JPET Fast Forward. Published on May 17, 2013 as DOI: 10.1124/jpet.112.202895 JPETThis Fast article Forward. has not been Published copyedited andon formatted. May 29, The 2013 final asversion DOI:10.1124/jpet.112.202895 may differ from this version.

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1) Title page

Monoamine transporter occupancy of a novel triple in baboon and human using Positron Emission Tomography

Robert A. Comley 1, Cristian A. Salinas 2, Mark Slifstein, Marcella Petrone 3, Carmine Marzano, Idriss Bennacef , Paul Shotbolt, Jasper Van der Aart 2, Marta Neve 3, Laura Iavarone, Roberto Gomeni 4, Marc Laruelle 5, Frank A. Gray, Roger N. Gunn 2, Eugenii A. Rabiner 2

Downloaded from Clinical Imaging Centre, GlaxoSmithKline, London, United Kingdom (R.A.C, C.A.S, C.M, I.B, P.S, J.V, R.N.G, M.L, E.A.R); Columbia University, New York, NY, USA (M.S); GlaxoSmithKline, Clinical Pharmacology Modelling and Simulation, Italy (M.P, M.N, L.L,

R.G); GlaxoSmithKline, Clinical Pharmacology and Discovery Medicine, UK (F.G) jpet.aspetjournals.org at ASPET Journals on September 28, 2021

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Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on May 17, 2013 as DOI: 10.1124/jpet.112.202895 This article has not been copyedited and formatted. The final version may differ from this version.

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2) Running title page a) Occupancy of a novel triple reuptake inhibitor using PET b) Name: Eugenii Rabiner

Address:

Imanova Limited

Burlington Danes Building, Downloaded from Imperial College London,

Hammersmith Hospital,

Du Cane Road, jpet.aspetjournals.org

London, W12 0NN

United Kingdom

Phone: +44 20 8008 6042 at ASPET Journals on September 28, 2021

Fax: +44 20 8008 6491

E-mail: [email protected]

c) Number of text pages: 32 Number of tables: 1 Number of figures: 2 Number of references: 48 Number of words in Abstract: 243 Number of words in Introduction: 544 Number of words in Discussion: 1130 d) BPND, In vivo PET binding potential, specific compared to nondisplaceable uptake; DAT: Dopamine reuptake transporter; MAOI, monoamine-oxidase inhibitor; MDD, Major Depressive Disorder; MRI, Magnetic Resonance Imaging; NET, Norepinephrine

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reuptake inhibitor; Occ, occupancy; PET, Positron Emission Tomography; ROI, region of interest; SERT, Serotonin reuptake transporter; SNDRI, serotonin–norepinephrine– dopamine reuptake inhibitor; SSRI, serotonin reuptake inhibitor; TCA, ; TOC time-occupancy-curve; TRI, Triple reuptake inhibitor; VND, PET volume of distribution non-displaceable ligand in tissue relative to plasma; VT, PET volume of distribution total radioligand in tissue relative to plasma e) Recommended section assignment: Neuropharmacology Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021

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3) Abstract Introduction: The selection of a therapeutically meaningful dose of a novel pharmaceutical is a crucial step in drug development. Positron emission tomography (PET) allows the in vivo estimation of the relationship between the plasma concentration of a drug and its target occupancy, optimising dose selection and reducing the time and cost of early development. Triple reuptake inhibitors (TRIs), also referred to as serotonin–norepinephrine–dopamine reuptake inhibitors enhance monoaminergic neurotransmission by blocking the action of the monoamine transporters, raising extracellular concentrations of those neurotransmitters. GSK1360707 is a novel TRI Downloaded from which until recently was under development for the treatment of major depressive disorder, its development having been put on hold for strategic reasons. We present the results of an in vivo assessment of the relationship between plasma exposure and Methods: Papio transporter blockade (occupancy). Studies were performed in baboon ( jpet.aspetjournals.org Anubis) to determine the relationship between plasma concentration and occupancy of brain SERT, DAT and NET, using the radioligands [11C]DASB, [11C]PE2I, and [11C]MeNER (also known as [11C]MRB), and in human using [11C]DASB and [11C]PE2I. Results: In Papio Anubis plasma concentrations resulting in half maximal occupancy at at ASPET Journals on September 28, 2021 SERT, DAT and NET were found to be 15.16, 15.56 and 0.97 ng/ml respectively. In human, the corresponding values for SERT and DAT were 6.80 and 18.00 ng/ml. Conclusion: GSK1360707 dose dependently blocked the signal of SERT, DAT and NET selective PET ligands, confirming its penetration across the blood brain barrier and blockade of all three monoamine transporters in vivo.

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4) Introduction

Triple reuptake inhibitors (TRI) are compounds designed to block the serotonin, nor- adrenaline and dopamine transporters, enhancing monoaminergic neurotransmission, and are seen as a promising advance in the treatment of neuropsychiatric disorders

(Marks et al., 2008). GSK1360707 is a novel TRI which has been shown to be potent and selective, with excellent in vitro and in vivo profiles (see compound 17 in Micheli et al. (Micheli et al., 2010); for structure see (Teller and Furstner, 2011)). Downloaded from GSK1360707 was until recently being developed for the treatment of Major Depressive

Disorder (MDD). Current therapies for MDD typically have response rates of 50-65%

(Weinmann et al., 2008) and clinically meaningful activity only after 2 to 4 weeks jpet.aspetjournals.org

(Montgomery, 1997). It is hypothesised that the blockade of the three mono-amine transporters will improve tolerability, speed of onset, response and/or remission rates.

An effective TRI could reduce the need to use multiple psychoactive medications in at ASPET Journals on September 28, 2021 individual patients, an issue of concern given the lack of data on the effectiveness of drug-drug combinations, likely poor compliance (due to complex dosing regimens), the possibility of cumulative toxicity, and subsequent vulnerability to adverse events. In addition a TRI could have therapeutic potential in other diseases such as Parkinson’s disease (Hauser et al., 2007; Rascol et al., 2008), pain (Basile et al., 2007; Al-Shamahi et al., 2009), and obesity (Axel et al., 2010; Hansen et al., 2010; Sjodin et al., 2010).

In contrast to the monoamine-oxidase inhibitors (MAOI) and tricyclic

(TCA), serotonin reuptake inhibitors (SSRIs) were synthesised and developed with a specific molecular target in mind; reaching the market in the late 1980s these drugs arguably represented the first major advance in the treatment of depression since the introduction of effective pharmacotherapy. However, despite their improved tolerability and safety profile, as compared to the TCA and MAOI, their efficacy and speed of therapeutic effect are not improved (as compared to the older agents). The observed

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antidepressant effects of selective norepinephrine reuptake inhibitors (e.g. ), seemingly independent of serotonergic mechanisms, have led to the development of dual reuptake inhibitors (e.g. ). More recently, the theory that enhanced dopaminergic neurotransmission may alleviate anhedonia, combined with evidence that several marketed antidepressants may possess pharmacologically relevant affinity for the DAT, has led to the development of TRI.

The selection of a therapeutically meaningful dose of a novel pharmaceutical such as a Downloaded from TRI is a crucial step in early drug development. Positron emission tomography (PET) allows the in vivo estimation of the relationship between the plasma concentration and

its target occupancy, optimising the dose selection process and reducing the time and jpet.aspetjournals.org cost of early phase drug development (Matthews et al., 2012).

Here we present results of in vivo assessment of central monoamine reuptake transporter occupancy by GSK1360707, using PET. The aim of these studies was to at ASPET Journals on September 28, 2021 measure the degree of occupancy of SERT, DAT and NET in the brain, and to relate those measures to plasma concentrations.

In doing so we also characterized the relationship between the plasma concentration time course and the transporter occupancy time course, which can be used to optimise the dose regimen and formulation of future clinical trials in order to stay within the anticipated therapeutic window (Nyberg et al., 1999).

In the first instance pre-clinical imaging experiments were conducted to evaluate target occupancies in the proposed clinical dose range, before Phase 1 testing in humans.

These data were then used to support the rationale for, and optimise the design of, a human dose-occupancy study (ClinicalTrials.gov identifier: NCT01153802) to obtain accurate estimates of PET IC50 values and support dose selection for subsequent clinical trials.

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5) Methods

Methods of quantification and test-rested variability for the radioligands used in this study have been previously reported in the literature; in brief, test-retest variability has been reported as <10% for [11C]DASB (Frankle et al., 2006); 9.2% to 15.6% for

[11C]PE2I (DeLorenzo et al., 2009); and <10% for [11C]MRB (also known as

[11C]MeNER) (Logan et al., 2007).

Pre-clinical PET imaging studies Downloaded from PET imaging of the baboon (Papio Anubis) brain was performed at Columbia University

Medical Center. All study procedures were approved by the IACUC (Institutional Animal

Care and Use Committees) of Columbia University and the New York State Psychiatric jpet.aspetjournals.org

Institute. Four male baboons were included in the study.

Experimental Design

SERT and DAT occupancy were evaluated in three animals (A, B, and C,) using the at ASPET Journals on September 28, 2021 radioligands [11C]DASB and [11C]PE2I. NET occupancy was evaluated in three animals

(A, C and D) using the radioligand [11C]MeNER. The time-occupancy characteristics of

GSK1360707 at the SERT and DAT were evaluated using an iterative approach, varying both the dose of GSK1360707 and the time of post-dose scans, in order to characterise the time-dependent relationship between plasma concentration of the drug and of the target occupancy (time-occupancy-curve, TOC). Evaluation of the NET occupancy was performed subsequent to the evaluation of the TOC at the SERT and DAT. Hence, the relationship between plasma concentration and NET occupancy was performed at one time-point only (approximately the time of maximal brain concentration of GSK1360707).

Since the relationship between plasma concentration and the occupancy at the SERT and DAT was determined to be direct (Derendorf and Meibohm, 1999) the examination of the TOC at the NET was not deemed to be essential.

Experimental Procedures

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Fasted animals were immobilized with (10mg/kg intramuscularly).

Anaesthesia during preparation and scans was maintained with 2 % isoflurane administered through an endotracheal tube. An intravenous catheter was inserted for radioligand administration, drug administration, and hydration. An arterial catheter was placed in a femoral artery for arterial blood sampling (including PK sampling) and continuous blood pressure monitoring. Vital signs (blood pressure, pulse, electrocardiograph, temperature, and respiration) were monitored continuously using a Downloaded from patient monitoring system (DataScope Corp, Paramus, NJ, USA). Temperature was maintained at 37°C with a heated water blanket.

jpet.aspetjournals.org

For all scanning sessions involving [11C]DASB and [11C]PE2I, acquisition of baseline scan data was followed by a constant intravenous infusion of GSK1360707 (formulated in saline and NaOH) lasting 30 minutes (up to a total dose (per session) of 0.125, 0.25 or at ASPET Journals on September 28, 2021

1 mg/kg; not exceeding 3 mg/kg for the study as a whole). The solution was delivered using a programmable syringe pump (Harvard Apparatus, Holliston, MA, USA) and this was followed by two post drug PET scans (commencing between 0.25 and 10.75 hours after the end of the drug infusion). In general doses were escalated from one session to the next, where this was not the case (in order to derive additional time-course data at a lower dose) a fresh baseline scan was acquired after an appropriate washout period.

For each subject, scanning sessions (either single-scans post- GSK1360707 or 2-scan sessions including baseline and post-GSK1360707) were separated by a minimum of 7 days. [11C]MeNER scanning sessions were similar, except that only a single scan was acquired following the administration of GSK1360707 (0.25 hours after the end of the drug infusion).

Radiochemistry

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[11C]DASB was prepared as previously described (Houle et al., 2000; Wilson et al.,

2002). [11C]PE2I was prepared as in (DeLorenzo et al., 2009). [11C]MeNER was prepared using a modification of the method in (Lin and Ding, 2004).

Image Acquisition

For each subject a structural T1-weighted magnetic resonance brain scan was acquired on a GE 1.5-T Signa Advantage system (General Electric, Fairfield, CT), to aid identification of regions of interest (ROI) for subsequent PET image analysis. Downloaded from All PET scanning was performed on an HR+ scanner (Siemens, Knoxville, TN, USA), operating in a three-dimensional mode. The animal's head was placed in the centre of

the field of view, and a 10-minute transmission scan was acquired for attenuation jpet.aspetjournals.org correction, prior to tracer injection. Radioligands were administered as a 30-second intravenous bolus. Emission data were binned into a sequence of frames of increasing length. Total scan durations were 120minutes for [11C]PE2I and [11C]MeNER, and at ASPET Journals on September 28, 2021

90minutes for [11C]DASB. PET data were corrected for attenuation, scatter, and randoms and reconstructed using filtered back-projection with a Shepp filter (cut-off 0.5 cycles per projection ray).

Blood sampling

Arterial plasma samples were collected using an automated sampling system during the first 4minutes (11 samples), and manually thereafter. A total of 22 for [11C]PE2I, 21 for

[11C]DASB, and 22 for [11C]MeNER arterial samples were collected for input function measurement. Six additional samples were collected during each scan for high- performance liquid chromatography analysis of the unmetabolized fraction of radiotracer.

During post GSK1360707 scans, six additional venous samples were also collected (at

7, 15, 30, 60, 95 and 185minutes post radiotracer injection) for subsequent analysis of the plasma concentration of GSK1360707 by LC/MS/MS.

Data Analysis

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ROIs were drawn manually on each animals MRI. The midbrain, thalamus, and the striatum were defined as the target regions of interest for SERT, the striatum for DAT, and the midbrain, thalamus and brain stem for NET. Dynamic PET data were corrected for motion and co-registered to each subjects MRI using a mutual information cost function. Decay corrected time activity curves were generated as the average ROI activity from each of the dynamically acquired PET frames.

Regional VT values for each ligand were derived from an appropriate compartmental Downloaded from model (1-tissue for [11C]DASB, 2-tissue for [11C]PE2I and [11C]MeNER) with metabolite corrected arterial plasma input function. For [11C]DASB and [11C]PE2I, regional binding

potential (BPND) values were derived from the PET volume of distribution (VT) values, jpet.aspetjournals.org with the cerebellar VT assumed equal to the VND (Non-displaceable)). Target occupancy was derived as the fractional decrease of baseline BPND following the administration of

GSK1360707. Occupancy values were averaged across all regions to provide an at ASPET Journals on September 28, 2021 occupancy estimate for each post-dose scan. Occupancy values derived in this way were compared to those derived using a simplified reference tissue model which has been found to be acceptable for the quantification of [11C]DASB and [11C]PE2I data, since the later would be employed in the human experiments. As anticipated the correlation was found to be acceptable, allowing the less invasive approach to be taken in the human study.

Equation 1:

Baseline GSK1360707 Baseline Occ = (BPND – BPND )/BPND

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In the absence of a well characterised reference region for [11C]MeNER, a modified

Lassen plot (Lassen et al., 1995; Cunningham et al., 2010) was used to estimate the VND and NET occupancy following each dose of GSK1360707.

Pharmacokinetic - Target Occupancy Analysis (pre-clinical)

The PK/PD population consisted of a total 7 measurements for SERT, 8 for DAT, and 7 for NET. Direct and Indirect models were evaluated (for SERT and DAT). A modified Hill Downloaded from equation (a direct model) was found to describe the data adequately (indirect data not shown). The model (equation 2), a semi-log form of the Hill equation, assumes that the

maximal occupancy at a given target is 100% and the minimum occupancy is 0. This jpet.aspetjournals.org model was fitted to the whole data set using GraphPad Prism, version 5.03 (GraphPad

Software, San Diego CA, USA). to obtain estimates of half-maximal occupancy (IC50).

at ASPET Journals on September 28, 2021

Equation 2:

Occ=100/ (1+10^((Log IC50-[GSK1360707])-Hill Slope))

Clinical PET imaging studies

Clinical studies were performed at the GlaxoSmithKline Clinical Imaging Centre (CIC),

London. The study was approved by the Capenhurst Independent Research Ethics

Committee (UK), and permission to administer radioisotopes was obtained from the

Administration of Radioactive Substances Advisory Committee of the United Kingdom

(ARSAC). 12 healthy male subjects were enrolled into the study meeting the protocol inclusion and exclusion criteria posted on the ClinicalTrials.gov database

(ClinicalTrials.gov Identifier: NCT01153802).

Experimental Design

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The aim of this study was to measure the time course of GSK1360707 occupancy at the

SERT and DAT in the human brain following oral dosing, and to relate those measures to plasma concentrations in order to investigate the relationship between the plasma concentration of GSK1360707 and its occupancy at each target. Human NET occupancy was not assessed.

Subjects were randomly assigned to one of two cohorts, for the assessment of SERT and DAT occupancy. Downloaded from Experimental Procedures

Each subject received a baseline [11C]DASB or [11C]PE2I PET scan (PET Scan 1).

Following a baseline scan, a single oral dose of GSK1360707 was administered followed jpet.aspetjournals.org by two further PET scans (PET Scans 2 and 3). PET Scans 2 and 3 were used to provide information on the time-course of brain SERT or DAT occupancy by

GSK1360707. The time of PET Scan 2 for the first two subjects was approximately 2 at ASPET Journals on September 28, 2021 hours post-dose (FTIH data indicated a plasma tmax of 1.5-3 hours post dose for

GSK1360707), and time of PET Scan 3 was approximately 12 hours post dose based on observed human pharmacokinetics. For subsequent subjects, the dose (of

GSK1360707) and timing of PET Scans 2 and 3 were adjusted following a review of the preceding subjects data, to ensure an adequate sampling of the TOC using an adaptive design approach (Zamuner et al., 2010; Abanades et al., 2011). Subjects returned for a follow-up visit approximately 7 to 14 days following their last dose of study medication

A range of doses of GSK1360707 were evaluated in this study (15 – 150 mg PO), following the demonstration of safety and tolerability in a previous placebo-controlled, single ascending dose study completed in healthy male subjects (report available at www.gsk-clinicalstudyregister.com, study identifier: SNV111914). Blood sampling was performed at the beginning of or immediately prior to each post-dose PET scan.

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Quantification of plasma GSK1360707 was performed by LC/MS/MS, with the lower limit of quantification at 1 ng/mL.

Radiochemistry

[11C]DASB

[11C]DASB was prepared as described previously (Abanades et al., 2011).

[11C] PE2I

[11C]PE2I was prepared as described in the supplementary data section. Downloaded from Image Acquisition

A structural T1-weighted magnetic resonance brain scan was acquired on a 3T MRI

scanner (Siemens Tim Trio 3T; Siemens AG, Medical Solutions Erlangen, Germany). jpet.aspetjournals.org

Data was acquired in the axial plane (FSPGR – IR PREPPED, TR=12.008, TE=5.1160, flip angle= 20o, slice thickness = 0.78 x 0.78 x 1.5 mm. All structural scans were inspected by an independent radiologist for unexpected findings of clinical significance. at ASPET Journals on September 28, 2021

All dynamic PET scans were acquired on a Siemens Biograph 6 PET/CT scanner with

TruePoint gantry (Siemens Healthcare). Subjects were positioned in the tomograph, following insertion of a venous cannula in an antecubital vein, and a head-fixation device was used to minimise head movements during data acquisition. A low dose CT scan was performed before each injection of the radioligand for subsequent attenuation and scatter correction. Dynamic emission data were collected continuously for DASB (100 min) and PE2I (120 min), following intravenous injection of an average of 231±56 MBq of

[11C]DASB or 202±82 MBq of [11C]PE2I. Image data were reconstructed using filtered backprojection with a 128 matrix, a zoom of 2.6, a transaxial Gaussian filter of 5 mm, scatter correction and attenuation correction.

Data Analysis

Imaging data was analysed within the GSK CIC Molecular imaging analysis pipeline as previously described (Abanades et al., 2011). The midbrain, thalamus, and the striatum

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were defined as the target regions of interest for SERT, and the striatum for DAT. The reference region time-activity curve derived from the cerebellum was used as an input to the simplified reference tissue model (Lammertsma and Hume, 1996) with a basis function implementation (Gunn et al., 1997), to quantify the binding potential (BPND) in all relevant target regions. Target occupancy in scans 2 and 3 was estimated as a fractional reduction in the relevant baseline BPND (see Equation 1):

Pharmacokinetic - Target Occupancy Analysis Downloaded from The PK/PD populations for SERT and DAT occupancy consisted of 10 subjects, providing a total of 10 occupancy measurements for SERT, and 8 for DAT. SERT

occupancy values were derived by averaging across midbrain, striatum and thalamus. jpet.aspetjournals.org

Data was treated as previously described above for pre-clinical imaging experiments. at ASPET Journals on September 28, 2021

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6) Results

Pre-clinical PET imaging studies

Four subjects provided data. The mean injected radioactivity for [11C]DASB, [11C]PE2I and [11C]MeNER were 160 ± 30, 155 ± 36 and 122 ± 44 MBq, respectively. The radiochemical purity of the injected [11C]DASB, [11C]PE2I and [11C]MeNER exceeded

95% for all scans. The injected masses of cold DASB, PE2I and MeNER varied between

0.43 and 1.22μg (mean 0.87 ± 0.29), 1.19 and 0.49μg (mean 0.84 ± 0.23), and 0.65 Downloaded from and 1.95μg (mean 0.86 ± 0.51) respectively. No serious adverse events were observed during the study.

Clinical PET imaging studies jpet.aspetjournals.org

A total of 10 evaluable subjects completed the study according to the protocol. The average age of the subjects was 42 years (range 35 to 50), with a mean body mass index (BMI) of 27 kg/m2 (range 23 to 29). The mean injected radioactivity was at ASPET Journals on September 28, 2021

231±56MBq for DASB and 221±63 for PE2I. The radiochemical purity of the injected

[11C]DASB and [11C]PE2I exceeded 95% for all scans. The injected masses of DASB and PE2I varied between 0.4 and 5.6μg (mean 1.2 ± 1.2), and 0.4 and 6.3μg (mean

1.9 ± 1.4), respectively. No serious adverse events were observed during the study.

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7) Discussion

We conducted PET imaging experiments in Papio Anubis and human s to es timate in vivo IC50 (half maximal target occupancy) of GSK1360707 for central SERT, DAT and

NET, and to ev aluate target occupancies in the l ikely clinical dose range. In Papio

Anubis in vivo IC50 values for SERT, DAT and NET were found to be 15. 16, 15.56 and

0.97 ng/ml respectively. In human values for SERT and DAT were 6.80 and 18.80 ng/ml.

Translational aspects Downloaded from In vitro studies had previously demonstrated a dose dependent occupancy of the SERT by GSK1360707, as well as the binding and blockade of functional responses at al l 3

targets (compound 17 in (Micheli et al., 2010). The rank order of pKi in binding assays jpet.aspetjournals.org was SERT > NET > DAT (Micheli et al., 2010). Our PET experiments in Papio Anubis suggest the IC50 at SERT and DAT is equivalent, and that the compound is more potent at NET (NET potency being judged in terms of the occupancy achieved at similar at ASPET Journals on September 28, 2021 concentrations, since the NET IC50 estimate may not be r eliable, see limitations). This difference between in vitro data obtained using cells transfected with human transporters, and the in vivo data from Papio Anubis could be due to species differences in the affinity of the transporters for GSK1360707, or because the in vitro assays do not adequately mimic the physiological context of the in vivo environment.

PET studies in human s howed a c lear separation in IC50 at SERT and DAT ; the inconsistency compared to Papio Anubis being driven by a l ower IC50 for SERT in humans, the IC50 for DAT being equivalent to that in Papio Anubis.

Clinical context – getting the “right” triple profile

Therapeutic effects at SER T are achieved after chronic treatment, generally when occupancy is ≥ 80% (Meyer et al., 2004). Whereas therapeutic effects mediated by DAT inhibition are generally achieved when occupancy is in the r egion of 30% (Learned-

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Coughlin et al., 2003); high levels of DAT inhibition having been linked to the pos itive reinforcing effects and abus e liability of c ocaine (Kuhar et al., 1991; W oolverton and

Johnson, 1992).

Less is known about the level of NET inhibition required for the clinical efficacy of SNRIs or NRIs. However, inhibition of NA uptak e or ligand binding at NET has been demonstrated in vitro for a range of antidepressants and their metabolites (Pristupa et al., 1994; Owens et al., 1997; T atsumi et al., 1997; Bymaster et al., 2001; Millan et al., Downloaded from 2001; Bymaster et al., 2002; Koch et al., 2003; Kuo et al., 2004; Vaishnavi et al., 2004), and , , reboxetine and to a lesser extent nortryptyline are highly

selective for NET (above SERT and DAT). and venlafaxine have been shown jpet.aspetjournals.org to act as NET antagonists in vivo at clinical doses (Gilmor et al., 2002; Davidson et al.,

2005; Owens et al., 2008). In addition it has been known for some time that the tricyclic antidepressant occupies 80% of SERT at dos es (and plasma at ASPET Journals on September 28, 2021 concentrations) much lower than those used clinically (Suhara et al., 2003), suggesting a role for NET inhibition (via its active metabolite desmethylclomipramine (Thomas and

Jones, 1977; Maj et al., 1982). Direct assessments of NET occupancy using PET have been made for , with a single dose of 75 mg (which is at the lower end of the clinically relevant range) producing in the region of 40% occupancy (Sekine et al., 2010).

With respect to nor epinephrine reuptake mediated side effects , an SNRI was withdrawn from the mar ket due to the l evel of ad verse cardiovascular events

(James et al., 2010). However, the dose of venlafaxine estimated to occupy 40% NET has minimal effects on he art rate and bl ood pressure. Taking all of t he above into consideration the i deal TRI profile is assumed to appr oximate to >70% SERT occupancy, 30-40% DAT occupancy and 30% NET occupancy, providing efficacy with minimal DAT and NET mediated AEs.

Profile of GSK1360707

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Human data i ndicates that i t is possible to pr oduce high levels of SE RT occupancy

(~70%) with moderate DAT occupancy (~30-40%) at doses of GSK1360707 that are well tolerated. However, a question remains as to what the measured NET occupancy would be in this dose range. At the time the study was conducted we did not have access to the NET ligand, [11C]MeNER (for human us e). GSK1360707 does appear to be mor e potent at NET (compared to the othe r targets) in Papio Anubis. However, the NET IC50 estimate is compromised by a lack of data at low occupancy levels,and, since all doses Downloaded from tested were well tolerated in humans, with only one s ubject experiencing a c linically significant increase in heart rate, and no s ubjects experiencing clinically significant

Papio Anubis changes in laboratory parameters or ECG, the data obtained from may not jpet.aspetjournals.org be predictive.

The discrepancy between data in Papio Anubis and human reinforces the importance of human occupancy studies have for decision making regarding compound progression, at ASPET Journals on September 28, 2021 and clinical dose selection.

Shortly after the conclusion of this study the development of the G SK1360707 was put on-hold following to a dec ision by GlaxoSmithKline to exit certain research areas. It is difficult to predict how the clinical development of GSK1360707 would have progressed, however, based on the data presented here it seems likely that the molecule would have progressed toward a proof of concept study, with the possible addition of an assessment of NET occupancy in human.

Limitations

The assessment of NET IC50 in baboon is compromised by a lack of data at exposures resulting in low NET occupancy values.

The clinical part of our study was conducted in healthy male control subjects. Whilst we are not aware of any evidence to support this hypothesis it is possible that physiological or genetic factors (e.g. transporter polymorphisms) could differ in a patient group and

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affect in vivo IC50 values. For instance differences in the affinity of the transporters, the concentration of GSK1360707 achieved in the brain, or endogenous neurotransmitter levels (such that a different level of transporter occupancy by the drug is required to raise endogenous neurotransmitter levels to the same degree). In addition if a given occupancy level is achieved at the same plasma exposure differences in second messenger activity, or the distribution and density of target or non-target transporters could still modify the downstream response e.g. NET blockade could raise dopamine Downloaded from levels in the PFC, as DA is transported by NET in the absence of DAT; or differences in organic cation transporter (OCT) or the plasma membrane monoamine transporter

(PMAT) systems could also affect clearance of biogenic amines. jpet.aspetjournals.org

For most patients antidepressant response to SSRIs can be expected to occur with

SERT blockade of >80%. We did not explore occupancy beyond that level in humans, in order to avoid excessive modulation of any of the 3 targets, particularly DAT. This could at ASPET Journals on September 28, 2021 result in inaccuracy in the IC50 estimates.

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8) Acknowledgments

The authors would like to gratefully acknowledge the contributions of Elizabeth Hackett,

John Castrillon, Sung A Baall at Columbia University (data acquisition in the non-clinical studies), Balu Easwararmoorthy also at Columbia University (chemistry for the non- clinical studies), Shaila Shabbir and Robert Lai from GlaxoSmithKline (clinical operations and medical monitoring); Gary Evoniuk also at GlaxoSmithKline (comments of the manuscript), staff from the PAREXEL International clinical pharmacology research unit in Downloaded from Harrow, and the study participants. jpet.aspetjournals.org at ASPET Journals on September 28, 2021

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9) Authorship contributions Participated in research design: Comley, Salinas, Slifstein, Petrone, Shotbolt, Neve,

Iavarone, Gomeni, Gray, Gunn, and Rabiner

Conducted experiments: Comley, Slifstein, Marzano, Shotbolt, Van der Aart, and

Rabiner

Performed data analysis: Comley, Salinas, Slifstein, Petrone, Shotbolt, Neve, Iavarone,

Gomeni, Gray, Gunn, and Rabiner Downloaded from Wrote or contributed to the writing of the manuscript: Comley, Salinas, Slifstein, Petrone,

Marzano, Shotbolt, Van der Aart, Neve, Iavarone, Gomeni, Gray, Gunn, and Rabiner jpet.aspetjournals.org at ASPET Journals on September 28, 2021

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10)

References

Abanades S, van der Aart J, Barletta JA, Marzano C, Searle GE, Salinas CA, Ahmad JJ,

Reiley RR, Pampols-Maso S, Zamuner S, Cunningham VJ, Rabiner EA, Laruelle

MA and Gunn RN (2011) Prediction of repeat-dose occupancy from single-dose

data: characterisation of the relationship between plasma pharmacokinetics and Downloaded from brain target occupancy. J Cereb Blood Flow Metab 31:944-952.

Al-Shamahi A, Kirkham K and Hookes L (2009) Society for Neuroscience - 39th Annual

Meeting. Part 1 - Novel therapies for the treatment of CNS disorders and pain. jpet.aspetjournals.org

IDrugs 12:731-733.

Axel AM, Mikkelsen JD and Hansen HH (2010) , a novel triple monoamine

reuptake inhibitor, induces appetite suppression by indirect stimulation of alpha1 at ASPET Journals on September 28, 2021

adrenoceptor and dopamine D1 receptor pathways in the diet-induced obese rat.

Neuropsychopharmacology 35:1464-1476.

Basile AS, Janowsky A, Golembiowska K, Kowalska M, Tam E, Benveniste M, Popik P,

Nikiforuk A, Krawczyk M, Nowak G, Krieter PA, Lippa AS, Skolnick P and

Koustova E (2007) Characterization of the antinociceptive actions of in

models of acute, persistent, and chronic pain. J Pharmacol Exp Ther 321:1208-

1225.

Bymaster FP, Dreshfield-Ahmad LJ, Threlkeld PG, Shaw JL, Thompson L, Nelson DL,

Hemrick-Luecke SK and Wong DT (2001) Comparative affinity of and

venlafaxine for serotonin and norepinephrine transporters in vitro and in vivo,

human serotonin receptor subtypes, and other neuronal receptors.

Neuropsychopharmacology 25:871-880.

- 22 - JPET Fast Forward. Published on May 17, 2013 as DOI: 10.1124/jpet.112.202895 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #202895

Bymaster FP, Zhang W, Carter PA, Shaw J, Chernet E, Phebus L, Wong DT and Perry

KW (2002) , but not other selective serotonin uptake inhibitors,

increases norepinephrine and dopamine extracellular levels in prefrontal cortex.

Psychopharmacology 160:353-361.

Cunningham VJ, Rabiner EA, Slifstein M, Laruelle M and Gunn RN (2010) Measuring

drug occupancy in the absence of a reference region: the Lassen plot re-visited.

J Cereb Blood Flow Metab 30:46-50. Downloaded from Davidson J, Watkins L, Owens M, Krulewicz S, Connor K, Carpenter D, Krishnan R and

Nemeroff C (2005) Effects of paroxetine and venlafaxine XR on heart rate

J Clin Psychopharmacol 25

variability in depression. :480-484. jpet.aspetjournals.org

DeLorenzo C, Kumar JSD, Zanderigo F, Mann JJ and Parsey RV (2009) Modeling

considerations for in vivo quantification of the dopamine transporter using

[(11)C]PE2I and positron emission tomography. J Cerebr Blood F Met 29:1332- at ASPET Journals on September 28, 2021

1345.

Derendorf H and Meibohm B (1999) Modeling of pharmacokinetic/pharmacodynamic

(PK/PD) relationships: concepts and perspectives. Pharm Res 16:176-185.

Frankle WG, Slifstein M, Gunn RN, Huang Y, Hwang DR, Darr EA, Narendran R, Abi-

Dargham A and Laruelle M (2006) Estimation of serotonin transporter

parameters with 11C-DASB in healthy humans: reproducibility and comparison of

methods. J Nucl Med 47:815-826.

Gilmor ML, Owens MJ and Nemeroff CB (2002) Inhibition of norepinephrine uptake in

patients with major depression treated with paroxetine. Am J Psychiatry

159:1702-1710.

Gunn RN, Lammertsma AA, Hume SP and Cunningham VJ (1997) Parametric imaging

of ligand-receptor binding in PET using a simplified reference region model.

Neuroimage 6:279-287.

- 23 - JPET Fast Forward. Published on May 17, 2013 as DOI: 10.1124/jpet.112.202895 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #202895

Hansen HH, Hansen G, Tang-Christensen M, Larsen PJ, Axel AM, Raben A and

Mikkelsen JD (2010) The novel triple monoamine reuptake inhibitor tesofensine

induces sustained weight loss and improves glycemic control in the diet-induced

obese rat: comparison to sibutramine and rimonabant. Eur J Pharmacol 636:88-

95.

Hauser RA, Salin L, Juhel N and Konyago VL (2007) Randomized trial of the triple

monoamine reuptake inhibitor NS 2330 (tesofensine) in early Parkinson's Downloaded from disease. Mov Disord 22:359-365.

Houle S, Ginovart N, Hussey D, Meyer JH and Wilson AA (2000) Imaging the serotonin

transporter with positron emission tomography: initial human studies with jpet.aspetjournals.org

[11C]DAPP and [11C]DASB. Eur J Nucl Med 27:1719-1722.

James WP, Caterson ID, Coutinho W, Finer N, Van Gaal LF, Maggioni AP, Torp-

Pedersen C, Sharma AM, Shepherd GM, Rode RA and Renz CL (2010) Effect of at ASPET Journals on September 28, 2021

sibutramine on cardiovascular outcomes in overweight and obese subjects. N

Engl J Med 363:905-917.

Koch S, Hemrick-Luecke SK, Thompson LK, Evans DC, Threlkeld PG, Nelson DL, Perry

KW and Bymaster FP (2003) Comparison of effects of dual transporter inhibitors

on monoamine transporters and extracellular levels in rats. Neuropharmacology

45:935-944.

Kuhar MJ, Ritz MC and Boja JW (1991) The dopamine hypothesis of the reinforcing

properties of . Trends Neurosci 14:299-302.

Kuo F, Gillespie TA, Kulanthaivel P, Lantz RJ, Ma TW, Nelson DL, Threlkeld PG,

Wheeler WJ, Yi P and Zmijewski M (2004) Synthesis and biological activity of

some known and putative duloxetine metabolites. Bioorg Med Chem Lett

14:3481-3486.

- 24 - JPET Fast Forward. Published on May 17, 2013 as DOI: 10.1124/jpet.112.202895 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #202895

Lammertsma AA and Hume SP (1996) Simplified reference tissue model for PET

receptor studies. Neuroimage 4:153-158.

Lassen NA, Bartenstein PA, Lammertsma AA, Prevett MC, Turton DR, Luthra SK,

Osman S, Bloomfield PM, Jones T, Patsalos PN and et al. (1995)

Benzodiazepine receptor quantification in vivo in humans using [11C]flumazenil

and PET: application of the steady-state principle. J Cereb Blood Flow Metab

15:152-165. Downloaded from Learned-Coughlin SM, Bergstrom M, Savitcheva I, Ascher J, Schmith VD and Langstrom

B (2003) In vivo activity of at the human dopamine transporter as

Biol Psychiatry 54

measured by positron emission tomography. :800-805. jpet.aspetjournals.org

Lin KS and Ding YS (2004) Synthesis, enantiomeric resolution, and selective C-11

methylation of a highly selective radioligand for imaging the norepinephrine

transporter with positron emission tomography. Chirality 16:475-481. at ASPET Journals on September 28, 2021

Logan J, Wang GJ, Telang F, Fowler JS, Alexoff D, Zabroski J, Jayne M, Hubbard B,

King P, Carter P, Shea C, Xu Y, Muench L, Schlyer D, Learned-Coughlin S,

Cosson V, Volkow ND and Ding YS (2007) Imaging the norepinephrine

transporter in humans with (S,S)-[11C]O-methyl reboxetine and PET: problems

and progress. Nucl Med Biol 34:667-679.

Maj J, Stala L, Gorka Z and Adamus A (1982) Comparison of the pharmacological

actions of desmethylclomipramine and clomipramine. Psychopharmacology

78:165-169.

Marks DM, Pae CU and Patkar AA (2008) Triple reuptake inhibitors: the next generation

of antidepressants. Curr Neuropharmacol 6:338-343.

Matthews PM, Rabiner EA, Passchier J and Gunn RN (2012) Positron emission

tomography molecular imaging for drug development. Br J Clin Pharmacol

73:175-186.

- 25 - JPET Fast Forward. Published on May 17, 2013 as DOI: 10.1124/jpet.112.202895 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #202895

Meyer JH, Wilson AA, Sagrati S, Hussey D, Carella A, Potter WZ, Ginovart N, Spencer

EP, Cheok A and Houle S (2004) Serotonin transporter occupancy of five

selective serotonin reuptake inhibitors at different doses: an [11C]DASB positron

emission tomography study. Am J Psychiatry 161:826-835.

Micheli F, Cavanni P, Andreotti D, Arban R, Benedetti R, Bertani B, Bettati M, Bettelini L,

Bonanomi G, Braggio S, Carletti R, Checchia A, Corsi M, Fazzolari E, Fontana S,

Marchioro C, Merlo-Pich E, Negri M, Oliosi B, Ratti E, Read KD, Roscic M, Downloaded from Sartori I, Spada S, Tedesco G, Tarsi L, Terreni S, Visentini F, Zocchi A, Zonzini L

and Di Fabio R (2010) 6-(3,4-dichlorophenyl)-1-[(methyloxy)methyl]-3-

J

azabicyclo[4.1.0]heptane: a new potent and selective triple reuptake inhibitor. jpet.aspetjournals.org

Med Chem 53:4989-5001.

Millan MJ, Gobert A, Lejeune F, Newman-Tancredi A, Rivet JM, Auclair A and Peglion

JL (2001) S33005, a novel ligand at both serotonin and norepinephrine at ASPET Journals on September 28, 2021

transporters: I. Receptor binding, electrophysiological, and neurochemical profile

in comparison with venlafaxine, reboxetine, , and clomipramine. J

Pharmacol Exp Ther 298:565-580.

Montgomery SA (1997) Fast-onset antidepressants. International clinical

psychopharmacology 12 Suppl 3:S1-5.

Nyberg S, Eriksson B, Oxenstierna G, Halldin C and Farde L (1999) Suggested minimal

effective dose of based on PET-measured D2 and 5-HT2A receptor

occupancy in schizophrenic patients. Am J Psychiatry 156:869-875.

Owens MJ, Krulewicz S, Simon JS, Sheehan DV, Thase ME, Carpenter DJ, Plott SJ and

Nemeroff CB (2008) Estimates of serotonin and norepinephrine transporter

inhibition in depressed patients treated with paroxetine or venlafaxine.

Neuropsychopharmacology 33:3201-3212.

- 26 - JPET Fast Forward. Published on May 17, 2013 as DOI: 10.1124/jpet.112.202895 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #202895

Owens MJ, Morgan WN, Plott SJ and Nemeroff CB (1997) Neurotransmitter receptor

and transporter binding profile of antidepressants and their metabolites. J

Pharmacol Exp Ther 283:1305-1322.

Pristupa ZB, Wilson JM, Hoffman BJ, Kish SJ and Niznik HB (1994) Pharmacological

heterogeneity of the cloned and native human dopamine transporter:

disassociation of [3H]WIN 35,428 and [3H]GBR 12,935 binding. Mol Pharmacol

45:125-135. Downloaded from Rascol O, Poewe W, Lees A, Aristin M, Salin L, Juhel N, Waldhauser L and Schindler T

(2008) Tesofensine (NS 2330), a monoamine reuptake inhibitor, in patients with

Arch

advanced Parkinson disease and motor fluctuations: the ADVANS Study. jpet.aspetjournals.org

Neurol 65:577-583.

Sekine M, Arakawa R, Ito H, Okumura M, Sasaki T, Takahashi H, Takano H, Okubo Y,

Halldin C and Suhara T (2010) Norepinephrine transporter occupancy by at ASPET Journals on September 28, 2021

antidepressant in human brain using positron emission tomography with (S,S)-

[18F]FMeNER-D2. Psychopharmacology 210:331-336.

Sjodin A, Gasteyger C, Nielsen AL, Raben A, Mikkelsen JD, Jensen JK, Meier D and

Astrup A (2010) The effect of the triple monoamine reuptake inhibitor tesofensine

on energy metabolism and appetite in overweight and moderately obese men. Int

J Obes (Lond) 34:1634-1643.

Suhara T, Takano A, Sudo Y, Ichimiya T, Inoue M, Yasuno F, Ikoma Y and Okubo Y

(2003) High levels of serotonin transporter occupancy with low-dose

clomipramine in comparative occupancy study with using positron

emission tomography. Arch Gen Psychiatry 60:386-391.

Tatsumi M, Groshan K, Blakely RD and Richelson E (1997) Pharmacological profile of

antidepressants and related compounds at human monoamine transporters. Eur

J Pharmacol 340:249-258.

- 27 - JPET Fast Forward. Published on May 17, 2013 as DOI: 10.1124/jpet.112.202895 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #202895

Teller H and Furstner A (2011) Concise synthesis of the antidepressive drug candidate

GSK1360707 by a highly enantioselective gold-catalyzed enyne

cycloisomerization reaction. Chemistry 17:7764-7767.

Thomas PC and Jones RB (1977) The effects of clomipramine and

desmethylclomipramine on the in vitro uptake of radiolabelled 5-HT and

noradrenaline into rat brain cortical slices. J Pharm Pharmacol 29:562-563.

Vaishnavi SN, Nemeroff CB, Plott SJ, Rao SG, Kranzler J and Owens MJ (2004) Downloaded from : a comparative analysis of human monoamine uptake and

transporter binding affinity. Biol Psychiatry 55:320-322.

Weinmann S, Becker T and Koesters M (2008) Re-evaluation of the efficacy and jpet.aspetjournals.org

tolerability of venlafaxine vs SSRI: meta-analysis. Psychopharmacology 196:511-

520; discussion 521-512.

Wilson AA, Ginovart N, Hussey D, Meyer J and Houle S (2002) In vitro and in vivo at ASPET Journals on September 28, 2021

characterisation of [11C]-DASB: a probe for in vivo measurements of the

serotonin transporter by positron emission tomography. Nucl Med Biol 29:509-

515.

Woolverton WL and Johnson KM (1992) Neurobiology of cocaine abuse. Trends

Pharmacol Sci 13:193-200.

Zamuner S, Di Iorio VL, Nyberg J, Gunn RN, Cunningham VJ, Gomeni R and Hooker

AC (2010) Adaptive-optimal design in PET occupancy studies. Clin Pharmacol

Ther 87:563-571.

- 28 - JPET Fast Forward. Published on May 17, 2013 as DOI: 10.1124/jpet.112.202895 This article has not been copyedited and formatted. The final version may differ from this version.

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11) Footnotes a) This work was funded by GlaxoSmithKline. b) van der Aart, J., R. A. Comley, C. A. Salinas, M. Slifstein, M. Petrone, M. Neve, L. E. Iavarone, R. O. Gomeni, F. A. Gray, R. N. Gunn and E. A. Rabiner (2011). Estimation of in vivo selectivity of a triple monoamine reuptake inhibitor in non-human primate and human. BrainPET '11: Xth International Conference on Quantification of Brain Function with PET. Barcelona, Spain

Petrone, M., R. A. Comley, C. A. Salinas, M. Neve, L. Iavarone, R. N. Gunn, R. Gomeni, Downloaded from E. Rabiner and F. A. Gray (2010). Assessment of the occupancy-exposure relationship of a triple monoamine re-uptake inhibitor in human, using Positron Emission Tomography Sixth International Symposium on Measurement & Kinetics of In Vivo Drug jpet.aspetjournals.org Effects. Leiden, The Netherlands. c) Eugenii Rabiner

Imanova Limited at ASPET Journals on September 28, 2021

Burlington Danes Building,

Imperial College London,

Hammersmith Hospital,

Du Cane Road,

London, W12 0NN

United Kingdom e-mail address: [email protected] d) Current affiliations where different to those at the time of study conduct:

1 F. Hoffmann La-Roche, Basel, Switzerland (R. C) 2 Imanova Limited, London, United Kingdom (C.A.S, J.V, R.N.G, E.A.R) 3 Aptuit, Verona, Italy (M. P, M. N) 4 Pharmacometrica, La Fouillade, France (R. G) 5 UCB S.A., Belgium (M. L)

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12) Figure Legends Figure 1, relationship between GSK1360707 plasma concentration, SERT, DAT and NET occupancy in Papio Anubis: Brain regions for transporter occupancies are the mean of midbrain, striatum and thalamus for SERT; striatum for DAT; midbrain for NET. Fitted curves show predicted occupancy based on a direct PK-TO relationship, using Equation 2. Figure 2, relationship between GSK1360707 plasma concentration, SERT and DAT occupancy in human: Brain regions for transporter occupancies are the mean of midbrain, striatum and thalamus for SERT; striatum for DAT. Fitted curves show Downloaded from predicted occupancy based on Equation 2. jpet.aspetjournals.org at ASPET Journals on September 28, 2021

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13) Tables

Table 1: Affinity and functional potency measures for GSK1360707

In vitro human In vivo PET Papio Anubis in vivo PET human

1 2 pKi pKi IC50 ng/mL (95%CI) Hill slope IC50 ng/mL (95%CI) Hill slope

SERT 9.20± 0.01 8.98 ± 0.02 15.16 (8.85-25.97) 0.91 6.80 (4.45-10.39) 0.87 Downloaded from

DAT 8.00± 0.01 7.92 ± 0.03 15.56 (5.73-42.22) 0.84 18.80 (13.97-25.29) 0.75

NET 8.10± 0.01 7.92 ± 0.02 0.97 (0.03-29.12) 0.46 N/A N/A jpet.aspetjournals.org

Relative molar mass of GSK1360707 = 322.665

1: Mean ± SEM from scintillation proximity binding assay for human recombinant SERT, at ASPET Journals on September 28, 2021

DAT and NET; pKi was calculated from pIC50 as reported by Micheli et al (Micheli et al., 2010). 2: Mean ± SEM from filtration binding assay for human recombinant SERT, DAT and NET; pKi was calculated from pIC50, as reported by Micheli et al (Micheli et al., 2010).

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Article title: Monoamine transporter occupancy of a novel triple reuptake inhibitor in baboon and human using Positron Emission Tomography

Author names: Robert A. Comley, Cristian A. Salinas, Mark Slifstein, Marcella Petrone,

Carmine Marzano, Paul Shotbolt, Jasper Van der Aart, Marta Neve, Laura Iavarone,

Roberto Gomeni, Marc Laruelle, Frank A. Gray, Roger N. Gunn, Eugenii A. Rabiner

Journal name: The Journal of Pharmacology and Experimental Therapeutics

15) Supplemental Data

[11C] PE2I for clinical studies

[11C]PE2I was prepared as follows: [11C]Carbon dioxide was produced using an Eclipse

RDS cyclotron (Siemens, USA) by the 14N(p,α)11C reaction and 11 MeV proton irradiation (typical bombardment of 55 µA, 50 min) of nitrogen gas containing 1%

Oxygen. [11C]Carbon dioxide was transformed to [11C]Methyliodide using a GE Microlab

(GE GEMS, Sweden) as previously described (Larsen et al., 1997). A semi-automated

Modular Lab Multifunctional Synthetic Module (Eckert & Ziegler, Germany) configured to trap the [11C]Methyl iodide was used for the radiolabeling and purification process.

The reference for [O-methyl] (E)-N-(3-iodoprop-2E-enyl)-2-carbomethoxy-3-(4’- methylphenyl)nortropane (PE2I) and precursor desmethyl PE2I (free base) were obtained from Pharmasynth (Estonia). The radiotracer [11C]PE2I was synthesized by reaction of [11C]Methyl iodide with the free base precursor in the presence of a base.

The [11C]Methyl iodide was delivered in a He flow to a 3 ml v-bottom vial containing 0.5-1 mg of the free base precursor in 350uL of DMF and 3uL of 0.1M TBAH (tetrabutyl ammonium hydroxide) cooled at - 5°C. Once all [11C]Methyl iodide was delivered the vial containing the reaction mixture was sealed and heated to 80°C for 4 minutes. After this time the reaction mixture was diluted with 1 ml of mobile phase and injected into a semi preparative HPLC for purification. The tracer purification was carried out using a uBondapack C-18 (Waters) 7.8*300mm 10um as stationary phase and as mobile phase

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55% methanol 45% Sodium dihydrogenophosphate (25mM, pH 4.1) at a flow of 8 ml/min. In this condition [11C]PE2I eluted between 4.8 and 5.5min. The radioactive peak was collected, diluted in 20 ml of water containing 80-100 mg of K2CO3. The fraction was loaded onto a tC2 Sep-pak plus (Waters) and washed with 10 ml of 35% ethanol solution. [11C]PE2I was eluted from the cartridge into a 20 ml mixing vial with 1.5ml of ethanol followed by 9.5 ml of 0.9% saline solution. In this way [11C]PE2I was obtained formulated in 11mL of 14 % (v/v) ethanol in 0.9 % saline solution for injection. The solution was then sterilized by passing it through a sterile filter (0.22um Posydine 33mm,

PALL); the final dose was collected in a sterile container.

The final product was tested for chemical and radiochemical purity by means of an analytical 1200 HPLC system (Agilent, West Lothian, UK) using a Zorbax SB Phenyl

4.6*100mm, 3.5 um (Agilent) and a mixture of Methanol (55%) and Ammonium Formate buffer (45%, 50mM at pH 4.00) as mobile phase at a flow of 1.2 mL/min. Desmethyl

PE2I (precursor, tR=3.1min) and [11C]PE2I (tR=3.9min) concentrations were determined by means of UV (230nm). The product identity was confirmed by co-injection of a fully characterized reference sample of PE2I and radiochemical purity was determined using a sodium iodide detector (Berthold, Harpenden, UK). Total analysis time per injection was 6.0 min. The end product was also tested for endotoxins, residual solvents and filter integrity.

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