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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.

Background: Extensive studies have suggested epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transformation (FMT) as major drivers of fibrosis. Both EMT and FMT are strongly regulated by TGFβ1. Objectives. The aim of this study was to assess a specific transcriptional analysis of a selected panel of genes involved in fibrogenesis by profiling TGFβ-stimulated human lung fibroblasts (HLFs) and human bronchial epithelial cells (HBECs) derived from IPF patients and healthy donors. Moreover, the effects of Nintedanib and the ALK5 inhibitor, SB525334, were evaluated. Methods: Cultured primary HLFs and HBECs were stimulated with TGF-β1 (1.25 and 5 ng/ml, respectively). IPF-derived cells were exposed to Nintedanib (3 µM) and SB525334 (1 µM). RNA was extracted and subsequently reverse transcribed and amplified. RT2 Profiler™ PCR Arrays Human Fibrosis (Qiagen) were used to gene expression analysis. Results: Approximately half of the assessed genes were endowed with a differential expression (≥1.5 fold) between non-stimulated IPF and control HLFs (e.g. GREM1, HGF) and HBECs (e.g. ITGB3, ENG). The above expression pattern was further modified when TGF-β1 was applied to IPF and control cells. Both Nintedanib and SB525334 mainly induced a significant effect on TGFβ1-induced differential expression of genes related to extracellular matrix remodelling (e.g. COL1A1 and COL3A1). Conclusions: Our study revealed a basal differential expression of a selected panel of genes among control and IPF cells. Furthermore, the subsequent validation step with Nintedanib may provide insights on new targets endowed with a potential anti-fibrotic effect.

Background: Evaluation of fibrosis and its severity is the commonest parameter to screen potential new medicines in rodents treated with bleomycin (BLM). Results are mainly reported by histological analysis and indirect measurement of collagen through hydroxyproline (HYP) quantification. Objectives: In the present study we performed a comparative analysis of different methods to assess collagens induced by BLM in the mouse lung. Methods: BLM (0.02 U/kg) was administered by oropharyngeal aspiration to male C57BL/6 mice on day 0 and day 4. Oral Nintedanib (60 mg/kg/day) started from day 7. Three and four weeks after the first BLM administration, histopathological examination and collagen genes expression analysis were applied. Moreover, HYP and collagen isoforms (LC-MS) quantification in lung homogenates were assessed. Results: Ashcroft score and automated measure of lung fibrosis revealed significant fibrotic lesions in BLM-treated animals. Specific collagen genes appeared up-regulated in BLM group. LC-MS quantification of collagen isoforms correlated with HYP levels although, interestingly, showed a higher therapeutic window between control and BLM group (1.5 vs. 0.5 fold, respectively). Nintedanib significantly affected all the evaluated readouts. Conclusions: Our study provides a comprehensive analysis of changes in collagen genes and proteins following BLM treatment in mice and the effect of Nintedanib. Importantly, our data indicate for the first time LC-MS as a sensitive and reliable method for collagen quantification in lung homogenate. This approach might implement the most employed HYP assay to achieve reliable predictions of efficacy for therapeutics in lung fibrosis.

Introduction Phenylketonuria (PKU MIM 261600) is a human metabolic disease caused by mutations in phenylalanine hydroxylase gene (PAH) and is inherited in an autosomal recessive Mendelian fashion. Phenylalanine hydroxylase (PAH also known as phenylalanine 4-monooxygenase EC 1,14,16,11) catalyzes the rate-limiting step in L-Phenylalanine (L-Phe) catabolism in liver, using tetrahydrobiopterin (BH4) and dioxygen as additional cosubstrates. Untreated PKU is associated with an abnormal phenotype which includes growth failure, poor skin pigmentation, microcephaly, seizures, global developmental delay and severe intellectual impairment. However, since the introduction of newborn screening programs and with early dietary intervention, children born with PKU can now expect to lead relatively normal lives.Dried Blood Spot, (DBS) is the golden method used to acquire specimen for neonatal screening of PKU. It is fast, not expensive and give the possibility to collect samples far from the reference centre and to quickly delivery to the analytical laboratory, where the Phe content is evaluated by Tandem Mass Chromatography. However, because PKU needs a continous followup, would be really worth to set a new, fast, reproducible and chipper biochemical approach to monitor patients and to optimize the dietary intervention. Here we describe a novel fast and non-destructive method to assay phenylalanine in human blood using Near Infra-Red (NIR) spectroscopy. Methods By using a mobile infrared micro sensor (SCIO sensor) calibrated in the Near Infrared spectrum (700nm to 1000nm), we acquired by scanning and elaborated spectra from DBS card alone, DBS card with a reference concentration scale for Phe alone, and Phe plus Tyr. Based on these data we selected the most sensitive wavelentgh window within the NIR and exploited this parameter to acquire spectra from DBS containingμ blood spiked with 0 to 1200 μM Phe, 0 to 1200 Tyr and with different Phe/Tyr.ratios. An additional experiment using 100 calibrators with 5 datapoints for phenylalaninemia was performed. For each sample, at least 20 scans in duplicate were acquired. Principal component analysis (PCA) was conducted. Results The analysis of the ‘scrubbed’ data both by a specific image elaboration software and by data point algorithms, clear show that it is possible to correlate the nature of the analyte, and its relative concentration with significative differences in the acquired spectra, to obtained the so called specific Phe NIR fingerprint of the sample These observation open the exciting possibility to design a chemiometric model to assay the amount of Phe in DBS without elution and the expensive chromatographic procedures.

In the last several years, NAD+ supplementation has emerged as an innovative and safe therapeutic strategy for a wide spectrum of disorders, including diabetes and neuropathy. However, critical questions remain as to how NAD+ and its precursors are taken up by cells, as well as the effects of long-lasting intracellular NAD+ (iNAD+) increases. Here, we investigated the kinetics of iNAD+ levels in different cell types challenged with prolonged exposure to extracellular NAD+ (eNAD+). Surprisingly, we found that after the initial increase, iNAD+ contents decreased back to control levels (iNAD+ resetting). Focusing our attention on HeLa cells, we found that oxygen and ATP consumption occurred with similar temporal kinetics after eNAD+ exposure. Using [3H]NAD+ and [14C]NAD+, we determined that NAD+ resetting was not due to increased dinucleotide extrusion but rather due to reduced uptake of cleaved NAD+ products. Indeed, eNAD+ exposure reduced the expression of the ecto-5′-nucleotidase CD73, the nicotinamide adenine mononucleotide transporter solute carrier family 12 member 8, and the nicotinamide riboside kinase. Interestingly, silencing the NAD+-sensor enzyme sirtuin 1 prevented eNAD+-dependent transcriptional repression of ecto-5′-nucleotidase, solute carrier family 12 member 8, and nicotinamide riboside kinase, as well as iNAD+ resetting. Our findings provide the first evidence for a sirtuin 1–mediated homeostatic response aimed at maintaining physiological iNAD+ levels in conditions of excess eNAD+ availability. These data may be of relevance for therapies designed to support the NAD+ metabolome via extracellular supplementation of the dinucleotide or its precursors.

Background & Objectives: The aim of this study was to investigate the time-course of the molecular and histological changes induced by a double intratracheal (IT) administration of bleomycin (BLM) on lung tissue. Methods: Male SD rats were injected with BLM (2 U/kg, IT) or saline on day 0 and day 4 and sacrificed at day 7, 14, 21, 28 and 56 after BLM first dose. Several inflammatory and fibrotic biomarkers were assessed in lung, BALF and plasma by colorimetric or immunometric assays. Histomorphometric analysis of lung fibrosis was performed on Masson’s trichrome stained sections. Results & Conclusions: In lungs of BLM-treated animals, several ECM proteins were significantly increased at day 7 (30-, 40- and 80-fold increase for TIMP1, PAI-1 and SPP1, respectively), and progressively declined over time, reaching control values at day 56. A significant increase in lung hydroxyproline levels was evident starting from day 14 (2.4-fold increase) and was maintained over the duration of the study. Histomorphometric evaluation revealed a sustained fibrosis during the first three weeks (fibrotic area: 50%), followed by a progressive reduction at day 28 and 56 (30%). Overall, these results indicate that the most suitable time point for assessing lung fibrosis and its targeted interventions is between 21 and 28 days post-BLM, where the fibrotic response appears to be more stable and consistent. Additional investigations, including transcriptomic and proteomic approaches will be essential to obtain a more comprehensive characterisation of the present model.

Background: Idiopathic Pulmonary Fibrosis (IPF), is a progressive lung disease characterized by basal and subpleural fibrosis associated with honeycomb changes. In experimental settings, single intratracheal (IT) administration of bleomycin (BLM) to rodents is the most commonly used model. In order to more closely mimic human IPF, a novel experimental procedure with two IT administrations of BLM in rats is described. Aims and objectives: Histological and biomolecular findings following single and double IT administration of BLM in the rat were compared. Furthermore, the effect of Nintedanib was assessed. Methods: Single (4U/Kg, day 1) or double (2 X 2U/Kg, day 1 and day 4) administration of BLM was given IT to male SD rats. Nintedanib (100 mg/kg, oral) was administered once daily for three weeks starting from day 7. Morphometric quantification of different patterns of lung fibrosis and cluster gene expression on tissue homogenate (Real Time PCR) were performed. Results: A single BLM administration induced focal collagen deposition mostly surrounding the main bronchi. In contrast, two BLM administrations seemed to evoke a more diffuse pattern of interstitial collagen deposition widening lung parenchyma. Reduction of lung fibrosis by Nintedanib was more effective in the 2 X 2U/Kg group (~46%) compared to 4U/Kg group (~11%). Moreover, normalisation by Nintedanib of differentially modulated IPF-associated genes was more evident in 2 X 2U/Kg group. Conclusions: Our study provides a novel improved BLM rat model of IPF that is sensitive to the effect of Nintedanib and functional to assess efficacy in drug development.

Background: In the last 10 years, advanced molecular biology approaches and profiling techniques have greatly contributed to identify key regulatory molecules and pathways involved in several pathological states. IPF is a relatively poorly understood chronic and progressive disease and much of the information regarding the involved etiopathogenetic mechanisms was generated in the rodent bleomycin (BLM) model. A comprehensive multidisciplinary approach is proposed here to study molecular alterations in the lungs of BLM-treated rats and to improve the predictive nature of this model. Aims and objectives: We established proteomic and genomic survey approaches to monitor molecular changes induced by BLM in the rat airways and the therapeutic effect by Nintedanib. Methods: Male SD rats were intratracheally injected with a single administration of BLM (4U/Kg). Nintedanib (100 mg/kg, oral) was administered once daily for three weeks starting 7 days after BLM administration. Proteomic and gene expression analysis (Real Time PCR) were assessed in BALFs and lung homogenates. Levels of some targeted proteins were also monitored by ELISA specific assays. Results and Conclusions: The results highlight the peculiar features of BALFs and lung homogenates for the comprehensive molecular profiling of the rat BLM model. Investigation of a set of signalling transduction pathways altered in the BLM model and affected by Nintedanib is disclosed. Perspectives on targeted monitoring of novel markers of lung fibrosis are also discussed for a better understanding of the molecular pathogenesis of IPF and the response to therapy. VP and BP equally contributed.

Therapeutic hypothermia is of relevance to treatment of increased body temperature and brain injury, but drugs inducing selective, rapid, and safe cooling in humans are not available. Here, we show that injections of adenosine 5′-monophosphate (AMP), an endogenous nucleotide, promptly triggers hypothermia in mice by directly activating adenosine A1 receptors (A1R) within the preoptic area (POA) of the hypothalamus. Inhibition of constitutive degradation of brain extracellular AMP by targeting ecto 5′-nucleotidase, also suffices to prompt hypothermia in rodents. Accordingly, sensitivity of mice and rats to the hypothermic effect of AMP is inversely related to their hypothalamic 5′-nucleotidase activity. Single-cell electrophysiological recording indicates that AMP reduces spontaneous firing activity of temperature-insensitive neurons of the mouse POA, thereby retuning the hypothalamic thermoregulatory set point towards lower temperatures. Adenosine 5′-monophosphate also suppresses prostaglandin E2-induced fever in mice, having no effects on peripheral hyperthermia triggered by dioxymetamphetamine (ecstasy) overdose. Together, data disclose the role of AMP, 5′-nucleotidase, and A1R in hypothalamic thermoregulation, as well and their therapeutic relevance to treatment of febrile illness.

During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N'-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N'-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a NAD-consuming enzyme with an emerging key role in epigenetic regulation of gene transcription. Although PARP-1 expression is characteristically restricted to the nucleus, a few studies report the mitochondrial localization of the enzyme and its ability to regulate organelle functioning. Here, we show that, despite exclusive nuclear localization of PARP-1, mitochondrial homeostasis is compromised in cell lines exposed to PARP-1 pharmacological inhibitors or small interfering RNA. PARP-1 suppression reduces integrity of mitochondrial DNA (mtDNA), as well as expression of mitochondria-encoded respiratory complex subunits COX-1, COX-2, and ND-2. Accordingly, PARP-1 localizes at promoters of nuclear genes encoding both the mtDNA repair proteins UNG1, MYH1, and APE1 and the mtDNA transcription factors TFB1M and TFB2M. It is noteworthy that poly(ADP-ribosyl)ation is required for nuclear gene expression of these mitochondrial proteins. Consistent with these findings, PARP-1 suppression impairs mitochondrial ATP production. Our results indicate that PARP-1 plays a central role in mitochondrial homeostasis by epigenetically regulating nuclear genes involved in mtDNA repair and transcription. These data might have important implications in pharmacology of PARP-1 inhibitors as well as clinical oncology and aging.

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

Background and purpose: Maintenance of poly(ADP-ribose) (PAR) polymers at homoeostatic levels by PAR glycohydrolase (PARG) is central in cell functioning and survival. Yet the pharmacological relevance of PARG inhibitors is still debated. Gallotannin, a complex mixture of hydrolysable tannins from oak gall, inhibits PARG but which of its constituents is responsible for the inhibition and whether the pharmacodynamic properties are due to its antioxidant properties, has not yet been established. Experimental approach: A structure–activity relationship study was conducted on different natural and synthetic tannins/galloyl derivatives as potential PARG inhibitors, using a novel in vitro enzymic assay. Cytotoxicity was assayed in cultured HeLa cells. Key results: Mono-galloyl glucose compounds were potent inhibitors of PARG, with activities similar to that of ADP-(hydroxymethyl) pyrrolidinediol, the most potent PARG inhibitor yet identified. When tested on HeLa cells exposed to the PAR polymerase (PARP)-1-activating compound 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), 3-galloyl glucose weakly inhibited PAR degradation. Conversely, the more lipophilic, 3-galloyl-1,2-O-isopropylidene glucose, despite being inactive on the pure enzyme, efficiently prolonged the half-life of the polymers in intact HeLa cells. Also, PARG inhibitors, but not radical scavengers, reduced, in part, cell death caused by MNNG. Conclusions and implications: Taken together, our findings identify mono-galloyl glucose derivatives as potent PARG inhibitors, and emphasize the active function of this enzyme in cell death. British Journal of Pharmacology (2008) 155, 1235–1249; doi:10.1038/bjp.2008.370; published online 22 September 2008

During the last decade, significant technological improvements in mass spectrometry have had a great impact on drug discovery. The development of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has set a new frontier for the study of the distribution of endogenous and exogenous molecules present within a tissue. MALDI-IMS is a surface sampling technique that allows not only the detection of multiple analytes but also gives the spatial distribution of those analytes. Active compounds for pulmonary disease need an optimal and well-studied delivery into the lungs, in order to assure distribution with greater penetration into the peripheral or the alveolar region of the lung to maximize the therapeutic effects. IMS is very useful in the field of drug discovery, showing drug delivery and distribution in the body and organs. In this study, we present a comparison between two different ways of carrying out pulmonary drug administration: inhalation of a nebulized aerosol of aqueous drug solutions and intratracheal administration, which is much simpler, not expensive and commonly used during in vivo screening. Tiotropium bromide is a long-acting anticholinergic medicine used for maintenance treatment of chronic obstructive pulmonary disease. In the present work, tiotropium was administered by nebulization and by intratracheal instillation to guinea pigs at doses able to induce significant anti-bronchoconstrictive activity. Lung samples were dissected, frozen, cryosectioned and coated with matrix ( α-hydroxy-cinnamic acid). IMS analyses were performed using a MALDI-LTQ-Orbitrap XL. Using this technique we were able to compare different distributions of the drug depending on the method of administration.

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

Arginase 2, inducible- and endothelial-nitric-oxide synthase (iNOS and eNOS), indoleamine 2,3-dioxygenase (IDO) and TGF-beta, might impair immune functions in prostate cancer (PCA) patients. However, their expression was not comparatively analysed in PCA and benign prostatic hyperplasia (BPH). We evaluated the expression of these genes in PCA and BPH tissues. Seventy-six patients (42 BPH, 34 PCA) were enrolled. Arginase 2, eNOS and iNOS gene expression was similar in BPH and PCA tissues. TGF-beta1 gene expression was higher in BPH than in PCA tissues (p=0.035). IDO gene expression was more frequently detectable (p=0.00007) and quantitatively higher (p=0.00001) in PCA tissues than in BPH. IDO protein, expressed in endothelial cells from both BPH and PCA, was detectable in tumour cells in PCA showing evidence of high specific gene expression. In these patients, IDO gene expression correlated with kynurenine/tryptophan ratio in sera. Thus high expression of IDO gene is specifically detectable in PCA.

A technique is described for quick urine specimen preparation with the use of a simple device to save time and laboratory material.