Valentina Cenacchi

CHF5074, a novel γ-secretase modulator, attenuates brain β-amyloid pathology and learning deficit in a mouse model of Alzheimer's disease

Background and purpose:  We evaluated the effects of 1‐(3′,4′‐dichloro‐2‐fluoro[1,1′‐biphenyl]‐4‐yl)‐cyclopropanecarboxylic acid (CHF5074), a new γ‐secretase modulator, on brain β‐amyloid pathology and spatial memory in transgenic mice expressing the Swedish and London mutations of human amyloid precursor protein (hAPP).

Bronchodilator Activity of (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino] carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407), a Potent, Long-Acting, and Selective Muscarinic M3 Receptor Antagonist

The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t½ = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [3H]CHF5407 dissociated quickly from hM2 receptors (t½ = 31 min), whereas [3H]tiotropium dissociated slowly from both hM3 (t½ = 163 min) and hM2 receptor (t½ = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC50 = 9.0–9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED50 value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.

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.

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.

Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory profile and suitable for topical pulmonary administration

A novel glucocorticoids series of (GCs), 6α,9α-di-Fluoro 3-substituted C-16,17-isoxazolines was designed, synthesised and their structure-activity relationship was evaluated with glucocorticoid receptor (GR) binding studies together with GR nuclear translocation cell-based assays. This strategy, coupled with in silico modelling analysis, allowed for the identification of Cpd #15, an isoxazoline showing a sub-nanomolar inhibitory potency (IC50=0.84 nM) against TNFα-evoked IL-8 release in primary human airways smooth muscle cells. In Raw264.7 mouse macrophages, Cpd #15 inhibited LPS-induced NO release with a potency (IC50=6 nM)>10-fold higher with respect to Dexamethasone. Upon intratracheal (i.t.) administration, Cpd #15, at 0.1 μmol/kg significantly inhibited and at 1 μmol/kg fully counteracted eosinophilic infiltration in a model of allergen-induced pulmonary inflammation in rats. Moreover, Cpd #15 proved to be suitable for pulmonary topical administration given its sustained lung retention (t1/2=6.5h) and high pulmonary levels (>100-fold higher than plasma levels) upon intratracheal administration in rats. In summary, Cpd #15 displays a pharmacokinetic and pharmacodynamic profile suitable for topical treatment of conditions associated with pulmonary inflammation such as asthma and COPD.

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

Abstract 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.

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.

CHF5074 reduces brain β-amyloid burden and hyperphosphorylated tau in a mouse model of Alzheimer's disease

peptides mainly generate two isoform, Aß40 and Aß42 by enzymatic proteolysis of amyloid precursor protein (APP). In particular, the Aß42 is believed to be the major etiologic agent in pathogenesis of AD due to its higher fibrillation or oligomerization properties than that of Aß40. Recently we have established conformation dependent human antibody, B6, which binds to Aß42 fibril, but not to soluble form of Aß42, inhibiting Aß42 fibril formation. Concurrently, we have identified a mimotope of B6, B6-C15, using the PhD.C7C phage library. We chemically synthesized TAT-conjugated B6-C15 peptide, TATB6-C15. This synthetic peptide has inhibitory activity on Aß42 fibrillation. Furthermore, TAT-B6-C15 specifically binds to the oligomer form of Aß42, but not to freshly prepared monomer Aß42 nor its fibril form. In this study, we investigated the effect of this TAT-B6-C15 peptide on Aß40 assembly. Methods: Aß42 or Aß40 was incubated at 37 C in the presence or absence of TAT-B6-C15 peptide. Aß fibrillation was monitored by amyloid specific fluorescence dye, Thioflavin T. To identify the Aß conformers which specifically bound to TAT-B6-C15 peptide, we performed dot blot analysis. Aß conformers were periodically sampled after the onset of Aß40 or 42 fibrillation assay and hand-spotted onto nitrocellulose membrane, followed by incubation with detection probes such as TAT-B6-C15 or anti-Aß antibody. Results: The TAT-B6-C15 peptide exhibited inhibitory effect on Aß42, but not Aß40 fibrillation. Furthermore, the TAT-B6-C15 showed binding activity to the Aß42 prefibrillar oligomer, but not any Aß40 conformers. Conclusions: The mimotope peptide which identified as conformation dependent antibody epitope, specifically binds to prefibrillar oligomers of Aß42, inhibiting Aß42 but not Aß40 fibril formation.

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.

P2-333: The gamma-secretase modulator CHF5074 acutely reduces brain Aβ42 in the guinea pig model of human β-amyloid production

acutely reduce A 40 levels in plasma. However, due to the fundamental role -secretase plays in the intramembrane proteolysis of a number of other proteins, the inhibitor approach can be associated with side-effects including Notch-based liabilities. More recently, modulation of -secretase cleavage of APP has been reported as exemplified by the NSAIDs. This approach is more favourable as modulators cause a shift in cleavage from the amyloidogenic A 42 site towards the less amyloidogenic A 37 and A 38 sites, whereas inhibitors abolish cleavage at all -sites . Thus, the modulators decrease neurotoxic A 42 peptide levels in the absence of any detrimental effect on Notch cleavage. Methods: We have profiled a number of -secretase modulators and inhibitors in vitro and in vivo for their effect on A levels. To determine whether an increase in A 38 could be disadvantageous, we investigated the potential neurotoxic effect of A 38 peptide in vitro on rat primary cortical neurons. Results: In SHSY5Y cells expressing APP Swedish variant, -secretase modulators concentration dependently decreased A 42 while concomitantly increasing A 38. Some compounds also lowered A 40 whereas others had no effect. In transgenic mice over-expressing mutant APP and PS-1 transgenes, administration of compounds decreased A 42 in the brain. The latter effect was associated with an increased level of A 38 and consistent with the profile of a modulator. In contrast, -secretase inhibitors decreased A 38, A 40 and A 42 in cells and in transgenic mice, consistent with the profile of an inhbitor. In rat primary cortical neurons, A 42 caused significant cell death whereas A 38 was not neurotoxic. Conclusions: These findings support the development of -secretase modulators for the treatment of AD.