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Project

Development of a radiolabelled probes for the in vivo visualisation of the TRPV1 receptor with PET

The transient receptor potential vanilloid subfamily member 1 (TRPV1) channel is classified as a member of the TRP family of ion channels. It is a pH- and heat-sensitive cation channel, but can also be activated by endogenous or naturally occurring or synthetic exogenous ligands. The most general known agonist of TRPV1 is capsaicin, the pungent compound of the red chilli pepper. TRPV1 is mainly expressed in a subset of nociceptive neurons, originating in the dorsal root and trigeminal ganglia that innervate virtually all tissues (e.g. skin, muscle, bone, internal organs and vascular system). Here it has an established role in pain sensation and the generation and progression of neurogenic inflammation. Therefore, TRPV1 became a target of interest for pharmaceutical companies for the development of a new class of analgesics and anti-inflammatory drugs.</>
Over the past years, intensive research has revealed the presence of TRPV1 channels in various brain regions. Yet, the extent to which these central TRPV1 channels are expressed and their exact (patho)physiological role remains to be elucidated.</> </>Hypothalamic TRPV1 receptorshave been suggested to play a role in thermoregulation and TRPV1-expressing hippocampal neurons may be involved in the control of emotions, learning and epileptic activity. Furthermore, there are hints that TRPV1 contributes to the pathogenesis of febrile seizures and neurodegenerative brain disorders such as Parkinsons disease. </></>
In the present study we aimed on developing carbon-11 (11C) and fluorine-18 (18F) labelled radioligands to visualise TRPV1 in vivo </>using positron emission tomography (PET). A successful PET-radioligand for TRPV1 could speed up theresearch to unravel its expression pattern and (patho)physiological role in the central nervous system (CNS), allow the clinical evaluation of therapeutics that target TRPV1 and provide new diagnostic opportunities for diseases in which TRPV1 expression or activity is altered. As a first approach, [11C]SB366791, a radiolabelled analogue of the potent cinnamic acid derivative SB366791, was successfully synthesised in good yields, high radiochemical purity and high specific activity. Its preliminary biological evaluation showed promising results including efficient blood-brain barrier (BBB) penetration, fast clearance from blood and TRPV1-selective binding in the trigeminal nerve. Yet, in our hands the experimental in vitro</> binding affinity of SB366791 for TRPV1 was found to be lower than earlier reported values. </>
Next, a subtle change in the structure of SB366791 led to the development of [11C]DVV24, which showed an improved in vitro</> binding affinity for TRPV1. As expected, the newtracer displayed a similar distribution pattern, comparable brain uptake and hepatobiliary elimination as observed for [11C]SB366791. Yet, [11C]DVV24 showed a lower specific accumulation in the trigeminal nerve, which may be due to its slightly higher lipophilicity and faster in vivo</>metabolism. </>
In a final attempt to develop high affinity tracers for TRPV1, several highly potent TRPV1 antagonists belonging to different structural classes including urea derivatives, aminoquinazolines and daphnane diterpenes have been selected from literature data to serve as lead compounds. Labelling of our urea precursor was unsuccessful because of stability problems, but the aminoquinazoline [18F]DVV54 and daphnane diterpene 123I-RTX were efficiently synthesised. Both compounds showed significantly higher in vitro</> binding affinities for TRPV1 compared tothe cinnamic acid derivatives. Biodistribution studies in mice showed that 123I-RTX was rapidly cleared from blood by the hepatobiliary pathway. But [18F]DVV54 showed much slower kinetics, high in vivo</> stability and significant non-specific binding, which may result in low target-to-background signals. The brain uptake was low for [18F]DVV54 and negligible for 123I-RTX, in accordance with their unfavourable logD and polar surface area (123I-RTX) values. Furthermore, 123I-RTX and [18F]DVV54 were not retained in the trigeminal nerve. Likely, the tracers do not reach their target in a sufficient concentration due to extensive plasma protein binding as a result of their too lipophilic nature. </>
An increasing body of evidence associates the TRPV1 channel with the endocannabinoid system (ECS). In a second part of this thesis we focused on this ECS, which is represented by the two types of cannabinoid receptors (CB1 and CB2), their endogenous lipid ligands called endocannabinoids (e.g. anandamide), and all proteins responsible for the transport, degradation and biosynthesis of the endocannabinoids. CB1 is predominantly expressed in brain, whereas CB2 receptors are mainly found in cells and organs related to the immune system such as β-lymphocytes, the spleen and lymph nodes. Yet, low levels of CB2 have been detected in the healthy brain, whereby its physiological role in the CNS may be underestimated. CB2 is, however, up-regulated in microglia, the resident immune cells of the brain, upon neuroinflammation. CB2-positive microglia have been detected inpatients and animal models with Alzheimers disease, multiple sclerosisand Huntingtons disease. </>
Fatty acid amide hydrolase (FAAH) is one of the metabolising enzymes of the ECS and is responsible for the degradation of anandamide. FAAH is co-expressed with CB1 and thus principally found in brain. Outside the CNS, FAAH is mainly found in liver and kidneys, but protein expression has also been demonstrated in the small intestines, lungs, pancreas, prostate and testes. Alterations in FAAH expression levels and disruption of its activity have been linked with the aetiology of several neuropathogenic disorders such as drug/alcohol abuse, anxiety, depression, epilepsy, Huntingtons disease and Parkinsons disease, amongst others.</>
The idea that modulation of this signallingsystem could introduce new therapies, led to the development of CB1 andCB2 (ant)agonists and several classes of FAAH inhibitors. Some of thesecompounds already served as lead molecules for the development of accompanying PET-tracers. However, more research is needed to elucidate the expression pattern of CB2 in the CNS and to clarify the role of FAAH and CB2 in the above mentioned neurological pathologies. Here we report the radiolabelling, in vitro</> and in vivo</> evaluation of two new tracerstargeting the ECS. </>
[11C]MA2 was successfully synthesised and evaluated for its potential to visualise CB2 in vivo</>. Its structure is based on that of an N-arylamide oxadiazole CB2-agonist with reported picomolar affinity for CB2 and good selectivity over CB1. Due to the high expression levels of CB2 in the spleen, retention of [11C]MA2 in this organ was anticipated. Yet, none of the studied organs showed accumulation of [11C]MA2, except the liver due to metabolism and/or excretion of the tracer. This lack of retention has, however, also been observed with someearlier developed PET-tracers for CB2, which in contrary were shown to bind the human CB2 receptor in vivo</>. As [11C]MA2 showed good BBB penetration, its potential to bind the human CB2 receptor was examined by performing a microPET study using an animal model with local overexpression of a hCB2 variant. Unfortunately, no difference in [11C]MA2 binding was observed between the left (control) and right (CB2-rich) striatum. In our hands, the binding affinity of MA2 was found to be rather low, whichmost likely explains the lack of CB2-specific retention of the tracer. It may be that the small structural change that was introduced in the lead molecule to allow radiolabelling has influenced its affinity for CB2 in a negative manner.</>
Finally, we synthesised a radiolabelled analogue ([11C]FI-02) of the potent FAAH inhibitor FI-02, developed by Merck. Biodistribution studies in mice with [11C]FI-02 showed high accumulation in the brain, liver, kidneys and pancreas, all tissues known to express high levels of FAAH. The accumulation was blocked by pretreatment with the FAAH inhibitor URB597, which suggests that the tracer selectively binds to FAAH. MicroPET experiments in rats confirmed the high brain uptake of [11C]FI-02 and showed that binding of the tracer was irreversible. Pretreatment of the animals with URB597 resulted in a significant increase of the tracer concentration in blood, whereby the time-activity-curves of the brain paralleled those of the blood pool. As URB597 has been shown to bind other hydrolases as well, additional studies in FAAH knockout mice are needed to confirm the in vivo</> selectivity of [11C]FI-02 for FAAH.  </></>
In conclusion, of all developed TRPV1-tracers,the cinnamic acid derivatives [11C]SB366791 and [11C]DVV24 displayed the best tracer characteristics including high brain uptake, efficient clearance from blood and TRPV1-specific binding. Hence, a major drawback istheir relatively low experimentally determined binding affinity for TRPV1, which makes their potential to visualise TRPV1 in vivo</> less likely. [11C]MA2 couldnt meet the expectations of a successful CB2 PET-tracer. Yet, MA2 provides a basis for the development of new, possibly high affinity derivatives, which may lead to new PET-tracers in the future. Finally, our data suggest that [11C]FI-02 is a promising PET-tracer for invivo</> mapping of FAAH. Further research will elucidate its potential for clinical applications. </>
Date:1 Oct 2008 →  24 Jan 2013
Keywords:TRPV1 receptor
Disciplines:Medical imaging and therapy, Other chemical sciences, Chemical product design and formulation, Biomaterials engineering, Medicinal products
Project type:PhD project