Assessment of Dopamine and Serotonin Release in the Non-Human Primate Brain using PET

The molecular imaging technique positron emission tomography (PET) allows for non-invasive examination of biochemical markers in the living brain. For over three decades PET studies have provided important insight into the relationship of monoaminergic neurotransmitter systems to brain functioning and psychiatric disorders. A more recent application of PET is the study of endogenous neurotransmitter release in vivo. Clinical relevance of such methods is found in studies demonstrating enhanced amphetamine-induced dopamine release in schizophrenia patients, whereas PET studies in non-human primates provide a translational model for evaluation of the pharmacological mechanisms before initiation of studies in man. The first aim of this thesis was to develop improved methods for measurement of endogenous dopamine levels. In study I the potent D2/D3 receptors agonist (R)-(-)-2-methoxy-N-n-propyl-norapomorphine (MNPA) was radiolabeled with carbon-11 and found suitable for in vivo characterization of the high affinity state. In study II, amphetamine-induced displacement of [11C]MNPA binding by dopamine was ~1.8 fold higher at four different doses than for the antagonist [11C]raclopride and demonstrated that an agonist radioligand has improved sensitivity to endogenous neurotransmitter level. Study III aimed to further obtain in vivo support for the existence of two affinity states for the D2/D3 receptors. Receptor occupancy of the exogenous agonist apomorphine was determined with [11C]MNPA and [11C]raclopride. Binding of [11C]MNPA and [11C]raclopride was inhibited monophasic and approached full saturation. ID50 and Ki values of apomorphine were indistinguishable when measured with the agonist or antagonist radioligand. Study III did not support the existence of two affinity states and a possible explanation could be that all D2/D3 receptors are in the high affinity state in vivo. In study IV, the new D1/D5 receptors partial agonist radioligand (S)-[11C]N-methyl-NNC 01-0259 was found insensitive to dopamine levels, and receptor binding was inferior to previously developed antagonist radioligands. Moreover, a COMT formed radiometabolite was found to enter the brain but the formation could be prevented with the use of a COMT inhibitor. COMT inhibition provides a methodology enabling quantitative PET measurements with (S)-[11C]N-methyl-NNC 01-0259. The second aim of this thesis was to evaluate the sensitivity of the new 5-HT1B receptor radioligand [11C]AZ10419369 to alterations in endogenous serotonin concentration. Previous serotonergic PET radioligands have ambiguously shown sensitivity to serotonin level. In study V the effective serotonin releaser fenfluramine decreased the binding of [11C]AZ10419369 in a dose-dependent manner. In study VI the effect of fenfluramine on [11C]AZ10419369 binding was confirmed using an equilibrium approach with a bolus infusion protocol. The further developed methodology is suitable for exploring the sensitivity limit to serotonin levels as measured using [11C]AZ10419369 and PET. In conclusion, the present thesis demonstrates that the D2/D3 receptors agonist radioligand [11C]MNPA is an improvement for measurement of dopamine release, when compared to previously used antagonist radioligands. Moreover, a novel methodology, using the 5-HT1B receptor antagonist [11C]AZ10419369 and PET, was developed for measurement of serotonin release in the living brain. These newly developed methodologies may help to further understand the treatment and pathophysiology of several major neurological and psychiatric disorders

ABC transporter-dependent brain uptake of the 5-HT1B receptor radioligand [C-11]AZ10419369:a comparative PET study in mouse, rat, and guinea pig

BACKGROUND: We have explored the possibility that the serotonin 1B receptor radioligand [(11)C]AZ10419369 is a substrate for adenosine triphosphate (ATP)-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), Mrp4, and Bcrp, in rodents and whether there is a species difference regarding its blood-brain barrier (BBB) penetration. METHODS: In a series of preclinical positron emission tomography measurements, we have administered [(11)C]AZ10419369 to mice, rats, and guinea pigs under baseline conditions and, on separate experimental days, after administration of the ABC transporter inhibitor, cyclosporin A (CsA). RESULTS: During baseline conditions, the brain uptake was low in mice and rats, but not in guinea pigs. After CsA pretreatment, the peak whole brain uptake values of [(11)C]AZ10419369 increased by 207% in mice, 94% in rats, and 157% in guinea pigs. Binding potentials (BP(ND)) could not be estimated during baseline conditions in mice and rats. After CsA pretreatment, the highest BP(ND) values were obtained in the striatum and thalamus (BP(ND) ≈ 0.4) in mice, while in rats, the highest binding areas were the striatum, thalamus, hypothalamus, and periaqueductal gray (BP(ND) ≈ 0.5). In guinea pigs, we did not find any significant changes in BP(ND) between baseline and CsA pretreatment, except in the striatum. CONCLUSIONS: The results indicate that BBB penetration of [(11)C]AZ10419369 was hindered by ABC transporter activity in mouse, rat, and guinea pig. This study highlights the importance of ABC transporters in the design of preclinical positron emission tomography (PET) studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13550-014-0064-0) contains supplementary material, which is available to authorized users

Application of In vitro transcytosis models to brain targeted biologics.

The blood brain barrier (BBB) efficiently limits the penetration of biologics drugs from blood to brain. Establishment of an in vitro BBB model can facilitate screening of central nervous system (CNS) drug candidates and accelerate CNS drug development. Despite many established in vitro models, their application to biologics drug selection has been limited. Here, we report the evaluation of in vitro transcytosis of anti-human transferrin receptor (TfR) antibodies across human, cynomolgus and mouse species. We first evaluated human models including human cerebral microvascular endothelial cell line hCMEC/D3 and human colon epithelial cell line Caco-2 models. hCMEC/D3 model displayed low trans-epithelial electrical resistance (TEER), strong paracellular transport, and similar transcytosis of anti-TfR and control antibodies. In contrast, the Caco-2 model displayed high TEER value and low paracellular transport. Anti-hTfR antibodies demonstrated up to 70-fold better transcytosis compared to control IgG. Transcytosis of anti-hTfR.B1 antibody in Caco-2 model was dose-dependent and saturated at 3 μg/mL. Enhanced transcytosis of anti-hTfR.B1 was also observed in a monkey brain endothelial cell based (MBT) model. Importantly, anti-hTfR.B1 showed relatively high brain radioactivity concentration in a non-human primate positron emission tomography study indicating that the in vitro transcytosis from both Caco-2 and MBT models aligns with in vivo brain exposure. Typically, brain exposure of CNS targeted biologics is evaluated in mice. However, antibodies, such as the anti-human TfR antibodies, do not cross-react with the mouse target. Therefore, validation of a mouse in vitro transcytosis model is needed to better understand the in vitro in vivo correlation. Here, we performed transcytosis of anti-mouse TfR antibodies in mouse brain endothelial cell-based models, bEnd3 and the murine intestinal epithelial cell line mIEC. There is a good correlation between in vitro transcytosis of anti-mTfR antibodies and bispecifics in mIEC model and their mouse brain uptake. These data strengthen our confidence in the predictive power of the in vitro transcytosis models. Both mouse and human in vitro models will serve as important screening assays for brain targeted biologics selection in CNS drug development

PET and SPECT imaging of the central dopamine system in humans

The neurotransmitter dopamine plays a role in many different functions of the human brain, ranging from psychomotor planning to cognition. This short review addresses which parts of the dopamine system can be imaged quantitatively in the living human brain using positron-emission tomography (PET) or single-photon emission computed tomography (SPECT). Nowadays, imaging of the nigrostriatal dopaminergic pathway in humans can be performed quantitatively using radiotracers like the aromatic amino acid decarboxylase (AADC) substrate [18F]FDOPA, vesicular monoamine transporter 2 (VMAT-2) radioligands derived from tetrabenazine or PET/SPECT radioligands that bind to the dopamine transporter (DAT). Using PET, also several other dopaminergic projection pathways (e.g. mesocortical projections) can be assessed in humans. Several antagonist PET radioligands for the dopamine D1 receptor have been developed successfully. In addition, well-validated antagonist PET and SPECT radioligands are available for imaging of dopamine D2/3 receptors in the living human brain. Recently, also agonist PET radioligands for the dopamine D2/3 receptors have become available, which afford the opportunity to evaluate the existence of the high-affinity state of these receptors in vivo. These agonist radiopharmaceuticals may also prove more sensitive to changes in dopamine concentrations (e.g. induced by the dopamine releaser amphetamine). Finally, selective antagonist PET radioligands for the dopamine D4 receptor have recently been synthesized and evaluated successfully in small laboratory animals, although these radioligands have not yet been reported as applied in human subjects. In conclusion, after almost three decades of research, several relevant parts of the central dopamine system can be assessed quantitatively in the living human brain using PET or SPECT. Future studies may include application of agonist radioligands and more dopamine receptor subtype selective radioligands

ABC transporter dependent brain uptake of the 5-HT1B receptor radioligand [11C]AZ10419369

BackgroundWe have explored the possibility that the serotonin 1B receptor radioligand [11C]AZ10419369 is a substrate for adenosine triphosphate (ATP)-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), Mrp4, and Bcrp, in rodents and whether there is a species difference regarding its blood-brain barrier (BBB) penetration.MethodsIn a series of preclinical positron emission tomography measurements, we have administered [11C]AZ10419369 to mice, rats, and guinea pigs under baseline conditions and, on separate experimental days, after administration of the ABC transporter inhibitor, cyclosporin A (CsA).ResultsDuring baseline conditions, the brain uptake was low in mice and rats, but not in guinea pigs. After CsA pretreatment, the peak whole brain uptake values of [11C]AZ10419369 increased by 207% in mice, 94% in rats, and 157% in guinea pigs. Binding potentials (BPND) could not be estimated during baseline conditions in mice and rats. After CsA pretreatment, the highest BPND values were obtained in the striatum and thalamus (BPND ≈ 0.4) in mice, while in rats, the highest binding areas were the striatum, thalamus, hypothalamus, and periaqueductal gray (BPND ≈ 0.5). In guinea pigs, we did not find any significant changes in BPND between baseline and CsA pretreatment, except in the striatum.ConclusionsThe results indicate that BBB penetration of [11C]AZ10419369 was hindered by ABC transporter activity in mouse, rat, and guinea pig. This study highlights the importance of ABC transporters in the design of preclinical positron emission tomography (PET) studies