Summary

ʟ-carnitine and its acyl derivatives are essential for a variety of biological functions and metabolic pathways, the most important of which is the transport of fatty acids into the mitochondria for β-oxidation, producing ATP. This process, known as ‘the carnitine shuttle’, proceeds via conjugation of fatty acids to carnitine by carnitine palmitoyltransferase (CPT1) on the outer mitochondrial membrane to generate acyl-carnitines. Subsequent carnitine-mediated transport of these fatty acids to the inner mitochondrial matrix and, finally, cleavage of the acyl-carnitine ester by CPT2 provides the fatty acid for β-oxidation and liberates free carnitine, which is recycled to continue the shuttling process.Cellular carnitine homeostasis is regulated by its de novo synthesis from gamma-butyrobetaine by gamma-butyrobetaine dioxygenase 1 (BBOX1) or by transport via the sodium-dependent cation transporter, OCTN2.
In addition to its vital role in facilitating β-oxidation, carnitine regulates acyl-CoA:CoA ratios, eliminates irregular organic acids, and acts as an antioxidant and cellular protectant. Concentrations of (acyl)carnitines and the ratio between ‘free’ carnitine and its acyl derivatives are widely used to identify inborn errors of fatty acid metabolism. Additionally, altered carnitine transport and aberrant carnitine metabolism are associated with a variety of diseases and disorders, including heart disease, cancer and insulin resistance. Carnitine has been established as an effective treatment in some cases, including for primary carnitine deficiency.
Despite carnitine's established importance for healthy energy metabolism, a quarter of a billion USD supplement market, and its approved application as both a pharmaceutical and a disease biomarker, there remains debate over the value and risks of carnitine supplementation. Indeed, mixed clinical results have been observed with carnitine supplementation in the context of Alzheimer's disease and dementia, cardiovascular disease (CVD) and peripheral artery disease, diabetes and insulin resistance, infertility, osteoarthritis, and athletic performance enhancement. New tools and techniques are therefore required to better understand healthy, supplemented, and dysregulated carnitine utilisation.
Fluorinated analogues of endogenous small molecules, such as amino acids and sugars, have proven to be invaluable tools for visualising metabolic processes. Labelling biomolecules with fluorine enables detection, identification, and quantification of the parent compound and its downstream metabolites using ¹⁹F nuclear magnetic resonance (NMR) and mass spectrometry (MS). Both ¹⁹F-NMR and MS rely on the low abundance of fluorine in living systems to provide high signal-to-noise detection of the exogenously administered ¹⁹F-labelled species within the complex biological milieux. The biotransformation of the labelled molecule can subsequently be identified and measured, enabling the interrogation of specific metabolic pathways.
The use of ¹⁸F-labelled biomolecules for in vivo imaging with positron emission tomography (PET) has had a considerable impact on our understanding of healthy and perturbed metabolism, as well as our ability to diagnose diseases. [¹⁸F]2-fluoro-2-deoxy-D-glucose (FDG) PET is perhaps the best example, providing a readout of aberrant glucose utilisation and used clinically for the staging and restaging of cancer. Building on this prior work, the authors hypothesized that the generation of an appropriately designed (radio)fluorinated carnitine derivative would provide a valuable molecular probe to interrogate carnitine metabolism, as well as a diagnostic tracer for pathologies exhibiting abnormal carnitine utilisation. Toward this goal, we developed a strategy that enables the labelling of carnitine with fluorine-19 or fluorine-18 without perturbing its biological activity. The strategy was inspired by the synthesis of [¹⁸F]fluoromethylcholine ([¹⁸F]FCH), a radiotracer with widespread utility for cancer imaging.
Herein, the authors report the design and synthesis of fluoromethylcarnitine (FMC) and its radiolabelled derivative, [¹⁸F]FMC, and demonstrate their ability to visualise carnitine transport and metabolism both in vitro and in vivo. Furthermore, carnitine utilisation in cancer was imaged for the first time with [¹⁸F]FMC PET in a xenograft model of non-small cell lung cancer (NSCLC), revealing increased carnitine utilisation in the transformed cells compared to healthy lung tissue.

Results from nanoScan® PET/CT

For all imaging studies, mice were maintained under anaesthesia with isoflurane (1.5–2% in O2) at 37 °C during tail vein cannulation and imaging. For healthy imaging, dynamic PET scans were acquired on a Mediso NanoScan PET/CT system (1–5 coincidence mode; 3D reconstruction; CT attenuation-corrected; scatter corrected) using the four-bed mouse hotel. Images were acquired for 120 min following a bolus intravenous injection of [¹⁸F]FMC (≈1.5–3 MBq in 100 µL) through a tail vein cannula. For the LC co-injection study, 400 µm of LC (Sigma) was simultaneously injected in 50 uL through a tail vein cannula. To determine radiotracer specificity in H460 tumour models, LC (50 mg kg⁻¹; n = 4/group) and meldonium (250 mg kg⁻¹; n = 4/group) were co-injected i.v. with [¹⁸F]FMC. CT images were obtained for anatomical reference and attenuation correction (180 projections; semicircular acquisition; 50 kVp; 300 ms exposure time). The acquired data were reconstructed into 15 bins of 4 × 15 s, 4 × 60 s, and 3 × 300 s, 4 x 20 min (Tera-Tomo 3D reconstructed algorithm; 4 iterations; 6 subjects; 400–600 keV; 0.3 mm³ voxel size). VivoQuant software (v 2.5, Invicro Ltd.) was used to analyse the reconstructed images. Regions of interest (ROIs) were drawn manually using CT images and 120-min dynamic PET images. Time verses radioactivity curves (TACs) were generated using the percentage injected dose per mL (%ID/g).

Given that [¹⁸F]FMC mapped carnitine metabolism in cells, the authors next assessed its ability to non-invasively probe carnitine utilisation in vivo. [¹⁸F]FMC was injected into healthy nude mice via the tail vein, and dynamic PET scans were acquired for 2 h, which was followed by biodistribution measurements ex vivo from excised tissue. [¹⁸F]FMC was rapidly cleared from the blood and reabsorbed by the kidneys before accumulating in the liver (Figure 3A,B). The observed reabsorption reflects the well-established mechanisms in place to maintain homeostasis of the carnitine pool. In humans, > 97% of carnitine is reabsorbed by the kidneys back into the blood when kidney function and carnitine blood levels are both normal.
To establish if [¹⁸F]FMC could be used to visualise disrupted kidney reabsorption, they co-injected carnitine (4.7 mg kg⁻¹) with [¹⁸F]FMC to generate a carnitine blood concentration of ≈400 µm – a concentration known to saturate carnitine reabsorption in the healthy kidney (≈10 times the kidney reabsorption threshold). Here, [¹⁸F]FMC was rapidly excreted to the bladder via the kidneys, which retained radioactivity up to 60 min before decreasing due to a combination of urinary excretion and reabsorption back into the blood (Figure 3C-E). The results align with clinical observations for indications characterised by reduced renal function and/or a deficiency of functional OCTN2 transporter, including primary carnitine deficiency. In addition to uptake in the kidney and liver, [¹⁸F]FMC also accumulated in the heart, which was significantly reduced by co-injection of carnitine (Figure 3E). [¹⁸F]FMC accumulation in the heart (without carnitine co-injection) showed clear delineation of the left ventricular myocardium (Figure 3F). Accumulation of [¹⁸F]FMC was well above background, with a heart-to-skeletal muscle ratio of 3.7 (Figure 3G).

Given that [¹⁸F]FMC mirrored carnitine utilisation in healthy mice, the authors next explored the use of [¹⁸F]FMC PET to visualise aberrant carnitine utilisation in cancer. In this setting, carnitine utilisation has previously been studied using ex vivo techniques, such as MS imaging. However, carnitine utilisation in tumours has yet to be visualised in living subjects. Here, [¹⁸F]FMC PET imaging was performed in H460 NSCLC tumour-bearing mice. Healthy tissue distribution of [¹⁸F]FMC closely matched that observed in healthy mice, with rapid extraction by the kidneys and redistribution to the liver (Figure 4). Tumour retention of [¹⁸F]FMC increased over the imaging time course (Figure S1, Supporting Information), with uptake in the tumour reaching 7.2 ± 2.8% ID g⁻¹ at 2 h (n = 7). [¹⁸F]FMC tumour uptake was considerably higher than blood and muscle, allowing clear delineation of the H460 tumour in the PET images (Figure 4A), yielding tumour-to-blood and tumour-to-muscle ratios of 3.2 and 4.9, respectively. Tumour [¹⁸F]FMC uptake was blocked by co-injection with either ʟ-carnitine (Figure 4B) or the selective OCTN2 inhibitor, meldonium (Figure S2, Supporting Information), suggesting [¹⁸F]FMC tumour uptake was mediated by OCTN2. [¹⁸F]FMC tissue distribution was corroborated by ex vivo biodistribution analysis (Figure 4C). The dynamic imaging of carnitine utilisation in tumours provides opportunities to improve our understanding of cancer metabolism in vivo. Visualisation of tumour metabolic plasticity during nutrient restriction or therapeutic pressure using [¹⁸F]FMC PET is an exciting possibility.
With PET, they can assess metabolism across the entire animal, which affords additional benefits. Carnitine or meldonium co-injection not only reduced [¹⁸F]FMC tumour accumulation, but also suppressed uptake in the heart, liver, kidney, and brown fat (Figure 4C-E). Meldonium, or mildronate, is used for the treatment of stable angina and has been evaluated clinically for the treatment of myocardial ischaemia. The results from our study suggest that [¹⁸F]FMC could be used to visualise meldonium inhibition of OCTN2 transport in cardiac tissue, potentially providing a readout of treatment efficacy or aiding dose optimisation.

• The authors have designed and synthesised (radio)fluorinated carnitine derivatives to interrogate carnitine utilisation in biological systems and living subjects. Both ¹⁹F- and ¹⁸F-labelled carnitine derivatives retained the essential biological activities of carnitine, concerning both transport and metabolism.
• The developed syntheses enabled quick access to the (radio)fluorinated compounds, with simple SPE purification to obtain the probes with excellent purity.
•  [¹⁸F]FMC PET was able to visualise both healthy and aberrant carnitine utilisation in tissues, revealing high tumour carnitine avidity in a xenograft model of NSCLC.
• Furthermore, ex vivo analysis revealed differential downstream fatty acid metabolism of [¹⁸F]FMC across tissues.
• Consequently, FMC and [¹⁸F]FMC are poised to improve our understanding of healthy, supplemented, and aberrant carnitine metabolism. With this in mind, they are developing an automated synthesis to enable high-activity clinical production of [¹⁸F]FMC.

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