Neutropenic Mouse Thigh Research Articles

In Vivo Pharmacodynamics of Torezolid Phosphate (TR-701), a New Oxazolidinone Antibiotic, against Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Strains in a Mouse Thigh Infection Model

Torezolid phosphate (TR-701) is the phosphate monoester prodrug of the oxazolidinone TR-700 which demonstrates potent in vitro activity against Gram-positive bacteria, including methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). The pharmacodynamics of TR-701 or TR-700 (TR-701/700) against S. aureus is incompletely defined. Single-dose pharmacokinetic studies were conducted in mice for TR-701/700. Forty-eight-hour dose range and 24-hour dose fractionation studies were conducted in a neutropenic mouse thigh model of S. aureus infection using MRSA ATCC 33591 to identify the dose and schedule of administration of TR-701/700 that was linked with optimized antimicrobial effect. Additional dose range studies compared the efficacies of TR-701/700 and linezolid for one MSSA strain and one community-associated MRSA strain. In dose range studies, TR-701/700 was equally bactericidal against MSSA and MRSA. Mean doses of 37.6 and 66.9 mg/kg of body weight/day of TR-701/700 resulted in stasis and 1 log CFU/g decreases in bacterial densities, respectively, at 24 h, and mean doses of 35.3, 46.6, and 71.1 mg/kg/day resulted in stasis and 1 and 2 log CFU/g reductions, respectively, at 48 h. Linezolid administered at doses as high as 150 mg/kg/day did not achieve stasis at either time point. Dose fractionation studies demonstrated that the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) was the pharmacodynamic index for TR-701/700 that was linked with efficacy. TR-701/700 was highly active against MSSA and MRSA, in vivo, and was substantially more efficacious than linezolid, although linezolid's top exposure has half the human exposure. Dose fractionation studies showed that AUC/MIC was the pharmacodynamic index linked with efficacy, indicating that once-daily dosing in humans is feasible.

Generic Vancomycin Products Fail In Vivo despite Being Pharmaceutical Equivalents of the Innovator

Generic versions of intravenous antibiotics are not required to demonstrate therapeutic equivalence with the innovator because therapeutic equivalence is assumed from pharmaceutical equivalence. To test such assumptions, we studied three generic versions of vancomycin in simultaneous experiments with the innovator and determined the concentration and potency of the active pharmaceutical ingredient by microbiological assay, single-dose pharmacokinetics in infected mice, antibacterial effect by broth microdilution and time-kill curves (TKC), and pharmacodynamics against two wild-type strains of Staphylococcus aureus by using the neutropenic mouse thigh infection model. The main outcome measure was the comparison of magnitudes and patterns of in vivo efficacy between generic products and the innovator. Except for one product exhibiting slightly greater concentration, vancomycin generics were undistinguishable from the innovator based on concentration and potency, protein binding, in vitro antibacterial effect determined by minimal inhibitory or bactericidal concentrations and TKC, and serum pharmacokinetics. Despite such similarities, all generic products failed in vivo to kill S. aureus, while the innovator displayed the expected bactericidal efficacy: maximum antibacterial effect (Emax) (95% confidence interval [CI]) was 2.04 (1.89 to 2.19), 2.59 (2.21 to 2.98), and 3.48 (2.92 to 4.04) versus 5.65 (5.52 to 5.78) log10 CFU/g for three generics and the innovator product, respectively (P<0.0001, any comparison). Nonlinear regression analysis suggests that generic versions of vancomycin contain inhibitory and stimulatory principles within their formulations that cause agonistic-antagonistic actions responsible for in vivo failure. In conclusion, pharmaceutical equivalence does not imply therapeutic equivalence for vancomycin.

In vitro and in vivo comparison of the anti-staphylococcal efficacy of generic products and the innovator of oxacillin

BackgroundOxacillin continues to be an important agent in the treatment of staphylococcal infections; many generic products are available and the only requirement for their approval is demonstration of pharmaceutical equivalence. We tested the assumption that pharmaceutical equivalence predicts therapeutic equivalence by comparing 11 generics with the innovator product in terms of concentration of the active pharmaceutical ingredient (API), minimal inhibitory (MIC) and bactericidal concentrations (MBC), and antibacterial efficacy in the neutropenic mouse thigh infection model.MethodsThe API in each product was measured by a validated microbiological assay and compared by slope (potency) and intercept (concentration) analysis of linear regressions. MIC and MBC were determined by broth microdilution according to Clinical and Laboratory Standard Institute (CLSI) guidelines. For in vivo efficacy, neutropenic ICR mice were inoculated with a clinical strain of Staphylococcus aureus. The animals had 4.14 ± 0.18 log10 CFU/thigh when treatment started. Groups of 10 mice per product received a total dose ranging from 2.93 to 750 mg/kg per day administered q1h. Sigmoidal dose-response curves were generated by nonlinear regression fitted to Hill equation to compute maximum effect (Emax), slope (N), and the effective dose reaching 50% of the Emax (ED50). Based on these results, bacteriostatic dose (BD) and dose needed to kill the first log of bacteria (1LKD) were also determined.Results4 generic products failed pharmaceutical equivalence due to significant differences in potency; however, all products were undistinguishable from the innovator in terms of MIC and MBC. Independently of their status with respect to pharmaceutical equivalence or in vitro activity, all generics failed therapeutic equivalence in vivo, displaying significantly lower Emax and requiring greater BD and 1LKD, or fitting to a non-sigmoidal model.ConclusionsPharmaceutical or in vitro equivalence did not entail therapeutic equivalence for oxacillin generic products, indicating that criteria for approval deserve review to include evaluation of in vivo efficacy.

Determination of Therapeutic Equivalence of Generic Products of Gentamicin in the Neutropenic Mouse Thigh Infection Model

BackgroundDrug regulatory agencies (DRA) support prescription of generic products of intravenous antibiotics assuming therapeutic equivalence from pharmaceutical equivalence. Recent reports of deaths associated with generic heparin and metoprolol have raised concerns about the efficacy and safety of DRA-approved drugs.Methodology/Principal FindingsTo challenge the assumption that pharmaceutical equivalence predicts therapeutic equivalence, we determined in vitro and in vivo the efficacy of the innovator product and 20 pharmaceutically equivalent generics of gentamicin. The data showed that, while only 1 generic product failed in vitro (MIC = 45.3 vs. 0.7 mg/L, P<0.05), 10 products (including gentamicin reference powder) failed in vivo against E. coli due to significantly inferior efficacy (Emax = 4.81 to 5.32 vs. 5.99 log10 CFU/g, P≤0.043). Although the design lacked power to detect differences in survival after thigh infection with P. aeruginosa, dissemination to vital organs was significantly higher in animals treated with generic gentamicin despite 4 days of maximally effective treatment.ConclusionPharmaceutical equivalence does not predict therapeutic equivalence of generic gentamicin. Stricter criteria based on solid experimental evidence should be required before approval for human use.

Elucidation of the Pharmacokinetic/Pharmacodynamic Determinant of Colistin Activity against Pseudomonas aeruginosa in Murine Thigh and Lung Infection Models

Colistin is increasingly used as last-line therapy against Gram-negative pathogens. The pharmacokinetic (PK)/pharmacodynamic (PD) index that best correlates with the efficacy of colistin remains undefined. The activity of colistin against three strains of Pseudomonas aeruginosa was studied in neutropenic mouse thigh and lung infection models. The PKs of unbound colistin were determined from single-dose PK studies together with extensive plasma protein binding analyses. Dose-fractionation studies were conducted over 24 h with a dose range of 5 to 160 mg/kg of body weight/day. The bacterial burden in the thigh or lung was measured at 24 h after the initiation of treatment. Relationships between antibacterial effect and measures of exposure to unbound (f) colistin (area under the concentration-time curve [fAUC/MIC], maximum concentration of drug in plasma [fC(max)]/MIC, and the time that the concentration in plasma is greater than the MIC [fT > MIC]) were examined by using an inhibitory sigmoid maximum-effect model. Nonlinearity in the PKs of colistin, including its plasma protein binding, was observed. The PK/PD index that correlated best with its efficacy was fAUC/MIC in both the thigh infection model (R(2) = 87%) and the lung infection model (R(2) = 89%). The fAUC/MIC targets required to achieve 1-log and 2-log kill against the three strains were 15.6 to 22.8 and 27.6 to 36.1, respectively, in the thigh infection model, while the corresponding values were 12.2 to 16.7 and 36.9 to 45.9 in the lung infection model. The findings of this in vivo study indicate the importance of achieving adequate time-averaged exposure to colistin. The results will facilitate efforts to define the more rational design of dosage regimens for humans.

Pharmacodynamics of RWJ-54428 against Staphylococcus aureus , Streptococcus pneumoniae , and Enterococcus faecalis in a Neutropenic Mouse Thigh Infection Model

RWJ-54428 (also known as MC-02,479) is a new cephalosporin with promising activity against gram-positive bacteria. The pharmacodynamics (PDs) of RWJ-54428 against Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis were studied in a neutropenic mouse thigh infection model. The RWJ-54428 MICs ranged from 0.25 to 1 mg/liter. Mice with ca. 10(6) CFU/thigh at the initiation of therapy were treated intraperitoneally with RWJ-54428 at doses that ranged from 3 to 1,200 mg/kg of body weight/day (in 2, 3, 4, 6, or 12 divided doses) for 24 h. The maximal reductions in bacterial counts in thigh tissues at 24 h for the methicillin-resistant S. aureus, penicillin-resistant S. pneumoniae, and E. faecalis strains were -2.8, -3.8, and -1.7 log10 CFU/thigh, respectively. The percentage of a 24-h dosing interval that the unbound serum RWJ-54428 concentrations exceeded the MIC (fT>MIC) was the pharmacokinetic (PK)-PD parameter that best described the efficacy of RWJ-54428. The fT>MICs for a bacteriostatic effect (no net change in the numbers of CFU/thigh over 24 h) ranged from 14 to 20% for staphylococci and streptococci; for maximal reductions in the numbers of CFU/thigh, the fT>MICs ranged from 22 to 36% for these strains. For E. faecalis, the ranges of fT>MICs for static and maximal effects were 30 to 46% and 55 to 60%, respectively. These data show that treatment with RWJ-54428 results in marked antibacterial effects in vivo, with the PK-PD parameters for efficacy being comparable to those for the efficacy of penicillins and carbapenems active against staphylococci and pneumococci.

Impact of Resistance Selection and Mutant Growth Fitness on the Relative Efficacies of Streptomycin and Levofloxacin for Plague Therapy

Yersinia pestis, the bacterium that causes plague, is a potential agent of biowarfare and bioterrorism. The aminoglycoside antibiotic streptomycin is the gold standard for treatment. However, this recommendation is based on scant animal and clinical data. We used an in vitro pharmacodynamic infection model to compare the efficacies of 10-day regimens of streptomycin versus the fluoroquinolone antibiotic levofloxacin for the treatment of Y. pestis infection and to evaluate for emergence of resistance. The human serum concentration-time profiles for standard clinical regimens of 1 g of streptomycin given every 12 h and 500 mg of levofloxacin given every 24 h were simulated. The growth fitness of drug-resistant mutants was examined in neutropenic and immunocompetent mouse thigh infection models. In the in vitro infection system, untreated bacteria grew from 10(7) to 10(10) CFU/ml. Streptomycin therapy caused a 10(5) CFU/ml reduction in the number of bacteria over 24 h, followed by regrowth with streptomycin-resistant mutants. Levofloxacin resulted in a 10(7) CFU/ml reduction in the number of bacteria within 12 h, ultimately sterilizing the culture without resistance selection. In both the normal and neutropenic mouse infection models, streptomycin-resistant and wild-type strains were equally fit. However, 90% of levofloxacin-resistant isolates, cultured from the control in vitro infection arm, did not proliferate in the mouse models. Thus, the fluoroquinolone antibiotic levofloxacin was superior to streptomycin in our in vitro infection model. The majority of levofloxacin-resistant mutants were less fit than streptomycin-resistant and wild-type Y. pestis.

Bactericidal Activities of Meropenem and Ertapenem against Extended-Spectrum-β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae in a Neutropenic Mouse Thigh Model

The purpose of this study was to examine the in vivo efficacies of meropenem and ertapenem against extended-spectrum-beta-lactamase (ESBL)-producing isolates with a wide range of MICs. Human-simulated dosing regimens in mice were designed to approximate the free drug percent time above the MIC (fT>MIC) observed for humans following meropenem at 1 g every 8 h and ertapenem at 1 g every 24 h. An in vivo neutropenic mouse thigh infection model was used to examine the bactericidal effects against 31 clinical ESBL Escherichia coli and Klebsiella pneumoniae isolates and 2 non-ESBL isolates included for comparison at a standard 10(5) inoculum. Three isolates were examined at a high 10(7) inoculum as well. Meropenem displayed greater in vitro potency, with a median MIC (range) (microg/ml) of 0.125 (0.03 to 32), than did ertapenem, with 0.5 (0.012 to 128). Seven of the 31 ESBL isolates were removed from the efficacy analysis due to their inability to establish infection in the mouse model. When MICs were<or=1.5 microg/ml for ertapenem (<or=0.5 microg/ml for meropenem), similar reductions in CFU (approximately 2-log kill) were observed for both ertapenem (fT>MIC>or=23%) and meropenem (fT>MIC>or=75%). Ertapenem showed bacterial regrowth for seven of eight isolates, with MICs of>or=2 microg/ml (fT>MIC<or=20%), while meropenem displayed antibacterial potency that varied from a static effect to a 1-log bacterial reduction in these isolates (fT>MIC=30 to 65%). At a 10(7) inoculum, both agents eradicated bacteria due to adequate exposures (fT>MIC=20 to 45%). Due to low MICs, no difference in bacterial kill was noted for the majority of ESBL isolates tested. However, for isolates with raised ertapenem MICs of>or=2 microg/ml, meropenem displayed sustained efficacy due to its greater in vitro potency and higher resultant fT>MIC.

Pharmacodynamics of Levofloxacin against Pseudomonas aeruginosa with Reduced Susceptibility Due to Different Efflux Pumps: Do Elevated MICs Always Predict Reduced In Vivo Efficacy?

The Pseudomonas aeruginosa efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN play an important role in susceptibility to fluoroquinolones in vitro. To determine if levofloxacin MICs arising from different levels of expression of efflux pumps result in a proportional reduction in the response to levofloxacin in vivo, isogenic strains of P. aeruginosa were tested with levofloxacin in two mouse models of infection (sepsis and neutropenic mouse thigh models). The levofloxacin 50% effective doses (ED(50)s) increased proportionally with the MICs for most strains. Similarly, the 24-h area under the concentration-time curve (AUC)/MIC ratio that resulted in 90% of the maximum bactericidal activity (90% E(max)) exceeded 75 for all strains except those with elevated MICs due to MexEF-OprN overexpression. In these strains, levofloxacin ED(50)s were 2- to 10-fold lower than the ED(50)/MIC ratios in the other strains and 90% E(max) AUC/MIC ratios were 2- to 4-fold lower than those predicted from pharmacodynamic modeling of efficacy against other strains. These data show that while the MexEF-OprN efflux pump can provide P. aeruginosa resistance to levofloxacin in vitro, it appears to be less efficient in providing resistance to levofloxacin in animal models of infection.

Systemic Antibacterial Activity of Novel Synthetic Cyclic Peptides

Cyclic peptides with an even number of alternating d,l-alpha-amino acid residues are known to self-assemble into organic nanotubes. Such peptides previously have been shown to be stable upon protease treatment, membrane active, and bactericidal and to exert antimicrobial activity against Staphylococcus aureus and other gram-positive bacteria. The present report describes the in vitro and in vivo pharmacology of selected members of this cyclic peptide family. The intravenous (i.v.) efficacy of six compounds with MICs of less than 12 microg/ml was tested in peritonitis and neutropenic-mouse thigh infection models. Four of the six peptides were efficacious in vivo, with 50% effective doses in the peritonitis model ranging between 4.0 and 6.7 mg/kg against methicillin-sensitive S. aureus (MSSA). In the thigh infection model, the four peptides reduced the bacterial load 2.1 to 3.0 log units following administration of an 8-mg/kg i.v. dose. Activity against methicillin-resistant S. aureus was similar to MSSA. The murine pharmacokinetic profile of each compound was determined following i.v. bolus injection. Interestingly, those compounds with poor efficacy in vivo displayed a significantly lower maximum concentration of the drug in serum and a higher volume of distribution at steady state than compounds with good therapeutic properties. S. aureus was unable to easily develop spontaneous resistance upon prolonged exposure to the peptides at sublethal concentrations, in agreement with the proposed interaction with multiple components of the bacterial membrane canopy. Although additional structure-activity relationship studies are required to improve the therapeutic window of this class of antimicrobial peptides, our results suggest that these amphipathic cyclic d,l-alpha-peptides have potential for systemic administration and treatment of otherwise antibiotic-resistant infections.

Identification of antimicrobial compounds active against intracellular Staphylococcus aureus

Small-colony variants (SCVs) of Staphylococcus aureus exhibit characteristics of bacteria that can penetrate mammalian cells and remain intracellular and innocuous for indefinite periods. These properties make SCVs a convenient tool that can be used to identify new antibacterial agents having activity against intracellular, quiescent bacteria. Agents active against SCVs could be useful in the treatment of chronic staphylococcal infections such as bovine mastitis. An hemB deletion mutant of S. aureus Newbould, a bovine mastitis isolate, having a stable, genetically defined SCV phenotype, was used in a screening program to identify compounds active against intracellular, gram-positive bacteria. Out of more than 260,000 compounds screened, nine compounds having the desired properties were identified. The range of MICs against gram-positive bacteria was ⩽0.12–32 μg ml −1. One of the compounds (no. 8) showed excellent activity against gram-positive (MICs ⩽0.12 μg ml −1) and gram-negative (MICs ⩽0.12–4 μg ml −1) bacteria. Each of the nine compounds demonstrated efficacy in a neutropenic mouse thigh infection model. Two compounds, including compound no. 8, reduced numbers of bacteria in a mouse mastitis model of infection. Application of a stepwise screening process has identified lead compounds that may be useful for treating persistent, intracellular infections.

Pharmacodynamic Profile of Telithromycin against Macrolide- and Fluoroquinolone-Resistant Streptococcus pneumoniae in a Neutropenic Mouse Thigh Model

The new ketolide telithromycin has potent in vitro activity against Streptococcus pneumoniae, including strains resistant to penicillin, macrolides, and fluoroquinolones. The aim of the present study was to define the pharmacodynamic profile of telithromycin against S. pneumoniae strains with various resistance profiles in an in vivo system. Ten S. pneumoniae strains were studied; seven exhibited penicillin resistance, six demonstrated macrolide resistance, and two exhibited gatifloxacin resistance. The telithromycin MICs for all isolates were < or =0.5 microg/ml. Using the murine thigh infection model, CD-1/ICR mice were rendered neutropenic and were then inoculated with 10(5) to 10(6) CFU of S. pneumoniae per thigh. Telithromycin was administered orally at doses ranging from 25 to 800 mg/kg of body weight/day, with the doses administered one, two, three, or four times a day. The activity of telithromycin was assessed by determination of the change in the bacterial density in thigh tissue after 24 h of treatment for each treatment group and the untreated controls. Pharmacokinetic studies of telithromycin were conducted in infected, neutropenic animals. The levels of protein binding by telithromycin in mice ranged from 70 to 95% over the observed range of pharmacokinetic concentrations. By using either the total or the free concentrations of telithromycin, the area under the concentration-time curve (AUC)/MIC ratio was a strong determinant of the response against S. pneumoniae, regardless of the phenotypic resistance profile. The maximal efficacy (the 95% effective dose) against this cohort of S. pneumoniae strains and bacterial inhibition (stasis) of telithromycin were predicted by ratios of the AUC for the free drug concentration/MIC of approximately 1,000 and 200, respectively.

Pharmacodynamic Profile of Ertapenem against Klebsiella pneumoniae and Escherichia coli in a Murine Thigh Model

The pharmacodynamic profile of ertapenem was evaluated in a neutropenic mouse thigh infection model. Extended-spectrum beta-lactamase (ESBL)-positive and ESBL-negative clinical strains of Escherichia coli and Klebsiella pneumoniae were studied. MICs ranged from 0.0078 to 0.06 microg/ml with standard inoculum tests. Ertapenem doses were administered once to five times daily to achieve various exposures, reported as the percentage of the dosing interval that the concentration of free ertapenem was in excess of the MIC (%T>MIC(free)). Mean values for the static exposure and 80% maximally effective exposure (ED(80)) were 19% (range, 2 to 38%) and 33% (range, 13 to 65%) T>MIC(free), respectively. Differences in exposure requirements based on the presence of an ESBL resistance mechanism or bacterial species were not evident. In addition, experiments using a 100-fold higher inoculum did not decrease the magnitude of the reduction in bacterial density from baseline achieved compared to lower-inoculum studies. The pharmacodynamic parameter of %T>MIC(free) correlated well with bactericidal activity for all isolates, and the static and ED(80) exposures are consistent with those reported previously for carbapenems.

Pharmacology of novel heteroaromatic polycycle antibacterials.

Heteroaromatic polycycle (HARP) compounds are a novel class of small (M(w), 600 to 650) DNA-binding antibacterials. HARP compounds exhibit a novel mechanism of action by preferentially binding to AT-rich sites commonly found in bacterial promoters and replication origins. Noncovalent binding in the minor groove of DNA results in inhibition of DNA replication and DNA-dependent RNA transcription and subsequent bacterial growth. HARP compounds have previously been shown to have potent in vitro activities against a broad spectrum of gram-positive organisms. The present report describes the extensive profiling of the in vitro and in vivo pharmacology of HARP antibacterials. The efficacies of representative compounds (GSQ-2287, GSQ-10547, and GSQ-11203), which exhibited good MIC activity, were tested in murine lethal peritonitis and neutropenic thigh infection models following intravenous (i.v.) administration. All compounds were efficacious in vivo, with potencies generally correlating with MICs. GSQ-10547 was the most potent compound in vitro and in vivo, with a 50% effective dose in the murine lethal peritonitis model of 7 mg/kg of body weight against methicillin-sensitive Staphylococcus aureus (MSSA) and 13 mg/kg against methicillin-resistant S. aureus (MRSA). In the neutropenic mouse thigh infection model, GSQ-11203 reduced the bacterial load (MRSA and MSSA) 2 log units following administration of a 25-mg/kg i.v. dose. In a murine lung infection model, treatment with GSQ-10547 at a dose of 50 mg/kg resulted in 100% survival. In addition to determination of efficacy in animals, the pharmacokinetic and tissue disposition profiles in animals following administration of an i.v. dose were determined. The compounds were advanced into broad safety screening studies, including screening for safety pharmacology, genotoxicity, and rodent toxicity. The results support further development of this novel class of antibiotics.

Pharmacodynamics of oritavancin (LY333328) in a neutropenic-mouse thigh model of Staphylococcus aureus infection.

The pharmacokinetics and pharmacodynamics of oritavancin (LY333328), a glycopeptide antibiotic with concentration-dependent bactericidal activity against gram-positive pathogens, in a neutropenic-mouse thigh model of Staphylococcus aureus infection were studied. Plasma radioequivalent concentrations of oritavancin were determined by using [(14)C]oritavancin at doses ranging from 0.5 to 20 mg/kg of body weight. Peak plasma radioequivalent concentrations after an intravenous dose were 7.27, 12.56, 69.29, and 228.83 micro g/ml for doses of 0.5, 1, 5, and 20 mg/kg, respectively. The maximum concentration of drug in serum (C(max)) and the area under the concentration-time curve (AUC) increased linearly in proportion to the dose. Neither infection nor neutropenia was seen to affect the pharmacokinetics of oritavancin. Intravenous administration resulted in much higher concentrations in plasma than the concentrations obtained with subcutaneous administration. Single-dose dose-ranging studies suggested a sigmoid maximum effect (E(max)) dose-response relationship, with a maximal effect evident at single doses exceeding 2 mg/kg. The oritavancin dose (stasis dose) that resulted in a 24-h colony count similar to the pretreatment count was 1.53 (standard error [SE], 0.35) mg/kg. The single oritavancin dose that resulted in 50% of maximal bacterial killing (ED(50)) was 0.95 (SE, 0.20) mg/kg. Dose fractionation studies suggested that single doses of 0.5, 1, 2, 4, and 16 mg/kg appeared to have greater bactericidal efficacy than the same total dose subdivided and administered multiple times during the 24-h treatment period. When using an inhibitory E(max) model, C(max) appears to correlate better with bactericidal activity than do the time during which the concentration in plasma exceeds the MIC (T>MIC) and AUC. These data suggest that optimal oritavancin dosing strategies will require regimens that favor high C(max) concentrations rather than long periods during which unbound concentrations in plasma exceed the MIC.

In vivo antibacterial activity of RWJ-54428, a new cephalosporin with activity against gram-positive bacteria.

RWJ-54428 (MC-02,479) is a new cephalosporin with activity against resistant gram-positive organisms, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and penicillin-resistant Streptococcus pneumoniae. The in vivo efficacy of RWJ-54428 was evaluated against gram-positive bacteria in four mouse models of infection. RWJ-54428 was effective in vivo against methicillin-susceptible and -resistant S. aureus in a mouse model of sepsis, with 50% effective doses being similar to those of vancomycin. In a single-dose neutropenic mouse thigh model of infection, RWJ-54428 at 30 mg/kg of body weight showed activity similar to that of vancomycin at 30 mg/kg against a strain of methicillin-resistant S. aureus. RWJ-54428 also showed a prolonged in vivo postantibiotic effect in this model. In a mouse model of pneumonia due to a penicillin-susceptible strain of Streptococcus pneumoniae, RWJ-54428 displayed efficacy and potency superior to those of penicillin G and cefotaxime. In a mouse model of pyelonephritis due to Enterococcus faecalis, RWJ-54428 had bactericidal effects similar to those of vancomycin and ampicillin, but at two- to threefold lower total daily doses. These studies show that RWJ-54428 is active in experimental mouse models of infection against gram-positive organisms, including strains resistant to earlier cephalosporins and penicillin G.

Peptide Deformylase Inhibitors, Potential for a New Class of Broad Spectrum Antibacterials

Peptide deformylase (PDF) has emerged as one of the most promising new targets for antimicrobial chemotherapy. PDF is an essential bacterial metalloenzyme that deformylates the N-formylmethionine of newly synthesised bacterial polypeptides. Although the enzyme has been known for over 30 years, it has proved difficult to isolate and characterise due to its apparent instability. However, recent progress in understanding the structure and function of PDF has greatly facilitated the drug discovery process. In this article we review the potential of PDF as an antibacterial target and highlight progress in the development of inhibitors of PDF. The field has evolved particularly rapidly through the use of high throughput screening and combinatorial techniques and the diverse series of structures exploited previously in other metalloenzyme programmes. Although many different structural classes of compounds have been reported as inhibitors of PDF, the real challenge has been in obtaining molecules with good a ntibacterial activity. Several PDF inhibitors have now been described that demonstrate potent in vitro antibacterial activity against a range of Gram-negative and Gram-positive drug resistant pathogens. Results in stringent animal models of infection suggest that for some of these molecules good in vitro activity translates into good in vivo efficacy. One of the most encouraging compounds reported, BB-83698, has shown activity against Streptococcus pneumoniae in both the neutropenic mouse thigh and lung infection models. These data suggest that PDF inhibitors are a promising new class of antibacterial agents with opportunities to treat respiratory and other bacterial infections. Keywords: peptide deformylase, broad spectrum antibacterial, bacterial polypeptode, bb-83698, pdf inhibitor

Pharmacodynamics of daptomycin in a murine thigh model of Staphylococcus aureus infection.

Daptomycin is a lipopeptide antibiotic with activity against gram-positive bacteria, including Staphylococcus aureus. We defined the pharmacodynamic parameters that determine the activity of daptomycin for S. aureus using in vitro methods and the Craig (W. A. Craig, J. Redington, and S. C. Ebert, J. Antimicrob. Chemother. 27[Suppl. C]:29--40, 1991) neutropenic mouse thigh infection model. In Mueller-Hinton broth, the MICs for three S. aureus isolates were 0.1 to 0.2 microg/ml. In mouse serum, the MICs were 1.0 microg/ml. The protein binding of daptomycin was 90 to 92.5% in mouse serum. Single-dose intraperitoneal (i.p.) pharmacokinetic studies with infected mice showed a linear relationship between dose versus the maximum concentration of drug in serum and dose versus the area under the concentration-time curve (AUC). The serum half-life of daptomycin in infected mice was approximately 1.8 h. In single-dose, dose-ranging studies using mice, daptomycin showed a dose-response effect described by an inhibitory sigmoid E(max) (maximum effect) curve (r = 0.974; P << 0.001). The density of S. aureus in untreated controls was 8.26 log(10) CFU/g, and the E(max) was 3.97 log(10) CFU/g. The 50% effective dose (ED(50)) was 3.7 mg/kg of body weight i.p. and the stasis dose was 7.1 mg/kg. Dose fractionation studies at schedules of Q6h, Q12h, and Q24h, for total 24-h ED(30), ED(60), and ED(80) doses of 2.5, 5.6, and 15 mg/kg i.p., showed no difference in effect at each total 24-h dose level by schedule, indicating that the AUC/MIC ratio is the dynamically linked variable.

Validation of a Noninvasive, Real-Time Imaging Technology Using Bioluminescent Escherichia coli in the Neutropenic Mouse Thigh Model of Infection

A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. The lux gene cluster of Photorhabdus luminescens was introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.

Combined effects of vancomycin and imipenem against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo.

Infectious disease caused by methicillin-resistant Staphylococcus aureus (MRSA) often develops in compromised hosts in whom a single administration of vancomycin is usually not effective. We assessed the combined antimicrobial effects of vancomycin and imipenem against MRSA growth in vitro as well as in vivo using a neutropenic mouse thigh infection model. Synergic and additive effects of the two drugs were observed for 34 and two, respectively, of the 36 clinical isolates of MRSA, as determined by the chequerboard method. For MRSA strain N, postantibiotic effect (PAE) values obtained in vitro were 1.9-2.6 h for vancomycin and 2.6-3.5 h for imipenem, while higher values of 2.7-4.4 h were obtained for the combination of vancomycin and imipenem. In vivo, a single administration of vancomycin at 1 or 2 mg/kg or of imipenem/cilastatin at 5 mg/kg produced growth curves similar to those in controls, with a suppressive time of 0 h. Imipenem/cilastatin at 10 mg/kg demonstrated a suppressive time of 3.1 h. Combinations of vancomycin 1 mg/kg plus imipenem/cilastatin 5 mg/kg, vancomycin 1 mg/kg plus imipenem/cilastatin 10 mg/k and vancomycin 2 mg/kg plus imipenem/cilastatin 10 mg/kg demonstrated suppressive times of 2.9, 3.9 and 5.2 h, respectively, indicating that combined administration was effective. Simultaneous administration of the two drugs was more effective than sequential administration. Thus, combined administration of vancomycin and imipenem/cilastatin proved effective for MRSA in vivo. The combined effects seemed to be synergic, since a single administration of either drug did not show anti-MRSA effects at the doses used in the combined regimen.