Mycobacterium tuberculosis sterilizing activity of faropenem, pyrazinamide and linezolid combination and failure to shorten the therapy duration.

BACKGROUND: Faropenem (F), an orally bioavailable ß-lactam, kills Mycobacterium tuberculosis (Mtb) without the help of a ß-lactamase inhibitor. This study explored the sterilizing effect of adding F once or twice daily to a linezolid (L) plus pyrazinamide (Z) backbone regimen. METHODS: In vitro studies were performed using the hollow fiber model of tuberculosis (HFS-TB) to compare the kill rates of: 1) ZL two-drug combination; 2) F administered once daily plus ZL (F1ZL); 3) F administered twice-daily plus once daily ZL (F2ZL); 4) F2ZL with high-dose Z (F2ZhiL); 5) standard therapy of isoniazid, rifampin and Z; and 6) non-treated controls. The study was performed over 56 days with three HFS-TB replicates for each regimen. RESULTS: Mtb in the non-treated HFS-TB grew at a rate of 0.018 ± 0.007 log10 CFU/mL/day. The exponential kill rates for standard therapy were 6.6-13.2-fold higher than ZL dual therapy. The F1ZL and F2ZL regimens ranked third. The pre-existing isoniazid-resistant sub-population in the inoculum (1.34 ± 0.57 log10 CFU/mL) grew to 4.21 ± 0.58 log10 CFU/mL in 56 days in non-treated HFS-TB. However, no isoniazid-resistant sub-population was recorded in any of the FZL combination regimens. CONCLUSION: Due to the slow kill rate compared to standard therapy, FZL regimens are unlikely to shorten therapy duration. Efficacy of these regimens against drug-resistant tuberculosis needs to be determined.

A 'shock and awe' thioridazine and moxifloxacin combination-based regimen for pulmonary Mycobacterium avium-intracellulare complex disease.

OBJECTIVES: To develop a thioridazine/moxifloxacin-based combination regimen for treatment of pulmonary infection due to Mycobacterium avium-intracellulare complex (MAC) that kills bacteria faster than the standard treatment regimen. METHODS: Monocytes were infected with MAC and inoculated into the hollow-fibre system model for pulmonary MAC disease (HFS-MAC). We co-administered ethambutol plus azithromycin daily for 28 days, to achieve the same human concentration-time profiles that result from standard doses, in three HFS-MAC systems. Two experimental regimens consisted of thioridazine at an exposure associated with optimal kill, given intermittently on days 0, 3, 7 and 10. Regimen A consisted of thioridazine in combination with standard dose azithromycin for the entire study duration. Regimen B was thioridazine plus moxifloxacin at concentration-time profiles achieved by the standard daily dose administered for 14 days, followed by daily azithromycin. Each HFS-MAC was sampled for bacterial burden every 7 days. RESULTS: The bacteria in the non-treated HFS-MAC grew at a rate of 0.11 ±âŸ0.01 log10 cfu/mL/day. The azithromycin/ethambutol regimen decreased bacterial burden by 1.21 ±âŸ0.74 log10 cfu/mL below baseline during the first 7 days, after which it failed. Regimen A killed 3.28 ±âŸ0.32 log10 cfu/mL below baseline up to day 14, after which regrowth occurred once thioridazine treatment stopped. Regimen B killed bacteria to below the limits of detection in 7 days (≥5.0 log10 cfu/mL kill), with rebound in the azithromycin continuation phase. CONCLUSIONS: The thioridazine/moxifloxacin regimen demonstrated that rapid microbial kill could be achieved within 7 days. This is a proof of principle that short-course chemotherapy for pulmonary MAC is possible.

Linezolid Dose That Maximizes Sterilizing Effect While Minimizing Toxicity and Resistance Emergence for Tuberculosis.

Linezolid has an excellent sterilizing effect in tuberculosis patients but high adverse event rates. The dose that would maximize efficacy and minimize toxicity is unknown. We performed linezolid dose-effect and dose-scheduling studies in the hollow fiber system model of tuberculosis (HFS-TB) for sterilizing effect. HFS-TB units were treated with several doses to mimic human-like linezolid intrapulmonary pharmacokinetics and repetitively sampled for drug concentration, total bacterial burden, linezolid-resistant subpopulations, and RNA sequencing over 2 months. Linezolid-resistant isolates underwent whole-genome sequencing. The expression of genes encoding efflux pumps in the first 1 to 2 weeks revealed the same exposure-response patterns as the linezolid-resistant subpopulation. Linezolid-resistant isolates from the 2nd month of therapy revealed mutations in several efflux pump/transporter genes and a LuxR-family transcriptional regulator. Linezolid sterilizing effect was linked to the ratio of unbound 0- to 24-h area under the concentration-time curve (AUC0-24) to MIC. Optimal microbial kill was achieved at an AUC0-24/MIC ratio of 119. The optimal sterilizing effect dose for clinical use was identified using Monte Carlo simulations. Clinical doses of 300 and 600 mg/day (or double the dose every other day) achieved this target in 87% and >99% of 10,000 patients, respectively. The susceptibility breakpoint identified was 2 mg/liter. The simulations identified that a 300-mg/day dose did not achieve AUC0-24s associated with linezolid toxicity, while 600 mg/day achieved those AUC0-24s in <20% of subjects. The linezolid dose of 300 mg/day performed well and should be compared to 600 mg/day or 1,200 mg every other day in clinical trials.

A Long-term Co-perfused Disseminated Tuberculosis-3D Liver Hollow Fiber Model for Both Drug Efficacy and Hepatotoxicity in Babies.

Treatment of disseminated tuberculosis in children≤6years has not been optimized. The pyrazinamide-containing combination regimen used to treat disseminated tuberculosis in babies and toddlers was extrapolated from adult pulmonary tuberculosis. Due to hepatotoxicity worries, there are no dose-response studies in children. We designed a hollow fiber system model of disseminated intracellular tuberculosis with co-perfused three-dimensional organotypic liver modules to simultaneously test for efficacy and toxicity. We utilized pediatric pharmacokinetics of pyrazinamide and acetaminophen to determine dose-dependent pyrazinamide efficacy and hepatotoxicity. Acetaminophen concentrations that cause hepatotoxicity in children led to elevated liver function tests, while 100mg/kg pyrazinamide did not. Surprisingly, pyrazinamide did not kill intracellular Mycobacterium tuberculosis up to fourfold the standard dose as monotherapy or as combination therapy, despite achieving high intracellular concentrations. Host-pathogen RNA-sequencing revealed lack of a pyrazinamide exposure transcript signature in intracellular bacteria or of phagolysosome acidification on pH imaging. Artificial intelligence algorithms confirmed that pyrazinamide was not predictive of good clinical outcomes in children≤6years who had extrapulmonary tuberculosis. Thus, adding a drug that works inside macrophages could benefit children with disseminated tuberculosis. Our in vitro model can be used to identify such new regimens that could accelerate cure while minimizing toxicity.

Amikacin Pharmacokinetics/Pharmacodynamics in a Novel Hollow-Fiber Mycobacterium abscessus Disease Model.

The treatment of pulmonary Mycobacterium abscessus disease is associated with very high failure rates and easily acquired drug resistance. Amikacin is the key drug in treatment regimens, but the optimal doses are unknown. No good preclinical model exists to perform formal pharmacokinetics/pharmacodynamics experiments to determine these optimal doses. We developed a hollow-fiber system model of M. abscessus disease and studied amikacin exposure effects and dose scheduling. We mimicked amikacin human pulmonary pharmacokinetics. Both amikacin microbial kill and acquired drug resistance were linked to the peak concentration-to-MIC ratios; the peak/MIC ratio associated with 80% of maximal kill (EC80) was 3.20. However, on the day of the most extensive microbial kill, the bacillary burden did not fall below the starting inoculum. We performed Monte Carlo simulations of 10,000 patients with pulmonary M. abscessus infection and examined the probability that patients treated with one of 6 doses from 750 mg to 4,000 mg would achieve or exceed the EC80. We also examined these doses for the ability to achieve a cumulative area under the concentration-time curve of 82,232 mg · h/liter × days, which is associated with ototoxicity. The standard amikacin doses of 750 to 1,500 mg a day achieved the EC80 in ≤ 21% of the patients, while a dose of 4 g/day achieved this in 70% of the patients but at the cost of high rates of ototoxicity within a month or two. The susceptibility breakpoint was an MIC of 8 to 16 mg/liter. Thus, amikacin, as currently dosed, has limited efficacy against M. abscessus. It is urgent that different antibiotics be tested using our preclinical model and new regimens developed.

Rapid drug tolerance and dramatic sterilizing effect of moxifloxacin monotherapy in a novel hollow-fiber model of intracellular Mycobacterium kansasii disease.

Mycobacterium kansasii is the second most common mycobacterial cause of lung disease. Standard treatment consists of rifampin, isoniazid, and ethambutol for at least 12 months after negative sputum. Thus, shorter-duration therapies are needed. Moxifloxacin has good MICs for M. kansasii. However, good preclinical models to identify optimal doses currently are lacking. We developed a novel hollow fiber system model of intracellular M. kansasii infection. We indexed the efficacy of the standard combination regimen, which was a kill rate of -0.08 ± 0.05 log10 CFU/ml/day (r(2) = 0.99). We next performed moxifloxacin dose-effect and dose-scheduling studies at a half-life of 11.1 ± 6.47 h. Some systems also were treated with the efflux pump inhibitor reserpine. The highest moxifloxacin exposure, as well as lower exposures plus reserpine, sterilized the cultures by day 7. This suggests that efflux pump-mediated tolerance at low ratios of the area under the concentration-time curve from 0 to 24 h (AUC0 - 24) to MICs is an early bacterial defense mechanism but is overcome by higher exposures. The highest rate of moxifloxacin monotherapy sterilization was -0.82 ± 0.15 log10 CFU/ml/day (r(2) = 0.97). The moxifloxacin exposure associated with 80% of maximal kill (EC80) was an AUC0-24/MIC of 317 (the non-protein-bound moxifloxacin AUC0-24/MIC was 158.5). We performed Monte Carlo simulations of 10,000 patients in order to identify the moxifloxacin dose that would achieve or exceed the EC80. The simulations revealed an optimal moxifloxacin dose of 800 mg a day. The MIC susceptibility breakpoint at this dose was 0.25 mg/liter. Thus, moxifloxacin, at high enough doses, is suitable to study in patients for the potential to add rapid sterilization to the standard regimen.

Pharmacokinetic mismatch does not lead to emergence of isoniazid- or rifampin-resistant Mycobacterium tuberculosis but to better antimicrobial effect: a new paradigm for antituberculosis drug scheduling.

Multidrug resistant-tuberculosis is a pressing problem. One of the major mechanisms proposed to lead to the emergence of drug resistance is pharmacokinetic mismatch. Stated as a falsifiable hypothesis, the greater the pharmacokinetic mismatch between rifampin and isoniazid, the higher the isoniazid- and rifampin-resistant subpopulation sizes become with time. To test this, we performed hollow-fiber-system studies for both bactericidal and sterilizing effects in experiments of up to 42 days. We mimicked pharmacokinetics of 600-mg/day rifampin and 300-mg/day isoniazid administered to patients. Rifampin was administered first, followed by isoniazid 0, 6, 12, and 24 h later. The treatment was for drug-susceptible Mycobacterium tuberculosis in some experiments and hollow fiber systems with inoculum preseeded with isoniazid- and rifampin-resistant isogenic Mycobacterium tuberculosis strains in others. Analysis of variance revealed that the 12-h and 24-h-mismatched regimens always killed better than the matched regimens during both bactericidal and sterilizing effects (P < 0.05). This means that either the order of scheduling or the sequential administration of drugs in combination therapy may lead to significant improvement in microbial killing. Rifampin-resistant and isoniazid-resistant subpopulations were not significantly higher with increased mismatching in numerous analysis-of-variance comparisons. Thus, the pharmacokinetic mismatch hypothesis was rejected. Instead, sequential administration of anti-tuberculosis (TB) drugs (i.e., deliberate mismatch) following particular schedules suggests a new paradigm for accelerating M. tuberculosis killing. We conclude that current efforts aimed at better pharmacokinetic matching to decrease M. tuberculosis resistance emergence are likely futile and counterproductive.

Efflux-pump-derived multiple drug resistance to ethambutol monotherapy in Mycobacterium tuberculosis and the pharmacokinetics and pharmacodynamics of ethambutol.

BACKGROUND: Ethambutol is used for the treatment of tuberculosis in cases where there is isoniazid resistance. We examined the emergence of drug resistance to ethambutol monotherapy in pharmacokinetic-pharmacodynamic studies of a hollow-fiber system. METHODS: Dose-effect and dose-scheduling studies were performed with ethambutol and log-phase growth Mycobacterium tuberculosis to identify exposures and schedules linked to optimal kill and resistance suppression. In one study, after 7 days of daily ethambutol, 300 mg isoniazid per day was administered to each system to determine its early bactericidal activity. RESULTS: Efflux-pump blockage reduced the mutation frequency to ethambutol 64-fold. In dose-effect studies, ethambutol had a maximal early bactericidal activity of 0.22 log10 colony-forming units/mL/day, as is encountered in patients. By day 7, resistance to both ethambutol and isoniazid had increased. Previous exposure to ethambutol halted isoniazid early bactericidal activity. Daily therapy, as opposed to more intermittent therapy, was associated with the least proportion of efflux-pump-driven resistance, consistent with a time-driven effect. Microbial kill was best explained by the ratio of area under the concentration-time curve to minimum inhibitory concentration (r2 = 0.90). CONCLUSION: The induction of an efflux pump that reduces the effect of multiple drugs provides an alternative pathway to sequential acquisition of mutations in the development of multiple drug resistance.