Blocking of efflux transporters in rats improves translational validation of brain radioligands.

BACKGROUND: Positron emission tomography (PET) is a molecular imaging technique that can be used to investigate the in vivo pharmacology of drugs. Initial preclinical evaluation of PET tracers is often conducted in rodents due to the accessibility of disease models as well as economic considerations. Compared to larger species, rodents display a higher expression and/or activity of efflux transporters such as the P-glycoprotein (P-gp). Low brain uptake could, therefore, be species-specific and uptake in rodents not be predictive for that in humans. We hypothesized that a better prediction from rodent data could be achieved when a tracer is evaluated under P-gp inhibition. Consequently, we compared the performance of eight neuroreceptor tracers in rats with and without P-gp inhibition including a specific binding blockade. This data set was then used to predict the binding of these eight tracers in pigs. METHODS: PET tracers targeting serotonin 5-HT2A receptors ([18F]MH.MZ, [18F]Altanserin, [11C]Cimbi-36, [11C]Pimavanserin), serotonin 5-HT7 receptors ([11C]Cimbi-701, [11C]Cimbi-717 and [11C]BA-10) and dopamine D2/3 receptors ([18F]Fallypride) were used in the study. The brain uptake and target-specific binding of these PET radiotracers were evaluated in rats with and without inhibition of P-gp. Rat data were subsequently compared to the results obtained in pigs. RESULTS: Without P-gp inhibition, the amount of target-specific binding in the rat brain was sufficient to justify further translation for three out of eight evaluated tracers. With P-gp inhibition, results for five out of eight tracers justified further translation. The performance in pigs could correctly be predicted for six out of eight tracers when rat data obtained under P-gp inhibition were used, compared to four out of eight tracers without P-gp inhibition. CONCLUSIONS: P-gp strongly affects the uptake of PET tracers in rodents, but false prediction outcomes can be reduced by evaluating a tracer under P-gp inhibition.

Radiosynthesis and preclinical evaluation of [11 C]Cimbi-701 - Towards the imaging of cerebral 5-HT7 receptors.

The serotonin 7 (5-HT7 ) receptor is suggested to be involved in a broad variety of CNS disorders, but very few in vivo tools exist to study this important target. Molecular imaging with positron emission tomography (PET) would enable an in vivo characterization of the 5-HT7 receptor. However, no clinical PET radiotracer exists for this receptor, and thus we aimed to develop such a tracer. In this study, we present the preclinical evaluation of [11 C]Cimbi-701. Cimbi-701 was synthesized in a one-step procedure starting from SB-269970. Its selectivity profile was determined using an academic screening platform (NIMH Psychoactive Drug Screening Program). Successful radiolabeling of [11 C]Cimbi-701 and subsequent in vivo evaluation was conducted in rats, pigs and baboon. In vivo specificity was investigated by 5-HT7 and σ receptor blocking studies. P-gp efflux transporter dependency was investigated using elacridar. [11 C]Cimbi-701 could successfully be synthesized. Selectivity profiling revealed high affinity for the 5-HT7 (Ki = 18 nM), σ-1 (Ki = 9.2 nM) and σ-2 (Ki = 1.6 nM) receptors. In rats, [11 C]Cimbi-701 acted as a strong P-gp substrate. After P-gp inhibition, rat brain uptake could specifically be blocked by 5-HT7 and σ receptor ligands. In pig, high brain uptake and specific 5-HT7 and σ-receptor binding was found for [11 C]Cimbi-701 without P-gp inhibition. Finally, low brain uptake was found in baboons. Both the specific σ-receptor binding and the low brain uptake of [11 C]Cimbi-701 displayed in baboon discouraged further translation to humans. Instead, we suggest exploration of this structural class as results indicate that selective 5-HT7 receptor imaging might be possible when more selective non-P-gp substrates are identified.

Towards selective CNS PET imaging of the 5-HT7 receptor system: Past, present and future.

Since its discovery in 1993, the serotonin receptor subtype 7 (5-HT7) has attracted significant attention as a potential drug target; due to its elucidated roles in conditions such as insomnia, schizophrenia, and more. Therefore, it is unsurprising that there has been relatively early efforts undertaken to develop a positron emission tomography (PET) imaging agent for said receptor system. PET can be clinically used to probe receptor systems in vivo, permitting information such as a drug's occupancy against this system to be investigated. This review focuses on the efforts towards the development of a 5-HT7R selective PET CNS tracer over the last 20 years, critically reflecting on applied strategies and commonly employed chemical frameworks and suggests future considerations that are needed to successfully develop a PET tracer for this clinically relevant target. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

The Chemogenetic Receptor Ligand Clozapine N-Oxide Induces in vivo Neuroreceptor Occupancy and Reduces Striatal Glutamate Levels.

Chemogenetic studies with the ligand clozapine N-oxide (CNO) are predicated upon the assumption that CNO is devoid of actions at natural neuroreceptors. However, recent evidence shows that CNO may be converted back to clozapine (CLZ) in vivo, which could yield plasma concentrations that may be sufficient to occupy inter alia dopamine D2/3 and serotonin 5HT2A receptors in living brain. To test this phenomenon, we measured striatal dopamine D2/3 receptor occupancy with [18F]fallypride PET and serotonin 5HT2A occupancy ex vivo using [18F]MH.MZ. We found a CNO dose-dependent effect on the availability of both neuroreceptor sites. In parallel MR spectroscopy experiments, we found that CNO reduced creatine + phosphcreatine (Cr+PCr) and increased N-acetylaspartate + N-acetylaspartylglutamate (NAA+NAAG) signals in the prefrontal cortex, and also reduced the glutamate signal in dorsal striatum, with peak effect at 2 mg/kg. Thus, our findings suggest that conversion of CNO to CLZ in living rats imparts significant occupancy at endogenous neuroreceptors and significant changes to neurometabolite levels.

Development and Evaluation of Two Potential 5-HT7 Receptor PET Tracers: [18F]ENL09 and [18F]ENL10.

The latest addition to the serotonin (5-HT) receptor family is the 5-HT7 receptor (5-HT7R). This receptor has gained interest as a drug target due to its involvement in various disorders such as depression or schizophrenia. There is currently no clinically validated positron emission tomography (PET) tracer for the 5-HT7R available. But, the (arylpiperazinyl-butyl)oxindole scaffold provides a promising lead structure for this purpose. Here, we synthesized 12 (arylpiperazinyl-butyl)oxindole derivatives and in vitro affinity screening identified two structures with suitable affinity and selectivity to be radiolabeled and tested as 5-HT7R selective PET tracers. Next, the radiolabeled products [18F]ENL09 and [18F]ENL10 were evaluated as PET tracers in rats. Both tracers were found to be P-gp substrates, but after P-gp inhibition the brain uptake showed a regional distribution in line with the known 5-HT7R distribution.  The [18F]ENL10 brain binding was displaceable with a 5-HT7R selective ligand, whereas [18F]ENL09 was not. We find that [18F]ENL10 is a promising 5-HT7R selective PET tracer candidate that should be investigated in higher species.