Tuberculosis (TB) is one of the oldest known human diseases and is still a major cause of mortality worldwide. The disease is caused by Mycobacterium tuberculosis (MTB) which is typically transferred from human to human through inhalation of aerosolized respiratory droplets. The techniques currently employed for diagnosis are generally insensitive and non-specific. Positron emission tomography (PET) combined with computed tomography (CT) is used extensively in clinics and is among the advanced molecular imaging modalities for disease diagnosis and for monitoring the response to treatment. However, the PET radio tracer 2-Deoxy-2-[18F] fluoro-D-glucose ([18F]FDG) images areas of high metabolism and thus is also non-specific.
Herein, we describe development of new PET probes based on sugars. 2-Fluoro-2-deoxy-trehalose (FDT), previously developed in our group has shown great promise as a specific PET imaging agent for the diagnosis of TB. We have used biocatalytic approaches for the synthesis of FDT, testing various enzymes. We found that the combination of hexokinase, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) for biosynthesis was faster and higher yielding than alternative approaches using enzyme fusions of TPS-TPP used with hexokinase or the use of alpha, alpha trehalose synthase (TreT) enzymes alone. We then sought to develop an LC-MS based method for the detection of FDT in vitro and in vivo. The enzyme kinetic parameters were also determined for various enzymes used in the FDT synthesis.
FDT administered to naïve marmosets labels very little tissue in the lung, but when the animals are infected MTB, the tubercular lesions take up the radiolabelled FDT and [18F] Trehalose monomycolate has been isolated from tubercular lesions indicating that the probe reaches the site of bacterial incorporation. The uptake of the probe into lesions can be blocked by administration of the unlabelled or “cold” FDT. In addition, FDT labelling of tubercular lesions is reduced with 4-drug standard TB treatment in marmosets. We have optimized and scaled up syntheses and developed GMP-standard methods suitable for producing the probe for clinical use. Toxicity of the synthesised sugar compound was assessed in two different animal models to support an IND application. Once toxicity studies are completed, we aim to file an IND application for testing in humans to determine if allows for specific and early diagnosis of TB. Through planned future work, we aim to optimise the conditions of the radio-synthesis of FDT for use in clinical trials of anti-tuberculosis agents.