender difference.CHF6366 was observed fully hydrolysed to alcohol (CHF6387) and carboxylic acid (CHF6361) in plasma and urine after IV and IT administration, and mainly unchanged in faeces only after oral administration. An important number of metabolites containing aminobutanolic moiety was excreted via urine, whereas carbamate-containing derivatives were excreted mainly by bile.The major metabolic routes of the alcoholic moiety (CHF6387) included isomerisation (Ma7), conjugation with glucuronic acid and dehydrogenation, while the carboxylic acid moiety (CHF6361) was mainly metabolised through oxidation, glucuronide conjugation and, in both pathways, combinations of those metabolic reactions.No major differences arose also from in vitro metabolism profiles investigated using liver microsomes and hepatocytes of different species.

CHF6001 II: a novel phosphodiesterase 4 inhibitor, suitable for topical pulmonary administration--in vivo preclinical pharmacology profile defines a potent anti-inflammatory compound with a wide therapeutic window.

CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide] is a novel phosphodiesterase 4 (PDE4) inhibitor designed for use in pulmonary diseases by inhaled administration. Intratracheal administration of CHF6001 to ovalbumin-sensitized Brown-Norway rats suppressed the antigen-induced decline of lung functions (ED50 = 0.1 µmol/kg) and antigen-induced eosinophilia (ED50 = 0.03 µmol/kg) when administered (0.09 µmol/kg) up to 24 hours before antigen challenge, in agreement with CHF6001-sustained lung concentrations up to 72 hours after intratracheal treatment (mean residence time 26 hours). Intranasal, once daily administration of CHF6001 inhibited neutrophil infiltration observed after 11 days of tobacco smoke exposure in mice, both upon prophylactic (0.15-0.45 µmol/kg per day) or interventional (0.045-0.45 µmol/kg per day) treatment. CHF6001 was ineffective in reversing ketamine/xylazine-induced anesthesia (a surrogate of emesis in rat) up to 5 µmol/kg administered intratracheally, a dose 50- to 150-fold higher than anti-inflammatory ED50 observed in rats. When given topically to ferrets, no emesis and nausea were evident up to 10 to 20 µmol/kg, respectively, whereas the PDE4 inhibitor GSK-256066 (6-[3-(dimethylcarbamoyl)phenyl]sulfonyl-4-(3-methoxyanilino)-8-methylquinoline-3-carboxamide) induced nausea at 1 µmol/kg intratracheally. A 14-day inhalation toxicology study in rats showed a no-observed-adverse-effect level dose of 4.4 µmol/kg per day for CHF6001, lower than the 0.015 µmol/kg per day for GSK-256066. CHF6001 was found effective and extremely well tolerated upon topical administration in relevant animal models, and may represent a step forward in PDE4 inhibition for the treatment of asthma and chronic obstructive respiratory disease.

In vitro and in vivo metabolism of CHF 6001, a selective phosphodiesterase (PDE4) inhibitor.

1. The metabolism of CHF 6001, a novel PDE4 inhibitor, was determined in vitro in mouse, rat, dog, monkey and human microsomes and hepatocytes and in vivo in plasma, urine, feces and bile of rats after intravenous and intratracheal administration. 2. The behavior of CHF 6001 in microsomes and hepatocytes changed across species. CYP3A4/5 isoenzymes were identified to be the primary enzymes responsible for the metabolism of CHF 6001 in human liver microsomes. 3. In the rat, CHF 6001 was found extensively metabolized in urine, feces and bile, but not in plasma, where CHF 6001 was the main compound present. The metabolite profiles were different in the four biological matrices from both qualitative and quantitative point of view. 4. CHF 6001 was metabolized through hydrolysis with the formation of the alcohol CHF 5956, loss of a chlorine atom, loss of the N-oxide, hydroxylation, loss of the cyclopropylmethyl group in the alcohol moiety, conjugation with glucuronic acid, glutathione and cysteine-glycine. 5. The major metabolite present in the bile was isolated and characterized by nuclear magnetic resonance analysis. It derived from CHF 6001 through contraction of the pyridine-N-oxide ring to N-hydroxy pyrrole and conjugation with glucuronic acid.

Co-ultraPEALut in Subjective Cognitive Impairment Following SARS-CoV-2 Infection: An Exploratory Retrospective Study.

Neurological involvement following coronavirus disease 19 (COVID-19) is thought to have a neuroinflammatory etiology. Co-ultraPEALut (an anti-inflammatory molecule) and luteolin (an anti-oxidant) have shown promising results as neuroinflammation antagonists. The aim of this study was to describe cognitive impairment in patients with post-COVID-19 treated with co-ultraPEALut. The Montreal Cognitive Assessment (MoCA), the Prospective-Retrospective Memory Questionnaire (PRMQ), the Fatigue Severity Scale (FSS), and a subjective assessment were administered at baseline and after 10 months. Patients treated with co-ultraPEALut were retrospectively compared with controls. Twenty-six patients treated with co-ultraPEALut showed a significant improvement in PRMQ (T0: 51.94 ± 10.55, T1: 39.67 ± 13.02, p < 0.00001) and MoCA raw score (T0: 25.76 ± 2.3, T1: 27.2 ± 2, p 0.0260); the MoCA-adjusted score and the FSS questionnaires also showed an improvement, even though it was not statistically significant; and 80.77% of patients reported a subjective improvement. In the control subjects (n = 15), the improvement was not as pronounced (PRMQ T0: 45.77 ± 13.47, T1: 42.33 ± 16.86, p 0.2051; FSS T0: 4.95 ± 1.57, T1: 4.06 ± 1.47, p 0.1352). Patients treated with co-ultraPEALut and corticosteroids were not statistically different from those treated with co-ultraPEALut alone. Neuro-post-COVID-19 patients treated with co-ultraPEALut scored better than controls in MoCA and PRMQ questionnaires after 10 months: this may support the importance of neuroinflammation modulation for neuro-long-COVID-19.

Using illusions to understand hallucinations: differences in perceptual performances on illusory figures may underscore specific visuoperceptual impairments in Parkinson's disease.

Visual hallucinations are prevalent, potentially disabling symptoms of Parkinson's Disease. Multiple impairments in bottom-up sensory processing and top-down perceptual modulation are implicated in the pathophysiology of these phenomena. In healthy individuals, visual illusions are elicited by illusory figures through parametric manipulations of geometrical configurations, contrast, color, or spatial relationships between stimuli. These illusory percepts provide insight on the physiologic processes subserving conscious and unconscious perception. In this exploratory, cross-sectional, controlled study, perceptual performance on illusory figures was assessed on 11 PD patients with hallucinations, 10 non-hallucinating PD patients, and 10 age-matched healthy individuals. In order to characterize potential neural substrates of perceptual performances, patients' brain metabolic patterns on FDG PET were also analyzed. Illusions relying on attentional modulation and global perception were attenuated in PD patients without hallucinations. This pattern was no longer recognizable in hallucinating patients. Conversely, illusory effects normally counteracted by figure to background segregation and overlapping figures recognition were enhanced in PD patients with hallucinations. FDG PET findings further suggest that perceptual differences between PD patients might be linked to abnormal top-down perceptual modulation.

Optimization of M3 Antagonist-PDE4 Inhibitor (MAPI) Dual Pharmacology Molecules for the Treatment of COPD.

Aiming at the inhaled treatment of pulmonary diseases, the optimization process of the previously reported MAPI compound 92a is herein described. The project was focused on overcoming the chemical stability issue and achieving a balanced bronchodilator/anti-inflammatory profile in rats in order to be confident in a clinical effect without having to overdose at one of the biological targets. The chemical strategy was based on fine-tuning of the substitution pattern in the muscarinic and PDE4 structural portions of the dual pharmacology compounds, also making use of the analysis of a proprietary crystal structure in the PDE4 catalytic site. Compound 10f was identified as a chemically stable, potent, and in vivo balanced MAPI lead compound, as assessed in bronchoconstriction and inflammation assays in rats after intratracheal administration. After the in-depth investigation of the pharmacological and solid-state profile, 10f proved to be safe and suitable for development.

A potential protective role of magnesium in neuroCOVID

Several recent studies support a role of dysregulated magnesium homeostasis in COVID-19. In the present narrative review, we focus on the neurological aspects of this disease, collectively known as neuroCOVID, and we propose some mechanisms by which alterations of magnesium may contribute to the involvement of the nervous system in the context of SARS-CoV-2 infection. Further fundamental, translational, and clinical research is needed to underpin the potential relationships between altered magnesium status and neuro-COVID, with potentially novel therapeutic implications.

From the Lab to Real Life: Monitoring Cardiorespiratory Fitness during the COVID-19 Pandemic through Wearable Devices. An Exploratory Longitudinal Study on Healthy Participants.

COVID-19 containment measures hampered population cardiorespiratory fitness (which can be quantified as peak oxygen consumption (V.O2peak)) and the possibility to assess it using laboratory-based techniques. Although it is useful to ascertain the V.O2peak recovery after lockdowns, the community and most scientific institutions were unable to evaluate it. Wearable devices may provide the opportunity to estimate cardiorespiratory fitness outside of the laboratory, without breaking self-isolation; herein, we explore the feasibility of this approach. Fifteen healthy participants were tested every 2 weeks for 10 weeks during a reduction of containment measures after a strict lockdown. Physical activity levels were measured using the International Physical Activity Questionnaire-Short Form (IPAQ-SF). V.O2peak was estimated through a previously validated test based on the speed of a 60 m sprint run, the baseline-to-peak heart rate (HR) variation, and the velocity of HR decay after the sprint, and measured through a wearable HR monitor. Participants increased physical activity from the end of lockdown (1833 [917-2594] MET-min/week; median [1st quartile-3rd quartile]) until the end of follow-up (2730 [1325-3380] MET-min/week). The estimated V.O2peak increased by 0.24 ± 0.19 mL/(min*kg*week) (regression coefficient ± standard error). Based on previous knowledge on the impact of inactivity on V.O2peak, our study indicates that a 10-week period of reducing the stringency of containment measures may not be sufficient to counteract the detrimental effects of the preceding lockdown.

COVID-19 lockdown: Physical activity, sedentary behaviour and sleep in Italian medicine students.

AbstractCOVID-19 pandemic led many countries to implement lockdown measures. Italy declared lockdown from 9th March to 3rd May 2020, and universities shifted to online classes. Home confinement could prevent students from achieving the physical activity and sleep levels recommended for their psychophysical health, and medicine students are already known to be at risk of inactivity and reduced sleep due to their time-consuming curricula. This study aimed at describing medicine students' behaviours during lockdown and comparing them with pre-lockdown data and current recommendations. A cross-sectional questionnaire survey was conducted among 6th-year Italian medicine students (n = 714; age=25 ± 2 y; female: 62%; male: 38%) in October-November 2019. The same survey was repeated in 6th-year students during lockdown (n = 394; age=25 ± 2 y; female: 73%; male: 27%), and extended to 1st-5th year (total 1st-6th-year sample during lockdown: n = 1471; age=23 ± 2 y; female: 70%; male: 30%). International Physical Activity Questionnaire Short Form (IPAQ) and selected questions from Pittsburgh Sleep Quality Index were administered to evaluate physical activity, sitting and sleep time. Decreased physical activity, and increased sitting and sleep time were observed from pre- to during lockdown in 6th-year students (p<0.01). 1st-6th-year students featured 10 [8-12] hours sitting (median [Q1-Q3]) and an IPAQ score of 1170 [400-2348] MET-min/week. Even participants with higher physical activity featured high sitting time. Sleeping less than recommended (<7 h/night) was associated with more sitting time and less energies to perform daily activities. Strategies fostering compliance with current guidelines for physical activity, sedentary behaviour and sleep should be implemented, especially in case of a repeated or intermittent lockdown.

Discovery of M3 Antagonist-PDE4 Inhibitor Dual Pharmacology Molecules for the Treatment of Chronic Obstructive Pulmonary Disease.

In this paper, we report the discovery of dual M3 antagonist-PDE4 inhibitor (MAPI) compounds for the inhaled treatment of pulmonary diseases. The identification of dual compounds was enabled by the intuition that the fusion of a PDE4 scaffold derived from our CHF-6001 series with a muscarinic scaffold through a common linking ring could generate compounds active versus both the transmembrane M3 receptor and the intracellular PDE4 enzyme. Two chemical series characterized by two different muscarinic scaffolds were investigated. SAR optimization was aimed at obtaining M3 nanomolar affinity coupled with nanomolar PDE4 inhibition, which translated into anti-bronchospastic efficacy ex vivo (inhibition of rat trachea contraction) and into anti-inflammatory efficacy in vitro (inhibition of TNFα release). Among the best compounds, compound 92a achieved the goal of demonstrating in vivo efficacy and duration of action in both the bronchoconstriction and inflammation assays in rat after intratracheal administration.

Novel Pyrrolidine Derivatives of Budesonide as Long Acting Inhaled Corticosteroids for the Treatment of Pulmonary Inflammatory Diseases.

Inhaled corticosteroids (ICSs) represent the first line therapy for the treatment of asthma and are also extensively utilized in chronic obstructive pulmonary disease. Our goal was to develop a new ICS with a basic group, which can allow solid state feature modulation, achieving at the same time high local anti-inflammatory effect and low systemic exposure. Through a rational drug design approach, a new series of pyrrolidine derivatives of budesonide was identified. Within the series, several compounds showed nanomolar binding affinity ( Ki) with GR that mostly correlated with the effect in inducing GR nuclear translocation in CHO cells and anti-inflammatory effects in macrophagic cell lines. Binding and functional cell-based assays allowed identifying compound 17 as a potent ICS agonist with a PK profile showing an adequate lung retention and low systemic exposure in vivo. Finally, compound 17 proved to be more potent than budesonide in a rat model of acute pulmonary inflammation.

Investigation of Lung Pharmacokinetic of the Novel PDE4 Inhibitor CHF6001 in Preclinical Models: Evaluation of the PreciseInhale Technology.

BACKGROUND: Preclinical evaluation of new chemical entities (NCEs) designed to be administered by inhalation route requires lung administration to rodents, especially in the discovery phase. Different administration methods have been used until now, but more efforts are required to obtain controlled and reproducible lung deposition when only small amounts of neat powder material are available. METHODS: The PreciseInhale platform used in the present study enables well-controlled powder aerosol exposures with only small amounts of micronized neat material, providing data on inhalation pharmacokinetic (PK) of NCEs at a very early stage. The DustGun aerosol technology uses compressed air to generate a respirable aerosol from milligram-amounts of powder that is delivered to one animal at a time. The new methodology was used to investigate the inhalation PK and lung retention in the rat of the novel Chiesi PDE4 inhibitor CHF6001 in three exposure models of the PreciseInhale platform: nose-only, intratracheally intubated rat, and the isolated, ventilated, and perfused rat lung. Results were compared with data from two other pulmonary delivery systems commonly used in preclinical studies: liquid instillation and powder insufflation. RESULTS: Administration of micronized CHF6001 using the PreciseInhale system yielded lung exposures in the same range as the other tested devices, but the reproducibility in lung deposition was improved. The initial amount of CHF6001 in lungs at the first sampling time point was close to the predetermined target dose. Tracheal deposition with PreciseInhale (0.36 ± 0.22 µg) was significantly less than with other tested delivery systems: PennCentury (23.7 ± 3.2 µg) and Airjet (25.6 ± 7.2 µg). CONCLUSIONS: The PreciseInhale platform enabled the administration of CHF6001 powder with good accuracy and reproducibility, with low tracheal deposition. The new platform can be used at an early discovery stage to obtain inhalatory PK data for respirable aerosols of neat NCE powder without excipients and with minimal use of dry powder formulation work.

Discovery and Optimization of Thiazolidinyl and Pyrrolidinyl Derivatives as Inhaled PDE4 Inhibitors for Respiratory Diseases.

Phosphodiesterase 4 (PDE4) is a key cAMP-metabolizing enzyme involved in the pathogenesis of inflammatory disease, and its pharmacological inhibition has been shown to exert therapeutic efficacy in chronic obstructive pulmonary disease (COPD). Herein, we describe a drug discovery program aiming at the identification of novel classes of potent PDE4 inhibitors suitable for pulmonary administration. Starting from a previous series of benzoic acid esters, we explored the chemical space in the solvent-exposed region of the enzyme catalytic binding pocket. Extensive structural modifications led to the discovery of a number of heterocycloalkyl esters as potent in vitro PDE4 inhibitors. (S*,S**)-18e and (S*,S**)-22e, in particular, exhibited optimal in vitro ADME and pharmacokinetics properties and dose-dependently counteracted acute lung eosinophilia in an experimental animal model. The optimal biological profile as well as the excellent solid-state properties suggest that both compounds have the potential to be effective topical agents for treating respiratory inflammatory diseases.

Synthesis and biological activity of flurbiprofen analogues as selective inhibitors of beta-amyloid(1)(-)(42) secretion.

Flurbiprofen, a nonsteroidal antiinflammatory drug (NSAID), has been recently described to selectively inhibit beta-amyloid(1)(-)(42) (Abeta42) secretion, the most toxic component of the senile plaques present in the brain of Alzheimer patients. The use of this NSAID in Alzheimer's disease (AD) is hampered by a significant gastrointestinal toxicity associated with cyclooxygenase (COX) inhibition. New flurbiprofen analogues were synthesized, with the aim of increasing Abeta42 inhibitory potency while removing anti-COX activity. In vitro ADME developability parameters were taken into account in order to identify optimized compounds at an early stage of the project. Appropriate substitution patterns at the alpha position of flurbiprofen allowed for the complete removal of anti-COX activity, while modifications at the terminal phenyl ring resulted in increased inhibitory potency on Abeta42 secretion. In rats, some of the compounds appeared to be well absorbed after oral administration and to penetrate into the central nervous system. Studies in a transgenic mice model of AD showed that selected compounds significantly decreased plasma Abeta42 concentrations. These new flurbiprofen analogues represent potential drug candidates to be developed for the treatment of AD.

Novel class of benzoic acid ester derivatives as potent PDE4 inhibitors for inhaled administration in the treatment of respiratory diseases.

The first steps in the selection process of a new anti-inflammatory drug for the inhaled treatment of asthma and chronic obstructive pulmonary disease are herein described. A series of novel ester derivatives of 1-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3,5-dichloropyridin-4-yl) ethanol have been synthesized and evaluated for inhibitory activity toward cAMP-specific phosphodiesterase-4 (PDE4). In particular, esters of variously substituted benzoic acids were extensively explored, and structural modification of the alcoholic and benzoic moieties were performed to maximize the inhibitory potency. Several compounds with high activity in cell-free and cell-based assays were obtained. Through the evaluation of opportune in vitro ADME properties, a potential candidate suitable for inhaled administration in respiratory diseases was identified and tested in an in vivo model of pulmonary inflammation, proving its efficacy.

Single and multiple ascending dose studies of a novel tissue-selective oestrogen receptor modulator, CHF 4227, in healthy postmenopausal women.

AIMS: We evaluated the tolerability, adverse events profile, pharmacokinetics, and pharmacodynamics of CHF 4227, a new selective oestrogen receptor modulator (SERM), in healthy postmenopausal women. METHODS: Two phase I studies were conducted according to a double-bind, placebo-controlled design. Subjects were randomized to receive six single (5-400 mg) or five multiple oral doses of CHF 4227 for 28 days (5-100 mg). RESULTS: No vaginal bleeding and no changes in either endometrial thickness or the placenta protein 14 marker were found after 4 weeks of treatment. The compound did not induce negative effects on the fibrinolytic system. After 28 days of treatment, CHF 4227 decreased both total and LDL cholesterol concentrations (maximum decreases from baseline of 17.4% (95% CI 7.0, 27.7) and 27.6% (95% CI 9.0, 46.3), respectively). Decreases in both serum and urinary type-I C-terminal collagen telopeptide were also observed producing maximum changes of 40.6% (95% CI 29.5, 51.7), and 41.7% (95% CI 20.3, 56.8), respectively. CHF4227 (5 and 10 mg) induced near maximal oestrogen-like effects on bone markers and serum lipids without causing hot flushes. The pharmacokinetics of CHF 4227 were characterized by a slow absorption, a long elimination half-life (31-42 h after single administration) and dose linearity with respect to C(max) and AUC up to 100 mg. CONCLUSIONS: CHF 4227 is a well-tolerated SERM when administered once daily for 28 days. It is potentially active on bone resorption and serum lipids, without affecting the endometrium and without worsening hot flushes. CHF 4227 is a promising agent for the treatment of several conditions in postmenopausal women.

In vitro and in vivo profiling of CHF5022 and CHF5074 Two beta-amyloid1-42 lowering agents.

Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may delay or prevent the onset of Alzheimer's disease (AD). A subset of NSAIDs, including flurbiprofen, has been shown to selectively inhibit the production of beta-amyloid(1-42) (Abeta42), independently from their cyclooxygenase (COX) inhibiting activity. We evaluated the in vitro and in vivo profiles of CHF5022 and CHF5074, two flurbiprofen analogues. The in vitro Abeta inhibiting activity was evaluated in a human neuroglioma cell line (H4) carrying the double Swedish mutation (K595N/M596L) of the human amyloid precursor protein (APPsw). The in vitro anti-COX activity was evaluated using human recombinant enzymes isolated from transfected Sf-9 cells. The in vivo pharmacokinetic and pharmacodynamic profiles of the two compounds were evaluated in young APPsw transgenic mice (Tg2576) after oral gavage (100 or 300mgkg(-1) day(-1) for 4-5 days) and after medicated diet (375ppm for 4 weeks). R-Flurbiprofen was used as comparator. In vitro, CHF5022 and CHF5074 were found to be 3- and 7-fold more potent than R-flurbiprofen in inhibiting Abeta42 secretion (IC(50)s of 92, 40 and 268microM, respectively). Differently from R-flurbiprofen, CHF5022 and CHF5074 did not affect COX-1 (at 100microM) and COX-2 (at 300microM) activity. Similarly to R-flurbiprofen, no significant alteration in the expression profile of a subset of Notch intracellular domain-responsive genes was observed with either CHF5022 or CHF5074. In Tg2576 mice, CHF5022 was well tolerated when administered by oral gavage (100mgkg(-1) day(-1) for 5 days) or by medicated diet (56mg kg(-1) day(-1) for 4 weeks). R-Flurbiprofen was poorly tolerated in the diet (32mgkg(-1) day(-1)) with 55% of the animals dying during the first week of treatment. After 4-5 days of oral gavage, CHF5022 and CHF5074 plasma and brain levels at 3h were found to increase with the dose, leading to brain concentrations of about 10% and 5% of the corresponding plasma concentrations, respectively. In animals fed for 4 weeks with compound-supplemented diet, mean plasma (580microM) and brain (20microM) Cyrillic) concentrations of CHF5022 were 8 and 15 times higher than those of R-flurbiprofen. Plasma Abeta42 concentration was dose-dependently decreased by CHF5022 and CHF5074. Brain Abeta levels (formic acid-extractable) were not significantly affected by either compound, although Abeta42 levels tended to inversely correlate (P=0.105) with CHF5022 concentration in the brain. CHF5022 and CHF5074 thus appear to have a promising in vitro and in vivo profile. This warrants further evaluation of their long-term effects on Abeta brain pathology.