Evaluation of Novel PET ligands for imaging of Neuroinflammation

Neuroinflammation is a complex well-orchestrated response to various stimuli aiming to restore tissue homeostasis but under pathological conditions, it can unexpectedly increase tissue damage. NI is observed in several CNS pathologies like AD, PD, stroke, MS, ALS, HD, neoplasia and brain trauma. Microglia are resident CNS cells having similar functions to macrophages. The activation of microglia is a primary adaptation reaction of the brain innate immune system, found in physiological and pathological conditions [2]. During brain damage, microglia rapidly migrate to the site of injury relying on the directional guidance provided by the expressed chemotactic mediators. During activation, microglia undergo functional and morphological changes which are linked to the modulation of expression of several receptors, enzymes, and ion channels [7]. Hence, it is highly relevant to quantify these as biomarkers for diagnosis and the evaluation of progression of neuroinflammation in disease and after therapy. A non-invasive accurate and reliable detection of NI is of fundamental clinical interest. Quantification of microglial activation in vivo is possible with positron emission tomography (PET) provided that there is a combination of a relevant molecular target and a suitable radiotracer for this target. The present study aimed to develop a carbon-11 labelled radioligand to visualise the endocannabinoid degradation enzyme MAGL in vivo using PET. A PET ligand can be useful to measure the expression of MAGL in various pathologies like (neuro)inflammation and various cancer types. MA-PB-1 is a structural analogue of JW642 where substitution of a phenoxy group with methoxy group provides (1) better affinity towards hMAGL, (2) lower clog D values and (3) enhanced brain penetration of MA-PB-1. [11C]MA-PB-1 was successfully synthesised with good radiochemical yields, high radiochemical purity, and molar activities. Significant brain uptake of [11C]MA-PB-1 was observed in biodistribution and microPET imaging experiments in mice, rats, and monkey. Self-blocking experiments confirmed the specificity of binding. Blocking and chasing studies with the structurally non-related MAGL inhibitor MJN110 in mice, rats and monkey established [11C]MA-PB-1 as a MAGL specific irreversible tracer. Another exciting target we studied is an endocannabinoid receptor CB2R. Low expression of CB2R is found in the healthy brain, whereas this expression is upregulated in microglia under neuroinflammatory conditions. Our group has previously reported two brain penetrable high-affinity agonist PET tracers [11C]MA2 and [18F]MA3 for CB2R. [18F]MA3 has 100-fold higher affinity for hCB2R compared to [11C]MA2. We further decided to study [18F]MA3 extensively in an animal model with local overexpression of hCB2R variant. To validate the developed hCB2R overexpressed animal model, we performed a microPET study with [11C]NE40 as a reference tracer and confirmed a high-intensity signal in the hCB2R injected striatum compared to control. Evaluation of [18F]MA3 in a rat model with local striatal overexpression of hCB2R showed specific and reversible tracer binding in the hCB2 vector injected striatum, indicating that this tracer has the potential for in vivo imaging of the active state fraction of CB2 receptors with PET. Next potential target for visualization of neuro(inflammation), cancers and rheumatoid arthritis is CSF-1R. It is expressed on various cell types including macrophages, microglia, and osteoclasts. The activation and differentiation of microglia and macrophages depend on CSF-1R. From literature, we found a high affinity, favourable brain penetrable CSF-1R small molecule synthesised by Johnson and Johnson. [11C]BA-1 was synthesised with good radiochemical yields, high purities, and high molar activity. The preliminary biological evaluation confirmed high brain uptake in mouse, rat and monkey brains. Blocking experiments performed with non-radioactive reference did not result in tracer blocking in the rodent brain. We hypothesize that the high brain uptake is thus due to lysosomal trapping. Monkey brain PET studies confirmed high brain penetration of [11C]BA-1. GPR84 is a potential target for neuroinflammation. GPR84 expression in the healthy brain is low but is upregulated during inflammatory conditions. To date, no antibodies are available for GPR84, hence it might be interesting to develop a PET probe to visualise GPR-84 in vivo. G611 is a structural analogue of GLPG1205 (GPR84 antagonist), [11C]G611 was synthesised in good radiochemical yields, high radiochemical purities, and high molar activities. The specificity of [11C]G611 was verified by cell binding experiments with GPR84 transduced HEK 293 cells and with control cells. A biodistribution study confirmed good uptake of [11C]G611 into mouse brain. LPS pretreatment induced increase tracer binding in mouse brain in vivo and in THP-1 cells in vitro. These preliminary results suggest a role for GPR84 in the neuroinflammatory process and warrant further studies to evaluate [11C]G611 in other models of neuroinflammation. In conclusion, the thesis demonstrated synthesis and preclinical evaluation of four radiotracers for biomarkers that have been associated with neuroinflammation.nrpages: 15