Crystal structure of the Mycobacterium tuberculosis VirS regulator reveals its interaction with the lead compound SMARt751.

Ethionamide (ETO) is a prodrug that is primarily used as a second-line agent in the treatment of tuberculosis. Among the bacterial ETO activators, the monooxygenase MymA has been recently identified, and its expression is regulated by the mycobacterial regulator VirS. The discovery of VirS ligands that can enhance mymA expression and thereby increase the antimycobacterial efficacy of ETO, has led to the development of a novel therapeutic strategy against tuberculosis. This strategy involves the selection of preclinical candidates, including SMARt751. We report the first crystal structure of the AraC-like regulator VirS, in complex with SMARt751, refined at 1.69 Å resolution. Crystals were obtained via an in situ proteolysis method in the requisite presence of SMARt751. The elucidated structure corresponds to the ligand-binding domain of VirS, adopting an α/ß fold with structural similarities to H-NOX domains. Within the VirS structure, SMARt751 is situated in a completely enclosed hydrophobic cavity, where it forms hydrogen bonds with Asn11 and Asn149 as well as van der Waals contacts with various hydrophobic amino acids. Comprehensive structural comparisons within the AraC family of transcriptional regulators are conducted and analyzed to figure out the effects of the SMARt751 binding on the regulatory activity of VirS.

Treatment of Multidrug-resistant or Rifampicin-resistant Tuberculosis With an All-oral 9-month Regimen Containing Linezolid or Ethionamide in South Africa: A Retrospective Cohort Study.

BACKGROUND: In 2019, the South African tuberculosis program replaced ethionamide with linezolid as part of an all-oral 9-month regimen. We evaluated treatment outcomes for patients assigned to regimens including linezolid in 2019 and ethionamide in 2017. METHODS: This retrospective cohort study included patients treated for multidrug-resistant/rifampicin-resistant tuberculosis throughout South Africa between 1 January and 31 December 2017 and 1 January to 31 December 2019. The cohort treated with a 9-month regimen containing ethionamide for four months, was compared with a cohort treated with a 9-month regimen containing linezolid for 2 months. The regimens were otherwise identical. Inverse probability weighting of propensity scores was used to adjust for potential confounding. A log-binomial regression model was used to estimate adjusted relative risk (aRR) comparing 24-month outcomes between cohorts including treatment success, death, loss to follow up, and treatment failure. Adverse event data were available for the linezolid cohort. FINDINGS: In total, 817 patients were included in the cohort receiving ethionamide and 4244 in the cohort receiving linezolid. No evidence for a difference was observed between linezolid and ethionamide regimens for treatment success (aRR = 0.96, 95% confidence interval [CI] .91-1.01), death (aRR = 1.01, 95% CI .87-1.17) or treatment failure (aRR = 0.87, 95% CI .44-1.75). Loss to follow-up was more common in the linezolid group, although estimates were imprecise (aRR = 1.22, 95% CI .99-1.50). CONCLUSIONS: No significant differences in treatment success and survival were observed with substitution of linezolid for ethionamide as a part of an all-oral 9-month regimen. Linezolid is an acceptable alternative to ethionamide in this shorter regimen for treatment of multidrug-resistant/rifampicin-resistant tuberculosis.

Loss-of-function mutations in ndh do not confer delamanid, ethionamide, isoniazid, or pretomanid resistance in Mycobacterium tuberculosis.

Results from clinical strains and knockouts of the H37Rv and CDC1551 laboratory strains demonstrated that ndh (Rv1854c) is not a resistance-conferring gene for isoniazid, ethionamide, delamanid, or pretomanid in Mycobacterium tuberculosis. This difference in the susceptibility to NAD-adduct-forming drugs compared with other mycobacteria may be driven by differences in the absolute intrabacterial NADH concentration.

Permeability of TB drugs through the mycolic acid monolayer: a tale of two force fields.

Tuberculosis (TB) treatment becomes challenging due to the unique cell wall structure of Mycobacterium tuberculosis (M. tb). Among various components of the M.tb cell wall, mycolic acid (MA) is of particular interest because it is speculated to exhibit extremely low permeability for most of the drug molecules, thus helping M.tb to survive against medical treatment. However, no quantitative assessment of the thermodynamic barrier encountered by various well-known TB drugs in the mycolic acid monolayer has been performed so far using computational tools. On this premise, our present work aims to probe the permeability of some first and second line TB drugs, namely ethambutol, ethionamide, and isoniazid, through the modelled mycolic acid monolayer, using molecular dynamics (MD) simulation with two sets of force field (FF) parameters, namely GROMOS 54A7-ATB (GROMOS) and CHARMM36 (CHARMM) FFs. Our findings indicate that both FFs provide consistent results in terms of the mode of drug-monolayer interactions but significantly differ in the drug permeability through the monolayer. The mycolic acid monolayer generally exhibited a higher free energy barrier of crossing with CHARMM FF, while with GROMOS FF, better stability of drug molecules on the monolayer surface was observed, which can be attributed to the greater electrostatic potential at the monolayer-water interface, found for the later. Although both the FF parameters predicted the highest resistance against ethambutol (permeability values of 8.40 × 10-34 cm s-1 and 9.61 × 10-31 cm s-1 for the CHARMM FF and the GROMOS FF, respectively), results obtained using GROMOS were found to be consistent with the water solubility of drugs, suggesting it to be a slightly better FF for modelling drug-mycolic acid interactions. Therefore, this study enhances our understanding of TB drug permeability and highlights the potential of the GROMOS FF in simulating drug-mycolic acid interactions.

MarR-Dependent Transcriptional Regulation of mmpSL5 Induces Ethionamide Resistance in Mycobacterium abscessus.

Mycobacterium abscessus (Mabs) is an emerging nontuberculosis mycobacterial (NTM) pathogen responsible for a wide variety of respiratory and cutaneous infections that are difficult to treat with standard antibacterial therapy. Mabs has a high degree of both innate and acquired antibiotic resistance to most clinically relevant drugs, including standard anti-mycobacterial agents. Ethionamide (ETH), an inhibitor of mycolic acid biosynthesis, is currently utilized as a second-line agent for treating multidrug-resistant tuberculosis infections. Here, we show that ETH displays activity against clinical strains of Mabs in vitro at concentrations that are >100× lower than other mycolic acid targeting drugs. Using transposon mutagenesis followed by transposon sequencing (Tn-Seq) and whole-genome sequencing of spontaneous ETH-resistant mutants, we identified MAB_2648c as a genetic determinant of ETH sensitivity in Mabs. MAB_2648c encodes a MarR family transcriptional regulator of the TetR class of regulators. We show that MAB_2648c represses expression of MAB_2649 (mmpS5) and MAB_2650 (mmpL5). Further, we show that derepression of these genes in MAB_2648c mutants confers resistance to ETH, but not other antibiotics. To identify determinants of resistance that may be shared across antibiotics with distinct mechanisms of action, we also performed Tn-Seq during treatment with amikacin and clarithromycin, drugs currently used clinically to treat Mabs. We found very little overlap in genes that modulate the sensitivity of Mabs to all three antibiotics, suggesting a high degree of specificity for resistance mechanisms in this emerging pathogen.

Genotypic and phenotypic comparison of drug resistance profiles of clinical multidrug-resistant Mycobacterium tuberculosis isolates using whole genome sequencing in Latvia.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) remains a major public health problem in many high tuberculosis (TB) burden countries. Phenotypic drug susceptibility testing (DST) take several weeks or months to result, but line probe assays and Xpert/Rif Ultra assay detect a limited number of resistance conferring gene mutations. Whole genome sequencing (WGS) is an advanced molecular testing method which theoretically can predict the resistance of M. tuberculosis (Mtb) isolates to all anti-TB agents through a single analysis. METHODS: Here, we aimed to identify the level of concordance between the phenotypic and WGS-based genotypic drug susceptibility (DS) patterns of MDR-TB isolates. Overall, data for 12 anti-TB medications were analyzed. RESULTS: In total, 63 MDR-TB Mtb isolates were included in the analysis, representing 27.4% of the total number of MDR-TB cases in Latvia in 2012-2014. Among them, five different sublineages were detected, and 2.2.1 (Beijing group) and 4.3.3 (Latin American-Mediterranean group) were the most abundant. There were 100% agreement between phenotypic and genotypic DS pattern for isoniazid, rifampicin, and linezolid. High concordance rate (> 90%) between phenotypic and genotypic DST results was detected for ofloxacin (93.7%), pyrazinamide (93.7%) and streptomycin (95.4%). Phenotypic and genotypic DS patterns were poorly correlated for ethionamide (agreement 56.4%), ethambutol (85.7%), amikacin (82.5%), capreomycin (81.0%), kanamycin (85.4%), and moxifloxacin (77.8%). For capreomycin, resistance conferring mutations were not identified in several phenotypically resistant isolates, and, in contrary, for ethionamide, ethambutol, amikacin, kanamycin, and moxifloxacin the resistance-related mutations were identified in several phenotypically sensitive isolates. CONCLUSIONS: WGS is a valuable tool for rapid genotypic DST for all anti-TB agents. For isoniazid and rifampicin phenotypic DST potentially can be replaced by genotypic DST based on 100% agreement between the tests. However, discrepant results for other anti-TB agents limit their prescription based solely on WGS data. For clinical decision, at the current level of knowledge, there is a need for combination of genotypic DST with modern, validated phenotypic DST methodologies for those medications which did not showed 100% agreement between the methods.

Thermodynamic integration combined with molecular dynamic simulations to explore the cross-resistance mechanism of isoniazid and ethionamide.

Tuberculosis is an ancient disease of mankind, and its causative bacterium is Mycobacterium tuberculosis. Isoniazid is one of the most effective first-line antituberculosis drugs. As prodrugs, it and its derivative ethionamide act on enoyl-acyl carrier protein reductase (InhA) after being oxidized in bacteria, and kill the bacteria by inhibiting the formation of M. tuberculosis cell walls. However, the S94A mutation of InhA causes M. tuberculosis to develop cross-resistance to isoniazid and ethionamide. This work is dedicated to studying the cross-resistance mechanism of isoniazid and ethionamide through theoretical calculations. First, thermodynamic integral simulations are used to accurately calculate the relative binding energy of two drugs in the mutant and wild-type system. Furthermore, through classic molecular dynamic simulations and molecular mechanics generalized-Born surface area calculation, some key residues are identified and the binding affinity of isoniazid and ethionamide reduced by 9-13 kcal/mol due to S94A mutation. The hydrogen bond between Ala94 and isoniazid (ethionamide) disappeared and the energy contribution of Ala94 decreased after the mutation. In addition, the dynamic network analysis indicated that the mutation of Ser94 also indirectly affected the conformation of key residues such as Met147, Thr196, and Leu97, resulting in a reduction in the energy contribution of these residues. Finally, the binding conformation of isoniazid and ethionamide has also undergone major changes. The obtained results could provide valuable information for the future molecular design to overcome the drug resistance.

Pharmacokinetics of standard versus high-dose isoniazid for treatment of multidrug-resistant tuberculosis.

BACKGROUND: The WHO-endorsed shorter-course regimen for MDR-TB includes high-dose isoniazid. The pharmacokinetics of high-dose isoniazid within MDR-TB regimens has not been well described. OBJECTIVES: To characterize isoniazid pharmacokinetics at 5-15 mg/kg as monotherapy or as part of the MDR-TB treatment regimen. METHODS: We used non-linear mixed-effects modelling to evaluate the combined data from INHindsight, a 7 day early bactericidal activity study with isoniazid monotherapy, and PODRtb, an observational study of patients on MDR-TB treatment including terizidone, pyrazinamide, moxifloxacin, kanamycin, ethionamide and/or isoniazid. RESULTS: A total of 58 and 103 participants from the INHindsight and PODRtb studies, respectively, were included in the analysis. A two-compartment model with hepatic elimination best described the data. N-acetyltransferase 2 (NAT2) genotype caused multi-modal clearance, and saturable first-pass was observed beyond 10 mg/kg dosing. Saturable isoniazid kinetics predicted an increased exposure of approximately 50% beyond linearity at 20 mg/kg dosing. Participants treated with the MDR-TB regimen had a 65.6% lower AUC compared with participants on monotherapy. Ethionamide co-administration was associated with a 29% increase in isoniazid AUC. CONCLUSIONS: Markedly lower isoniazid exposures were observed in participants on combination MDR-TB treatment compared with monotherapy. Isoniazid displays saturable kinetics at doses >10 mg/kg. The safety implications of these phenomena remain unclear.

Design, synthesis and molecular docking studies of imidazole and benzimidazole linked ethionamide derivatives as inhibitors of InhA and antituberculosis agents.

To explore effective antituberculosis agents, a new class of imidazoles and benzimidazoles linked ethionamide analogs were designed and synthesized. The elemental analysis, 1H NMR, 13C NMR and mass spectral data were used to characterize all of the novel analogs. In vitro activity against Mycobacterium tuberculosis (Mtb) H37Rv was assessed for all of the target compounds. The hydroxy and nitrile moieties on the imidazole ring, as well as the hydroxy and methoxy groups on the benzimidazole ring connected to the ethionamide side chain, were shown to be advantageous. In our cell viability experiment against the Vero cell line, all of the compounds were non-cytotoxic even at 100 µM. To confirm the powerful analogs target identification, we investigated their in vitro inhibitory action on an M. tuberculosis InhA over-expressing (Mtb InhA-OE) strain, which yielded MICs nearly twice those of the Mtb H37Rv strain. Furthermore, the results of molecular docking confirmed the experimental findings. Additionally, the molecules were evaluated in silico for ADMET and drug similarity features. The experimental observation enables the newly generated ethionamide derivatives to be attractive candidates for the creation of newer and better anti-TB agents.

Evaluation of the broth microdilution plate methodology for susceptibility testing of Mycobacterium tuberculosis in Peru.

BACKGROUND: Tuberculosis (TB) is a communicable, preventable and curable disease caused by the bacterium Mycobacterium tuberculosis (MTB). Peru is amongst the 30 countries with the highest burden of multidrug-resistant tuberculosis (MDR-TB) worldwide. In the fight against drug-resistant tuberculosis, the UKMYC6 microdilution plate was developed and validated by the CRyPTIC project. The objective of the study was to evaluate the use of the broth microdilution (BMD) plate methodology for susceptibility testing of drug-resistant MTB strains in Peru. METHODS: MTB strains isolated between 2015 and 2018 in Peru were used. 496 nationally-representative strains determined as drug-resistant by the routine 7H10 Agar Proportion Method (APM) were included in the present study. The Minimum Inhibitory Concentration (MIC) of 13 antituberculosis drugs were determined for each strain using the UKMYC6 microdilution plates. Diagnostic agreement between APM and BMD plate methodology was determined for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. Phenotypes were set using binary (or ternary) classification based on Epidemiological cut-off values (ECOFF/ECV) proposed by the CRyPTIC project. Whole Genome Sequencing (WGS) was performed on strains with discrepant results between both methods. RESULTS: MIC distributions were determined for 13 first- and second-line anti-TB drugs, including new (bedaquiline, delamanid) and repurposed (clofazimine, linezolid) agents. MIC results were available for 80% (397/496) of the strains at 14 days and the remainder at 21 days. The comparative analysis determined a good agreement (0.64 ≤ k ≤ 0.79) for the drugs rifampicin, ethambutol, ethionamide and kanamycin, and the best agreement (k > 0.8) for isoniazid and levofloxacin. Overall, 12% of MIC values were above the UKMYC6 plate dilution ranges, most notably for the drugs rifampicin and rifabutin. No strain presented MICs higher than the ECOFF/ECV values for the new or repurposed drugs. Discrepant analysis using genotypic susceptibility testing by WGS supported half of the results obtained by APM (52%, 93/179) and half of those obtained by BMD plate methodology (48%, 86/179). CONCLUSIONS: The BMD methodology using the UKMYC6 plate allows the complete susceptibility characterization, through the determination of MICs, of drug-resistant MTB strains in Peru. This methodology shows good diagnostic performances for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. It also allows for the characterization of MICs for other drugs used in previous years against tuberculosis, as well as for new and repurposed drugs recently introduced worldwide.

Xpert MTB/XDR for detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin.

BACKGROUND: The World Health Organization (WHO) End TB Strategy stresses universal access to drug susceptibility testing (DST). DST determines whether Mycobacterium tuberculosis bacteria are susceptible or resistant to drugs. Xpert MTB/XDR is a rapid nucleic acid amplification test for detection of tuberculosis and drug resistance in one test suitable for use in peripheral and intermediate level laboratories. In specimens where tuberculosis is detected by Xpert MTB/XDR, Xpert MTB/XDR can also detect resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin. OBJECTIVES: To assess the diagnostic accuracy of Xpert MTB/XDR for pulmonary tuberculosis in people with presumptive pulmonary tuberculosis (having signs and symptoms suggestive of tuberculosis, including cough, fever, weight loss, night sweats). To assess the diagnostic accuracy of Xpert MTB/XDR for resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin in people with tuberculosis detected by Xpert MTB/XDR, irrespective of rifampicin resistance (whether or not rifampicin resistance status was known) and with known rifampicin resistance. SEARCH METHODS: We searched multiple databases to 23 September 2021. We limited searches to 2015 onwards as Xpert MTB/XDR was launched in 2020. SELECTION CRITERIA: Diagnostic accuracy studies using sputum in adults with presumptive or confirmed pulmonary tuberculosis. Reference standards were culture (pulmonary tuberculosis detection); phenotypic DST (pDST), genotypic DST (gDST),composite (pDST and gDST) (drug resistance detection). DATA COLLECTION AND ANALYSIS: Two review authors independently reviewed reports for eligibility and extracted data using a standardized form. For multicentre studies, we anticipated variability in the type and frequency of mutations associated with resistance to a given drug at the different centres and considered each centre as an independent study cohort for quality assessment and analysis. We assessed methodological quality with QUADAS-2, judging risk of bias separately for each target condition and reference standard. For pulmonary tuberculosis detection, owing to heterogeneity in participant characteristics and observed specificity estimates, we reported a range of sensitivity and specificity estimates and did not perform a meta-analysis. For drug resistance detection, we performed meta-analyses by reference standard using bivariate random-effects models. Using GRADE, we assessed certainty of evidence of Xpert MTB/XDR accuracy for detection of resistance to isoniazid and fluoroquinolones in people irrespective of rifampicin resistance and to ethionamide and amikacin in people with known rifampicin resistance, reflecting real-world situations. We used pDST, except for ethionamide resistance where we considered gDST a better reference standard. MAIN RESULTS: We included two multicentre studies from high multidrug-resistant/rifampicin-resistant tuberculosis burden countries, reporting on six independent study cohorts, involving 1228 participants for pulmonary tuberculosis detection and 1141 participants for drug resistance detection. The proportion of participants with rifampicin resistance in the two studies was 47.9% and 80.9%. For tuberculosis detection, we judged high risk of bias for patient selection owing to selective recruitment. For ethionamide resistance detection, we judged high risk of bias for the reference standard, both pDST and gDST, though we considered gDST a better reference standard. Pulmonary tuberculosis detection - Xpert MTB/XDR sensitivity range, 98.3% (96.1 to 99.5) to 98.9% (96.2 to 99.9) and specificity range, 22.5% (14.3 to 32.6) to 100.0% (86.3 to 100.0); median prevalence of pulmonary tuberculosis 91.3%, (interquartile range, 89.3% to 91.8%), (2 studies; 1 study reported on 2 cohorts, 1228 participants; very low-certainty evidence, sensitivity and specificity). Drug resistance detection People irrespective of rifampicin resistance - Isoniazid resistance: Xpert MTB/XDR summary sensitivity and specificity (95% confidence interval (CI)) were 94.2% (87.5 to 97.4) and 98.5% (92.6 to 99.7) against pDST, (6 cohorts, 1083 participants, moderate-certainty evidence, sensitivity and specificity). - Fluoroquinolone resistance: Xpert MTB/XDR summary sensitivity and specificity were 93.2% (88.1 to 96.2) and 98.0% (90.8 to 99.6) against pDST, (6 cohorts, 1021 participants; high-certainty evidence, sensitivity; moderate-certainty evidence, specificity). People with known rifampicin resistance - Ethionamide resistance: Xpert MTB/XDR summary sensitivity and specificity were 98.0% (74.2 to 99.9) and 99.7% (83.5 to 100.0) against gDST, (4 cohorts, 434 participants; very low-certainty evidence, sensitivity and specificity). - Amikacin resistance: Xpert MTB/XDR summary sensitivity and specificity were 86.1% (75.0 to 92.7) and 98.9% (93.0 to 99.8) against pDST, (4 cohorts, 490 participants; low-certainty evidence, sensitivity; high-certainty evidence, specificity). Of 1000 people with pulmonary tuberculosis, detected as tuberculosis by Xpert MTB/XDR: - where 50 have isoniazid resistance, 61 would have an Xpert MTB/XDR result indicating isoniazid resistance: of these, 14/61 (23%) would not have isoniazid resistance (FP); 939 (of 1000 people) would have a result indicating the absence of isoniazid resistance: of these, 3/939 (0%) would have isoniazid resistance (FN). - where 50 have fluoroquinolone resistance, 66 would have an Xpert MTB/XDR result indicating fluoroquinolone resistance: of these, 19/66 (29%) would not have fluoroquinolone resistance (FP); 934 would have a result indicating the absence of fluoroquinolone resistance: of these, 3/934 (0%) would have fluoroquinolone resistance (FN). - where 300 have ethionamide resistance, 296 would have an Xpert MTB/XDR result indicating ethionamide resistance: of these, 2/296 (1%) would not have ethionamide resistance (FP); 704 would have a result indicating the absence of ethionamide resistance: of these, 6/704 (1%) would have ethionamide resistance (FN). - where 135 have amikacin resistance, 126 would have an Xpert MTB/XDR result indicating amikacin resistance: of these, 10/126 (8%) would not have amikacin resistance (FP); 874 would have a result indicating the absence of amikacin resistance: of these, 19/874 (2%) would have amikacin resistance (FN). AUTHORS' CONCLUSIONS: Review findings suggest that, in people determined by Xpert MTB/XDR to be tuberculosis-positive, Xpert MTB/XDR provides accurate results for detection of isoniazid and fluoroquinolone resistance and can assist with selection of an optimised treatment regimen. Given that Xpert MTB/XDR targets a limited number of resistance variants in specific genes, the test may perform differently in different settings. Findings in this review should be interpreted with caution. Sensitivity for detection of ethionamide resistance was based only on Xpert MTB/XDR detection of mutations in the inhA promoter region, a known limitation. High risk of bias limits our confidence in Xpert MTB/XDR accuracy for pulmonary tuberculosis. Xpert MTB/XDR's impact will depend on its ability to detect tuberculosis (required for DST), prevalence of resistance to a given drug, health care infrastructure, and access to other tests.

Phosphatidylcholine (18:0/20:4), a potential biomarker to predict ethionamide-induced hepatic steatosis in rats.

Ethionamide (ETH), a second-line drug for multidrug-resistant tuberculosis, is known to cause hepatic steatosis in rats and humans. To investigate predictive biomarkers for ETH-induced steatosis, we performed lipidomics analysis using plasma and liver samples collected from rats treated orally with ETH at 30 and 100 mg/kg for 14 days. The ETH-treated rats developed hepatic steatosis with Oil Red O staining-positive vacuolation in the centrilobular hepatocytes accompanied by increased hepatic contents of triglycerides (TG) and decreased plasma TG and total cholesterol levels. A multivariate analysis for lipid profiles revealed differences in each of the 35 lipid species in the plasma and liver between the control and the ETH-treated rats. Of those lipids, phosphatidylcholine (PC) (18:0/20:4) decreased dose-dependently in both the plasma and liver. Moreover, serum TG-rich very low-density lipoprotein (VLDL) levels, especially the large particle fraction of VLDL composed of PC containing arachidonic acid (20:4) involved in hepatic secretion of TG, were decreased dose-dependently. In conclusion, the decreased PC (18:0/20:4) in the liver, possibly leading to suppression of hepatic TG secretion, was considered to be involved in the pathogenesis of the ETH-induced hepatic steatosis. Therefore, plasma PC (18:0/20:4) levels are proposed as mechanism-related biomarkers for ETH-induced hepatic steatosis.

Effect of Isoniazid Intake on Ethionamide Pharmacokinetics and Target Attainment in Multidrug-Resistant Tuberculosis Patients.

Ethionamide is recommended as part of regimens to treat multidrug-resistant and rifampicin-resistant tuberculosis. This study was conducted to (i) describe the distribution of ethionamide MICs, (ii) describe the pharmacokinetics of ethionamide, and (iii) determine the probability of attaining target area under the concentration-time curve from 0 to 24 h (AUC0-24)/MIC values associated with suppression of resistant subpopulation and microbial kill. Participants received 15 to 20 mg of drug/kg of body weight of ethionamide daily (in 500- or 750-mg doses) as part of a multidrug regimen. Pretreatment MICs of ethionamide for Mycobacterium tuberculosis sputum isolates were determined using Sensititre MYCOTB MIC plates. Plasma concentrations of ethionamide (measured predose and at 2, 4, 6, 8, and 10 h postdose) were available for 84 patients. A one-compartment disposition model, including a liver compartment capturing hepatic extraction, best described ethionamide pharmacokinetics. Clearance and volume were allometrically scaled using fat-free mass. Isoniazid coadministration reduced ethionamide clearance by 31%, resulting in a 44% increase in AUC0-24. The median (range) MIC (n = 111) was 2.5 mg/liter (<0.3 to >40 mg/liter). Simulations showed increased daily doses of ethionamide (1,250 mg, 1,500 mg, and 1,750 mg for patients weighing ≤45 kg, 46 to 70 kg, and >70 kg, respectively) resulted in the probability of attaining an area under the concentration-time curve from 0 to 24 h for the free, unbound fraction of a drug (fAUC0-24)/MIC ratio of ≥42 in more than 90% of patients only at the lowest MIC of 0.3 mg/liter. The WHO-recommended doses of ethionamide do not achieve target concentrations even for the lowest MIC measured in the cohort.

Further Insights into the Oxidative Pathway of Thiocarbonyl-Type Antitubercular Prodrugs: Ethionamide, Thioacetazone, and Isoxyl.

A chemical activation study of the thiocarbonyl-type antitubercular prodrugs, ethionamide (ETH), thioacetazone (TAZ), and isoxyl (ISO), was performed. Biomimetic oxidation of ethionamide using H2O2 (1 equiv) led to ETH-SO as the only stable S-oxide compound, which was found to occur in solution in the preferential form of a sulfine (ETH═S═O vs the sulfenic acid tautomer ETH-S-OH), as previously observed in the crystal state. It was also demonstrated that ETH-SO is capable of reacting with amines, as the putative sulfinic derivative (ETH-SO2H) was supposed to do. Unlike ETH, oxidation of TAZ did not allow observation of the mono-oxygenated species (TAZ-SO), leading directly to the more stable sulfinic acid derivative (TAZ-SO2H), which can then lose a SOxH group after further oxidation or when placed in a basic medium. It was also noticed that the unstable TAZ-SO intermediate can lead to the carbodiimide derivative as another electrophilic species. It is suggested that TAZ-SOH, TAZ-SO2H, and the carbodiimide compound can also react with NH2-containing nucleophilic species, and therefore be involved in toxic effects. Finally, ISO showed a very complex reactivity, here assigned to the coexistence of two mono-oxygenated structures, the sulfine and sulfenic acid tautomers. The mono- and dioxygenated derivatives of ISO are also highly unstable, leading to a panel of multiple metabolites, which are still reactive and likely contribute to the toxicity of this prodrug.

Ethionamide population pharmacokinetics/pharmacodynamics and therapeutic implications in South African adult patients with drug-resistant tuberculosis.

INTRODUCTION: Ethionamide is part of the drug-resistant tuberculosis regimen whose pharmacokinetic (PK) and pharmacodynamic (PD) information is limited. The aim of the study was to describe the PK and simulate doses to assess PD attainment. METHODS: This was an observational population PK study of patients admitted for drug-resistant tuberculosis at a hospital in South Africa. Nonlinear mixed-effects modelling implemented in Monolix 2019R2 was used to estimate population pharmacokinetic parameters. We performed Monte Carlo simulations to assess and optimise the dose regimen. The target Cmax range was 2.5-5 µg/mL, which is within the minimum inhibitory concentration (MIC) range. The target AUC0-24h was 140.5 µg*h/mL, which corresponds to the PK/PD target ratio AUC0-24h /MIC of 56.2. RESULTS: A one-compartment pharmacokinetic model with a lag-time, first-order absorption and elimination best described the PK of ethionamide. The lag-time, absorption rate constant (ka), volume of distribution (V/F) and clearance (Cl/F) were 0.66 hours, 0.434 h-1 , 180 L and 99.5 L/h, respectively, for a typical individual weighing 52.6 kg. Between-subject variability in lag-time, ka, V/F and Cl/F were 38%, 92%, 168% and 120%, respectively. Simulation of the recommended doses of 15-20 mg/kg, 500 mg, 750 mg and 1000 mg for patients in the weight bands <33, 33-50, 51-70 and >70 kg resulted in <17% and 3% of the patients achieving the target Cmax and AUC0-24h , respectively. CONCLUSION: There is high variability in ethionamide PK and very few patients attain the desired target exposure at standard or optimised doses. We propose individualised dose regimen optimisation.

New ethionamide boosters and EthR2: structural and energetic analysis.

Ethionamide (ETH) is a high-profile drug for the treatment of patients with multidrug-resistant Mycobacterium tuberculosis and, in order to produce its inhibitory effects, it needs to be bioactivated by monooxygenase EthA. This process is under the control of the transcriptional repressors EthR and EthR2, so that their inhibition results in the boosting of ethionamide activation. Herein, through crystallographic data and computer simulations, we calculated the interaction binding energies of four inhibitors with improved in vitro potency, namely BDM76060 (PDB ID: 6HS1), BDM72201 (PDB ID: 6HRX), BDM76150 (PDB ID: 6HS2) and BDM72719 (PDB ID: 6HRY), in complexes with the transcriptional repressor EthR2, using density functional theory (DFT) within the molecular fractionation with conjugated caps (MFCC) approach. It was observed that these ligands share the same binding site within a 10.0 Å radius of the EthR2 protein; however, their structural particularities have a significant impact on the global energies of systems. The BDM72201 and BDM72719 components are weakly attached to the binding site, while BDM76060 and BDM76150 components produce stronger bonds, corroborating with experimental studies demonstrating that BDM76060 and BDM76150 are more successful in producing inhibitory effects. BDM76060 and BDM76150 have many functional groups that increase the contact surface with the protein and attract a more significant number of amino acid residues, being able to produce polarities that generate stronger interactions. In the current scenario of a growing number of cases of bacterial resistance, the obtained data can be used to guide clinical trials of these inhibitors and other inhibitors that act on the alternative EthR2 pathway, focusing on improving the activity of ethionamide, its effectiveness, a reduction in the treatment time and exposure to cytotoxic effects.

Ethionamide Population Pharmacokinetic Model and Target Attainment in Multidrug-Resistant Tuberculosis.

Ethionamide (ETA), an isonicotinic acid derivative, is part of the multidrug-resistant tuberculosis (MDR-TB) regimen. The current guidelines have deprioritized ETA because it is potentially less effective than other agents. Our aim was to develop a population pharmacokinetic (PK) model and simulate ETA dosing regimens in order to assess target attainment. This study included subjects from four different sites, including healthy volunteers and patients with MDR-TB. The TB centers included were two in the United States and one in Bangladesh. Patients who received ETA and had at least one drug concentration reported were included. The population PK model was developed, regimens with a total of 1,000 to 2,250 mg daily were simulated, and target attainment using published MICs and targets of 1.0-log kill and resistance suppression was assessed with the Pmetrics R package. We included 1,167 ethionamide concentrations from 94 subjects. The final population model was a one-compartment model with first-order elimination and absorption with a lag time. The mean (standard deviation [SD]) final population parameter estimates were as follows: absorption rate constant, 1.02 (1.11) h-1; elimination rate constant, 0.69 (0.46) h-1; volume of distribution, 104.16 (59.87) liters; lag time, 0.43 (0.32) h. A total daily dose of 1,500 mg or more was needed for ≥90% attainment of the 1.0-log kill target at a MIC of 1 mg/liter, and 2,250 mg/day led to 80% attainment of the resistance suppression target at a MIC of 0.5 mg/liter. In conclusion, we developed a population PK model and assessed target attainment for different ETA regimens. Patients may not be able to tolerate the doses needed to achieve the predefined targets supporting the current recommendations for ETA deprioritization.

Population Pharmacokinetics and Dosing of Ethionamide in Children with Tuberculosis.

Ethionamide has proven efficacy against both drug-susceptible and some drug-resistant strains of Mycobacterium tuberculosis Limited information on its pharmacokinetics in children is available, and current doses are extrapolated from weight-based adult doses. Pediatric doses based on more robust evidence are expected to improve antituberculosis treatment, especially in small children. In this analysis, ethionamide concentrations in children from 2 observational clinical studies conducted in Cape Town, South Africa, were pooled. All children received ethionamide once daily at a weight-based dose of approximately 20 mg/kg of body weight (range, 10.4 to 25.3 mg/kg) in combination with other first- or second-line antituberculosis medications and with antiretroviral therapy in cases of HIV coinfection. Pharmacokinetic parameters were estimated using nonlinear mixed-effects modeling. The MDR-PK1 study contributed data for 110 children on treatment for multidrug-resistant tuberculosis, while the DATiC study contributed data for 9 children treated for drug-susceptible tuberculosis. The median age of the children in the studies combined was 2.6 years (range, 0.23 to 15 years), and the median weight was 12.5 kg (range, 2.5 to 66 kg). A one-compartment, transit absorption model with first-order elimination best described ethionamide pharmacokinetics in children. Allometric scaling of clearance (typical value, 8.88 liters/h), the volume of distribution (typical value, 21.4 liters), and maturation of clearance and absorption improved the model fit. HIV coinfection decreased the ethionamide bioavailability by 22%, rifampin coadministration increased clearance by 16%, and ethionamide administration by use of a nasogastric tube increased the rate, but the not extent, of absorption. The developed model was used to predict pediatric doses achieving the same drug exposure achieved in 50- to 70-kg adults receiving 750-mg once-daily dosing. Based on model predictions, we recommend a weight-banded pediatric dosing scheme using scored 125-mg tablets.

Ethionamide Preconditioning Enhances the Proliferation and Migration of Human Wharton's Jelly-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are a useful source for cell-based therapy of a variety of immune-mediated diseases, including neurodegenerative disorders. However, poor migration ability and survival rate of MSCs after brain transplantation hinder the therapeutic effects in the disease microenvironment. Therefore, we attempted to use a preconditioning strategy with pharmacological agents to improve the cell proliferation and migration of MSCs. In this study, we identified ethionamide via the screening of a drug library, which enhanced the proliferation of MSCs. Preconditioning with ethionamide promoted the proliferation of Wharton's jelly-derived MSCs (WJ-MSCs) by activating phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2 signaling. Preconditioning with ethionamide also enhanced the migration ability of MSCs by upregulating expression of genes associated with migration, such as C-X-C motif chemokine receptor 4 (CXCR4) and C-X-C motif chemokine ligand 12 (CXCL12). Furthermore, preconditioning with ethionamide stimulated the secretion of paracrine factors, including neurotrophic and growth factors in MSCs. Compared to naïve MSCs, ethionamide-preconditioned MSCs (ETH-MSCs) were found to survive longer in the brain after transplantation. These results suggested that enhancing the biological process of MSCs induced by ethionamide preconditioning presents itself as a promising strategy for enhancing the effectiveness of MSCs-based therapies.

[Cross-resistance between isoniazid and ethionamide and its strong association with mutation C-15T in Mycobacterium tuberculosis isolates from Peru]. / Resistencia cruzada entre isoniacida y etionamida y su alta correlación con la mutación C-15T en aislamientos de Mycobacterium tuberculosis de Perú.

Several studies have shown cross-resistance between isoniazid and ethionamide, 2of the drugs used in the treatment of multidrug-resistant tuberculosis. The objective of this study was to determine the cross-resistance between both drugs in Mycobacterium tuberculosis isolates from a hospital with high incidence of tuberculosis in Lima, Peru. The frequency of mutations to isoniazid in the katG gene and the inhA promoter region was identified by the Genotype MTBDRplus v2.0 molecular test. The gold standard Agar Proportion method (APM) allowed todetect resistance to isoniazid and ethionamide. Of 107 isoniazid-resistant isolates (54 multidrug-resistant isolates identified by the Genotype MTBDRplus test, 45.8% (49/107) were also resistant to ethionamide by the APM. Mutations were found in the katG gene in 50.5% (54/107), in the promoter region inhA in 23.3% (25/107) and 14.0% (15/107) that share both mutations in the resistant isolates to INH. The absence of the wild type and mutation bands indicated that 12.1% (13/107) of the isolates were resistant to INH. The mutation C-15T in the inhA promoter region showed a strong association with resistance to ethionamide in 73.4% (36/49) of the isolates analyzed. The results of the present study suggest that the identification of mutations related to resistance to isoniazid, especially in the inhA promoter region, could be very useful to identify cross-resistance to ethionamide and improve the treatment of individuals suffering from this disease.