Exploiting the potential of gallium for the treatment of Pseudomonas aeruginosa pulmonary infection

Project Summary (English) - Background Morbidity and mortality in cystic fibrosis (CF) patients is ultimately attributable to persistent pulmonary infection mainly caused by bacteria belonging to different species, the most worrisome one being Pseudomonas aeruginosa (Pa). The emergence of antibiotic resistance in CF pathogens has become a serious problem that must be addressed by developing new antimicrobials and identifying new drug targets. Gallium (Ga3+) inhibits bacterial growth, acting as an iron mimetic, and is a drug already used in clinical practice (brand name Ganite®) for the treatment of non-infectious diseases. The pharmacological properties of Ga3+ rely on the chemical resemblance between the Ga3+ and the ferric (Fe3+) ions. - Hypothesis and objectives Bacterial cells are unable to discriminate between these two ions, and erroneously incorporates Ga3+ instead of Fe3+ within essential molecules, resulting in suppression of bacterial growth. The main aims of this project is to: i) generate new gallium complexes with pyochelin-based deriovatives; ii) perform a comparative evaluation of the antibacterial activity of Ga(III) complexes/formulations against CF pathogens, and to investigate the toxicology and pharmacokinetics of the Ga(III) complexes/formulations. - Material, patients, methods We shall capitalize upon expertise in organic synthesis, pharmaceutical chemistry, microbiology and pharmacology to: i) synthesize gallium complexes ex novo; ii) evaluate the effect of Ga(III) complexes/formulations on CF pathogens; iii) determine their toxicity and pharmacological properties. In this project, the antibacterial properties of Ga3+ will be potentiated by conjugation with ligands that facilitate the stability/solubility, in order to lower the therapeutic dosage. The pharmacological activities of the new compounds will be assessed by: i) inhibition of CF-pathogens growth in vitro; ii) lack of toxicity in cellular and animal systems; iii) evaluation of biodistribution in animal models. - Expected results and spin-offs We expect to identify new Ga3+ formulations endowed with more potent antibacterial properties, high bioavailability, low toxicity and suited for aerosol administration, thus ready for clinical testing. In the worrying scenario of increasing antibiotic resistance CF pathogens, the identification of new antimicrobials such as gallium-based compounds, is highly desirable. These compounds hold great promise for the progression into drugs with potential clinical applicability in the short-medium perspective

Uterine Dysfunction in Diabetic Mice: The Role of Hydrogen Sulfide

It is well-known that the physiological uterine peristalsis, related to several phases of reproductive functions, plays a pivotal role in fertility and female reproductive health. Here, we have addressed the role of hydrogen sulfide (H2S) signaling in changes of uterine contractions driven by diabetes in non-obese diabetic (NOD) mice, a murine model of type-1 diabetes mellitus. The isolated uterus of NOD mice showed a significant reduction in spontaneous motility coupled to a generalized hypo-contractility to uterotonic agents. The levels of cyclic nucleotides, cAMP and cGMP, notoriously involved in the regulation of uterus homeostasis, were significantly elevated in NOD mouse uteri. This increase was well-correlated with the higher levels of H2S, a non-specific endogenous inhibitor of phosphodiesterases. The exposure of isolated uterus to L-cysteine (L-Cys), but not to sodium hydrogen sulfide, the exogenous source of H2S, showed a weak tocolytic effect in the uterus of NOD mice. Western blot analysis revealed a reorganization of the enzymatic expression with an upregulation of 3-mercaptopyruvate-sulfurtransferase (3-MST) coupled to a reduction in both cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) expression. In conclusion, the increased levels of cyclic nucleotides dysregulate the uterus peristalsis and contractility in diabetic mice through an increase in basal H2S synthesis suggesting a role of 3-MST

Involvement of Hydrogen Sulfide (H2S) in Dexamethasone (DEX) Induced Hypertension in Rat

Introduction: Glucocorticoid (GC) excess is related to hypertension. The deletion of endothelial GC-receptors abrogates the blood pressure increase, suggesting GC-induced hypertension is endothelium-dependent. In response to shear stress endothelium releases nitric oxide, endothelial derived hyperpolarizing factor (EDHF) and prostacyclin. Recently H2S has been proposed as a candidate for EDHF. H2S is mainly produced by the enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) from L-cysteine. The aim of this study was to investigate the EDHF/H2S signaling in GC-hypertension. Methods: Male Wistar rats were treated with DEX (1.5 mg/kg/sc) or vehicle (VEH) for 8 days. Systolic blood pressure (SBP) was monitored every 2 days. EDHF was evaluated in mesenteric plexus and carotid artery performing a concentration-effect curve of acetylcholine in presence of indomethacin (INDO) and nitro-L-arginine methyl ester (L-NAME). Apamin (APA) plus charibdotoxin (CTX), SKCa and BKCa inhibitors, or propargylglycine (PAG), CSE inhibitor, were used. CBS and CSE levels were analyzed by immunoblot. H2S levels were measured by a colorimetric assay. Results: DEX treatment significantly increased SBP compared to VEH (*p<0.05, **p<0.01, ***p<0.001 at days 2-4, 6, 8 respectively). EDHF-mediated relaxation of mesenteric bed or carotid artery was markedly reduced in DEX group compared to VEH (***p<0.001). APA and CTX as well as PAG abolished EDHF-mediated relaxation in DEX or VEH group (***,°°°p<0.001 respectively). CBS and CSE levels were significantly reduced in mesenteric plexus and carotid artery in DEX group (*p<0.05). The H2S production was markedly reduced in mesenteric plexus and carotid artery (*p<0.05, **p<0.01 respectively) as well as plasmatic H2S levels (*p<0.05) in DEX rats compared to VEH. Conclusions: Our data demonstrate that GC-excess induces an impairment of H2S/EDHF signaling indicating an additional cause of GC-mediated hypertension