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\begin{document}

\title{Pharmacokinetics and brain sigma 1 (\selectlanguage{greek}σ\selectlanguage{english}1) receptor occupancy of MR309, a
selective \selectlanguage{greek}σ\selectlanguage{english}1 receptor antagonist\selectlanguage{english}}



\author[1]{Eugenii  Rabiner}%
\author[2]{Kevin Smith}%
\author[2]{Carla Bennet}%
\author[1]{Gaia Rizzo}%
\author[1]{Yvonne Lewis}%
\author[2]{Gill Mundin}%
\author[2]{Helen Dooner}%
\author[2]{Alexander  Oksche  }%
\affil[1]{Invicro}%
\affil[2]{Mundipharma Research}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and purpose: Preclinical studies of MR309, a selective sigma1
receptor (\selectlanguage{greek}σ\selectlanguage{english}1R) antagonist, support a potential role in treating
neuropathic pain. We report two studies that provide insight into the
pharmacokinetics (PK) and brain \selectlanguage{greek}σ\selectlanguage{english}1R binding of MR309. Experimental
approach: Steady-state PK of MR309 (400 mg QD and 200 mg BID for 10
days; EudraCT 2015-001818-99 {[}PK study{]}) and the relationship
between MR309 plasma exposure and brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy (EudraCT
2017-000670-11 {[}PET study{]}) were investigated in healthy volunteers.
Positron emission tomography (PET) using the \selectlanguage{greek}σ\selectlanguage{english}1R ligand {[}11C{]}SA4503
was conducted at baseline, 2h and 8h after a single dose of MR309
(200--800 mg). The relationship between brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy and MR309
exposure was explored using data-driven model fitting. Key results:
MR309 was well tolerated, brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy ranged between 30.5\% and
74.9\% following single-dose MR309 (n=7). MR309 BID provided a plasma PK
profile with less fluctuation than QD dosing (n=16). MR309 200 mg BID
yielded average steady state plasma concentrations between 2000 and 4000
ng/mL in the PK study, which corresponded to an estimated brain \selectlanguage{greek}σ\selectlanguage{english}1R
occupancy of 59--74\%. Conclusions and implications: MR309 200 mg BID
dose was below the 75\% \selectlanguage{greek}σ\selectlanguage{english}1R occupancy threshold expected to elicit
maximal antinociceptive effect as observed in neuropathic pain models.
Further investigations of MR309 for neuropathic pain will require higher
brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy, and establish the optimal dose by elucidating the
clinical impact of a broad range of brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy across different
neuropathic pain indications.%
\end{abstract}%



\sloppy


\selectlanguage{greek}\textbf{Pharmacokinetics and brain sigma 1 (σ1) receptor occupancy of
MR309, a selective σ1 receptor antagonist}\selectlanguage{english}

Eugenii A. Rabiner,\textsuperscript{1a} Kevin
Smith,\textsuperscript{2a,b} Carla Bennett,\textsuperscript{2} Gaia
Rizzo\textsuperscript{1}, Yvonne Lewis\textsuperscript{1}

Gill Mundin,\textsuperscript{2b} Helen
Dooner,\textsuperscript{2b}Alexander Oksche\textsuperscript{2b,3,4}

1. Invicro(former Imanova Ltd), A Konica Minolta Company, London, UK

2. Mundipharma Research Limited, Cambridge, UK

3. Institut f\selectlanguage{ngerman}ür medizinische und pharmazeutische Prüfungsfragen, Mainz,
Germany

4. Rudolf-Buchheim Institute of Pharmacology, Giessen, Germany

\textsuperscript{a}ER and KS are co-lead authors

\textsuperscript{b}Affiliation reflects the period when the study was
conducted (AO, HD, GM and KS are no longer employees of Mundipharma
Research Limited).

\textbf{Corresponding author}

Carla Bennett Mundipharma Research Limited Cambridge Science Park Milton
Road Cambridge CB4 0AB UK

Email: Carla.Bennett@mundipharma-rd.eu

Telephone +44 (0)1223 397946

\textbf{Word Count:} 3,542 (excludes 1515 words in methods)

\selectlanguage{greek}\textbf{Running title: Pharmacokinetics and brain σ1 receptor occupancy
of MR309}\selectlanguage{english}

\textbf{Key words:} MR309, sigma receptor, brain, neuropathy,
pharmacokinetic, positron emission tomography

\textbf{Data Availability Statement}

The data that support the findings of this study are available from
Mundipharma Research Limited Restrictions apply to the availability of
these data, which were used under license for this study. Data are
available from the authors at www.mundipharma-rd.eu with the permission
of Mundipharma Research Limited.

\textbf{Author contributions}

All authors critically reviewed each draft of the manuscript for
important intellectual content, approved the final submitted version,
and agree to be accountable for all aspects of the work. In addition,

ER contributed to study design and data interpretation

AO contributed to study design and data interpretation

CB contributed to study design and data interpretation

GR analysed the PET data and contributed to data interpretation

YL managed the study and contributed to data interpretation

HD contributed to study design and data interpretation

GM contributed to study design and data interpretation

KS contributed to study design and data interpretation

\textbf{Acknowledgements and funding}

MR309 was developed by Esteve (Barcelona, Spain). Mundipharma Research
Ltd. funded this research.

Medical writing support was provided by Si\selectlanguage{ngerman}ân Marshall, of SIANTIFIX
Ltd., Cambridge, UK, funded by Mundipharma Research Limited.

\textbf{Conflict of interest}

ER is an employee of Invicro (former Imanova Ltd.), there are no other
conflict of interests

AO was an employee of Mundipharma Research Limited at the time the study
was conducted, there are no other conflict of interests

CB is an employee of Mundipharma Research Limited, there are no other
conflict of interests

GR is an employee of Invicro (former Imanova Ltd.), there are no other
conflict of interests

YL is an employee of Invicro (former Imanova Ltd.), there are no other
conflict of interests

HD was an employee of Mundipharma Research Limited at the time the study
was conducted, there are no other conflict of interests

GM was an employee of Mundipharma Research Limited at the time the study
was conducted, there are no other conflict of interests

KS was an employee of Mundipharma Research Limited at the time the study
was conducted, there are no other conflict of interests



\textbf{Abstract}

\emph{Background and purpose} : Preclinical studies of MR309, a
selective sigma1 receptor (\selectlanguage{greek}σ\selectlanguage{english}1R) antagonist, support a potential role in
treating neuropathic pain. We report two studies that provide insight
into the pharmacokinetics (PK) and brain \selectlanguage{greek}σ\selectlanguage{english}1R binding of MR309.

\emph{Experimental approach} : Steady-state PK of MR309 (400 mg QD and
200 mg BID for 10 days; EudraCT 2015-001818-99 {[}PK study{]}) and the
relationship between MR309 plasma exposure and brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy
(EudraCT 2017-000670-11 {[}PET study{]}) were investigated in healthy
volunteers. Positron emission tomography (PET) using the \selectlanguage{greek}σ\selectlanguage{english}1R ligand
{[}\textsuperscript{11}C{]}SA4503 was conducted at baseline, 2h and 8h
after a single dose of MR309 (200--800 mg). The relationship between
brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy and MR309 exposure was explored using data-driven
model fitting.

\emph{Key results:} MR309 was well tolerated, brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy ranged
between 30.5\% and 74.9\% following single-dose MR309 (n=7). MR309 BID
provided a plasma PK profile with less fluctuation than QD dosing
(n=16). MR309 200 mg BID yielded average steady state plasma
concentrations between 2000 and 4000 ng/mL in the PK study, which
corresponded to an estimated brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy of 59--74\%.

\emph{Conclusions and implications:} MR309 200 mg BID dose was below the
75\% \selectlanguage{greek}σ\selectlanguage{english}1R occupancy threshold expected to elicit maximal antinociceptive
effect as observed in neuropathic pain models. Further investigations of
MR309 for neuropathic pain will require higher brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy, and
establish the optimal dose by elucidating the clinical impact of a broad
range of brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy across different neuropathic pain
indications.

\textbf{Bullet point summary}

\emph{What is already known?}

MR309, a selective \selectlanguage{greek}σ\selectlanguage{english}1R antagonist, has a potential role in the treatment
of neuropathic pain

\emph{What this study adds}

Twice-daily (BID) dosing of MR309 provided a plasma PK profile with less
fluctuation than once-daily dosing

A monotonic relationship between MR309 plasma concentration and brain
\selectlanguage{greek}σ\selectlanguage{english}1R occupancy was seen, but 200 mg BID did not achieve 75\% \selectlanguage{greek}σ\selectlanguage{english}1R
occupancy required for maximal antinociception in animal models

\emph{What is the clinical significance?}

The relationship between \selectlanguage{greek}σ\selectlanguage{english}1R occupancy across a range of levels and the
clinical efficacy in neuropathic pain remains to be elucidated

Establishing MR309 optimal dose requires further investigation across
various neuropathic pain indications



\textbf{Introduction}

Neuropathic pain is caused by a lesion or disease of the somatosensory
system that results in altered and disordered transmission of sensory
signals into the spinal cord and brain. There are numerous causes of
neuropathic pain including spinal cord injury or other trauma to the
nervous system, peripheral diabetic neuropathy, HIV and herpes virus
infections, autoimmune disorders and chemotherapy-induced peripheral
neuropathy (CIPN). (Colloca et al., 2017) CIPN has a complex and
multifactorial aetiology and can present as either acute or chronic
neuropathy. For example, acute neuropathy associated with oxaliplatin is
characterised by painful cold hyperalgesia or laryngeal dysaesthesia and
cramps, while chronic oxaliplatin-induced neuropathy is characterised by
numbness in hands and feet which can be functionally disabling. (Saif \&
Reardon, 2005; Zajaczkowska, Kocot-Kepska, Leppert, Wrzosek, Mika \&
Wordliczek, 2019) The prevalence of chronic oxaliplatin-induced
polyneuropathy is approximately 26--46\% at 12 month follow-up.
(Zajaczkowska, Kocot-Kepska, Leppert, Wrzosek, Mika \& Wordliczek, 2019)
Depending on the duration and severity of neurosensory toxicity, the
chemotherapy dose may need to be reduced, delayed or the treatment
terminated. (Accord Healthcare Limited, October 2019) Current treatments
for acute, painful neuropathy provide modest benefit at best and
treatment of established chronic neuropathy is largely ineffective.
Thus, effective new treatment approaches for CIPN are needed. (Hou, Huh,
Kim, Kim \& Abdi, 2018)

The sigma1 receptor (\selectlanguage{greek}σ\selectlanguage{english}1R) is a ligand-operated chaperone at the
endoplasmic reticulum which regulates multiple processes, including
modulation of intracellular signalling cascades related to noxious
stimuli, and sensitization and trafficking of proteins involved in
nociception. (Ortiz-Renteria et al., 2018; Pabba et al., 2014; Vela,
Merlos \& Almansa, 2015) The \selectlanguage{greek}σ\selectlanguage{english}1R is expressed at high levels in areas of
the central and peripheral nervous system involved in pain transmission
and transduction and neuropathic pain-like behaviours are attenuated in
\selectlanguage{greek}σ\selectlanguage{english}1R -knockout mice. (Bravo-Caparros et al., 2019; Davis, 2015;
Sanchez-Fernandez, Entrena, Baeyens \& Cobos, 2017) MR309 (previously
E-52862) is a selective \selectlanguage{greek}σ\selectlanguage{english}1R antagonist with a high affinity for human
\selectlanguage{greek}σ\selectlanguage{english}1R (Ki=17 nM). (Romero et al., 2012) Data from animal studies indicate
MR309 crosses the blood--brain barrier and binds to \selectlanguage{greek}σ\selectlanguage{english}1R in the central
nervous system (CNS) and support a potential role in the treatment of
neuropathic pain. (Romero et al., 2012) For example, MR309 inhibited
formalin-induced nociception, capsaicin-induced mechanical
hypersensitivity and sciatic nerve injury-induced mechanical and thermal
hypersensitivity in a dose-dependent manner. (Bravo-Caparros et al.,
2019; Romero et al., 2012) Knockout animal studies have also implicated
\selectlanguage{greek}σ\selectlanguage{english}1R activation in sensory nerve mitochondrial damage and the development
of neuropathic pain induced by paclitaxel treatment, indicating a
potential role for \selectlanguage{greek}σ\selectlanguage{english}1R antagonists for preventing CIPN. (Nieto et al.,
2014) Indeed, in a model of oxaliplatin-induced neuropathy, MR309
prevented hypersensitivity to cold stimuli, and twice daily (BID)
administration for 1 week increased the antinociceptive effect. (Gris et
al., 2016) Based on preclinical pharmacological studies and animal
neuropathic pain models, \selectlanguage{greek}σ\selectlanguage{english}1R occupancy \textgreater{}75\% is needed to
obtain maximal antinociceptive effect. (Romero et al., 2012)

In early clinical trials MR309 was well tolerated by healthy volunteers,
with no serious adverse events (AEs) reported with single doses up to
800 mg, or 400 mg once daily (QD) for 8 days, and some mild-to-moderate
transient CNS effects observed with the highest dose. (Abadias,
Escriche, Vaque, Sust \& Encina, 2013) MR309 400 mg QD was also well
tolerated by patients with colorectal cancer receiving oxaliplatin
within a FOLFOX regimen, with preliminary signs of efficacy for
oxaliplatin-induced peripheral neuropathy and hyperexcitability motor
symptoms. (Bruna et al., 2018) A four-week study with MR309 400 mg QD in
patients with chronic post-surgical neuropathic pain (PSNP) for
\textgreater{} 6 months reduced the primary end point (average pain
intensity between baseline and last week of dosing) significantly better
than placebo (EU Clinical Trials Register (EudraCT 2012-000402-30)).
Pharmacokinetic (PK) data in healthy volunteers indicate that MR309 is
absorbed rapidly, and systemic exposure (AUC\(\tau\))
increased proportionally with ascending dose up to 100 mg QD. At higher
doses extent of exposure increased in a greater than dose proportional
manner. (Abadias, Escriche, Vaque, Sust \& Encina, 2013) Based on Cmax,
the estimated therapeutic dose range of MR309 was 100--400 mg. (Abadias,
Escriche, Vaque, Sust \& Encina, 2013) Although not investigated to
date, BID dosing might be expected to result in less fluctuation of
MR309 plasma concentrations compared with QD administration, thereby
potentially improving tolerability and efficacy.

While clinical data are needed to provide insight into the efficacy of
MR309, (Bruna et al., 2018) pharmacodynamic (PD) markers to guide dose
selection for agents targeting the \selectlanguage{greek}σ\selectlanguage{english}1R pathway are currently lacking.
{[}\textsuperscript{11}C{]}SA-4503 is a selective \selectlanguage{greek}σ\selectlanguage{english}1R positron emission
tomography (PET) radioligand suitable for quantifying \selectlanguage{greek}σ\selectlanguage{english}1R density in the
human brain, and has been used to estimate brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy in
healthy volunteers administered haloperidol and fluvoxamine. (Ishikawa
et al., 2007; Ishiwata et al., 2006; Mansur et al., 2019) Assessment of
the relationship between \selectlanguage{greek}σ\selectlanguage{english}1R occupancy and the plasma concentration of
MR309 will confirm the drug target interaction in the human brain, and
help establish a suitable dose range for future clinical studies.

Here, we report two exploratory studies which assessed the relationship
between MR309 given BID and QD and plasma concentration at steady-state
(PK study), and the relationship between plasma exposure of MR309 and
occupancy of \selectlanguage{greek}σ\selectlanguage{english}1R in the brain, using PET with the selective \selectlanguage{greek}σ\selectlanguage{english}1R
radioligand, {[}\textsuperscript{11}C{]}SA-4503 (PET study).

\textbf{Methods}

\emph{PK study design}

In this single centre, open-label cross-over study (EudraCT
2015-001818-99) volunteers received oral MR309 at doses of 400 mg QD for
10 days and 200 mg BID (12 h intervals) for 10 days, with a washout
period of [?]10 days between the two dosing schedules. Allocation to
dosing regimen sequence (QD--BID or BID--QD) was randomised using an
automated random numbers scheme. MR309 was taken following an overnight
fast for the morning doses on Days 1 and 10; at other time points
participants fasted for 2 hr before and after dosing. Individuals
included in this study were 18--45 years old with a body weight and body
mass index (BMI) of 55--100 kg and 18.5--30 kg/m\textsuperscript{2},
respectively. All participants were considered healthy based on their
medical history (no significant illness during the 4 weeks prior to
study entry), physical examination, laboratory parameters and
electrocardiogram (ECG) tests. All participants agreed to use an
effective method of contraception throughout the study duration, and
pregnant and breastfeeding women were excluded. Other key exclusion
criteria included a history of drug or alcohol abuse, use of opioid or
opioid antagonist medication [?]30 days prior to study start,
participation in a clinical trial [?]90 days prior to study start, and a
history of frequent nausea or vomiting of any aetiology.

\emph{PK study assessments}

Blood samples for PK assessment were collected on Days 1, 2, 7, 8 and 9
(once or twice daily). On Day 10 (steady state) blood samples were
collected pre-dose and at 20 min, 40 min, 1 h, 1 h 20 min, 1 h 40 min, 2
h, 2 h 20 min, 2 h 40 min, 3 h, 4 h, 6 h, 8 h and 12 h following QD and
BID dosing and also at 16 h post dosing for the QD schedule. Plasma
concentrations of MR309 were measured using a validated liquid
chromatography--tandem mass spectrometry method.

Safety was assessed using 12-lead ECGs (monitored predose and on each
study day with multiple assessments on Days 1 and 10), vital signs
(monitored predose and at regular intervals during each study period),
and AEs which were recorded throughout the study (coded per Medical
Dictionary for Regulatory Activities {[}MedDRA{]} v18.1).

The primary objective was to compare the steady state PK of MR309 given
QD and BID. The primary endpoints were area under the plasma
concentration time curve from the time of dosing over one dosing
interval (24 h QD or 12 h BID) at steady state (AUCtau), maximum plasma
concentration at steady state (Cmaxss) and minimum plasma concentration
at steady state (Cminss) for MR309. Secondary PK endpoints for MR309
included time to Cmaxss (tmaxss), fluctuation, and relative
bioavailability (ratio of dose-corrected AUCtau values, and expressed as
a percentage), Cmaxss ratio and Cminss ratio (calculated from derived
parameters).

\emph{PET study design}

In this single centre, open-label study (EudraCT 2017-000670-11), male
participants (25--60 years old, body weight 55--100 kg, BMI 18.5--30
kg/m\textsuperscript{2}) received a single oral dose of MR309 across 4
dosing cohorts (200--800 mg). All participants were considered healthy
and subject to similar inclusion and exclusion criteria as described for
the PK study, with the additional exclusion of any contraindications to
radial artery cannulation and magnetic resonance imaging (MRI).

Prior to study treatment, participants underwent a single brain MRI scan
to confirm suitability to take part in the study and obtain anatomical
information to support PET scan analysis.

Each subject underwent three PET scans with
{[}\textsuperscript{11}C{]}SA-4503 at baseline, 2h and 8h after a single
oral dose of MR309. Participants fasted for [?]8 hours prior to receiving
MR309. Arterial blood samples were collected from a radial artery
cannula throughout each PET scan to estimate the time-course of total
blood and plasma radioactivity and the concentration of unmetabolized
{[}\textsuperscript{11}C{]}SA-4503.

\emph{PET data quantification}

The primary objectives were to evaluate brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy following a
single oral dose of MR309 and to describe the relationship between \selectlanguage{greek}σ\selectlanguage{english}1R
occupancy and MR309 plasma concentration at the time of the PET scan.
The primary quantification parameter was the total volume of
distribution (V\textsubscript{T}) for {[}\textsuperscript{11}C{]}SA-4503
at each PET scan. V\textsubscript{T} represents the partition
coefficient for SA-4503 between brain and plasma at equilibrium, and is
proportional to the total binding of SA-4503 (displaceable and
non-displaceable) in the relevant brain region (Innis et al., 2007). \selectlanguage{greek}σ\selectlanguage{english}1R
occupancy was calculated by comparing V\textsubscript{T} at baseline and
post-dose {[}\textsuperscript{11}C{]}SA-4503 for each subject, and \selectlanguage{greek}σ\selectlanguage{english}1R
occupancy for each scan was related to the plasma concentration of
MR309.

Brain MRI images underwent brain extraction, grey matter segmentation
and co-registration to a standard reference space (MNI152 template brain
image and associated CIC atlas), (Grabner, Janke, Budge, Smith,
Pruessner \& Collins, 2006; Tziortzi et al., 2011) and were nonlinearly
warped to each participant's MRI image to enable automated definition of
regions of interest (ROIs). ROIs included the frontal, temporal and
occipital lobes, caudate nucleus, putamen, thalamus, hippocampus and
cerebellum. Representative orthogonal cross sections of co-registered
PET and MRI images are shown in Figure 1.

Dynamic PET images were corrected for motion using a frame-to-frame
registration process with a normalized mutual information cost function,
and co-registered to each participant's MRI scan. ROIs defined on the
MRI images were applied to the dynamic PET data to derive regional
time-activity curves (TACs), with activity concentrations expressed as
standardised uptake values (SUV), calculated by normalising the measured
radioactivity concentration to the injected radioactivity and the
participant's bodyweight.

Regional TACs were analysed using the alternative multilinear analysis 1
(MA1) kinetic model with a metabolite-corrected arterial plasma input
function, to generate regional V\textsubscript{T} values. \selectlanguage{greek}σ\selectlanguage{english}1R occupancy
for each post-dose PET scan was calculated from the regional
V\textsubscript{T} estimates using an occupancy plot analysis
(Cunningham, Rabiner, Slifstein, Laruelle \& Gunn, 2010). The occupancy
plot method makes the assumption that the volume of distribution of the
non-displaceable component (V\textsubscript{ND}) is uniform across an
individual's brain, and that the fractional occupancy of the target in
each post-dose scan is uniform across all ROI. V\textsubscript{ND} was
constrained to be the same for each post-dose PET scan in each
individual.

The relationship between \selectlanguage{greek}σ\selectlanguage{english}1R occupancy and drug exposure was then
explored using data-driven model fitting. To characterize the
relationship between MR309 plasma concentration and \selectlanguage{greek}σ\selectlanguage{english}1R occupancy, the
\selectlanguage{greek}σ\selectlanguage{english}1R occupancy estimates were plotted against MR309 plasma concentration
measured at the start of each post-dose PET scan. Four models were
fitted and compared: the optimal model to describe the relationship
between MR309 plasma concentration and \selectlanguage{greek}σ\selectlanguage{english}1R occupancy was selected based
on goodness of fit and AIC values (Table 1). For each model,
EC\textsubscript{50} , E\textsubscript{max}, Hill slope and Akaike
information criterion (AIC) values (measure of goodness of fit) were
obtained. (Bozdogan, 1987)

PK of single-dose MR309 and its metabolites were assessed as a secondary
objective. Plasma concentrations of MR309 and its metabolites were
assessed as described in the PK study with the endpoints of Cmax, Tmax,
AUCt (area under the plasma concentration time curve from the time of
dosing to the last measurable concentration), AUCINF (area under the
plasma concentration time curve from the time of dosing to infinity),
LambdaZ (estimate of terminal elimination rate constant) and t1/2Z
(apparent terminal phase half-life). Safety and tolerability were
assessed pre-dose and at regular intervals up to Day 4 post-dose, as
described in the PK study.

\emph{Data and statistical analysis (PK study and PET study)}

The data and statistical analysis comply with the recommendations of the
British Journal of Pharmacology on experimental design and analysis.
(Curtis et al., 2018)

In both studies, PK parameters were calculated using Phoenix WinNonlin
version 6.2 or later (Certara L.P., Princeton, NJ, US) and statistical
analyses were performed using SAS\selectlanguage{ngerman}® version 9.3 or later (SAS Institute,
Cary, NC, USA). The PK population comprised all subjects who received \selectlanguage{english}[?]1
dose of MR309 and provided data for at least 1 PK measurement while the
safety population included all subjects who received at \selectlanguage{english}[?]1 dose of
MR309.

Formal sample size calculations were not performed for either of these
exploratory studies. Recruitment of 16 individuals was planned for the
PK study, anticipating PK data would then be available from \selectlanguage{english}[?]12 subjects
completing the study. A sample size of up to 8 participants (1 or 2
individuals per cohort) was planned for the PET study.

Safety parameters were described using summary statistics. Differences
in PK parameters between QD and BID dosing schedules were assessed using
an ANOVA model which included fixed terms for treatment regimen (QD or
BID), sequence, period, and subject within the treatment sequence. PK
parameters were not summarized by cohort or dose level in the PET study
due to the small sample size. PET data analysis was performed by Imanova
Ltd. (London, UK) using in-house MIAKAT\textsuperscript{TM} software
(version 4.2.6.1), implemented using MATLAB (The Mathworks Inc. Natick,
MA, US).

Both studies were conducted in accordance with the Declaration of
Helsinki, International Conference on Harmonization Guidelines for Good
Clinical Practice and the European Union Clinical Trials Directive.
Procedures were approved by local ethics committees, and all subjects
provided informed, written consent.

\emph{Materials}

MR309 was provided by Esteve (Barcelona, Spain). 4-O-demethyl SA4503 for
the manufacture of {[}\textsuperscript{11}C{]}SA-4503 was purchased from
Onyx Scientific Limited,

\textbf{Results}

\emph{Participant characteristics}

In the PK study 8 participants were randomized to both QD--BID and
BID--QD dosing. In the QD--BID cohort mean (SD) age was higher than the
BID--QD cohort (35.6 {[}7.8{]} years vs 28.0 {[}10.1{]} years) and there
were fewer female participants (25\% vs 50\%), while mean (SD) BMI was
comparable across the two groups (25.0 {[}2.5{]} kg/m\textsuperscript{2}
vs 24.7 {[}3.2{]} kg/m\textsuperscript{2}). All participants completed
the assigned study treatment.

Of 28 individuals enrolled in the PET study, 7 received a single dose of
MR309 and completed the study (Cohort 1: 400 mg n=2, Cohort 2: 200 mg
n=2, Cohort 3: 800 mg n=2, Cohort 4: 200 mg n=1; selection of doses for
Cohorts 2, 3 and 4 were based binding data associated with the prior
dose). One additional subject underwent a baseline PET scan only, and
their participation in the study was terminated prior to treatment with
MR309 due to a positive drug test. All the participants who received
MR309 were male, median (range) age was 49 (26--59) years and median
(range) BMI was 24.4 (22.7, 29.9) kg/m\textsuperscript{2}.

\emph{Pharmacokinetic outcomes: PK study}

MR309 200 mg BID dosing was associated with lower geometric mean (CV,
\%) Cmaxss (4143 {[}17.2{]} ng/mL vs 5222 {[}18.3{]} ng/mL) and higher
Cminss (1915 {[}29.8{]} ng/mL vs 1764 {[}29.9{]} ng/mL) compared with
400 mg QD dosing (Table 2; Figure 2). Bioavailability comparisons
yielded geometric mean ratios (BID/QD dosing) of 79.3\% (90\% CI 73.5,
85.7\%) and 108.5 (90\% CI 104.6, 112.6\%) for Cmaxss and Cminss,
respectively. Correspondingly, less fluctuation was observed with BID
dosing (mean 87\% {[}SD 22\%{]}) compared with the QD regimen (145\%
{[}49\%{]}; Table 2). The ratio of dose-adjusted AUCtau (BID/QD dosing)
indicated broadly equivalent bioavailability across the two dosing
schedules (107\% {[}90\% CI 103, 111.5{]}).

\selectlanguage{greek}\emph{σ1 Receptor occupancy: PET study}\selectlanguage{english}

Kinetic modelling using a two-tissue compartment model for
{[}\textsuperscript{11}C{]}SA-4503 performed poorly in some ROIs
(including the caudate, the region with the lowest uptake). (Sakata et
al., 2007) The alternative MA1 model produced an acceptable fit for all
198 TACs (corresponding to 9 ROIs in 22 PET scans obtained from the 7
participants who received MR309 and one additional subject with baseline
PET scan only) in accordance with prior studies. (Ichise, Toyama, Innis
\& Carson, 2002; Mansur et al., 2019) Representative TACs and MA1 model
fits are shown in Figure 3.

In all ROIs a dose-dependent reduction in
{[}\textsuperscript{11}C{]}SA-4503 V\textsubscript{T} was observed
following MR309 administration. The occupancy plot method produced
broadly consistent estimates of {[}\textsuperscript{11}C{]}SA-4503
V\textsubscript{ND} across subjects (range of 11.0 to 23.2
mL/cm\textsuperscript{3}). Brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy ranged between 30.5\% and
74.9\% across the participants who received a single oral dose of
200--800 mg MR309 (Table 3).

Single-dose MR309 PK data obtained in the PET study are summarized in
the supplementary appendix. Of the four PK-- \selectlanguage{greek}σ\selectlanguage{english}1R occupancy models, Model
1 best described the relationship between MR309 plasma concentration and
corresponding \selectlanguage{greek}σ\selectlanguage{english}1R occupancy and was selected to describe
{[}\textsuperscript{11}C{]}SA-4503 PK/brain kinetics (Tables 1, 4).
Using Model 1, the estimated EC\textsubscript{50} was 1380 ng/mL (95\%
CI: 778 to 1981 ng/mL), with E\textsubscript{max} fixed to 100\% and
Hill slope fixed to 1 (Figure 4).

In the PK study, MR309 200 mg BID, the proposed dose for further
evaluation, yielded steady state plasma concentrations within an
approximate range of 2000--4000 ng/mL (Table 1). Using the plasma
concentration--\selectlanguage{greek}σ-\selectlanguage{english}1R occupancy model, this was calculated to correspond
to an estimated brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy ranging between 59--74\% (Figure 4).

\emph{Safety outcomes}

In both studies all AEs were mild with the exception of upper limb
fracture (n=1 in PK study; considered unrelated to study medicine). No
serious AEs were reported, ECG findings revealed no QT prolongations or
conduction abnormalities, and there were no clinically significant
changes in haematology or blood chemistry parameters. Isolated,
out-of-range blood pressure values were noted in both studies but were
considered not clinically significant.

In the PK study, overall exposure to MR309 was the same for patients
receiving 400 QD and 200 BID dosing (10 days, each). Treatment-emergent
AEs (n=9, 56.3\% vs n=6, 37.5\%) and treatment-related AEs (n=7, 43.8 vs
n=3, 18.8\%) were more frequent with MR309 400 mg QD versus 200 mg BID.
The only AEs reported in [?]10\% of individuals were dizziness (400 mg QD:
n=3, 18.8\%; 200 mg BID: n=1, 6.3\%) and symptomatic orthostatic
tachycardia (400 mg QD: n=2, 12.5\%; 200 mg BID: n=1, 6.3\%). All cases
of symptomatic orthostatic tachycardia were transient and, while
considered related to study medication, a direct causal relation was
unclear as in two of three individuals the event was reported pre-dose
when plasma levels of MR309 were unquantifiable. One case of
asymptomatic orthostatic tachycardia was also observed during screening
(pre-dose, Day 1) and considered unrelated to study medication.

In the PET study, 4 participants (57.1\%) reported 6 AEs: dizziness (3
instances in n=2; all considered related to study medication), contusion
and dysgeusia (2 instances in n=2 and 1 instance in n=1, respectively;
all AEs were considered unrelated to study medication).

\textbf{Discussion and Conclusion}

A clear understanding of drug exposure at the site of action, target
binding and functional pharmacological activity is key for optimal
design of phase II clinical trials. (Morgan et al., 2012) MR309, a
selective \selectlanguage{greek}σ\selectlanguage{english}1R antagonist, was well tolerated in early clinical trials at
a daily dose of 400 mg and signals of preliminary efficacy for acute
CIPN were observed in patients with colorectal cancer receiving an
oxaliplatin-containing regimen (Abadias, Escriche, Vaque, Sust \&
Encina, 2013; Bruna et al., 2018) and in patients with chronic PSNP
lasting for \textgreater{}6 months. (EU Clinical Trials Register
(EudraCT 2012-000402-30)) Here, we confirm the occupancy of brain \selectlanguage{greek}σ\selectlanguage{english}1R by
MR309 following administration of clinically meaningful doses, and
demonstrate a quantitative relationship between plasma concentration of
MR309 and target occupancy, enabling optimised dose selection in future
studies.

The PK study demonstrated that BID dosing of MR309 offers the
opportunity to lower fluctuation of drug plasma levels compared with QD
dosing. At steady state lower peak and higher trough concentrations of
MR309 were observed with BID compared with QD dosing: geometric mean
{[}CV, \%{]} Cmaxss (4143 {[}17.2{]} ng/mL vs 5222 {[}18.3{]} ng/mL);
Cminss (1915 {[}29.8{]} ng/mL vs 1764 {[}29.9{]} ng/mL). Consequently,
it is feasible that BID dosing of MR309 may be better tolerated than QD
dosing, with the potential to reduce the incidence and/or severity of
AEs associated with high peak plasma concentrations; this may have
particular relevance as MR309 is a CNS active compound. Indeed, in this
study treatment-emergent AEs (56.3\% vs 37.5\%) and treatment-related
AEs (43.8 vs 18.8\%) were more frequent with MR309 400~mg QD compared
with 200 mg BID. Based on these PK findings, BID dosing of MR309 was
recommended for evaluation in future studies.

In the second study, the relationship between plasma concentrations of
MR309 and \selectlanguage{greek}σ\selectlanguage{english}1R occupancy in the human brain was estimated successfully.
Modelling the relationship between MR309 plasma concentration and \selectlanguage{greek}σ\selectlanguage{english}1R
occupancy indicated that for MR309 200 mg BID dose, which produces
steady state plasma concentrations of approximately 2000--4000 ng/mL,
brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy will be in the range of 59--74\%. The data suggest
that meaningful levels of \selectlanguage{greek}σ\selectlanguage{english}1R occupancy can be achieved with steady
state dosing of 400 mg MR309 daily dose. Further, the signs of clinical
efficacy in the CIPN and in the PSNP studies seen with 400 mg daily
dosing could be interpreted as further support for the mechanism of
action. It is apparent that higher \selectlanguage{greek}σ\selectlanguage{english}1R occupancy rates will need to be
investigated to explore the full potential of \selectlanguage{greek}σ\selectlanguage{english}1R in neuropathic pain.
Notably, nonclinical data suggest that a higher degree of brain \selectlanguage{greek}σ\selectlanguage{english}1R
occupancy may be required to elicit a maximal effect. When MR309 was
administered to animal models of neuropathic pain, \emph{ex vivo}binding
in brain section autoradiograms using\textsuperscript{3}H-pentazocine
and analysis of dose--response curves indicated maximal antinociception
was achieved at brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy levels \textgreater{}75\%. (Romero
et al., 2012)

In addition to achieving higher brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy, it is also
important to elucidate the clinical impact of 59--74\% brain \selectlanguage{greek}σ\selectlanguage{english}1R
occupancy by a selective \selectlanguage{greek}σ\selectlanguage{english}1R antagonist. Plasma steady-state was
previously reported after 8 days of MR309 QD (based on no significant
difference in Cmin values on days 6, 7 and 8; p\textless{}0.05)
(Abadias, Escriche, Vaque, Sust \& Encina, 2013). Consequently, the
5-day dosing regimen for MR309 utilized in the Phase II study of CIPN
study is anticipated to result in \selectlanguage{greek}σ\selectlanguage{english}1R occupancy below 59--74\%. Further
studies are warranted to fully explore the potential of MR309 to
mitigate signs of oxaliplatin-induced acute sensory and motor
neurotoxicity, and reduce the frequency and severity of chronic sensory
neuropathy. These would require higher dosing of MR309 or a pre-dosing
regimen in order to achieve steady state levels resulting in \selectlanguage{greek}σ\selectlanguage{english}1R
occupancy \textgreater{}75\%.

Investigation of \selectlanguage{greek}σ\selectlanguage{english}1R occupancy by MR309 in peripheral nerves is also
warranted, as it is feasible that receptor occupancies may differ
between the peripheral and central nervous systems, given that
concentrations of MR309 were approximately 2-fold greater in brain
compared with plasma in animal studies. (Romero et al., 2012) To our
knowledge, the only PET study analysing \selectlanguage{greek}σ\selectlanguage{english}1R in peripheral tissue was a
case report of a patient with chronic knee pain.
Using\textsuperscript{18}F-FTC-146 \selectlanguage{greek}σ\selectlanguage{english}1R radioligand, \selectlanguage{greek}σ\selectlanguage{english}1 receptor
upregulation was reported in a subject with an inflamed intra-articular
synovial lipoma. Removal of the lipoma resulted in cessation of pain.
(Cipriano et al., 2018)

It is noteworthy that MR309 was well tolerated by the healthy volunteers
participating in the present two studies. AEs were mild in severity,
with dizziness being the most frequently reported event. Orthostatic
pulse rate changes were also reported by some individuals in the PK
study, although were not correlated with plasma concentrations of MR309
and were not consistent with postural orthostatic tachycardia syndrome.
(Stewart, 2004) While dizziness was also a common side effect reported
by healthy volunteers in a prior Phase I study of MR309, it was reported
less frequently by patients with colorectal cancer in the Phase II study
of MR309 compared with the present study. (Abadias, Escriche, Vaque,
Sust \& Encina, 2013; Bruna et al., 2018) Variations in the reported
safety profile may relate in part to the different characteristics of
the study participants, including underlying disease and chemotherapy.

The reported studies are associated with some limitations. Both studies
were exploratory and the PK-- \selectlanguage{greek}σ\selectlanguage{english}1R model fitting did not account for
potential variation in the precision with which individual data points
were determined, or that the data set comprised multiple points for each
participant rather than independent samples. Consequently, while the
choice of model used in the present study had only a modest effect on
EC\textsubscript{50} estimates (ranged from 1268--1380 ng/mL), it is
feasible that more sophisticated model fitting may have produced a
different EC\textsubscript{50} estimate. Also, based on data that were
available at the time the PK study was designed, 9 days of dosing with
MR309 was considered sufficient to achieve steady-state PK. However, in
the present study small increases in trough concentrations of MR309 were
observed prior to Day 10, when full PK sampling was obtained (data not
shown). This was not anticipated, given the previously reported
half-life of MR309 was approximately 18 hours. (Abadias, Escriche,
Vaque, Sust \& Encina, 2013) As trough concentrations were not formally
compared to establish whether steady-state had been achieved in the
present study, further evaluation of the time to establish steady state
is recommended in future studies of MR309. With regard to the PET study,
an underestimation of receptor occupancy achieved with MR309 may occur
if SA4503 had limited selectivity for the \selectlanguage{greek}σ\selectlanguage{english}1R. SA4503 has been reported
to have \selectlanguage{greek}σ\selectlanguage{english}2R / \selectlanguage{greek}σ\selectlanguage{english}1R ratio of approximately 100-fold, with no significant
affinity to 36 other receptors, transporters or channels. (Kawamura et
al., 2000; Matsuno, Nakazawa, Okamoto, Kawashima \& Mita, 1996) However,
using masking protocols with selective ligands, the \selectlanguage{greek}σ\selectlanguage{english}2R / \selectlanguage{greek}σ\selectlanguage{english}1R ratio was
reported to be 14-fold in guinea pig brain membranes, although such
masking protocols were shown to carry their own challenges and these
data should be treated with caution. (Abbas, Borde, Willars, Ferry \&
Safrany, 2020; Lever, Gustafson, Xu, Allmon \& Lever, 2006). The
magnitude of the PET signal is proportional to the ratio of target
density and affinity (B\textsubscript{max}/K\textsubscript{d}). The \selectlanguage{greek}σ\selectlanguage{english}2R,
a transmembrane ER-resident regulator of the Nieman-Pick-Carrier
protein, (Alon, Schmidt, Wood, Sahn, Martin \& Kruse, 2017) contributed
to the PET signals obtained in this study. While sigma receptor
expression is qualitatively similar between various animal species and
man, there are notable species-dependent differences in expression
levels. For example, Bmax for \selectlanguage{greek}σ\selectlanguage{english}1R in cerebellum differs between s mouse
strains and is lower for Rhesus compared with guinea pig and mice.
(Brust, Deuther-Conrad, Lehmkuhl, Jia \& Wunsch, 2014) While human data
on brain \selectlanguage{greek}σ\selectlanguage{english}1R binding has not been published, \selectlanguage{greek}σ\selectlanguage{english}2R expression was found to
be much lower in human cortical membranes than in rat brain. (Brust,
Deuther-Conrad, Lehmkuhl, Jia \& Wunsch, 2014) Data so far suggest
comparable expression levels of \selectlanguage{greek}σ\selectlanguage{english}1R and \selectlanguage{greek}σ\selectlanguage{english}2R. Based on the assumption of
comparable \selectlanguage{greek}σ\selectlanguage{english}1R and \selectlanguage{greek}σ\selectlanguage{english}2R expression levels and using the lowest reported
\selectlanguage{greek}σ\selectlanguage{english}2R / \selectlanguage{greek}σ\selectlanguage{english}1R selectivity of 14, the contribution of non- \selectlanguage{greek}σ\selectlanguage{english}1R component to
the total {[}11C{]}SA4503 will be at most \textasciitilde{}7\%. Assuming
that MR309 has no affinity for \selectlanguage{greek}σ\selectlanguage{english}2R, and use this estimate to adjust for
the potential bias in the estimation of \selectlanguage{greek}σ\selectlanguage{english}1R occupancy, we find a minor
effect with the expected EC50 at \selectlanguage{greek}σ\selectlanguage{english}1R being 1225 ng/mL and a range of
62-77\% receptor occupancy expected in the plasma concentration range of
2000-4000 ng/mL.

In conclusion, our data indicate MR309 200 mg BID dosing facilitates
less fluctuation of plasma levels of this selective \selectlanguage{greek}σ\selectlanguage{english}1R antagonist
compared with 400 mg QD dosing. However, PK-- \selectlanguage{greek}σ\selectlanguage{english}1R occupancy modelling
indicated that at steady state a daily dose of MR309 200 mg BID would be
associated with a level of brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy \textless{}75\%, which
may be insufficient to elicit maximal efficacy in neuropathic pain.
Furthermore, the 5-day MR309 regimen utilized in the previous study of
CIPN would likely be associated with lower brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy compared
to that observed in the present study of healthy volunteers. (Bruna et
al., 2018) Consequently, further clinical studies of MR309 in
neuropathic pain indications are needed to inform efficacy and
tolerability across a broad range of brain \selectlanguage{greek}σ\selectlanguage{english}1R occupancy levels in order
to establish the optimal dose ranges in different patient populations.

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\par\null

Table 1: Pharmacokinetic--\selectlanguage{greek}σ-\selectlanguage{english}1 brain receptor occupancy models\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Model} & \textbf{Model description}\tabularnewline
\midrule
\endhead
1 & \(\selectlanguage{greek}\text{ΔBP}\selectlanguage{english}=100\ x\ \frac{C}{C+\text{EC}_{50}}\)\tabularnewline
2 & \(\selectlanguage{greek}\text{ΔBP}\selectlanguage{english}=E_{\text{max\ }}\text{x\ }\frac{C}{C+\text{EC}_{50}}\)\tabularnewline
3 & \(\selectlanguage{greek}\text{ΔBP}\selectlanguage{english}=100\ x\ \frac{C^{\delta}}{C^{\delta}+\text{EC}_{50\ }^{\delta}}\)\tabularnewline
4 & \(\selectlanguage{greek}\text{ΔBP}\selectlanguage{english}=E_{\text{max\ }}x\frac{C^{\delta}}{C^{\delta}+\text{EC}_{50\ }^{\delta}}\)\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{greek}Δ\selectlanguage{english}BP, estimated receptor signal change (\%); C, MR309 plasma
concentration at the start of the post-dose PET-CT scan,
E\textsubscript{max}, maximum attainable signal change;
EC\textsubscript{50} , MR309 plasma concentration corresponding to 50\%
of the maximal signal change; \selectlanguage{greek}δ, \selectlanguage{english}Hill slope. Model 1 was selected as the
optimal model to describe the relationship between MR309 plasma
concentration and \selectlanguage{greek}σ\selectlanguage{english}1R occupancy based on goodness of fit and Akaike
information criterion (AIC) values. While Model 3 (which involved
fitting an EC\textsubscript{50} and Hill slope) was favoured by AIC
analysis, the precision associated with the estimated parameters was
lower than that associated with the simpler Model 1. Of note, the choice
of model had a modest effect on the estimated EC\textsubscript{50}: 1380
ng/mL (Model 1) to 1268 ng/mL (Model 3).

Table 2 : Summary statistics for MR309 PK parameters (PK study)

\textbf{MR309 200 mg BID}

(N=16)

\textbf{MR309 400 mg QD}

(N=16)

\textbf{AUCtau (ng.h/mL)}

\textbf{AUCtau (ng.h/mL)}

\textbf{AUCtau (ng.h/mL)}

Geometric mean (log SD)

31279 (0.2)

58412 (0.2)

CV (\%)

23.9

20.9

Arithmetic mean (SD)

32113 (7744)

59572 (12097)

Median (min, max)

33378 (20631, 52275)

60822 (39532)

\textbf{Cmaxss (ng/mL)}

\textbf{Cmaxss (ng/mL)}

\textbf{Cmaxss (ng/mL)}

Geometric mean (log SD)

4143 (0.2)

5222 (0.2)

CV (\%)

17.2

18.3

Arithmetic mean (SD)

4200 (719)

5304 (969)

Median (min, max)

4395 (3010, 5940)

5300 (3920, 7140)

\textbf{Cminss (ng/mL)}

\textbf{Cminss (ng/mL)}

\textbf{Cminss (ng/mL)}

Geometric mean (log SD)

1915 (0.3)

1764 (0.3)

CV (\%)

29.8

29.9

Arithmetic mean (SD)

1991 (580)

1832 (493)

Median (min, max)

2055 (1070, 3510)

1870 (856, 2860)

\textbf{tmaxss (h)}

\textbf{tmaxss (h)}

\textbf{tmaxss (h)}

Arithmetic mean (SD)

1.28 (0.47)

1.40 (0.38)

Median (min, max)

1.00 (0.67, 2.33)

1.33 (0.63, 2.0)

\textbf{Fluctuation (\%)\textsuperscript{a}}

\textbf{Fluctuation (\%)\textsuperscript{a}}

\textbf{Fluctuation (\%)\textsuperscript{a}}

Arithmetic mean (SD)

86 (22)

145 (49)

AUCtau area under the plasma concentration time curve from the time of
dosing over one dosing interval (24 h QD or 12 h BID) at steady state;
Cmaxss, maximum plasma concentration at steady state; Cminss minimum
plasma concentration at steady state; CV, coefficient of variation
(calculated using log transformed data); SD, standard deviation; tmaxss,
time to Cmaxss

\textsuperscript{b}Fluctuation was calculated as 100*(Cmax minus
Cmin/Cavg), with Cavg derived from AUC from dose time to tau divided by
tau)

Table 3: \selectlanguage{greek}σ\selectlanguage{english}1 Receptor occupancy estimates and MR309 PK (PET study)\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Participant} & \textbf{MR309 dose (mg/day)} & \textbf{PET scan
time (hours post MR309 dose)} &
\textbf{{[}\textsuperscript{11}C{]}SA-4503 V\textsubscript{ND}
(mL/cm\textsuperscript{3})} & \selectlanguage{greek}\textbf{σ1 Receptor ΔBP (\%)}\selectlanguage{english} &
\textbf{MR309 at start of PET scan (ng/mL)}\tabularnewline
\midrule
\endhead
1 & 400 & 2 & 11.0 & 59.8 & 2280\tabularnewline
& & 8 & & 46.1 & 750\tabularnewline
2 & 400 & 2 & 23.2 & 73.8 & 1630\tabularnewline
& & 8 & & 52.0 & 946\tabularnewline
3 & 200 & 2 & 15.0 & 30.5 & 1640\tabularnewline
& & 8 & & 30.7 & 457\tabularnewline
4 & 200 & 2 & 11.8 & 48.3 & 2720\tabularnewline
& & 8 & & 46.7 & 1270\tabularnewline
5 & 800 & 2 & 16.8 & 74.9 & 3780\tabularnewline
& & 8 & & 36.6 & 1320\tabularnewline
6 & 800 & 2 & 20.0 & 64.9 & 4410\tabularnewline
& & 8 & & 32.7 & 1360\tabularnewline
7 & 200 & 2 & 22.9 & 55.1 & 999\tabularnewline
& & 8 & & 55.5 & 289\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{greek}σ\selectlanguage{english}1, sigma-1 receptor; \selectlanguage{greek}Δ\selectlanguage{english}BP, change in binding potential; PET, positron
emission tomography; V\textsubscript{ND}, volume of distribution of the
non-displaceable component

Table 4. Pharmacokinetic--\selectlanguage{greek}σ-\selectlanguage{english}1 brain receptor occupancy model fit
parameters\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Model} & \textbf{AIC} & \textbf{EC\textsubscript{50} (ng/mL)} &
\textbf{E\textsubscript{MAX} (\%)} & \textbf{Hill slope}\tabularnewline
\midrule
\endhead
1 & 80.3 & 1380 (778, 1981) & -- & --\tabularnewline
2 & 77.1 & 165 (-214, 544) & 58 (40, 76) & --\tabularnewline
3 & 75.5 & 1268 (21, 2516) & -- & 0.3 (-0.1, 0.8)\tabularnewline
4 & 77.6 & 1249 (-71327, 73824) & 100 (-859, 1059) & 0.3 (-2.9,
3.6)\tabularnewline
\bottomrule
\end{longtable}

Values in parentheses are 95\% confidence intervals

AIC, Akaike information criterion; E\textsubscript{max}, maximum
attainable signal change; EC\textsubscript{50} , MR309 plasma
concentration corresponding to 50\% of the maximal signal change.

\textbf{Figure Legends}

\textbf{Figure 1} . Representative orthogonal cross sections of
co-registered PET and MRI images

MRI, magnetic resonance imaging; PET, positron emission tomography; SUV,
standardised uptake value

\textbf{Figure 2} : MR309 mean plasma--concentration time curves (PK
study)

\textbf{Figure 3} . Representative time--activity curves and multilinear
analysis 1 method for selected regions of interest (PET study)

PET, positron emission tomography; ROI, region of interest; SUV,
standardised uptake value

A. PET baseline scan data; B. PET scan data, acquired \textasciitilde{}2
hours after MR309 400 mg; C. PET scan data, acquired \textasciitilde{}8
hours after MR309 400 mg

\textbf{Figure 4:} Brain \selectlanguage{greek}σ\selectlanguage{english}1 receptor occupancy and plasma concentrations
of MR309 (PET study)

Dotted line represents a model fit to the data using
pharmacokinetic--\selectlanguage{greek}σ-\selectlanguage{english}1 brain receptor Model 1 (Table 1). Vertical hashed
lines show the corresponding estimated EC\textsubscript{50} and its 95\%
confidence interval

\textbf{Hosted file}

\verb`MR309 fig1 v2.eps`  available at \url{https://authorea.com/users/725986/articles/708788-pharmacokinetics-and-brain-sigma-1-%CF%831-receptor-occupancy-of-mr309-a-selective-%CF%831-receptor-antagonist}

\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/MR309-fig2-v1/MR309-fig2-v1}

\end{center}
\end{figure}

\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/MR309-fig3-v3/MR309-fig3-v3}

\end{center}
\end{figure}

\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/MR309-fig4-v1/MR309-fig4-v1}

\end{center}
\end{figure}

\selectlanguage{english}
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