Quantitation of Commercially Available API Solid Forms by Application of the NMR-qSRC Approach: An Optimization Strategy Based on In Silico Simulations.

Physical forms of active pharmaceutical ingredients (APIs) play a crucial role in drug discovery since 85% of API molecules exhibit polymorphism and sometimes complicated phase behavior, often resulting in important differences in the respective biochemical and physical properties. Characterization and quantitation of the different forms are becoming more and more essential in the pharmaceutical industry: once these characteristics are known, it is easier to choose the best solid form for development, formulation, manufacturing, and storage. Time domain-nuclear magnetic resonance (TD-NMR) has recently been used to develop a quantitation protocol for solid mixtures, named qSRC, based on the linear combination of T1 saturation recovery curves (SRCs) collected on a bench-top instrument. Despite its potentials and ease of use, a limited number of application cases have been reported in the literature since its development and many aspects remain to be clarified for the technique to be adopted as a robust routinely industrial analytical tool. In the present work, the reliability of the qSRC approach has been studied by focusing on the role played by key experimental variables, including mixture composition, signal-to-noise ratio, and T1 differences. In silico simulations were carried out for a wide range of theoretical cases to predict the expected level of accuracy obtainable for a given sample-parameter acquisition set and to clearly define the range of applicability of the method. Results of the simulation are presented alongside a comparison with three real-case studies of commercially available APIs: piroxicam, naproxen sodium, and benzocaine.

Discovery of a novel class of inhaled dual pharmacology muscarinic antagonist and ß2 agonist (MABA) for the treatment of chronic obstructive pulmonary disease (COPD).

The targeting of both the muscarinic and ß-adrenergic pathways is a well validated therapeutic approach for the treatment of chronic obstructive pulmonary disease (COPD). In this communication we report our effort to incorporate two pharmacologies into a single chemical entity, whose characteristic must be suitable for a once daily inhaled administration. Contextually, we aimed at a locally acting therapy with limited systemic absorption to minimize side effects. Our lung-tailored design of bifunctional compounds that combine the muscarinic and ß-adrenergic pharmacologies by the elaboration of the muscarinic inhibitor 7, successfully led to the potent, pharmacologically balanced muscarinic antagonist and ß2 agonist (MABA) 13.

Sample preparation strategy for the detection of steroid-like compounds using MALDI mass spectrometry imaging: pulmonary distribution of budesonide as a case study.

Corticosteroids as budesonide can be effective in reducing topic inflammation processes in different organs. Therapeutic use of budesonide in respiratory diseases, like asthma, chronic obstructive pulmonary disease, and allergic rhinitis is well known. However, the pulmonary distribution of budesonide is not well understood, mainly due to the difficulties in tracing the molecule in lung samples without the addition of a label. In this paper, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging protocol that can be used to visualize the pulmonary distribution of budesonide administered to a surfactant-depleted adult rabbit. Considering that budesonide is not easily ionized by MALDI, we developed an on-tissue derivatization method with Girard's reagent P followed by ferulic acid deposition as MALDI matrix. Interestingly, this sample preparation protocol results as a very effective strategy to raise the sensitivity towards not only budesonide but also other corticosteroids, allowing us to track its distribution and quantify the drug inside lung samples.

Mass spectrometry imaging as a tool for evaluating the pulmonary distribution of exogenous surfactant in premature lambs.

BACKGROUND: The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb. METHODS: Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH2O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant. RESULTS: Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p < 0.05). Nevertheless, the physiological response to surfactant treatment was not uniform across all animals. SP-C analog and SP-B analog were successfully imaged and quantified by means of MSI in the peripheral lungs of all surfactant-treated animals. The intensity of the signal was remarkably low in untreated lambs, corresponding to background noise. The signal intensity of SP-B analog in each surfactant-treated animal, which represents the surfactant distributed to the peripheral right lung, correlated well with the physiologic response as assessed by the area under the curves of the individual arterial partial oxygen pressure and dynamic lung compliance curves of the lambs. CONCLUSIONS: Applying MSI, we were able to detect, locate and quantify the amount of exogenous surfactant distributed to the lower right lung of surfactant-treated lambs. The distribution pattern of SP-B analog correlated well with the pulmonary physiological outcomes of the animals. MSI is a valuable label-free technique which is able to simultaneously evaluate qualitative and quantitative drug distribution in the lung.

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 and Optimization of Pyridazinones as PI3Kδ Selective Inhibitors for Administration by Inhalation.

A hit-to-lead campaign pursuing the identification of novel inhalant small-molecule phosphatidylinositol 3-kinase (PI3K) inhibitors for the treatment of inflammatory respiratory diseases is disclosed. A synthetically versatile pyridazin-3(2H)-one scaffold was designed, and three exit vectors on the core moiety were used to explore chemical diversity and optimize pharmacological and absorption, distribution, metabolism, and excretion (ADME) properties. Desired modulation of PI3Kδ selectivity and cellular potency as well as ADME properties in view of administration by inhalation was achieved. Intratracheal administration of lead compound 26 resulted in a promising pharmacokinetic profile, thus demonstrating that the optimization strategy of in vitro profiles successfully translated to an in vivo setting.

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.

Identification of two cannabimimetic compounds WIN48098 and AM679 in illegal products.

Two synthetic cannabinoids have been identified, during a survey, as new adulterants; they might have been intended to be used as ingredients for smart drugs. The characterization of these compounds has been made by gas chromatography-mass spectrometry (GC-MS), Orbitrap high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR), leading to the identification of WIN48098, a compound disclosed as a new adulterant in herbal and powder products, and AM679, identified in Italy for the first time. Taking into account the high number of synthetic cannabinoids seized during the last year in Italy, how quickly they appear on the illegal market and the rapidity required for analytical results, a method was developed for the simultaneous quantitation of several synthetic cannabinoids, using gas chromatography-flame ionization detection (GC-FID).

Residual Dipolar Coupling Based Conformational Analysis Allows the Configurational Assessment of Steroids with up to Eight Stereocenters.

Residual dipolar couplings (RDCs) induced by anisotropic media have been proved as a powerful tool for the structure elucidation of organic molecules in solution in nuclear magnetic resonance (NMR) based analysis. The value of dipolar couplings to solve complex conformational and configurational problems represents indeed an appealing analytical tool for the pharmaceutical industry particularly focusing on the stereochemistry characterization of NCEs since the early phase of the drug development process. In our work, RDCs were used for the conformational and configurational study of synthetic steroids with multiple stereocenters - prednisone and beclomethasone dipropionate (BDP) -. For both molecules the correct relative configuration was identified among all the possible diastereoisomers (32 and 128 respectively) arising from the compounds stereogenic carbons. Only for prednisone the use of additional experimental data (i. e. rOes) was necessary to resolve the right stereochemical structure.

Designing Soluble PROTACs: Strategies and Preliminary Guidelines.

Solubility optimization is a crucial step to obtaining oral PROTACs. Here we measured the thermodynamic solubilities (log S) of 21 commercial PROTACs. Next, we measured BRlogD and log kwIAM (lipophilicity), EPSA, and Δ log kwIAM (polarity) and showed that lipophilicity plays a major role in governing log S, but a contribution of polarity cannot be neglected. Two-/three-dimensional descriptors calculated on conformers arising from conformational sampling and steered molecular dynamics failed in modeling solubility. Infographic tools were used to identify a privileged region of soluble PROTACs in a chemical space defined by BRlogD, log kwIAM and topological polar surface area, while machine learning provided a log S classification model. Finally, for three pairs of PROTACs we measured the solubility, lipophilicity, and polarity of the building blocks and identified the limits of estimating PROTAC solubility from the synthetic components. Overall, this paper provides promising guidelines for optimizing PROTAC solubility in early drug discovery programs.

Discovery of Clinical Candidate CHF-6366: A Novel Super-soft Dual Pharmacology Muscarinic Antagonist and ß2 Agonist (MABA) for the Inhaled Treatment of Respiratory Diseases.

The development of molecules embedding two distinct pharmacophores acting as muscarinic antagonists and ß2 agonists (MABAs) promises to be an excellent opportunity to reduce formulation issues and boost efficacy through cross-talk and allosteric interactions. Herein, we report the results of our drug discovery campaign aimed at improving the therapeutic index of a previous MABA series by exploiting the super soft-drug concept. The incorporation of a metabolic liability, stable at the site of administration but undergoing rapid systemic metabolism, to generate poorly active and quickly eliminated fragments was pursued. Our SAR studies yielded MABA 29, which demonstrated a balanced in vivo profile up to 24 h, high instability in plasma and the liver, as well as sustained exposure in the lung. In vitro safety and non-GLP toxicity studies supported the nomination of 29 (CHF-6366) as a clinical candidate, attesting to the successful development of a novel super-soft MABA compound.

Surfactant-Assisted Distal Pulmonary Distribution of Budesonide Revealed by Mass Spectrometry Imaging.

Direct lung administration of budesonide in combination with surfactant reduces the incidence of bronchopulmonary dysplasia. Although the therapy is currently undergoing clinical development, the lung distribution of budesonide throughout the premature neonatal lung has not yet been investigated. Here, we applied mass spectrometry imaging (MSI) to investigate the surfactant-assisted distal lung distribution of budesonide. Unlabeled budesonide was either delivered using saline as a vehicle (n = 5) or in combination with a standard dose of the porcine surfactant Poractant alfa (n = 5). These lambs were ventilated for one minute, and then the lungs were extracted for MSI analysis. Another group of lambs (n = 5) received the combination of budesonide and Poractant alfa, followed by two hours of mechanical ventilation. MSI enabled the label-free detection and visualization of both budesonide and the essential constituent of Poractant alfa, the porcine surfactant protein C (SP-C). 2D ion intensity images revealed a non-uniform distribution of budesonide with saline, which appeared clustered in clumps. In contrast, the combination therapy showed a more homogeneous distribution of budesonide throughout the sample, with more budesonide distributed towards the lung periphery. We found similar distribution patterns for the SP-C and budesonide in consecutive lung tissue sections, indicating that budesonide was transported across the lungs associated with the exogenous surfactant. After two hours of mechanical ventilation, the budesonide intensity signal in the 2D ion intensity maps dropped dramatically, suggesting a rapid lung clearance and highlighting the relevance of achieving a uniform surfactant-assisted lung distribution of budesonide early after delivery to maximize the anti-inflammatory and maturational effects throughout the lung.

Characterization of the phosphoproteome in human bronchoalveolar lavage fluid.

Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL) from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphoproteins, including protein identities and exact description of their phosphorylation sites. We used a gel-free bioanalytical workflow that included whole-proteome digestion of depleted BAL proteins, enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC), LC-MS/MS analyses with a linear ion trap mass spectrometer, and searches of a protein sequence database to generate a panel of BAL phosphoproteins and their sites of phosphorylation. Based on sequence-diagnostic MS/MS fragmentation patterns, we identified a collection of 36 phosphopeptides that contained 26 different phosphorylation sites. These phosphopeptides mapped to 21 phosphoproteins including, for example, vimentin, plastin-2, ferritin heavy chain, kininogen-1, and others. The characterized phosphoproteins have diverse characteristics in terms of cellular origin and biological function. To the best of our knowledge, results of this study represent the first description of the human BAL phosphoproteome.