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D) receptor antagonist, GSK588045, using positron emission tomography
Neuropharmacology 92 (2015) 44e48
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In vivo occupancy of the 5-HT1A receptor by a novel pan 5-HT1(A/B/D) receptor antagonist, GSK588045, using positron emission tomography zs Gulya s c, Martine Garnier b, 2, Robert A. Comley a, Jasper van der Aart a, 1, Bala Laura Iavarone b, 3, Christer Halldin c, Eugenii A. Rabiner a, * a b c
Clinical Imaging Centre, GlaxoSmithKline, London, United Kingdom Psychiatry Centre for Excellence in Drug Discovery, GlaxoSmithKline, Verona, Italy Department of Neuroscience, Karolinska Institute, Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2014 Received in revised form 27 October 2014 Accepted 25 November 2014 Available online 2 December 2014
5-hydroxytryptamine 1 (5-HT1) receptor blockade in combination with serotonin reuptake inhibition may provide a more rapid elevation of synaptic 5-HT compared to serotonin reuptake alone, by blocking the inhibitory effect of 5-HT1 receptor activation on serotonin release. GSK588045 is a novel compound with antagonist activity at 5-HT1A/1B/1D receptors and nanomolar affinity for the serotonin transporter, which was in development for the treatment of depression and anxiety. Here we present the results of an in vivo assessment of the relationship between plasma exposure and 5-HT1A receptor occupancy. Six Cynomolgus monkeys (Macaca fascicularis) were scanned using the PET ligand [11C]WAY100635 before and after dosing with GSK588045 (0.03, 0.1 and 0.3 mg/kg 60 min i.v. infusion). Data was quantified using a simplified reference tissue model, with the cerebellar timeeactivity curve used as an input function. Plasma levels of GSK588045 were measured, and the EC50 of GSK588045 for 5-HT1A receptor occupancy was estimated. An Emax model described the relationship between the GSK588045 plasma concentration and 5-HT1A receptor occupancy data well. EC50 estimates (and 95% confidence intervals) for raphe nuclei and the frontal cortex were 6.99 (2.48 to 11.49) and 7.80 (2.84 to 12.76) ng/ml respectively. GSK588045 dose dependently blocked the signal of the PET ligand [11C]WAY100635, confirming its brain entry and occupancy of 5-HT1A receptors in the primate brain. The estimated EC50 at the postsynaptic heteroreceptors and somatodendritic autoreceptors is similar. 5-HT1 receptor blockade by compounds such as GSK588045 may provide a faster alternate mechanism of antidepressant and anxiolytic action than standard SSRI treatment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Antidepressive agents Anti-anxiety agents Drug discovery 5-HT1 receptors Positron emission tomography
1. Introduction The role of serotonergic neurotransmission in the aetiology of mood disorders has been the subject of discussion since the late
* Corresponding author. Imanova Limited, Burlington Danes Building, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 0NN, United Kingdom. E-mail address: [email protected] (E.A. Rabiner). 1 Centre for Human Drug Research, Leiden, Netherlands. 2 Independent professional, Medical Biological Science Value Innovation, Verona, Italy, Italy. 3 PAREXEL, United Kingdom. http://dx.doi.org/10.1016/j.neuropharm.2014.11.017 0028-3908/© 2014 Elsevier Ltd. All rights reserved.
1960s (Coppen, 1967). Treatment of depression and anxiety with drugs acting on the serotonergic system is supported by numerous pre-clinical and clinical studies, and pharmacological treatment strategies now target various aspects of the serotonergic system (for reviews see: Albert and Benkelfat, 2013; Alenina and Klempin, 2014; Carr and Lucki, 2011; Stahl et al., 2013). For the last two decades selective serotonin reuptake inhibitors (SSRIs) have been the treatment of choice for depression and anxiety. However, improved efficacy, tolerability, and faster onset of response remain important unmet medical needs. Up to 50% of patients are non-responders to available antidepressant treatments (Weinmann et al., 2008) while a significant number also suffer from drug-induced side effects which can affect compliance and lead to early discontinuation of treatment. Clinicians generally report a
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2e3 week delay between the start of SSRI treatment and significant clinical response in patients with depression (Montgomery, 1997). In addition some patients report a worsening of symptoms of anxiety at the start of treatment (Sinclair et al., 2009), which is thought to be due to a transient increase in extracellular 5-HT concentrations, activating 5-HT1 inhibitory autoreceptors (i.e. somatodendritic 5-HT1A and terminal 5-HT1B/1D receptors), attenuating the rise in the levels of synaptic 5-HT (Blier et al., 1998; Hughes and Dawson, 2004), and inhibiting the firing of 5-HT neurons (Pineyro and Blier, 1999). 5-HT1A, 5-HT1B and 5-HT1D are members of the 5-HT1 receptor (5-HT1 R) family which consists of 5 seven-transmembrane domain receptors (5-HT1A, 5-HT1B, 5-HT1D, 5-ht1e, 5-HT1F). There is no 5-HT1C receptor, 1C having been reclassified as 5-HT2C, In addition since 5-ht1e is yet to achieve full receptor status a lowercase naming convention is used (Andrade et al., 2014). 5-HT1 R couple to Gi/Go proteins to inhibit adenylyl cyclase activity and reduce local cAMP concentration, and are therefore predominantly inhibitory. 5-HT1 receptors are situated both post-synaptically (e.g. on glutamatergic pyramidal neurons in the frontal cortex, and pre-synaptically as autoreceptors on presynaptic membranes (5-HT1B) and on serotonergic cell bodies (5HT1A in the midbrain raphe nuclei), playing a major role in modulating the activity of serotonergic neurons and 5-HT release. In the human brain the 5-HT1A R is distributed in the hippocampal formation, neocortex, and raphe nuclei (Hall et al., 1997). 5-HT1A R are expressed as somatodendritic autoreceptors in the raphe nuclei, and post-synaptically as heteroreceptors on glutamatergic and GABAergic neurons in other brain regions (Descarries et al in Roth, 2008). In the past similarities between 5-HT1B and 5-HT1D have been a cause for confusion. Both receptors appear to have a similar pharmacological profile, except in the rodent, and were thought to be species homologues of each other (Hoyer and Middlemiss, 1989) until cloning and expression of the two receptors in the human revealed a low amino acid sequence homology despite a similar ligand-binding profile (for review see Mengod et al in Roth, 2008). A clarification to the nomenclature was proposed by Hartig et al. (1996), but has not been universally adopted. In the human brain the highest level of 5-HT1B expression is found in the basal ganglia, followed by the striatum, amygdala, hippocampus, septal nuclei, hypothalamus, and cerebral cortex €s et al., 2001). 5-HT1B R are predominately pre-synaptic. 5(Varna HT1D R are thought to be much less abundant, and possibly localised to the ventral pallidum (Varn€ as et al., 2001). However, further studies are required in light of the aforementioned species differences, a known mismatch between the localisation of 5-HT1B/D mRNA and receptors, and a lack of selective 5-HT1D ligands. In vitro evidence suggests that chronic administration of SSRIs desensitises 5-HT autoreceptors, which over time allows significant increases in synaptic 5-HT levels (Newman et al., 2004). For example, antagonist activity at the somatodendric autoreceptor in the raphe nuclei reduces the inhibition of terminal 5-HT release, resulting in more serotonergic activity in these projection areas (Kreiss and Lucki, 1994; Wang and Aghajanian, 1977). The blockade of 5-HT1A (Blier and Bergeron, 1998) and 5-HT1B (Shalom et al., 2004) receptors has been shown to enhance antidepressant action in preclinical models. A combination of 5-HT1A, 5-HT1B and 5HT1D receptor blockade, using selective antagonists, causes an immediate increase in extracellular 5-HT levels, as measured by microdialysis, in the dentate gyrus and frontal cortex of both guinea pigs and rats (Hughes and Dawson, 2004). It has been hypothesised therefore, that the combined blockade of 5-HT1 autoreceptors will result in acute elevations in synaptic 5-HT levels, which normally require chronic administration of an SSRI (for review see Dawson and Bromidge, 2008). A link between 5-HT1 autoreceptor
45
activation and psychological dysfunction is further supported by genetic association studies which have identified a C(-1019)G polymorphism in the promoter region of the human 5-HT1A receptor gene, which has been associated with major depression and suicide. The C(-1019)G polymorphism was shown to abolish repression of 5-HT1A autoreceptor expression (Lemonde et al., 2003), supporting the hypothesis that antagonism of this receptor could be beneficial in the treatment of depression and/or anxiety. Genetic, pharmacological, neuroendocrine, behavioural and postmortem studies all provide evidence for the involvement of 5HT1B in the pathophysiology of depression (for review see Ruf and Bhagwagar, 2009). In addition pre-clinical behavioural pharmacology studies (Sari, 2004), and more recently human positron emission tomography (PET) studies suggest a role for 5-HT1B in the aetiology and treatment of post-traumatic stress disorder (Murrough et al., 2011), whilst genetic studies have suggested an association between 5-HT1D and anorexia nervosa (Bergen et al., 2003). Accordingly 5-HT1A/1B/1D receptor antagonists such as GSK588045 were designed to augment synaptic levels 5-HT levels by mimicking SSRI activity on 5-HT1A desensitization through a direct inhibition of the 5-HT1A receptors, and potentiating the effect through the simultaneous blockade of 5-HT1B/1D receptors. GSK588045 is a 5-HT1A/1B/1D antagonist which has been characterised functionally in vitro using [35S]-GTPgS-SPA binding to h5HT1A (HEK), h5-HT1B (CHO), and h5-HT1D (CHO) cell membranes. It has a high affinity for 5-HT1 autoreceptors (in vitro human 5-HT1A/ 1B/1D pKi 9.9, 9.1 and 10.0 respectively), with moderate activity at the 5-HT transporter (in vitro human 5-HTT pKi 7.5), which elevates brain 5-HT release acutely in animal models (Bromidge et al., 2010). Positron emission tomography (PET) is an imaging technique that allows the quantification of the availability of a molecular target in vivo, which has proven to be a useful tool in drug development (Matthews et al., 2011). PET imaging with selective 5-HT1A radioligands can be used to image 5-HT1 R in the living human brain, and in other species. A number of ligands exist, [11C]WAY100635 (Pike et al., 1995), [18F]MPPF (Shiue et al., 1997), [18F]FCWAY (Carson et al., 2000), and [11C]CUMI-101 (Milak et al., 2008). [11C] WAY100635 in particular has been used to aid early drug development by defining the relationship between administered dose and 5-HT1A receptor occupancy (Rabiner et al., 2000, 2002). More recently, a number of PET radioligands have become available for 5HT1B: [11C]P943 (Nabulsi et al., 2010) and [11C]AZ10419369 (Varnas et al., 2011). However, these ligands were not available to us at the time our study was conducted. At present there is no 5-HT1D selective PET ligand. In this paper, we present the results of an in vivo assessment of the relationship between GSK588045 plasma exposure and the degree of 5-HT1A receptor occupancy in the brain of the Cynomolgus monkey (Macaca fascicularis), conducted to support the selection of doses for testing in humans.
2. Experimental procedures PET imaging was performed at the Department of Clinical Neuroscience at the Karolinska Institute, after approval from the Animal Ethics Committee of Northern Stockholm, Sweden (Dnr. 147/05) and keeping in line with the “Guidelines for planning, conducting and documenting experimental research” of the Karolinska Institutet (Dnr. 4820/06-600). Principles of laboratory animal care were followed, as per NIH publication No. 85-23, revised 1985.
2.1. Compounds and radiochemistry GSK588045B, the chloride salt of 6-{2-[4-(2-methyl-5-quinolinyl)-1piperazinyl]ethyl-4H-imidazo[5,1-C][1,4]benoxazine-3-carboxamide, was supplied by GlaxoSmithKline, UK. GSK588045 was prepared for intravenous administration as a stock solution batch according to good laboratory practice routines. [11C] WAY100635 was prepared as previously described (Shchukin et al., 2005).
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2.2. Subject preparation Six female Cynomolgus monkeys (weight: 3.5 ± 0.8 kg (mean ± SD), range 2.6e4.6 kg) were supplied by the Swedish Institute for Infectious Disease Control, SMI, Solna, Sweden. Anaesthesia was induced and maintained by repeated intramuscular injections of a mixture of ketamine (3e7 mg/(kg h) Ketalar™, Pfizer) and xylazine hydrochloride (0.5e2 mg/(kg h) Rompun™ Vet. Bayer). A venous cannula was inserted into a sural vein for administration of [11C]WAY-100635. A head fixation system was used to secure a fixed position of the monkey's head during the PET measurements. The positioning of the imaging planes was parallel to the canthomeatal line. Heart and breath rate of the monkeys were monitored during the PET examinations. Rectal temperature was continuously monitored by an electric thermometer during the PET examinations (Precision 4600, Harvard Applications, MA, USA), and was maintained at 37 C by a heating blanket (Bair HuggerdModel 505, Arizant Healthcare Inc., MN, USA). [11C]WAY100635 was administered in 8 mL of phosphate buffered saline. Two animals were examined at each of the 3 dose levels of GSK588045 (0.03, 0.1 and 0.3 mg/kg 60 min i.v. infusion). Post-dose scans started 1 h after dosing and continued for 93 min.
of GSK588045 was calculated as the difference between BPND measured following treatment (BPND post) and BPND measured in the baseline condition (BPND pre), and expressed as percentage occupancy (Equation (1)). Occupancy ¼ ðBPND pre BPND postÞ=ðBPND preÞ 100
(1)
2.6. Pharmacokinetic e target occupancy analysis GSK588045 plasma concentrations measured at the start of post-dose scans were fitted to regional occupancy estimates obtained using Equation (1). A simple Emax model (Equation (2)) was found to describe the data adequately. This model was fitted to the data set using GraphPad Prism, version 5.03 (GraphPad Software, San Diego CA, USA) to obtain estimates of half-maximal occupancy (EC50) and maximal occupancy (Emax). RO ¼ Emax C=ðEC50 þ CÞ þ E0
(2)
3. Results
2.3. Image acquisition and processing PET Imaging was performed on an ECAT EXACT HR þ system (CTI/Siemens, Knoxville, USA) in three-dimensional mode. In 3D mode, this camera provides an inplane resolution of 4.3 mm, 4.5 mm, 5.4 mm and 8.0 mm full width at half maximum (FWHM) at a distance of 0, 1, 10 and 20 cm from the centre of the field of view, respectively (Brix et al., 1997). Prior to each emission scan a transmission scan of 10 min was performed using 3 rotating 68Gee68Ga sources, this information was used for attenuation correction. Radioactivity in brain was measured for 93 min after IV injection of radioligand as 20 successive frames of increasing duration (3 1 min, 4 3 min, 13 6 min). Emission data were attenuation-corrected, and frames were reconstructed using a 2 mm Hann filter. Dynamic PET images in ECAT7 format were transferred from the Karolinska Institute to GSK, and converted to Analyze format and made isotropic using Convert2 and AVWisotropic, respectively (in-house software). Summed (integral) images for frames 1e8 (3 1 min, 4 3 min, 1 6 min) and 9e20 (12 6 min) were created using dyn2static (in-house software). In each of the six monkeys a baseline PET measurement was performed in the morning. A sterile physiological phosphate buffer (pH ¼ 7.4) solution containing 54 ± 2 (average ± SD) (range: 51e56) MBq of [carbonyl-11C]WAY100635 was injected as a bolus into a sural vein during 2 s. The PET measurements were repeated on the same day 1 h after IV infusion administration of GSK588045. 2.4. Blood sampling Venous blood samples were acquired from the femoral vein for assessment of plasma concentration of GSK588045. Samples were taken at 1.0, 1.3, 1.5, 2.0 and 2.5 h post infusion. 500 mL venous blood was collected into potassium EDTA tubes, which were spun at 4 C, and 150 mL plasma were collected and stored at 80 C. Plasma samples were returned to GSK and assayed using tandem mass spectrometry (HPLCeMSeMS). 2.5. Data analysis To allow determination of regional radioligand binding, regions of interest (ROIs) were manually delineated on integrated isotropic PET images (voxel size 1 1 1 mm) for each subject (Analyze version 4.0, Biomedical Imaging Resource, Mayo Foundation). The ROIs were defined in order to enable the examination of a region of post-synaptic 5-HT1A receptors (frontal cortex), a region of somatodendritic autoreceptors (midbrain raphe nuclei), and a region devoid of specific binding for [11C]WAY100635, to serve as a reference region (cerebellum). The cerebellum ROI was defined on summed images of the early time frames (0e21 min) in the transverse orientation. A rectangle width 5 mm, height 2 mm, tilted to fit was placed on 2 slices below the gap between the cerebellum and frontal lobes. The frontal cortex and midbrain raphe nuclei ROIs were defined on summed images of late time frames (22e93 min). The frontal cortex was delineated in the transverse orientation, by an ellipse width 6 mm; height 4 mm, and was placed on 3 slices below the corpus callosum. The midbrain raphe nuclei was defined in the sagittal plane. A rectangle width 2 mm; height 4 mm; tilted to fit, and was placed on 2 slices. Binding potential (BPND) relative to the reference region was used as the outcome parameter for quantification of 5-HT1A receptor availability. A simplified reference tissue model (Gunn et al., 1997) was used to determine BPND values on a voxel by voxel basis using the basis pursuit method (Gunn et al., 2002). The cerebellum ROI was applied to dynamic PET data to generate timeeactivity curves (TACs), which were used as an input function; minimal 5-HT1A receptor binding having been previously demonstrated in this region (Pazos et al., 1987; Pike et al., 1996). Parametric BP images were created by a basis pursuit denoising approach using the software package DEPICT version 1.2 (Gunn et al., 2001, 2002). Frontal cortex and midbrain raphe nuclei ROIs were applied to parametric BPND images to estimate regional BPND (Analyze Software version 4.0, Biomedical Imaging Resource, Mayo Foundation). The relative change in BPND elicited by administration
All six subjects provided data. Plasma concentrations of GSK588045 are presented in Table 1. There was no difference in the mean injected radioactivity of the baseline scans (54 ± 2.1 MBq) and the blocked scans (53.5 ± 1.2 MBq), p ¼ 0.62. The radiochemical purity of the injected [11C]WAY100635 exceeded 95% for all scans. The injected mass of cold WAY100635 was <10 mg. No serious adverse events were observed during the study. Examination of the [11C]WAY100635 time activity curves for the cerebellum revealed a consistent reduction in binding in the cerebellum following dosing with GSK588045 (across all subjects and doses). There are a number of possible explanations for this finding: (1) There could be a specific binding component of the [11C] WAY100635 signal in the cerebellum, which is reduced following a blocking dose of GSK588045. This is thought to be unlikely since the cerebellum is believed to have a low level of specific binding of [11C]WAY-100635 (Burnet et al., 1997; Hall et al., 1997), and whilst 5-HT1A is present in the vermis, the cerebellar hemispheres which are composed of largely of white matter have been shown to be devoid of 5-HT1A receptor binding (Parsey et al., 2005; Pazos et al., 1987; Pike et al., 1996); (2) There could be a “spill-over” of signal from brain adjacent areas into the cerebellum. This effect would be more pronounced during the baseline scans, and diminished in the blocked condition, resulting in an artificially high baseline signal in the cerebellum. As the cerebellar TAC is used to calculate the input function used for the calculation of BPND, a falsely high baseline input function could result in an underestimation of receptor occupancy. Accordingly in order to evaluate the significance of this change in the cerebellar TAC, input functions for the baseline condition were derived using both baseline (Method 1) and blocked (Method 2) cerebellar TACs for each subject. Results of PK-receptor occupancy modelling are shown in Fig. 1. EC50 estimates (and 95% confidence intervals) for raphe nuclei and the frontal cortex using method 1 are 23.23 (2.46 to 48.91) and 48.56 (10.68 to 107.8) ng/ml respectively. EC50 estimates (and 95% Table 1 Concentration of GSK588045 plasma. Subject
1 2 3 4 5 6
Dose of GSK588045 (mg/kg 60 min i.v. infusion)
[GSK588045] in plasma ng/mL (time relative to start of infusion) 1 ha
1.3 h
1.5 h
2h
2.5 h
0.03 0.03 0.3 0.3 0.1 0.1
13.8 17.9 161.2 213.6 66.3 48.0
10.8 10.9 112.0 164.7 60.4 31.8
10.3 7.7 100.4 141.8 38.3 33.3
8.5 10.1 91.8 124.7 51.2 32.2
11.0 8.6 129.4 NA 46.2 31.1
NA: sample not available. a Start of post-dose PET scan.
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Fig. 1. Relationship between plasma GSK588045 concentration and 5-HT1A occupancy in Cynomolgus monkey. Brain regions are raphe nuclei and frontal cortex. Fitted curves show predicted occupancy based on a simple Emax model.
confidence intervals) for raphe nuclei and the frontal cortex using method 2 are 6.99 (2.48e11.49) and 7.80 (2.84e12.76) ng/ml respectively. Based on the narrower 95% confidence intervals around the EC50 estimates obtained using method 2, and the points discussed above, we are inclined to place greater confidence in the estimates obtained using method 2.
pharmacokinetics of [11C]WAY-100635 or GSK588045. Whilst such an effect cannot be excluded, a clear dose response relationship was observed between the plasma concentration of GSK588045 and 5HT1A occupancy, which suggests that this is unlikely.
References 4. Discussion This study evaluated 5-HT1A receptor occupancy of GSK588045 in the Cynomolgus monkey brain, at doses expected to be therapeutically efficacious. Following intravenous administration, GSK588045 enters the brain and occupies 5-HT1A receptor sites in a dose-dependent manner. EC50 estimates (and 95% confidence intervals) for raphe nuclei and the frontal cortex are 6.99 (2.48e11.49) and 7.80 (2.84e12.76) ng/ml respectively, consistent with data from ex vivo radioligand binding to guinea-pig cortical 5-HT1A receptors with an IC50 of 9.0 ng/mL in plasma (Bromidge et al., 2010). In this study we have shown that GSK588045 penetrates the blood brain barrier (in non-human primates) and results in measurable dose-dependent occupancy of the 5-HT1A receptor in vivo. The results confirmed the 5-HT1A doseeresponse relationship observed in animal models of efficacy using ex vivo binding techniques, and provided useful data to support the selection of doses for testing in humans. In addition together with the data obtained with the front-runner molecule GSK163090 (Leslie et al., 2010), the data supports the proposed novel mechanism of antidepressant action. 4.1. Limitations During the conduct of this work the development of GSK588045 was terminated for strategic reasons. The study was stopped before an assessment of the time course of occupancy at the 5-HT1A receptor was conducted. Occupancy at 5-HT1B and 5-HT1D was not assessed since at the time this study was conducted we did not have access to a 5-HT1B or 5-HT1D selective PET ligands. The in vivo 5-HT transporter EC50 occupancy for GSK588045 was not evaluated as part of this study. All scans took place with animals under anaesthesia, which can effect pharmacokinetics and ligand binding (Seeman and Kapur, 2003). However, we would expect any effect on [11C]WAY-100635 binding to be equivalent in both the pre- and post GSK588045 scans, and therefore not influence occupancy by this mechanism. Anaesthesia could also affect occupancy measures by changing the
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Pike, V.W., McCarron, J.A., Lammertsma, A.A., Osman, S., Hume, S.P., Sargent, P.A., et al., 1996. Exquisite delineation of 5-HT1A receptors in human brain with PET and [carbonyl-11C]WAY-100635. Eur. J. Pharmacol. 301 (1e3), R5eR7. Pineyro, G., Blier, P., 1999. Autoregulation of serotonin neurons: role in antidepressant drug action. Pharmacol. Rev. 51 (3), 533e591. Rabiner, E.A., Gunn, R.N., Wilkins, M.R., Sargent, P.A., Mocaer, E., Sedman, E., et al., 2000. Drug action at the 5-HT(1A) receptor in vivo: autoreceptor and postsynaptic receptor occupancy examined with PET and [carbonyl-(11)C]WAY100635. Nucl. Med. Biol. 27 (5), 509e513. Rabiner, E.A., Wilkins, M.R., Turkheimer, F., Gunn, R.N., Udo de Haes, J., de Vries, M., et al., 2002. 5-Hydroxytryptamine1A receptor occupancy by novel full antagonist 2-[4-[4-(7-chloro-2,3-dihydro-1,4-benzdioxyn-5-yl)-1-piperazinyl]butyl]1, 2-benzisothiazol-3-(2H)-one-1,1-dioxide: a[11C][O-methyl-3H]-N-(2-(4-(2methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-py ridinyl)cyclohexanecarboxamide trihydrochloride (WAY-100635) positron emission tomography study in humans. J. Pharmacol. Exp. Ther. 301 (3), 1144e1150. Roth, B.L., 2008. The Serotonin Receptors: from Molecular Pharmacology to Human Therapeutics. Humana Press. Ruf, B.M., Bhagwagar, Z., 2009. The 5-HT1B receptor: a novel target for the pathophysiology of depression. Curr. Drug Targets 10 (11), 1118e1138. Sari, Y., 2004. Serotonin1B receptors: from protein to physiological function and behavior. Neurosci. Biobehav. Rev. 28 (6), 565e582. Seeman, P., Kapur, S., 2003. Anesthetics inhibit high-affinity states of dopamine D2 and other G-linked receptors. Synapse 50 (1), 35e40. Shalom, G., Gur, E., Van de Kar, L.D., Newman, M.E., 2004. Repeated administration of the 5-HT(1B) receptor antagonist SB-224289 blocks the desensitisation of 5HT(1B) autoreceptors induced by fluoxetine in rat frontal cortex. NaunynSchmiedeberg's Arch. Pharmacol. 370 (2), 84e90. Shchukin, E., Krasikova, R., Andersson, J., Truong, P., Christer, H., 2005. A fully automated one-pot synthesis of [carbonyl-11C] WAY-100635: validation in routine PET studies. J. Label. Compd. Radiopharm. 48, S208. Shiue, C.Y., Shiue, G.G., Mozley, P.D., Kung, M.P., Zhuang, Z.P., Kim, H.J., et al., 1997. P[18F]-MPPF: a potential radioligand for PET studies of 5-HT1A receptors in humans. Synapse 25 (2), 147e154. Sinclair, L.I., Christmas, D.M., Hood, S.D., Potokar, J.P., Robertson, A., Isaac, A., et al., 2009. Antidepressant-induced jitteriness/anxiety syndrome: systematic review. Br. J. Psychiatry 194 (6), 483e490. Stahl, S.M., Lee-Zimmerman, C., Cartwright, S., Morrissette, D.A., 2013. Serotonergic drugs for depression and beyond. Curr. Drug Targets 14 (5), 578e585. €s, K., Hall, H., Bonaventure, P., Sedvall, G., 2001. Autoradiographic mapping of Varna 5-HT1B and 5-HT1D receptors in the post mortem human brain using [3H]GR 125743. Brain Res. 915 (1), 47e57. Varnas, K., Nyberg, S., Karlsson, P., Pierson, M.E., Kagedal, M., Cselenyi, Z., et al., 2011. Dose-dependent binding of AZD3783 to brain 5-HT1B receptors in non-human primates and human subjects: a positron emission tomography study with [11C] AZ10419369. Psychopharmacology 213 (2e3), 533e545. Wang, R.Y., Aghajanian, G.K., 1977. Physiological evidence for habenula as major link between forebrain and midbrain raphe. Science 197 (4298), 89e91. Weinmann, S., Becker, T., Koesters, M., 2008. Re-evaluation of the efficacy and tolerability of venlafaxine vs SSRI: meta-analysis. Psychopharmacology 196 (4), 511e520 discussion 521e512.
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
In vivo occupancy of the 5-HT1A receptor by a novel pan 5-HT1(A/B/D) receptor antagonist, GSK588045, using positron emission tomography zs Gulya s c, Martine Garnier b, 2, Robert A. Comley a, Jasper van der Aart a, 1, Bala Laura Iavarone b, 3, Christer Halldin c, Eugenii A. Rabiner a, * a b c
Clinical Imaging Centre, GlaxoSmithKline, London, United Kingdom Psychiatry Centre for Excellence in Drug Discovery, GlaxoSmithKline, Verona, Italy Department of Neuroscience, Karolinska Institute, Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 October 2014 Received in revised form 27 October 2014 Accepted 25 November 2014 Available online 2 December 2014
5-hydroxytryptamine 1 (5-HT1) receptor blockade in combination with serotonin reuptake inhibition may provide a more rapid elevation of synaptic 5-HT compared to serotonin reuptake alone, by blocking the inhibitory effect of 5-HT1 receptor activation on serotonin release. GSK588045 is a novel compound with antagonist activity at 5-HT1A/1B/1D receptors and nanomolar affinity for the serotonin transporter, which was in development for the treatment of depression and anxiety. Here we present the results of an in vivo assessment of the relationship between plasma exposure and 5-HT1A receptor occupancy. Six Cynomolgus monkeys (Macaca fascicularis) were scanned using the PET ligand [11C]WAY100635 before and after dosing with GSK588045 (0.03, 0.1 and 0.3 mg/kg 60 min i.v. infusion). Data was quantified using a simplified reference tissue model, with the cerebellar timeeactivity curve used as an input function. Plasma levels of GSK588045 were measured, and the EC50 of GSK588045 for 5-HT1A receptor occupancy was estimated. An Emax model described the relationship between the GSK588045 plasma concentration and 5-HT1A receptor occupancy data well. EC50 estimates (and 95% confidence intervals) for raphe nuclei and the frontal cortex were 6.99 (2.48 to 11.49) and 7.80 (2.84 to 12.76) ng/ml respectively. GSK588045 dose dependently blocked the signal of the PET ligand [11C]WAY100635, confirming its brain entry and occupancy of 5-HT1A receptors in the primate brain. The estimated EC50 at the postsynaptic heteroreceptors and somatodendritic autoreceptors is similar. 5-HT1 receptor blockade by compounds such as GSK588045 may provide a faster alternate mechanism of antidepressant and anxiolytic action than standard SSRI treatment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Antidepressive agents Anti-anxiety agents Drug discovery 5-HT1 receptors Positron emission tomography
1. Introduction The role of serotonergic neurotransmission in the aetiology of mood disorders has been the subject of discussion since the late
* Corresponding author. Imanova Limited, Burlington Danes Building, Imperial College London, Hammersmith Hospital, Du Cane Road, London, W12 0NN, United Kingdom. E-mail address: [email protected] (E.A. Rabiner). 1 Centre for Human Drug Research, Leiden, Netherlands. 2 Independent professional, Medical Biological Science Value Innovation, Verona, Italy, Italy. 3 PAREXEL, United Kingdom. http://dx.doi.org/10.1016/j.neuropharm.2014.11.017 0028-3908/© 2014 Elsevier Ltd. All rights reserved.
1960s (Coppen, 1967). Treatment of depression and anxiety with drugs acting on the serotonergic system is supported by numerous pre-clinical and clinical studies, and pharmacological treatment strategies now target various aspects of the serotonergic system (for reviews see: Albert and Benkelfat, 2013; Alenina and Klempin, 2014; Carr and Lucki, 2011; Stahl et al., 2013). For the last two decades selective serotonin reuptake inhibitors (SSRIs) have been the treatment of choice for depression and anxiety. However, improved efficacy, tolerability, and faster onset of response remain important unmet medical needs. Up to 50% of patients are non-responders to available antidepressant treatments (Weinmann et al., 2008) while a significant number also suffer from drug-induced side effects which can affect compliance and lead to early discontinuation of treatment. Clinicians generally report a
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2e3 week delay between the start of SSRI treatment and significant clinical response in patients with depression (Montgomery, 1997). In addition some patients report a worsening of symptoms of anxiety at the start of treatment (Sinclair et al., 2009), which is thought to be due to a transient increase in extracellular 5-HT concentrations, activating 5-HT1 inhibitory autoreceptors (i.e. somatodendritic 5-HT1A and terminal 5-HT1B/1D receptors), attenuating the rise in the levels of synaptic 5-HT (Blier et al., 1998; Hughes and Dawson, 2004), and inhibiting the firing of 5-HT neurons (Pineyro and Blier, 1999). 5-HT1A, 5-HT1B and 5-HT1D are members of the 5-HT1 receptor (5-HT1 R) family which consists of 5 seven-transmembrane domain receptors (5-HT1A, 5-HT1B, 5-HT1D, 5-ht1e, 5-HT1F). There is no 5-HT1C receptor, 1C having been reclassified as 5-HT2C, In addition since 5-ht1e is yet to achieve full receptor status a lowercase naming convention is used (Andrade et al., 2014). 5-HT1 R couple to Gi/Go proteins to inhibit adenylyl cyclase activity and reduce local cAMP concentration, and are therefore predominantly inhibitory. 5-HT1 receptors are situated both post-synaptically (e.g. on glutamatergic pyramidal neurons in the frontal cortex, and pre-synaptically as autoreceptors on presynaptic membranes (5-HT1B) and on serotonergic cell bodies (5HT1A in the midbrain raphe nuclei), playing a major role in modulating the activity of serotonergic neurons and 5-HT release. In the human brain the 5-HT1A R is distributed in the hippocampal formation, neocortex, and raphe nuclei (Hall et al., 1997). 5-HT1A R are expressed as somatodendritic autoreceptors in the raphe nuclei, and post-synaptically as heteroreceptors on glutamatergic and GABAergic neurons in other brain regions (Descarries et al in Roth, 2008). In the past similarities between 5-HT1B and 5-HT1D have been a cause for confusion. Both receptors appear to have a similar pharmacological profile, except in the rodent, and were thought to be species homologues of each other (Hoyer and Middlemiss, 1989) until cloning and expression of the two receptors in the human revealed a low amino acid sequence homology despite a similar ligand-binding profile (for review see Mengod et al in Roth, 2008). A clarification to the nomenclature was proposed by Hartig et al. (1996), but has not been universally adopted. In the human brain the highest level of 5-HT1B expression is found in the basal ganglia, followed by the striatum, amygdala, hippocampus, septal nuclei, hypothalamus, and cerebral cortex €s et al., 2001). 5-HT1B R are predominately pre-synaptic. 5(Varna HT1D R are thought to be much less abundant, and possibly localised to the ventral pallidum (Varn€ as et al., 2001). However, further studies are required in light of the aforementioned species differences, a known mismatch between the localisation of 5-HT1B/D mRNA and receptors, and a lack of selective 5-HT1D ligands. In vitro evidence suggests that chronic administration of SSRIs desensitises 5-HT autoreceptors, which over time allows significant increases in synaptic 5-HT levels (Newman et al., 2004). For example, antagonist activity at the somatodendric autoreceptor in the raphe nuclei reduces the inhibition of terminal 5-HT release, resulting in more serotonergic activity in these projection areas (Kreiss and Lucki, 1994; Wang and Aghajanian, 1977). The blockade of 5-HT1A (Blier and Bergeron, 1998) and 5-HT1B (Shalom et al., 2004) receptors has been shown to enhance antidepressant action in preclinical models. A combination of 5-HT1A, 5-HT1B and 5HT1D receptor blockade, using selective antagonists, causes an immediate increase in extracellular 5-HT levels, as measured by microdialysis, in the dentate gyrus and frontal cortex of both guinea pigs and rats (Hughes and Dawson, 2004). It has been hypothesised therefore, that the combined blockade of 5-HT1 autoreceptors will result in acute elevations in synaptic 5-HT levels, which normally require chronic administration of an SSRI (for review see Dawson and Bromidge, 2008). A link between 5-HT1 autoreceptor
45
activation and psychological dysfunction is further supported by genetic association studies which have identified a C(-1019)G polymorphism in the promoter region of the human 5-HT1A receptor gene, which has been associated with major depression and suicide. The C(-1019)G polymorphism was shown to abolish repression of 5-HT1A autoreceptor expression (Lemonde et al., 2003), supporting the hypothesis that antagonism of this receptor could be beneficial in the treatment of depression and/or anxiety. Genetic, pharmacological, neuroendocrine, behavioural and postmortem studies all provide evidence for the involvement of 5HT1B in the pathophysiology of depression (for review see Ruf and Bhagwagar, 2009). In addition pre-clinical behavioural pharmacology studies (Sari, 2004), and more recently human positron emission tomography (PET) studies suggest a role for 5-HT1B in the aetiology and treatment of post-traumatic stress disorder (Murrough et al., 2011), whilst genetic studies have suggested an association between 5-HT1D and anorexia nervosa (Bergen et al., 2003). Accordingly 5-HT1A/1B/1D receptor antagonists such as GSK588045 were designed to augment synaptic levels 5-HT levels by mimicking SSRI activity on 5-HT1A desensitization through a direct inhibition of the 5-HT1A receptors, and potentiating the effect through the simultaneous blockade of 5-HT1B/1D receptors. GSK588045 is a 5-HT1A/1B/1D antagonist which has been characterised functionally in vitro using [35S]-GTPgS-SPA binding to h5HT1A (HEK), h5-HT1B (CHO), and h5-HT1D (CHO) cell membranes. It has a high affinity for 5-HT1 autoreceptors (in vitro human 5-HT1A/ 1B/1D pKi 9.9, 9.1 and 10.0 respectively), with moderate activity at the 5-HT transporter (in vitro human 5-HTT pKi 7.5), which elevates brain 5-HT release acutely in animal models (Bromidge et al., 2010). Positron emission tomography (PET) is an imaging technique that allows the quantification of the availability of a molecular target in vivo, which has proven to be a useful tool in drug development (Matthews et al., 2011). PET imaging with selective 5-HT1A radioligands can be used to image 5-HT1 R in the living human brain, and in other species. A number of ligands exist, [11C]WAY100635 (Pike et al., 1995), [18F]MPPF (Shiue et al., 1997), [18F]FCWAY (Carson et al., 2000), and [11C]CUMI-101 (Milak et al., 2008). [11C] WAY100635 in particular has been used to aid early drug development by defining the relationship between administered dose and 5-HT1A receptor occupancy (Rabiner et al., 2000, 2002). More recently, a number of PET radioligands have become available for 5HT1B: [11C]P943 (Nabulsi et al., 2010) and [11C]AZ10419369 (Varnas et al., 2011). However, these ligands were not available to us at the time our study was conducted. At present there is no 5-HT1D selective PET ligand. In this paper, we present the results of an in vivo assessment of the relationship between GSK588045 plasma exposure and the degree of 5-HT1A receptor occupancy in the brain of the Cynomolgus monkey (Macaca fascicularis), conducted to support the selection of doses for testing in humans.
2. Experimental procedures PET imaging was performed at the Department of Clinical Neuroscience at the Karolinska Institute, after approval from the Animal Ethics Committee of Northern Stockholm, Sweden (Dnr. 147/05) and keeping in line with the “Guidelines for planning, conducting and documenting experimental research” of the Karolinska Institutet (Dnr. 4820/06-600). Principles of laboratory animal care were followed, as per NIH publication No. 85-23, revised 1985.
2.1. Compounds and radiochemistry GSK588045B, the chloride salt of 6-{2-[4-(2-methyl-5-quinolinyl)-1piperazinyl]ethyl-4H-imidazo[5,1-C][1,4]benoxazine-3-carboxamide, was supplied by GlaxoSmithKline, UK. GSK588045 was prepared for intravenous administration as a stock solution batch according to good laboratory practice routines. [11C] WAY100635 was prepared as previously described (Shchukin et al., 2005).
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2.2. Subject preparation Six female Cynomolgus monkeys (weight: 3.5 ± 0.8 kg (mean ± SD), range 2.6e4.6 kg) were supplied by the Swedish Institute for Infectious Disease Control, SMI, Solna, Sweden. Anaesthesia was induced and maintained by repeated intramuscular injections of a mixture of ketamine (3e7 mg/(kg h) Ketalar™, Pfizer) and xylazine hydrochloride (0.5e2 mg/(kg h) Rompun™ Vet. Bayer). A venous cannula was inserted into a sural vein for administration of [11C]WAY-100635. A head fixation system was used to secure a fixed position of the monkey's head during the PET measurements. The positioning of the imaging planes was parallel to the canthomeatal line. Heart and breath rate of the monkeys were monitored during the PET examinations. Rectal temperature was continuously monitored by an electric thermometer during the PET examinations (Precision 4600, Harvard Applications, MA, USA), and was maintained at 37 C by a heating blanket (Bair HuggerdModel 505, Arizant Healthcare Inc., MN, USA). [11C]WAY100635 was administered in 8 mL of phosphate buffered saline. Two animals were examined at each of the 3 dose levels of GSK588045 (0.03, 0.1 and 0.3 mg/kg 60 min i.v. infusion). Post-dose scans started 1 h after dosing and continued for 93 min.
of GSK588045 was calculated as the difference between BPND measured following treatment (BPND post) and BPND measured in the baseline condition (BPND pre), and expressed as percentage occupancy (Equation (1)). Occupancy ¼ ðBPND pre BPND postÞ=ðBPND preÞ 100
(1)
2.6. Pharmacokinetic e target occupancy analysis GSK588045 plasma concentrations measured at the start of post-dose scans were fitted to regional occupancy estimates obtained using Equation (1). A simple Emax model (Equation (2)) was found to describe the data adequately. This model was fitted to the data set using GraphPad Prism, version 5.03 (GraphPad Software, San Diego CA, USA) to obtain estimates of half-maximal occupancy (EC50) and maximal occupancy (Emax). RO ¼ Emax C=ðEC50 þ CÞ þ E0
(2)
3. Results
2.3. Image acquisition and processing PET Imaging was performed on an ECAT EXACT HR þ system (CTI/Siemens, Knoxville, USA) in three-dimensional mode. In 3D mode, this camera provides an inplane resolution of 4.3 mm, 4.5 mm, 5.4 mm and 8.0 mm full width at half maximum (FWHM) at a distance of 0, 1, 10 and 20 cm from the centre of the field of view, respectively (Brix et al., 1997). Prior to each emission scan a transmission scan of 10 min was performed using 3 rotating 68Gee68Ga sources, this information was used for attenuation correction. Radioactivity in brain was measured for 93 min after IV injection of radioligand as 20 successive frames of increasing duration (3 1 min, 4 3 min, 13 6 min). Emission data were attenuation-corrected, and frames were reconstructed using a 2 mm Hann filter. Dynamic PET images in ECAT7 format were transferred from the Karolinska Institute to GSK, and converted to Analyze format and made isotropic using Convert2 and AVWisotropic, respectively (in-house software). Summed (integral) images for frames 1e8 (3 1 min, 4 3 min, 1 6 min) and 9e20 (12 6 min) were created using dyn2static (in-house software). In each of the six monkeys a baseline PET measurement was performed in the morning. A sterile physiological phosphate buffer (pH ¼ 7.4) solution containing 54 ± 2 (average ± SD) (range: 51e56) MBq of [carbonyl-11C]WAY100635 was injected as a bolus into a sural vein during 2 s. The PET measurements were repeated on the same day 1 h after IV infusion administration of GSK588045. 2.4. Blood sampling Venous blood samples were acquired from the femoral vein for assessment of plasma concentration of GSK588045. Samples were taken at 1.0, 1.3, 1.5, 2.0 and 2.5 h post infusion. 500 mL venous blood was collected into potassium EDTA tubes, which were spun at 4 C, and 150 mL plasma were collected and stored at 80 C. Plasma samples were returned to GSK and assayed using tandem mass spectrometry (HPLCeMSeMS). 2.5. Data analysis To allow determination of regional radioligand binding, regions of interest (ROIs) were manually delineated on integrated isotropic PET images (voxel size 1 1 1 mm) for each subject (Analyze version 4.0, Biomedical Imaging Resource, Mayo Foundation). The ROIs were defined in order to enable the examination of a region of post-synaptic 5-HT1A receptors (frontal cortex), a region of somatodendritic autoreceptors (midbrain raphe nuclei), and a region devoid of specific binding for [11C]WAY100635, to serve as a reference region (cerebellum). The cerebellum ROI was defined on summed images of the early time frames (0e21 min) in the transverse orientation. A rectangle width 5 mm, height 2 mm, tilted to fit was placed on 2 slices below the gap between the cerebellum and frontal lobes. The frontal cortex and midbrain raphe nuclei ROIs were defined on summed images of late time frames (22e93 min). The frontal cortex was delineated in the transverse orientation, by an ellipse width 6 mm; height 4 mm, and was placed on 3 slices below the corpus callosum. The midbrain raphe nuclei was defined in the sagittal plane. A rectangle width 2 mm; height 4 mm; tilted to fit, and was placed on 2 slices. Binding potential (BPND) relative to the reference region was used as the outcome parameter for quantification of 5-HT1A receptor availability. A simplified reference tissue model (Gunn et al., 1997) was used to determine BPND values on a voxel by voxel basis using the basis pursuit method (Gunn et al., 2002). The cerebellum ROI was applied to dynamic PET data to generate timeeactivity curves (TACs), which were used as an input function; minimal 5-HT1A receptor binding having been previously demonstrated in this region (Pazos et al., 1987; Pike et al., 1996). Parametric BP images were created by a basis pursuit denoising approach using the software package DEPICT version 1.2 (Gunn et al., 2001, 2002). Frontal cortex and midbrain raphe nuclei ROIs were applied to parametric BPND images to estimate regional BPND (Analyze Software version 4.0, Biomedical Imaging Resource, Mayo Foundation). The relative change in BPND elicited by administration
All six subjects provided data. Plasma concentrations of GSK588045 are presented in Table 1. There was no difference in the mean injected radioactivity of the baseline scans (54 ± 2.1 MBq) and the blocked scans (53.5 ± 1.2 MBq), p ¼ 0.62. The radiochemical purity of the injected [11C]WAY100635 exceeded 95% for all scans. The injected mass of cold WAY100635 was <10 mg. No serious adverse events were observed during the study. Examination of the [11C]WAY100635 time activity curves for the cerebellum revealed a consistent reduction in binding in the cerebellum following dosing with GSK588045 (across all subjects and doses). There are a number of possible explanations for this finding: (1) There could be a specific binding component of the [11C] WAY100635 signal in the cerebellum, which is reduced following a blocking dose of GSK588045. This is thought to be unlikely since the cerebellum is believed to have a low level of specific binding of [11C]WAY-100635 (Burnet et al., 1997; Hall et al., 1997), and whilst 5-HT1A is present in the vermis, the cerebellar hemispheres which are composed of largely of white matter have been shown to be devoid of 5-HT1A receptor binding (Parsey et al., 2005; Pazos et al., 1987; Pike et al., 1996); (2) There could be a “spill-over” of signal from brain adjacent areas into the cerebellum. This effect would be more pronounced during the baseline scans, and diminished in the blocked condition, resulting in an artificially high baseline signal in the cerebellum. As the cerebellar TAC is used to calculate the input function used for the calculation of BPND, a falsely high baseline input function could result in an underestimation of receptor occupancy. Accordingly in order to evaluate the significance of this change in the cerebellar TAC, input functions for the baseline condition were derived using both baseline (Method 1) and blocked (Method 2) cerebellar TACs for each subject. Results of PK-receptor occupancy modelling are shown in Fig. 1. EC50 estimates (and 95% confidence intervals) for raphe nuclei and the frontal cortex using method 1 are 23.23 (2.46 to 48.91) and 48.56 (10.68 to 107.8) ng/ml respectively. EC50 estimates (and 95% Table 1 Concentration of GSK588045 plasma. Subject
1 2 3 4 5 6
Dose of GSK588045 (mg/kg 60 min i.v. infusion)
[GSK588045] in plasma ng/mL (time relative to start of infusion) 1 ha
1.3 h
1.5 h
2h
2.5 h
0.03 0.03 0.3 0.3 0.1 0.1
13.8 17.9 161.2 213.6 66.3 48.0
10.8 10.9 112.0 164.7 60.4 31.8
10.3 7.7 100.4 141.8 38.3 33.3
8.5 10.1 91.8 124.7 51.2 32.2
11.0 8.6 129.4 NA 46.2 31.1
NA: sample not available. a Start of post-dose PET scan.
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47
Fig. 1. Relationship between plasma GSK588045 concentration and 5-HT1A occupancy in Cynomolgus monkey. Brain regions are raphe nuclei and frontal cortex. Fitted curves show predicted occupancy based on a simple Emax model.
confidence intervals) for raphe nuclei and the frontal cortex using method 2 are 6.99 (2.48e11.49) and 7.80 (2.84e12.76) ng/ml respectively. Based on the narrower 95% confidence intervals around the EC50 estimates obtained using method 2, and the points discussed above, we are inclined to place greater confidence in the estimates obtained using method 2.
pharmacokinetics of [11C]WAY-100635 or GSK588045. Whilst such an effect cannot be excluded, a clear dose response relationship was observed between the plasma concentration of GSK588045 and 5HT1A occupancy, which suggests that this is unlikely.
References 4. Discussion This study evaluated 5-HT1A receptor occupancy of GSK588045 in the Cynomolgus monkey brain, at doses expected to be therapeutically efficacious. Following intravenous administration, GSK588045 enters the brain and occupies 5-HT1A receptor sites in a dose-dependent manner. EC50 estimates (and 95% confidence intervals) for raphe nuclei and the frontal cortex are 6.99 (2.48e11.49) and 7.80 (2.84e12.76) ng/ml respectively, consistent with data from ex vivo radioligand binding to guinea-pig cortical 5-HT1A receptors with an IC50 of 9.0 ng/mL in plasma (Bromidge et al., 2010). In this study we have shown that GSK588045 penetrates the blood brain barrier (in non-human primates) and results in measurable dose-dependent occupancy of the 5-HT1A receptor in vivo. The results confirmed the 5-HT1A doseeresponse relationship observed in animal models of efficacy using ex vivo binding techniques, and provided useful data to support the selection of doses for testing in humans. In addition together with the data obtained with the front-runner molecule GSK163090 (Leslie et al., 2010), the data supports the proposed novel mechanism of antidepressant action. 4.1. Limitations During the conduct of this work the development of GSK588045 was terminated for strategic reasons. The study was stopped before an assessment of the time course of occupancy at the 5-HT1A receptor was conducted. Occupancy at 5-HT1B and 5-HT1D was not assessed since at the time this study was conducted we did not have access to a 5-HT1B or 5-HT1D selective PET ligands. The in vivo 5-HT transporter EC50 occupancy for GSK588045 was not evaluated as part of this study. All scans took place with animals under anaesthesia, which can effect pharmacokinetics and ligand binding (Seeman and Kapur, 2003). However, we would expect any effect on [11C]WAY-100635 binding to be equivalent in both the pre- and post GSK588045 scans, and therefore not influence occupancy by this mechanism. Anaesthesia could also affect occupancy measures by changing the
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