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Candida auris is an emerging fungal pathogen that exhibits resistance to multiple drugs, including the most commonly prescribed antifungal, fluconazole. Here, we use a combinatorial screening approach to identify a bis-benzodioxolylindolinone (azoffluxin) that synergizes with fluconazole against C. auris. Azoffluxin enhances fluconazole activity through the inhibition of efflux pump Cdr1, thus increasing intracellular fluconazole levels. This activity is conserved across most C. auris clades, with the exception of clade III. Azoffluxin also inhibits efflux in highly azole-resistant strains of Candida albicans, another human fungal pathogen, increasing their susceptibility to fluconazole. Furthermore, azoffluxin enhances fluconazole activity in mice infected with C. auris, reducing fungal burden. Our findings suggest that pharmacologically targeting Cdr1 in combination with azoles may be an effective strategy to control infection caused by azole-resistant isolates of C. auris.

Omadacycline is an effective therapy for community-acquired bacterial pneumonia (CABP). Given its potent activity against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA), we sought to determine the pharmacodynamic activity and target pharmacokinetic/pharmacodynamic (PK/PD) exposures associated with a therapeutic effect in the neutropenic mouse pneumonia model against 10 MSSA/MRSA strains. The area under the concentration-time curve (AUC)/MIC associated with 1-log kill was noted at 24-h epithelial lining fluid (ELF) and plasma AUC/MIC exposures of ∼2 (ELF range, <0.93 to 19; plasma range, <1.06 to 17) and 2-log kill was noted at 24-h ELF and plasma AUC/MIC exposures of ∼12 (ELF range, 2.5 to 130; plasma range, 3.5 to 151)., (Copyright © 2020 American Society for Microbiology.)

Delafloxacin is a broad-spectrum anionic fluoroquinolone that has completed a phase 3 study for community-acquired bacterial pneumonia. We investigated the pharmacodynamic target for delafloxacin against 12 Klebsiella pneumoniae and 5 Pseudomonas aeruginosa strains in the neutropenic murine lung infection model. The median 24-h free-drug area under the curve ( f AUC)/MIC values associated with net stasis and 1-log kill were 28.6 and 64.1 for K. pneumoniae , respectively. The 24-h f AUC/MIC values associated with net stasis and 1-log kill for P. aeruginosa were 5.66 and 14.3, respectively., (Copyright © 2019 American Society for Microbiology.)

Omadacycline is a novel aminomethylcycline antibiotic with potent activity against Staphylococcus aureus , including methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). We investigated the pharmacodynamic activity of omadacycline against 10 MSSA/MRSA strains in a neutropenic murine thigh model. The median 24-h area under the concentration-time curve (AUC)/MIC values associated with net stasis and 1-log kill were 21.9 and 57.7, respectively., (Copyright © 2019 American Society for Microbiology.)

APX001, the prodrug of APX001A, is a first-in-class antifungal agent that has a potent activity against Aspergillus fumigatus The goal of current study was to determine the pharmacodynamic (PD) index and target of APX001 in an immunocompromised murine model of invasive pulmonary aspergillosis against 6  A. fumigatus isolates. Minimum effective concentration (MEC) values ranged from 0.03 to 0.06 mg/liter. Dose fractionation was performed against isolate AF293 using total doses of APX001 ranging from 81 to 768 mg/kg of body weight/day fractionated into every 3-, 6-, and 8-h regimens over a 96-h treatment duration. Efficacy was assessed by A. fumigatus quantitative PCR (qPCR) of conidial equivalents from lung homogenates. Nonlinear regression analysis using the Hill equation demonstrated that the 24-h area under the concentration-time curve (AUC)/MEC ratio was the pharmacokinetic (PK)/PD index that best correlated with efficacy (coefficient of determination [ R 2 ] = 0.79). Treatment studies with the remaining strains utilized regimens of 40 to 1,536 mg/kg of APX001 administered every 3 h for a 96-h duration. Exposure-response relationships for all strains were similar, and the median free drug AUC/MEC PK/PD targets for stasis and 1-log-kill endpoints were 47.6 and 89.4, respectively. The present studies demonstrated in vitro and in vivo APX001A/APX001 potency against A. fumigatus These results have potential relevance for clinical dose selection and evaluation of susceptibility breakpoints., (Copyright © 2019 American Society for Microbiology.)

NOSO-502 is a novel odilorhabdin antibiotic with potent activity against Enterobacteriaceae The goal of these studies was to determine which pharmacokinetic/pharmacodynamic (PK/PD) indices and magnitude best correlated with efficacy in the murine thigh infection model. Six Escherichia coli and 6 Klebsiella pneumoniae isolates were utilized. MICs were determined using CLSI methods and ranged from 1 to 4 mg/liter. A neutropenic murine thigh infection model was utilized for all treatment studies. Single-dose plasma pharmacokinetics were determined in mice after subcutaneous administration of 7.81, 31.25, 125, and 500 mg/kg of body weight. Pharmacokinetic studies exhibited peak concentration ( C max ) values of 1.49 to 84.6 mg/liter, area under the concentration-time curve from 0 h to infinity (AUC 0-∞ ) values of 1.94 to 352 mg · h/liter, and beta elimination half-lives of 0.41 to 1.1 h. Dose fractionation studies were performed using total drug doses of 7.81 mg/kg to 2,000 mg/kg fractionated into regimens of every 3 h (q3h), q6h, q12h, or q24h. Nonlinear regression analysis demonstrated that AUC/MIC was the PK/PD parameter that best correlated with efficacy ( R 2 , 0.86). In subsequent studies, we used the neutropenic murine thigh infection model to determine the magnitude of NOSO-502 AUC/MIC needed for the efficacy against a diverse group of Enterobacteriaceae Mice were treated with 4-fold-increasing doses (range, 3.91 to 1,000 mg/kg) of NOSO-502 every 6 h. The mean 24-h free-drug AUC/MIC ( f AUC)/MIC) magnitudes associated with net stasis and 1-log kill endpoint for K. pneumoniae were 4.22 and 17.7, respectively. The mean f AUC/MIC magnitude associated with net stasis endpoint for E. coli was 10.4. NOSO-502 represents a promising novel, first-in-class odilorhabdin antibiotic with in vivo potency against Enterobacteriaceae ., (Copyright © 2018 American Society for Microbiology.)

APX001 is the prodrug of APX001A, which is a first-in-class small molecule with a unique mechanism of action that inhibits the fungal enzyme Gwt1 in the glycosylphosphatidylinositol (GPI) biosynthesis pathway. The goal of the present study was to determine which pharmacokinetic/pharmacodynamic (PK/PD) index and magnitude best correlated with efficacy in the murine disseminated candidiasis model for Candida albicans ( n = 5), C. glabrata ( n = 5), and C. auris ( n = 4). MIC values ranged from 0.002 to 0.03 mg/liter for C. albicans , from 0.008 to 0.06 mg/liter for C. glabrata , and from 0.004 to 0.03 mg/liter for C. auris Plasma APX001A pharmacokinetic measurements were performed in mice after oral administration of 4, 16, 64, and 256 mg/kg of body weight APX001. Single-dose pharmacokinetic studies exhibited maximum plasma concentration ( C max ) values of 0.46 to 15.6 mg/liter, area under the concentration-time curve (AUC) from time zero to infinity (AUC 0-inf ) values of 0.87 to 70.0 mg · h/liter, and half-lives of 1.40 to 2.75 h. A neutropenic murine disseminated candidiasis model was utilized for all treatment studies, and drug dosing was by the oral route. Dose fractionation was performed against C. albicans K1, with total doses ranging from 4 to 1,024 mg/kg/day of APX001 fractionated into regimens of dosing every 3, 6, 8, and 12 h for a 24-h treatment duration. Nonlinear regression analysis was used to determine which PK/PD index best correlated with efficacy on the basis of the reduction in the number of CFU/kidney at 24 h. The 24-h free-drug AUC/MIC ratio ( f AUC 0-24 /MIC) was the PK/PD index that best correlated with efficacy (coefficient of determination [ R 2 ] = 0.88). Treatment studies with the remaining strains utilized regimens of 1 to 256 mg/kg of APX001 administered every 6 h for a 24-h duration with C. albicans and a 96-h study duration with C. glabrata and C. auris The dose required to achieve 50% of the maximum effect (ED 50 ) and stasis f AUC/MIC targets were as follows: for C. albicans , 3.67 ± 3.19 and 20.60 ± 6.50, respectively; for C. glabrata , 0.38 ± 0.21 and 1.31 ± 0.27, respectively; and for C. auris , 7.14 ± 4.54 and 14.67 ± 8.30, respectively. The present studies demonstrated in vitro and in vivo APX001A and APX001 potency, respectively, against C. albicans , C. glabrata , and C. auris. These results have potential relevance for clinical dose selection and evaluation of susceptibility breakpoints. The identification of a lower AUC/MIC ratio target for C. glabrata suggests that species-specific susceptibility breakpoints should be explored., (Copyright © 2018 American Society for Microbiology.)

The neutropenic murine thigh infection model was used to define the pharmacokinetic/pharmacodynamic index linked to efficacy of iclaprim against Staphylococcus aureus ATCC 29213 and Staphylococcus pneumoniae ATCC 10813. The 24-h area under the curve (AUC)/MIC index was most closely linked to efficacy for S. aureus ( R 2 , 0.65), while both the 24-h AUC/MIC and the percentage of time that drug concentrations remain above the MIC (% T >MIC) were strongly associated with effect ( R 2 , 0.86 for both parameters) for S. pneumoniae ., (Copyright © 2018 American Society for Microbiology.)

Eravacycline is a novel fluorocycline antibiotic with potent activity against a broad range of pathogens, including strains with tetracycline and other drug resistance phenotypes. The goal of the studies was to determine which pharmacokinetic/pharmacodynamic (PK/PD) parameter and magnitude best correlated with efficacy in the murine thigh infection model. Six Escherichia coli isolates were utilized for the studies. MICs were determined using CLSI methods and ranged from 0.125 to 0.25 mg/liter. A neutropenic murine thigh infection model was utilized for all treatment studies. Single-dose plasma pharmacokinetics were determined in mice after administration of 2.5, 5, 10, 20, 40, and 80 mg/kg of body weight. Pharmacokinetic studies exhibited maximum plasma concentration ( C max ) values of 0.34 to 2.58 mg/liter, area under the concentration-time curve (AUC) from time zero to infinity (AUC 0-∞ ) values of 2.44 to 57.6 mg · h/liter, and elimination half-lives of 3.9 to 17.6 h. Dose fractionation studies were performed using total drug doses of 6.25 mg/kg to 100 mg/kg fractionated into 6-, 8-, 12-, or 24-h regimens. Nonlinear regression analysis demonstrated that the 24-h free drug AUC/MIC ( f AUC/MIC) was the PK/PD parameter that best correlated with efficacy ( R 2 = 0.80). In subsequent studies, we used the neutropenic murine thigh infection model to determine if the magnitude of the AUC/MIC needed for the efficacy of eravacycline varied among pathogens. Mice were treated with 2-fold increasing doses (range, 3.125 to 50 mg/kg) of eravacycline every 12 h. The mean f AUC/MIC magnitudes associated with the net stasis and the 1-log-kill endpoints were 27.97 ± 8.29 and 32.60 ± 10.85, respectively., (Copyright © 2017 American Society for Microbiology.)

Omadacycline is a novel aminomethylcycline antibiotic in clinical development for community-acquired bacterial pneumonia (CABP). We used a neutropenic murine pneumonia infection model to characterize the in vivo pharmacodynamic activity of omadacycline against Streptococcus pneumoniae Four strains with various phenotypic resistances to other antimicrobials, including tetracyclines, were utilized. Drug concentration measurements were performed in the plasma and epithelial lining fluid (ELF) after administration of 0.5, 2, 8, and 32 mg/kg. Pharmacokinetic parameters were calculated using a noncompartmental model and were linear over the dose range. Penetration into ELF ranged from 72 to 102%. Omadacycline demonstrated net cidal activity in relation to the initial burden against all four strains. The pharmacokinetic/pharmacodynamic index AUC/MIC correlated well with efficacy ( R 2 = 0.74). The plasma 24-h static dose AUC/MIC values were 16 to 20 (24-h ELF AUC/MIC of 14 to 18). A 1-log 10 kill was achieved at 24-h plasma AUC/MIC values of 6.1 to 180 (24-h ELF AUC/MIC values 6.0 to 200). A 2-log 10 kill was achieved at 24-h plasma AUC/MIC values of 19 to 56 (24-h ELF AUC/MIC of 17 to 47). The targets identified in this study in combination with in vitro potency and favorable human pharmacokinetics make omadacycline an attractive candidate for further development and study in patients with CABP., (Copyright © 2017 American Society for Microbiology.)

New drug targets are of great interest for the treatment of fungal biofilms, which are routinely resistant to antifungal therapies. We theorized that the interaction of Candida albicans with matricellular host proteins would provide a novel target. Here, we show that an inhibitory protein (FUD) targeting Candida-fibronectin interactions disrupts biofilm formation in vitro and in vivo in a rat venous catheter model. The peptide appears to act by blocking the surface adhesion of Candida, halting biofilm formation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Candida albicans is the most prevalent cause of hospital-acquired fungal infections and forms biofilms on indwelling medical devices that are notoriously difficult to treat or remove. We recently demonstrated that the colonization of C. albicans on the surfaces of catheter tube segments can be reduced in vitro by coating them with polyelectrolyte multilayers (PEMs) that release a potent antifungal β-peptide. Here, we report on the impact of polymer structure and film composition on both the inherent and β-peptide-mediated ability of PEM-coated catheters to prevent or reduce the formation of C. albicans biofilms in vitro and in vivo using a rat model of central venous catheter infection. Coatings fabricated using polysaccharide-based components [hyaluronic acid (HA) and chitosan (CH)] and coatings fabricated using polypeptide-based components [poly-l-lysine (PLL) and poly-l-glutamic acid (PGA)] both served as reservoirs for the loading and sustained release of β-peptide, but differed substantially in loading and release profiles and in their inherent antifungal properties (e.g., the ability to prevent colonization and biofilm growth in the absence of β-peptide). In particular, CH/HA films exhibited inherent antifungal and antibiofilm behaviors in vitro and in vivo, a result we attribute to the incorporation of CH, a weak polycation demonstrated to exhibit antimicrobial properties in other contexts. The antifungal properties of both types of films were improved substantially when β-peptide was incorporated. Catheter segments coated with β-peptide-loaded CH/HA and PLL/PGA films were both strongly antifungal against planktonic C. albicans and the formation of surface-associated biofilms in vitro and in vivo. Our results demonstrate that PEM coatings provide a useful platform for the design of new antifungal materials, and suggest opportunities to design multifunctional or dual-action platforms to prevent or reduce the severity of fungal infections in applied biomedical contexts or other areas in which fungal biofilms are endemic.

Among the most fascinating virulence attributes of Candida is the ability to transition to a biofilm lifestyle. As a biofilm, Candida cells adhere to a surface, such as a vascular catheter, and become encased in an extracellular matrix. During this mode of growth, Candida resists the normal immune response, often causing devastating disease. Based on scanning electron microscopy images, we hypothesized that host cells and proteins become incorporated into clinical biofilms. As a means to gain an understanding of these host-biofilm interactions, we explored biofilm-associated host components by using microscopy and liquid chromatography-mass spectrometry. Here we characterize the host proteins associated with several in vivo rat Candida albicans biofilms, including those from vascular catheter, denture, and urinary catheter models as well as uninfected devices. A conserved group of 14 host proteins were found to be more abundant during infection at each of the niches. The host proteins were leukocyte and erythrocyte associated and included proteins involved in inflammation, such as C-reactive protein, myeloperoxidase, and alarmin S100-A9. A group of 59 proteins were associated with both infected and uninfected devices, and these included matricellular and inflammatory proteins. In addition, site-specific proteins were identified, such as amylase in association with the denture device. Cellular analysis revealed neutrophils as the predominant leukocytes associating with biofilms. These experiments demonstrate that host cells and proteins are key components of in vivo Candida biofilms, likely with one subset associating with the device and another being recruited by the proliferating biofilm., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Antibiotics with novel mechanisms of action are urgently needed. Processes of cellular division are attractive targets for new drug development. FtsZ, an integral protein involved in cell cytokinesis, is a representative example. In the present study, the pharmacodynamic (PD) activity of an FtsZ inhibitor, TXA-709, and its active metabolite, TXA-707, was evaluated in the neutropenic murine thigh infection model against 5 Staphylococcus aureus isolates, including both methicillin-susceptible and methicillin-resistant isolates. The pharmacokinetics (PK) of the TXA-707 active metabolite were examined after oral administration of the TXA-709 prodrug at 10, 40, and 160 mg/kg of body weight. The half-life ranged from 3.2 to 4.4 h, and the area under the concentration-time curve (AUC) and maximum concentration of drug in serum (Cmax) were relatively linear over the doses studied. All organisms exhibited an MIC of 1 mg/liter. Dose fractionation demonstrated the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) to be the PD index most closely linked to efficacy (R(2) = 0.72). Dose-dependent activity was demonstrated against all 5 isolates, and the methicillin-resistance phenotype did not alter the pharmacokinetic/pharmacodynamic (PK/PD) targets. Net stasis was achieved against all isolates and a 1-log10 kill level against 4 isolates. PD targets included total drug 24-h AUC/MIC values of 122 for net stasis and 243 for 1-log10 killing. TXA-709 and TXA-707 are a promising novel antibacterial class and compound for S. aureus infections. These results should prove useful for design of clinical dosing regimen trials., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Drugs that act more promiscuously provide fewer routes for the emergence of resistant mutants. This benefit, however, often comes at the cost of serious off-target and dose-limiting toxicities. The classic example is the antifungal amphotericin B (AmB), which has evaded resistance for more than half a century. We report markedly less toxic amphotericins that nevertheless evade resistance. They are scalably accessed in just three steps from the natural product, and they bind their target (the fungal sterol ergosterol) with far greater selectivity than AmB. Hence, they are less toxic and far more effective in a mouse model of systemic candidiasis. To our surprise, exhaustive efforts to select for mutants resistant to these more selective compounds revealed that they are just as impervious to resistance as AmB. Thus, highly selective cytocidal action and the evasion of resistance are not mutually exclusive, suggesting practical routes to the discovery of less toxic, resistance-evasive therapies.

Echinocandins inhibit the synthesis of β-1,3-D-glucan in Candida and are the first-line therapy in numerous clinical settings. Their use is limited by poor oral bioavailability, and they are available only as intravenous therapies. Derivatives of enfumafungin are novel orally bioavailable glucan synthase inhibitors. We performed an in vivo pharmacodynamic (PD) evaluation with a novel enfumafungin derivative, SCY-078 (formerly MK-3118), in a well-established neutropenic murine model of invasive candidiasis against C. albicans, C. glabrata, and C. parapsilosis. The SCY-078 MICs varied 8-fold. Oral doses of 3.125 to 200 mg/kg SCY-078 salt in sterile water produced peak levels of 0.04 to 2.66 μg/ml, elimination half-lives of 5.8 to 8.5 h, areas under the concentration-time curve from 0 to 24 h (AUC0-24 h) of 0.61 to 41.10 μg·h/ml, and AUC from 0 to infinity (AUC0-∞) values of 0.68 to 40.31 μg·h/ml. The pharmacokinetics (PK) were approximately linear over the dose range studied. Maximum response (Emax) and PK/PD target identification studies were performed with 4 C. albicans, 4 C. glabrata, and 3 C. parapsilosis isolates. The PD index AUC/MIC was explored by using total (tAUC) and free (fAUC) drug concentrations. The maximum responses were 4.0, 4.0, and 4.3 log10 CFU/kidney reductions for C. albicans, C. glabrata, and C. parapsilosis, respectively. The AUC/MIC was a robust predictor of efficacy (R2, 0.53 to 0.91). The 24-h PD targets were a static dose of 63.5 mg/kg, a tAUC/MIC of 500, and an fAUC/MIC of 1.0 for C. albicans; a static dose of 58.4 mg/kg, a tAUC/MIC of 315, and an fAUC/MIC of 0.63 for C. glabrata; and a static dose of 84.4 mg/kg, a tAUC/MIC of 198, and an fAUC/MIC of 0.40 for C. parapsilosis. The mean fAUC/MIC values associated with a 1-log kill endpoint against these species were 1.42, 1.26, and 0.91 for C. albicans, C. glabrata, and C. parapsilosis, respectively. The static and 1-log kill endpoints were measured relative to the burden at the start of therapy. The static and 1-log kill doses, as well as the total and free drug AUC/MIC PD targets, were not statistically different between species but were numerically lower than those observed for echinocandins. SCY-078 is a promising novel oral glucan synthase inhibitor against Candida species, and further investigation is warranted., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

NAI-107 is a novel lantibiotic compound with potent in vitro activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The purpose of this study was to examine the activity of NAI-107 against S. aureus strains, including MRSA, in the neutropenic murine thigh infection model. Serum pharmacokinetics were determined and time-kill studies were performed following administration of single subcutaneous doses of 5, 20, and 80 mg/kg body weight. The dose fractionation included total doses ranging from 1.56 to 400 mg/kg/72 h, divided into 1, 2, 3, or 6 doses. Studies of treatment effects against 9 S. aureus strains (4 methicillin-susceptible Staphylococcus aureus [MSSA] and 5 MRSA) using a 12-h dosing interval and total dose range of 1.56 to 400 mg/kg/72 h were also performed. A maximum effect (Emax) model was used to determine the pharmacokinetic/pharmacodynamic (PK/PD) index that best described the dose-response data and to estimate the doses required to achieve a net bacteriostatic dose (SD) and a 1-log reduction in CFU/thigh. The pharmacokinetic studies demonstrated an area under the concentration-time curve (AUC) range of 26.8 to 276 mg·h/liter and half-lives of 4.2 to 8.2 h. MICs ranged from 0.125 to 0.5 μg/ml. The 2 highest single doses produced more than a 2-log kill and prolonged postantibiotic effects (PAEs) ranging from 36 to >72 h. The dose fractionation-response curves were similar, and the AUC/MIC ratio was the most predictive PD index (AUC/MIC, coefficient of determination [R2]=0.89; maximum concentration of drug in serum [Cmax]/MIC, R2=0.79; time [T]>MIC, R2=0.63). A ≥2-log kill was observed against all 9 S. aureus strains. The total drug 24-h AUC/MIC values associated with stasis and a 1-log kill for the 9 S. aureus strains were 371±130 and 510±227, respectively. NAI-107 demonstrated concentration-dependent killing and prolonged PAEs. The AUC/MIC ratio was the predictive PD index. Extensive killing was observed for S. aureus organisms, independent of the MRSA status. The AUC/MIC target should be useful for the design of clinical dosing regimens., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Indwelling urinary catheters are commonly used in the management of hospitalized patients. Candida can adhere to the device surface and propagate as a biofilm. These Candida biofilm communities differ from free-floating Candida, exhibiting high tolerance to antifungal therapy. The significance of catheter-associated candiduria is often unclear, and treatment may be problematic considering the biofilm drug-resistant phenotype. Here we describe a rodent model for the study of urinary catheter-associated Candida albicans biofilm infection that mimics this common process in patients. In the setting of a functioning, indwelling urinary catheter in a rat, Candida proliferated as a biofilm on the device surface. Characteristic biofilm architecture was observed, including adherent, filamentous cells embedded in an extracellular matrix. Similar to what occurs in human patients, animals with this infection developed candiduria and pyuria. Infection progressed to cystitis, and a biofilmlike covering was observed over the bladder surface. Furthermore, large numbers of C. albicans cells were dispersed into the urine from either the catheter or bladder wall biofilm over the infection period. We successfully utilized the model to test the efficacy of antifungals, analyze transcriptional patterns, and examine the phenotype of a genetic mutant. The model should be useful for future investigations involving the pathogenesis, diagnosis, therapy, prevention, and drug resistance of Candida biofilms in the urinary tract., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Invasive pulmonary aspergillosis (IPA) continues to rise in concert with increasing numbers of immune suppression techniques to treat other medical conditions and transplantation. Despite these advances, morbidity and mortality rates remain unacceptably high. One strategy used to optimize outcomes is antifungal pharmacodynamic (PD) examination. We explored the pharmacodynamics of a new triazole in development, isavuconazole, in a murine neutropenic IPA model. Ten A. fumigatus isolates were used, including four wild-type isolates and six cyp51 mutants. The MIC range was 0.125 to 8 mg/liter. Following infection, groups of mice were treated orally with the prodrug (BAL8557) at 40 to 640 mg/kg/12 h for 7 days. Efficacy was determined by quantitative PCR of lung homogenates. At the start of therapy, mice had 4.97 log10 conidial equivalents (CE)/ml of lung homogenate, and this increased to 6.82 log10 CE/ml of lung homogenate in untreated animals. The infection model was uniformly lethal in untreated control mice. The PD target endpoints examined included the static-dose AUC/MIC ratio and the 1-log10 killing AUC/MIC ratio. A stasis endpoint was achieved for all isolates with an MIC of ≤1 mg/liter and 1-log10 killing in all isolates with an MIC of ≤0.5 mg/liter, regardless of the presence or absence of the cyp51 mutation. The static-dose range was 65 to 617 mg/kg/12 h. The corresponding median free-drug AUC/MIC ratio was near 5. The 1-log10 killing dose range was 147 to 455 mg/kg/12 h, and the corresponding median free-drug AUC/MIC ratio was 11.1. These values are similar to those previously reported for other triazoles.

Previous studies examining combination therapy for invasive pulmonary aspergillosis (IPA) have revealed conflicting results, including antagonism, indifference, and enhanced effects. The most commonly employed combination for this infection includes a mold-active triazole and echinocandin. Few studies have evaluated combination therapy from a pharmacodynamic (PD) perspective, and even fewer have examined combination therapy against both wild-type and azole-resistant Cyp51 mutant isolates. The current studies aim to fill this gap in knowledge. Four Aspergillus fumigatus isolates were utilized, including a wild-type strain, an Fks1 mutant (posaconazole susceptible and caspofungin resistant), and two Cyp51 mutants (posaconazole resistant). A neutropenic murine model of IPA was used for the treatment studies. The dosing design included monotherapy with posaconazole, monotherapy with caspofungin, and combination therapy with both. Efficacy was determined using quantitative PCR, and results were normalized to known quantities of conidia (conidial equivalents [CE]). The static dose, 1-log kill dose, and associated PD target area under the curve (AUC)/MIC ratio were determined for monotherapy and combination therapy. Monotherapy experiments revealed potent activity for posaconazole, with reductions of 3 to 4 log10 Aspergillus CE/ml with the two "low"-MIC isolates. Posaconazole alone was less effective for the two isolates with higher MICs. Caspofungin monotherapy did not produce a significant decrease in fungal burden for any strain. Combination therapy with the two antifungals did not enhance efficacy for the two posaconazole-susceptible isolates. However, the drug combination produced synergistic activity against both posaconazole-resistant isolates. Specifically, the combination resulted in a 1- to 2-log10 decline in burden that would not have been predicted based on the monotherapy results for each drug. This corresponded to a reduction in the free-drug posaconazole AUC/MIC ratio needed for stasis of up to 17-fold. The data suggest that combination therapy using a triazole and an echinocandin may be a beneficial treatment strategy for triazole-resistant isolates.

Pharmacodynamic (PD) studies with triazoles in the neutropenic murine disseminated candidiasis model have been performed extensively for Candida albicans. They have consistently shown that the pharmacodynamic index most closely correlated with efficacy is the ratio of the 24-h area under the concentration-time curve (AUC) to the MIC, and a target 24-h free-drug AUC/MIC ratio near 25 is associated with 50% of maximal microbiologic efficacy. We utilized this model to investigate the pharmacodynamics of isavuconazole. Isavuconazole pharmacokinetics were linear over the dose range studied. Oral-gastric doses of 640, 160, 40, and 10 mg of prodrug/kg of body weight produced peak levels of 0.51 to 25.4 mg/liter, an elimination half-life of 1 to 5 h, and an AUC from 0 h to infinity (AUC0-∞) of 0.9 to 287 mg · h/liter. The AUC/MIC ratio was the pharmacodynamic index that correlated best with efficacy (R(2), 0.84). Pharmacodynamic target studies were performed using 4 C. albicans isolates with both a 24-h and a 96-h treatment duration. The strains were chosen to include previously characterized fluconazole-resistant strains. The mean 50% effective doses (ED50) (expressed in mg/kg of body weight/12 h) and associated 24-h free-drug AUC/MIC ratios were 89.3 ± 46.7 and 67.7 ± 35 for the 24-h treatment and 59.6 ± 22 and 33.3 ± 25.5 for the 96-h treatment. These differences were not statistically significant. Pharmacodynamic targets for two non-albicans Candida species were also explored. The mean ED50 (expressed in mg/kg/12 h) and associated 24-h free-drug AUC/MIC ratios were 31.2 and 6.2 for Candida tropicalis (n = 1) and 50.5 and 1.6 for Candida glabrata (n = 2). These PD targets were significantly different from C. albicans targets (P, 0.04). Isavuconazole PD targets for C. albicans are similar to those observed in this model with other triazoles. However, the PD targets for non-albicans Candida species were more than 10-fold lower than those for C. albicans (P, 0.04). This difference is similar to the species-specific PD relationships for the echinocandins. The lower PD targets for these species in this model will be important to consider in the analysis of clinical trial data and during the development of susceptibility breakpoints.

A set of 37 doxycycline neoglycosides were prepared, mediated via a C-9 alkoxyamino-glycyl-based spacer reminiscent of that of tigecycline. Subsequent in vitro antibacterial assays against representative drug-resistant Gram negative and Gram positive strains revealed a sugar-dependent activity profile and one doxycycline neoglycoside, the 2'-amino-α-D-glucoside conjugate, to rival that of the parent pharmacophore. In contrast, the representative tetracycline-susceptible strain E. coli 25922 was found to be relatively responsive to a range of doxycycline neoglycosides. This study also extends the use of aminosugars in the context of neoglycosylation via a simple two-step strategy anticipated to be broadly applicable for neoglycorandomization.

In vivo biofilms grown on medical devices are necessary to understand the interactions of the fungal biofilm and the host environment in which it is most commonly found. This protocol describes a way to grow Candida albicans biofilms on the interior lumen of central venous catheters surgically implanted into rats, which mimics quite well the clinical cases of biofilms found on human central venous catheters. These infected catheters can then be studied via a multitude of different experiments, including cell counting by plating, imaging the catheters under light or electron microscopy, or comparing the relative content of in vivo biofilms to in vitro biofilms and planktonic cultures. These biofilms also provide enough high quality RNA for transcriptional profiling.

Invasive pulmonary aspergillosis (IPA) is a devastating disease of immunocompromised patients. Pharmacodynamic (PD) examination of antifungal drug therapy in IPA is one strategy that may improve outcomes. The current study explored the PD target of posaconazole in an immunocompromised murine model of IPA against 10 A. fumigatus isolates, including 4 Cyp51 wild-type isolates and 6 isolates carrying Cyp51 mutations conferring azole resistance. The posaconazole MIC range was 0.25 to 8 mg/liter. Following infection, mice were given 0.156 to 160 mg/kg of body weight of oral posaconazole daily for 7 days. Efficacy was assessed by quantitative PCR (qPCR) of lung homogenate and survival. At the start of therapy, mice had 5.59 ± 0.19 log(10) Aspergillus conidial equivalents (CE)/ml of lung homogenate, which increased to 7.11 ± 0.29 log(10) CE/ml of lung homogenate in untreated animals. The infection was uniformly lethal prior to the study endpoint in control mice. A Hill-type dose response function was used to model the relationship between posaconazole free drug area under the concentration-time curve (AUC)/MIC and qPCR lung burden. The static dose range was 1.09 to 51.9 mg/kg/24 h. The free drug AUC/MIC PD target was 1.09 ± 0.63 for the group of strains. The 1-log kill free drug AUC/MIC was 2.07 ± 1.02. The PD target was not significantly different for the wild-type and mutant organism groups. Mortality mirrored qPCR results, with the greatest improvement in survival noted at the same dosing regimens that produced static or cidal activity. Consideration of human pharmacokinetic data and the current static dose PD target would predict a clinical MIC threshold of 0.25 to 0.5 mg/liter.

Tedizolid phosphate (TR-701) is a novel oxazolidinone prodrug (converted to the active form tedizolid [TR-700]) with potent Staphylococcus aureus activity. The current studies characterized and compared the in vivo pharmacokinetic/pharmacodynamic (PD) characteristics of TR-701/TR-700 and linezolid against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in the neutropenic murine pneumonia model. The pharmacokinetic properties of both drugs were linear over a dose range of 0.625 to 40 mg/kg of body weight. Protein binding was 30% for linezolid and 85% for TR-700. Mice were infected with one of 11 isolates of S. aureus, including MSSA and community- and hospital-acquired MRSA strains. Each drug was administered by oral-gastric gavage every 12 h (q12h). The dosing regimens ranged from 1.25 to 80 mg/kg/12 h for linezolid and 0.625 to 160 mg/kg/12 h for TR-701. At the start of therapy, mice had 6.24 ± 0.40 log(10) CFU/lungs, which increased to 7.92 ± 1.02 log(10) CFU/lungs in untreated animals over a 24-h period. A sigmoid maximum-effect (E(max)) model was used to determine the antimicrobial exposure associated with net stasis (static dose [SD]) and 1-log-unit reduction in organism relative to the burden at the start of therapy. The static dose pharmacodynamic targets for linezolid and TR-700 were nearly identical, at a free drug (non-protein-bound) area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) of 19 and 20, respectively. The 1-log-unit kill endpoints were also similar, at 46.1 for linezolid and 34.6 for TR-700. The exposure targets were also comparable for both MSSA and MRSA isolates. These dosing goals support further clinical trial examination of TR-701 in MSSA and MRSA pneumonia.