Combination Therapy to Kill Mycobacterium tuberculosis in Its Nonreplicating Persister Phenotype.

Mycobacterium tuberculosis (Mtb) exists in various metabolic states, including a nonreplicating persister (NRP) phenotype which may affect response to therapy. We have adopted a model-informed strategy to accelerate discovery of effective Mtb treatment regimens and previously found pretomanid (PMD), moxifloxacin (MXF), and bedaquiline (BDQ) to readily kill logarithmic- and acid-phase Mtb. Here, we studied multiple concentrations of each drug in flask-based, time-kill studies against NRP Mtb in single-, two- and three-drug combinations, including the active M2 metabolite of BDQ. We used nonparametric population algorithms in the Pmetrics package for R to model the data and to simulate the 95% confidence interval of bacterial population decline due to the two-drug combination regimen of PMD + MXF and compared this to observed declines with three-drug regimens. PMD + MXF at concentrations equivalent to average or peak human concentrations effectively eradicated Mtb. Unlike other states for Mtb, we observed no sustained emergence of less susceptible isolates for any regimen. The addition of BDQ as a third drug significantly (P < 0.05) shortened time to total bacterial suppression by 3 days compared to the two-drug regimen, similar to our findings for Mtb in logarithmic or acid growth phases.

Ceftazidime-Avibactam in Combination With Fosfomycin: A Novel Therapeutic Strategy Against Multidrug-Resistant Pseudomonas aeruginosa.

Previously, by targeting penicillin-binding protein 3, Pseudomonas-derived cephalosporinase (PDC), and MurA with ceftazidime-avibactam-fosfomycin, antimicrobial susceptibility was restored among multidrug-resistant (MDR) Pseudomonas aeruginosa. Herein, ceftazidime-avibactam-fosfomycin combination therapy against MDR P. aeruginosa clinical isolate CL232 was further evaluated. Checkerboard susceptibility analysis revealed synergy between ceftazidime-avibactam and fosfomycin. Accordingly, the resistance elements present and expressed in P. aeruginosa were analyzed using whole-genome sequencing and transcriptome profiling. Mutations in genes that are known to contribute to ß-lactam resistance were identified. Moreover, expression of blaPDC, the mexAB-oprM efflux pump, and murA were upregulated. When fosfomycin was administered alone, the frequency of mutations conferring resistance was high; however, coadministration of fosfomycin with ceftazidime-avibactam yielded a lower frequency of resistance mutations. In a murine infection model using a high bacterial burden, ceftazidime-avibactam-fosfomycin significantly reduced the P. aeruginosa colony-forming units (CFUs), by approximately 2 and 5 logs, compared with stasis and in the vehicle-treated control, respectively. Administration of ceftazidime-avibactam and fosfomycin separately significantly increased CFUs, by approximately 3 logs and 1 log, respectively, compared with the number at stasis, and only reduced CFUs by approximately 1 log and 2 logs, respectively, compared with the number in the vehicle-treated control. Thus, the combination of ceftazidime-avibactam-fosfomycin was superior to either drug alone. By employing a "mechanism-based approach" to combination chemotherapy, we show that ceftazidime-avibactam-fosfomycin has the potential to offer infected patients with high bacterial burdens a therapeutic hope against infection with MDR P. aeruginosa that lack metallo-ß-lactamases.

Evaluation of Daptomycin Exposure and Efficacy and Safety Endpoints To Support Risk-versus-Benefit Considerations.

The choice of an antimicrobial agent must balance optimization of efficacy endpoints with the minimization of safety events. The risk versus benefit of daptomycin for patients with Staphylococcus aureus bacteremia with or without infective endocarditis receiving daptomycin at 6, 8, and 10 mg/kg of body weight/day was assessed. The relationships between the area under the concentration-time curve over 24 h (AUC)/MIC ratio and both clinical response and time to decreased susceptibility were evaluated using data from patients with such infections who received daptomycin at 6 mg/kg/day. Using these relationships, plus the previously identified relationship between the minimum concentration and an elevation in the creatine phosphokinase (CPK) concentration (CPK elevation) (S. M. Bhavnani, C. M. Rubino, P. G. Ambrose, and G. L. Drusano, Clin Infect Dis 50:1568-1574, 2010) and Monte Carlo simulation, the probability of each outcome by MIC for daptomycin at 6, 8, and 10 mg/kg/day was calculated. The function for exposure-response relationships for clinical response (P = 0.06) and time to decreased susceptibility (P = 0.01) resembled U and inverted U shapes, respectively. Multivariable analyses demonstrated AUC/MIC ratio, creatinine clearance, albumin concentration, and disease category to be predictors of clinical response. The results of simulations failed to demonstrate large improvements in the probabilities of clinical success among cohorts of simulated patients defined by the above-described predictive factors or the probability of decreased susceptibility at 30 days when the daptomycin dose was increased from 6 to 10 mg/kg/day. The probability of CPK elevation increased from 0.073 to 0.156 over this dose range. These data can be used to inform risk-versus-benefit decisions for daptomycin dose selection in patients with S. aureus bacteremia with or without infective endocarditis. The risk of CPK elevation, which is reversible, should be weighed in the context of the mortality and severe morbidity associated with these types of serious staphylococcal infections.

Preclinical Evaluations To Identify Optimal Linezolid Regimens for Tuberculosis Therapy.

UNLABELLED: Linezolid is an oxazolidinone with potent activity against Mycobacterium tuberculosis. Linezolid toxicity in patients correlates with the dose and duration of therapy. These toxicities are attributable to the inhibition of mitochondrial protein synthesis. Clinically relevant linezolid regimens were simulated in the in vitro hollow-fiber infection model (HFIM) system to identify the linezolid therapies that minimize toxicity, maximize antibacterial activity, and prevent drug resistance. Linezolid inhibited mitochondrial proteins in an exposure-dependent manner, with toxicity being driven by trough concentrations. Once-daily linezolid killed M. tuberculosis in an exposure-dependent manner. Further, 300 mg linezolid given every 12 hours generated more bacterial kill but more toxicity than 600 mg linezolid given once daily. None of the regimens prevented linezolid resistance. These findings show that with linezolid monotherapy, a clear tradeoff exists between antibacterial activity and toxicity. By identifying the pharmacokinetic parameters linked with toxicity and antibacterial activity, these data can provide guidance for clinical trials evaluating linezolid in multidrug antituberculosis regimens. IMPORTANCE: The emergence and spread of multidrug-resistant M. tuberculosis are a major threat to global public health. Linezolid is an oxazolidinone that is licensed for human use and has demonstrated potent activity against multidrug-resistant M. tuberculosis. However, long-term use of linezolid has shown to be toxic in patients, often resulting in thrombocytopenia. We examined therapeutic linezolid regimens in an in vitro model to characterize the exposure-toxicity relationship. The antibacterial activity against M. tuberculosis was also assessed for these regimens, including the amplification or suppression of resistant mutant subpopulations by the chosen regimen. Higher exposures of linezolid resulted in greater antibacterial activity, but with more toxicity and, for some regimens, increased resistant mutant subpopulation amplification, illustrating the trade-off between activity and toxicity. These findings can provide valuable insight for designing optimal dosage regimens for linezolid that are part of the long combination courses used to treat multidrug-resistant M. tuberculosis.

Pharmacokinetic-pharmacodynamic analysis for efficacy of ceftaroline fosamil in patients with acute bacterial skin and skin structure infections.

Ceftaroline is a cephalosporin with broad-spectrum in vitro activity against pathogens commonly associated with acute bacterial skin and skin structure infections (ABSSSI), including methicillin-resistant Staphylococcus aureus. Ceftaroline fosamil, the prodrug of ceftaroline, is approved for the treatment of patients with ABSSSI. Using data from the microbiologically evaluable population from two phase 2 and two phase 3 randomized, multicenter, double-blind studies of patients with ABSSSI, an analysis examining the relationship between drug exposure, as measured by the percentage of time during the dosing interval that free-drug steady-state concentrations remain above the MIC (f%T>MIC), and clinical and microbiological responses was undertaken. The analysis population included 526 patients, of whom 423 had infections associated with S. aureus. Clinical and microbiological success percentages were 94.7 and 94.5%, respectively, among all of the patients and 95.3 and 95.7%, respectively, among those with S. aureus infections. Univariable analysis based on data from all of the patients and those with S. aureus infections demonstrated significant relationships between f%T>MIC and microbiological response (P < 0.001 and P = 0.026, respectively). Multivariable logistic regression analyses demonstrated other patient factors in addition to f%T>MIC to be significant predictors of microbiological response, including age and infection type for all of the patients evaluated and age, infection type, and the presence of diabetes mellitus for patients with S. aureus infections. Results of these analyses confirm that a ceftaroline fosamil dosing regimen of 600 mg every 12 h provides exposures associated with the upper plateau of the pharmacokinetic-pharmacodynamic relationship for efficacy.

Combination treatment with meropenem plus levofloxacin is synergistic against Pseudomonas aeruginosa infection in a murine model of pneumonia.

BACKGROUND: Meropenem plus levofloxacin treatment was shown to be a promising combination in our in vitro hollow fiber infection model. We strove to validate this finding in a murine Pseudomonas pneumonia model. METHODS: A dose-ranging study with meropenem and levofloxacin alone and in combination against Pseudomonas aeruginosa was performed in a granulocytopenic murine pneumonia model. Meropenem and levofloxacin were administered to partially humanize their pharmacokinetic profiles in mouse serum. Total and resistant bacterial populations were estimated after 24 hours of therapy. Pharmacokinetic profiling of both drugs was performed in plasma and epithelial lining fluid, using a population model. RESULTS: Meropenem and levofloxacin penetrations into epithelial lining fluid were 39.3% and 64.3%, respectively. Both monotherapies demonstrated good exposure responses. An innovative combination-therapy analytic approach demonstrated that the combination was statistically significantly synergistic (α = 2.475), as was shown in the hollow fiber infection model. Bacterial resistant to levofloxacin and meropenem was seen in the control arm. Levofloxacin monotherapy selected for resistance to itself. No resistant subpopulations were observed in any combination therapy arm. CONCLUSIONS: The combination of meropenem plus levofloxacin was synergistic, producing good bacterial kill and resistance suppression. Given the track record of safety of each agent, this combination may be worthy of clinical trial.

Evaluation of imipenem for prophylaxis and therapy of Yersinia pestis delivered by aerosol in a mouse model of pneumonic plague.

It has been previously shown that mice subjected to an aerosol exposure to Yersinia pestis and treated with ß-lactam antibiotics after a delay of 42 h died at an accelerated rate compared to controls. It was hypothesized that endotoxin release in antibiotic-treated mice accounted for the accelerated death rate in the mice exposed to aerosol Y. pestis. Imipenem, a ß-lactam antibiotic, binds to penicillin binding protein 2 with the highest affinity and produces rounded cells. The binding of imipenem causes cells to lyse quickly and thereby to release less free endotoxin. Two imipenem regimens producing fractions of time that the concentration of free, unbound drug was above the MIC (fT>MIC) of approximately 25% (6/24 h) and 40% (9.5/24 h) were evaluated. In the postexposure prophylaxis study, the 40% and 25% regimens produced 90% and 40% survivorship, respectively. In the 42-h treatment study, both regimens demonstrated a 40 to 50% survivorship at therapy cessation and some deaths thereafter, resulting in a 30% survivorship. As this was an improvement over the results with other ß-lactams, a comparison of both endotoxin and cytokine levels in mice treated with imipenem and ceftazidime (a ß-lactam previously demonstrated to accelerate death in mice during treatment) was performed and supported the original hypotheses; however, the levels observed in animals treated with ciprofloxacin (included as an unrelated antibiotic that is also bactericidal but should cause little lysis due to a different mode of action) were elevated and significantly (7-fold) higher than those with ceftazidime.

Impact of spores on the comparative efficacies of five antibiotics for treatment of Bacillus anthracis in an in vitro hollow fiber pharmacodynamic model.

Bacillus anthracis, the bacterium that causes anthrax, is an agent of bioterrorism. The most effective antimicrobial therapy for B. anthracis infections is unknown. An in vitro pharmacodynamic model of B. anthracis was used to compare the efficacies of simulated clinically prescribed regimens of moxifloxacin, linezolid, and meropenem with the "gold standards," doxycycline and ciprofloxacin. Treatment outcomes for isogenic spore-forming and non-spore-forming strains of B. anthracis were compared. Against spore-forming B. anthracis, ciprofloxacin, moxifloxacin, linezolid, and meropenem reduced the B. anthracis population by 4 log(10) CFU/ml over 10 days. Doxycycline reduced the population of this B. anthracis strain by 5 log(10) CFU/ml (analysis of variance [ANOVA] P = 0.01 versus other drugs). Against an isogenic non-spore-forming strain, meropenem killed the vegetative B. anthracis the fastest, followed by moxifloxacin and ciprofloxacin and then doxycycline. Linezolid offered the lowest bacterial kill rate. Heat shock studies using the spore-producing B. anthracis strain showed that with moxifloxacin, ciprofloxacin, and meropenem therapies the total population was mostly spores, while the population was primarily vegetative bacteria with linezolid and doxycycline therapies. Spores have a profound impact on the rate and extent of killing of B. anthracis. Against spore-forming B. anthracis, the five antibiotics killed the total (spore and vegetative) bacterial population at similar rates (within 1 log(10) CFU/ml of each other). However, bactericidal antibiotics killed vegetative B. anthracis faster than bacteriostatic drugs. Since only vegetative-phase B. anthracis produces the toxins that may kill the infected host, the rate and mechanism of killing of an antibiotic may determine its overall in vivo efficacy. Further studies are needed to examine this important observation.

Dose range evaluation of Mycograb C28Y variant, a human recombinant antibody fragment to heat shock protein 90, in combination with amphotericin B-desoxycholate for treatment of murine systemic candidiasis.

Systemic candidiasis causes significant mortality in patients despite amphotericin B (AMB) therapy. Mycograb C28Y variant, a human recombinant antibody fragment to heat shock protein 90, is closely related to Mycograb, which showed a survival advantage in combination with AMB in a phase III human trial. The Mycograb C28Y variant could potentially increase the antifungal effect of AMB. In our study, the interaction between AMB-desoxycholate (DAMB) and the Mycograb C28Y variant was characterized in vitro by using a checkerboard method. Quantitative cultures of kidneys, livers, and spleens of neutropenic mice with systemic Candida albicans infections were used to assess the in vivo interaction between 1.4 mg/kg of body weight/day of DAMB and 0.15, 1.5, and 15 mg/kg/day of the Mycograb C28Y variant after 1, 3, and 5 days of therapy. DAMB and Mycograb C28Y variant monotherapies, vehicle, and a no-treatment arm served as controls. Also, single- and multidose pharmacokinetics for the Mycograb C28Y variant were determined. Indifference or synergy between DAMB and the Mycograb C28Y variant was seen in two trials by the checkerboard method. The pharmacokinetics of the Mycograb C28Y variant was best described by a 2-compartment model with a median serum t(1/2)(α) of ~0.198 h and a t(1/2)(ß) of ~1.77 h. In mice, DAMB together with the Mycograb C28Y variant was no more effective than AMB alone (P > 0.05 by analysis of variance). The Mycograb C28Y variant alone had no antifungal activity. We therefore conclude that the Mycograb C28Y variant in combination with DAMB offered no benefit over DAMB monotherapy in a neutropenic murine model of systemic candidiasis.

Use of pharmacodynamic endpoints for the evaluation of levofloxacin for the treatment of acute maxillary sinusitis.

Sinusitis remains 1 of the most common reasons for antimicrobial prescriptions in the United States, with health care costs approaching $4 billion annually. We utilized the serial sinus aspirate sampling (SSAS) technique to obtain daily specimens to evaluate the time course of drug effect in patients with acute maxillary sinusitis. Eighteen patients with a radiologically confirmed acute maxillary sinusitis were enrolled into a study evaluating the relationship between levofloxacin exposure and the time course of antimicrobial effect using SSAS. SSAS was performed daily during therapy for bacteriologic evaluation. Six steady-state levofloxacin concentrations were obtained. Levofloxacin plasma and sinus aspirate concentrations were modeled using Monte Carlo Parametric Expectation Maximization algorithm implemented in S-ADAPT 1.53. Endpoints evaluated included time to resolution of signs and symptoms and time to sinus sterilization. Among the 18 enrolled patients, 15 were clinically evaluable. From these, 1 Streptococcus pneumoniae, 3 Haemophilus influenzae, 1 Moraxella catarrhalis, 1 Corynebacterium spp., and 1 coagulase-negative Staphylococcus organisms were isolated, with the latter 2 organisms being likely contaminants. For the pathogens, levofloxacin MIC values ranged from 0.03 to 2 mg/L. All pathogens were eradicated by the 4th day of therapy. The median and mean time to sinus sterilization (pathogens only) was 1 and 1.4 days, respectively. The median time to resolution of each sign and symptom ranged from 1.5 to 12-19 days, with the 83% of total signs and symptoms resolved by the end of therapy (day 5). The mean plasma area under the concentration-time curve (AUC) (mg x h/L) was 100.1 (n = 14, %CV = 27). Plasma AUC/MIC ratios ranged from 33.9 to 1696 for isolated pathogens. In this pilot SSAS study, levofloxacin rapidly eradicated isolated pathogens from the maxillary sinus.

The pharmacokinetics and pharmacodynamics of micafungin in experimental hematogenous Candida meningoencephalitis: implications for echinocandin therapy in neonates.

BACKGROUND: Hematogenous Candida meningoencephalitis (HCME) is a relatively frequent manifestation of disseminated candidiasis in neonates and is associated with significant mortality and neurodevelopmental abnormalities. The outcome after antifungal therapy is often suboptimal, with few therapeutic options. Limited clinical data suggest that echinocandins may have role to play in the treatment of HCME. METHODS: We studied the pharmacokinetics and pharmacodynamics of micafungin in a rabbit model of neonatal HCME and bridged the results to neonates by use of population pharmacokinetics and Monte Carlo simulation. RESULTS: Micafungin exhibited linear plasma pharmacokinetics in the range of 0.25-16 mg/kg. Micafungin penetrated most compartments of the central nervous system (CNS), but only with doses >2 mg/kg. Micafungin was not reliably found in cerebrospinal fluid. With few exceptions, drug penetration into the various CNS subcompartments was not statistically different between infected and noninfected rabbits. A dose-microbiological response relationship was apparent in the brain, and near-maximal effect was apparent with doses of 8 mg/kg. Monte Carlo simulations revealed that near-maximal antifungal effect was attained at human neonatal doses of 12-15 mg/kg. CONCLUSIONS: These results provide a foundation for clinical trials of micafungin in neonates with HCME and a model for antimicrobial bridging studies from bench to bedside in pediatric patients.

Comparison of 2 antibiotics that inhibit protein synthesis for the treatment of infection with Yersinia pestis delivered by aerosol in a mouse model of pneumonic plague.

INTRODUCTION: Intentional release of Yersinia pestis will likely be propagated by aerosol exposure. We explored the effects of neutropenia on the outcome of doxycycline and gentamicin therapy. METHODS: Female BALB/c mice were exposed to 20 LD(50) of Y. pestis CO92 by aerosol. Treatments were saline (negative control), levofloxacin at 15 mg/kg every 12 h (positive control), doxycycline at 40 mg/kg every 6 h, and gentamicin at 12 mg/kg every 6 h, 24 mg/kg every 12 h, and 48 mg/kg every 24 h in cohorts of normal and neutropenic mice for 5 days. RESULTS: Control mice died. Positive control mice (levofloxacin) had 100% survivorship in both neutropenic and nonneutropenic groups. Doxycycline treatment in the presence of granulocytes yielded 90% survivorship; all neutropenic mice died after the termination of treatment (P<<.001). For gentamicin, survivorship of mice receiving drug every 24, 12, and 6 h was, respectively, 80%, 80%, and 90% for normal mice and 80%, 100%, and 70% for neutropenic mice. No significant differences were seen in the neutropenia versus normal mouse comparison or by schedule. CONCLUSIONS: Doxycycline behaves in vivo as a bacteriostatic drug, requiring an intact immune system for clearance of the infection after aerosol challenge with Y. pestis. Gentamicin is bactericidal, even when given on a daily schedule. Neutropenia did not significantly affect survivorship.

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.

Once-weekly micafungin therapy is as effective as daily therapy for disseminated candidiasis in mice with persistent neutropenia.

The effect of micafungin dose scheduling on the treatment of candidemia is unknown. Neutropenic mice with disseminated Candida glabrata infection were treated with single intraperitoneal micafungin doses of 0 to 100 mg/kg of body weight and sacrificed 7 days later. The maximal decline in kidney fungal burden was 5.8 log(10) CFU/g. A 1-week pharmacokinetic-pharmacodynamic study revealed a micafungin serum half-life of 6.13 h. In mice treated with > or =50 mg/kg, there was maximal fungal decline without regrowth during the 1-week dosing interval. Next, doses associated with 34% (34% effective dose [ED(34)]) and 50% (ED(50)) of maximal kill were administered at one of three dose schedules: a single dose at t = 0, two equal doses at t = 0 and t = 3.5 days, and 7 equal doses daily. Some mice received a single dose of 100 mg/kg. Fungal burden was examined on days 1, 5, and 7. In mice treated with the ED(34), microbial kill with the daily therapy initially lagged behind the intermittent doses but exceeded it by day 7. In mice treated with the ED(50), daily and intermittent doses had equivalent day 7 effects. In mice treated with 100 mg/kg, there was no regrowth. The relative likelihoods that the area under the concentration-time curve/MIC ratio was linked to microbial kill versus peak concentration/MIC ratio or time above the MIC was 10.3 and 10,161.2, respectively. In all the experiments, no paradoxical increase in fungal burden was observed with high micafungin doses. However, only a single Candida isolate was tested. Regimens that simulated micafungin concentration-time profiles in patients treated with a single micafungin dose of 1,400 mg once a week demonstrated maximal fungal decline. Once-weekly micafungin therapy is as efficacious as daily therapy in a murine model of disseminated candidiasis.

Effect of neutropenia and treatment delay on the response to antifungal agents in experimental disseminated candidiasis.

Disseminated candidiasis is associated with a high rate of morbidity and mortality. The presence of neutrophils and the timely administration of antifungal agents are likely to be critical factors for a favorable therapeutic outcome of this syndrome. The effect of neutropenia on the temporal profile of the burden of Candida albicans in untreated mice and those treated with amphotericin B was determined using a pharmacodynamic model of disseminated candidiasis. A mathematical model was developed to describe the rate and extent of the C. albicans killing attributable to neutrophils and to amphotericin B. The consequences of a delay in the administration of amphotericin B, flucytosine, or micafungin were studied by defining dose-response relationships. Neutrophils caused a logarithmic decline in fungal burden in treated and untreated mice. The combination of amphotericin B and neutrophils resulted in a high rate of Candida killing and a sustained anti-C. albicans effect. In neutropenic mice, 5 mg/kg of body weight of amphotericin B was required to prevent progressive logarithmic growth. An increased delay in drug administration resulted in a reduction in the maximum effect to a point at which no drug effect could be observed. Neutrophils and the timely initiation of antifungal agents are critical determinants in the treatment of experimental disseminated candidiasis.

Pharmacokinetic considerations and efficacy of levofloxacin in an inhalational anthrax (postexposure) rhesus monkey model.

Because the treatment of inhalational anthrax cannot be studied in human clinical trials, it is necessary to conduct efficacy studies using a rhesus monkey model. However, the half-life of levofloxacin was approximately three times shorter in rhesus monkeys than in humans. Computer simulations to match plasma concentration profile, area under the concentration-time curve (AUC), and time above MIC for a human oral dose of 500 mg levofloxacin once a day identified a dosing regimen in rhesus monkeys that would most closely match human exposure: 15 mg/kg followed by 4 mg/kg administered 12 h later. Approximately 24 h following inhalational exposure to approximately 49 times the 50% lethal doses of Bacillus anthracis (Ames strain), monkeys were treated daily with vehicle, levofloxacin, or ciprofloxacin for 30 days. Ciprofloxacin was administered at 16 mg/kg twice a day. Following the 30-day treatment, monkeys were observed for 70 days. Nine of 10 control monkeys died within 9 days of exposure. No clinical signs were observed in fluoroquinolone-treated monkeys during the 30 treatment days. One monkey died 8 days after levofloxacin treatment, and two monkeys from the ciprofloxacin group died 27 and 36 days posttreatment, respectively. These deaths were probably related to the germination of residual spores. B. anthracis was positively cultured from several tissues from the three fluoroquinolone-treated monkeys that died. MICs of levofloxacin and ciprofloxacin from these cultures were comparable to those from the inoculating strain. These data demonstrate that a humanized dosing regimen of levofloxacin was effective in preventing morbidity and mortality from inhalational anthrax in rhesus monkeys and did not select for resistance.

Anidulafungin pharmacokinetics and microbial response in neutropenic mice with disseminated candidiasis.

Candidemia is often fatal, especially in patients with persistent neutropenia. New therapies are needed. We performed 24-h pharmacodynamic studies to compare the efficacies of anidulafungin, fluconazole, and amphotericin B in neutropenic mice with disseminated candidiasis caused by one of three strains of Candida glabrata. Anidulafungin produced a maximal fungal kill (E(max)) of 1.4 to 1.9 log(10) CFU/g in kidneys and was not influenced by resistance to either fluconazole or amphotericin B. Fluconazole produced an E(max) of 1.3 log(10) CFU/g in mice infected with fluconazole-susceptible C. glabrata, but the E(max) was 0 for mice infected with a C. glabrata strain that had a fluconazole MIC of >/=32 mg/liter. Amphotericin B achieved an E(max) of 4.2 log(10) CFU/g in mice infected with amphotericin B-susceptible C. glabrata, but the E(max) was 0 for mice infected with a C. glabrata strain with an amphotericin B MIC of 2 mg/liter. In all instances, anidulafungin's maximal microbial kill was superior to that of fluconazole. Next, we performed a 96-h anidulafungin pharmacokinetic-pharmacodynamic study. Anidulafungin exhibited delayed peak concentrations in kidneys compared to those in serum, after which the concentrations declined, with a serum terminal half-life of 21.6 (+/-4.6) h. This was accompanied by a persistent 96-h decrease in the kidney fungal burden after treatment with a single anidulafungin dose of >/=8 mg/kg of body weight. This pharmacokinetic-pharmacodynamic picture of anidulafungin persistence in tissues and the resultant persistent fungal decline should be exploited to improve the efficacy of anidulafungin therapy for candidemia.

Infection site concentrations: their therapeutic importance and the macrolide and macrolide-like class of antibiotics.

Much confusion exists over the pharmacodynamics of macrolides, azalides, and ketolides, as the concentration-time profile for these agents is low relative to the minimum inhibitory concentration (MIC) of the pathogens for which they are used. Studies of respiratory tract infection have highlighted the importance of drug concentrations at the site of infection and have demonstrated a role for white blood cells in the delivery of drug to the infection site. Population mathematical modeling and Monte Carlo simulation have shown that the ability of macrolides, azalides, and ketolides to concentrate at the infection site has a considerable effect on microbial activity. Studies of the pharmacodynamics of these agents in animal models have centered on the mouse thigh model; however, the suitability of this model for investigation of respiratory tract infections for macrolides and macrolide-like drugs is questionable. Comparison of the mouse thigh model with the mouse lung model shows immediate discrepancies, such as a need for higher area under the concentration-time curve (AUC):MIC ratios in the mouse thigh. There are obvious failings in the use of a thigh model, as it does not take into account the accumulation of white blood cells in the epithelial lining fluid and therefore ignores the impact of white blood cell delivery to the site of infection and release of significant amounts of drug during phagocytosis. Ultimately, whereas the mouse pneumonia model is useful in identifying pharmacodynamically linked variables and the magnitude of variable required for a successful microbiologic outcome, extrapolation to human dosing must involve the use of human epithelial lining fluid penetration data.

Surface response modeling to examine the combination of amphotericin B deoxycholate and 5-fluorocytosine for treatment of invasive candidiasis.

The strategy of combining antifungal drugs in a treatment regimen may improve the outcome of invasive candidiasis. Using a well-validated pharmacodynamic murine model of invasive candidiasis, we defined the effect of the combination of amphotericin B deoxycholate (AmB) and 5-fluorocytosine (5FC) by use of the Greco model of drug interaction. The combination was additive, meaning that the experimental effect did not deviate in a statistically significant manner from the null reference model (or additive surface) of the combined effect. From a clinical perspective, the addition of 5FC to a regimen of AmB may enable the near-maximum effect to be reached in circumstances in which the administration of a given dose of AmB alone produces a submaximum effect but an increase in the dose is not possible, because of dose-related toxicity. Our methods provide a way in which some of the complex issues surrounding antifungal combination treatment can be addressed.