Effects of Levofloxacin, Aztreonam, and Colistin on Enzyme Synthesis by P. aeruginosa Isolated from Cystic Fibrosis Patients.

(1) Background: Cystic fibrosis (CF) is characterized by chronic pulmonary inflammation and persistent bacterial infections. P. aeruginosa is among the main opportunistic pathogens causing infections in CF. P. aeruginosa is able to form a biofilm, decreasing antibiotic permeability. LOX, a lipoxygenase enzyme, is a virulence factor produced by P. aeruginosa and promotes its persistence in lung tissues. The aim of this study is to evaluate if antibiotics currently used for aerosol therapy in CF are able to interfere with the production of lipoxygenase from open isolates of P. Aeruginosa from patients with CF. (2) Methods: Clinical isolates of P. aeruginosa from patients with CF were grown in Luria broth (LB). Minimum inhibitory concentration (MIC) was performed and interpreted for all isolated strains according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. We selected four antibiotics with different mechanisms of action: aztreonam, colistin, amikacin, and levofloxacin. We used human pulmonary epithelial NCI-H929 cells to evaluate LOX activity and its metabolites according to antibiotic action at increasing concentrations. (3) Results: there is a correlation between LOX secretion by clinical isolates of P. aeruginosa and biofilm production. Levofloxacin exhibits highly significant inhibitory activity compared to the control. Amikacin also exhibits significant inhibitory activity against LOX production. Aztreonam and colistin do not show inhibitory activity. These results are also confirmed for LOX metabolites. (4) Conclusions: among the evaluated antibiotics, levofloxacin and amikacin have an activity on LOX secretion.

Pharmacodynamics of D-mannose in the prevention of recurrent urinary infections.

Recurring urinary tract infections (rUTIs) are frequently caused by Escherichia coli, which invades urothelial cells and forms quiescent bacterial reservoirs. D-mannose, an inert monosaccharide, represents a notable agent for rUTI prevention; however, there is no agreement on its dosage. To provide pharmacological basis for an effective dose, we evaluated its ability to inhibit adhesion of E. coli to urothelial cells. E. coli strains isolated from the urine of a woman with recurrent urinary tract infections were selected according to adhesion capacity. Anti-adhesive efficacy and invasion were tested using the TCC-5637 urothelial cell line. The IC50 for the anti-adhesive efficacy and anti-invasion activity of D-mannose were 0.51 mg/ml and 0.30 mg/ml, respectively, both with concentration-dependent inhibition. Lastly, the biofilm interference of D-mannose was evaluated to be 50 mg/ml. D-mannose inhibited the adhesion of E. coli to urothelial cells at high concentrations, whereas inhibition of invasion occurred at much lower concentrations.

Erdosteine enhances antibiotic activity against bacteria within biofilm.

Bacterial biofilms form on inert and living surfaces and display high levels of resistance to antibiotics, making it difficult to eradicate biofilm-related infections. Erdosteine, a thiol-based drug used in the treatment of acute and chronic respiratory diseases, has multiple pharmacodynamic properties (mucolytic, anti-inflammatory, antioxidant), suggesting that it may have potential in controlling biofilm-related infections. This in vitro study aimed to evaluate the effects of erdosteine in combination with different antibiotics against methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) biofilms. Biofilm production/mass and bacterial viability were measured using crystal violet absorbance and resorufin resonance, respectively, in young (6 h) and mature (24 h) biofilms incubated with antibiotics [at concentrations from 0 to 200 times the minimum inhibitory concentration (MIC)] for 24 h in the absence or presence of erdosteine (2, 5 and 10 mg/L). In 6-h MRSA biofilms, vancomycin and linezolid displayed concentration-dependent reductions in biofilm mass and viability, which was enhanced in the presence of increasing concentrations of erdosteine. Similar results were seen for amoxicillin/clavulanate and levofloxacin against 6-h MSSA biofilms. Antibiotics alone had reduced efficacy against 24-h biofilms, while the effect of the erdosteine-antibiotic combination was significantly greater against 24-h biofilms (MRSA and MSSA). These results suggest that erdosteine enhances the activity of the antibiotic by facilitating its penetration into biofilms and by disrupting the extracellular polymeric substance matrix, which should be confirmed with further studies. The potential clinical value of erdosteine in treating biofilm-related infections warrants further investigation.

Identification of Bivalent Ligands with Melatonin Receptor Agonist and Fatty Acid Amide Hydrolase (FAAH) Inhibitory Activity That Exhibit Ocular Hypotensive Effect in the Rabbit.

Activation of melatonin receptors and inhibition of fatty acid amide hydrolase (FAAH) have both shown potential benefits for the treatment of glaucoma. To exploit the combination of these biological activities in single therapeutic agents, we designed dual-acting compounds sharing the pharmacophore elements required for the two targets, in search for balanced potencies as MT1/MT2 agonists and FAAH inhibitors. In particular, the N-anilinoethylamide scaffold, previously developed for melatonergic ligands, was decorated at meta position with a polymethylene linker bound to an O-arylcarbamate group, substituted according to known structure-activity relationships for FAAH inhibition. For the most active series, the N-anilinoethylamide portion was also replaced with the indole scaffold of melatonin. O-Biphenyl-3-ylcarbamate derivatives were characterized by remarkable and balanced activity at both targets, in the nanomolar range for compound 29. Topical administration reduced elevated intraocular pressure in rabbits, with a longer action and improved efficacy compared to the reference compounds melatonin and URB597.

Melatonin Analogue Antiproliferative and Cytotoxic Effects on Human Prostate Cancer Cells.

Melatonin has been indicated as a possible oncostatic agent in different types of cancer, its antiproliferative role being demonstrated in several in vitro and in vivo experimental models of tumors. Specifically, melatonin was proven to inhibit cell growth of both androgen-dependent and independent prostate cancer cells, through various mechanisms. A number of melatonin derivatives have been developed and tested for their role in the prevention and treatment of neoplastic diseases. We recently proved the in vitro and in vivo anticancer activity of UCM 1037, a newly-synthetized melatonin analogue, on melanoma and breast cancer cells. In this study we evaluated UCM 1037 effects on cell proliferation, cell cycle distribution, and cytotoxicity in LNCaP, PC3, DU145, and 22Rv1 prostate cancer cells. We demonstrated significant dose- and time-dependent UCM 1037 antiproliferative effects in androgen-sensitive LNCaP and 22Rv1 cells. Data from flow cytometric studies suggest that UCM 1037 is highly cytotoxic in androgen-sensitive prostate cancer cells, although no substantial increase in the apoptotic cell fraction has been observed. UCM 1037 cytotoxic effects were much less evident in androgen-insensitive PC3 and DU145 cells. Experiments performed to gain insights into the possible mechanism of action of the melatonin derivative revealed that UCM 1037 down-regulates androgen receptor levels and Akt activation in LNCaP and 22Rv1 cells.

Comparative Emergence of Resistance to Clofoctol, Erythromycin, and Amoxicillin against Community-Acquired Bacterial Respiratory Tract Pathogens in Italy.

Due to increasing bacterial resistance and poor availability of new antibiotics, physicians need to use old, still active antibiotics more frequently. In this study, we focused on clo-foctol and aimed to verify the emergence of clofoctol resistance over time. Additionally, the ability of clofoctol to induce resistance under static and dynamic conditions was evaluated. The minimum inhibitory concentration (MIC) values measured in pathogens isolated from 1990 to 1995 were compared to those isolated from 2017 to 2018. The behaviour of clofoctol is similar to that of amoxicillin, while erythromycin shows a different behaviour with an increase in MIC. A rapid decline in CFUs with complete eradication at 96 and 120 h in the case of clofoctol and amoxicillin, respectively, was observed in a dynamic in vitro model of a pharmacokinetic simulation. Erythromycin provides a reduction in CFUs of approximately one order of magnitude for up to 72 h, and then re-growth is observed. The MIC trend was observed during 5 days of kinetic simulation. The clofoctol MICs remain almost stable up to 96 h, after which the colonies are no longer detectable. The MICs of amoxicillin show a 2-fold increase starting from 36 h; however, at 120 h the colonies are no longer detectable. The MICs of erythromycin show a progressive increase starting from 72 h and reaching 32-fold. Clofoctol maintains its activity towards the common pathogens of respiratory tract infections and, similarly to amoxicillin, does not induce resistance in a strain of Streptococcus pneumoniae, resulting in complete eradication, while erythromycin was able to select resistant mutants.

Antiproliferative and pro-apoptotic activity of melatonin analogues on melanoma and breast cancer cells.

Melatonin plays different physiological functions ranging from the regulation of circadian rhythms to tumor inhibition, owing to its antioxidant, immunomodulatory and anti-aging properties. Due to its pleiotropic functions, melatonin has been shown to elicit cytoprotective processes in normal cells and trigger pro-apoptotic signals in cancer cells. The therapeutic potential of melatonin analogues prompted us to investigate the in vitro and in vivo antitumor activity of new melatonin derivatives and explore the underlying molecular mechanisms. The experiments revealed that the new melatonin analogues inhibited the growth of melanoma and breast cancer cells in a dose- and time-dependent manner. In addition, our results indicated that melatonin derivative UCM 1037 could induce apoptosis in melanoma and breast cancer cells, as well as cell necrosis, in MCF-7. Together, apoptosis and necrosis could be two possible mechanisms to explain the cytotoxic effect of the melatonin analogue against cancer cells. The suppression of tumor growth by the melatonin analogues was further demonstrated in vivo in a xenograft mice model. A decrease in the activation of MAPK pathway was observed in all cancer cells following UCM 1037 treatment. Overall, this study describes a promising antitumor compound showing antiproliferative and cytotoxic activity in melanoma and breast cancer cells.

Antibiotics counteract the worsening of airway remodelling induced by infections in asthma.

Asthma is associated with structural remodelling processes, including basement membrane thickening, increased vascularity and smooth muscle alterations. It is known that respiratory infections are associated with asthma exacerbation; infections can worsen asthma symptoms and influence susceptibility to asthma onset. How infections affect asthma is not fully elucidated. It is possible that the immune response, due to recurrent infections, leads to the pathogen's eradication but also increases bronchial inflammation, which induces airway remodelling in asthmatic subjects. We evaluated how infection affects lung remodelling and inflammatory responses and assessed the impact of antibiotic treatment in a murine model of asthma. Ovalbumin-sensitised BALB/c mice were divided into control, mild and chronic asthmatics. A subset of animals in each group was infected with Streptococcus pneumoniae and was treated with antibiotics. The results show an increase in key lung remodelling factors in mice with chronic asthma, particularly those infected with S. pneumoniae. Notably, antibiotic therapy attenuated these effects. These findings demonstrate for the first time that prompt antibiotic therapy may be useful to reduce lung remodelling progression in infected asthmatic subjects.

In vitro and in vivo pharmacokinetic/pharmacodynamic activity of clofoctol.

The aim of the study was to examine the In vitro susceptibility of clinical isolates of respiratory pathogens to clofoctol compared with amoxicillin and erythromycin, and to characterize the pharmacokinetic/pharmacodynamic (PK/PD) relationships of clofoctol using a murine pneumonia infection model. Strains clinically isolated from patients between 2005 and 2009 were used to examine susceptibility: penicillin-susceptible Streptococcus pneumoniae, penicillin-resistant S. pneumoniae, Streptococcus pyogenes, methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, and Haemophilus influenzae. The In vitro activity of clofoctol against clinical isolates has essentially remained unchanged over recent years. The MIC50 and MIC90 of clofoctol against penicillin-resistant S. pneumoniae are lower than that of amoxicillin and erythromycin. The area under curve/minimum inhibitory concentration (AUC/MIC) ratio is the PK/PD parameter that best correlates with in vivo clofoctol efficacy; the value of AUC/MIC required to achieve the maximum effect in this study was 75.5.

MT1-selective melatonin receptor ligands: synthesis, pharmacological evaluation, and molecular dynamics investigation of N-{[(3-O-substituted)anilino]alkyl}amides.

The design of compounds selective for the MT1 melatonin receptor is still a challenging task owing to the limited knowledge of the structural features conferring selectivity for the MT1 subtype, and only few selective compounds have been reported so far. N-(Anilinoalkyl)amides are a versatile class of melatonin receptor ligands that include nonselective MT1/MT2 agonists and MT2-selective antagonists. We synthesized a new series of N-(anilinoalkyl)amides bearing 3-arylalkyloxy or 3-alkyloxy substituents at the aniline ring, looking for new potent and MT1-selective ligands. To evaluate the effect of substituent size and shape on binding affinity and intrinsic activity, both flexible and conformationally constrained derivatives were prepared. The phenylbutyloxy substituent gave the best result, providing the partial agonist 4 a, which was endowed with high MT1 binding affinity (pKi=8.93) and 78-fold selectivity for the MT1 receptor. To investigate the molecular basis for agonist recognition, and to explain the role of the 3-arylalkyloxy substituent, we built a homology model of the MT1 receptor based on the ß2 adrenergic receptor crystal structure in its activated state. A binding mode for MT1 agonists is proposed, as well as a hypothesis regarding the receptor structural features responsible for MT1 selectivity of compounds with lipophilic arylalkyloxy substituents.

Toward the definition of stereochemical requirements for MT2-selective antagonists and partial agonists by studying 4-phenyl-2-propionamidotetralin derivatives.

New derivatives of 4-phenyl-2-propionamidotetralin (4-P-PDOT) were prepared and tested on cloned MT1 and MT2 receptors, with the purpose of merging previously reported pharmacophores for nonselective agonists and for MT2-selective antagonists. A 8-methoxy group increases binding affinity of both (±)-cis- and (±)-trans-4-P-PDOT, and it can be bioisosterically replaced by a bromine. Conformational analysis of 8-methoxy-4-P-PDOT by molecular dynamics, supported by NMR data, revealed an energetically favored conformation for the (2S,4S)-cis isomer and a less favorable conformation for the (2R,4S)-trans one, fulfilling the requirements of a pharmacophore model for nonselective melatonin receptor agonists. A new superposition model, including features characteristic of MT2-selective antagonists, suggests that MT1/MT2 agonists and MT2 antagonists can share the same arrangement for their pharmacophoric elements. The model correctly predicted the eutomers of (±)-cis- and (±)-trans-4-P-PDOT. The model was validated by preparing three dihydronaphthalene derivatives, either able or not able to reproduce the putative active conformation of 4-P-PDOT.