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Through his works, Celestino Testore gave an important reflection on the construction of otherness by the “natural definition”. The missionary discourse ends up confirming or invalidating, through the play of representation, the identity of the young protagonists. In order to understand the linguistic and cultural issues of missionary discourse in a given intercultural context, this article is centred on a corpus of 23 missionary narratives for youth. These texts – published in Quebec and edited by l’Apostolat de la Presse – present a classical ethnological approach for which the identification of the Other results in a closed network of meanings that use comparisons based on an ethno-centred perspective. However, in this kind of discourse we can also observe some unexpected signs of interculturality.

Marco Micone is among the most important French-speaking writers in Quebec. Through his works, he gave an important reflection on literature as a means of mediation and expression of intercultural communication. He also translated some classics of Italian literature. In our work, we intend to investigate how much the translation of Carlo Goldoni’s La Locandiera can become a paradigm through which to investigate the characteristics of intercultural communication. His Locandiera can be defined as a migrant translation, in the sense that, through it, its author expresses the complex approach to diversity and inclusion.

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 ultrahigh-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. [ABSTRACT FROM AUTHOR]

CHF6366, a dual action β2-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

Lungs are pharmacologically active organs and the pulmonary drug metabolism is of interest for inhaled drugs design. Carboxylesterases (CESs) are enzymes catalyzing the hydrolysis of many structurally different ester, amide and carbamate chemicals, including prodrugs. For the first time, the presence, kinetics, inhibition and inter-individual variations of the major liver CES isozymes (CES1 and CES2) were investigated in cytosol and microsomes of human lungs from 20 individuals using 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD) as substrates the rates of hydrolysis (V max ) for pNPA and 4-MUA, unlike FD, were double in microsomes than in cytosol. In these cellular fractions, the V max of pNPA, as CES1 marker, were much greater (30–50-fold) than those of FD, as a specific CES2 marker. Conversely, the K m values were comparable suggesting the involvement of the same enzymes. Inhibition studies revealed that the FD hydrolysis was inhibited by bis-p-nitrophenylphosphate, phenylmethanesulfonyl fluoride, and loperamide (specific for CES2), whereas the pNPA and 4-MUA hydrolysis inhibition was limited. Inhibitors selective for other esterases missed having any effect on above-mentioned activities. In cytosol and microsomes of 20 lung samples, inter-individual variations were found for the hydrolysis of pNPA (2.5–5-fold), FD or 4-MUA (8–15-fold). Similar variations were also observed in CES1 and CES2 gene expression, although determined in a small number (n = 9) of lung samples. The identification of CES1 and CES2 and their variability in human lungs are important for drug metabolism and design of prodrugs which need to be activated in this organ. [ABSTRACT FROM AUTHOR]

Lysinuric protein intolerance (LPI) is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y+L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA), a significant induction of the expression and release of the inflammatory mediators IL1β and TNFa was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1β has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. The p -nitrophenyl acetate (p NPA) 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. 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 K m values were found in both living cells and S9 fractions. Conversely, kinetic parameters relative to the p NPA 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, p NPA 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. These findings add information to esterase knowledge in pulmonary cells that could be used as in vitro models for toxicological and pharmacological studies. [Display omitted] • Lung cells comparison toward carboxylesterase and arylacetamide deacetylase enzymes • Esterase activities estimation in human A549, H460, and H727 pulmonary cells • CES1 and CES2 genes were expressed by all analyzed cell lines. • AADAC gene was found expressed by only A549 and H460 cell lines. • Comparable CESs activities were detected among all analyzed lung cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • SLC6A14/ATB0,+ is strongly expressed at the apical side of alveolar epithelium. • OCT1, OCT3, and OCTN2 are active at the basolateral side. • All these transporters are involved in the interaction with respiratory drugs. [ABSTRACT FROM AUTHOR]

Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[ 3 H]carnitine transport. Carnitine uptake was strictly Na + -dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (K m < 2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (K m ~ 20 μM) and a low affinity (K m > 1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB 0,+ , a Na + , and Cl – -coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB 0,+ silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects. [ABSTRACT FROM AUTHOR]

Organic cation transporters (OCT1–3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1–3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [ 3 H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE 2 ) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component ( K m ~ 50 μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE 2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high ( K m < 20 μM) and a low affinity ( K m > 0.6 mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air–liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. [ABSTRACT FROM AUTHOR]

Abstract: Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may delay or prevent the onset of Alzheimer''s disease (AD). A subset of NSAIDs, including flurbiprofen, has been shown to selectively inhibit the production of β-amyloid1–42 (Aβ42), independently from their cyclooxygenase (COX) inhibiting activity. We evaluated the in vitro and in vivo profiles of CHF5022 and CHF5074, two flurbiprofen analogues. The in vitro Aβ inhibiting activity was evaluated in a human neuroglioma cell line (H4) carrying the double Swedish mutation (K595N/M596L) of the human amyloid precursor protein (APPsw). The in vitro anti-COX activity was evaluated using human recombinant enzymes isolated from transfected Sf-9 cells. The in vivo pharmacokinetic and pharmacodynamic profiles of the two compounds were evaluated in young APPsw transgenic mice (Tg2576) after oral gavage (100 or 300mgkg−1 day−1 for 4–5 days) and after medicated diet (375ppm for 4 weeks). R-Flurbiprofen was used as comparator. In vitro, CHF5022 and CHF5074 were found to be 3- and 7-fold more potent than R-flurbiprofen in inhibiting Aβ42 secretion (IC50s of 92, 40 and 268μM, respectively). Differently from R-flurbiprofen, CHF5022 and CHF5074 did not affect COX-1 (at 100μM) and COX-2 (at 300μM) activity. Similarly to R-flurbiprofen, no significant alteration in the expression profile of a subset of Notch intracellular domain-responsive genes was observed with either CHF5022 or CHF5074. In Tg2576 mice, CHF5022 was well tolerated when administered by oral gavage (100mgkg−1 day−1 for 5 days) or by medicated diet (56mg kg−1 day−1 for 4 weeks). R-Flurbiprofen was poorly tolerated in the diet (32mgkg−1 day−1) with 55% of the animals dying during the first week of treatment. After 4–5 days of oral gavage, CHF5022 and CHF5074 plasma and brain levels at 3h were found to increase with the dose, leading to brain concentrations of about 10% and 5% of the corresponding plasma concentrations, respectively. In animals fed for 4 weeks with compound-supplemented diet, mean plasma (580μM) and brain (20μМ) concentrations of CHF5022 were 8 and 15 times higher than those of R-flurbiprofen. Plasma Aβ42 concentration was dose-dependently decreased by CHF5022 and CHF5074. Brain Aβ levels (formic acid-extractable) were not significantly affected by either compound, although Aβ42 levels tended to inversely correlate (P =0.105) with CHF5022 concentration in the brain. CHF5022 and CHF5074 thus appear to have a promising in vitro and in vivo profile. This warrants further evaluation of their long-term effects on Aβ brain pathology. [Copyright &y& Elsevier]

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.

Clinical guidelines for COPD and asthma recommend inhaled β-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 β 2 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.

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.

CHF6366, a dual action β 2 -receptor agonist and M3-muscarinic receptor antagonist developed for chronic obstructive pulmonary disease (COPD) was [ 14 C]-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.

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., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The development of molecules embedding two distinct pharmacophores acting as muscarinic antagonists and β 2 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.

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.

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 β-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., Competing Interests: The authors have declared that no competing interests exist.

Human arylacetamide deacetylase (AADAC) is a single microsomal serine esterase involved in the hydrolysis of many acetyl-containing drugs. To date, the presence and activity of the AADAC enzyme in human lungs has been scarcely examined. We investigated its gene and protein expression as well as interindividual variations in AADAC activities in a large number of human lungs ( n = 25) using phenacetin as a selective substrate. The kinetic parameters K m and V max were determined. Our findings highlighted a high interindividual variability in both AADAC mRNA levels and hydrolysis activities. Furthermore, for the first time we demonstrated the presence of the AADAC protein in various lung samples by means of immunoblot analysis. As a comparison, phenacetin hydrolysis was detected in pooled human liver microsomes. Lung activities were much lower than those found in the liver. However, similar K m values were found, which suggests that this hydrolysis could be due to the same enzyme. Pulmonary phenacetin hydrolysis proved to be positively correlated with AADAC mRNA (* P < 0.05) and protein (* P < 0.05) levels. Moreover, the average values of AADAC activity in smokers was significantly higher than in nonsmoker subjects (* P < 0.05), and this might have an important role in the administration of some drugs. These findings add more information to our knowledge of pulmonary enzymes and could be particularly useful in the design and preclinical development of inhaled drugs. SIGNIFICANCE STATEMENT: This study investigated the presence and activity of the AADAC enzyme in several human lungs. Our results highlight high interindividual variability in both AADAC gene and protein expression as well as in phenacetin hydrolysis activity. These findings add more information to our knowledge of pulmonary enzymes and could be particularly useful in the design and preclinical development of inhaled drugs., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Recently, mesenchymal stromal stem cells (MSCs) have been proposed as therapeutic agents because of their promising preclinical features and good safety profile. However, their introduction into clinical practice has been associated with a suboptimal therapeutic profile. In this review, we address the biodistribution of MSCs in preclinical studies with a focus on the current understanding of the pharmacodynamics (PD) and pharmacokinetics (PK) of MSCs as key aspects to overcome unsatisfactory clinical benefits of MSC application. Beginning with evidence of MSC biodistribution and highlighting PK and PD factors, a new PK-PD model is also proposed. According to this theory, MSCs and their released factors are key players in PK, and the efficacy biomarkers are considered relevant for PD in more predictive preclinical investigations. Accounting for the PK-PD relationship in MSC translational research and proposing new models combined with better biodistribution studies could allow realization of the promise of more robust MSC clinical translation.

The interest in using physiologically-based pharmacokinetic (PBPK) models as a support to the drug development decision making process has rapidly increased in the last years. These kind of models are examples of the "bottom up" modelling strategy, which progressively integrates into a mechanistic framework different information as soon as they become available along the drug development. For this reason PBPK models can be used with different aims, from the early stages of drug development up to the clinical phases. Different software tools are nowadays available. They can be categorized in "designed software" and "open software". The first ones typically include commercial platforms expressly designed to implement PBPK models, in which the model structure is pre-defined, assumptions are generally not explicitly declared and equations are hidden to the user. Even if the software is validated and routinely used in the pharmaceutical industry, sometimes they do not allow working with the flexibility needed to cope with specific applications/tasks. For this reason, some scientists prefer to define and implement their own PBPK tool in "open" software. This paper shows how to build an in-house PBPK tool from species-related physiological information available in the literature and a limited number of drug specific parameters generally made available by the drug development process. It also reports the results of an evaluation exercise that compares simulated plasma concentration-time profiles and related pharmacokinetic (PK) parameters (i.e., AUC, C max and T max ) with literature and in-house data. This evaluation involved 25 drugs with different physico-chemical properties, intravenously or orally administrated in three different species (i.e., rat, dog and man). The comparison shows that model predictions have a good degree of accuracy, since the average fold error for all the considered PK parameters is close to 1 and only in few cases the fold error is greater than 2. In summary, the paper demonstrates that addressing specific aims when needed is possible by creation of in-house PBPK tools with satisfactory performances and it provides some suggestions how to do that., Competing Interests: Conflict of interest: N. Cesari, G. Brogin and P. Puccini are employees of Chiesi Farmaceutici spa, the sponsor of the study. The authors report no other conflicts of interest in this work., (Copyright © 2018 by the authors.)

l-Carnitine, in addition to playing a fundamental role in the β-oxidation of fatty acids, has been recently identified as a modulator of immune function, although the mechanisms that underlie this role remain to be clarified. In this study, we addressed the modulation of l-carnitine transport and expression of related transporters during differentiation of human monocytes to macrophages. Whereas monocytes display a modest uptake of l-carnitine, GM-CSF-induced differentiation massively increased the saturable Na + -dependent uptake of l-carnitine. Kinetic and inhibition analyses demonstrate that in macrophage l-carnitine transport is mediated by a high-affinity component (K m ∼4 µM) that is identifiable with the operation of OCTN2 transporter and a low-affinity component (K m > 10 mM) that is identifiable with system A for neutral amino acids. Consistently, both SLC22A5/OCTN2 and SLC38A2/SNAT2 are induced during the differentiation of monocytes to macrophages at gene and protein levels. Elucidation of GM-CSF signaling demonstrates that the cytokine causes the activation of mTOR kinase, leading to the phosphorylation and activation of STAT3, which, in turn, is responsible for OCTN2 transcription. SLC22A5/OCTN2 therefore emerges as a novel member of the set of genes markers of macrophage differentiation., (© Society for Leukocyte Biology.)

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.)

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.

Flurbiprofen, a nonsteroidal antiinflammatory drug (NSAID), has been recently described to selectively inhibit beta-amyloid(1)(-)(42) (Abeta42) secretion, the most toxic component of the senile plaques present in the brain of Alzheimer patients. The use of this NSAID in Alzheimer's disease (AD) is hampered by a significant gastrointestinal toxicity associated with cyclooxygenase (COX) inhibition. New flurbiprofen analogues were synthesized, with the aim of increasing Abeta42 inhibitory potency while removing anti-COX activity. In vitro ADME developability parameters were taken into account in order to identify optimized compounds at an early stage of the project. Appropriate substitution patterns at the alpha position of flurbiprofen allowed for the complete removal of anti-COX activity, while modifications at the terminal phenyl ring resulted in increased inhibitory potency on Abeta42 secretion. In rats, some of the compounds appeared to be well absorbed after oral administration and to penetrate into the central nervous system. Studies in a transgenic mice model of AD showed that selected compounds significantly decreased plasma Abeta42 concentrations. These new flurbiprofen analogues represent potential drug candidates to be developed for the treatment of AD.

A cell-based assay using homogeneous time-resolved fluorescence has been developed for high throughput screening of putative beta-amyloid (Abeta)production inhibitors. In this assay, total Abeta is detected by simply adding two commercially available antibody complexes. The first was a biotinylated monoclonal antibody (4G8), specifically recognizing an epitope comprising the residues 17-24 of the Abetapeptide, complexed with europium cryptate-streptavidin conjugate. The second was a polyclonal antibody (BioS-N), raised against the N-terminus of the Abeta peptide, complexed with an allophycocyanin-anti rabbit antibody conjugate. Binding of the two complexes to the Abeta peptide brought europium cryptate (fluorescence donor) and allophycocyanin (fluorescence acceptor) into close proximity, consequently a fluorescent resonance energy transfer signal was produced upon excitation at 337 nm. The resulting fluorescence signal (665 nm) was then detected using a Discovery or a ViewLux reader. Detection of Abeta by the proposed method is possible at concentrations of approximately 1 nM. The method was employed for the detection of Abeta secreted from a stable transfected human neuroglioma cell line (H4) overexpressing a mutated form of the human amyloid precursor protein (APP695NL) and developed for robotic automation. At optimized conditions, signal-to-background ratios exceeding 5 and Z' factors around 0.7 were achieved in a 384-well format. High throughput screening of 56,913 potential Abeta production inhibitors led to identification of new non-cytotoxic and cell permeable compounds with potencies in the submicromolar range.