Ceftazidime-avibactam has potent sterilizing activity against highly drug-resistant tuberculosis

Abstract

There are currently many patients with multidrug-resistant and extensively drug-resistant tuberculosis. Ongoing transmission of the highly drug-resistant strains and high mortality despite treatment remain problematic. The current strategy of drug discovery and development takes up to a decade to bring a new drug to clinical use. We embarked on a strategy to screen all antibiotics in current use and examined them for use in tuberculosis. We found that ceftazidime-avibactam, which is already used in the clinic for multidrug-resistant Gram-negative bacillary infections, markedly killed rapidly growing, intracellular, and semidormant Mycobacterium tuberculosis in the hollow fiber system model. Moreover, multidrug-resistant and extensively drug-resistant clinical isolates demonstrated good ceftazidime-avibactam susceptibility profiles and were inhibited by clinically achievable concentrations. Resistance arose because of mutations in the transpeptidase domain of the penicillin-binding protein PonA1, suggesting that the drug kills M. tuberculosis bacilli via interference with cell wall remodeling. We identified concentrations (exposure targets) for optimal effect in tuberculosis, which we used with susceptibility results in computer-aided clinical trial simulations to identify doses for immediate clinical use as salvage therapy for adults and young children. Moreover, this work provides a roadmap for efficient and timely evaluation of antibiotics and optimization of clinically relevant dosing regimens.

Fig. 1. Program for rapid screening and translation for reuse of antibiotics in TB.

In the first step of the program, we examined the effect of CAV in comparison to standard first-line agents in intracellular and extracellular assays in a biosafety level 2 (BSL2) laboratory using avirulent Mtb. After demonstrating potential effectiveness, we then identified the MIC distribution in X/MDR-TB clinical strains from South Africa, in a BSL3 laboratory. In this first step, static concentrations of CAV were used. In step 2, we examined the efficacy of intrapulmonary concentration-time profiles of the CAV in the HFS-TB in several strains, for both bactericidal and sterilizing effect. These studies with dynamic concentrations of CAV against different Mtb metabolic subpopulations identified the concentrations and exposures associated with optimal kill and resistance suppression. They also generated CAV-resistant isolates, which then underwent whole-genome sequencing (WGS) to explore for mechanisms of effect. Step 3 takes place in silico, and uses output of step 2 as well as population-level pharmacokinetic parameters and measures of between-patient pharmacokinetic variability, plus MIC distributions from step 1, in Monte Carlo experiments to identify optimal clinical doses for use in patients with drug-resistant TB and for susceptibility breakpoints for decision-making of whom should be treated with the drug. Step 4 involves handing over of the clinical dose for immediate clinical trial studies and salvage therapy.

Fig. 2. Exposure effect screening study of CAV against Mtb.

Each concentration was examined in triplicate, and results shown are for mean and SD (error bar). For each study, concentration versus log₁₀ CFU/ml was examined using the inhibitory sigmoid E_max model. (A) Pyrazinamide failed to kill intracellular Mtb in 7 days. We acidified broth, which enabled pyrazinamide to kill Mtb but had poor fit of the model to the data (r² = 0.718), associated with maximal kill (E_max) of 1.43 ± 1.07 log₁₀ CFU/ml. (B) Isoniazid E_max was 3.56 ± 0.10 log₁₀ CFU/ml against intracellular Mtb (r² = 0.988), similar to the E_max of 3.32 ± 0.33 log₁₀ CFU/ml (r² = 0.845) for extracellular Mtb. (C) Rifampin E_max for intracellular Mtb was 5.68 ± 0.30 log₁₀ CFU/ml (r² = 0.976), and that for extracellular bacteria was 5.30 ± 0.09 log₁₀ CFU/ml (r² = 0.996). (D) Ceftazidime alone did not kill Mtb. (E) There was also poor fit of the model to the data for avibactam because of minimal to no microbial kill by the avibactam (r² = 0.357). (F) CAV achieved an E_max of 7.05 ± 0.00 log₁₀ CFU/ml and an EC₅₀ (median effective concentration) of 104.50 ± 13.44 mg/liter (r² = 0.910) against extracellular Mtb. This EC₅₀ is easily achieved with standard doses. (G) CAV achieved an E_max of 4.19 ± 0.29 log₁₀ CFU/ml and an EC₅₀ of 74.92 ± 9.03 mg/liter (r² = 0.960) against intracellular Mtb. (H) CAV MICs for five laboratory isolates are shown by arrows. The modal MIC in clinical isolates was 32 mg/liter.

Fig. 3. Bactericidal effect of CAV in the HFS-TB.

Data for each unit in replicate HFS-TB are shown separately. (A) In the first HFS-TB study, CAV was administered three times per day to each HFS-TB system, to produce concentration-time profiles similar to the stated human doses. The lowest human equivalent dose of 0.75 g demonstrated significant kill below the day 0 bacterial burden (stasis) of 2.78 log₁₀ CFU/ml. At a dose of 6 g, the day 0 burden of 6.2 log₁₀ CFU/ml was reduced to 0, indicating marked speed of bactericidal effect that is greater than isoniazid’s 1.8 log₁₀ CFU/ml in the HFS-TB and in patients, which is the most effective first-line drug against log-phase growth Mtb. (B to D) In the second HFS-TB study, triplicate HFS-TBs were treated with CAV in a dose-fractionation design. Concentration-time profiles achieved with once-a-day dosing schedule (B) had the lowest proportion of time above MIC (%T_MIC), compared to the twice-a-day dosing schedule (C) that had matching peak concentrations; %T_MIC was highest with the dosing schedule of every 8 hours (D). (E) The inhibitory sigmoid E_max model for CFU/ml versus %T_MIC had an r² of 0.94 on day 8. (F) The relationship between AUC/MIC and CAV-resistant CFU/ml had an r² of 0.90; the ratio associated with resistance suppression can be read off the graph as a ratio of 250.

Fig. 4. Mutations in the Mtb genome of resistant mutants.

Shown are the mutations that were common to all 12 drug-resistant isolates. Cell wall and cell membrane component genes are in magenta, including those for Rv0012 (PonA1).

Fig. 5. Pharmacokinetics and efficacy of CAV in intracellular Mtb.

Mean values and SDs (error bars) are shown in three replicates each. Given the range of concentrations, we used a log₁₀ scale for drug concentrations and intracellular to extracellular ratios for pharmacokinetic parameters and concentrations. (A) There were much higher concentrations achieved for both avibactam and ceftazidime inside the infected monocytes compared to those outside. (B) TTP increased 2.2-fold (maximal) on CAV alone, consistent with decreased bacterial burden. (C) Mtb CFU/ml reveals ~3.0 log₁₀ CFU/ml decline below day 0 burden with CAV treatment.

Fig. 6. Sterilizing effect of CAV versus standard combination therapy.

We compared the sterilizing effect of CAV to standard first-line drug combination in three replicates of hollow fiber systems for each treatment condition. Standard therapy consists of rifampin, isoniazid, and pyrazinamide, with ethambutol added in the beginning in case there is isoniazid resistance, and is dropped when susceptibility results become available; with some new point of care diagnostics, isoniazid susceptibility is actually known quickly, and ethambutol was excluded. In the figures, the mean value is shown with SD error bars. On many sampling days, the error bars were too small (SD was very low) and are obscured by the symbol. (A) First-line drug exposures achieved in the replicate HFS-TB. The MICs were 0.125 mg/liter for rifampin, 0.06 mg/liter for isoniazid, and 12.5 mg/liter for pyrazinamide, in Mtb H7Rv. (B) The mean Mtb burden progressively declined throughout the course of treatment with CAV to a kill of 4.72 log₁₀ CFU/ml below stasis (day 0 bacterial burden) at the end of the study on day 42. It can also be seen that starting on day 35, the CAV effect was similar to the standard three-drug regimen.

Fig. 7. CAV dose target attainment in childhood TB.

Each data point is shown as the proportion of 10,000 children. (A) Pharmacokinetic parameters and concentrations of ceftazidime and avibactam in 10,000 simulated children treated with a CAV dose of 50/12.5 mg/kg are compared to those observed in the clinic in children treated with this same dose for other indications. Our simulated children achieved concentrations that are the same as those that were observed in the clinic by others (used here to benchmark), meaning that the simulations faithfully recapitulated concentrations that are achieved in children by the different doses. (B) The dose of 100 mg/kg achieved good target attainment for %T_MIC of 47% up to an MIC of 64 mg/liter. (C) For the %T_MIC of 63% target, only the dose of 200 mg/kg achieved good target attainment up to an MIC of 64 mg/liter. (D) A summary of the proportion of 10,000 children with TB who achieved each of the two target %T_MIC values of 47 and 63% over the entire MIC range for each dose. The dose of 100 mg/kg achieves the target that gives the same kill rates as standard first-line drugs in 90% of the children and even higher kill rates in at least 60% of the children. This means that the dose of 100 mg/kg is recommended for clinical testing in children with TB.

Fig. 8. CAV dose target attainment in adult-type TB.

Each data point is shown as the proportion of 10,000 adult patients. (A) To validate that the simulations reflected clinical reality, we compared the pharmacokinetic parameters of clearance and volume, as well as drug concentrations such as peak and AUC concentrations achieved in the 10,000 simulated patients treated with the CAV dose of 2000/500 mg, to those observed in clinical pharmacokinetic studies published by others. It can be seen that the values in the simulated subjects are almost exactly the same as those observed in actual patients by others, meaning that the simulations worked. (B) The proportion of patients at each MIC who achieved a %T_MIC of 47%, termed target attainment probability, in simulated subjects treated with doses between 2 and 16 g. As MIC increases, the target attainment probability falls, but higher doses perform better until a dose of 12 g, above which there is no further improvement. (C) Target attainment probability for the %T_MIC target of 63% was poor for all doses examined in adults. (D) Proportion of 10,000 adult TB patients treated with different doses who achieved %T_MIC of 47 or 63% over the entire MIC range for each dose. The dose of 12 g achieves the target that gives the same kill rates as standard first-line drugs in 90% of the patients. This means that the dose of 12 g is recommended for clinical testing in adults with TB.