S. Dugnani

A New Model Examining Intracellular and Extracellular Activity of Amoxicillin, Azithromycin, and Clarithromycin in Infected Cells

An in vitro infection model was created using a suspension of macrophages, polymorphonuclear leukocytes, lymphocytes, fibroblasts, and human serum to which pathogen and antibiotic were added. Separate intracellular and extracellular antibiotic concentrations and activity against Staphylococcus aureus and Legionella pneumophila were assessed for three antimicrobial agents: amoxicillin, azithromycin and clarithromycin. Amoxicillin was found almost exclusively in extracellular fluid, where it was active; intracellularly, it was ineffective. Azithromycin, in contrast, was primarily concentrated and active intracellularly, with little activity in extracellular fluid. Clarithromycin was present in both compartments and possessed significant activity both intracellularly and extracellularly.

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 β2 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.

Immunostimulation by clarithromycin in healthy volunteers and chronic bronchitis patients.

The immune response to infecting pathogens may be either enhanced or depressed by therapeutic antimicrobial agents. Some macrolides have been shown to enhance aspects of the immune response. This study evaluates the effects of clarithromycin, a new broad-spectrum macrolide antibiotic, on leukocyte function in both healthy volunteers (single 500 mg dose) and chronic bronchitis patients (500 mg b.i.d.) by testing blood samples collected at baseline and at 2, 4, 8 and 12 hours after clarithromycin administration. Clarithromycin did not affect leukocyte chemotaxis but did significantly increase (P < 0.01) phagocytosis (both frequency and index), intracellular killing and natural killer activity, in healthy volunteers. In patients, clarithromycin enhanced phagocytosis frequency, index and intracellular killing. Clarithromycin appears to enhance the human immune response; the mechanism, pharmacodynamics, and clinical significance of this enhancement remain unknown.

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.

Bactericidal Kinetics of an in vitro Infection Model of Once-Daily Ceftriaxone plus Amikacin against Gram-Positive and Gram-Negative Bacteria

The in vitro efficacy of ceftriaxone plus amikacin combination against gram-positive and gram-negative bacteria, clinically isolated from patients affected by pneumonia in intensive care units, was compared to that of the 2 drugs used alone. The study was performed using a dynamic model in which the human kinetics of the drugs after intramuscular administration was simulated. The antibacterial activity was tested by determining the bacterial cell count (CFU/ml). Killing curves came out from plotting the log CFU/ml versus time. In the same way, ceftriaxone and amikacin concentrations were assayed by HPLC and fluorescence polarization immunoassay, respectively. The results show that ceftriaxone plus amikacin combination exert a high killing activity against all tested strains. The two antibiotics alone initially have a good killing activity but this is followed by bacterial regrowth for all tested isolates. This data supports the results of several clinical studies which have shown a good therapeutic efficacy of ceftriaxone plus amikacin combination in the treatment of severe infections caused by organisms intermediately sensitive to these drugs.

Penetration of Clarithromycin into Oral and Respiratory Tissues

SummaryThis report summarises the results of 5 studies carried out to determine the penetration of clarithromycin and its 14-hydroxy metabolite into oral and respiratory tissues. 128 patients of both sexes undergoing dental surgery, rhinoplasty, lung resection or tonsil resection, or presenting with an acute exacerbation of chronic bronchitis, were administered clarithromycin (generally 250mg twice daily; 500mg twice daily for lung resections) for 3 days prior to sample collection. Serum, tissue and secretion samples were assayed for clarithromycin and 14-hydroxy clarithromycin using an agar diffusion bioassay or high performance liquid chromatography. Concentrations were assayed in triplicate and standard curves created using computerised linear regression analysis. Following oral administration, the pharmacodynamics of clarithromycin in oral and respiratory tissues and serum include therapeutic levels that exceed the minimum inhibitory concentrations for common respiratory pathogens for up to 12 hours following administration.

Miokamycin and leukocyte activity in man

The effects of miokamycin on the immune system were studied in healthy volunteers by means of the following assays: phagocytosis, intracellular killing and natural killer activity. The tests were performed before, 2 and 6 h after oral administration of 600 mg of miokamycin. The findings suggest that miokamycin stimulates phagocytosis and intracellular killing. These effects could be clearly shown 2 h after drug intake and disappeared at the 6th hour. On the contrary, the natural killer activity was not significantly stimulated by miokamycin.

Effects of Lincomycin on the Immune System

The effects of lincomycin on the immune system were studied on patients suffering from chronic bronchitis by means of phagocytosis, chemotaxis and natural-killer tests. The tests were performed before and 2, 4, 8, 12 and 24 h after administration of 600 mg lincomycin i.m. in a single dose. The results indicate that lincomycin stimulates phagocytosis, expressed as enhanced superoxide production (O2-), chemotaxis and the activity of natural killer cells, measured on the basis of their ability to perform a lysis on the K562 tumoral target labelled with 51Cr. The stimulating effects on chemotaxis appear already 2 h after the administration of the drug, while the same effect on phagocytosis and natural-killer activity occurs after 4 h. The maximal stimulating activity on all three parameters can be shown at 8 h and disappears at 24 h.

Effect of antibiotics on Bordetella pertussis adhering activity: hypothesis regarding mechanism of action

Microbial adherence to epithelial cell surfaces has been implicated as the first step in the initiation of several infectious diseases. The ability of antibiotics to affect the properties of bacterial adherence to cell surfaces may be a criterion in selecting antibiotics for therapy. This study was performed in order to investigate the activity of amoxicillin, chloramphenicol, and clarithromycin in modifying the adhering activity of Bordetella pertussis to human epithelial cells. The actions of antibiotics, alone or combined with aprotinin, were compared with that of trypsin, aprotinin and trypsin+aprotinin, to investigate the chemical nature of the ligand where antibiotics could act. The adhering activity was evaluated on human epithelial cells, collected from the oral mucosa, challenged with B. pertussis A2963 previously incubated in the presence of the tested substances for 1 h at 37 degrees C in a shaker incubator. After staining, the percentage of mucosal cells with more than 50 adhering bacteria was evaluated. Under the described experimental conditions, trypsin significantly reduced the adherence of B. pertussis. Aprotinin had no effect but was able to counteract the inhibitory action of trypsin. Both clarithromycin and chloramphenicol markedly reduced adhering activity and their actions were not counteracted by aprotinin. Amoxicillin was without effect. It was hypothesized that chloramphenicol and clarithromycin, exerting their antimicrobial action by inhibiting bacterial protein synthesis, affected bacterial adhesion through an unknown mechanism without proteolytic effect.