Bactericidal Activity and Mechanism of Action of AZD5847, a Novel Oxazolidinone for Treatment of Tuberculosis

Treatment of tuberculosis (TB) is impaired by the long duration and complexity of therapy and the rising incidence of drug resistance. There is an urgent need for new agents with improved efficacy, safety, and compatibility with combination chemotherapies. Oxazolidinones offer a potential new class of TB drugs, and linezolid—the only currently approved oxazolidinone—has proven highly effective against extensively drug-resistant (XDR) TB in experimental trials. However, widespread use of linezolid is prohibited by its significant toxicities. AZD5847, a novel oxazolidinone, demonstrates improved in vitro bactericidal activity against both extracellular and intracellular M. tuberculosis compared to that of linezolid. Killing kinetics in broth media and in macrophages indicate that the rate and extent of kill obtained with AZD5847 are superior to those obtained with linezolid. Moreover, the efficacy of AZD5847 was additive when tested along with a variety of conventional TB agents, indicating that AZD5847 may function well in combination therapies. AZD5847 appears to function similarly to linezolid through impairment of the mycobacterial 50S ribosomal subunit. Future studies should be undertaken to further characterize the pharmacodynamics and pharmacokinetics of AZD5847 in both in vitro and animal models as well is in human clinical trials.     

Pharmacokinetics and Pharmacodynamics of Clofazimine in a Mouse Model of Tuberculosis

The antileprosy drug clofazimine has shown potential for shortening tuberculosis treatment; however, the current dosing of the drug is not evidence based, and the optimal dosing is unknown. Our objective was to conduct a preclinical evaluation of the pharmacokinetics and pharmacodynamics of clofazimine in the mouse model of tuberculosis, with the goal of providing useful information on dosing for future studies. Pharmacokinetic parameters were evaluated in infected and uninfected BALB/c mice. Pharmacodynamic parameters were evaluated in Mycobacterium tuberculosis-infected mice that were treated for 12 weeks with one of six different clofazimine dosing regimens, i.e., doses of 6.25, 12.5, and 25 mg/kg of body weight/day and 3 regimens with loading doses. Clofazimine progressively accumulated in the lungs, livers, and spleens of the mice, reaching levels of greater than 50 μg/g in all tissues by 4 weeks of administration, while serum drug levels remained low at 1 to 2 μg/ml. Elimination of clofazimine was extremely slow, and the half-life was dependent on the duration of drug administration. Clofazimine exhibited dose-dependent tissue and serum concentrations. At any dose, clofazimine did not have bactericidal activity during the first 2 weeks of administration but subsequently demonstrated potent, dose-independent bactericidal activity. The antituberculosis activity of clofazimine was dependent on neither the dose administered nor the drug concentrations in the tissues, suggesting that much lower doses could be effectively used for tuberculosis treatment.     

In Vitro Activity of AZD5847 against Geographically Diverse Clinical Isolates of Mycobacterium tuberculosis

The MIC of the novel antituberculosis (anti-TB) drug AZD5847 was determined against 146 clinical isolates from diverse geographical regions, including eastern Europe, North America, Africa, and Asia, using the automated Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system. These isolates originated from specimen sources such as sputum, bronchial alveolar lavage fluid, pleural fluid, abscess material, lung biopsies, and feces. The overall MIC₉₀ was 1.0 mg/liter (range, 0.125 to 4 mg/liter). The MICs of AZD5847 for isolates of Mycobacterium tuberculosis were similar among drug-sensitive strains, multidrug-resistant (MDR) strains, and extensively drug resistant (XDR) strains. The good in vitro activity of AZD5847 against M. tuberculosis and the lack of cross-resistance make this agent a promising anti-TB drug candidate.     

Caspase-Based PET for Evaluating Pro-Apoptotic Treatments in a Tuberculosis Mouse Model

Purpose

Despite recent advances in antimicrobial treatments, tuberculosis (TB) remains a major global health threat. Mycobacterium tuberculosis proliferates in macrophages, preventing apoptosis by inducing anti-apoptotic proteins leading to necrosis of the infected cells. Necrosis then leads to increased tissue destruction, reducing the penetration of antimicrobials and immune cells to the areas where they are needed most. Pro-apoptotic drugs could be used as host-directed therapies in TB to improve antimicrobial treatments and patient outcomes.

Procedure

We evaluated [¹⁸F]-ICMT-11, a caspase-3/7-specific positron emission tomography (PET) radiotracer, in macrophage cell cultures and in an animal model of pulmonary TB that closely resembles human disease.

Results

Cells infected with M. tuberculosis and treated with cisplatin accumulated [¹⁸F]-ICMT-11 at significantly higher levels compared with that of controls, which correlated with levels of caspase-3/7 activity. Infected mice treated with cisplatin with increased caspase-3/7 activity also had a higher [¹⁸F]-ICMT-11 PET signal compared with that of untreated infected animals.

Conclusions

[¹⁸F]-ICMT-11 PET could be used as a noninvasive approach to measure intralesional pro-apoptotic responses in situ in pulmonary TB models and support the development of pro-apoptotic host-directed therapies for TB.

     

Pharmacokinetic/Pharmacodynamic Analysis of the Influence of Inoculum Size on the Selection of Resistance in Escherichia coli by a Quinolone in a Mouse Thigh Bacterial Infection Model

Maintaining quinolone concentrations outside the mutant selection window (MSW) between the MIC and mutant prevention concentration (MPC) was suggested by in vitro and in vivo studies to prevent the selection of resistant mutants. However, selection also may depend on the presence of resistant bacterial mutants at the start of treatment, which is highly dependent on the initial inoculum size. In this study, a mouse thigh bacterial infection model was used to test the influence of different exposures to marbofloxacin on the selection of resistant bacteria after infection with a low (10⁵ CFU) or high (10⁸ CFU) initial inoculum of Escherichia coli. The inoculum size was shown to influence the exposure to marbofloxacin and the values of pharmacokinetic/pharmacodynamic indices. When the abilities of the indices time within the MSW (T_MSW), area under the concentration-time curve of 0 to 24 h divided by the MIC, and the maximum concentration of drug in plasma divided by the MIC to predict the selection of resistant bacteria were compared, only T_MSW appeared to be a good predictor of the prevention of resistance for values less than 30%. When the T_MSW was higher than 34%, the selection of resistant bacteria occurred less often in thighs initially infected with the low inoculum (11/24; 46%) than in those infected with the high inoculum (30/36; 80%), suggesting that the selection of resistant mutants depends on both the T_MSW and inoculum size. The relevance of these results merits further investigation to test different strategies of antibiotic therapy depending on the expected bacterial burden at the infectious site.Resistances to fluoroquinolones can occur spontaneously in bacterial populations at a frequency of about 10⁻⁶ to 10⁻⁸ (5) by following a stepwise process that involves mutations in genes coding for the targets DNA gyrase and topoisomerase IV (23, 29). Consequently, if the bacterial load at the infectious site exceeds the inverse of the mutation frequency, it can be presumed that a small resistant subpopulation already coexists with a larger susceptible population before any antimicrobial treatment is administered. Traditionally, in vitro antimicrobial studies and animal infection models have been used to assess the reduction in the total bacterial population at an infectious site while often ignoring the impact of drug pressure on the amplification of the drug-resistant subpopulation (2, 8). Thus, the values of pharmacokinetic/pharmacodynamic (PK/PD) indices determined from these experiments were selected previously to predict the bacteria killing and not the selection of resistant bacteria. The PK/PD indices fAUC/MIC (area under the free-plasma concentration curve divided by the MIC), fC_max/MIC (peak free-plasma concentration divided by the MIC), and fT_>MIC (the time the free concentrations are above the MIC) all are expressed as a function of the MIC, which is the pharmacodynamic parameter used to describe the susceptibility of the major drug-susceptible population. From experiments carried out with fluoroquinolones, the mutant prevention concentration (MPC) has been proposed to assess the susceptibility of the resistant subpopulation (12, 24, 30). MIC and MPC then define the boundaries of the so-called mutant selection window (MSW), which is the range of antibiotic concentrations that would favor the selection of first-step mutants (30). First-step mutants of Staphylococcus aureus and Streptococcus pneumoniae were selected by ciprofloxacin, levofloxacin, or moxifloxacin when antibiotic concentrations fell within the MSW in vitro (6, 7, 17, 31). A study carried out in a rabbit lung infection model with Streptococcus pneumoniae showed that the selection of resistant bacteria occurred systematically when concentrations of gatifloxacin were within the MSW (T_MSW) for more than 45% of the treatment duration (10). Another experiment in rabbits infected by Staphylococcus aureus also showed that drug concentrations needed to be at the bottom of the MSW (just above the MIC) for only 33% of the time to enrich mutants (11). These in vivo results, which suggest that fluoroquinolone concentrations need to be outside the MSW for most of the time to prevent the selection of resistant mutants, were observed with a large inoculum size (more than 10⁹ CFU). However, the bacterial burdens to be eradicated at the infectious site may be very low or null when antibiotics are used preventively, as in the prophylaxis of gram-negative bacteremia in immunocompromised patients (25) or metaphylaxis in veterinary medicine. For both human and veterinary medicine, prophylaxis is the administration of antimicrobials to exposed individuals who are considered at risk but before the expected onset of disease. Metaphylaxis consists of treating all animals at the herd level when there is a clinical disease in only some animals (27). In the case of prophylaxis and metaphylaxis, the bacterial inocula at the beginning of the treatment presumably are much smaller than those targeted when patients express clinical signs or are critically ill, and therefore the likelihood of a mutant appearing may be low, particularly if the bacterial load is less than the inverse of the mutation rate.We previously observed in vitro that the emergence of resistance was more frequent when both the T_MSW and the bacterial inoculum size increased (15), and the aim of this study was to test, in vivo, the ability of T_MSW and also of the area under the concentration-time curve of 0 to 24 h divided by the MIC (AUC_0-24/MIC) and C_max/MIC indices to predict the selection of resistant bacteria in a low and a high inoculum. We assessed the selection of a preexisting resistant subpopulation of Escherichia coli in mouse thighs infected with the two bacterial inoculum sizes after different exposures to marbofloxacin, a fluoroquinolone extensively used in veterinary medicine.     

Pharmacokinetic and Pharmacodynamic Considerations in Antimalarial Dose Optimization

Antimalarial drugs have usually been first deployed in areas of malaria endemicity at doses which were too low, particularly for high-risk groups such as young children and pregnant women. This may accelerate the emergence and spread of resistance, thereby shortening the useful life of the drug, but it is an inevitable consequence of the current imprecise method of dose finding. An alternative approach to dose finding is suggested in which phase 2 studies concentrate initially on pharmacokinetic-pharmacodynamic (PK-PD) characterization and in vivo calibration of in vitro susceptibility information. PD assessment is facilitated in malaria because serial parasite densities are readily assessed by microscopy, and at low densities by quantitative PCR, so that initial therapeutic responses can be quantitated accurately. If the in vivo MIC could be characterized early in phase 2 studies, it would provide a sound basis for the choice of dose in all target populations in subsequent combination treatments. Population PK assessments in phase 2b and phase 3 studies which characterize PK differences between different age groups, clinical disease states, and human populations can then be combined with the PK-PD observations to provide a sound evidence base for dose recommendations in different target groups.     

Evaluation of Once-Weekly Therapy for Tuberculosis Using Isoniazid plus Rifamycins in the Mouse Aerosol Infection Model

Once-weekly therapy with combinations of isoniazid plus a rifamycin was tested in the mouse low-dose aerosol infection model against two strains of Mycobacterium tuberculosis. Combinations of isoniazid and rifalizil and isoniazid and rifapentine were both highly effective. These animal model data thus support the evaluation of these regimens under clinical conditions.     

In Vitro Pharmacokinetic and Pharmacodynamic Evaluation of S-013420 against Haemophilus influenzae and Streptococcus pneumoniae

The pharmacokinetic (PK)/pharmacodynamic (PD) parameters and the antibacterial activity of S-013420, a novel bicyclolide, against Haemophilus influenzae and Streptococcus pneumoniae, including macrolide-resistant isolates, were investigated using an in vitro PD model. Various time-concentration curves were artificially constructed by modifying the PK data obtained in phase I studies. The activity against H. influenzae was evaluated using two parameters, that is, the area above the killing curve (AAC) and the viable cell reduction at 24 h. The relationships between the antibacterial activity of S-013420 and the three PK/PD parameters were investigated by fitting the data to the sigmoid maximum effective concentration model. The square of the correlation coefficient (R²) values for AAC versus the area under the concentration-time curve from 0 to 24 h (AUC_0-24)/MIC, the peak concentration (C_max)/MIC, and the cumulative percentage of a 24-h period that the drug concentration exceeded the MIC under steady-state PK conditions (%T_MIC) were 0.92, 0.87, and 0.49, respectively. The R² values for viable cell reduction at 24 h versus AUC_0-24/MIC, C_max/MIC, and %T_MIC were 0.93, 0.61, and 0.56, respectively. These results demonstrated that AUC_0-24/MIC is the most significant parameter for evaluation of the antibacterial activity of S-013420. The values of AUC_0-24/MIC required for maximum and static efficacy were 10.8 and 9.63, respectively, for H. influenzae and 16.3 to 22.3 and 4.66 to 9.01, respectively, for S. pneumoniae. This analysis is considered useful for determining the AUC value at the infection site, which would be required for efficacy in clinical use.In antimicrobial chemotherapy, the pharmacokinetic (PK)/pharmacodynamic (PD) concept has recently been used during drug development to determine susceptibility breakpoints, predict clinical efficacy, and optimize the antimicrobial regimen (1, 4, 13). Generally, antimicrobials can be categorized according to the dominant parameter of three PK/PD parameters correlated with antibacterial activity. These three parameters (1) are the area under the concentration-time curve from 0 to 24 h (AUC_0-24)/MIC, the peak concentration (C_max)/MIC, and the cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state PK conditions (%T_MIC). Nonclinical PK/PD characterizations of antimicrobials using in vitro PD models or in vivo infection models have been reported (2, 5, 26, 29). In general, AUC_0-24/MIC is the dominant PK/PD parameter for macrolides, ketolides, and quinolones, while C_max/MIC is the main parameter for quinolones and aminoglycosides and %T_MIC is that for β-lactams (1, 4). Overall, nonclinical PK/PD data have agreed well with data obtained for infected patients, and a PK/PD evaluation would be helpful for cutting costs, addressing ethical issues, and shortening the duration of clinical trials (1, 4).Community-acquired respiratory tract infections (RTIs), such as community-acquired pneumonia, acute exacerbations of chronic bronchitis, sinusitis, and acute otitis media, are among the most common infectious diseases for which oral antibiotics are prescribed in primary clinical settings and are mainly caused by Streptococcus pneumoniae and Haemophilus influenzae (6). The macrolide antimicrobials, including clarithromycin and azithromycin, are often used for the treatment of community-acquired RTIs. However, the decreased susceptibility of S. pneumoniae to macrolide agents is a key problem worldwide. Recent reports have suggested that pneumococcal macrolide resistance rates were about 35% in some regions (7, 14, 23). In S. pneumoniae, two main mechanisms of macrolide resistance were reported, i.e., active efflux due to mef genes and ribosomal modification mediated by erm genes (11). S-013420 (EDP-420, EP-013420) is a novel bicyclolide (bridged bicyclic macrolide) that is characterized by a 6,11-O-bridged structure discovered by Enanta Pharmaceuticals, Inc. (Fig. ​(Fig.1),1), and that shows a broad spectrum of activity against a variety of the RTI pathogens, including erythromycin-resistant streptococci. (12a, 28).Open in a separate windowFIG. 1.Chemical structure of S-013420.The purpose of the present study was to investigate the pharmacodynamic relationship between the activity of S-013420 against H. influenzae and S. pneumoniae, including macrolide-resistant isolates, and the well-established PK/PD parameters AUC_0-24/MIC, C_max/MIC, and %T_MIC by using an in vitro PD model.     

抗焦虑药布格呋喃的药代动力学和药效学研究

  目的  研究单次和多次服用60或120 mg抗焦虑药布格呋喃的药代动力学及药效学特征。  方法  试验为随机、双盲、安慰剂对照、平行组设计, 在布格呋喃60 mg组和120 mg组分别纳入14名中国健康受试者, 男女各7名。每个剂量组中, 男性和女性受试者随机接受布格呋喃胶囊或安慰剂治疗的比例均为5:2。受试者在研究第1天给药1次, 48 h后, 在研究第3天起每天2次给药, 连续4.5 d。在首次及末次给药后, 分别按照方案规定的时间点连续采集血液和尿液药代动力学样本至给药后48 h, 同时进行躯体摆动、选择反应时间、数字广度、视觉类比量表(visual analogue scale, VAS)等药效学测试。  结果  单次口服60或120 mg布格呋喃后, 其药代动力学参数的平均值分别为:血浆峰浓度C_max(37.7±18.4)和(95.8±34.8)ng/ml, 零至最后一个可定量时间点血浆浓度-时间曲线下面积AUC_0-t为(108±46)和(336±104)h·ng/ml, 表观清除率为(581±203)L/h和(367±122)L/h, 消除相半衰期t_1/2为(10.4±7.1)和(19.8±6.5)h。在每日2次重复给药4.5 d后, 60 mg组和120 mg组布格呋喃的平均C_max分别为(48.5±32.2)和(118.0±20.3)ng/ml, AUC_0-t分别为(241±122)和(656±135)h·ng/ml。除VAS清醒度、VAS外在感受和VAS内在感受外, 本研究检测的绝大多数药效学指标在单次和多次给药后与其他给药组间的差异均无统计学意义(P均 > 0.05)。  结论  每日2次、连续口服布格呋喃约24 h后, 其血浆暴露水平达到稳态, 较单次给药后有2~3倍蓄积。口服60或120 mg布格呋喃在健康受试者中的安全性和耐受性良好。研究选用的药效学指标呈阴性, 可能与药效学方法验证欠充分相关。     

A Physiologically Based Pharmacokinetic Model of Isoniazid and Its Application in Individualizing Tuberculosis Chemotherapy

Due to its high early bactericidal activity, isoniazid (INH) plays an essential role in tuberculosis treatment. Genetic polymorphisms of N-acetyltransferase type 2 (NAT2) cause a trimodal distribution of INH pharmacokinetics in slow, intermediate, and fast acetylators. The success of INH-based chemotherapy is associated with acetylator and patient health status. Still, a standard dose recommended by the FDA is administered regardless of acetylator type or immune status, even though adverse effects occur in 5 to 33% of all patients. Slow acetylators have a higher risk of development of drug-induced toxicity, while fast acetylators and immune-deficient patients face lower treatment success rates. To mechanistically assess the trade-off between toxicity and efficacy, we developed a physiologically based pharmacokinetic (PBPK) model describing the NAT2-dependent pharmacokinetics of INH and its metabolites. We combined the PBPK model with a pharmacodynamic (PD) model of antimycobacterial drug effects in the lungs. The resulting PBPK/PD model allowed the simultaneous simulation of treatment efficacies at the site of infection and exposure to toxic metabolites in off-target organs. Subsequently, we evaluated various INH dosing regimens in NAT2-specific immunocompetent and immune-deficient virtual populations. Our results suggest the need for acetylator-specific dose adjustments for optimal treatment outcomes. A reduced dose for slow acetylators substantially lowers the exposure to toxic metabolites and thereby the risk of adverse events, while it maintains sufficient treatment efficacies. Vice versa, intermediate and fast acetylators benefit from increased INH doses and a switch to a twice-daily administration schedule. Our analysis outlines how PBPK/PD modeling may be used to design and individualize treatment regimens.     

Pharmacokinetic/Pharmacodynamic Investigation of Colistin against Pseudomonas aeruginosa Using an In Vitro Model

Colistin plays a key role in treatment of serious infections by Pseudomonas aeruginosa. The aims of this study were to (i) identify the pharmacokinetic/pharmacodynamic (PK/PD) index (i.e., the area under the unbound concentration-time curve to MIC ratio [ƒAUC/MIC], the unbound maximal concentration to MIC ratio [ƒC_max/MIC], or the cumulative percentage of a 24-h period that unbound concentrations exceed the MIC [ƒT_>MIC]) that best predicts colistin efficacy and (ii) determine the values for the predictive PK/PD index required to achieve various magnitudes of killing effect. Studies were conducted in a one-compartment in vitro PK/PD model for 24 h using P. aeruginosa ATCC 27853, PAO1, and the multidrug-resistant mucoid clinical isolate 19056 muc. Six intermittent dosing intervals, with a range of ƒC_max colistin concentrations, and two continuous infusion regimens were examined. PK/PD indices varied from 0.06 to 18 for targeted ƒC_max/MIC, 0.36 to 312 for ƒAUC/MIC, and 0 to 100% for ƒT_>MIC. A Hill-type model was fit to killing effect data, which were expressed as the log₁₀ ratio of the area under the CFU/ml curve for treated regimens versus control. With ƒC_max values equal to or above the MIC, rapid killing was observed following the first dose; substantial regrowth occurred by 24 h with most regimens. The overall killing effect was best correlated with ƒAUC/MIC (R² = 0.931) compared to ƒC_max/MIC (R² = 0.868) and ƒT_>MIC (R² = 0.785). The magnitudes of ƒAUC/MIC required for 1- and 2-log₁₀ reductions in the area under the CFU/ml curve relative to growth control were 22.6 and 30.4, 27.1 and 35.7, and 5.04 and 6.81 for ATCC 27853, PAO1, and 19056 muc, respectively. The PK/PD targets identified will assist in designing optimal dosing strategies for colistin.Globally there is a growing threat from the emergence of multidrug-resistant (MDR) microorganisms (38), especially among a number of important Gram-negative bacterial pathogens (16, 29, 38). Colistin (polymyxin E) still retains significant activity against many of these MDR Gram-negative pathogens, including Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, which often leaves it as the only therapeutic option available (19, 26). With very few new chemical entities against Gram-negative infections in the drug development pipeline (29, 30, 38), particularly against P. aeruginosa (38), the use of colistin, a once-neglected antibiotic, has increased dramatically over the last 5 years (11, 26).Colistin is available commercially as colistin sulfate (hereafter referred to as colistin) and sodium colistin methanesulfonate (CMS), which is administered parenterally. CMS is an inactive prodrug of colistin (3) and, after parenteral administration, colistin is formed in vivo (21, 27, 33). Despite its newfound importance in therapy, there is a dearth of information on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of colistin, a situation of significant concern given that resistance to colistin is beginning to emerge (1, 15, 18, 26, 28). Thus, the aims of the present study were to utilize an in vitro PK/PD model to (i) identify the PK/PD index (i.e., the area under the unbound concentration-time curve to MIC ratio [ƒAUC/MIC], the unbound maximal concentration to MIC ratio [ƒC_max/MIC], or the cumulative percentage of a 24-h period that unbound concentrations exceed the MIC [ƒT_>MIC]) that best predicts colistin efficacy and (ii) determine the magnitude of the predictive PK/PD index required to achieve various magnitudes of killing effect.(Parts of the present study were presented at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy [ICAAC], Washington, DC, 25 to 28 October 2008, and at the Second American Conference on Pharmacometrics, Mashantucket, CT, 4 to 7 October 2009.)     

Pharmacokinetic and Pharmacodynamic Evaluation of Liposomal Amikacin for Inhalation in Cystic Fibrosis Patients with Chronic Pseudomonal Infection

The pharmacokinetics and pharmacodynamics of a novel liposomal amikacin for inhalation were evaluated in cystic fibrosis patients with chronic pseudomonas infection. Twenty-four patients from two studies received 500 mg of liposomal amikacin by inhalation once daily for 14 days. Serum, sputum, and 24-h urine samples were collected on days 1 and 14 of therapy; pulmonary function tests (PFT) and sputum for quantitative microbiology were assessed at baseline and serially for 14 days. Relationships between amikacin exposure in serum and sputum and absolute change in PFT endpoints and log₁₀ CFU of Pseudomonas aeruginosa from baseline on days 7 and 14 of therapy were assessed. On days 7 and 14, absolute change from baseline in forced expiratory volume in 1 s (FEV₁), percent predicted forced expiratory volume in 1 s (FEV₁ % predicted), and forced expiratory flow between 25 and 75% of forced vital capacity (FEF_25-75%) increased by 0.24 (P = 0.002) and 0.13 (P = 0.10) liters, 7.49 (P < 0.001) and 4.38 (P = 0.03), and 0.49 (P < 0.001) and 0.42 (P = 0.02) liters/s, respectively. In addition, relative change from baseline in FEV₁ % predicted was 10.8% (P < 0.001) and 5.62% (P = 0.073) on days 7 and 14, respectively. While significant relationships between absolute change in PFT endpoints and the ratio of serum or sputum area under the concentration-time curve to the MIC (AUC/MIC) were not observed, relationships between change in log₁₀ CFU and serum AUC/MIC ratio and change in log₁₀ CFU and absolute changes in all PFT endpoints were significant. Together, these findings likely represent drug effect and warrant the further development of liposomal amikacin for inhalation.Cystic fibrosis (CF) is a genetic chronic disease that affects approximately 60,000 people worldwide (10). The genetic defect occurs in the CF transmembrane regulator gene, resulting in abnormal production and/or function of its protein. CF transmembrane regulator is an essential protein that is found in the epithelial cells of the body''s internal passageways, such as the lung and gastrointestinal tract. This defective protein results in the production of thick and sticky mucus that impairs normal lung function and leads to chronic life-threatening infections, often involving Pseudomonas aeruginosa (3).Despite the observed benefits of frequent antimicrobial therapy in CF patients with P. aeruginosa infections, eradication of this organism is a difficult endpoint to achieve. Mucus plugs, inactivation of agents by sputum of CF patients, and development of bacterial biofilm are factors which contribute to the poor lung penetration of antimicrobial agents and, ultimately, the emergence of multidrug bacterial resistance. To increase drug exposure at the infection site and minimize systemic exposure (thereby reducing the potential for systemic adverse events), drug delivery via inhalation has been a preferred route for agents such as the aminoglycosides in CF patients (13). Sustained release of such agents via a liposome delivery system may provide the opportunity to maintain prolonged targeted lung exposures and enhance the uptake of drug to the site of infection. Liposomal amikacin for inhalation is a sustained-release formulation of amikacin encapsulated inside nanoscale liposomal carriers designed for administration via inhalation. Given the promise of enhanced delivery and prolonged therapeutic effect at the infection site, CF patients may benefit from this formulation of amikacin for inhalation.In the analysis described herein, we evaluated the pharmacokinetics (PK) of liposomal amikacin for inhalation using data from two phase 1b/2a studies in which CF patients who were chronically infected with P. aeruginosa received 14 daily doses of the study drug. Subsequently, PK-pharmacodynamic (PD) relationships between exposure of liposomal amikacin and the changes relative to baseline in pulmonary function (change in forced expiratory volume in 1 s [FEV₁], percent predicted forced expiratory volume in 1 s [FEV₁ % predicted], forced expiratory flow between 25 and 75% of forced vital capacity [FEF_25-75%], and forced vital capacity [FVC]) and values of P. aeruginosa CFU in sputum were evaluated.     

In Vitro Pharmacokinetic/Pharmacodynamic Modeling of Voriconazole Activity against Aspergillus Species in a New In Vitro Dynamic Model

The pharmacodynamics (PD) of voriconazole activity against Aspergillus spp. were studied using a new in vitro dynamic model simulating voriconazole human pharmacokinetics (PK), and the PK-PD data were bridged with human drug exposure to assess the percent target (near-maximum activity) attainment of different voriconazole dosages. Three Aspergillus clinical isolates (1 A. fumigatus, 1 A. flavus, and 1 A. terreus isolate) with CLSI MICs of 0.5 mg/liter were tested in an in vitro model simulating voriconazole PK in human plasma with C(max) values of 7, 3.5, and 1.75 mg/liter and a t(1/2) of 6 h. The area under the galactomannan index-time curve (AUC(GI)) was used as the PD parameter. In vitro PK-PD data were bridged with population human PK of voriconazole exposure, and the percent target attainment was calculated. The in vitro PK-PD relationship of fAUC(0-24)-AUC(GI) followed a sigmoid pattern (global R(2) = 0.97), with near-maximum activities (10% fungal growth) observed at an fAUC(0-24) (95% confidence interval [CI]) of 18.9 (14.4 to 23.1) mg · h/liter against A. fumigatus, 26.6 (21.1 to 32.9) mg · h/liter against A. flavus, and 36.2 (27.8 to 45.7) mg · h/liter against A. terreus (F test; P < 0.0001). Target attainment for 3, 4, and 5 mg/kg-of-body-weight voriconazole dosages was 24% (11 to 45%), 80% (32 to 97%), and 93% (86 to 97%) for A. fumigatus, 12% (5 to 26%), 63% (17 to 93%), and 86% (73 to 94%) for A. flavus, and 4% (2 to 11%), 36% (6 to 83%), and 68% (47 to 83%) for A. terreus. Based on the in vitro exposure-effect relationships, a standard dosage of voriconazole may be adequate for most patients with A. fumigatus but not A. flavus and A. terreus infections, for which a higher drug exposure may be required. This could be achieved using a higher voriconazole dosage, thus highlighting the usefulness of therapeutic drug monitoring in patients receiving a standard dosage.     

Impact of cyp51A Mutations on the Pharmacokinetic and Pharmacodynamic Properties of Voriconazole in a Murine Model of Disseminated Aspergillosis

The in vivo efficacy of voriconazole against 4 clinical Aspergillus fumigatus isolates with MICs ranging from 0.125 to 2 mg/liter (CLSI document M38A) was assessed in a nonneutropenic murine model of disseminated aspergillosis. The study involved TR/L98H, M220I, and G54W mutants and a wild-type control isolate. Oral voriconazole therapy was started 24 h after intravenous infection of mice and was given once daily for 14 consecutive days, with doses ranging from 10 to 80 mg/kg of body weight, using survival as the endpoint. Survival for all isolates was dependent on the voriconazole dose level (R² value of 0.5 to 0.6), but a better relationship existed for the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) or the AUC for the free, unbound fraction of the drug divided by the MIC (fAUC/MIC ratio) (R² value of 0.95 to 0.98). The 24-h fAUC/MIC ratio showed a clear relationship to effect, with an exposure index for amount of free drug required for 50% of maximum effectiveness (fEI₅₀) of 11.17 at day 7. Maximum effect was reached at values of around 80 to 100, comparable to that observed for posaconazole and A. fumigatus. Mice infected with an isolate having a MIC of 2 mg/liter required an exposure that was inversely correlated with the increase in MIC compared to that of the wild-type control, but due to nonlinear pharmacokinetics, this required only doubling of the voriconazole dose. The efficacy of voriconazole for isolates with high MICs for other triazoles but voriconazole MICs within the wild-type population range was comparable to that for the wild-type control. Finally, we used a grapefruit juice-free murine model of aspergillosis and concluded that this model is appropriate to study pharmacokinetic/pharmacodynamic relationships of voriconazole.Aspergillus fumigatus is a ubiquitous fungus and is the most common cause of invasive fungal infections in severely immunocompromised patients. Invasive aspergillosis remains associated with significant morbidity and mortality, but the outcome of this disease in hematopoietic stem cell recipients has improved significantly in recent years (22). Although improved survival coincided with multiple changes in transplantation practices, the introduction and use of voriconazole were independently associated with protection from invasive aspergillosis-related death (22). Voriconazole is recommended for primary therapy for most clinical manifestations of invasive aspergillosis, including central nervous system aspergillosis (28). However, the efficacy of voriconazole might be hampered by the development of azole resistance in aspergilli. Several studies have indicated that acquired resistance is an emerging problem (20) and that in vitro resistance is associated with treatment failure (8, 20, 23, 24, 27). Most of the recently reported mechanisms of resistance to azoles include mutations in the fungal mitochondrial cyp51 genes, leading to alterations in the target enzyme 14α-sterol demethylase. The identified different mutations in the fungal cyp51A gene are associated with distinct phenotypes in vitro. These phenotypes are commonly characterized by partial or complete loss of susceptibility to one or more of the mold-active azoles. For instance, a phenotype for amino acid substitutions at glycine 54 (G54) is expressed in vitro by resistance to itraconazole and posaconazole (10, 14), but the activity of voriconazole remains comparable to that of isolates without cyp51A mutations. Isolates harboring the M220I substitution are resistant to itraconazole, while the MIC of posaconazole is elevated compared to that for wild-type isolates. The susceptibility of M220I mutant isolates to voriconazole is comparable to that of wild-type isolates. (12). Another A. fumigatus resistance mechanism involves an amino acid substitution in cyp51A, at codon 98, in combination with the duplication in tandem of a 34-bp sequence in the cyp51A promoter (TR/L98H) (13). The phenotypic profile for this mechanism shows in vitro resistance to itraconazole and reduced susceptibility to posaconazole. The activity of voriconazole in strains with a TR/L98H mutation is variable, ranging from susceptibility of isolates to a complete lack of activity of voriconazole.Given the prominent role of voriconazole in the primary therapy of invasive aspergillosis, it is important to understand the impact of the presence of resistance mechanisms on the efficacy of the drug. Furthermore, it is important to investigate the efficacy of voriconazole in those isolates that are resistant to other azole compounds but remain susceptible to voriconazole in vitro. Using a nonneutropenic murine model of disseminated aspergillosis, we recently showed that the efficacy of posaconazole was dependent on the drug exposure and MIC of the A. fumigatus isolate (11), underscoring the hypothesis that the presence of cyp51 mutations is clinically relevant.The aim of our research was to investigate the pharmacodynamic and pharmacokinetic properties of voriconazole against clinical A. fumigatus isolates with cyp51A mutations and to determine whether the efficacy of the drug was attenuated in isolates with a mutation in the cyp51A gene but a voriconazole-susceptible phenotype.     

Pharmacokinetic Determinants of Virological Response to Raltegravir in the In Vitro Pharmacodynamic Hollow-Fiber Infection Model System

Daily administration (q24h) of raltegravir has been shown to be as efficacious as twice-daily administration (q12h) in the hollow-fiber infection model (HFIM) system. However, q24h regimens were not noninferior to q12h dosing in a clinical trial. We hypothesized that between-patient variability in raltegravir pharmacokinetics (PK) was responsible for the discordance in conclusions between the in vitro and in vivo studies. Hollow-fiber cartridges were inoculated with HIV-infected H9 cells and uninfected CEM-SS cells. Four cartridges received the total daily exposure (800 mg) q24h and four received half the daily exposure (400 mg) q12h. PK profiles with half-lives of 8, 4, 3, and 2 h were simulated for each dosing interval. Cell-to-cell viral spread was assessed by flow cytometry. Viral inhibition was similar between q24h and q12h dosing at the 8- and 4-h half-lives. The q24h dosing was not as efficacious as the q12h dosing when faster half-lives were simulated; a lack of viral suppression was observed at days 3 and 4 for the 2- and 3-h half-lives, respectively. The discrepancy in conclusions between the in vitro HFIM system studies and clinical trials is likely due to the large interindividual variation in raltegravir PK.     

In Vivo Pharmacokinetic/Pharmacodynamic Profiles of Valnemulin in an Experimental Intratracheal Mycoplasma gallisepticum Infection Model

Valnemulin, a semisynthetic pleuromutilin antibiotic derivative, is greatly active against Mycoplasma. The objective of our study was to evaluate the effectiveness of valnemulin against Mycoplasma gallisepticum in a neutropenic intratracheal model in chickens using a pharmacokinetic/pharmacodynamic (PK-PD) method. The PK of valnemulin after intramuscular (i.m.) administration at doses of 1, 10, and 20 mg/kg of body weight in M. gallisepticum-infected neutropenic chickens was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Real-time PCR (RT-PCR) was used for quantitative detection of M. gallisepticum. The ratio of the 24-h area under the concentration-time curve divided by the MIC (AUC₂₄/MIC) correlated well with the in vivo antibacterial effectiveness of valnemulin (R² = 0.9669). The AUC₂₄/MIC ratios for mycoplasmastasis (a reduction of 0 log₁₀ color-changing unit [CCU] equivalents/ml), a reduction of 1 log₁₀ CCU equivalents/ml, and a reduction of 2.5 log₁₀ CCU equivalents/ml are 28,820, 38,030, and 56,256, respectively. In addition, we demonstrated that valnemulin at a dose of 6.5 mg/kg resulted in a reduction of 2.5 log₁₀ CCU equivalents/ml. These investigations provide a solid foundation for the usage of valnemulin in poultry with M. gallisepticum infections.     

Single-Dose Pharmacodynamics of Amphotericin B against Aspergillus Species in an In Vitro Pharmacokinetic/Pharmacodynamic Model

Conventional MIC testing of amphotericin B results in narrow MIC ranges challenging the detection of resistant strains. In order to discern amphotericin B pharmacodynamics, the in vitro activity of amphotericin B was studied against Aspergillus isolates with the same MICs by using a new in vitro pharmacokinetic/pharmacodynamic (PK/PD) model that simulates amphotericin B human plasma levels. Clinical isolates of Aspergillus fumigatus, A. terreus, and A. flavus with the same Clinical and Laboratory Standards Institute modal MICs of 1 mg/liter were exposed to amphotericin B concentrations following the plasma concentration-time profile after single-bolus administration with C_max values of 0.6, 1.2, 2.4, and 4.8 mg/liter. Fungal growth was monitored for up to 72 h based on galactomannan production. Complete growth inhibition was observed only against A. fumigatus with amphotericin B with a C_max of ≥2.4 mg/liter. At the lower C_max values 0.6 and 1.2 mg/liter, significant growth delays of 34 and 52 h were observed, respectively (P < 0.001). For A. flavus, there was no complete inhibition but a progressive growth delay of 1 to 50 h at an amphotericin B C_max of 0.6 to 4.8 mg/liter (P < 0.001). For A. terreus, the growth delay was modest (up to 8 h) at all C_maxs (P < 0.05). The C_max (95% confidence interval) associated with 50% activity for A. fumigatus was 0.60 (0.49 to 0.72) mg/liter, which was significantly lower than for A. flavus 3.06 (2.46 to 3.80) mg/liter and for A. terreus 7.90 (5.20 to 12.29) mg/liter (P < 0.001). A differential in vitro activity of amphotericin B was found among Aspergillus species despite the same MIC in the order A. fumigatus > A. flavus > A. terreus in the in vitro PK/PD model, possibly reflecting the different concentration- and time-dependent inhibitory/killing activities amphotericin B exerted against these species.     

Clofazimine Contributes Sustained Antimicrobial Activity after Treatment Cessation in a Mouse Model of Tuberculosis Chemotherapy

Experimental and clinical studies have indicated that the antileprosy drug clofazimine may contribute treatment-shortening activity when included in tuberculosis treatment regimens. Clofazimine accumulates to high levels in tissues, has a long half-life, and remains in the body for months after administration is stopped. We hypothesized that in tuberculosis treatment, accumulated clofazimine may contribute sustained antimicrobial activity after treatment cessation, and we used the BALB/c mouse model of chronic tuberculosis chemotherapy to address this hypothesis. Mycobacterium tuberculosis-infected mice were treated for 4 weeks or 8 weeks with either isoniazid alone, clofazimine alone, the first-line regimen rifampin-isoniazid-pyrazinamide-ethambutol, or a first-line regimen where clofazimine was administered in place of ethambutol. To evaluate posttreatment antimicrobial activity, bacterial regrowth in the lungs and spleens was assessed at the day of treatment cessation and 2, 4, 6, and 8 weeks after treatment was stopped. Bacterial regrowth was delayed in all mice receiving clofazimine, either alone or in combination, compared to the mice that did not receive clofazimine. This effect was especially evident in mice receiving multidrug therapy. In mice not receiving clofazimine, bacterial regrowth began almost immediately after treatment was stopped, while in mice receiving clofazimine, bacterial regrowth was delayed for up to 6 weeks, with the duration of sustained antimicrobial activity being positively associated with the time that serum clofazimine levels remained at or above the 0.25-μg/ml MIC for M. tuberculosis. Thus, sustained activity of clofazimine may be important in the treatment-shortening effect associated with this drug.