"A combination of oral treatment with high dosage of rifamycins,
Professor Denny Mitchison. 2010
Eight years have passed since the idea that a weak acid could be used by inhalation as an adjuvant to oral pyrazinamide, as a method able to increase the bactericidal activity of pyrazinamide, and this is an update of the progress being made in developing a therapeutic product.
The basis of this concept, is that pyrazinamide has dose-dependant activity against M. tuberculosis, but dose size is unable to be increased further above the recommended amount in TB therapy, because it raises the risk of hepatic Injury. A novel solution to this problem, is to acidify pulmonary lesions from an external source, where just a small shift towards a lower pH will result in a potentially huge increase in PZA / POA activity.
The steps that I took to explore this idea further was to first contact Prof Clive Page, head of the Sackler Lung Institute to confirm if it was safe to introduce a weak acid into the lung, and was then able to work on a plan that the weak acid was to be formulated as dry powder particles for use by inhalation.
I sent out an expertise request flyer through the London Technology Network, that resulted in a collaboration with Professor Denny Mitchison, a renown TB bacteriologist. The hypothesis was that a weak acid could be administered by inhalation to further lower the existing pH in TB lesions that would help increase the bactericidal activity of oral pyrazinamide against TB bacilli internalised within alveola macrophages.
Following some in vitro tests that produced predictable results, the most likely acid able to shift pH was identified as being pyrazinoic acid, the moiety of pyrazinamide that has been used in TB therapy for over 50 years already and with a pKa=2.9 is capable of lowering pH in lesions to make the required difference in increasing PZA activity.
This concept was first revealed at the INTERTB symposium in London 2009, and the hypothesis is outlined in an article by Mitchison, 'Improving the activity of rifamycins and pyrazinamide' (2010) where Mitchison postulates that using POA by inhalation as an adjunct to oral PZA, high dosage of rifamycins, and TMC, (where pretomanid is now a better drug choice) will result in TB therapy being able to be completed in about one month, from what is now about six months, without increasing risk of recurrent TB through incomplete therapy.
PROOF OF CONCEPT EVIDENCE.
There are distinct therapeutic objectives in lowering acid pH to increase the bactericidal activity of pyrazinamide, and the use of POA to have activity against MTB itself. However, there may be an emergence of objectives in the use of POA esters, that has yet to be discovered.
POA is poorly active in antimycobacterial tests, while it cannot pass through mycobacterial cell walls due to its high hydrophilic and ionizable characteristics. Cynamon et al, proposed the esterification of POA to obtain more lipophilic compounds of the active agent, able to cross the cell wall. Several compounds exhibiting activity were synthesized, with 5-chloropyrazinoates and 5-methylpyrazinoates being the most active analogs. Since then, a number of formulations of POA esters have been produced, and could be capable of lowering the pH of TB lesions to increase the activity of oral PZA, and also have activity against MTB.
With this in mind, the first evidence that this concept might work was through a proof of concept animal study undertaken in 2016, where aerosolised pyrazinoic acid esters have been tested for efficacy. In this study, PAE’s were used as a supplement to oral therapy and significantly reduced the organ bacterial burden in comparison to infected, untreated control animals. The team suggest that PAE aerosol therapy is a potentially significant addition to the regimen for PZA resistant MDR-TB and XDR-TB treatment.
This study group have a direct relationship with my original collaborators, and the results publication is shown in the links below.
What's changed over the last eight years is that the original concept of using pyrazinoic acid in inhalation therapy, has resulted in studies with POA salts and esters, and several new drugs have appeared and are in trial, with the main interest drug being pretomanid, being unique in that it has two separate mechanisms of action able to kill both hypoxic non replicating and replicating bacilli, at the same time has a low MIC for M. tuberculosis, comparable to that of Isoniazid.
Studies reveal a synergy exists between PA and PZA making them ideal companions in standard TB therapy. However, its the treatment of X/MDR-TB therapy that is left most wanting for new antibiotics and treatment solutions. The loss of rifampicin in particular, means the clearance of MTB within caseous lesions might suffer, where liquefied caseum is the main route for TB pathogenesis during active disease, having a high pH necessary for MTB replication, that can result in cavity formation where extracellular growth of tubercle bacilli in cavities cause lung damage and bronchial spread of MTB.
Early treatment of drug resistant disease is essenintial to prevent irreversible lung damage caused by cavitation, that may be provided by the use of aerosolised POA ester formulations, in combination with oral pretomanid in a novel therapeutic strategy.
In standard TB therapy PZA has no activity inside liquified caseum because the pH is too high. This is not the case for pyrazinoic acid esters when administered by pulmonary delivery, the only way in which it is bioavailable, and represents a new therapeutic opportunity in X/MDR-TB therapy. The use of aerosolised pyrazinoic acid esters, if able to penetrate and accumulate within liquefied caseum, the effect is to lower acid pH to inhibit growth of replicating bacilli within, as part of a stepped process to prevent cavity formations.
Various formulations of pyrazinoic acid for pulmonary delivery are being developed. The proposal is to have a suitable formulation of POA esters for use by inhalation directly into the lung. On delivery, the intention is that POA esters will have a bacteriostatic effect on TB bacilli within liquefied caseum, by lowering the typical neutral pH7.2 to pH6.6. How low the pH can be reduced is dependent on the uptake of POA within caseum and subsequent accumulation. The next parameter being frequency of administration; all of these are unknown currently. Pyrazionic acid also has a mechanism of action similar to that of bedaquiline, so it has in fact two modes of action.
The therapeutic aims of a combination of aerosolised POA esters and oral pretmomanid, is POA will first inhibit bacterial growth by lowering acid pH within liquefied caseum and then have synergistic action with pretomonid against MTB. The expected outcome of this action is the clearance of TB bacilli within liquefied caseum so preventing new cavity formations where extracellular growth of tubercule bacilli cause lung damage and bronchial spread of MTB. At the same time, POA esters in combination with pretomanid will also have synergistic activity against TB bacilli within activated macrophages.
The acidification of TB lesions may alter the efficacy of some antibiotics, that will need to be examined.
Aerosolised POA esters has had a first success in the animal model, that is going to lead to more studies. While this approach differs from the original concept, the movement towards pulmonary delivery of drugs is gaining in popularity, but the use of POA in whatever therapeutic forms, is only bioavailable through inhalation, and its a question of how important POA as an agent proves to be within TB therapy that needs to be discovered.
FIRST PUBLIC DISCLOSURE: INTERTB Symposium London Sept 2009.
The near future: Improving the activity of rifamycins and pyrazinamide 2010
Authors: D.A. Mitchison. P.B. Fourie
The chemotherapy of tuberculosis: past, present and future. 2012
Schematic poster of the concept.
Spray Dried Aerosol Particles of Pyrazinoic Acid Salts for Tuberculosis Therapy
POA Esters history
First animal proof of concept study for aerosolised pyrazinoic acid esters
My thanks to Professor Clive Page, Professor of Pharmacology and Head of Sackler Institute of Pulmonary Pharmacology, and Professor Paul Francis, Kings College London, for arranging my meeting with experts.
Copyright © Gino Francesco 2016
Update 19th February 2018