Design, synthesis, antibacterial activity and toxicity of novel quaternary ammonium compounds based on pyridoxine and fatty acids.

A diverse series of 43 novel "soft antimicrobials" based on quaternary ammonium pyridoxine derivatives which include six-membered acetals and ketals of pyridoxine bound via cleavable linker moieties (amide, ester) with a fragment of fatty carboxylic acid was designed. Nine compounds exhibited in vitro promising antibacterial activity against Gram-positive and Gram-negative bacterial strains with MIC values comparable with reference antiseptics miramistin, benzalkonium chloride and chlorohexidine. On various clinical isolates, the lead compounds 6i and 12a exhibited antibacterial activity comparable with that of benzalkonium chloride while higher than that of miramistin. Moreover, 6i and 12a were able to kill bacteria embedded into the matrix of mono- and dual species biofilms. The treatment of bacterial cells by either 6i and 12a lead to fast depolarization of the membrane suggesting that the membrane is an apparent molecular target of compounds. 6i and 12a were non mutagenic neither in SOS-chromotest nor in Ames test and non-toxic in vivo at acute oral (LD50 > 2000 mg/kg) and cutaneous administration (LD50 > 2500 mg/kg) on mice. Taken together, our data allow suggesting described active compounds as promising starting point for the new antibacterial agents development.

Anti-biofilm and wound-healing activity of chitosan-immobilized Ficin.

Biofouling is among the key factors slowing down healing of acute and chronic wounds. Here we report both anti-biofilm and wound-healing properties of the chitosan-immobilized Ficin. The proposed chitosan-adsorption approach allowed preserving ~90% of the initial total activity of the enzyme (when using azocasein as a substrate) with stabilization factor of 4.9, and ~70% of its specific enzymatic activity. In vitro, the chitosan-immobilized Ficin degraded staphylococcal biofilms, this way increasing the efficacy of antimicrobials against biofilm-embedded bacteria. In vivo, in the presence of Ficin (either soluble or immobilized), the S.aureus-infected skin wound areas in rats reduced twofold after 4 instead of 6 days treatment. Moreover, topical application of the immobilized enzyme resulted in a 3-log reduction of S. aureus cell count on the wound surfaces in 6 days, compared to more than 10 days required to achieve the same effect in control. Additional advantages include smoother reepithelisation, and new tissue formation exhibiting collagen structure characteristics closely reminiscent of those observed in the native tissue. Taken together, our data suggest that both soluble and immobilized Ficin appear beneficial for the treatment of biofilm-associated infections, as well as speeding up wound healing and microbial decontamination.

Novel Bis-Ammonium Salts of Pyridoxine: Synthesis and Antimicrobial Properties.

A series of 108 novel quaternary bis-ammonium pyridoxine derivatives carrying various substituents at the quaternary nitrogen's and acetal carbon was synthesized. Thirteen compounds exhibited antibacterial and antifungal activity (minimum inhibitory concentration (MIC) 0.25-16 µg/mL) comparable or superior than miramistin, benzalkonium chloride, and chlorhexidine. A strong correlation between the lipophilicity and antibacterial activity was found. The most active compounds had logP values in the range of 1-3, while compounds with logP > 6 and logP < 0 were almost inactive. All active compounds demonstrated cytotoxicity comparable with miramistin and chlorhexidine on HEK-293 cells and were three-fold less toxic when compared to benzalkonium chloride. The antibacterial activity of leading compound 5c12 on biofilm-embedded Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli or Pseudomonas aeruginosa was comparable or even higher than that of the benzalkonium chloride. In vivo 5c12was considerably less toxic (LD50 1705 mg/kg) than benzalkonium chloride, miramistine, and chlorhexidine at oral administration on CD-1 mice. An aqueous solution of 5c12 (0.2%) was shown to be comparable to reference drugs efficiency on the rat's skin model. The molecular target of 5c12 seems to be a cellular membrane as other quaternary ammonium salts. The obtained results make the described quaternary bis-ammonium pyridoxine derivatives promising and lead molecules in the development of the new antiseptics with a broad spectrum of antimicrobial activity.

Antibacterial activity profile of miramistin in in vitro and in vivo models.

BACKGROUND: Miramistin is a widely used antiseptic, disinfectant and preservative, and one of the most popular antimicrobial agents on pharmaceutical market of the Russian Federation (http://www.dsm.ru/en/news/385/). However, there is a lack of reported systematic data on antibacterial efficacy of this agent obtained in accordance with the international standards. AIM: This paper represents a systematic study of antibacterial properties of miramistin. Another objective of this work is to evaluate and compare the exploratory performance of in vitro and in vivo protocols of antiseptics' efficacy testing using miramistin as the reference antiseptic. METHODS: Antibacterial activity of 0.1% and 0.2% aqueous solutions of miramistin against two museum strains of S. aureus (ATCC 209p) and E. coli (CDC F-50) was studied. Three standard in vitro laboratory tests (microdilution test, suspension test, and metal surface test), and one in vivo test (on rat's skin) were used. The study was conducted in accordance with the international regulatory documents. RESULTS: Miramistin showed high bactericidal activity against the studied bacterial pathogens in the standard in vitro tests. Thus, in the microdilution test it showed expressed activity against S. aureus (MIC 8 µg/ml, MBC 16 µg/ml) and E. coli (MIC 32 µg/ml, MBC 128 µg/ml). In the suspension test, miramistin decreased the amount of colony forming units by at least 6 log10 units for S. aureus, and by at least 4.5 log10 units for E. coli. Transition to the metal surface test led to significant decrease of antibacterial activity by 1-3 log10 units as compared to the suspension test. Further dramatic reduction of antiseptic activity (by 3-4 log10 units) was observed in in vivo rat skin test. Addition of a protein contaminant (bovine serum albumin) led to a general decrease in the effectiveness of miramistin against the test pathogens (typically, by 1-2 log10 units). An interesting effect of exposure time-dependent reversal of miramistin's specificity to the studied Gram-positive S. aureus and the Gram-negative E. coli organisms was observed in the metal surface test. CONCLUSIONS: The results of this work provide systematic data on antibacterial efficacy of miramistin. They also underscore the need in relevant in vivo models for evaluation of antiseptics' efficacy. While the existing in vitro methods can be successfully applied at the discovery stages, it is necessary to use more realistic in vivo models at more advanced development stages. The observed selectivity reversal effect should be taken into account when carrying out the antiseptics' efficacy testing and surface disinfection procedures.