In Vitro Activity Analysis of a New Polymyxin, SPR741, Tested in Combination with Antimicrobial Agents against a Challenge Set of Enterobacteriaceae, Including Molecularly Characterized Strains.

The activities of azithromycin, fusidic acid, vancomycin, doxycycline, and minocycline were evaluated alone and in combination with SPR741. A total of 202 Escherichia coli and 221 Klebsiella pneumoniae isolates were selected, and they included a genome-sequenced subset (n = 267), which was screened in silico for ß-lactamase, macrolide-lincosamide-streptogramin (MLS), and tetracycline (tet) genes. Azithromycin (>16 mg/liter), fusidic acid (>64 mg/liter), vancomycin (>16 mg/liter), and SPR741 (>8 mg/liter) showed off-scale MICs when each was tested alone against all isolates. MIC50/90 results of 0.5/8 mg/liter, 4/>32 mg/liter, 16/>16 mg/liter, 2/32 mg/liter, and 0.25/4 mg/liter were obtained for azithromycin-SPR741, fusidic acid-SPR741, vancomycin-SPR741, doxycycline-SPR741 and minocycline-SPR741, respectively, against all isolates. Overall, azithromycin-SPR741 (MIC90, 2 to 4 mg/liter) and minocycline-SPR741 (MIC90, 0.5 to 2 mg/liter) showed the lowest MIC90 values against different subsets of E. coli isolates, except for azithromycin-SPR741 (MIC90, 16 mg/liter) against the AmpC and metallo-ß-lactamase subsets. In general, minocycline-SPR741 (MIC90, 2 to 8 mg/liter) had the lowest MIC90 against K. pneumoniae isolates producing different groups of ß-lactamases. The azithromycin-SPR741 MIC (MIC50/90, 2/32 mg/liter) was affected by MLS genes (MIC50/90 of 0.25/2 mg/liter against isolates without MLS genes), whereas doxycycline-SPR741 (MIC50/90, 0.5/2 versus 8/32 mg/liter) and minocycline-SPR741 (MIC50/90, 0.25/1 versus 1/8 mg/liter) MIC results were affected when tested against isolates carrying tet genes in general. However, minocycline-SPR741 inhibited 88.2 to 92.9% of tet-positive isolates regardless of the tet gene. The azithromycin-SPR741 MIC results (MIC50/90, 1/16 mg/liter) against isolates with enzymatic MLS mechanisms were lower than against those with ribosomal protection (MIC50/90, 16/>32 mg/liter). SPR741 increased the in vitro activity of tested codrugs at different levels and seemed to be dependent on the species and resistance mechanisms of the respective codrug.

In Vitro Activity of Tebipenem (SPR859) against Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria.

Tebipenem (SPR859) is the microbiologically active form of SPR994 (tebipenem-pivoxil), an orally available carbapenem with activity against extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae Measurement of the relative binding of SPR859 to the bacterial cell targets revealed that it is a potent inhibitor of multiple penicillin-binding proteins (PBPs) but primarily a Gram-negative PBP 2 inhibitor, similar to other compounds in this class. These data support further clinical development of SPR994.

Oritavancin Pharmacokinetics and Bone Penetration in Rabbits.

The pharmacokinetics and bone concentrations of oritavancin were investigated after a single intravenous dose was administered to rabbits. The pharmacokinetic profile of oritavancin in rabbits showed that it is rapidly distributed to bone tissues, with concentrations remaining stable for up to 168 h, the last measured time point. Based on these findings, further evaluation of oritavancin for the treatment of infections in bone tissues is warranted.

Assessment of oritavancin serum protein binding across species.

Biophysical methods to study the binding of oritavancin, a lipoglycopeptide, to serum protein are confounded by nonspecific drug adsorption to labware surfaces. We assessed oritavancin binding to serum from mouse, rat, dog, and human by a microbiological growth-based method under conditions that allow near-quantitative drug recovery. Protein binding was similar across species, ranging from 81.9% in human serum to 87.1% in dog serum. These estimates support the translation of oritavancin exposure from nonclinical studies to humans.

Oritavancin disrupts membrane integrity of Staphylococcus aureus and vancomycin-resistant enterococci to effect rapid bacterial killing.

Oritavancin is an investigational lipoglycopeptide in clinical development for the treatment of acute bacterial skin and skin structure infections. In this study, we demonstrate that oritavancin causes bacterial membrane depolarization and permeabilization leading to cell death of Gram-positive pathogens and that these effects are attributable to the 4'-chlorobiphenylmethyl group of the molecule.

Synthesis and in vitro evaluation of bisphosphonated glycopeptide prodrugs for the treatment of osteomyelitis.

As therapeutic agents of choice in the treatment of complicated infections, glycopeptide antibiotics are often preferentially used in cases of osteomyelitis, an infection located in bone and notoriously difficult to successfully manage. Yet frequent and heavy doses of these systemically administered antibiotics are conventionally prescribed to obtain higher antibiotic levels in the bone and reduce the high recurrence rates. Targeting antibiotics to the bone after systemic administration would present at least three potential advantages: (i) greater efficacy, by concentrating the therapeutic agent in bone; (ii) greater convenience, through a reduction in the frequency of administration; and (iii) greater safety, by reducing the levels of systemic drug exposure. We present here the design, synthesis and in vitro evaluation of eight prodrugs of the glycopeptide antibacterial agents vancomycin and oritavancin taking advantage of the affinity of the bisphosphonate group for bone for delivery to osseous tissues.

Oritavancin kills stationary-phase and biofilm Staphylococcus aureus cells in vitro.

Slow-growing bacteria and biofilms are notoriously tolerant to antibiotics. Oritavancin is a lipoglycopeptide with multiple mechanisms of action that contribute to its bactericidal action against exponentially growing gram-positive pathogens, including the inhibition of cell wall synthesis and perturbation of membrane barrier function. We sought to determine whether oritavancin could eradicate cells known to be tolerant to many antimicrobial agents, that is, stationary-phase and biofilm cultures of Staphylococcus aureus in vitro. Oritavancin exhibited concentration-dependent bactericidal activity against stationary-phase inocula of methicillin-susceptible S. aureus (MSSA) ATCC 29213, methicillin-resistant S. aureus (MRSA) ATCC 33591, and vancomycin-resistant S. aureus (VRSA) VRS5 inoculated into nutrient-depleted cation-adjusted Mueller-Hinton broth. As has been described for exponential-phase cells, oritavancin induced membrane depolarization, increased membrane permeability, and caused ultrastructural defects including a loss of nascent septal cross walls in stationary-phase MSSA. Furthermore, oritavancin sterilized biofilms of MSSA, MRSA, and VRSA at minimal biofilm eradication concentrations (MBECs) of between 0.5 and 8 mug/ml. Importantly, MBECs for oritavancin were within 1 doubling dilution of their respective planktonic broth MICs, highlighting the potency of oritavancin against biofilms. These results demonstrate a significant activity of oritavancin against S. aureus in phases of growth that exhibit tolerance to other antimicrobial agents.

Impact of human serum albumin on oritavancin in vitro activity against enterococci.

Oritavancin is a lipoglycopeptide with activity against gram-positive pathogens including vancomycin-resistant enterococci. The impact of human serum albumin (HSA) on oritavancin activity against enterococci was compared to those of vancomycin, daptomycin, teicoplanin, and linezolid in vitro using MIC and time-kill methods. Oritavancin MICs increased between 0- and 8-fold in the presence of HSA. In time-kill assays with HSA, oritavancin retained activity, killing or inhibiting enterococci more rapidly than did comparators when peak concentrations were simulated.

Comparative in vitro activity profile of oritavancin against recent gram-positive clinical isolates.

Oritavancin activity was tested against 15,764 gram-positive isolates collected from 246 hospital centers in 25 countries between 2005 and 2008. Organisms were Staphylococcus aureus (n = 9,075), coagulase-negative staphylococci (n = 1,664), Enterococcus faecalis (n = 1,738), Enterococcus faecium (n = 819), Streptococcus pyogenes (n = 959), Streptococcus agalactiae (n = 415), group C, G, and F streptococci (n = 84), and Streptococcus pneumoniae (n = 1,010). Among the evaluated staphylococci, 56.7% were resistant to oxacillin. The vancomycin resistance rate among enterococci was 21.2%. Penicillin-resistant and -intermediate rates were 14.7% and 21.4%, respectively, among S. pneumoniae isolates. Among nonpneumococcal streptococci, 18.5% were nonsusceptible to erythromycin. Oritavancin showed substantial in vitro activity against all organisms tested, regardless of resistance profile. The maximum oritavancin MIC against all staphylococci tested (n = 10,739) was 4 microg/ml; the MIC(90) against S. aureus was 0.12 microg/ml. Against E. faecalis and E. faecium, oritavancin MIC(90)s were 0.06 and 0.12, respectively. Oritavancin was active against glycopeptide-resistant enterococci, including VanA strains (n = 486), with MIC(90)s of 0.25 and 1 microg/ml against VanA E. faecium and E. faecalis, respectively. Oritavancin showed potent activity against streptococci (n = 2,468); MIC(90)s for the different streptococcal species were between 0.008 and 1 microg/ml. These data are consistent with previous studies with respect to resistance rates of gram-positive isolates and demonstrate the spectrum and in vitro activity of oritavancin against a wide variety of contemporary gram-positive pathogens, regardless of resistance to currently used drugs. The data provide a foundation for interpreting oritavancin activity and potential changes in susceptibility over time once oritavancin enters into clinical use.

Assessment by time-kill methodology of the synergistic effects of oritavancin in combination with other antimicrobial agents against Staphylococcus aureus.

Oritavancin is a semisynthetic lipoglycopeptide in clinical development for serious gram-positive infections. This study describes the synergistic activity of oritavancin in combination with gentamicin, linezolid, moxifloxacin, or rifampin in time-kill studies against methicillin-susceptible, vancomycin-intermediate, and vancomycin-resistant Staphylococcus aureus.

Effect of polysorbate 80 on oritavancin binding to plastic surfaces: implications for susceptibility testing.

Oritavancin, a semisynthetic lipoglycopeptide with activity against gram-positive bacteria, has multiple mechanisms of action, including the inhibition of cell wall synthesis and the perturbation of the membrane potential. Approved guidelines for broth microdilution MIC assays with dalbavancin, another lipoglycopeptide, require inclusion of 0.002% polysorbate 80. To investigate the potential impact of polysorbate 80 on oritavancin susceptibility assays, we quantified the recovery of [(14)C]oritavancin from susceptibility assay plates with and without polysorbate 80 and examined the effect of the presence of polysorbate 80 on the oritavancin MICs for 301 clinical isolates from the genera Staphylococcus, Enterococcus, and Streptococcus. In the absence of polysorbate 80, [(14)C]oritavancin was rapidly lost from solution in susceptibility assay test plates: 9% of the input drug was recovered in broth at 1 h when [(14)C]oritavancin was tested at 1 mug/ml. Furthermore, proportionately greater losses were observed at lower oritavancin concentrations, suggesting saturable binding of oritavancin to surfaces. The inclusion of 0.002% polysorbate 80 or 2% lysed horse blood permitted the recovery of 80 to 100% [(14)C]oritavancin at 24 h for all drug concentrations tested. Concordantly, oritavancin MIC(90)s for streptococcal isolates, as determined in medium containing 2% lysed horse blood, were identical with and without polysorbate 80. In stark contrast, polysorbate 80 reduced the oritavancin MIC(90)s by 16- to 32-fold for clinical isolates of enterococci and staphylococci, which are typically cultured without blood. The results presented here provide evidence that the MIC data for oritavancin in the current literature significantly underestimate the potency of oritavancin in vitro. Moreover, the combination of data from MIC and [(14)C]oritavancin recovery studies supports the revision of the oritavancin broth microdilution method to include polysorbate 80 throughout the assay.

Newly defined in vitro quality control ranges for oritavancin broth microdilution testing and impact of variation in testing parameters.

A 9-laboratory M23-A2 quality control (QC) study was performed to evaluate reproducibility of oritavancin MICs against reference strains of Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae using broth microdilution assays in the presence of polysorbate 80. Polysorbate 80 has previously been shown to be required for accurate measurement of oritavancin broth microdilution MICs. Greater than 95% of replicate results (n = 270/organism) fell within the following QC ranges (in micrograms per milliliter): S. aureus ATCC 29213, 0.015 to 0.12; E. faecalis ATCC 29212, 0.008 to 0.03; and S. pneumoniae ATCC 49619, 0.001 to 0.004. Oritavancin MIC QC ranges were, thus, narrow and reproducible. Parameters affecting testing results in the presence of polysorbate 80 were also evaluated. Oritavancin MICs were equivalent to or within 1 doubling dilution of those obtained under standard Clinical and Laboratory Standards Institute testing conditions, regardless of incubation time (18, 24, or 48 h), Ca(2+) concentration, pH, or frozen panel storage time (up to 6 months).

Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for the prevention of osteomyelitis.

Osteomyelitis is a difficult to treat bacterial infection of the bone. Delivering antibacterial agents to the bone may overcome the difficulties in treating this illness by effectively concentrating the antibiotic at the site of infection and by limiting the toxicity that may result from systemic exposure to the large doses conventionally used. Using bisphosphonates as osteophilic functional groups, different forms of fluoroquinolone esters were synthesized and evaluated for their ability to bind bone and to release the parent antibacterial agent. Bisphosphonated glycolamide fluoroquinolone esters were found to present a profile consistent with effective and rapid bone binding and efficient release of the active drug moiety. They were assessed for their ability to prevent bone infection in vivo and were found to be effective when the free fluoroquinolones were not.

Tebipenem, the first oral carbapenem antibiotic.

INTRODUCTION: Infections caused by antibiotic-resistant pathogens, particularly Gram-negative bacteria, have become increasingly challenging to successfully treat. The beta-lactam antibiotic subclass, the carbapenems, have proven valuable for the treatment of such Gram-negative bacterial infections due to their spectrum and ß-lactamase stability properties. However, all marketed carbapenems to date are parenterally administered to adult patients. Areas covered: One carbapenem, tebipenem-pivoxil (TBPM-PI), is an oral prodrug that was approved in Japan for pediatric use only in 2009. This review summarizes preclinical and clinical data for TBPM-PI, which is now in clinical development again this time for use as the first oral carbapenem available for treatment of bacterial infections in adult patients. Expert commentary: There is an urgent unmet need with an increasing prevalence of fluoroquinolone-resistant and ESBL-producing Gram-negative pathogens in the hospital and community setting. Carbapenems have traditionally been considered the drugs of choice for infections caused by enterobacteria producing ESBL and AmpC enzymes because they are not affected by these resistance mechanisms. The carbapenem, TBPM-PI, offers an oral option, particularly as step-down therapy, for use of this class in the treatment of serious Gram-negative infections.

Treatment of Gram-negative bacterial infections by potentiation of antibiotics.

Infections caused by antibiotic-resistant pathogens, particularly Gram-negative bacteria, represent significant treatment challenges for physicians resulting in high rates of morbidity and mortality. The outer membrane of Gram-negative bacteria acts as a permeability barrier to many compounds that would otherwise be effective antibacterial agents, including those effective against Gram-positive pathogens. Potentiator molecules disrupt this barrier allowing entry of otherwise impermeant molecules, thus providing a strategy to render multi-drug resistant pathogens susceptible to a broader range of antibiotics. Potentiator molecules are cationic and the mechanism of disruption involves interaction with the negatively charged outer membrane. This physical attribute, along with an often high degree of lipophilicity typically endears these molecules with unacceptable toxicity. Presented herein are examples of advanced potentiator molecules being evaluated for use in combination therapy for the treatment of resistant Gram-negative infections.

Structural and Kinetic Characterization of Diazabicyclooctanes as Dual Inhibitors of Both Serine-ß-Lactamases and Penicillin-Binding Proteins.

Avibactam is a diazabicyclooctane ß-lactamase inhibitor possessing outstanding but incomplete efficacy against multidrug-resistant Gram-negative pathogens in combination with ß-lactam antibiotics. Significant pharmaceutical investment in generating derivatives of avibactam warrants a thorough characterization of their activity. We show here through structural and kinetic analysis that select diazabicyclooctane derivatives display effective but varied inhibition of two clinically important ß-lactamases (CTX-M-15 and OXA-48). Furthermore, these derivatives exhibit considerable antimicrobial activity (MIC ≤ 2 µg/mL) against clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Enterobacter spp. Imaging of cell phenotype along with structural and biochemical experiments unambiguously demonstrate that this activity, in E. coli, is a result of targeting penicillin-binding protein 2. Our results suggest that structure-activity relationship studies for the purpose of drug discovery must consider both ß-lactamases and penicillin-binding proteins as targets. We believe that this approach will yield next-generation combination or monotherapies with an expanded spectrum of activity against currently untreatable Gram-negative pathogens.

4-Substituted D-glutamic acid analogues: the first potent inhibitors of glutamate racemase (MurI) enzyme with antibacterial activity.

The first potent inhibitors of glutamate racemase (MurI) enzyme that show whole cell antibacterial activity are described. Optically pure 4-substituted D-glutamic acid analogues with (2R,4S) stereochemistry and bearing aryl-, heteroaryl-, cinnamyl-, or biaryl-methyl substituents represent a novel class of glutamate racemase inhibitors. Exploration of the D-Glu core led to the identification of lead compounds (-)-8 and 10. 2-Naphthylmethyl derivative 10 was found to be a potent competitive inhibitor of glutamate racemase activity (K(i) = 16 nM, circular dichroism assay; IC(50) = 0.1 microg/mL high-performance liquid chromatography (HPLC) assay). Thorough structure-activity relationship (SAR) studies led to benzothienyl derivatives such as 69 and 74 with increased potency (IC(50) = 0.036 and 0.01 microg/mL, respectively, HPLC assay). These compounds showed potent whole cell antibacterial activity against S. pneumoniae PN-R6, and good correlation with the enzyme assay. Compounds 69, 74 and biaryl derivative 52 showed efficacy in an in vivo murine thigh infection model against Streptococcus pneumoniae. Data described herein suggest that glutamate racemase may be a viable target for developing new antibacterial agents.

Activity of oritavancin and comparators in vitro against standard and high inocula of Staphylococcus aureus.

In this study, the impact of inoculum density on the growth inhibitory and killing activities of oritavancin and comparators (vancomycin, daptomycin and linezolid) in vitro against four Staphylococcus aureus strains at clinically relevant drug concentrations was studied. Broth microdilution and time-kill assays were performed using a standard inoculum [ca. 10(5)colony-forming units (CFU)/mL as per Clinical and Laboratory Standards Institute (CLSI) guidelines] and a high inoculum (ca. 10(7)CFU/mL). Whereas minimal inhibitory concentrations (MICs) of comparators were 2-8-fold higher when tested at high inoculum, oritavancin MICs were 16-fold higher for all strains at the high inoculum relative to the standard inoculum. However, in time-kill assays, when tested at its fC(min) [trough concentration of free (non-protein-bound) drug] and fC(max) (peak concentration of non-protein-bound drug), oritavancin retained its bactericidal activity against a vancomycin-susceptible, meticillin-susceptible S. aureus (VS-MSSA) strain and a vancomycin-susceptible, meticillin-resistant S. aureus (VS-MRSA) strain both at standard and high inocula. At its fC(max), oritavancin was bactericidal at standard inoculum but not at high inoculum against two vancomycin-intermediate S. aureus (VISA) strains. Against both VISA strains at standard inoculum, oritavancin at its fC(min) reduced cell density by between 2 and 3 log (bacteriostatic), predicting that it will retain activity against certain VISA infections. However, oritavancin had no substantial growth inhibitory effect against either VISA strain at high inoculum, suggesting that in rare VISA infections with an anticipated high bacterial burden such as endocarditis, alternative oritavancin dosing strategies, including combinations with other agents, may be explored.

In vitro characterization of oritavancin clearance from human blood by low-flux, high-flux, and continuous renal replacement therapy dialyzers.

BACKGROUND: Oritavancin is an investigational lipoglycopeptide antibiotic under clinical development for the treatment of gram-positive bacterial infections. The impact of hemodialysis on plasma concentrations of oritavancin is unknown and may be important in making dosage adjustments in such patients. The present study sought to determine the clearance of oritavancin from human blood by various commercially available dialyzers in an in vitro hemodialysis model. METHODS: Three types each of low-flux (Dicea 130, F6, and Polyflux 14L) and high-flux (Revaclear, Exeltra 150, and Optiflux F160NR) dialyzers and one type of continuous renal replacement therapy (CRRT) dialyzer (Prismaflex M100) were studied. Heparinized human blood containing oritavancin (200 mg/L) was circulated from a reservoir to the dialyzers and back to the reservoir. Fresh dialysate was pumped through the dialyzers in a countercurrent manner. Blood samples from each side of the dialyzers and contaminated dialysate samples were collected at periodic intervals. Oritavancin levels were analyzed by a liquid chromatography/mass spectrometry method with a limit of quantification of 12.5 ng/mL and plasma clearances of oritavancin were calculated. RESULTS: The mean dialytic clearance of oritavancin was insignificant for each of the low-flux, high-flux and CRRT dialyzers. Clinically significant amounts of oritavancin were not detected in dialysate during any of the experimental dialysis sessions. CONCLUSIONS: The clearance of oritavancin from human blood by the dialyzers used in this study is insignificant. Further clinical studies would be required before making changes in dosage of oritavancin in hemodialysis patients.