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1.  Pharmacodynamic Analysis of a Serine Protease Inhibitor, MK-4519, against Hepatitis C Virus Using a Novel In Vitro Pharmacodynamic System 
The development of new antiviral compounds active against hepatitis C virus (HCV) has surged in recent years. In order for these new compounds to be efficacious in humans, optimal dosage regimens for each compound must be elucidated. We have developed a novel in vitro pharmacokinetic/pharmacodynamic system, the BelloCell system, to identify optimal dosage regimens for anti-HCV compounds. In these experiments, genotype 1b HCV replicon-bearing cells (2209-23 cells) were inoculated onto carrier flakes in BelloCell bottles and treated with MK-4519, a serine protease inhibitor. Our dose-ranging studies illustrated that MK-4519 inhibited replicon replication in a dose-dependent manner, yielding a 50% effective concentration (EC50) of 1.8 nM. Dose-fractionation studies showed that shorter dosing intervals resulted in greater replicon suppression, indicating that the time that the concentration is greater than the EC50 is the pharmacodynamic parameter for MK-4519 linked with inhibition of replicon replication. Mutations associated with resistance to serine protease inhibitors were detected in replicons harvested from all treatment arms. These data suggest that MK-4519 is highly active against genotype 1b HCV, but monotherapy is not sufficient to prevent the amplification of resistant replicons. In summary, our findings show that the BelloCell system is a useful and clinically relevant tool for predicting optimal dosage regimens for anti-HCV compounds.
doi:10.1128/AAC.05383-11
PMCID: PMC3294889  PMID: 22155837
2.  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 log10 CFU/ml over 10 days. Doxycycline reduced the population of this B. anthracis strain by 5 log10 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 log10 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.
doi:10.1128/AAC.01109-10
PMCID: PMC3294912  PMID: 22155821
3.  Evaluation of Once-Daily Vancomycin against Methicillin-Resistant Staphylococcus aureus in a Hollow-Fiber Infection Model 
For methicillin-resistant Staphylococcus aureus (MRSA) infections, data suggest that the clinical response is significantly better if the total vancomycin area under the concentration-time curve (AUC)/MIC ratio is ≥400. While the AUC/MIC ratio is the accepted pharmacokinetic/pharmacodynamic (PK/PD) index for vancomycin, this target has been achieved using multiple daily doses. We are unaware of a systematically designed dose fractionation study to compare the bactericidal activity of once-daily administration to that of traditional twice-daily administration. A dose fractionation study was performed with vancomycin in an in vitro hollow-fiber infection model against an MRSA USA300 strain (MIC of 0.75 μg/ml) using an inoculum of ∼106 CFU/ml. The three vancomycin regimens evaluated for 168 h were 2 g every 24 h (q24h) as a 1-h infusion, 1 g q12h as a 1-h infusion, and 2 g q24h as a continuous infusion. Free steady-state concentrations (assuming 45% binding) for a total daily AUC/MIC ratio of ≥400 were simulated for all regimens. A validated liquid chromatography-tandem mass spectrometry method was used to determine vancomycin concentrations. Although once-daily and twice-daily dosage regimens exhibited total trough concentrations of <15 μg/ml, all regimens achieved similar bactericidal activities between 24 and 168 h and suppressed the amplification of nonsusceptible subpopulations. No colonies were found on agar plates with 3× MIC for any of the treatment arms. Overall, the results suggest that once-daily vancomycin administration is feasible from a PK/PD perspective and merits further inquiry in the clinical arena.
doi:10.1128/AAC.05664-11
PMCID: PMC3264248  PMID: 22083484
4.  Ertapenem Pharmacokinetics and Pharmacodynamics during Continuous Ambulatory Peritoneal Dialysis 
Scant data exist for the pharmacokinetics (PK) of ertapenem in patients on continuous ambulatory peritoneal dialysis (CAPD). The goals of this study were to characterize the PK profile of ertapenem during CAPD, determine the extent of ertapenem penetration into the peritoneal cavity, and quantify the probability of the target attainment (PTA) profile in the serum and peritoneal cavity. A single-dose PK study was conducted in seven patients on CAPD. Population PK modeling and Monte Carlo simulation determined the probability that ertapenem at 500 mg intravenously (i.v.) every 24 h (q24h) would achieve concentrations in excess of the MIC for 40% of the dosing interval (40% T>MIC, where T is time) in the serum and peritoneal cavity. Monte Carlo simulation was also used to calculate the peritoneal cavity/serum mean and median penetration ratios by estimating the area under the concentration-time curve in the peritoneal cavity and serum (AUCPeritoneal and AUCSerum, respectively) from zero to infinity after a single simulated dose. The population mean (± standard deviation [SD]) values for the apparent volume in the central compartment, clearance, and apparent volume in the peritoneal cavity were 2.78 (0.62) liters, 0.24 (0.07) liters/hr, and 5.81 (2.05) liters, respectively. The mean (SD) AUCPeritoneal/AUCSerum ratio was 1.039 (0.861), and the median penetration ratio was 0.801 (interquartile range, 0.486 to 1.317). In both the serum and peritoneal cavity, ertapenem at 500 mg i.v. q24h was very likely (>90%) to achieve the pharmacodynamic target for MICs of ≤2 mg/liter. The simulations suggest that 500 mg of ertapenem i.v. q24h is very likely to achieve the exposure target associated with clinical efficacy in both the serum and peritoneal cavity against the range of MIC values deemed susceptible by the Clinical and Laboratory Standards Institute.
doi:10.1128/AAC.05515-11
PMCID: PMC3264279  PMID: 22083473
5.  Differential Effects of Linezolid and Ciprofloxacin on Toxin Production by Bacillus anthracis in an In Vitro Pharmacodynamic System 
Bacillus anthracis causes anthrax. Ciprofloxacin is a gold standard for the treatment of anthrax. Previously, using the non-toxin-producing ΔSterne strain of B. anthracis, we demonstrated that linezolid was equivalent to ciprofloxacin for reducing the total (vegetative and spore) bacterial population. With ciprofloxacin therapy, the total population consisted of spores. With linezolid therapy, the population consisted primarily of vegetative bacteria. Linezolid is a protein synthesis inhibitor, while ciprofloxacin is not. Since toxins are produced only by vegetative B. anthracis, the effect of linezolid and ciprofloxacin on toxin production is of interest. The effect of simulated clinical regimens of ciprofloxacin and linezolid on the vegetative and spore populations and on toxin production was examined in an in vitro pharmacodynamic model over 15 days by using the toxin-producing Sterne strain of B. anthracis. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. With ciprofloxacin therapy, the total Sterne population consisted of spores. With linezolid therapy, >90% of the population was vegetative B. anthracis. With ciprofloxacin therapy, toxin was first detectable at 3 h and remained detectable for at least 5 h. Toxin was never detected with linezolid therapy. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. However, the B. anthracis population was primarily spores with ciprofloxacin therapy and was primarily vegetative bacteria with linezolid therapy. Toxin production was detected for at least 5 h with ciprofloxacin therapy but was never detected with linezolid treatment. Linezolid may have an advantage over ciprofloxacin for the treatment of B. anthracis infections.
doi:10.1128/AAC.05724-11
PMCID: PMC3256020  PMID: 22064542
6.  Resistance Emergence Mechanism and Mechanism of Resistance Suppression by Tobramycin for Cefepime for Pseudomonas aeruginosa 
The panoply of resistance mechanisms in Pseudomonas aeruginosa makes resistance suppression difficult. Defining optimal regimens is critical. Cefepime is a cephalosporin whose 3′ side chain provides some stability against AmpC β-lactamases. We examined the activity of cefepime against P. aeruginosa wild-type strain PAO1 and its isogenic AmpC stably derepressed mutant in our hollow-fiber infection model. Dose-ranging studies demonstrated complete failure with resistance emergence (both isolates). Inoculum range studies demonstrated ultimate failure for all inocula. Lower inocula failed last (10 days to 2 weeks). Addition of a β-lactamase inhibitor suppressed resistance even with the stably derepressed isolate. Tobramycin combination studies demonstrated resistance suppression in both the wild-type and the stably derepressed isolates. Quantitating the RNA message by quantitative PCR demonstrated that tobramycin decreased the message relative to that in cefepime-alone experiments. Western blotting with AmpC-specific antibody for P. aeruginosa demonstrated decreased expression. We concluded that suppression of β-lactamase expression by tobramycin (a protein synthesis inhibitor) was at least part of the mechanism behind resistance suppression. Monte Carlo simulation demonstrated that a regimen of 2 g of cefepime every 8 h plus 7 mg/kg of body weight of tobramycin daily would provide robust resistance suppression for Pseudomonas isolates with cefepime MIC values up to 8 mg/liter and tobramycin MIC values up to 1 mg/liter. For P. aeruginosa resistance suppression, combination therapy is critical.
doi:10.1128/AAC.05252-11
PMCID: PMC3256024  PMID: 22005996
7.  Pharmacodynamics of β-Lactamase Inhibition by NXL104 in Combination with Ceftaroline: Examining Organisms with Multiple Types of β-Lactamases 
New broad-spectrum β-lactamases such as KPC enzymes and CTX-M-15 enzymes threaten to markedly reduce the utility of our armamentarium of β-lactam agents, even our most potent drugs, such as carbapenems. NXL104 is a broad-spectrum non-β-lactam β-lactamase inhibitor. In this evaluation, we examined organisms carrying defined β-lactamases and identified doses and schedules of NXL104 in combination with the new cephalosporin ceftaroline, which would maintain good bacterial cell kill and suppress resistance emergence for a clinically relevant period of 10 days in our hollow-fiber infection model. We examined three strains of Klebsiella pneumoniae and one isolate of Enterobacter cloacae. K. pneumoniae 27-908M carried KPC-2, SHV-27, and TEM-1 β-lactamases. Its isogenic mutant, K. pneumoniae 4207J, was “cured” of the plasmid expressing the KPC-2 enzyme. K. pneumoniae 24-1318A carried a CTX-M-15 enzyme, and E. cloacae 2-77C expressed a stably derepressed AmpC chromosomal β-lactamase. Dose-ranging experiments for NXL104 administered as a continuous infusion with ceftaroline at 600 mg every 8 h allowed identification of a 24-h area under the concentration-time curve (AUC) for NXL104 that mediated bactericidal activity and resistance suppression. Dose fractionation experiments identified that “time > threshold” was the pharmacodynamic index linked to cell kill and resistance suppression. Given these results, we conclude that NXL104 combined with ceftaroline on an 8-hourly administration schedule would be optimal for circumstances in which highly resistant pathogens are likely to be encountered. This combination dosing regimen should allow for optimal bacterial cell kill (highest likelihood of successful clinical outcome) and the suppression of resistance emergence.
doi:10.1128/AAC.05005-11
PMCID: PMC3256033  PMID: 22024819
8.  Impact of Granulocytes on the Antimicrobial Effect of Tedizolid in a Mouse Thigh Infection Model▿ 
Antimicrobial Agents and Chemotherapy  2011;55(11):5300-5305.
Tedizolid (TR-700, formerly torezolid) is the active component of the new oxazolidinone prodrug tedizolid phosphate (TR-701). We had previously demonstrated that tedizolid possessed potent antistaphylococcal activity superior to that of linezolid in a neutropenic mouse thigh infection model (A. Louie, W. Liu, R. Kulawy, and G. L. Drusano, Antimicrob. Agents Chemother. 55:3453-3460, 2011). In the current investigation, we used a mouse thigh infection model to delineate the effect of an interaction of TR-700 and granulocytes on staphylococcal cell killing. We compared the antistaphylococcal killing effect of doses of TR-701 equivalent to human exposures ranging from 200 to 3,200 mg/day in both granulocytopenic and normal mice. The mice were evaluated at 24, 48, and 72 h after therapy initiation. In granulocytopenic mice, a clear exposure response in which, depending on the time point of evaluation, stasis was achieved at “human-equivalent” doses of slightly below 2,300 mg/day (at 24 h) to slightly below 2,000 mg/day (at 72 h) was observed. In immune-normal animals, stasis was achieved at human-equivalent doses of slightly greater than 100 mg/day or less. The variance in bacterial cell killing results was attributable to the presence of granulocytes (without drug), the direct effect of TR-700 on Staphylococcus aureus, and the effect of the drug on Staphylococcus aureus mediated through granulocytes. The majority of the bacterial cell killing in normal animals was attributable to the effect of TR-700 mediated through granulocytes. Additional studies need to be undertaken to elucidate the mechanism underlying this observation.
doi:10.1128/AAC.00502-11
PMCID: PMC3195040  PMID: 21911576
9.  In Vivo Pharmacodynamics of Torezolid Phosphate (TR-701), a New Oxazolidinone Antibiotic, against Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Strains in a Mouse Thigh Infection Model ▿ 
Torezolid phosphate (TR-701) is the phosphate monoester prodrug of the oxazolidinone TR-700 which demonstrates potent in vitro activity against Gram-positive bacteria, including methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). The pharmacodynamics of TR-701 or TR-700 (TR-701/700) against S. aureus is incompletely defined. Single-dose pharmacokinetic studies were conducted in mice for TR-701/700. Forty-eight-hour dose range and 24-hour dose fractionation studies were conducted in a neutropenic mouse thigh model of S. aureus infection using MRSA ATCC 33591 to identify the dose and schedule of administration of TR-701/700 that was linked with optimized antimicrobial effect. Additional dose range studies compared the efficacies of TR-701/700 and linezolid for one MSSA strain and one community-associated MRSA strain. In dose range studies, TR-701/700 was equally bactericidal against MSSA and MRSA. Mean doses of 37.6 and 66.9 mg/kg of body weight/day of TR-701/700 resulted in stasis and 1 log CFU/g decreases in bacterial densities, respectively, at 24 h, and mean doses of 35.3, 46.6, and 71.1 mg/kg/day resulted in stasis and 1 and 2 log CFU/g reductions, respectively, at 48 h. Linezolid administered at doses as high as 150 mg/kg/day did not achieve stasis at either time point. Dose fractionation studies demonstrated that the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) was the pharmacodynamic index for TR-701/700 that was linked with efficacy. TR-701/700 was highly active against MSSA and MRSA, in vivo, and was substantially more efficacious than linezolid, although linezolid's top exposure has half the human exposure. Dose fractionation studies showed that AUC/MIC was the pharmacodynamic index linked with efficacy, indicating that once-daily dosing in humans is feasible.
doi:10.1128/AAC.01565-10
PMCID: PMC3122459  PMID: 21502615
10.  Comparative Efficacies of Candidate Antibiotics against Yersinia pestis in an In Vitro Pharmacodynamic Model▿ 
Yersinia pestis, the bacterium that causes plague, is a potential agent of bioterrorism. Streptomycin is the “gold standard” for the treatment of plague infections in humans, but the drug is not available in many countries, and resistance to this antibiotic occurs naturally and has been generated in the laboratory. Other antibiotics have been shown to be active against Y. pestis in vitro and in vivo. However, the relative efficacies of clinically prescribed regimens of these antibiotics with streptomycin and with each other for the killing of Yersinia pestis are unknown. The efficacies of simulated pharmacokinetic profiles for human 10-day clinical regimens of ampicillin, meropenem, moxifloxacin, ciprofloxacin, and gentamicin were compared with the gold standard, streptomycin, for killing of Yersinia pestis in an in vitro pharmacodynamic model. Resistance amplification with therapy was also assessed. Streptomycin killed the microbe in one trial but failed due to resistance amplification in the second trial. In two trials, the other antibiotics consistently reduced the bacterial densities within the pharmacodynamic systems from 108 CFU/ml to undetectable levels (<102 CFU/ml) between 1 and 3 days of treatment. None of the comparator agents selected for resistance. The comparator antibiotics were superior to streptomycin against Y. pestis and deserve further evaluation.
doi:10.1128/AAC.01374-10
PMCID: PMC3101461  PMID: 21486959
11.  Effect of Half-Life on the Pharmacodynamic Index of Zanamivir against Influenza Virus Delineated by a Mathematical Model▿  
Intravenous zanamivir is recommended for the treatment of hospitalized patients with complicated oseltamivir-resistant influenza virus infections. In a companion paper, we show that the time above the 50% effective concentration (time>EC50) is the pharmacodynamic (PD) index predicting the inhibition of viral replication by intravenous zanamivir. However, for other neuraminidase inhibitors, the ratio of the area under the concentration-time curve to the EC50 (AUC/EC50) is the most predictive index. Our objectives are (i) to explain the dynamically linked variable of intravenous zanamivir by using different half-lives and (ii) to develop a new, mechanism-based population pharmacokinetic (PK)/PD model for the time course of viral load. We conducted dose fractionation studies in the hollow-fiber infection model (HFIM) system with zanamivir against an oseltamivir-resistant influenza virus. A clinical 2.5-h half-life and an artificially prolonged 8-h half-life were simulated for zanamivir. The values for the AUC from 0 to 24 h (AUC0-24) of zanamivir were equivalent for the two half-lives. Viral loads and zanamivir pharmacokinetics were comodeled using data from the present study and a previous dose range experiment via population PK/PD modeling in S-ADAPT. Dosing every 8 h (Q8h) suppressed the viral load better than dosing Q12h or Q24h at the 2.5-h half-life, whereas all regimens suppressed viral growth similarly at the 8-h half-life. The model provided unbiased and precise individual (Bayesian) (r2, >0.96) and population (pre-Bayesian) (r2, >0.87) fits for log10 viral load. Zanamivir inhibited viral release (50% inhibitory concentration [IC50], 0.0168 mg/liter; maximum extent of inhibition, 0.990). We identified AUC/EC50 as the pharmacodynamic index for zanamivir at the 8-h half-life, whereas time>EC50 best predicted viral suppression at the 2.5-h half-life, since the trough concentrations approached the IC50 for the 2.5-h but not for the 8-h half-life. The model explained data at both half-lives and holds promise for optimizing clinical zanamivir dosage regimens.
doi:10.1128/AAC.01629-10
PMCID: PMC3067163  PMID: 21263045
12.  Zanamivir, at 600 Milligrams Twice Daily, Inhibits Oseltamivir-Resistant 2009 Pandemic H1N1 Influenza Virus in an In Vitro Hollow-Fiber Infection Model System▿  
In 2009, a novel H1N1 influenza A virus emerged and spread worldwide, initiating a pandemic. Various isolates obtained from disparate parts of the world were shown to be uniformly resistant to the adamantanes but sensitive to the neuraminidase inhibitors oseltamivir and zanamivir. Over time, resistance to oseltamivir became more prevalent among pandemic H1N1 virus isolates, while most remained susceptible to zanamivir. The government has proposed the use of intravenous (i.v.) zanamivir to treat serious influenza virus infections among hospitalized patients. To use zanamivir effectively for patients with severe influenza, it is necessary to know the optimal dose and schedule of administration of zanamivir that will inhibit the replication of oseltamivir-sensitive and -resistant influenza viruses. Therefore, we performed studies using the in vitro hollow-fiber infection model system to predict optimal dosing regimens for zanamivir against an oseltamivir-sensitive and an oseltamivir-resistant virus. Our results demonstrated that zanamivir, at a dose of 600 mg given twice a day (Q12h), inhibited the replication of oseltamivir-sensitive and oseltamivir-resistant influenza viruses throughout the course of the experiment. Thus, our findings suggest that intravenous zanamivir, at a dose of 600 mg Q12h, could be used to treat hospitalized patients suffering from serious infections with oseltamivir-sensitive or -resistant influenza viruses.
doi:10.1128/AAC.01628-10
PMCID: PMC3067184  PMID: 21263046
13.  Randomized Pharmacokinetic and Pharmacodynamic Comparison of Fluoroquinolones for Tuberculous Meningitis ▿ † ‡ 
Tuberculous meningitis (TBM) is the most lethal form of tuberculosis, and new treatments that improve outcomes are required. We randomly assigned adults with TBM to treatment with standard antituberculosis treatment alone or in combination with ciprofloxacin (750 mg/12 h), levofloxacin (500 mg/12 h), or gatifloxacin (400 mg/24 h) for the first 60 days of therapy. Fluoroquinolone concentrations were measured with plasma and cerebrospinal fluid (CSF) specimens taken at predetermined, randomly assigned times throughout treatment. We aimed to describe the pharmacokinetics of each fluoroquinolone during TBM treatment and evaluate the relationship between drug exposure and clinical response over 270 days of therapy (Controlled Trials number ISRCTN07062956). Sixty-one patients with TBM were randomly assigned to treatment with no fluoroquinolone (n = 15), ciprofloxacin (n = 16), levofloxacin (n = 15), or gatifloxacin (n = 15). Cerebrospinal fluid penetration, measured by the ratio of the plasma area under the concentration-time curve from 0 to 24 h (AUC0–24) to the cerebrospinal fluid AUC0–24, was greater for levofloxacin (median, 0.74; range, 0.58 to 1.03) than for gatifloxacin (median, 0.48; range, 0.47 to 0.50) or ciprofloxacin (median, 0.26; range, 0.11 to 0.77). Univariable and multivariable analyses of fluoroquinolone exposure against a range of different treatment responses revealed worse outcomes among patients with lower and higher plasma and CSF exposures than for patients with intermediate exposures (a U-shaped exposure-response). TBM patients most likely to benefit from fluoroquinolone therapy were identified, along with exposure-response relationships associated with improved outcomes. Fluoroquinolones add antituberculosis activity to the standard treatment regimen, but to improve outcomes of TBM, they must be started early, before the onset of coma.
doi:10.1128/AAC.00064-11
PMCID: PMC3122453  PMID: 21502621
14.  Pharmacodynamic Modeling of Anti-Cancer Activity of Tetraiodothyroacetic Acid in a Perfused Cell Culture System 
PLoS Computational Biology  2011;7(2):e1001073.
Unmodified or as a poly[lactide-co-glycolide] nanoparticle, tetraiodothyroacetic acid (tetrac) acts at the integrin αvβ3 receptor on human cancer cells to inhibit tumor cell proliferation and xenograft growth. To study in vitro the pharmacodynamics of tetrac formulations in the absence of and in conjunction with other chemotherapeutic agents, we developed a perfusion bellows cell culture system. Cells were grown on polymer flakes and exposed to various concentrations of tetrac, nano-tetrac, resveratrol, cetuximab, or a combination for up to 18 days. Cells were harvested and counted every one or two days. Both NONMEM VI and the exact Monte Carlo parametric expectation maximization algorithm in S-ADAPT were utilized for mathematical modeling. Unmodified tetrac inhibited the proliferation of cancer cells and did so with differing potency in different cell lines. The developed mechanism-based model included two effects of tetrac on different parts of the cell cycle which could be distinguished. For human breast cancer cells, modeling suggested a higher sensitivity (lower IC50) to the effect on success rate of replication than the effect on rate of growth, whereas the capacity (Imax) was larger for the effect on growth rate. Nanoparticulate tetrac (nano-tetrac), which does not enter into cells, had a higher potency and a larger anti-proliferative effect than unmodified tetrac. Fluorescence-activated cell sorting analysis of harvested cells revealed tetrac and nano-tetrac induced concentration-dependent apoptosis that was correlated with expression of pro-apoptotic proteins, such as p53, p21, PIG3 and BAD for nano-tetrac, while unmodified tetrac showed a different profile. Approximately additive anti-proliferative effects were found for the combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab. Our in vitro perfusion cancer cell system together with mathematical modeling successfully described the anti-proliferative effects over time of tetrac and nano-tetrac and may be useful for dose-finding and studying the pharmacodynamics of other chemotherapeutic agents or their combinations.
Author Summary
Clinical treatment protocols for specific solid cancers have favorable response rates of 20%–25%. Cancer cells frequently become resistant to treatment. Therefore, novel anti-cancer drugs and combination regimens need to be developed. Conducting enough clinical trials to evaluate combinations of anti-cancer agents in several regimens to optimize treatment is not feasible. We showed that tetrac inhibits the growth of various cancer cell lines. Our newly developed in vitro system allowed studying the effects of tetrac over time in various human cancer cell lines. Our mathematical model could distinguish two effects of tetrac and may be used to predict effects of other than the studied dosage regimens. Human breast cancer cells were more sensitive to the effect on success of replication than the effect on growth rate, whereas the maximum possible effect was larger for the latter effect. Nanoparticulate tetrac, which does not enter into cells, had a larger effect than unmodified tetrac. The combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab showed approximately additive effects. Our in vitro perfusion system together with mathematical modeling may be useful for dose-finding, translation from in vitro to animal and human studies, and studying effects of other chemotherapeutic agents or their combinations.
doi:10.1371/journal.pcbi.1001073
PMCID: PMC3033367  PMID: 21304935
15.  Impact of Different Carbapenems and Regimens of Administration on Resistance Emergence for Three Isogenic Pseudomonas aeruginosa Strains with Differing Mechanisms of Resistance▿  
We compared drugs (imipenem and doripenem), doses (500 mg and 1 g), and infusion times (0.5 and 1.0 [imipenem], 1.0 and 4.0 h [doripenem]) in our hollow-fiber model, examining cell kill and resistance suppression for three isogenic strains of Pseudomonas aeruginosa PAO1. The experiments ran for 10 days. Serial samples were taken for total organism and resistant subpopulation counts. Drug concentrations were determined by high-pressure liquid chromatography-tandem mass spectrometry (LC/MS/MS). Free time above the MIC (time > MIC) was calculated using ADAPT II. Time to resistance emergence was examined with Cox modeling. Cell kill and resistance emergence differences were explained, in the main, by differences in potency (MIC) between doripenem and imipenem. Prolonged infusion increased free drug time > MIC and improved cell kill. For resistance suppression, the 1-g, 4-h infusion was able to completely suppress resistance for the full period of observation for the wild-type isolate. For the mutants, control was ultimately lost, but in all cases, this was the best regimen. Doripenem gave longer free time > MIC than imipenem and, therefore, better cell kill and resistance suppression. For the wild-type organism, the 1-g, 4-h infusion regimen is preferred. For organisms with resistance mutations, larger doses or addition of a second drug should be studied.
doi:10.1128/AAC.01721-09
PMCID: PMC2876389  PMID: 20308371
16.  The Combination of Meropenem and Levofloxacin Is Synergistic with Respect to both Pseudomonas aeruginosa Kill Rate and Resistance Suppression▿  
New approaches are needed for the treatment of Pseudomonas aeruginosa infections. All available single agents are suboptimal, especially for resistance suppression. Classical β-lactam/aminoglycoside combinations are not used often enough at least in part because of concern for nephrotoxicity. We evaluated the combination of meropenem and levofloxacin against the P. aeruginosa PAO1 wild type and its isogenic MexAB pump-overexpressed mutant. The drugs were studied using an in vitro hollow-fiber pharmacodynamic infection model. There were 16 different regimens evaluated for both isolates. Both total population and resistant subpopulations were quantified. Drug concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The impact of monotherapy versus that of combination therapy for attainment of a 3-log cell kill and for resistance suppression was examined using Kaplan-Meier analysis. Drug exposures were calculated by fitting the concentration-time data using the ADAPT II package of programs. For both isolates, monotherapy allowed resistance emergence with all but the largest exposure or with all exposures. In contrast, there was no resistance emergence with any combination regimen. Kaplan-Meier analysis showed significant differences in time to attainment of a 3-log cell kill as well as time to resistance emergence for monotherapy and combination therapy for both isolates, in favor of the combination regimens. Determination of the pharmacodynamic indices associated with resistance suppression demonstrated a 2- to 3-fold reduction with the use of combinations. Combination therapy with meropenem and levofloxacin provides a significantly faster time to attain a 3-log cell kill and significantly better resistance suppression than does either monotherapy. This combination should be evaluated in a clinical trial.
doi:10.1128/AAC.00065-10
PMCID: PMC2876409  PMID: 20368395
17.  The Combination of Rifampin plus Moxifloxacin Is Synergistic for Suppression of Resistance but Antagonistic for Cell Kill of Mycobacterium tuberculosis as Determined in a Hollow-Fiber Infection Model 
mBio  2010;1(3):e00139-10.
Moxifloxacin is under development for expanded use against Mycobacterium tuberculosis. Rifampin is a mainstay of therapy. We examined the interaction of moxifloxacin plus rifampin for log-phase and nonreplicating persister (NRP) organisms. For this evaluation, we employed our hollow-fiber infection model, in which organisms are exposed to clinically relevant drug concentration-time profiles and the impact on bacterial cell kill and resistant subpopulation amplification is determined. In log phase, resistance emergence was observed in all monotherapy regimens and in no combination therapy regimen. No difference was seen in time to a 3-log reduction in the bacterial burden; there was a significant difference in time to resistance emergence (P = 0.0006). In the NRP experiment, no resistance emergence was seen. There was a significant difference between the monotherapy and combination therapy regimens in time to a 3-log reduction in the bacterial burden (P = 0.042). The combination is efficacious for suppressing resistant organisms but is antagonistic for cell kill.
IMPORTANCE
M. tuberculosis infects one-third of the world’s population. Multiresistant organisms have become more frequent, threatening our ability to provide adequate chemotherapy. Moxifloxacin has been seen as an important new agent with the potential to supplant isoniazid or add to the rifampin/isoniazid combination. M. tuberculosis also exists in different physiological states, including the nonreplicating persister phenotype. We examined the moxifloxacin/rifampin combination in a new in vitro system to allow judgment of how moxifloxacin would interact with rifampin and allow its performance in clinical trials to be placed into perspective. Importantly, the combination suppressed resistance emergence, but at the price of slightly slowing bacterial cell kill. This new combination is a welcome addition to the physician’s armamentarium.
doi:10.1128/mBio.00139-10
PMCID: PMC2925073  PMID: 20802826
18.  Pharmacodynamics of Levofloxacin in a Murine Pneumonia Model of Pseudomonas aeruginosa Infection: Determination of Epithelial Lining Fluid Targets▿  
The dose choice for Pseudomonas aeruginosa remains a matter of debate. The actual exposure targets required for multilog killing of organisms at the primary infection site have not been delineated. We studied Pseudomonas aeruginosa PAO1 using a murine model of pneumonia. We employed a large mathematical model to fit all the concentration-time data in plasma and epithelial lining fluid (ELF) as well as colony counts in lung simultaneously for all drug doses. Penetration into ELF was calculated to be approximately 77.7%, as indexed to the ratio of the area under the concentration-time curve for ELF (AUCELF) to the AUCplasma. We determined the ELF concentration-time profile required to drive a stasis response as well as 1-, 2-, or 3-log10(CFU/g) kill. AUC/MIC ratios of 12.4, 31.2, 62.8, and 127.6 were required to drive these bacterial responses. Emergence of resistance was seen only at the two lowest doses (three of five animals at 50 mg/kg [body weight] and one of five animals at 100 mg/kg). The low exposure targets were likely driven by a low mutational frequency to resistance. Bridging to humans was performed using Monte Carlo simulation. With a 750-mg levofloxacin dose, target attainment rates fell below 90% at 4 mg/liter, 1 mg/liter, and 0.5 mg/liter for 1-, 2-, and 3-log kills, respectively. Given the low exposure targets seen with this strain, we conclude that levofloxacin at a 750-mg dose is not adequate for serious Pseudomonas aeruginosa pneumonia as a single agent. More isolates need to be studied to make these observations more robust.
doi:10.1128/AAC.00006-09
PMCID: PMC2715581  PMID: 19364849
19.  Differing Effects of Combination Chemotherapy with Meropenem and Tobramycin on Cell Kill and Suppression of Resistance of Wild-Type Pseudomonas aeruginosa PAO1 and Its Isogenic MexAB Efflux Pump-Overexpressed Mutant▿  
The drug interaction terminology (synergy, additivity, antagonism) relates to bacterial kill. The suppression of resistance requires greater drug exposure. We examined the combination of meropenem and tobramycin for kill and resistance suppression (wild-type Pseudomonas aeruginosa PAO1 and its isogenic MexAB-overexpressed mutant). The drug interaction was additive. The introduction of MexAB overexpression significantly altered the 50% inhibitory concentration of meropenem but not that of tobramycin, resulting in the recovery of a marked increase in colony numbers from drug-containing plates. For the wild type, more tobramycin-resistant isolates than meropenem-resistant isolates were present, and the tobramycin-resistant isolates were harder to suppress. MexAB overexpression unexpectedly caused a significant increase in the number of tobramycin-resistant mutants, as indexed to the area under the curve of slices through the inverted U resistance mountain. The differences were significant, except in the absence of meropenem. We hypothesize that the pump resulted in the presence of less meropenem for organism inhibition, allowing more rounds of replication and also affecting the numbers of tobramycin-resistant mutants. When resistance suppression is explored by combination chemotherapy, it is important to examine the impacts of differing resistance mechanisms for both agents.
doi:10.1128/AAC.01680-08
PMCID: PMC2687233  PMID: 19289521
20.  Prediction of the Pharmacodynamically Linked Variable of Oseltamivir Carboxylate for Influenza A Virus Using an In Vitro Hollow-Fiber Infection Model System▿  
MDCK cells transfected with the human β-galactoside α-2,6-sialyltransferase 1 gene (AX-4 cells) were used to determine the drug susceptibility and pharmacodynamically linked variable of oseltamivir for influenza virus. For dose-ranging studies, five hollow-fiber units were charged with 102 A/Sydney/5/97 (H3N2) influenza virus-infected AX-4 cells and 108 uninfected AX-4 cells. Each unit was treated continuously with different oseltamivir carboxylate concentrations in virus growth medium for 6 days. For dose fractionation studies, one hollow-fiber unit received no drug, one unit received a 1× 50% effective concentration (EC50) exposure to oseltamivir by continuous infusion, one unit received the same AUC0-24 (area under the concentration-time curve from 0 to 24 h) by 1-h infusion every 24 h, one unit received the same total exposure in two equal fractions every 12 h, and one unit received the same total exposure in three equal fractions every 8 h. Each infusion dose was followed by a no-drug washout, producing the appropriate half-life for this drug. The effect of the drug on virus replication was determined by sampling the units daily, measuring the amount of released virus by plaque assay, and performing a hemagglutination assay. The drug concentration in the hollow-fiber infection model systems was determined at various times by liquid chromatography-tandem mass spectrometry. The dose-ranging study showed that the EC50s for oseltamivir carboxylate for the A/Sydney/5/97 strain of influenza virus was about 1.0 ng/ml. The dose fractionation study showed that all treatment arms suppressed virus replication to the same extent, indicating that the pharmacodynamically linked variable was the AUC0-24/EC50 ratio. This implies that it may be possible to treat influenza virus infection once daily with a dose of 150 mg/day.
doi:10.1128/AAC.00167-09
PMCID: PMC2687202  PMID: 19364864
21.  Is 60 Days of Ciprofloxacin Administration Necessary for Postexposure Prophylaxis for Bacillus anthracis?▿  
Antimicrobial Agents and Chemotherapy  2008;52(11):3973-3979.
Sixty days of ciprofloxacin administration at 500 mg every 12 h is currently recommended for the prophylaxis of inhalational exposure to Bacillus anthracis. We examined Bacillus anthracis (Δ-Sterne strain) in our hollow-fiber infection model. We measured the ciprofloxacin concentrations achieved and the number of organisms present before heat shock (total population) and after heat shock (spore population). We fit a mathematical model to these data. Monte Carlo simulation with differing initial spore burdens (3, 5, and 6.9 log10 CFU/ml) demonstrated that 35 days of this regimen would completely clear the spore burden in 95% of patients. Durations of 110 days did not achieve 99.9% eradication, irrespective of initial burden, because of between-patient variance in drug pharmacokinetics. Given the absence of person-to-person transmission for Bacillus anthracis, adverse drug effects with long-term ciprofloxacin administration, and the possibility of engendering resistance in bodily flora, shorter prophylaxis duration should be given consideration, along with careful monitoring of all exposed individuals.
doi:10.1128/AAC.00453-08
PMCID: PMC2573157  PMID: 18725437
22.  In Vitro Infection Model Characterizing the Effect of Efflux Pump Inhibition on Prevention of Resistance to Levofloxacin and Ciprofloxacin in Streptococcus pneumoniae▿  
Antimicrobial Agents and Chemotherapy  2007;51(11):3988-4000.
The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae is slowly rising as a consequence of the increased use of fluoroquinolone antibiotics to treat community-acquired pneumonia. We tested the hypothesis that increased efflux pump (EP) expression by S. pneumoniae may facilitate the emergence of fluoroquinolone resistance. By using an in vitro pharmacodynamic infection system, a wild-type S. pneumoniae strain (Spn-058) and an isogenic strain with EP overexpression (Spn-RC2) were treated for 10 days with ciprofloxacin or levofloxacin in the presence or absence of the EP inhibitor reserpine to evaluate the effect of EP inhibition on the emergence of resistance. Cultures of Spn-058 and Spn-RC2 were exposed to concentration-time profiles simulating those in humans treated with a regimen of ciprofloxacin at 750 mg orally once every 12 h and with regimens of levofloxacin at 500 and 750 mg orally once daily (QD; with or without continuous infusions of 20 μg of reserpine/ml). The MICs of ciprofloxacin and levofloxacin for Spn-058 were both 1 μg/ml when susceptibility testing was conducted with each antibiotic alone and with each antibiotic in the presence of reserpine. For Spn-RC2, the MIC of levofloxacin alone and with reserpine was also 1 μg/ml; the MICs of ciprofloxacin were 2 and 1 μg/ml, respectively, when determined with ciprofloxacin alone and in combination with reserpine. Reserpine, alone, had no effect on the growth of Spn-058 and Spn-RC2. For Spn-058, simulated regimens of ciprofloxacin at 750 mg every 12 h or levofloxacin at 500 mg QD were associated with the emergence of fluoroquinolone resistance. However, the use of ciprofloxacin at 750 mg every 12 h and levofloxacin at 500 mg QD in combination with reserpine rapidly killed Spn-058 and prevented the emergence of resistance. For Spn-RC2, levofloxacin at 500 mg QD was associated with the emergence of resistance, but again, the resistance was prevented when this levofloxacin regimen was combined with reserpine. Ciprofloxacin at 750 mg every 12 h also rapidly selected for ciprofloxacin-resistant mutants of Spn-RC2. However, the addition of reserpine to ciprofloxacin therapy only delayed the emergence of resistance. Levofloxacin at 750 mg QD, with and without reserpine, effectively eradicated Spn-058 and Spn-RC2 without selecting for fluoroquinolone resistance. Ethidium bromide uptake and efflux studies demonstrated that, at the baseline, Spn-RC2 had greater EP expression than Spn-058. These studies also showed that ciprofloxacin was a better inducer of EP expression than levofloxacin in both Spn-058 and Spn-RC2. However, in these isolates, the increase in EP expression by short-term exposure to ciprofloxacin and levofloxacin was transient. Mutants of Spn-058 and Spn-RC2 that emerged under suboptimal antibiotic regimens had a stable increase in EP expression. Levofloxacin at 500 mg QD in combination with reserpine, an EP inhibitor, or at 750 mg QD alone killed wild-type S. pneumoniae and strains that overexpressed reserpine-inhibitable EPs and was highly effective in preventing the emergence of fluoroquinolone resistance in S. pneumoniae during therapy. Ciprofloxacin at 750 mg every 12 h, as monotherapy, was ineffective for the treatment of Spn-058 and Spn-RC2. Ciprofloxacin in combination with reserpine prevented the emergence of resistance in Spn-058 but not in Spn-RC2, the EP-overexpressing strain.
doi:10.1128/AAC.00391-07
PMCID: PMC2151412  PMID: 17846144
23.  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 log10 CFU/g. A 1-week pharmacokinetic-pharmacodynamic study revealed a micafungin serum half-life of 6.13 h. In mice treated with ≥50 mg/kg, there was maximal fungal decline without regrowth during the 1-week dosing interval. Next, doses associated with 34% (34% effective dose [ED34]) and 50% (ED50) 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 ED34, microbial kill with the daily therapy initially lagged behind the intermittent doses but exceeded it by day 7. In mice treated with the ED50, 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.
doi:10.1128/AAC.01337-06
PMCID: PMC1803141  PMID: 17194830

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