Ken Bartizal

Platensimycin is a selective FabF inhibitor with potent antibiotic properties

Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of β-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

Multicenter evaluation of a broth macrodilution antifungal susceptibility test for yeasts.

Thirteen laboratories collaborated to optimize interlaboratory agreement of results of a broth macrodilution procedure for testing three classes of antifungal drugs against pathogenic yeasts. The activities of amphotericin B, flucytosine, and ketoconazole were tested against 100 coded isolates of Candida albicans, Candida tropicalis, Candida parapsilosis, Candida lusitaniae, Torulopsis (Candida) glabrata, and Cryptococcus neoformans. Two starting yeast inoculum sizes (5 x 10(4) and 2.5 x 10(3) cells per ml) were compared, and readings were taken after 24 and 48 h of incubation. All other test conditions were standardized. The resultant turbidities in all tubes were estimated visually on a scale from 0 to 4+ turbidity, and MIC-0, MIC-1, and MIC-2 were defined as the lowest drug concentrations that reduced growth to 0, 1+, or 2+ turbidity, respectively. For flucytosine, agreement among laboratories varied between 57 and 87% for different inocula, times of incubation, and end point criteria. Agreement was maximized (85%) when the lower inoculum was incubated for 2 days and the MICs were defined as 1+ turbidity or less. For amphotericin B, variations in test conditions produced much smaller differences in interlaboratory agreement. For ketoconazole, interlaboratory agreement was poorer by all end point criteria. However, MIC-2 endpoints distinguished T. glabrata as resistant compared with the other species. Overall, the studies indicated that readings from the lower inoculum obtained on the second day of reading result in the greatest interlaboratory agreement. In combination with data from previous multicenter studies (National Committee for Clinical Laboratory Standards, Antifungal Susceptibility Testing: Committee Report, Vol. 5, No. 17, 1988; M. A. Pfaller, L. Burmeister, M. S. Bartlett, and M. G. Rinaldi, J. Clin. Microbiol. 26:1437-1441, 1988; M. A. Pfaller, M. G. Rinaldi, J. N. Galgiani, M. S. Bartlett, B.A. Body, A. Espinel-Ingroff, R.A. Fromtling, G.S. Hall, C.E. Hughes, F. C. Odds, and A. M. SUgar, J. Clin. Microbiol. 34:1648-1654, 1990), these findings will be used by the National Committee for Clinical Laboratory Standards to develop a standardized method for in vitro antifungal susceptibility testing for yeasts.

In Vitro, In Vivo, and Clinical Studies of Tedizolid To Assess the Potential for Peripheral or Central Monoamine Oxidase Interactions

ABSTRACT Tedizolid phosphate is a novel oxazolidinone prodrug whose active moiety, tedizolid, has improved potency against Gram-positive pathogens and pharmacokinetics, allowing once-daily administration. Given linezolid warnings for drug-drug and drug-food interactions mediated by monoamine oxidase (MAO) inhibition, including sporadic serotonergic toxicity, these studies evaluated tedizolid for potential MAO interactions. In vitro, tedizolid and linezolid were reversible inhibitors of human MAO-A and MAO-B; the 50% inhibitory concentration (IC50) for tedizolid was 8.7 μM for MAO-A and 5.7 μM for MAO-B and 46.0 and 2.1 μM, respectively, with linezolid. Tedizolid phosphate was negative in the mouse head twitch model of serotonergic activity. Two randomized placebo-controlled crossover clinical studies assessed the potential of 200 mg/day tedizolid phosphate (at steady state) to enhance pressor responses to coadministered oral tyramine or pseudoephedrine. Sensitivity to tyramine was determined by comparing the concentration of tyramine required to elicit a ≥30-mmHg increase in systolic blood pressure (TYR30) when administered with placebo versus tedizolid phosphate. The geometric mean tyramine sensitivity ratio (placebo TYR30/tedizolid phosphate TYR30) was 1.33; a ratio of ≥2 is considered clinically relevant. In the pseudoephedrine study, mean maximum systolic blood pressure was not significantly different when pseudoephedrine was coadministered with tedizolid phosphate versus placebo. In summary, tedizolid is a weak, reversible inhibitor of MAO-A and MAO-B in vitro. Provocative testing in humans and animal models failed to uncover significant signals that would suggest potential for hypertensive or serotonergic adverse consequences at the therapeutic dose of tedizolid phosphate. Clinical studies are registered at www.clinicaltrials.gov as NCT01539473 (tyramine interaction study conducted at Covance Clinical Research Center, Evansville, IN) and NCT01577459 (pseudoephedrine interaction study conducted at Vince and Associates Clinical Research, Overland Park, KS).

Results of the Surveillance of Tedizolid Activity and Resistance Program: in vitro susceptibility of Gram-positive pathogens collected in 2011 and 2012 from the United States and Europe

The in vitro activity and spectrum of tedizolid and comparators were analyzed against 6884 Gram-positive clinical isolates collected from multiple US and European sites as part of the Surveillance of Tedizolid Activity and Resistance Program in 2011 and 2012. Organisms included 4499 Staphylococcus aureus, 537 coagulase-negative staphylococci (CoNS), 873 enterococci, and 975 β-hemolytic streptococci. The MIC values that inhibited 90% of the isolates within each group (MIC90) were 0.25 μg/mL for Staphylococcus epidermidis and β-hemolytic streptococci and 0.5 μg/mL for S. aureus, other CoNS, and enterococci. Of 16 isolates with elevated tedizolid or linezolid MIC values (intermediate or resistant isolates), 10 had mutations in the genes encoding 23S rRNA (primarily G2576T), 5 had mutations in the genes encoding ribosomal proteins L3 or L4, and 5 carried the cfr multidrug resistance gene. Overall, tedizolid showed excellent activity against Gram-positive bacteria and was at least 4-fold more potent than linezolid against wild-type and linezolid-resistant isolates. Given the low overall frequency of isolates that would be resistant to tedizolid at the proposed break point of 0.5 μg/mL (0.19%) and potent activity against contemporary US and European isolates, tedizolid has the potential to serve as a valuable therapeutic option in the treatment of infections caused by Gram-positive pathogens.

The isolation and structure elucidation of zaragozic acid C, a novel potent squalene synthase inhibitor.

Abstract The novel zaragozic acid C ( 1 ) has been isolated as a potent inhibitor of squalene synthase. It was found to be a competitive inhibitor of rat liver squalene synthase with an apparent K i of 45 ± 15 pM, and a broad spectrum antifungal agent against both yeast and filamentous fungi.

Preclinical Evaluation of the Stability, Safety and Efficacy of CD101, a Novel Echinocandin.

ABSTRACT Fungal infections pose a significant public health burden with high morbidity and mortality. CD101 is a novel echinocandin under development for the treatment and prevention of systemic Candida infections. Preclinical studies were conducted to evaluate the metabolic stability, plasma protein binding, pharmacokinetics, toxicity, and efficacy of CD101 at various dose levels. CD101 was stable to biotransformation in rat, monkey, and human liver microsomes and rat, monkey, dog, and human hepatocytes. In vitro studies suggest minimal interaction with recombinant cytochrome P450 enzymes (50% inhibitory concentrations [IC50s] of >10 μM). Similar to anidulafungin, CD101 bound avidly (>98%) to human, mouse, rat, and primate plasma proteins. In a 2-week repeat-dose comparison study, CD101 was well tolerated in rats (no effects on body weight, hematology, coagulation, or urinalysis). In contrast, administration of anidulafungin (at comparable exposure levels) resulted in reduced body weight, decreases in red blood cell, hemoglobin, hematocrit, mean cell volume, mean corpuscular hemoglobin, platelet, and reticulocyte counts, increases in neutrophil and eosinophil counts, polychromasia, and decreased activated partial thromboplastin time. Elevated plasma transaminases, total bilirubin, cholesterol, and globulin, dark and enlarged spleens, and single-cell hepatocyte necrosis were also observed for anidulafungin but not CD101. Hepatotoxicity may be due to the inherent chemical lability of anidulafungin generating potentially reactive intermediates. A glutathione trapping experiment confirmed the formation of a reactive species from anidulafungin, whereas CD101 did not exhibit instability or reactive intermediates. CD101 showed antifungal activity against Candida and Aspergillus infections in neutropenic mice. These preclinical studies demonstrated that CD101 is chemically and metabolically stable, well tolerated with no hepatotoxicity, and efficacious as an antifungal agent.

CD101: a novel long-acting echinocandin

CD101 is a novel echinocandin drug being developed to treat severe fungal infections including invasive candidiasis. We have performed a series of studies to evaluate the antifungal properties of CD101 against both echinocandin‐susceptible and ‐resistant Candida strains. Antifungal susceptibility testing performed on a collection of 95 Candida strains including 30 caspofungin‐resistant isolates containing fks mutations demonstrated comparable antifungal potency of CD101 relative to micafungin (MCF) across different Candida species. Comparable kinetic inhibition of glucan synthase activity was also observed for CD101 and MCF on both wild‐type (WT) and resistant fks mutant Candida strains. Similarly, both drugs yielded nearly identical values for a mutant prevention concentration. In a murine model of invasive candidiasis, CD101 displayed better or at least comparable efficacy relative to MCF in treating WT or fks mutant Candida albicans. An exceptional long‐lived pharmacokinetic profile was observed in mice following a single dose of CD101. Collectively, CD101 has great potential not only in treating invasive Candida infections but also in preventing emergence of resistance to currently approved echinocandin drugs.

Activity of Tedizolid Phosphate (TR-701) in Murine Models of Infection with Penicillin-Resistant and Penicillin-Sensitive Streptococcus pneumoniae

ABSTRACT The in vitro activity of tedizolid (previously known as torezolid, TR-700) against penicillin-resistant Streptococcus pneumoniae (PRSP) clinical isolates and the in vivo efficacy of tedizolid phosphate (torezolid phosphate, TR-701) in murine models of PRSP systemic infection and penicillin-susceptible S. pneumoniae (PSSP) pneumonia were examined using linezolid as a comparator. The MIC90 against 28 PRSP isolates was 0.25 μg/ml for tedizolid, whereas it was 1 μg/ml for linezolid. In mice infected systemically with a lethal inoculum of PRSP 1 h prior to a single administration of either antimicrobial, oral tedizolid phosphate was equipotent to linezolid (1 isolate) to 2-fold more potent than linezolid (3 isolates) for survival at day 7, with tedizolid phosphate 50% effective dose (ED50) values ranging from 3.19 to 11.53 mg/kg of body weight/day. In the PSSP pneumonia model, the ED50 for survival at day 15 was 2.80 mg/kg/day for oral tedizolid phosphate, whereas it was 8.09 mg/kg/day for oral linezolid following 48 h of treatment with either agent. At equivalent doses (10 mg/kg once daily tedizolid phosphate or 5 mg/kg twice daily linezolid), pneumococcal titers in the lungs at 52 h postinfection were approximately 3 orders of magnitude lower with tedizolid phosphate treatment than with linezolid treatment or no treatment. Lung histopathology showed less inflammatory cell invasion into alveolar spaces in mice treated with tedizolid phosphate than in untreated or linezolid-treated mice. These results demonstrate that tedizolid phosphate is effective in murine models of PRSP systemic infection and PSSP pneumonia.

Comparative in vitro activities of ertapenem against aerobic and facultative bacterial pathogens from patients with complicated skin and skin structure infections

This study compared the in vitro activities of ertapenem (Merck & Co., Inc.), ceftriaxone, amoxicillin-clavulanate, and piperacillin-tazobactam against 518 aerobic and facultative bacterial pathogens isolated from 340 patients with complicated skin and skin structure infections. Ciprofloxacin was also tested against Gram-negative isolates. Gram-positive cocci accounted for 68.1% of the aerobic bacteria; Staphylococcus aureus was the most common isolate (45.6%). The ertapenem MIC was < or = 2 microg/ml for 80.9% of isolates and > or = 8 microg/ml for 16.2% (including isolates of enterococci, methicillin-resistant S. aureus, Pseudomonas aeruginosa, and other nonfermentative Gram-negative bacteria). Against methicillin-susceptible S. aureus, ertapenem had the most potent activity. Ertapenem was the most active drug against Enterobacteriaceae (100% susceptible), whereas amoxicillin-clavulanate was least active (66% susceptible). Piperacillin-tazobactam was the most active drug against P. aeruginosa (100% susceptible), followed by ciprofloxacin (87% susceptible). In summary, ertapenem was highly active in vitro against many aerobic and facultative bacterial pathogens commonly recovered from patients with complicated skin and skin structure infections.

Pneumocandin antifungal lipopeptides. The phenolic hydroxyl is required for 1,3-β-glucan synthesis inhibition.

Abstract Pneumocandin B 0 1 undergoes selective oxidation / reduction chemistry at the homotyrosine ( hty ) residue. Removal of the phenolic hydroxyl gives >140-fold loss in activity against a Candida albicans 1,3-β-glucan synthetase enzyme preparation and a significant loss of antifungal activity. Inversion of the C4- hty hydroxyl causes about a 70-fold decrease in potency, while removal of this hydroxyl yields a more potent inhibitor.