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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.

In this paper, we report the discovery of dual M 3 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 M 3 receptor and the intracellular PDE4 enzyme. Two chemical series characterized by two different muscarinic scaffolds were investigated. SAR optimization was aimed at obtaining M 3 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.

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.

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.

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., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

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.

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.

A series of pyrazoles and pyrazolo[3,4-d]pyridazinones were synthesized and evaluated for their PDE4 inhibitory activity. All the pyrazoles were found devoid of activity, whereas some of the novel pyrazolo[3,4-d]pyridazinones showed good activity as PDE4 inhibitors. The most potent compounds in this series showed an IC(50) in the nanomolar range. The ability to inhibit TNF-alpha release in human PBMCs was determined for two representative compounds, finding values in the sub-micromolar range. SARs studies demonstrated that the best arranged groups around the heterocyclic core are 2-chloro-, 2-methyl- and 3-nitrophenyl at position 2, an ethyl ester at position 4 and a small alkyl group at position 6. Molecular modeling studies performed on a representative compound allowed to define its binding mode to the PDE4B isoform., (Copyright 2010 Elsevier Ltd. All rights reserved.)