Substance P radiolabeled with 68Ga as a novel-imaging agent for increased Neurokinin-1 Receptor availability in chronic pain disorders

Abstract

The World Health Organization estimates that up to 33% of the world’s population suffers from chronic pain originating from an underlying musculoskeletal disorder. The prevalence of this class of diseases is on the rise with a high estimated economic impact world-wide, also in South Africa. The clinical challenge associated with chronic pain is a lack of understanding of the core pathology of the disease mechanisms involved coupled with mostly non-specific diagnostic tools and ineffective chronic pain treatment. The emerging neuropeptide Substance P (SP) targets the neurokinin-1 receptor (NK1R) and is intimately involved in the processes of inflammation and chronic pain. This is evident from histology reports of elevated levels of SP and NK1R in chronically painful tendon tissue. Molecular imaging modalities such as Positron Emission Tomography/Computed Tomography (PET/CT) have the ability to visualise and provide a quantitative measurement in vivo of the function of cellular and biological processes. Visualisation of the normal NK1R expression in vivo as well as its pathological expression on non-neural cells and tissue cells could improve understanding of the behaviour of the receptor. In this study the tracer biodistribution, pharmacokinetic and targeted in vivo PET/CT imaging of [68Ga]Ga-DOTA-[Thi8,Met(O2)11]SP was evaluated for the assessment of NK1R expression in healthy dogs and in dogs with suspected osteoarthritic conditions. The radiolabelling approach used 68Ga-activity from a tin-dioxide-based 68Ge/68Ga generator, supporting optimal radiosynthesis of [68Ga]Ga-DOTA-[Thi8,Met(O2)11]SP by way of varying parameters such as 68Ga-peptide acidity (pH) of radiolabelling solution, peptide concentration, reaction time, the benefit of a heated step, purification and the introduction of freeze dried aliquots of the peptide. Formulation was carried out to ensure physiological pH, sterility, low salt and reduced ethanol content. The potential to adapt the radiosynthesis proposed for [68Ga]Ga-DOTA-[Thi8,Met(O2)11]SP to its therapeutic counterpart [213Bi]Bi-DOTA-[Thi8,Met(O2)11]SP was determined. The modification of the peptide sequence warranted in vitro assessment of the SP analogue testing DOTA-[Thi8,Met(O2)11]SP mode of action towards the tachykinin receptor family and its dose dependent agonist/antagonist behaviour at NK1R in particular. Healthy and diseased (suspected osteoarthritis) outbreed dogs were the animal of choice because their physiology is known to be closer to humans. Furthermore, a larger animal model is a more suitable fit to a clinical PET/CT camera. Ethical approval was obtained from the Animal Ethics Committee at the University of Pretoria. Animals that met the inclusion criteria were injected with the radiotracer and each animal was allowed three static whole-body PET/CT scans at 30, 60 and 120 min post injection. Blood samples were obtained at certain time points and the bladder of the animal voided prior to each image. Time-activity curves facilitated the calculation of the pharmacological half-life and urinary elimination rate of the radiopharmaceutical. Radiosynthesis, optimisation, and a safe for administration formulation of [68Ga]Ga-DOTA-[Thi8,Met(O2)11]SP (0.05 mg/28 nmol, pH 6.5, <5% ethanol) was achieved by way of a robust, repeatable method (95 ºC, 15 min, C18 purification) that used freeze-dried aliquots of the peptide. The optimised parameters were improved when compared with other 68Ga-peptide preparations suitable for pre-clinical or clinical application. Reliably high radiolabelling efficiencies were achieved (>90%) with colloids <6% and uncomplexed 68Ga <3% (n > 3). Excellent radiochemical purity >99% could be achieved following C18-based solid phase extraction. [68Ga]Ga DOTA-[Thi8,Met(O2)11]SP was prepared with high specific activity (13.5 ± 3.9 MBq/nmol) providing sufficient yields to serve multiple doses for pre-clinical imaging studies. [213Bi]Bi-DOTA-[Thi8,Met(O2)11]SP was prepared using a simple, robust radiolabelling method similar to that employed for [68Ga]Ga-DOTA-[Thi8,Met(O2)11]SP. The theranostic pair of [68Ga]Ga-/[213Bi]Bi-DOTA-[Thi8,Met(O2)11]SP may not have application in chronic pain disorders, however the safe to administer formulation may be of benefit for other applications. The optimised radiosynthesis of [213Bi]Bi DOTA-[Thi8,Met(O2)11]SP achieved a RCP of 65.7%, 91.2% and 97.5% (using 0.05 mg, 222-259 MBq, n = 3) after 5, 10 and 15 min incubation, respectively. A simpler radiolabelling method applicable to [68Ga]Ga-/[213Bi]Bi-DOTA-[Thi8,Met(O2)11]SP was successfully established. [68Ga]Ga-DOTA-[Thi8,Met(O2)11]SP was found to have similar efficacy and potency as native SP but greater selectivity for the NK1R in vitro. The DOTA conjugated peptide functions as an agonist at the receptor with no antagonistic behavior. Activation of NK1R may result in undesired pharmacological effects which must be carefully considered when attempting translation into the clinical setting. The radiotracer was unable to elicit significant activation of any of the other tachykinin receptors at high concentrations and also did not have any antagonistic behaviour at these receptors. Favourable pharmacokinetics and biodistribution was determined in outbreed dogs for [68Ga]Ga-DOTA-[Thi8,Met(O2)11]SP which was deemed suitable for imaging of osteoarthritic pain. The pharmacological half-life was determined to be around 15 min. Excretion was predominantly renal with acceptable, transient liver uptake. Areas synonymous with osteoarthritis including the legs, paws, hips and shoulders presented with unilateral uptake of the tracer. This was observed in bone and soft tissue. Elevated and persistent uptake in characteristic NK1R-dense tissues such as the gut mucosa could be visualised. [68Ga]Ga-DOTA-[Thi8,Met(O2)11]SP-PET/CT imaging may be a powerful tool to detect NK1R-mediated tissue pain. However further investigations should focus on correlating the NK1R expression in non-neuronal tissues with the accumulation of [68Ga]Ga-DOTA-[Thi8,Met(O2)11]SP in vivo.