Omadacycline pharmacokinetics/pharmacodynamics and efficacy against multidrug-resistant Mycobacterium tuberculosis in the hollow fiber system model.

Seventy-five years ago, first-generation tetracyclines demonstrated limited efficacy in the treatment of tuberculosis but were more toxic than efficacious. We performed a series of pharmacokinetic/pharmacodynamic (PK/PD) experiments with a potentially safer third-generation tetracycline, omadacycline, for the treatment of multidrug-resistant tuberculosis (MDR-TB). Mycobacterium tuberculosis (Mtb) H37Rv and an MDR-TB clinical strain (16D) were used in the minimum inhibitory concentration (MIC) and static concentration-response studies in test tubes, followed by a PK/PD study using the hollow fiber system model of TB (HFS-TB) that examined six human-like omadacycline doses. The inhibitory sigmoid maximal effect (Emax) model and Monte Carlo experiments (MCEs) were used for data analysis and clinical dose-finding, respectively. The omadacycline MIC for both Mtb H37Rv and MDR-TB clinical strain was 16 mg/L but dropped to 4 mg/L with daily drug supplementation to account for omadacycline degradation. The Mycobacteria Growth Indicator Tube MIC was 2 mg/L. In the test tubes, omadacycline killed 4.39 log10 CFU/mL in 7 days. On Day 28 of the HFS-TB study, the Emax was 4.64 log10 CFU/mL, while exposure mediating 50% of Emax (EC50) was an area under the concentration-time curve to MIC (AUC0-24/MIC) ratio of 22.86. This translates to PK/PD optimal exposure or EC80 as AUC0-24/MIC of 26.93. The target attainment probability of the 300-mg daily oral dose was 90% but fell at MIC ≧4 mg/L. Omadacycline demonstrated efficacy and potency against both drug-susceptible and MDR-TB. Further studies are needed to identify the omadacycline effect in combination therapy for the treatment of both drug-susceptible and MDR-TB.

Meropenem-vaborbactam restoration of first-line drug efficacy and comparison of meropenem-vaborbactam-moxifloxacin versus BPaL MDR-TB regimen.

BACKGROUND: Meropenem in combination with ß-lactamase inhibitors (BLIs) and other drugs was tested to identify alternative treatment regimens for multidrug-resistant tuberculosis (MDR-TB). METHODS: The following were performed: (1) MIC experiments; (2) static time-kill studies (STKs) with different BLIs; and (3) a hollow fibre model system of TB (HFS-TB) studies with meropenem-vaborbactam combined with human equivalent daily doses of 20 mg/kg or 35 mg/kg rifampin, or moxifloxacin 400 mg, or linezolid 600 mg vs. bedaquiline-pretonamid-linezolid (BPaL) for MDR-TB. The studies were performed using Mycobacterium tuberculosis (M. tuberculosis) H37Rv and an MDR-TB clinical strain (named M. tuberculosis 16D) that underwent whole genome sequencing. Exponential decline models were used to calculate the kill rate constant (K) of different HFS-TB regimens. RESULTS: Whole genome sequencing revealed mutations associated with resistance to rifampin, isoniazid, and cephalosporins. The meropenem-vaborbactam MIC of M. tuberculosis was H37Rv 2 mg/L and > 128 mg/L for M. tuberculosis 16D. Relebactam and vaborbactam improved both the potency and efficacy of meropenem in STKs. Meropenem-vaborbactam alone failed to kill M. tuberculosis 16D but killed below day 0 burden when combined with isoniazid and rifampin, with the moxifloxacin combination being the most effective and outranking bedaquiline and pretomanid. In the HFS-TB, meropenem-vaborbactam-moxifloxacin and BPaL had the highest K (log10 cfu/mL/day) of 0.31 (95% CI 0.17-0.58) and 0.34 (95% CI 0.21-0.56), while meropenem-vaborbactam-rifampin (35 mg/kg) had a K of 0.18 (95% CI 0.12-0.25). The K for meropenem-vaborbactam-moxifloxacin-linezolid demonstrated antagonism. CONCLUSION: Adding meropenem-vaborbactam could potentially restore the efficacy of isoniazid and rifampin against MDR-TB. The meropenem-vaborbactam-moxifloxacin backbone regimen has implications for creating a new effective MDR-TB regimen.

Isoniazid pharmacokinetics/pharmacodynamics as monotherapy and in combination regimen in the hollow fiber system model of Mycobacterium kansasii.

BACKGROUND: There is limited high quality evidence to guide the optimal doses of drugs for the treatment of Mycobacterium kansasii pulmonary disease (Mkn-PD). METHODS: We performed (1) minimum inhibitory concentration experiment, (2) isoniazid dose-response study using the hollow fiber system model (HFS-Mkn) to determine PK/PD optimized exposure, and (3) another HFS-Mkn study to determine the efficacy of high dose isoniazid (15 mg/kg/day) with standard dose rifampin (10 mg/kg/day) and ethambutol (15 mg/kg/day). Inhibitory sigmoid maximal effect model and linear regression was used for data analysis. RESULTS: MIC of the 20 clinical isolates ranged between 0.5 mg/L to 32 mg/L. In the HFS-Mkn, isoniazid monotherapy failed to control the bacterial growth beyond day 7. On day 7, when the maximal Mkn kill was observed, the optimal isoniazid exposure for Mkn kill was calculated as 24hr area under the concentration-time curve to the MIC of 12.41. Target attainment probability of 300 mg/day dose fell below 90% above the MIC of 1 mg/L. High dose isoniazid combination sterilized the HFS-Mkn in 30-days with a kill rate of -0.15 ± 0.02 log10 CFU/mL/day. CONCLUSION: Despite initial kill, isoniazid monotherapy failed due to resistance emergence. Our pre-clinical model derived results suggest that higher than currently recommended 300 mg/day isoniazid dose may achieve better clinical efficacy against Mkn-PD.

Minocycline intra-bacterial pharmacokinetic hysteresis as a basis for pharmacologic memory and a backbone for once-a-week pan-tuberculosis therapy.

Background: There is need for shorter duration regimens for the treatment of tuberculosis, that can treat patients regardless of multidrug resistance status (pan-tuberculosis). Methods: We combined minocycline with tedizolid, moxifloxacin, and rifampin, in the hollow fiber system model of tuberculosis and mimicked each drugs' intrapulmonary pharmacokinetics for 28 days. Minocycline-tedizolid was administered either as a once-a-week or a daily regimen. In order to explore a possible explanation for effectiveness of the once-a-week regimen, we measured systemic and intra-bacterial minocycline pharmacokinetics. Standard daily therapy (rifampin, isoniazid, pyrazinamide) was the comparator. We then calculated γ f or kill slopes for each regimen and ranked the regimens by time-to-extinction predicted in patients. Results: The steepest γ f and shortest time-to-extinction of entire bacterial population was with daily minocycline-rifampin combination. There was no difference in γ f between the minocycline-tedizolid once-a-week versus the daily therapy (p = 0.85). Standard therapy was predicted to cure 88% of patients, while minocycline-rifampin would cure 98% of patients. Minocycline concentrations fell below minimum inhibitory concentration after 2 days of once-weekly dosing schedule. The shape of minocycline intra-bacterial concentration-time curve differed from the extracellular pharmacokinetic system and lagged by several days, consistent with system hysteresis. Hysteresis explained the persistent microbial killing after hollow fiber system model of tuberculosis concentrations dropped below the minimum inhibitory concentration. Conclusion: Minocycline could form a backbone of a shorter duration once-a-week pan-tuberculosis regimen. We propose a new concept of post-antibiotic microbial killing, distinct from post-antibiotic effect. We propose system hysteresis as the basis for the novel concept of pharmacologic memory, which allows intermittent dosing.

Rifampin Pharmacokinetics/Pharmacodynamics in the Hollow-Fiber Model of Mycobacterium kansasii Infection.

There is limited high-quality evidence to guide the optimal treatment of Mycobacterium kansasii pulmonary disease. We retrospectively collected clinical data from 33 patients with M. kansasii pulmonary disease to determine the time-to-sputum culture conversion (SCC) upon treatment with a standard combination regimen consist of isoniazid-rifampin-ethambutol. Next, MIC experiments with 20 clinical isolates were performed, followed by a dose-response study with the standard laboratory strain using the hollow-fiber system model of M. kansasii infection (HFS-Mkn). The inhibitory sigmoid maximum effect (Emax) model was used to describe the relationship between the bacterial burden and rifampin concentrations. Finally, in silico clinical trial simulations were performed to determine the clinical dose to achieve the optimal rifampin exposure in patients. The SCC rate in patients treated with combination regimen containing rifampin at 10 mg/kg of body weight/day was 73%, the mean time to SSC was 108 days, and the mean duration of therapy was 382 days. The MIC of the M. kansasii laboratory strain was 0.125 mg/L, whereas the MICs of the clinical isolates ranged between 0.5 and 4 mg/L. In the HFS-Mkn model, a maximum kill (Emax) of 7.82 log10 CFU/mL was recorded on study day 21. The effective concentration mediating 80% of the Emax (EC80) was calculated as the ratio of the maximum concentration of drug in serum for the free, unbound fraction (fCmax) to MIC of 34.22. The target attainment probability of the standard 10-mg/kg/day dose fell below 90% even at the MIC of 0.0625 mg/L. Despite the initial kill, there was M. kansasii regrowth with the standard rifampin dose in the HFS-Mkn model. Doses higher than 10 mg/kg/day, in combination with other drugs, need to be evaluated for better treatment outcome.

Erratum: Cefdinir and ß-Lactamase Inhibitor Independent Efficacy Against Mycobacterium tuberculosis.

[This corrects the article DOI: 10.3389/fphar.2021.677005.].

Cefdinir and ß-Lactamase Inhibitor Independent Efficacy Against Mycobacterium tuberculosis.

Background: There is renewed interest in repurposing ß-lactam antibiotics for treatment of tuberculosis (TB). We investigated efficacy of cefdinir, that withstand the ß-lactamase enzyme present in many bacteria, against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis (Mtb). Methods: Minimum inhibitory concentration (MIC) experiments were performed with Mtb H37Ra, eight drug-susceptible, and 12 MDR-TB clinical isolates with and without the ß-lactamase inhibitor, avibactam at 15 mg/L final concentration. Next, we performed dose-response study with Mtb H37Ra in test-tubes followed by a sterilizing activity study in the pre-clinical hollow fiber model of tuberculosis (HFS-TB) study using an MDR-TB clinical strain. Inhibitory sigmoid Emax model was used to describe the relationship between the drug exposure and bacterial burden. Results: Cefdinir MIC for Mtb H37Ra was 4 and 2 mg/L with or without avibactam, respectively. The MIC of the clinical strains ranged between 0.5 and 16 mg/L. In the test-tube experiments, cefdinir killed 4.93 + 0.07 log10 CFU/ml Mtb H37Ra in 7 days. In the HFS-TB studies, cefdinir showed dose-dependent killing of MDR-TB, without combination of avibactam. The cefdinir PK/PD index linked to the Mtb sterilizing efficacy was identified as the ratio of area under the concentration-time curve to MIC (AUC0-24/MIC) and optimal exposure was calculated as AUC0-24/MIC of 578.86. There was no resistance emergence to cefdinir in the HFS-TB. Conclusion: In the HFS-TB model, cefdinir showed efficacy against both drug susceptible and MDR-TB without combination of ß-lactamase inhibitor. However, clinical validation of these findings remains to be determined.

Modeling of Effective Antimicrobials to Reduce Staphylococcus aureus Virulence Gene Expression Using a Two-Compartment Hollow Fiber Infection Model.

Toxins produced by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) contribute to virulence. We developed a statistical approach to determine an optimum sequence of antimicrobials to treat CA-MRSA infections based on an antimicrobial's ability to reduce virulence. In an in vitro pharmacodynamic hollow fiber model, expression of six virulence genes (lukSF-PV, sek, seq, ssl8, ear, and lpl10) in CA-MRSA USA300 was measured by RT-PCR at six time points with or without human-simulated, pharmacokinetic dosing of five antimicrobials (clindamycin, minocycline, vancomycin, linezolid, and trimethoprim/sulfamethoxazole (SXT)). Statistical modeling identified the antimicrobial causing the greatest decrease in virulence gene expression at each time-point. The optimum sequence was SXT at T0 and T4, linezolid at T8, and clindamycin at T24-T72 when lukSF-PV was weighted as the most important gene or when all six genes were weighted equally. This changed to SXT at T0-T24, linezolid at T48, and clindamycin at T72 when lukSF-PV was weighted as unimportant. The empirical p-value for each optimum sequence according to the different weights was 0.001, 0.0009, and 0.0018 with 10,000 permutations, respectively, indicating statistical significance. A statistical method integrating data on change in gene expression upon multiple antimicrobial exposures is a promising tool for identifying a sequence of antimicrobials that is effective in sustaining reduced CA-MRSA virulence.

Tedizolid, Faropenem, and Moxifloxacin Combination With Potential Activity Against Nonreplicating Mycobacterium tuberculosis.

Background: Mycobacterium tuberculosis [Mtb] could be present in different metabolic population in the lung lesions, and nonreplicating persisters [NRP], associated with latent tuberculosis [TB], are the most difficult to kill. Objective: Test the combination of tedizolid, moxifloxacin, and faropenem for activity against NRP using Mtb SS18b in the hollow fiber model [HFS-TB]. Methods: Tedizolid and moxifloxacin were tested as, first, two-drug combination against log-phase growth [LPG] and, second, slowly replicating bacilli [SRB] under acidic condition and with faropenem to create a three-drug combination regimen. Finally, standard regimen [isoniazid-rifampin-pyrazinamide] was used as comparator in the HFS-TB experiment with NRP Mtb. HFS-TB units were sampled for drug-concentration measurement as well as for estimation of bacterial burden using solid agar and mycobacterial growth indicator tube [MGIT] method. Linear regression was used to calculate the kill slopes with each treatment regimen and analysis of variance (ANOVA) to compare the regimen. Results: Tedizolid at standard dose in combination with high-dose moxifloxacin killed 3.05 log10 CFU/ml LPG Mtb and 7.37 log10 CFU/ml SRB in the bactericidal and sterilizing activity HFS-TB experiments, respectively. There was no statistical difference between tedizolid-moxifloxacin-faropenem combination and the standard regimen as both killed 7.35 log10 CFU/ml NRP Mtb in 21 days. There was no emergence of resistance to any of the drugs studied in the three HFS-TB experiments. Conclusion: The experimental regimen of tedizolid, moxifloxacin, and faropenem could effectively kill NRP population of Mtb, and given the efficacy against different metabolic population of Mtb could serve as a pan-TB regimen. Clinical studies are warranted to validate the in vitro findings.

Exploring the Pharmacokinetic/Pharmacodynamic Relationship of Relebactam (MK-7655) in Combination with Imipenem in a Hollow-Fiber Infection Model.

Resistance to antibiotics among bacterial pathogens is rapidly spreading, and therapeutic options against multidrug-resistant bacteria are limited. There is an urgent need for new drugs, especially those that can circumvent the broad array of resistance pathways that bacteria have evolved. In this study, we assessed the pharmacokinetic/pharmacodynamic relationship of the novel ß-lactamase inhibitor relebactam (REL; MK-7655) in a hollow-fiber infection model. REL is intended for use with the carbapenem ß-lactam antibiotic imipenem for the treatment of Gram-negative bacterial infections. In this study, we used an in vitro hollow-fiber infection model to confirm the efficacy of human exposures associated with the phase 2 doses (imipenem at 500 mg plus REL at 125 or 250 mg administered intravenously every 6 h as a 30-min infusion) against imipenem-resistant strains of Pseudomonas aeruginosa and Klebsiella pneumoniae Dose fractionation experiments confirmed that the pharmacokinetic parameter that best correlated with REL activity is the area under the concentration-time curve, consistent with findings in a murine pharmacokinetic/pharmacodynamic model. Determination of the pharmacokinetic/pharmacodynamic relationship between ß-lactam antibiotics and ß-lactamase inhibitors is complex, as there is an interdependence between their respective exposure-response relationships. Here, we show that this interdependence could be captured by treating the MIC of imipenem as dynamic: it changes with time, and this change is directly related to REL levels. For the strains tested, the percentage of the dosing interval time that the concentration remains above the dynamic MIC for imipenem was maintained at the carbapenem target of 30 to 40%, required for maximum efficacy, for imipenem at 500 mg plus REL at 250 mg.

Systematic Review and Meta-analyses of the Effect of Chemotherapy on Pulmonary Mycobacterium abscessus Outcomes and Disease Recurrence.

In pharmacokinetic/pharmacodynamic models of pulmonary Mycobacterium abscessus complex, the recommended macrolide-containing combination therapy has poor kill rates. However, clinical outcomes are unknown. We searched the literature for studies published between 1990 and 2017 that reported microbial outcomes in patients treated for pulmonary M. abscessus disease. A good outcome was defined as sustained sputum culture conversion (SSCC) without relapse. Random effects models were used to pool studies and estimate proportions of patients with good outcomes. Odds ratios (OR) and 95% confidence intervals (CI) were computed. Sensitivity analyses and metaregression were used to assess the robustness of findings. In 19 studies of 1,533 patients, combination therapy was administered to 508 patients with M. abscessus subsp. abscessus, 204 with M. abscessus subsp. massiliense, and 301 with M. abscessus with no subspecies specified. Macrolide-containing regimens achieved SSCC in only 77/233 (34%) new M. abscessus subsp. abscessus patients versus 117/141 (54%) M. abscessus subsp. massiliense patients (OR, 0.108 [95% CI, 0.066 to 0.181]). In refractory disease, SSCC was achieved in 20% (95% CI, 7 to 36%) of patients, which was not significantly different across subspecies. The estimated recurrent rates per month were 1.835% (range, 1.667 to 3.196%) for M. abscessus subsp. abscessus versus 0.683% (range, 0.229 to 1.136%) for M. abscessus subsp. massiliense (OR, 6.189 [95% CI, 2.896 to 13.650]). The proportion of patients with good outcomes was 52/223 (23%) with M. abscessus subsp. abscessus versus 118/141 (84%) with M. abscessus subsp. massiliense disease (OR, 0.059 [95% CI, 0.034 to 0.101]). M. abscessus subsp. abscessus pulmonary disease outcomes with the currently recommended regimens are atrocious, with outcomes similar to those for extensively drug-resistant tuberculosis. Therapeutically, the concept of nontuberculous mycobacteria is misguided. There is an urgent need to craft entirely new treatment regimens.

Linezolid for Infants and Toddlers With Disseminated Tuberculosis: First Steps.

BACKGROUND: Infants and toddlers often present with disseminated and lymph node tuberculosis, in which Mycobacterium tuberculosis (Mtb) is predominantly intracellular. Linezolid, used to treat tuberculosis in adults, has not been formally studied in infants. Infants clear linezolid 5 times faster than adults and achieve lower 0- to 24-hour area under the concentration-time curves (AUC0-24). METHODS: To mimic intracellular disease, we infected human-derived THP-1 macrophages with Mtb and inoculated hollow fiber systems. We performed dose-effect and dose-scheduling studies in which we recapitulated the linezolid half-life of 3 hours encountered in infants. Repetitive sampling for linezolid pharmacokinetics, Mtb intracellular burden, viable monocyte count, and RNA sequencing reads were performed up to 28 days. RESULTS: The linezolid extracellular half-life was 2.64 ± 0.38 hours, whereas intracellular half-life was 8.93 ± 1.30 hours (r2 = 0.89). Linezolid efficacy was linked to the AUC0-24 to minimum inhibitory concentration (MIC) ratio (r2 = 0.98). The exposure associated with maximal Mtb kill was an AUC0-24/MIC of 23.37 ± 1.16. We identified a 414-gene transcript on exposure to toxic linezolid doses. The largest number of genes mapped to ribosomal proteins, a signature hitherto not associated with linezolid toxicity. The second-largest number of differentially expressed genes mapped to mitochondrial enzyme inhibition. Linezolid AUC0-24 best explained the mitochondrial gene inhibition, with 50% inhibition at 94 mg × hour/L (highest r2 = 0.98). CONCLUSIONS: We identified the linezolid AUC0-24/MIC target for optimal efficacy against pediatric intracellular tuberculosis, and an AUC0-24 threshold associated with mitochondrial inhibition. These constitute a therapeutic window to be targeted for optimal linezolid doses in children with tuberculosis.

A Faropenem, Linezolid, and Moxifloxacin Regimen for Both Drug-Susceptible and Multidrug-Resistant Tuberculosis in Children: FLAME Path on the Milky Way.

BACKGROUND: The regimen of linezolid and moxifloxacin was found to be efficacious in the hollow fiber system model of pediatric intracellular tuberculosis. However, its kill rate was slower than the standard 3-drug regimen of isoniazid, rifampin, and pyrazinamide. We wanted to examine the effect of adding a third oral agent, faropenem, to this dual combination. METHODS: We performed a series of studies in the hollow fiber system model of intracellular Mycobacterium tuberculosis, by mimicking pediatric pharmacokinetics of each antibiotic. First, we varied the percentage of time that faropenem persisted above minimum inhibitory concentration (TMIC) on the moxifloxacin-linezolid regimen. After choosing the best faropenem exposure, we performed experiments in which we varied the moxifloxacin and linezolid doses in the triple regimen. Finally, we performed longer-duration therapy validation experiments. Bacterial burden was quantified using both colony-forming units per milliliter (CFU/mL) and time to positivity (TTP). Kill slopes were modeled using exponential regression. RESULTS: TTP was a more sensitive measure of bacterial burden than CFU/mL. A faropenem TMIC > 62% was associated with steepest microbial kill slope. Regimens of standard linezolid and moxifloxacin plus faropenem TMIC > 60%, as well as higher-dose moxifloxacin, achieved slopes equivalent to those of the standard regimen based by both TTP and CFU/mL over 28 days of treatment. CONCLUSIONS: We have developed an oral faropenem-linezolid-moxifloxacin (FLAME) regimen that is free of first-line drugs. The regimen could be effective against both multidrug-resistant and drug-susceptible tuberculosis in children.

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.

Challenges in the clinical assessment of novel tuberculosis drugs.

To tackle the global TB epidemic effectively, novel treatment strategies are critically needed to shorten the duration of TB therapy and treat drug-resistant TB. Drug development for TB, stymied for decades, has enjoyed a renaissance over the past several years. However, the development of new TB regimens is hindered by the limitations in our understanding and use of preclinical models; the paucity of accurate, early surrogate markers of cure, and challenges in untangling the individual contributions of drugs to multidrug regimens in a complex, multi-compartment disease. Lack of profit motive, advocacy, and imagination has contributed mightily to the dearth of drugs we have on the shelf to treat this ancient disease. Areas that will speed the development of new regimens for TB include novel murine and in vitro pharmacodynamics models, clinical endpoints that are not culture-based, innovative clinical trial designs, and an infusion of much-needed funding.