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.

New insight on porcine carboxylesterases expression and activity in lung tissues.

Over the course of the last twenty years, there has been a growing recognition of the pig's potential as a valuable model for studying human drug metabolism. This study aimed to investigate the expression, enzymatic activity, inhibitory susceptibility, and cellular localization of carboxylesterases (CES) in porcine lung tissue not yet explored. Our results showed that CESs hydrolysis activity followed Michaelis-Menten kinetics in both cytosolic and microsomal fractions of porcine lung tissues (N = 8), with comparable hydrolysis rates for tested substrates, namely 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD). We also determined the CESs hydrolysis activity in a representative sample of the porcine liver that, as expected, displayed higher activity than the lung ones. The study demonstrated variable levels of enzyme activities and interindividual variability in both porcine lung fractions. Inhibition studies used to assess the CESs' involvement in the hydrolysis of pNPA, 4-MUA, and FD suggested that CESs may be the enzymes primarily involved in the metabolism of ester compounds in the pig lung tissue. Overall, this study provides insight into the distribution and diversity of CES isoforms involved in substrate hydrolysis across different cellular fractions (cytosol and microsomes) in porcine lungs.

Electron FLASH radiotherapy in vivo studies. A systematic review.

FLASH-radiotherapy delivers a radiation beam a thousand times faster compared to conventional radiotherapy, reducing radiation damage in healthy tissues with an equivalent tumor response. Although not completely understood, this radiobiological phenomenon has been proved in several animal models with a spectrum of all kinds of particles currently used in contemporary radiotherapy, especially electrons. However, all the research teams have performed FLASH preclinical studies using industrial linear accelerator or LINAC commonly employed in conventional radiotherapy and modified for the delivery of ultra-high-dose-rate (UHDRs). Unfortunately, the delivering and measuring of UHDR beams have been proved not to be completely reliable with such devices. Concerns arise regarding the accuracy of beam monitoring and dosimetry systems. Additionally, this LINAC totally lacks an integrated and dedicated Treatment Planning System (TPS) able to evaluate the internal dose distribution in the case of in vivo experiments. Finally, these devices cannot modify dose-time parameters of the beam relevant to the flash effect, such as average dose rate; dose per pulse; and instantaneous dose rate. This aspect also precludes the exploration of the quantitative relationship with biological phenomena. The dependence on these parameters need to be further investigated. A promising advancement is represented by a new generation of electron LINAC that has successfully overcome some of these technological challenges. In this review, we aim to provide a comprehensive summary of the existing literature on in vivo experiments using electron FLASH radiotherapy and explore the promising clinical perspectives associated with this technology.

A bioconvergence study on platinum-free concurrent chemoradiotherapy for the treatment of HPV-negative head and neck carcinoma.

Locally advanced head and neck squamous cell carcinoma (LA-HNSCC) is characterized by high rate of recurrence, resulting in a poor survival. Standard treatments are associated with significant toxicities that impact the patient's quality of life, highlighting the urgent need for novel therapies to improve patient outcomes. On this regard, noble metal nanoparticles (NPs) are emerging as promising agents as both drug carriers and radiosensitizers. On the other hand, co-treatments based on NPs are still at the preclinical stage because of the associated metal-persistence.In this bioconvergence study, we introduce a novel strategy to exploit tumour chorioallantoic membrane models (CAMs) in radio-investigations within clinical equipment and evaluate the performance of non-persistent nanoarchitectures (NAs) in combination with radiotherapy with respect to the standard concurrent chemoradiotherapy for the treatment of HPV-negative HNSCCs. A comparable effect has been observed between the tested approaches, suggesting NAs as a potential platinum-free agent in concurrent chemoradiotherapy for HNSCCs. On a broader basis, our bioconvergence approach provides an advance for the translation of Pt-free radiosensitizer to the clinical practice, positively shifting the therapeutic vs. side effects equilibrium for the management of HNSCCs.

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.

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.

Discovery of a Potent, Selective, and Orally Bioavailable Tool Compound for Probing the Role of Lysophosphatidic Acid Type 2 Receptor Antagonists in Fibrotic Disorders.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by lung fibrosis leading to an irreversible decline of lung function. Current antifibrotic drugs on the market slow down but do not prevent the progression of the disease and are associated with tolerability issues. The involvement of lysophosphatidic acid receptor 2 (LPA2) in IPF is supported by LPA2 knockdown studies. To further validate the role of LPA2 receptors in modulating IPF and potentially other fibrotic processes, a potent and selective LPA2 receptor antagonist with a good pharmacokinetic (PK) profile is needed. Herein, we report the medicinal chemistry exploration that led to the discovery of a new class of highly potent and selective LPA2 antagonists. Among them, compound 58 exhibits excellent potency, selectivity, and oral PK profile, making it a suitable tool for probing the involvement of LPA2 receptors in IPF and other fibrotic processes.

Lymph-vascular Space Involvement and/or p53 Overexpression Correlated With the Clinical Outcome of Early-stage Endometrial Cancer Patients Treated With Adjuvant Vaginal Brachytherapy.

BACKGROUND/AIM: The majority of patients with endometrial cancer (EC) are diagnosed at an early stage and undergo primary surgery, followed by observation or adjuvant therapy according to risk factors on surgical samples. The objective of this study was to assess the correlation between a risk profile represented by the presence of substantial lymph-vascular space involvement (LVSI) and/or p53 overexpression and the clinical outcome of patients with early-stage endometrial cancer (EC) who received adjuvant vaginal brachytherapy (BT). PATIENTS AND METHODS: This investigation assessed 79 patients who underwent hysterectomy, bilateral salpingo-oophorectomy, and pelvic and/o aortic lymphadenectomy or sentinel lymph node biopsy followed by hypofractionated (HDR)-vaginal BT, using 192Ir source, for stage I-II endometrioid (n=70) or non-endometrioid (n=9) EC. Thirty-four patients (43.0%) were considered to have an unfavorable risk profile defined by the presence of substantial LVSI and /or p53 overexpression. RESULTS: Five-year disease-free survival (DFS) and five-year overall survival (OS) were 93.7% and 95%, respectively. There was a significant correlation between unfavorable risk-profile and pelvic recurrence rate (p=0.002) and distant recurrence rate (p=0.017). Patients with abnormal p53 had a higher risk of local relapse (p=0.041). Substantial LVSI was strongly associated with pelvic recurrence (p=0.001) and distant metastasis (p<0.001). CONCLUSION: The presence of substantial LVSI and/or p53 overexpression strictly correlated with poor outcome of patients with early-stage EC and should be taken into consideration for better planning adjuvant treatment in this clinical setting.

Expression and Function of ABC Transporters in Human Alveolar Epithelial Cells.

ATP-binding cassette (ABC) transporters are a large superfamily of membrane transporters that facilitate the translocation of different substrates. While ABC transporters are clearly expressed in various tumor cells where they can play a role in drug extrusion, the presence of these transporters in normal lung tissues is still controversial. Here, we performed an analysis of ABC transporters in EpiAlveolarTM, a recently developed model of human alveoli, by defining the expression and activity of MDR1, BCRP, and MRPs. Immortalized primary epithelial cells hAELVi (human alveolar epithelial lentivirus-immortalized cells) were employed for comparison. Our data underline a close homology between these two models, where none of the ABC transporters here studied are expressed on the apical membrane and only MRP1 is clearly detectable and functional at the basolateral side. According to these findings, we can conclude that other thus-far-unidentified transporter/s involved in drug efflux from alveolar epithelium deserve investigations.

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.

Design, Synthesis, and Biological Characterization of Inhaled p38α/ß MAPK Inhibitors for the Treatment of Lung Inflammatory Diseases.

The identification of novel inhaled p38α/ß mitogen-activated protein kinases (MAPK) (MAPK14/11) inhibitors suitable for the treatment of pulmonary inflammatory conditions has been described. A rational drug design approach started from the identification of a novel tetrahydronaphthalene series, characterized by nanomolar inhibition of p38α with selectivity over p38γ and p38δ isoforms. SAR optimization of 1c is outlined, where improvements in potency against p38α and ligand-enzyme dissociation kinetics led to several compounds showing pronounced anti-inflammatory effects in vitro (inhibition of TNFα release). Targeting of the defined physicochemical properties allowed the identification of compounds 3h, 4e, and 4f, which showed, upon intratracheal instillation, low plasma levels, prolonged lung retention, and anti-inflammatory effects in a rat acute model of a bacterial endotoxin-induced pulmonary inflammation. Compound 4e, in particular, displayed remarkable efficacy and duration of action and was selected for progression in disease models of asthma and chronic obstructive pulmonary disease (COPD).

Organic cation transporters (OCTs/OCTNs) in human primary alveolar epithelial cells.

Alveolar epithelium, besides exerting a key role in gas exchange and surfactant production, plays important functions in host defense and inflammation. Pathological conditions associated to alveolar dysfunction include Acute Respiratory Distress Syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The use of predictive in vitro models of human alveolar epithelium is nowadays required for the study of disease mechanisms, as well as of pharmacokinetic parameters of pulmonary drugs delivery. Here, we employed a novel 3D model of human alveoli, namely EpiAlveolar™, consisting of primary alveolar epithelial cells, pulmonary endothelial cells and fibroblasts, that reflects properly the in vivo-like conditions. In EpiAlveolar™ we performed a characterization of Organic Cation Transporters (OCTs and OCTNs) expression and activity and we found that OCTN2, OCT1 and OCT3 are expressed on the basolateral membrane; instead, ATB0,+ transporter for cationic and neutral amino acids, which shares with OCTN2 the affinity for carnitine as substrate, is readily detectable and functional at the apical side. We also show that these transporters differentially interact with anticholinergic drugs. Overall, our findings reveal close similarities of EpiAlveolar™ with the tracheal/bronchial epithelium (EpiAirway™ model) and entrust this alveolar tissue as a potential tool for the screening of biopharmaceuticals molecules.

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.

Carboxylesterases and arylacetamide deacetylase comparison in human A549, H460, and H727 pulmonary cells.

AIMS: Human carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) are serine-esterase enzymes catalyzing the hydrolysis of many compounds containing esters, amides, thioesters, or acetyl groups. This study aimed to investigate the presence, kinetic parameters, and inhibition of CES1, CES2, and AADAC in A549, H460, and H727 pulmonary cells in both living cells and S9 fractions. MATERIALS AND METHODS: The p-nitrophenyl acetate (pNPA) and 4-methylumbelliferyl acetate (4-MUA) were used as non-selective esterase substrates, whereas phenacetin as selective AADAC substrate. CESs activities were also investigated in living cells by cellular bioimaging using selective fluorescent probes. KEY FINDINGS: AADAC gene was detected in A549 and H460 cells; nevertheless, arylesterase activity was not found in relative S9 fractions. Besides, CES1 and CES2 were expressed to a different extent by all lung cells, and enzymatic activities were quite overlapping each other. All enzymes exhibited a typical Michaelis-Menten saturation curve and, regarding 4-MUA, similar Km values were found in both living cells and S9 fractions. Conversely, kinetic parameters relative to the pNPA hydrolysis by S9 fractions were significantly lower than those detected in living cells. Inhibition studies revealed that 4-MUA hydrolysis was inhibited by bis-p-nitrophenyl phosphate and phenylmethanesulfonyl fluoride more than loperamide; on the contrary, pNPA hydrolysis inhibition was limited with similar inhibition profiles being obtained in both living cells and S9 fractions. The presence of carboxylesterases was definitely confirmed by cellular bioimaging. SIGNIFICANCE: These findings add information to esterase knowledge in pulmonary cells that could be used as in vitro models for toxicological and pharmacological studies.

Inter-compound and Intra-compound Global Sensitivity Analysis of a Physiological Model for Pulmonary Absorption of Inhaled Compounds.

In recent years, global sensitivity analysis (GSA) has gained interest in physiologically based pharmacokinetics (PBPK) modelling and simulation from pharmaceutical industry, regulatory authorities, and academia. With the case study of an in-house PBPK model for inhaled compounds in rats, the aim of this work is to show how GSA can contribute in PBPK model development and daily use. We identified two types of GSA that differ in the aims and, thus, in the parameter variability: inter-compound and intra-compound GSA. The inter-compound GSA aims to understand which are the parameters that mostly influence the variability of the metrics of interest in the whole space of the drugs' properties, and thus, it is useful during the model development. On the other hand, the intra-compound GSA aims to highlight how much the uncertainty associated with the parameters of a given drug impacts the uncertainty in the model prediction and so, it is useful during routine PBPK use. In this work, inter-compound GSA highlighted that dissolution- and formulation-related parameters were mostly important for the prediction of the fraction absorbed, while the permeability is the most important parameter for lung AUC and MRT. Intra-compound GSA highlighted that, for all the considered compounds, the permeability was one of the most important parameters for lung AUC, MRT and plasma MRT, while the extraction ratio and the dose for the plasma AUC. GSA is a crucial instrument for the quality assessment of model-based inference; for this reason, we suggest its use during both PBPK model development and use.

A Hybrid In Silico/In Vitro Target Fishing Study to Mine Novel Targets of Urolithin A and B: A Step Towards a Better Comprehension of Their Estrogenicity.

SCOPE: Urolithin A and B are gut metabolites of ellagic acid and ellagitannins associated with many beneficial effects. Evidence in vitro pointed to their potential as estrogenic modulators. However, both molecular mechanisms and biological targets involved in such activity are still poorly characterized, preventing a comprehensive understanding of their bioactivity in living organisms. This study aimed at rationally identifying novel biological targets underlying the estrogenic-modulatory activity of urolithins. METHODS AND RESULTS: The work relies on an in silico/in vitro target fishing study coupling molecular modeling with biochemical and cell-based assays. Estrogen sulfotransferase and 17ß-hydroxysteroid dehydrogenase are identified as potentially subject to inhibition by the investigated urolithins. The inhibition of the latter undergoes experimental confirmation either in a cell-free or cell-based assay, validating computational outcomes. CONCLUSIONS: The work describes target fishing as an effective tool to identify unexpected targets of food bioactives detailing the interaction at a molecular level. Specifically, it described, for the first time, 17ß-hydroxysteroid dehydrogenase as a target of urolithins and highlighted the need of further investigations to widen the understanding of urolithins as estrogen modulators in living organisms.

Organic Cation Transporters (OCTs) in EpiAirway™, A Cellular Model of Normal Human Bronchial Epithelium.

Organic cation transporters (OCTs) and novel organic cation transporters (OCTNs) are responsible for drug delivery in the intestine and kidney; in the lung, OCTs mediate inhaled drugs' transport, although their physiological role in airways remains poorly understood. The studies addressing OCTs/OCTNs in human airways were mostly performed in immortal or transformed cell lines; here, we studied OCTs in EpiAirway™, a recently developed in vitro model of normal bronchial epithelium. Calu-3 monolayers were used for comparison. The activity of OCTs was evaluated by measuring the uptake of 1-methyl-4-phenylpyridinium (MPP+) at the apical and basolateral side of monolayers and protein expression through Western Blot analysis. OCTs and OCTNs expression, along with that of Amino acid Transporter B0,+ (ATB0,+)transporter, was determined by measuring the number of mRNA molecules through quantitative Polymerase Chain Reaction (qPCR). The interaction of the transporters with bronchodilators was also assessed. Results highlight significant differences between Calu-3 cells and EpiAirway™, since, in the latter, OCTs are active only on the basolateral membrane where they interact with the bronchodilator ipratropium. No activity of OCTs is detectable at the apical side; there, the most abundant carrier is, instead, SLC6A14/ATB0,+, that can thus be potentially listed among organic cation transporters responsible for drug delivery in the lung.

Characterization of ABC Transporters in EpiAirway™, a Cellular Model of Normal Human Bronchial Epithelium.

The ATP-binding cassette (ABC) transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) play a crucial role in the translocation of a broad range of drugs; data about their expression and activity in lung tissue are controversial. Here, we address their expression, localization and function in EpiAirway™, a three-dimensional (3D)-model of human airways; Calu-3 cells, a representative in vitro model of bronchial epithelium, are used for comparison. Transporter expression has been evaluated with RT-qPCR and Western blot, the localization with immunocytochemistry, and the activity by measuring the apical-to-basolateral and basolateral-to-apical fluxes of specific substrates in the presence of inhibitors. EpiAirway™ and Calu-3 cells express high levels of MRP1 on the basolateral membrane, while they profoundly differ in terms of BCRP and MDR1: BCRP is detected in EpiAirway™, but not in Calu-3 cells, while MDR1 is expressed and functional only in fully-differentiated Calu-3; in EpiAirway™, MDR1 expression and activity are undetectable, consistently with the absence of the protein in specimens from human healthy bronchi. In summary, EpiAirway™ appears to be a promising tool to study the mechanisms of drug delivery in the bronchial epithelium and to clarify the role of ABC transporters in the modulation of the bioavailability of administered drugs.

Correction: Functional analysis of OCTN2 and ATB0,+ in normal human airway epithelial cells.

[This corrects the article DOI: 10.1371/journal.pone.0228568.].

Functional analysis of OCTN2 and ATB0,+ in normal human airway epithelial cells.

In human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by ß-oxidation. Aim of the present study was to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI for different times (8d or 21d of culture) were used as comparison. OCTN2 transporter was equally expressed in both models and functional at the basolateral side. ATB0,+ was, instead, highly expressed and active on the apical membrane of EpiAirway™ and only in early-cultures of Calu-3 (8d but not 21d ALI). In both cell models, L-carnitine uptake on the apical side was significantly inhibited by the bronchodilators glycopyrrolate and tiotropium, that hence can be considered substrates of ATB0,+; ipratropium was instead effective on the basolateral side, indicating its interaction with OCTN2. Inflammatory stimuli, such as LPS or TNFα, caused an induction of SLC6A14/ATB0,+ expression in Calu-3 cells, along with a 2-fold increase of L-carnitine uptake only at the apical side; on the contrary SLC22A5/OCTN2 was not affected. As both OCTN2 and ATB0,+, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the study of drug inhalation and pulmonary delivery.