Discovery of a Novel Muscarinic Receptor PET Radioligand with Rapid Kinetics in the Monkey Brain

Article abstract of DOI:10.1021/acschemneuro.7b00340

Positron emission tomography (PET), together with a suitable radioligand, is one of the more prominent methods for measuring changes in synaptic neurotransmitter concentrations in vivo. The radioligand of choice for such measurements on the cholinergic sy

Full text of DOI:10.1021/acschemneuro.7b00340

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Letter
Discovery of a Novel Muscarinic Receptor PET
Radioligand with Rapid Kinetics in the Monkey Brain
Jonas Malmquist, Katarina Varnas, Marie Svedberg, Frédéric
Vallée, Jeffery S. Albert, Sjoerd J Finnema, and Magnus Schou
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00340 • Publication Date (Web): 26 Oct 2017
Downloaded from http://pubs.acs.org on October 31, 2017
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Discovery of a Novel Muscarinic Receptor PET Radioligand with Rapid
Kinetics in the Monkey Brain
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Jonas Malmquist1, Katarina Varnäs1, Marie Svedberg1, Frédéric Vallée3, Jeffrey S. Albert3,
Sjoerd J. Finnema1 and Magnus Schou1,2*
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1Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet
and Stockholm Country Council, SEꢀ171 76 Stockholm, Sweden
2PET Science Centre at Karolinska Institutet, Personalised Healthcare and Biomarkers,
AstraZeneca R&D, Stockholm, Sweden
3IntelliSyn Pharma, Montreal, Canada
*Author to whom correspondence should be addressed. Magnus.schou@astrazeneca.com, tel: +46 8
51775598 fax: +46 8 517 717 53
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Abstract
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Positron emission tomography (PET), together with a suitable radioligand, is one of the more
prominent methods for measuring changes in synaptic neurotransmitter concentrations in vivo.
The radioligand of choice for such measurements on the cholinergic system has been the
muscarinic receptor antagonist Nꢀ[1ꢀ11C]propylꢀ3ꢀpiperidyl benzilate (PPB). In an effort to
overcome the shortcomings with the technically cumbersome synthesis of [11C]PPB, we
designed and synthesized four structurally related analogs of PPB, of which (S,R)ꢀ1ꢀ
methylpiperidinꢀ3ꢀyl)2ꢀcyclopentylꢀ2ꢀhydroxyꢀ2ꢀphenylacetate (1) was found to bind
muscarinic receptors with similar affinity as PPB (3.5 vs. 7.9 nM, respectively). (S,R)-1 was
radiolabeled via Nꢀ11Cꢀmethylation at high radiochemical purity (>99%) and high specific
radioactivity (>130 GBq/µmol). In vitro studies by autoradiography on human brain tissue
and in vivo studies by PET in nonꢀhuman primates demonstrated excellent signal to noise
ratios and a kinetic profile in brain comparable to that of [11C]PBB. (S,R)ꢀ[11C]1 is a
promising candidate for measuring changes in endogenous acetylcholine concentrations.
Keywords
PET, radioligand, acetylcholine, muscarinic, receptor, carbon-11
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Positron emission tomography (PET) is a nonꢀinvasive imaging technique increasingly used
in translational biomedical research and drug development. A key feature of PET is that it
facilitates investigations of endogenous synaptic neurotransmitter concentrations in the living
brain.1, 2 This can provide critical understanding of the pathophysiology of neuropsychiatric
and neurodegenerative disease, while aiding the development of new central nervous system
drugs that aim to increase synaptic neurotransmitter concentrations. Although this
experimental paradigm has been successfully applied for several receptor systems,3 there is a
general lack of radioligands that are sensitive to changes in endogenous neurotransmitter
concentration.
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Muscarinic acetylcholine (mACh) receptors were imaged in the human brain over three
decades ago using Nꢀ[11C]methyl benztropine.4 Since then, a number of PET radioligands for
mACh receptors have been reported, including [11C]scopolamine,5 [11C]quinuclidinylꢀ
benzylate ([11C]ꢀQNB),6 Nꢀ[11C]methylpiperidylꢀbenzylate ([11C]MPB)7 and Nꢀ[11C]propylꢀ3ꢀ
piperidyl benzilate ([11C]PPB) (Figure 1),8 among which only a few have demonstrated
sensitivity towards endogenous ACh concentrations8. One of the frontrunner radioligands for
this purpose is [11C]PPB which was identified from a homologous series of Nꢀalkyl analogs
that also included its N-methyl and N-ethyl analogs, [11C]MPB and [11C]EPB.8 Interestingly,
in this series of molecules, the sensitivity towards endogenous ACh was inversely correlated
to binding affinity and the least potent analog, [11C]PPB, was found to be most sensitive to
endogenous neurotransmitter concentrations. A drawback with [11C]PPB is that its synthesis
proceeds via N-11Cꢀpropylation; this is technically cumbersome and thus hampers widespread
use of the radioligand. The aim of this work was to develop an equipotent analog of PBB that
could be labeled using the most commonly used 11Cꢀlabeled precursor, 11Cꢀmethyl iodide, and
evaluate it as a candidate radioligand for mACh receptors in vitro and in vivo.
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Figure 1. Chemical structures of muscarinic receptor antagonists used in PET imaging.
Our lead molecule in this study, PBB, was reported along with a series of psychotomimetic
glycolate esters by Baumgold et al. in 1975.9 Evaluation of the described structureꢀactivity
relationship on this scaffold indicated that substitution of one of the phenyls with a
cyclopentyl group resulted in a reduction in affinity towards mACh.9 Although only racemic
mixtures were evaluated in the original publication, the substitution of a phenyl with a
cyclopentyl group was considered attractive since it would introduce an additional
stereocenter. The chemistry would thus provide four stereomers that could be evaluated
against PBB for muscarinic receptor in vitro affinity. Thus, a chemical route was designed
that provided the four stereomers of 1 in two steps from phenylacetic acid 2, via esterification
and chiral separation using supercritical fluid chromatography (SFC) (Scheme 1).
Scheme 1. Synthesis of the four stereomers of 1, here exemplified with the preparation of
(S,R)-1; i) methanol, sulfuric acid, reflux then SFC on ChiralCel ODꢀH with 95:5% heptane /
iꢀpropanol with 0.1% dimethylꢀethylamine; ii) sodium hydride, heptane, reflux.
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A high affinity assay for human muscarinic 1 receptor (hM1) was performed to identify a
potential candidate radioligand from the newly synthesized compounds. Gratifyingly, three
stereomers of 1 showed similar or higher potency as compared to (+)ꢀPPB (see Table 1) and
were selected for further evaluation in vivo. The affinity of the (S,S)ꢀisomer was deemed to
low to enable imaging of mAChR in vivo.
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Table 1. Results from a high affinity assay for the human muscarinic 1 receptor (hM1)
Compound Ki (nM)
(S,S)-1
(R,S)-1
(+)ꢀPPB
(S,R)-1
(R,R)-1
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3.5
0.5
Next, a stereoselective route was designed to provide the desired optically pure N-desmethyl
precursor materials for the three most potent stereomers of 1. Starting from phenyl hydroxy
acid 5, dioxolanone 7 was obtained via stereospecific alkylation of intermediate 6 (>99.9%
ee). Dehydration, saponification and catalytic hydrogenation furnished compound 10, which
was converted into 11 by reaction with (S)ꢀ1ꢀBocꢀ3ꢀhydroxypiperidine under Mitsunobu
conditions. This reaction proceeded with a modest yield of 50% and hence was the lowest
yielding reaction in the synthetic sequence. Acidic deprotection finally yielded the target
molecule in >99.9% diastereomeric excess and in a 20% overall yield from 5.
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H3C CH3
CH3
H3C
O
H3C
O
CH3
CH3
CH3
CH3
OH
OH
O
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c
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d
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O
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HCl
H
N
NBoc
O
O
OH
f
OH
e
g
OH
OH
O
O
OH
OH
O
O
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Scheme 2. Synthesis of the Nꢀdesmethyl precursor to 1, here exemplified by the (S,R)ꢀisomer.
Reagents and conditions: (a) pivalaldehyde, cat. TfOH, pentane, reflux (85%); (b)
cyclopentanone, LHMDS, THF, ꢀ78oC, >99.9% de (65%); (c) SOCl2, pyridine, THF, 0oC
(99%); (d) (i) KOH, MeOH, reflux, (ii) HCl (100%); (e) H2, Pd/C, MeOH, rt, >99.9% ee
(80%); (f) (S)ꢀ1ꢀBocꢀ3ꢀhydroxypiperidine, DIAD, PPh₃, THF, 25oC (50%); (g) (i) TFA,
CH₂Cl₂, rt, (ii) HCl, >99.9% de (93%).
The three more potent stereomers of [11C]1 ((S,R), (R,S) and (R,R)) were synthesized by N-
[11C]methylation of their corresponding desꢀmethyl precursors (12) with [11C]methyl triflate
(Scheme 1). The radiochemical conversion of [11C]methyl triflate into [11C]1 was greater than
80% and the total time of synthesis was approximately 32 min, producing on average 3 GBq
of (S,R)ꢀ[11C]1, (R,S)ꢀ[11C]1, and (R,R)ꢀ[11C]1 at high specific radioactivity (130ꢀ230
GBq/µmol). The products were formulated in a solution of ethanol (5%) in phosphate
buffered saline (pH 7.4) for subsequent PET imaging experiments in nonꢀhuman primates.
The formulations of [11C]1 were radiochemically pure (>99%) and stable for at least 60 min
after completed synthesis.
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Scheme 3. Radiolabeling of [11C]1, here exemplified by the (S,R)ꢀisomer. Reagents and
conditions: a) [11C]MeOTf, acetone, NaOH, rt
The three [11C]1 stereomers were evaluated by subsequent PET measurements in the same
cynomolgus monkey on the same experimental day. Following intravenous administration of
the individual radiolabeled stereomers, radioactivity readily entered the brain and distributed
heterogeneously in tissue (Fig 3). One major radiometabolite was observed in monkey plasma
that was more polar than the parent compound. For all three stereomers the concentration of
radioactivity was higher in mACh receptor rich regions (cortex, striatum) than in cerebellum,
a region known to be almost devoid of mACh receptors.10, 11 The ratio of radioactivity
between target regions and cerebellum was proportional to the binding affinity of the
individual stereomers. Thus, the greatest radioactivity ratio was observed for (R,R)ꢀ[11C]1 and
the lowest ratio was observed for (R,S)ꢀ[11C]1. Based on the work by Tsukada et al.,8 it was
hypothesized that the binding kinetics of the radioligand were important for sensitivity
towards endogenous ACh. Timeꢀactivity curves (TACs) for the regional distribution of
radioactivity following administration of the three stereomers were generated and compared.
The shape of the TACs for target regions was found to be related to binding affinity, with the
highest affinity stereomer, (R,R)ꢀ[11C]1 showing near absence of washꢀout from target regions
during the timeꢀframe of the PET measurement (123 min). Gratifyingly, however, the TACs
for (S,R)ꢀ[11C]1 showed a rapid decrease in target regions as well as high contrast between
target and nonꢀtarget regions. Thus, (S,R)ꢀ[11C]1 showed a similar kinetic profile in brain as
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that reported for [11C]PBB and was therefore selected for further evaluation by in vitro and in
vivo challenge studies.
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A series of in vitro autoradiography studies were next performed to investigate (S,R)ꢀ[11C]1
binding to whole hemispheres from the postꢀmortem human brain. First, the binding was fully
blocked with the nonꢀselective mACh antagonist scopolamine, thus demonstrating binding
specificity towards muscarinic receptors in the human brain. Second, the binding was found to
be partially blocked by the M1 selective antagonist pirenzepine, indicating that there is a
degree of M1 selectivity in the binding of (S,R)ꢀ[11C]1. Third, the binding was found to be
unaffected by coꢀincubation with the selective ACh esterase inhibitor, donepezil. The last
observation was important to rule out any competition between donepezil and (S,R)ꢀ[11C]1 at
mACh receptors. An effect of donepezil on the binding of (S,R)ꢀ[11C]1 in vivo might thus be
attributed to the increase of synaptic ACh and would not be a result from a direct interaction
of donepezil with a shared binding site in brain tissue.
Finally, the specific binding of (S,R)ꢀ[11C]1 in the nonꢀhuman primate brain was investigated
in a preꢀtreatment PET experiment. In this experiment, a baseline PET measurement with
(S,R)ꢀ[11C]1 was followed by a preꢀtreatment PET measurement. In the latter experiment, the
injection of (S,R)ꢀ[11C]1 was preceded by treatment with the mACh receptor antagonist
scopolamine (0.05 mg/kg). Following scopolamine, the radioactivity in target regions was
markedly reduced to the same level as cerebellum, thus confirming high level of binding
specificity towards muscarinic receptors in vivo (Fig. 3 B,D).
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Conclusions
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(S,R)ꢀ[11C]1 has good characteristics for visualization of mACh receptors in vitro and in vivo.
Further studies are required to elucidate its sensitivity towards endogenous ACh in vivo.
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Figure 2. Autoradiograms of horizontal hemispheres from the postꢀmortem human brain
incubated with (S,R)ꢀ[11C]1.
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Figure 3. PET images and timeꢀactivity curves (TACs) following intravenous injection of
[11C]1 in cynomolgus monkey. (A) Color coded PET images showing distribution of
radioactivity in the monkey brain after radioligand injection. Summation images from 3 to
123 minutes are shown. Image intensity was normalized for injected radioactivity and body
weight. (B) Representative TACs for brain regions of interest during baseline measurements.
(C) Color coded PET images showing distribution of radioactivity in the monkey brain after
injection of (S,R)ꢀ[11C]1 and after pretreatment with scopolamine. Summation images from 3
to 123 minutes are shown. Image intensity was normalized for injected radioactivity and body
weight. (D) Timeꢀactivity curves for brain regions following pretreatment with scopolamine
(0.05 mg/kg). CAU, Caudate; PUT, putamen; PFC, prefrontal cortex; THA, thalamus; CER,
cerebellum.
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Methods
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Animals and study design
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The study was approved by the Animal Ethics Committee of the Swedish Animal Welfare
Agency (Dnr 452/11) and was performed according to the “Guidelines for planning,
conducting and documenting experimental research” (Dnr 4820/06ꢀ600) of Karolinska
Institutet, the “Guide for the Care and Use of Laboratory Animals”,12 the AstraZeneca
Bioethical Policy and the EU Directive 2010/63/EU on the protection of animals used for
scientific purposes. Two female cynomolgus monkeys (body weights, 5.6 and 7.1 kg) were
examined in this study. The monkeys were owned by the Centre for Psychiatry Research,
Department of Clinical Neuroscience, Karolinska Institutet, and housed in the Astrid Fagraeus
Laboratory of the Swedish Institute for Infectious Disease Control, Solna, Sweden.
Five PET measurements were performed in two experimental sessions. In the first session,
baseline PET measurements were performed after intravenous injection of (S,R)ꢀ[11C]1, (R,S)ꢀ
[11C]1, and (R,R)ꢀ[11C]1 (202ꢀ212 MBq). In the second session, a baseline PET measurement
was first performed with (R,S)ꢀ[11C]1 (208 MBq). This was followed by a preꢀtreatment PET
experiment, in which the PET measurement with (R,S)ꢀ[11C]1 (208 MBq) was preceded by the
intravenous infusion of a solution of scopolamine (0.05 mg/kg) over 10 minutes.
PET experimental procedure
Anaesthesia was induced by intramuscular injection of ketamine hydrochloride
(approximately 10 mg/kg) and maintained by the administration of a mixture of sevoflurane
(2ꢀ8%), oxygen and medical air after endotracheal intubation. The head was immobilized with
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a fixation device.13 Body temperature was maintained by a Bair Hugger model 505 (Arizant
Healthcare, MN) and monitored by an esophageal thermometer. Heart rate, respiratory rate
and oxygen saturation were continuously monitored throughout the experiments. Blood
pressure was monitored every 15 min. Fluid balance was maintained by a continuous infusion
of saline.
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In each PET measurement, a sterile physiological phosphate buffered (pH 7.4) saline (PBS)
solution containing radiolabeled drug, in a volume not exceeding 5 mL, was injected as a
bolus into a sural vein during 5 s. PET data acquisition started at time of the bolus injection.
PET measurements
PET measurements were conducted using the High Resolution Research Tomograph (HRRT)
(Siemens Molecular Imaging). Listꢀmode data were reconstructed using the ordinary Poissonꢀ
3Dꢀordered subset expectation maximization (OPꢀ3DꢀOSEM) algorithm, with 10 iterations
and 16 subsets including modelling of the point spread function (PSF). The corresponding inꢀ
plane resolution with OPꢀ3DꢀOSEM PSF was 1.5 mm fullwidth at halfꢀmaximum (FWHM)
in the centre of the field of view (FOV) and 2.4 mm at 10ꢀcm offꢀcentre directions.14
A transmission scan of 6 min using a single 137Cs source was performed immediately before
radioligand injection. List mode data were acquired continuously for 123 min immediately
after i.v. injection of the radioligand. Images were reconstructed with a series of frames (10
sec × 9, 15 sec × 2, 20 sec × 3, 30 sec × 4, 1 min × 4, 3 min × 4 and 6 min × 17).
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In vitro autoradiography
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The human brain used in this study was obtained from the National Institute of Forensic
Medicine, Karolinska Institutet (Stockholm, Sweden). The brain had been removed at clinical
autopsy and was handled in a manner similar to that previously described.15 Horizontal
sections including the striatum were selected for the binding experiments.
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The sections were incubated for 20 min at RT with 0.2 MBq/mL [11C]1 in a TRIS buffer (50
mM; pH 7.4) containing sodium chloride (100 mM), and ascorbic acid (0.1% (w/v). The
sections were then washed (same buffer) twice for 5 min each and briefly dipped in cold
distilled water before being exposed to phosphor imaging plates (Fujifilm Plate BAS SR2025,
Fujifilm, Tokyo, Japan). The phosphor imaging plates were scanned and the resulting images
processed in a Fujifilm BASꢀ5000 phosphor imager (Fujifilm, Tokyo, Japan). Nonꢀspecific
binding was estimated by simultaneous incubation with scopolamine (10 ꢁM). Competition
studies were also performed with two further reference ligands: (i) pirenzepine, 100 nM, a
selective M1 receptor antagonist16 (ii) donepezil, 100 nM, a selective Ach esterase inhibitor.17
Radiochemistry
[11C]Methane was produced in a PETtrace medical cyclotron (General Electric Medical
Systems, Uppsala, Sweden) using 16 MeV protons in the 14N(p,α)11C reaction on a mixture of
nitrogen and hydrogen gas (10% hydrogen). [11C]Methane was isolated from the target gas on
a cryogenic trap and subsequently converted into [11C]methyl iodide by radical iodination of
[11C]methane in a recirculation system as previously described elsewhere.18 [11C]Methyl
triflate was in turn obtained by passing [11C]methyl iodide through a heated column
containing silver triflate impregnated on graphpac. Radiomethylation, purification and
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formulation was performed using a computer controlled automated system (Scansys,
Denmark). Semiꢀpreparative HPLC was performed using a reversedꢀphase ACE 5 Cꢀ18L
column (250 × 10 mm, 5 ꢁm, Advanced Chromatography Technologies). The column outlet
9
was connected with an UV absorbance detector (λ = 254 nm) in series with a detector for
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radioactivity. The radiochemical purity and identity of the formulated radiolabeled products
were determined by analytic reverse phase HPLC using a ZORBAX Eclipse XDBꢀC18
column (150 × 3 mm, 5
ꢀm; Agilent) and an UV absorbance detector (
λ = 254 nm) in series
with a
βꢀflow detector for radioactivity (Beckman).
[11C]1
[11C]methyl triflate was transferred to a solution of 12 (0.5 mg, 1 µmol) and sodium
hydroxide (6 µL, 0.5 M) in acetone (400 µL). After completed transfer, the crude reaction
mixture was diluted with mobile phase acetonitrile:ammonium formate (0.1 M) 45:55
(600 µL) and purified by semiꢀpreparative HPLC. The collected fraction containing the title
compound (retention time = 8 minutes) was evaporated to dryness and reꢀdissolved in a
solution of ethanol (5% v/v) in physiologically buffered saline (PBS, pH 7.4, 6.5 mL). The
formulated product was sterilized by membrane filtration (0.22 µm, Millipore) to yield the
final product in a solution ready for injection. [11C]1 coꢀeluted with an unlabeled reference
standard of 1 on analytical HPLC. Its identity was further confirmed by tandem mass
spectrometry (MS/MS) analysis of the carrier associated with 1 and comparison with an
authentic reference standard. The radiochemical purity was >98% by HPLC and the
determined average specific activity at injection was 147 GBq/ꢁmol.
Associated content
Experimental details for the chemical synthesis.
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Author information
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Corresponding Author
*Eꢀmail: magnus.schou@astrazeneca.com
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Author Contributions
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The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
Acknowledgements
The authors thank all members of the Karolinska Institutet PET Center. The work was funded
by AstraZeneca.
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Graphical TOC
89x41mm (300 x 300 DPI)
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