Discovery, Multiparametric Optimization, and Solid-State Driven Identification of CHF-6550, a Novel Soft Dual Pharmacology Muscarinic Antagonist and ß2 Agonist (MABA) for the Inhaled Treatment of Respiratory Diseases.

Clinical guidelines for COPD and asthma recommend inhaled ß-adrenergic agonists, muscarinic antagonists, and, for frequent exacerbators, inhaled corticosteroids, with the challenge of combining them into a single device. The MABA (muscarinic antagonist and ß2 agonist) concept has the potential to simplify this complexity while increasing the efficacy of both pharmacologies. In this article, we report the outcome of our solid-state driven back-up program that led to the discovery of the MABA compound CHF-6550. A soft drug approach was applied, aiming at high plasma protein binding and high hepatic clearance, concurrently with an early stage assessment of crystallinity through a dedicated experimental workflow. A new chemotype was identified, the diphenyl hydroxyacetic esters, able to generate crystalline material. Among this class, CHF-6550 demonstrated in vivo efficacy, suitability for dry powder inhaler development, favorable pharmacokinetics, and safety in preclinical settings and was selected as a back-up candidate, fulfilling the desired pharmacological and solid-state profile.

Application of an "inhalation by design" approach to the identification and in-vitro evaluation of novel purine based PI3Kδ inhibitors.

PI3Kδ is a lipid kinase which plays a key role in airway inflammatory conditions. Accordingly, the inhibition of PI3Kδ can be considered a valuable strategy for the treatment of chronic respiratory diseases such as Asthma and Chronic obstructive pulmonary disease (COPD). In this work, we describe our efforts to identify new PI3Kδ inhibitors following an "inhalation by design" strategy. Starting from the identification of a purine scaffold, we carried out a preliminary SAR expansion which led to the identification of a new hit characterized by a high enzymatic potency and moderate PI3Kδ selectivity. A subsequent optimization led to novel purine based derivatives with favorable in vitro ADME profiles, which might represent promising starting points for future development of new inhaled drug candidates.

Direct Evidence of the Presence of Cross-Linked Aß Dimers in the Brains of Alzheimer's Disease Patients.

Brain-derived amyloid-ß (Aß) dimers are associated with Alzheimer's disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link has been proposed to make brain-derived dimers (brain dimers) more synaptotoxic than Aß monomers and would also make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aß (CL Aß) dimers, obtained by means of the photoinduced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative brain CL Aß dimers. Second, we used these PICUP CL Aß dimers as standards to improve the isolation of brain Aß dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of brain Aß dimer samples allowing the detection of the CL [Aß(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aß dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease.

SDS-PAGE analysis of Aß oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS.

The characterization of amyloid-beta peptide (Aß) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aß40 and Aß42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aß42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aß oligomer samples.