Neutropenic Mouse Thigh Infection Model Research Articles

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.

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 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.

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.

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.

The post-antibiotic effect of antimicrobial combinations in a neutropenic murine thigh infection model.

The post-antibiotic effect (PAE) may allow for more widely spaced dosing of antibiotics than is currently employed without loss of efficacy. Antimicrobial combinations are widely used in clinical medicine. However, dosing schedules are usually based on pharmacological profiles of the drugs used alone. Previously we have demonstrated significant prolongation of the PAE induced by antimicrobial combinations in vitro as compared to PAEs induced by the agents alone. We examined this issue further in vivo in a neutropenic mouse thigh infection model, by exposing Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae to several antimicrobials, either singly or in combination. The PAE in vivo was defined as the difference in time needed for the organisms in the treated animals to grow 1 log10 as compared with controls after serum drug concentrations had fallen below the MIC. Drug concentrations exceeded the MIC for 1.2-3.2 h, but bactericidal activity occurred mainly during the first hour. When the agents were used singly a negative PAE was produced by ceftazidime against P. aeruginosa, a PAE of approximately 0 h by imipenem against E. coli and K. pneumoniae, a PAE of 2-4 h by cefazolin against S. aureus, gentamicin against E. coli and K. pneumoniae, and imipenem and tobramycin against P. aeruginosa, and a PAE of 6-7 h by gentamicin against S. aureus and rifampicin against P. aeruginosa. The beta-lactam/aminoglycoside combinations when used against S. aureus and P. aeruginosa prolonged the PAE by 1.0-3.3 h, compared with the longer of the individual drug PAEs, but no prolongation was observed against E. coli and K. pneumoniae. Ceftazidime reduced the PAE when used with tobramycin against P. aeruginosa. The long PAE of rifampicin against P. aeruginosa was 'carried over' to the combination, thus prolonging the growth suppression achieved by imipenem and tobramycin alone or in combination by 5.5-8.0 h. This effect on the PAE was additive only, and synergy was not observed. In conclusion, a potentially significant prolongation of the PAE by combination of drugs was observed in vivo, but only if both (or all) agents induced a PAE when used alone. The impact of this observation needs to be examined further in studies involving multiple and different dosing regimens in an infection model.

Integration of pharmacokinetics and pharmacodynamics of imipenem in a human-adapted mouse model

The relationship between the pharmacokinetics and bactericidal activity of imipenem against Pseudomonas aeruginosa and Escherichia coli was investigated in a neutropenic mouse thigh infection model. To circumvent the problem of short elimination time in small animals, imipenem was administered in fractionized, decreasing doses such that the pharmacokinetic profiles as observed in humans after intravenous and intramuscular injections were approximated in mice. The human-simulated kinetic profile corresponding to an intramuscular injection of 500 mg at 12-h intervals proved to be as effective as the human-simulated profile of the same dose injected intravenously every 6 h. In contrast, the human-simulated profile corresponding to only one intravenous injection every 12 h resulted in bacterial breakthrough growth between 8 and 12 h after the onset of treatment. The results of our investigations confirm the hypothesis that the bactericidal effect of imipenem against P. aeruginosa and E. coli in vivo depends mainly on the time during which drug levels remain above the MIC rather than on the plasma peak/MIC ratio.

Pharmacokinetic and experimental data on beta-lactam antibiotics in the treatment of patients.

The in vitro and animal model studies on optimal dosage of the newer beta-lactams are summarized and put into historical perspective. They provide a rationale for dosage schedules that continuously maintain inhibitory serum and tissue concentrations throughout the dosage interval. In vitro studies on the post-antibiotic effect (PAE) with beta-lactams revealed only short time periods of post-antibiotic growth suppression with gram-positive cocci and no post-antibiotic effect with gram-negative bacilli. A similar lack of persistent growth suppression was observed with beta-lactams in a neutropenic mouse thigh infection model for both gram-positive and gram-negative bacteria. In the same animal model, dosing regimens of beta-lactams which continuously provided serum concentrations above the MIC were more efficacious than those that did not. The newer third-generation cephalosporins have prolonged half-lives and can maintain serum levels above the MIC for most pathogens, even when dosed at widely spaced intervals.