Increased Brain Exposure of an Alpha-Synuclein Fibrillization Modulator by Utilization of an Activated Ester Prodrug Strategy

Article abstract of DOI:10.1021/acschemneuro.8b00236

Previous work in our laboratories has identified a series of peptidomimetic 2-pyridone molecules as modulators of alpha-synuclein (α-syn) fibrillization in vitro. As a first step toward developing molecules from this scaffold as positron emission tomography imaging agents, we were interested in evaluating their blood-brain barrier permeability in nonhuman primates (NHP) in vivo. For this purpose, 2-pyridone 12 was prepared and found to accelerate α-syn fibrillization in vitro. Acid 12, and its acetoxymethyl ester analogue 14, were then radiolabeled with ¹¹C (t_1/2 = 20.4 min) at high radiochemical purity (>99%) and high specific radioactivity (>37 GBq/μmol). Following intravenous injection of each compound in NHP, a 4-fold higher radioactivity in brain was observed for [¹¹C]14 compared to [¹¹C]12 (0.8 vs 0.2 SUV, respectively). [¹¹C]14 was rapidly eliminated from plasma, with [¹¹C]12 as the major metabolic product observed by radio-HPLC. The presented prodrug approach paves the way for future development of 2-pyridones as imaging biomarkers for in vivo imaging of α-synuclein deposits in brain.

Full text of DOI:10.1021/acschemneuro.8b00236

Letter
pubs.acs.org/chemneuro
Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
Increased Brain Exposure of an Alpha-Synuclein Fibrillization
Modulator by Utilization of an Activated Ester Prodrug Strategy
†
‡
‡
†
́
̈
Andrew G. Cairns, Ana Vazquez-Romero, Mohammad Mahdi-Moein, Jorgen Åden,
Charles S. Elmore,∥ Akihiro Takano,‡ Ryosuke Arakawa,‡ Andrea Varrone,‡ Fredrik Almqvist,*,†
and Magnus Schou*,‡,§
†
̊
̊
Department of Chemistry, Umea University, 901 87 Umea, Sweden
‡Department of Clinical Neuroscience, Center for Psychiatry Research, Karolinska Institutet and Stockholm County Council,
SE-171 76 Stockholm, Sweden
§PET Science Centre, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Karolinska Institutet, S-171 76
Stockholm, Sweden
∥Isotope Chemistry, Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
S
* Supporting Information
ABSTRACT: Previous work in our laboratories has identified
a series of peptidomimetic 2-pyridone molecules as modu-
lators of alpha-synuclein (α-syn) fibrillization in vitro. As a
first step toward developing molecules from this scaffold as
positron emission tomography imaging agents, we were
interested in evaluating their blood-brain barrier permeability
in nonhuman primates (NHP) in vivo. For this purpose, 2-
pyridone 12 was prepared and found to accelerate α-syn
fibrillization in vitro. Acid 12, and its acetoxymethyl ester
analogue 14, were then radiolabeled with 11C (t1/2 = 20.4
min) at high radiochemical purity (>99%) and high specific
radioactivity (>37 GBq/μmol). Following intravenous in-
jection of each compound in NHP, a 4-fold higher
radioactivity in brain was observed for [11C]14 compared to [11C]12 (0.8 vs 0.2 SUV, respectively). [11C]14 was rapidly
eliminated from plasma, with [11C]12 as the major metabolic product observed by radio-HPLC. The presented prodrug
approach paves the way for future development of 2-pyridones as imaging biomarkers for in vivo imaging of α-synuclein
deposits in brain.
KEYWORDS: Alpha-synuclein, PET, carbon-11
arkinson’s disease (PD) is a progressive neurodegener-
(PET). PET is a quantitative, noninvasive molecular imaging
Pative disease affecting approximately 0.1% of the technique that can be used in a translational fashion for a wide
range of purposes. These include studies of drug molecule
distribution (i.e., microdosing3), drug-target engagement
studies, and studies of disease pathology.4 The latter studies
relies on the development of a specific tracer molecule, which
needs to fulfill a set of strict requirements.5 Though the
development of a suitable imaging biomarker for α-syn
aggregates represents a significant challenge, it is crucial in
relation to the great unmet clinical need in PD.
FN075 (Figure 1) is a ring-fused thiazolo-2-pyridone
peptidomimetic notable for modulating amyloid formation,6,7
in particular accelerating the onset of α-synuclein fibrillization.8
The detailed mechanism for this acceleration is not known, but
it appears to involve increased formation of the soluble
population and about 1% of individuals over 60 years of
age.1 PD is characterized by the presence of Lewy body
inclusions in brain neurons as well as loss of dopaminergic
function.2 Although Lewy bodies may be identified by
histology on post mortem human brain tissue sections, it is
widely recognized that novel biomarkers are required to
quantify these inclusions in the brains of living patients
suffering from PD. Such biomarkers would not only be highly
valuable in relation to clinical research of disease pathophysi-
ology and progression, but also provide crucial patient
segmentation and end points for PD drug development.
Inspired by the successful development of imaging biomarkers
for Amyloid aggregates in Alzheimer’s disease (AD), we were
led to investigate the development of an imaging biomarker for
α-syn aggregates, the major constituent of Lewy bodies.2 In
analogy with previous imaging biomarkers developed for AD,
we directed our attention to positron emission tomography
Received: May 15, 2018
Accepted: June 14, 2018
Published: June 14, 2018
© XXXX American Chemical Society
A
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Letter
labile in vivo,14,15 easily synthesized, and have been previously
used as prodrugs for carboxylic acids in drug16 and probe
molecules.17−20
Both C4′ methoxy substituted standards and the labeling
precursor were synthesized by a common route. Benzylic
chloro intermediate13 4 was first synthesized by ketene−imine
cycloaddition,21−23 then used in an established Suzuki coupling
strategy12,24 with substituted naphthyl boronic esters 7 and 8,
themselves prepared using Miyaura borylation. Hydrolysis of
the C4′ methoxy precursor 10 and subsequent protection with
bromomethyl acetate gave the cold analogues of the acid (12)
and prodrug (14) probe forms, respectively. The same
sequence was performed on the C4′ O-benzyl analogue 9
and followed by hydrogenolysis to give the radiolabeling
precursor (15) (Scheme 1).
Figure 1. Chemical structure of FN075 (X = H) with the
peptidomimetic motif highlighted in red.
oligomer intermediates through which α-synuclein eventually
develops into fibrils.8,9 Applied in vivo, FN075 has been
observed to modulate the soluble α-synuclein oligomer
fraction, activity profile, and lifespan of Drosophila.10 In a
murine model, intranigral injection of FN075 resulted in
symptoms mimicking early stages Parkinson’s disease.11 These
included metabolomic profiles consistent with those observed
in Parkinson’s patients, loss of motor function, and decline in
the number of TH+ neurons.11 Given the evidence for FN075
interacting with α-syn and remaining incorporated in
oligomeric species9 which lead to fibrils, we decided to
investigate this molecule as a starting point in the search for an
in vivo α-syn aggregate imaging biomarker for PET. However,
because of the lipohilic nature of FN075, it was in itself
considered a poor candidate for in vivo imaging of α-syn
aggregates.
Scheme 1. Synthesis of the Radiolabelling Precursor and
a
Control Compounds
At the onset of this project, the C3 carboxylic acid moiety of
FN075 was identified as a potential risk factor for low blood-
brain barrier (BBB) permeability. Since this functionality was
considered necessary for fibrilization modulation,8 a strategic
decision was made to investigate the brain exposure of a tool
compound from the FN075 platform and to evaluate a
potential prodrug approach for delivering the molecule more
efficiently to brain. The aim of this project was thus threefold:
(i) to design and prepare an analogue of FN075 that contained
a handle for radiolabeling, while retaining activity as an α-syn
fibrillization accelerator, (ii) to radiolabel the compound, and
its prodrug analogue, with the positron emitting radionuclide
11C (t1/2 = 20.4 min), and (iii) to evaluate the brain exposure
of both radioligands by PET in vivo in nonhuman primates
(NHP).
RESULTS AND DISCUSSION
■
The first challenge addressed was the design of an analogue of
FN075 containing a suitable handle for facile introduction of
the 11C-label. The position of the radiolabel reflects
considerations of synthetic accessibility, but also a desire to
retain the acceleration effect observed with FN075. Although
not strictly necessary for this work, an analogue which retains
this activity can be more confidently assumed to properly
mimic the in vivo behavior of FN075. Furthermore, an FN075
analogue with such a handle would be a valuable intermediate
for future amyloid probe synthesis. Working from the
hypothesis that retention of the hydrogen-bonding peptidomi-
metic component was necessary, C8 modification was
considered as a possibility. However, the effects of modifying
this position are variable and can lead to loss of activity.
Therefore, we instead chose to investigate a C4′ modification,
as variation of the pendant naphthyl is tolerated in other
applications of ring fused pyridone peptidomimetics.12,13 The
strategy taken for improving brain delivery was to derivatize
the carboxylic acid. For this purpose, an acetoxymethyl ester
moiety was utilized; these are relatively stable on the bench but
a
A list of abbreviations can be found in the Supporting Information.
Compounds 12, 14, and 15 were then tested using an in
vitro fibrillization assay (Figure 2). This showed the methoxy
analogue 12 to accelerate fibril formation, as indicated by the
shortened lag phase, although to a lesser extent than FN075.
The two acetoxymethyl ester standards 14 and 15 were
inactive, as indicated by a lag phase close to that observed for
α-synuclein alone. Thus, any interaction with α-synuclein in
vivo following administration of the ester prodrug [11C]14
would be expected to originate from the acid [11C]12. It is
important to note that the in vitro binding affinities of these
B
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Letter
ethanol in propylene glycol, and sterile filtered prior to
administration.
With the two radioligands in hand, we next evaluated the
exposure of [11C]12 and [11C]14 in the NHP brain in vivo. A
total of four dynamic PET measurements were made (two with
each radioligand) while keeping the head of the NHP in the
field of view. Following intravenous injection of each
radioligand, radioactivity was distributed homogeneously in
brain, with a notably higher brain exposure observed for [11C]
14 than that observed for [11C]12 (Figure 3).
Figure 2. α-Synuclein fibril formation rates in the presence of
different additives, based on ThT fluorescence. Each line shown is an
average of six measurements (see the Supporting Information for
individual curves). The protein CsgC is an inhibitor of fibrillization.25
Vertical traces represent the lag time until fibrillization starts.
molecules to α-synuclein and other related protein aggregates
is unknown at this point. In addition, although 12 accelerates
α-synuclein aggregation in vitro, the high specific radioactivity
obtained with 11C leads to administration of a microdose of
unlabeled compound, which is expected to be pharmacolog-
ically inactive in vivo.
Encouraged by the acceleratory properties of 12, we
proceeded to radiolabeling with carbon-11 (Scheme 2). For
Figure 3. Summation (3−123 min) color-coded PET-MR images
showing the distribution of radioactivity in rhesus monkey brain
following the consecutive intravenous injections of [11C]12 (A) and
[11C]14 (B) in the same experimental session. Image intensity was
corrected for injected radioactivity.
Scheme 2. Radiolabeling of [11C]12 and Acetoxymethyl
Ester [11C]14
One of the advantages of PET imaging is that radioactivity
concentrations can be studied in the organ of interest as a
function of time. Thus, the time course for radioactivity in
brain was presented in Figure 4 in the form of time−activity
curves (TACs). Following intravenous injection of [11C]12,
peak brain radioactivity (0.2 SUV) was observed after
this purpose, we chose 11C-methylation, which is a standard
protocol for facile introduction of 11C into small druglike
molecules. Gratifyingly, the acetoxymethyl ester [11C]14 was
readily obtained via the reaction of phenolic precursor 15 with
[11C]methyl triflate in acetone under mild alkaline conditions.
[11C]14 in turn served as a convenient intermediate for the
preparation of [11C]12 via saponification. Both [11C]12 and
[11C]14 were obtained at high molar activity (>37 GBq/μmol)
and at high radiochemical purity (>97%) within 42 min after
the end of bombardment. The radioligands were formulated in
an isotonic mixture between phosphate buffered saline and
Figure 4. Time−activity curves for the concentration of radioactivity
in whole brain (0−123 min) following the intravenous injection of
[11C]12 and [11C]14 in NHP2.
C
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approximately 20 min. The maximum radioactivity observed in
brain after injection of the acetoxymethyl ester [11C]14 was
roughly 4-fold higher (0.8 SUV), but also notably earlier (at 10
min). After reaching peak uptake, the washout from brain was
slow for both radioligands, with approximately half of the
radioactivity present in brain at the end of the emission
measurement. Since the NHPs in this study were healthy, it is
unlikely that the slow wash-out from brain is a result of specific
binding to α-syn aggregates. One potential explanation for the
slow washout of the acetoxymethyl ester [11C]14 from brain is
that ester hydrolysis is partially taking place within the brain.
Since the acid [11C]12 has limited permeability across the
BBB, it could be trapped within the brain. Indeed, multiple
esterases have been identified in the primate brain that would
be capable of such a transformation,26 but future work is
required to test this hypothesis. Alternatively, the slow washout
from brain may reflect nonspecific binding to brain tissue,
especially considering the lipophilic nature of 14 (clogP =
6.127). Arterial blood sampling and kinetic analysis of the PET
data may help identify the most probable reason for the
observed slow wash-out. The synthesis of less lipophilic
fibrillization modulators from this scaffold is therefore
underway in our laboratories. Nevertheless, the 4-fold increase
of radioactivity delivered to brain was encouraging and
supports the use of the devised prodrug approach for
improving BBB permeability of radioligands derived from
this scaffold.
elimination of the ester from plasma can be viewed as both a
strength and a weakness; it may limit the radioactivity
concentration in brain, which in this case was on the lower
end for a useful PET radioligand. On the other hand, the
observed rapid elimination results in lower prodrug concen-
tration in plasma at later time-points when quantification of the
PET data is usually conducted. Lower plasma prodrug
concentrations will drive washout of the ester [11C]14 from
brain and increase the probability that a larger fraction of the
radioactivity in brain is constituted by the acid metabolite
[11C]12. This is important in relation to modeling of the data,
since it is well-known that the presence of multiple
radiochemical entities in brain hampers accurate quantification
of the PET signal.
We herein report the first in vivo imaging of a radiolabeled
2-pyridone α-synuclein fibrillization modulator. As expected at
the onset of this study, the 2-pyridone 12 was unable to cross
the BBB in meaningful concentrations for in vivo PET imaging
and a derivatization approach was required. Although the tool
compounds used in this study by no means should be
considered optimal imaging biomarker candidates, we expect
the identified derivatization strategy to be applicable also on
other molecules from the scaffold. The focus for future
development of imaging biomarkers from the 2-pyridone
scaffold is to improve the physicochemical properties and to
optimize their affinity and selectivity for aggregated alpha-
synuclein in relation to other protein aggregates.
Discrete blood samples were taken during the time course of
each PET measurement for the purpose of analyzing
radioactive metabolites in plasma. After injection of [11C]12,
the amount of unchanged radioligand in plasma decreased
from approximately 96% of plasma radioactivity at 4 min to
approximately 20% at 60 min (Figure 5). Based on the elution
CONCLUSION
■
In conclusion, a prodrug approach was developed that
delivered a 2-pyridone fibrillization modulator to the nonhu-
man primate brain with 4-fold higher concentration of
radioactivity. This approach paves the way for the future
development of 2-pyridones as radioligands for in vivo imaging
of α-synuclein aggregates in brain.
METHODS
■
Radiochemistry. HPLC solvents were obtained from Fisher
(Sweden). Unless otherwise stated, all other reagents and solvents
were obtained from Sigma-Aldrich (Sweden) and used without
further purification.
General Procedure for Radiolabeling. No-carrier-added
[11C]CH4 was produced using 16.5 MeV protons in the 14N(p,α)11C
nuclear reaction on a mixture of nitrogen and hydrogen gas (10%
hydrogen). [11C]CH4 was converted to [11C]CH3I by radical
iodination in a gas-phase recirculation system and swept in a stream
of helium through a heated glass column containing silver triflate
impregnated on graphpac to produce [11C]CH3OTf. The radio-
labeling agent was bubbled through a solution of phenol 15 (0.5 mg)
and aqueous sodium hydroxide (0.5 M) in acetone at room
temperature. After 3 min, the reaction mixture was diluted with
mobile phase, purified and formulated using a computer controlled
automated radiochemistry system (Scansys AS, Denmark). Semi-
preparative HPLC was performed using a reversed-phase C-18
column column (ACE-C18, 5 μm, 7.8 × 300 mm, Advanced
Chromatography Technologies) eluted with MeCN- HCO2NH4 (0.1
mM) 70:30 v/v at 8 mL/min. The column outlet was connected to an
absorbance detector (λ = 254 nm) in a series with radiation detector.
Solvents were removed from the product fraction by solid phase
extraction (Oasis HLB 1 cm3 cartridge, Waters). [11C]14 was
formulated in a solution of ethanol (30%) in propylene glycol (3 mL)
and PBS (phosphate buffered saline, pH 7.4, 10 mL) and sterilized by
membrane filtration through a Millex-GV filter unit (0.22 μm)
(Millipore, Billerica, MA, USA).
Figure 5. Time course for unchanged radioligand in plasma following
intravenous administration of [11C]12 and [11C]14, respectively
(each point represents an average of two experiments).
profile on reverse phase radio-HPLC, all of the observed
radiometabolites of [11C]12 were more polar than the parent
compound. Following injection of the acetoxymethyl ester
[11C]14, rapid hydrolysis was observed. Approximately 40% of
the measured radioactivity in plasma corresponded to the acid
[11C]12 at 4 min post injection. At 60 min, approximately 10%
of the parent ester was observed in plasma. The rapid
D
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[11C]12 was prepared via hydrolysis of crude ester [11C]14, which
was prepared according to the above method and used without HPLC
and SPE purification. Following the radiolabeling reaction, a solution
of sodium hydroxide (0.125 M, 1 mL) was added and the mixture was
heated at 80 °C for 5 min. After cooling to room temperature, an
aqueous solution of TFA (0.15 M, 1 mL) was added and the reaction
mixture was purified using HPLC. Semipreparative HPLC was
performed using a reversed-phase C-18 column (ACE-C18, 5 μm, 7.8
× 300 mm, Advanced Chromatography Technologies) eluted with
MeCN-HCO2NH4 (0.1 mM) 40:60 v/v at 8 mL/min. [11C]12 was
isolated, formulated, and sterilized in an identical fashion as that
described for [11C]14 above.
PET Imaging in Nonhuman Primates. The study was approved
by the Animal Ethics Committee of the Swedish Animal Welfare
Agency (Dnr 145/08, 399/08, and 386/09) and was performed
according to the “Guidelines for planning, conducting and
documenting experimental research” (Dnr 4820/06-600) at the
Karolinska Institutet, the Guide for the Care and Use of Laboratory
Animals” the AstraZeneca bioethics policy and the EU Directive
2010/63/EU.
Four PET experiments were performed in three experiment
sessions. Session 1: PET measurements with 70 MBq of [11C]14 in
rhesus monkey 1 (6.1 kg). Session 2: PET measurement with 89 MBq
of [11C]12 followed by a PET measurement with [11C]14 (125 MBq)
in rhesus monkey 2 (6.5 kg). Session 3: PET measurement with 140
MBq of [11C]12 in rhesus monkey 1 (6.3 kg).
A head fixation system was used to secure a fixed position of the
monkey’s head throughout the PET measurements undertaken in
each experimental session. In each PET experiment, the radiotracer
was injected as a bolus into a sural vein during 5 s with simultaneous
start of PET data acquisition. Radioactivity in the brain was measured
continuously for 123 min according to a preprogrammed series of 34
frames.
Notes
The authors declare the following competing financial
interest(s): M.S. and C.E. are employees and/or shareholders
at AstraZeneca.
ACKNOWLEDGMENTS
The authors thank all members of Karolinska Institutet PET
Centre.
■
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschemneur-
o.8b00236.
Synthesis of tool compounds, protocol for fibrillization
assay, and quality control data for radiolabeled
compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: magnus.schou@astrazeneca.com. Tel: +46-8 517
75598.
*E-mail: Fredrik.almqvist@umu.se. Tel: +46-90 786 69 25.
ORCID
(11) Chermenina, M., Chorell, E., Pokrzywa, M., Antti, H., Almqvist,
Magnus Schou: 0000-0002-4314-2418
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small-molecule amyloid accelerator results in cell death of nigral
dopamine neurons in mice. npj Parkinson's Dis. 1, 15024.
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K. S., Salin, O., Engstrom, P., Svensson, R., Artursson, P., Gylfe, A.,
Bergstrom, S., and Almqvist, F. (2016) Thiazolino 2-Pyridone Amide
Inhibitors of Chlamydia trachomatis Infectivity. J. Med. Chem. 59 (5),
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Author Contributions
A.G.C., A.T., R.A., A.V., F.A. and M.S. contributed to the
experimental design. A.G.C., A.V.R., M.M.M., J.Å., C.S.E., R.A.
performed the experiments. A.G.C., M.M.M., A.V., F.A., M.S.
analyzed the data. The manuscript was written by A.G.C. and
M.S. and all authors have given approval to the final version.
(13) Singh, P., Chorell, E., Krishnan, K. S., Kindahl, T., Aden, J.,
Wittung-Stafshede, P., and Almqvist, F. (2015) Synthesis of Multiring
Fused 2-Pyridones via a Nitrene Insertion Reaction: Fluorescent
Modulators of alpha-Synuclein Amyloid Formation. Org. Lett. 17
(24), 6194−6197.
Funding
The authors are grateful to AstraZeneca, Vinnova, and the
Michael J. Fox Foundation for financial support. F.A. received
funding from the Swedish Research Council, the Goran
Gustafsson Foundation, the Swedish Foundation for Strategic
Research, and the Kempe Foundation.
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DOI: 10.1021/acschemneuro.8b00236
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX