Identification of 2-Aryl-Quinolone Inhibitors of Cytochrome bd and Chemical Validation of Combination Strategies for Respiratory Inhibitors against Mycobacterium tuberculosis.

Mycobacterium tuberculosis cytochrome bd quinol oxidase (cyt bd), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome bd. The heterologous M. tuberculosis cytochrome bd expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome bd inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt bd inhibitor CK-2-63 with either cyt bcc-aa3 inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and in vitro sterilization kinetics. In vivo combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the in vivo experiments compared to in vitro experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt bd inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy.

Remdesivir-ivermectin combination displays synergistic interaction with improved in vitro activity against SARS-CoV-2.

A key element for the prevention and management of coronavirus disease 2019 is the development of effective therapeutics. Drug combination strategies offer several advantages over monotherapies. They have the potential to achieve greater efficacy, to increase the therapeutic index of drugs and to reduce the emergence of drug resistance. We assessed the in vitro synergistic interaction between remdesivir and ivermectin, both approved by the US Food and Drug Administration, and demonstrated enhanced antiviral activity against severe acute respiratory syndrome coronavirus-2. Whilst the in vitro synergistic activity reported here does not support the clinical application of this combination treatment strategy due to insufficient exposure of ivermectin in vivo, the data do warrant further investigation. Efforts to define the mechanisms underpinning the observed synergistic action could lead to the development of novel treatment strategies.

Treatment responses to Azithromycin and Ciprofloxacin in uncomplicated Salmonella Typhi infection: A comparison of Clinical and Microbiological Data from a Controlled Human Infection Model.

BACKGROUND: The treatment of enteric fever is complicated by the emergence of antimicrobial resistant Salmonella Typhi. Azithromycin is commonly used for first-line treatment of uncomplicated enteric fever, but the response to treatment may be sub-optimal in some patient groups when compared with fluoroquinolones. METHODS: We performed an analysis of responses to treatment with azithromycin (500mg once-daily, 14 days) or ciprofloxacin (500mg twice-daily, 14 days) in healthy UK volunteers (18-60 years) enrolled into two Salmonella controlled human infection studies. Study A was a single-centre, open-label, randomised trial. Participants were randomised 1:1 to receive open-label oral ciprofloxacin or azithromycin, stratified by vaccine group (Vi-polysaccharide, Vi-conjugate or control Men-ACWY vaccine). Study B was an observational challenge/re-challenge study, where participants were randomised to challenge with Salmonella Typhi or Salmonella Paratyphi A. Outcome measures included fever clearance time, blood-culture clearance time and a composite measure of prolonged treatment response (persistent fever ≥38.0°C for ≥72 hours, persistently positive S. Typhi blood cultures for ≥72 hours, or change in antibiotic treatment). Both trials are registered with ClinicalTrials.gov (NCT02324751 and NCT02192008). FINDINGS: In 81 participants diagnosed with S. Typhi in two studies, treatment with azithromycin was associated with prolonged bacteraemia (median 90.8 hours [95% CI: 65.9-93.8] vs. 20.1 hours [95% CI: 7.8-24.3], p<0.001) and prolonged fever clearance times <37.5°C (hazard ratio 2.4 [95%CI: 1.2-5.0]; p = 0.02). Results were consistent when studies were analysed independently and in a sub-group of participants with no history of vaccination or previous challenge. A prolonged treatment response was observed significantly more frequently in the azithromycin group (28/52 [54.9%]) compared with the ciprofloxacin group (1/29 [3.5%]; p<0.001). In participants treated with azithromycin, observed systemic plasma concentrations of azithromycin did not exceed the minimum inhibitory concentration (MIC), whilst predicted intracellular concentrations did exceed the MIC. In participants treated with ciprofloxacin, the observed systemic plasma concentrations and predicted intracellular concentrations of ciprofloxacin exceeded the MIC. INTERPRETATION: Azithromycin at a dose of 500mg daily is an effective treatment for fully sensitive strains of S. Typhi but is associated with delayed treatment response and prolonged bacteraemia when compared with ciprofloxacin within the context of a human challenge model. Whilst the cellular accumulation of azithromycin is predicted to be sufficient to treat intracellular S. Typhi, systemic exposure may be sub-optimal for the elimination of extracellular circulating S. Typhi. In an era of increasing antimicrobial resistance, further studies are required to define appropriate azithromycin dosing regimens for enteric fever and to assess novel treatment strategies, including combination therapies. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02324751 and NCT02192008).

Intracellular Pharmacodynamic Modeling Is Predictive of the Clinical Activity of Fluoroquinolones against Tuberculosis.

Clinical studies of new antitubercular drugs are costly and time-consuming. Owing to the extensive tuberculosis (TB) treatment periods, the ability to identify drug candidates based on their predicted clinical efficacy is vital to accelerate the pipeline of new therapies. Recent failures of preclinical models in predicting the activity of fluoroquinolones underline the importance of developing new and more robust predictive tools that will optimize the design of future trials. Here, we used high-content imaging screening and pharmacodynamic intracellular (PDi) modeling to identify and prioritize fluoroquinolones for TB treatment. In a set of studies designed to validate this approach, we show moxifloxacin to be the most effective fluoroquinolone, and PDi modeling-based Monte Carlo simulations accurately predict negative culture conversion (sputum sterilization) rates compared to eight independent clinical trials. In addition, PDi-based simulations were used to predict the risk of relapse. Our analyses show that the duration of treatment following culture conversion can be used to predict the relapse rate. These data further support that PDi-based modeling offers a much-needed decision-making tool for the TB drug development pipeline.

Intra-individual effects of food upon the pharmacokinetics of rifampicin and isoniazid.

Background: Poor response to TB therapy might be attributable to subtherapeutic levels in drug-compliant patients. Pharmacokinetic parameters can be affected by comorbidities or the interaction of drugs with food. Objectives: This study aimed to determine the effect of food intake upon pharmacokinetics of rifampicin and isoniazid in a Peruvian population with TB. Methods: Rifampicin and isoniazid levels were analysed at 2, 4 and 6 h after drug intake in both fasting and non-fasting states using LC-MS methods. Results: Sixty patients participated in the study. The median rifampicin Cmax and AUC0-6 were higher during fasting than non-fasting: 7.02 versus 6.59 mg/L (P = 0.054) and 28.64 versus 24.31 mg·h/L (P = 0.002). There was a statistically significant delay overall of non-fasting Tmax compared with the fasting state Tmax (P = 0.005). In the multivariate analysis, besides the effect of fasting, Cmax for females was 20% higher than for males (P = 0.03). Concerning isoniazid, there were significant differences in the Cmax during non-fasting (median = 3.51 mg/L) compared with fasting (4.54 mg/L). The isoniazid dose received had an effect upon the isoniazid levels (1.26, P = 0.038). In the multivariate analysis, isoniazid exposure during fasting was found to be 14% higher than during non-fasting (CI = 1.02-1.28, P < 0.001). Neither radiological extent of the disease nor consumption of food with drug intake nor pharmacokinetics of rifampicin or isoniazid was associated with a poorer treatment outcome. Conclusions: Rifampicin in particular and isoniazid pharmacokinetics were significantly affected by the intake of the drug with food between and within individuals.

Pharmacokinetic-Pharmacodynamic modelling of intracellular Mycobacterium tuberculosis growth and kill rates is predictive of clinical treatment duration.

Tuberculosis (TB) treatment is long and complex, typically involving a combination of drugs taken for 6 months. Improved drug regimens to shorten and simplify treatment are urgently required, however a major challenge to TB drug development is the lack of predictive pre-clinical tools. To address this deficiency, we have adopted a new high-content imaging-based approach capable of defining the killing kinetics of first line anti-TB drugs against intracellular Mycobacterium tuberculosis (Mtb) residing inside macrophages. Through use of this pharmacokinetic-pharmacodynamic (PK-PD) approach we demonstrate that the killing dynamics of the intracellular Mtb sub-population is critical to predicting clinical TB treatment duration. Integrated modelling of intracellular Mtb killing alongside conventional extracellular Mtb killing data, generates the biphasic responses typical of those described clinically. Our model supports the hypothesis that the use of higher doses of rifampicin (35 mg/kg) will significantly reduce treatment duration. Our described PK-PD approach offers a much needed decision making tool for the identification and prioritisation of new therapies which have the potential to reduce TB treatment duration.

Rational Design, Synthesis, and Biological Evaluation of Heterocyclic Quinolones Targeting the Respiratory Chain of Mycobacterium tuberculosis.

A high-throughput screen (HTS) was undertaken against the respiratory chain dehydrogenase component, NADH:menaquinone oxidoreductase (Ndh) of Mycobacterium tuberculosis (Mtb). The 11000 compounds were selected for the HTS based on the known phenothiazine Ndh inhibitors, trifluoperazine and thioridazine. Combined HTS (11000 compounds) and in-house screening of a limited number of quinolones (50 compounds) identified ∼100 hits and four distinct chemotypes, the most promising of which contained the quinolone core. Subsequent Mtb screening of the complete in-house quinolone library (350 compounds) identified a further ∼90 hits across three quinolone subtemplates. Quinolones containing the amine-based side chain were selected as the pharmacophore for further modification, resulting in metabolically stable quinolones effective against multi drug resistant (MDR) Mtb. The lead compound, 42a (MTC420), displays acceptable antituberculosis activity (Mtb IC50 = 525 nM, Mtb Wayne IC50 = 76 nM, and MDR Mtb patient isolates IC50 = 140 nM) and favorable pharmacokinetic and toxicological profiles.

Effects of dosage, comorbidities, and food on isoniazid pharmacokinetics in Peruvian tuberculosis patients.

Poor response to tuberculosis (TB) therapy might be attributable to subtherapeutic levels in drug-compliant patients. Pharmacokinetic (PK) parameters can be affected by several factors, such as comorbidities or the interaction of TB drugs with food. This study aimed to determine the PK of isoniazid (INH) in a Peruvian TB population under observed daily and twice-weekly (i.e., biweekly) therapy. Isoniazid levels were analyzed at 2 and 6 h after drug intake using liquid chromatography mass spectrometric methods. A total of 107 recruited patients had available PK data; of these 107 patients, 42.1% received biweekly isoniazid. The mean biweekly dose (12.8 mg/kg of body weight/day) was significantly lower than the nominal dose of 15 mg/kg/day (P < 0.001), and this effect was particularly marked in patients with concurrent diabetes and in males. The median maximum plasma concentration (Cmax) and area under the concentration-time curve from 0 to 6 h (AUC0-6) were 2.77 mg/liter and 9.71 mg · h/liter, respectively, for daily administration and 8.74 mg/liter and 37.8 mg · h/liter, respectively, for biweekly administration. There were no differences in the Cmax with respect to gender, diabetes mellitus (DM) status, or HIV status. Food was weakly associated with lower levels of isoniazid during the continuation phase. Overall, 34% of patients during the intensive phase and 33.3% during the continuation phase did not reach the Cmax reference value. However, low levels of INH were not associated with poorer clinical outcomes. In our population, INH exposure was affected by weight-adjusted dose and by food, but comorbidities did not indicate any effect on PK. We were unable to demonstrate a clear relationship between the Cmax and treatment outcome in this data set. Twice-weekly weight-adjusted dosing of INH appears to be quite robust with respect to important potentially influential patient factors under program conditions.

Quantification of rifampicin in human plasma and cerebrospinal fluid by a highly sensitive and rapid liquid chromatographic-tandem mass spectrometric method.

A highly sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed to measure the levels of the antitubercular drug rifampicin (RIF) in human plasma and cerebrospinal fluid (CSF). The analyte and internal standard (IS) were isolated from plasma and CSF by a simple organic solvent based precipitation of proteins followed by centrifugation. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay was linear in the concentration range 25-6400 ng/mL with intra- and inter-day precision of <7% and <8%, respectively. The validated method was applied to the study of RIF pharmacokinetics in human CSF and plasma over 25 h period after a 10 mg/kg oral dose.

Pharmacokinetics of rifampin in Peruvian tuberculosis patients with and without comorbid diabetes or HIV.

For drug-compliant patients, poor responses to tuberculosis (TB) treatment might be attributable to subtherapeutic drug concentrations. An impaired absorption of rifampin was previously reported for patients with diabetes mellitus (DM) or HIV. The objectives of this study were to determine whether TB drug pharmacokinetics differed in Peruvian TB patients with DM or HIV. In this cross-sectional study, TB patients, recruited from health centers in Lima, Peru, had blood samples taken at 2 and 6 h after directly observed TB drug ingestion, to determine plasma concentrations of rifampin. Of 105 patients, 50 had TB without a comorbidity, 26 had coexistent DM, and 29 had coexistent HIV. Unexpectedly, the overall median 2- and 6-h levels of rifampin were 1.6 and 3.2 mg/liter, respectively, and the time to the peak concentration was 6 h (slow absorber) instead of 2 h (fast absorber) for 61 patients (62.2%). The geometric mean peak concentration of drug in serum (C(max)) was significantly higher in fast absorbers than in slow absorbers (5.0 versus 3.8 mg/liter; P = 0.05). The rifampin C(max) was significantly lower in male patients than in female patients (3.3 versus 6.3 mg/liter; P < 0.001). Neither slow nor fast absorbers with comorbidities (DM or HIV) had significantly different C(max) results compared to those of TB patients without comorbidities. An analysis of variance regression analysis showed that female gender (P < 0.001) and the time to maximum concentration of drug in serum (T(max)) at 2 h (P = 0.012) were independently correlated with increased exposure to rifampin. Most of this Peruvian study population exhibited rifampin pharmacokinetics different from those conventionally reported, with delayed absorption and low plasma concentrations, independent of the presence of an HIV or DM comorbidity.

A rapid and sensitive HPLC-MS method for the detection of plasma and cellular rifampicin.

Rifampicin is active against both intracellular and extracellular Mycobacterium tuberculosis. The ability to measure rifampicin drug concentrations in both plasma and in cells may be useful in evaluating the suitability of dosage regimens for populations and individuals. Here a novel simple, precise and accurate method for the quantification of rifampicin in both cells and plasma is reported. Sample proteins were precipitated with acetonitrile containing the internal standard and then diluted with water. Aliquots of supernatant were then injected into the HPLC-MS system for chromatographic separation and detection. Rifampicin calibration curves encompassed concentrations from 100 to 12,800 ng/mL. Intra- and inter-assay precision and accuracy were determined using low, medium and high concentration quality control samples and was found to be within 10% in all cases. Rifampicin concentrations were found to be unaffected by freeze-thaw cycles, but were significantly affected by heat-inactivation (58 degrees C, 40 min). This assay was successfully utilised to determine the pharmacokinetic profile of rifampicin in plasma and peripheral blood mononuclear cells (PBMC) in 8 tuberculosis patients receiving rifampicin over an 8h period.