A model-based analysis identifies differences in phenotypic resistance between in vitro and in vivo: implications for translational medicine within tuberculosis.

Proper characterization of drug effects on Mycobacterium tuberculosis relies on the characterization of phenotypically resistant bacteria to correctly establish exposure-response relationships. The aim of this work was to evaluate the potential difference in phenotypic resistance in in vitro compared to murine in vivo models using CFU data alone or CFU together with most probable number (MPN) data following resuscitation with culture supernatant. Predictions of in vitro and in vivo phenotypic resistance i.e. persisters, using the Multistate Tuberculosis Pharmacometric (MTP) model framework was evaluated based on bacterial cultures grown with and without drug exposure using CFU alone or CFU plus MPN data. Phenotypic resistance and total bacterial number in in vitro natural growth observations, i.e. without drug, was well predicted by the MTP model using only CFU data. Capturing the murine in vivo total bacterial number and persisters during natural growth did however require re-estimation of model parameter using both the CFU and MPN observations implying that the ratio of persisters to total bacterial burden is different in vitro compared to murine in vivo. The evaluation of the in vitro rifampicin drug effect revealed that higher resolution in the persister drug effect was seen using CFU and MPN compared to CFU alone although drug effects on the other bacterial populations were well predicted using only CFU data. The ratio of persistent bacteria to total bacteria was predicted to be different between in vitro and murine in vivo. This difference could have implications for subsequent translational efforts in tuberculosis drug development.

Novel classes of antibiotics or more of the same?

The world is running out of antibiotics. Between 1940 and 1962, more than 20 new classes of antibiotics were marketed. Since then, only two new classes have reached the market. Analogue development kept pace with the emergence of resistant bacteria until 10-20 years ago. Now, not enough analogues are reaching the market to stem the tide of antibiotic resistance, particularly among gram-negative bacteria. This review examines the existing systemic antibiotic pipeline in the public domain, and reveals that 27 compounds are in clinical development, of which two are new classes, both of which are in Phase I clinical trials. In view of the high attrition rate of drugs in early clinical development, particularly new classes and the current regulatory hurdles, it does not seem likely that new classes will be marketed soon. This paper suggests that, if the world is to return to a situation in which there are enough antibiotics to cope with the inevitable ongoing emergence of bacterial resistance, we need to recreate the prolific antibiotic discovery period between 1940 and 1962, which produced 20 classes that served the world well for 60 years. If another 20 classes and their analogues, particularly targeting gram-negatives could be produced soon, they might last us for the next 60 years. How can this be achieved? Only a huge effort by governments in the form of finance, legislation and providing industry with real incentives will reverse this. Industry needs to re-enter the market on a much larger scale, and academia should rebuild its antibiotic discovery infrastructure to support this effort. The alternative is Medicine without effective antibiotics.

Predictive in vitro models of the sterilizing activity of anti-tuberculosis drugs.

Sterilizing drugs kill Mycobacterium tuberculosis that persists during chemotherapy. Predictive models should mimic the conditions causing persistence in the lesions of cavitary disease, and should grade current anti-tuberculosis drugs according to their sterilizing activity determined in clinical trials. Models should start with old, stationary cultures grown micro-aerophilically. In these, persistent bacilli occur in different populations in which there is no appreciable cell division. Population 1. Grows in liquid culture medium but not on solid medium. Killed by rifampicin. Population 2. Grows on solid culture medium. Killed by rifampicin. Population 3. Grows in liquid medium but not on solid medium. Tolerant of rifampicin. Population 4. Bacilli from Cornell model mice, after treatment with pyrazinamide and isoniazid, cannot grow in liquid or on solid culture medium. Some of these populations are incorporated in models which start with 100-day liquid medium cultures. In model 1 (population 2) the new drug is added and colony counted after 7 days incubation. In models 2 and 3, 100 mg/L rifampicin is added to the 100-day culture when the bacilli lose their ability to grow on solid culture medium (population 3). After re-suspension in rifampicin-free liquid medium for 7 days, the bacilli recover growth on solid medium, when a colony count is done. The new drug is added during incubation with rifampicin in model 3 and at the start of recovery in drug-free medium in model 2. Models 1 and 3 grade isoniazid, rifampicin and pyrazinamide according to their sterilizing activity determined by clinical trials.