Determination of Organ Specific Pharmacokinetic Data for the Anti-Tuberculosis Drugs in Guinea Pigs Using Microdialysis

There is a need for innovative and optimised pre-clinical models in the TB drug development pipeline to better predict the pharmacokinetics of candidate anti-TB drugs in vivo to shorten the time taken for novel drugs and treatment combinations to be approved for clinical trial, Microdialysis can be used in awake freely moving animals to describe the pharmacokinetic behaviour of drugs in the organs as a continuous sampling technique. It is the only technique available that collects the unbound pharmacologically relevant portion of the drug. The aim of this PhD was to develop and optimise the microdialysis methodology in the guinea pig model to better understand the pharmacokinetics of anti-TB drugs in the lung. Meticulous in vitro experiments were performed with each drug before progressing into in vivo experiments because the recovery (concentration of the drug in the tissue fluid related to that in the collected dialysate) of the drug is dependent upon a variety of experimental conditions. Mass spectrometry of the dialysate was used to determine the impact of flow rate, perfusion fluid, and the molecular weight cut-off and membrane length of probes on the recovery of ethambutol, isoniazid and rifampicin at physiologically relevant concentrations. Following identification of a strong correlation between rifampicin concentrations in the clinically relevant lung and the sacrospinalis (site of probe implantation) and confirmation of the calculated probe efficiency (21±9%), an in vivo microdialysis experiment was designed to measure rifampicin concentrations in the sacrospinalis. Four guinea pigs were dialysed for four hours. Guinea pigs were ear bled and relevant organs taken at necropsy to compare total vs unbound drug concentrations. Blood concentrations did not reflect the concentrations of rifampicin in the organs

Preclinical assessment of a new live attenuated Mycobacterium tuberculosis Beijing-based vaccine for tuberculosis.

Tuberculosis still claims more lives than any other pathogen, and a vaccine better than BCG is urgently needed. One of the challenges for novel TB vaccines is to protect against all Mycobacterium tuberculosis lineages, including the most virulent ones, such as the Beijing lineage. Here we developed a live attenuated M. tuberculosis mutant derived from GC1237, a Beijing strain responsible for tuberculosis outbreaks in the Canary Islands. The mutant strain is inactivated both in the Rv1503c gene, responsible for surface glycolipid synthesis, and in the two-component global regulator PhoPR. This double mutant is as safe as BCG in immunodeficient SCID mice. In immune-competent mice and guinea pigs, the mutant is as protective as BCG against M. tuberculosis strains of common lineage 4 (Euro-American). By contrast, in mice the vaccine is protective against a M. tuberculosis strain of lineage 2 (East-Asian, Beijing), while BCG is not. These results highlight differences in protection efficacy of live attenuated M. tuberculosis-derived vaccine candidates depending on their genetic background, and provide insights for the development of novel live vaccines against TB, especially in East-Asian countries where M. tuberculosis strains of the Beijing family are highly dominant

Determination of the Pharmacokinetics and Pharmacodynamics of Isoniazid, Rifampicin, Pyrazinamide and Ethambutol in a Cross-Over Cynomolgus Macaque Model of Mycobacterium tuberculosis Infection

Innovative cross-over study designs were explored in non-human primate (NHP) studies to determine the value of this approach for the evaluation of drug efficacy against tuberculosis (TB). Firstly, the pharmacokinetics (PK) of each of the drugs Isoniazid (H), Rifampicin (R), Pyrazinamide (Z) and Ethambutol (E), that are standardly used for the treatment of tuberculosis, was established in the blood of macaques after oral dosing as a monotherapy or in combination. Two studies were conducted to evaluate the pharmacokinetics and pharmacodynamics of different drug combinations using cross-over designs. The first employed a balanced, three-period Pigeon design with an extra period; this ensured that treatment by period interactions and carry-over could be detected comparing the treatments HR, HZ and HRZ using H37Rv as the challenge strain of Mycobacterium tuberculosis (M. tb). Although the design accounted for considerable variability between animals, the three regimens evaluated could not be distinguished using any of the alternative endpoints assessed. However, the degree of pathology achieved using H37Rv in the model during this study was less than expected. Based on these findings, a second experiment using a classical AB/BA design comparing HE with HRZ was conducted using the M. tb Erdman strain. More extensive pathology was observed, and differences in computerized tomography (CT) scores and bacteriology counts in the lungs were detected, although due to the small group sizes, clearer differences were not distinguished. Type 1 T helper (Th1) cell response profiles were characterized using the IFN-γ ELISPOT assay and revealed differences between drug treatments that corresponded to decreases in disease burden. Therefore, the studies performed support the utility of the NHP model for the determination of PK/PD of TB drugs, although further work is required to optimize the use of cross-over study designs

A Rabbit Model to Study Antibiotic Penetration at the Site of Infection for Nontuberculous Mycobacterial Lung Disease: Macrolide Case Study

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is a potentially fatal infectious disease requiring long treatment duration with multiple antibiotics and against which there is no reliable cure. Among the factors that have hampered the development of adequate drug regimens is the lack of an animal model that reproduces the NTM lung pathology required for studying antibiotic penetration and efficacy. Given the documented similarities between tuberculosis and NTM immunopathology in patients, we first determined that the rabbit model of active tuberculosis reproduces key features of human NTM-PD and provides an acceptable surrogate model to study lesion penetration. We focused on clarithromycin, a macrolide and pillar of NTM-PD treatment, and explored the underlying causes of the disconnect between its favorable potency and pharmacokinetics and inconsistent clinical outcome. To quantify pharmacokinetic-pharmacodynamic target attainment at the site of disease, we developed a translational model describing clarithromycin distribution from plasma to lung lesions, including the spatial quantitation of clarithromycin and azithromycin in mycobacterial lesions of two patients on long-term macrolide therapy. Through clinical simulations, we visualized the coverage of clarithromycin in plasma and four disease compartments, revealing heterogeneous bacteriostatic and bactericidal target attainment depending on the compartment and the corresponding potency against nontuberculous mycobacteria in clinically relevant assays. Overall, clarithromycin's favorable tissue penetration and lack of bactericidal activity indicated that its clinical activity is limited by pharmacodynamic, rather than pharmacokinetic, factors. Our results pave the way toward the simulation of lesion pharmacokinetic-pharmacodynamic coverage by multidrug combinations to enable the prioritization of promising regimens for clinical trials

Image_2_Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.tif

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.</p

Image_8_Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.tif

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.</p

Image_1_Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.pdf

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.</p

Image_6_Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.tif

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.</p