Distributable, Metabolic PET Reporting of Tuberculosis

Abstract

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [¹⁸F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[¹⁸F]fluoro-2-deoxytrehalose ([¹⁸F]FDT) - can act as a mechanism-based enzyme reporter in vivo. Use of [¹⁸F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-specific processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [¹⁸F]FDT from the most globally-abundant organic ¹⁸F-containing molecule, [¹⁸F]FDG. The full, pre-clinical validation of both production method and [¹⁸F]FDT now creates a new, bacterium-specific, clinical diagnostic candidate. We anticipate that this distributable technology to generate clinical-grade [¹⁸F]FDT directly from the widely-available clinical reagent [¹⁸F]FDG, without need for either bespoke radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.

Figure 1.. A Strategy for Non-Invasive Imaging Reporters using Trehalose-based, TB-specific Probes Derived Directly from [¹⁸F]FDG.

A) Trehalose (blue) in Mtb is found in the outer portion of the mycobacterial cell envelope as its corresponding mycolate glycolipids. The biosynthesis of trehalose mycolates (lipidation) is catalyzed specifically in Mtb by abundant membrane-associated Antigen 85 (Ag85a, Ag85b and Ag85c) enzymes. The lack of naturally occurring trehalose in mammalian hosts as well as the uptake of exogenous trehalose by Mtb suggests that it could function as both a highly specific and sensitive probe, allowing here the development of an in vivo TB-specific, PET-radiotracer analogue [¹⁸F]FDT that selectively labels lesions (red) in infected organisms (right). Uninfected organisms (left) do not process trehalose and so probe is not retained. B) Prior work has established that Ag85s are sufficiently plastic in the substrate scope that they can process, for example, fluorescent analogues of trehalose, allowing them to be metabolically incorporated into the mycobacterial outer membrane in vitro for labeling. Fluorescence-based methods are not yet amenable to effective, non-invasive imaging in vivo. C) Four ¹⁸F-labeled variants of trehalose were tested in which each of the available hydroxyls (OH-2, 3, 4, 6) were converted in turn to ¹⁸F. D) Enzymatic synthesis of [¹⁸F]FDT. Three parallel routes (A, B and C, gray) were evaluated for efficiency, rate and yield. Route A: TreT-mediated synthesis was evaluated using enzymes from two different sources (from T. tenax or P. horikoshii). Both proved functional but gave lower turnover under a range of conditions (see pseudo-single substrate plot for TreT (from T. tenax), lower right – See Supplementary Figure S1 for further details of kinetics). [Reagents and Conditions: 50 mM HEPES, 100 mM NaCl and 10 mM MgCl₂, pH 7.5]. Route B: OtsAB-fusion enzyme-mediated synthesis was explored to allow use of fewer biocatalysts. A OtsAB fusion protein was constructed but proved to be more difficult to express and less stable under typical reaction conditions [Reagents and Conditions: 50 mM HEPES, 100 mM NaCl and 10 mM MgCl₂, pH 7.5 C] Route C: Although a three-step, three-enzyme route, Route C proved to be more flexible and reliable. The selectivity of the biocatalysts allowed this to be performed in a convenient one-pot manner. The stability of enzyme components and the ability to vary catalyst amounts to control flux led to its choice over Route B. The higher turnovers and efficiencies led to its choice over Route A (see pseudo-single substrate plot for OtsA, upper right – See Supplementary Figure S1 for further details of kinetics). [Reagents and Conditions: 50 mM HEPES, 100 mM NaCl and 10 mM MgCl₂, pH 7.5.]

Figure 2.. Reaction Optimization, Scale-up and Batch Synthesis in the Development of an Efficient Scaleable One-Pot Synthesis.

A) Chosen Route C (from Figure 1) was tested in a one-pot format [¹⁹F]FDT using pyrogen-free enzymes OtsA^Sf and OtsB^Sf. B) Example reaction optimization with fixed donor sugar Glc-UDP [30 mM] at different acceptor substrate quantities (6.25, 12.5, 25 and 50 mg) of [¹⁹F]FDG reactions were monitored in real time by calibrated ¹⁹F NMR to optimize reaction. C) Direct reaction monitoring of one-pot, 3-step [¹⁹F]FDT synthesis from [¹⁹F]FDG by ¹⁹F-NMR. NMR spectra: (i) ¹⁹F NMR spectrum of purified [¹⁹F]FDT; (ii) Crude ¹⁹F NMR spectrum of [¹⁹F]FDT with small amount of deoxy-fluoro-G6P ([¹⁹F]-1) still present; (iii) Crude ¹⁹F NMR spectrum of intermediates [¹⁹F]-1 and [¹⁹F]-2; (iv) Crude ¹⁹F NMR spectrum of [¹⁹F]-1 from [¹⁹F]FDG conversion; and (v) reference sample of starting material [¹⁹F]FDG. D) Representative, ¹⁹F-NMR spectra of crude reaction mixtures containing [¹⁹F]FDT from repeated batches synthesised by 3-enzymes-3-steps, one-pot syntheses, prior to purification.

Figure 3.. Pulmonary [¹⁸F]FDT PET uptake into Tuberculous Lung Lesions is Reproducible within 90 min with Correlated Signal in Lesions with Higher Bacterial Loads.

A) [¹⁸F]FDT PET/CT scan of a naïve marmoset lung (administered 1 mCi) [¹⁸F]FDT and imaged 90 min post-injection (transverse view). B) [¹⁸F]FDT PET/CT scan of a representative, Mtb-infected marmoset lung showing lesions outlined in green and red (transverse view). The iliocostalis muscle was used for normalization of PET uptake (region outlined in blue). The target dose was 2.2 mCi/kg (~1 mCi). C) The [¹⁸F]FDT PET signal from Mtb-infected marmoset lungs is significantly higher than the signal from uninfected (control) marmoset lungs. The mean Standard uptake values (SUVbw) are represented as boxplots where the central bars represent the median. At least 4 lung regions of interest (ROI) were measured from 3 infected and 2 uninfected marmosets (p<0.0001). D) and E) Transverse images of [¹⁸F]FDT PET uptake scans for unblocked (D) and blocked (E) uptake in the same infected marmoset. [¹⁸F]FDT uptake was blocked by administering excess blocker, split into two administrations 1 h and 5 min prior to radiotracer administration. F) Reduction of [¹⁸F]FDT uptake in marmosets administered cold [¹⁹F]FDT blocker prior to being administered [¹⁸F]FDT (1.2 mCi) compared to when administered [¹⁸F]FDT alone. Animals (n = 22) were randomized as to the order of blocked scan; two days later the same animals were imaged with the treatments reversed. Uptake was measured in images collected 60 min and 90 min after probe administration. In both cases the animal receiving [¹⁹F]FDT blocking dose showed significantly lower accumulation of [¹⁸F]FDT into tubercular lesions. G) Comparison of [¹⁸F]FDT (1.0 mCi) lesion uptake (SUV/CMR) at 60, 90, and 120 mins post injection (lesions from two infected, antibiotic-treated marmosets) demonstrates that signal does not increase after 90 minutes. H) [¹⁸F]FDT uptake (SUV/CMR) is reproducible (r² = 0.94), as illustrated by comparison of individual lesion SUV/CMR values from marmosets with imaging repeated two days apart (44 dpi and 46 dpi). I) [¹⁸F]FDT uptake into tubercular lesion tends to increase with higher mycobacterial loads (p = 0.001, ρ = 0.64). The total SUV of each lesion was compared with mycobacterial colony forming units measured (log CFU) in the lesions from two marmosets.

Figure 4.. Differential Uptake and Labeling of Tubercular Lesions by [¹⁸F]FDT and [¹⁸F]FDG in Mtb-Infected Marmosets.

Serial [¹⁸F]FDG (0.8 mCi) (A and B) and [¹⁸F]FDT (1.1 mCi) (D and E) scans of the same infected marmoset were collected at 90 minutes post injection and co-registered based on the PET/CT shown in (A) and (D), respectively. In (C) the three-dimensional maximum intensity projection (MIP) of the FDG signal in the lung (colored shades of yellow-to-gold (B)) was overlaid with the three-dimensional MIP of the [¹⁸F]FDT signal (colored shades of green-to-blue (E), green showing most intense labeling) to indicate the differences in the regions of intense labeling. Together these show clear differences in regions of intense radioactive signal indicating that the two probes are pooling in tissue regions with different characteristics.

Figure 5.. [¹⁸F]FDT Scans of an Mtb-infected Marmoset Correlate with 4-week HRZE Treatment.

A) Pretreatment scan 60 min post-injection with [¹⁸F]FDG PET (0.7 mCi) shows two lesions (indicated as 1 and 2). B) Marmoset imaged with [¹⁸F]FDG after 4 weeks of treatment showing the same 2 lesions. C) [¹⁸F]FDG SUV before and after treatment of the lung lesions was variable and occasionally increased. D) Total probe uptake as determined by total lesion glycolysis (TLG) of the lesions showed a lesion-dependent pattern suggestive of a highly refractory response to [¹⁸F]FDG uptake, despite treatment. E and F) By contrast, similarly-timed (pretreatment and 4 weeks of treatment) [¹⁸F]FDT scans 90 minutes post injection (E 0.7 mCi; F 1 mCi) showed reduced SUV and total [¹⁸F]FDT uptake in the lesions after 4 weeks of treatment. Low bacterial load was indeed observed in the lesions: lesion 1 = 2.6 logCFU, lesion 2 = 2.8 logCFU. G) After HRZE combination drug therapy, the [¹⁸F]FDT SUV/CMR of the lung lesions was significantly decreased (p< 0.001) and H) the total [¹⁸F]FDT uptake in the lesions, as determined by TLG, was also reduced (p<0.005). One representative image pair is shown of three similar treatment experiments.

Figure 6. [¹⁸F]FDT is also an Effective and Specific Radiotracer in an ‘Old World’ NHP TB Model

A) Axial and coronal views of the lung of a representative Mtb-infected cynomolgus macaque showing labeling of the tubercular lesion clusters labeling (green and yellow arrows) with [¹⁸F]FDT (5 mCi) scanned 60 minutes post-injection. Calibration scale in SUV. B) Axial and coronal views of the same lung lesion clusters labeled with [¹⁸F]FDG (5 mCi) 60 minutes post-injection. Calibration scale in SUV. C) The animal was necropsied and the individual lesions were plated. Ante mortum probe uptake of the lesions was compared to their culture status. In images captured at 60 minutes there was a trend toward lesions with culturable bacteria having higher probe uptake. D) By 120 minutes, the uptake of probe was significantly higher among lesions with culturable mycobacteria than among sterile lesions. Data from one of three representative animals are shown.

Figure 7.. Dynamic Biodistribution of [¹⁸F]FDT in Naïve Rhesus Macaques and Infected Marmosets to Estimate Organ Exposure.

A) Coronal maximum intensity projections of [¹⁸F]FDT PET activity in a representative naïve rhesus macaque collected over approximately 115 min in scan frames of increasing duration in a dynamic PET scan (from top left) after administration of 151 MBq of [¹⁸F]FDT. B) Organ radiation absorbed doses (mSv/MBq) calculated by extrapolating the rhesus macaque data to humans; organs receiving the highest doses were urinary bladder wall, kidneys, and adrenals. C) Similar to the rhesus experiments, the quantification of the radioactivity in marmoset tissues (using gamma counting of [¹⁸F]FDT radioactivity in excised tissues of euthanised marmosets) indicates the kidneys were the solid organs with the highest proportion of the injected dose (%ID/g) when marmosets were necropsied 120 min after being administered [¹⁸F]FDT.