Uptake of Ozenoxacin and Other Quinolones in Gram-Positive Bacteria.

The big problem of antimicrobial resistance is that it requires great efforts in the design of improved drugs which can quickly reach their target of action. Studies of antibiotic uptake and interaction with their target it is a key factor in this important challenge. We investigated the accumulation of ozenoxacin (OZN), moxifloxacin (MOX), levofloxacin (LVX), and ciprofloxacin (CIP) into the bacterial cells of 5 species, including Staphylococcus aureus (SA4-149), Staphylococcus epidermidis (SEP7602), Streptococcus pyogenes (SPY165), Streptococcus agalactiae (SAG146), and Enterococcus faecium (EF897) previously characterized.The concentration of quinolone uptake was estimated by agar disc-diffusion bioassay. Furthermore, we determined the inhibitory concentrations 50 (IC50) of OZN, MOX, LVX, and CIP against type II topoisomerases from S. aureus.The accumulation of OZN inside the bacterial cell was superior in comparison to MOX, LVX, and CIP in all tested species. The accumulation of OZN inside the bacterial cell was superior in comparison to MOX, LVX, and CIP in all tested species. The rapid penetration of OZN into the cell was reflected during the first minute of exposure with antibiotic values between 190 and 447 ng/mg (dry weight) of bacteria in all strains. Moreover, OZN showed the greatest inhibitory activity among the quinolones tested for both DNA gyrase and topoisomerase IV isolated from S. aureus with IC50 values of 10 and 0.5 mg/L, respectively. OZN intracellular concentration was significantly higher than that of MOX, LVX and CIP. All of these features may explain the higher in vitro activity of OZN compared to the other tested quinolones.

A novel high-throughput screening method for identifying compounds that inhibit plasmid conjugation.

Plasmid conjugation is an important contributing factor to the spread of antibiotic resistance among bacteria, posing a significant global health threat. Our method introduces an innovative high-throughput screening approach to identify compounds that inhibit or reduce conjugation, addressing the need for new strategies against the spread of antimicrobial resistance. Using Escherichia coli strains as donor and recipient, we screened 3500 compounds from a library provided by ABAC Therapeutics. Each 96 -well plate was loaded with 88 different compounds and bacterial cultures. Every plate also included negative and positive controls of conjugation. After an hour, cultures from wells were spotted on agar plates and assessed visually. Compounds that showed a visible effect on conjugation were retested. Six compounds targeting conjugation were found, showing promise for further analysis.

Potent antimalarial 4-pyridones with improved physico-chemical properties.

Antimalarial 4-pyridones are a novel class of inhibitors of the plasmodial mitochondrial electron transport chain targeting Cytochrome bc1 (complex III). In general, the most potent 4-pyridones are lipophilic molecules with poor solubility in aqueous media and low oral bioavailability in pre-clinical species from the solid dosage form. The strategy of introducing polar hydroxymethyl groups has enabled us to maintain the high levels of antimalarial potency observed for other more lipophilic analogues whilst improving the solubility and the oral bioavailability in pre-clinical species.

A novel class of fast-acting antimalarial agents: Substituted 15-membered azalides.

BACKGROUND AND PURPOSE: Efficacy of current antimalarial treatments is declining as a result of increasing antimalarial drug resistance, so new and potent antimalarial drugs are urgently needed. Azithromycin, an azalide antibiotic, was found useful in malaria therapy, but its efficacy in humans is low. EXPERIMENTAL APPROACH: Four compounds belonging to structurally different azalide classes were tested and their activities compared to azithromycin and chloroquine. in vitro evaluation included testing against sensitive and resistant Plasmodium falciparum, cytotoxicity against HepG2 cells, accumulation and retention in human erythrocytes, antibacterial activity, and mode of action studies (delayed death phenotype and haem polymerization). in vivo assessment enabled determination of pharmacokinetic profiles in mice, rats, dogs, and monkeys and in vivo efficacy in a humanized mouse model. KEY RESULTS: Novel fast-acting azalides were highly active in vitro against P. falciparum strains exhibiting various resistance patterns, including chloroquine-resistant strains. Excellent antimalarial activity was confirmed in a P. falciparum murine model by strong inhibition of haemozoin-containing trophozoites and quick clearance of parasites from the blood. Pharmacokinetic analysis revealed that compounds are metabolically stable and have moderate oral bioavailability, long half-lives, low clearance, and substantial exposures, with blood cells as the preferred compartment, especially infected erythrocytes. Fast anti-plasmodial action is achieved by the high accumulation into infected erythrocytes and interference with parasite haem polymerization, a mode of action different from slow-acting azithromycin. CONCLUSION AND IMPLICATIONS: The hybrid derivatives described here represent excellent antimalarial drug candidates with the potential for clinical use in malaria therapy.

Improved murine model of malaria using Plasmodium falciparum competent strains and non-myelodepleted NOD-scid IL2Rgammanull mice engrafted with human erythrocytes.

Murine models of Plasmodium falciparum malaria may become crucial tools in drug discovery. Here we show that non-myelodepleted NOD-scid IL2Rgamma(null) mice engrafted with human erythrocytes support an infectious burden up to tenfold higher than that supported by engrafted NOD-scid beta2microglobulin(null) mice. The new model was validated for drug discovery and was used to assess the therapeutic efficacy of 4-pyridones, selective inhibitors of P. falciparum cytochrome bc1.

Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence.

Flow cytometry is a powerful tool for measuring parasitemias in murine malaria models used to test new antimalarials. Measurement of the emission of the nonpermeable nucleic acid dye YOYO-1 (at 530 and 585 nm after excitation at 488 nm) allowed the unambiguous detection of low parasitemias (> or =0.01%) but required prolonged fixation and permeabilization of the sample. Thus, we tested whether this issue could be overcome by use of the cell-permeant dye SYTO-16 with this same bidimensional method. Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, or Plasmodium chabaudi or from NOD(scidbeta2m-/-) engrafted with human erythrocytes and infected with P. falciparum were stained with SYTO-16 in the presence or absence of TER-119 mAb (for engrafted mice) in 96-well plate format and acquired in Trucount tubes. Bidimensional analysis with SYTO-16 was quantitatively equivalent to YOYO-1. Moreover, by combining SYTO-16 with the use of TER-119-PE antimouse erythrocyte mAb and Trucount tubes, the measurement of the concentration of P. falciparum-infected erythrocytes over a range of five orders of magnitude was achieved. Bidimensional analysis using SYTO-16 can be used to accurately measure the concentration of Plasmodium spp.-infected erythrocytes in mice without complex sample preparation.

Microbiological profile of ozenoxacin.

Aim: To explore the antibacterial spectrum of ozenoxacin and compare its in vitro activity with that of other antibacterial agents. Materials & methods: In 2010, 10,054 isolates were collected from 128 centers worldwide. Minimum inhibitory concentrations against Gram-positive and Gram-negative isolates were determined for 23 and 13 antibacterial agents, respectively. Results: Ozenoxacin exhibited high in vitro activity against susceptible, and methicillin- or levofloxacin-resistant, Gram-positive bacteria. Ozenoxacin was one or two dilutions less active against Enterobacteriaceae isolates, except for Escherichia coli, than other quinolones. Conclusion: Ozenoxacin is a potent antimicrobial agent mainly against susceptible and resistant strains of Gram-positive isolates (staphylococci and streptococci), and shows activity against some Gram-negative isolates.

Synthesis and structure-activity relationships of 4-pyridones as potential antimalarials.

A series of diaryl ether substituted 4-pyridones have been identified as having potent antimalarial activity superior to that of chloroquine against Plasmodium falciparum in vitro and murine Plasmodium yoelii in vivo. These were derived from the anticoccidial drug clopidol through a systematic study of the effects of varying the side chain on activity. Relative to clopidol the most active compounds show >500-fold improvement in IC50 for inhibition of P. falciparum in vitro and about 100-fold improvement with respect to ED50 against P. yoelii in mice. These compounds have been shown elsewhere to act selectively by inhibition of mitochondrial electron transport at the cytochrome bc1 complex.

Comparative in vitro antibacterial activity of ozenoxacin against Gram-positive clinical isolates.

AIM: To compare the in vitro activity of the anti-impetigo agent, ozenoxacin, and other antimicrobial agents against Gram-positive clinical isolates from skin and soft tissue infections. MATERIALS & METHODS: Isolates were collected in two studies: 1097 isolates from 49 centers during 2009-2010 and 1031 isolates from ten centers during 2014. Minimum inhibitory concentrations were determined for 18 and 11 antimicrobials in these studies, respectively, using standard broth microdilution methods. Isolates were stratified by species and methicillin susceptibility/resistance and/or levofloxacin susceptibility/nonsusceptibility status. RESULTS: Ozenoxacin exhibited high in vitro activity against Staphylococcus aureus and coagulase-negative staphylococci isolates in both studies. Ozenoxacin was also highly active against Streptococcus pyogenes and Streptococcus agalactiae isolates. CONCLUSION: Ozenoxacin is a potent antimicrobial agent against staphylococci and streptococci.

Studies on articular and general toxicity of orally administered ozenoxacin in juvenile rats and dogs.

AIM: Ozenoxacin is a nonfluorinated quinolone antibacterial approved for topical treatment of impetigo. Because quinolones have known chondrotoxic effects in juvenile animals, the potential toxicity of ozenoxacin was assessed in preclinical studies. MATERIALS & METHODS: Ozenoxacin or ofloxacin (300 mg/kg/day for 5 days, for each compound) was orally administered to juvenile rats, and oral ozenoxacin (10-100 mg/kg/day for 14 days) was administered to juvenile dogs. RESULTS: In juvenile rats, ozenoxacin showed no chondrotoxicity, whereas ofloxacin produced typical quinolone-induced lesions in articular cartilage in three of ten rats. Oral ozenoxacin administration to juvenile dogs showed no chondrotoxicity or toxicologically relevant findings in selected target organs. CONCLUSION: Ozenoxacin was generally well-tolerated in juvenile rats and dogs, with no evidence of quinolone-induced arthropathy.

Characterization of variables that may influence ozenoxacin in susceptibility testing, including MIC and MBC values.

Ozenoxacin is a new des-fluoro-(6)-quinolone active against pathogens involved in skin and skin structure infections, including Gram-positives resistant to fluoroquinolones. The in vitro bacteriostatic and bactericidal activity of ozenoxacin, ciprofloxacin, and levofloxacin was studied against 40 clinical isolates and 16 ATCC quality control strains under different test conditions, including cation supplementation, pH, inoculum size, inoculum preparation, incubation time, human serum, and CO2 incubation. The activity of ozenoxacin was unaffected by cation test medium supplementation, inoculum preparation, incubation time, and the increasing CO2 environment. On the contrary, ozenoxacin activity decreased by high inoculum (10(7) CFU/mL), increased presence of human serum in the medium, and increased pH. The last effect was different for ciprofloxacin and levofloxacin, which decreased activity when pH decreased. The bactericidal mode of action of ozenoxacin and control drugs was consistently maintained (MBC/MIC ratios ≤4) in spite of variations of their activity under different test conditions.

Exploration of 4(1H)-pyridones as a novel family of potent antimalarial inhibitors of the plasmodial cytochrome bc1.

A novel family of antimalarials based on the 4(1H)-pyridone scaffold is described. The compounds display potent antimalarial activity against Plasmodium falciparum in vitro and in vivo. Like atovaquone, 4(1H)-pyridones exert their antimalarial action by inhibiting selectively the electron-transport chain in P. falciparum at the cytochrome bc1 level (complex III). However, despite the similar mechanism of action, no cross-resistance with atovaquone has been found, suggesting that the binding mode of 4(1H)-pyridones might be different from that of atovaquone. The medicinal chemistry program, focused on improving potency and physicochemical properties, ultimately led to the discovery of GSK932121, which was progressed efficiently into first time in human studies. However, progression of GSK932121 was terminated when new toxicology results were obtained in the rat with a soluble phosphate prodrug of the candidate, indicating a potentially narrow therapeutic index.

Thermodynamic concepts in the study of microbial populations: age structure in Plasmodium falciparum infected red blood cells.

Variability is a hallmark of microbial systems. On the one hand, microbes are subject to environmental heterogeneity and undergo changeable conditions in their immediate surroundings. On the other hand, microbial populations exhibit high cellular diversity. The relation between microbial diversity and variability of population dynamics is difficult to assess. This connection can be quantitatively studied from a perspective that combines in silico models and thermodynamic methods and interpretations. The infection process of Plasmodium falciparum parasitizing human red blood cells under laboratory cultivation conditions is used to illustrate the potential of Individual-based models in the context of predictive microbiology and parasitology. Experimental data from several in vitro cultures are compared to the outcome of an individual-based model and analysed from a thermodynamic perspective. This approach allows distinguishing between intrinsic and external constraints that give rise to the diversity in the infection forms, and it provides a criterion to quantitatively define transient and stationary regimes in the culture. Increasing the ability of models to discriminate between different states of microbial populations enhances their predictive capability which finally leads to a better the control over culture systems. The strategy here presented is of general application and it can substantially improve modelling of other types of microbial communities.

A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes.

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NOD(scid/beta2m-/-) mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NOD(scid/beta2m-/-) mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D7(0087/N9) as a reference strain for model development. Pf3D7(0087/N9) caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.

Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1.

BACKGROUND: Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. METHODS: Samples of blood from Plasmodium yoelii-infected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in logarithmic side/scatter plots were analyzed in bidimensional FL-2/YOYO-1 plots in comparison with unidimensional YOYO-1 analysis. RESULTS: The infected erythrocytes showed a characteristic pattern of staining different from that of noninfected erythrocytes. In routine evaluation, the limit of sensitivity was 0.01% and the measurements of parasitemia were linear at parasitemias above 0.1%. Interestingly, using this approach, infected reticulocytes could be differentiated from infected normocytes. CONCLUSIONS: The method described is robust, increases the specificity and sensitivity of detection in routine testing, and is especially well suited for detection of low parasitemias in murine models of malaria.

Therapeutic efficacies of GW471552 and GW471558, two new azasordarin derivatives, against pneumocystosis in two immunosuppressed-rat models.

Two new azasordarins, GW471552 and GW471558, were studied in vivo for treatment of Pneumocystis carinii pneumonia. In the Wistar rat spontaneous pneumonia model, both azasordarins significantly reduced the number of P. carinii cysts per gram of lung homogenate when administered at 1 mg/kg of body weight twice a day for 10 days. In a nude rat inoculation model, both compounds showed therapeutic efficacy at 0.25 mg/kg twice a day for 10 days.