11C Radiolabeling of anle253b: a Putative PET Tracer for Parkinson's Disease That Binds to α-Synuclein Fibrils in vitro and Crosses the Blood-Brain Barrier

Article abstract of DOI:10.1002/cmdc.201900689

There is an urgent clinical need for imaging of α-synuclein (αSyn) fibrils, the hallmark biomarker for Parkinson's disease, in neurodegenerative disorders. Despite immense efforts, promising tracer candidates for nuclear imaging of αSyn are rare. Diphenyl pyrazoles are known modulators of αSyn aggregation and thus bear potential for non-invasive detection of this biomarker in vivo. Here we demonstrate high-affinity binding of the family member anle253b to fibrillar αSyn and present a high-yielding site-selective radiosynthesis route for ¹¹C radiolabeling using in-situ generated [¹¹C]formaldehyde and reductive methylation. Radio-HPLC of the tracer after incubation with rat serum in vitro shows excellent stability of the molecule. Positron emission tomography in healthy animals is used to assess the pharmacokinetics and biodistribution of the tracer, showing good penetration of the blood–brain barrier and low background binding to the non-pathological brain.

Full text of DOI:10.1002/cmdc.201900689

Accepted Article
Title: 11C radiolabeling of anle253b: A putative PET tracer for
Parkinson’s disease that binds to alpha-synuclein fibrils in vitro
and crosses the blood-brain barrier
Authors: Andreas Maurer, Andrei Leonov, Sergey Ryazanov, Kristina
Herfert, Laura Kuebler, Sabrina Buss, Felix Schmidt, Daniel
Weckbecker, Ruth Linder, Dirk Bender, Armin Giese, Bernd J.
Pichler, and Christian Griesinger
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900689
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900689
A Journal of
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ChemMedChem
10.1002/cmdc.201900689
COMMUNICATION
1
1
C radiolabeling of anle253b: A putative PET tracer for
Parkinson’s disease that binds to alpha-synuclein fibrils in vitro
and crosses the blood-brain barrier.
[
a]
[b,d]
[b]
[a]
[a]
Andreas Maurer,* Andrei Leonov,
Sergey Ryazanov, Kristina Herfert, Laura Kuebler, Sabrina
[
a]
[c]
[c]
[b]
[e]
[c,d]
Buss, Felix Schmidt, Daniel Weckbecker, Ruth Linder, Dirk Bender, Armin Giese, Bernd J.
[
a]
[b,d]
Pichler, and Christian Griesinger*
[a]
[b]
[c]
Dr. A. Maurer, Dr. K. Herfert, L. Kübler, S. Buss, Prof. Dr. B. J. Pichler
Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy
Eberhard Karls University Tübingen
Röntgenweg 15, 72076 Tübingen (Germany)
E-mail: andreas.maurer@med.uni-tuebingen.de
Dr. A. Leonov, Dr. S. Ryazanov, R. Linder, Prof. Dr. C. Griesinger
Department of NMR-based Structural Biology
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Göttingen (Germany)
E-mail: cigr@nmr.mpibpc.mpg.de
Dr. F. Schmidt, Dr. D. Weckbecker, Prof. Dr. A. Giese
Center for Neuropathology and Prion Research
Ludwig-Maximilians-University, Munich
Feodor-Lynen-Str. 23, 81377 Munich (Germany)
Dr. A. Leonov, Prof. Dr. C. Griesinger, Prof. Dr. A. Giese
MODAG GmbH
Mikro-Forum-Ring 3, 55234 Wendelsheim (Germany)
Prof. Dr. D. Bender
[
d]
e]
[
Department for Nuclear Medicine and PET center
Aarhus University Hospital
Palle Juul-Jensens Boulevard 165, 8200 Aarhus (Denmark)
Supporting information for this article is given via a link at the end of the document.
Abstract: There is an urgent clinical need for imaging of α-synuclein
αSyn) fibrils, the hallmark biomarker for Parkinson’s disease, in
dementia with Lewy Bodies, multiple systems atrophy and other,
[4]
(
less frequent neurodegenerative diseases. Not only precise
diagnosis of these diseases but also deeper understanding of
the interplay between αSyn aggregation and progressive
neurodegenerative disorders. Despite immense efforts, promising
tracer candidates for nuclear imaging of αSyn are rare. Diphenyl
pyrazoles are known modulators of αSyn aggregation and thus bear
potential for non-invasive detection of this biomarker in vivo. Here
we demonstrate high-affinity binding of the family member anle253b
[
5]
neurodegeneration
and the crosstalk between the different
[
6]
αSyn, Aβ and tau pathologies
would benefit from sensitive
real-time detection and quantification of αSyn in patients. Most
importantly, αSyn imaging would be a major pillar besides the
clinical disease rating scales for evaluation of therapeutic
approaches.
to fibrillar αSyn and present
a
high-yielding site-selective
11
radiosynthesis route for C radiolabeling using in-situ generated
11
[
C]formaldehyde and reductive methylation. Radio-HPLC of the
tracer after incubation with rat serum in vitro shows excellent stability
of the molecule. Positron emission tomography in healthy animals is
This unmet demand for αSyn radiotracers comes up against the
only few known groups of small-molecule compounds interacting
used to assess the pharmacokinetics and biodistribution of the tracer, with aggregated αSyn with notable affinity and selectivity, mainly
[
7]
[8]
showing good penetration of the blood-brain-barrier and low
background binding to the non-pathological brain.
phenothiazines, other fluorescent dyes
and compounds
Diphenylpyrazoles
[
9]
based on 3-(benzylidene)indolin-2-one.
such as anle138b (1) were identified from
a large-scale
screening to inhibit pathological aggregation of prion protein
Due to set-backs with clinical trials in neurodegenerative
diseases, early disease detection and monitoring of disease-
modifying therapies are becoming increasingly important.
Groundbreaking work enabled nuclear (positron emission
tomography, PET) imaging and longitudinal quantification of
beta-amyloid (Aβ) plaques in Alzheimer’s disease, now routinely
sc
[10]
(
PrP ) and αSyn in vivo
and their direct binding to αSyn fibrils
[11]
was demonstrated in vitro by fluorescence spectroscopy. This
led us to the hypothesis that the anle138b family might represent
good candidates for the development of PET tracers for αSyn
aggregation. As anle253b (2) from the compound list originally
[12]
11
reported
bears a methyl group suitable for C-methylation
[1]
used in clinical research. This greatly contributed to advanced
understanding of the pathogenesis and development of the
(
t
1/2 = 20.3 min) we selected this compound as a starting point
for tracer development. Previous reports demonstrated αSyn
[
2]
disease. In contrast, despite great public attention and large-
scale initiatives, no clinically evaluated radiotracers are currently
available to detect aggregation of alpha-synuclein (αSyn) to
aggregation inhibition of 50 % in the reported scanning for
[13]
intensely fluorescent targets assay
and a brain uptake in
mice of 2.1 nmol/g four hours after oral application of 1 mg (ca.
0 mg/kg).
[
3]
Lewy Bodies, the pathological hallmark of Parkinson’s disease,
4
1
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COMMUNICATION
1
1
The most common approach for C radiolabeling is methylation
base added. Indeed, when we performed radiomethylation
reactions with Cs CO addition (Table 1, line 2), we found that
2 3
1
1
11
by means of [ C]methyl iodide (MeI) or [ C]methyl triflate
MeOTf). These methylating agents can be efficiently
synthesized from cyclotron-produced [ C]CO
(
the radioactivity was nearly quantitatively incorporated as
1
1
11
2
using automated
pyrazole-methylated [ C]4 and 5 (in a ratio of about 5:2,
gas-phase reactions on commercially available synthesizers.
The alternative synthon [ C]formaldehyde (CH O) is less
2
commonly applied since its preparation required the more
complicated “wet method” and thus specialized radiochemistry
respectively), while only minor radioactivity peaks were detected
at the retention time of 2 (Figure 2A and Table 1) under the
various conditions applied. By omitting additional base we were
able to completely suppress pyrazole methylations but still only
achieved low radiochemical conversions of <15 %. While further
optimization might be possible using longer beam-times, higher
temperatures, or MeOTf as methylating agent, we hypothesized
that reductive methylation with formaldehyde might be a more
1
1
[
14]
modules. Groundbreaking work from Hooker et al.
has
O from [ C]MeI by oxidation
with trimethyl-N-oxide (TMAO), making radiosynthesis with
1
1
11
enabled the synthesis of [ C]CH
2
1
1
[
C]CH
2
O an easily available and attractive alternative to the
11
classical methylating reagents. Reductive radiomethylation using
efficient route for radiosynthesis of [ C]2 due to the expected
selectivity for primary amines.
1
1
[
2
C]CH O was successfully applied to primary aromatic amines.
Among others, the Pittsburgh compound B, a PET tracer used in
Screening of suitable reducing agents (Suppl. Scheme 3) led to
the identification of sodium cyanoborohydride and a mixture of
phosphate buffer and dimethylformamide (DMF) as the best
studies of Alzheimer’s disease, was prepared using this
[
15]
method.
11
[15]
In this work, we describe the radiosynthesis of [ C]2 from its
desmethyl precursor 3 exploiting the distinct site-selectivity of
combination for this reaction. According to literature evidence
suggesting dimethylformamide as a possible reason of specific
activity erosion, diethylformamide (DEF) was selected as solvent.
Fine tuning of buffer pH led to a slightly increased yield of target
product 2 at a pH of 5.
1
1
11
[
[
C]CH
2
O and [ C]MeI. We show data for specific binding of
1
1
C]2 to αSyn fibrils in vitro and analyze the time-dependent
biodistribution of the tracer in vivo using PET imaging.
11
Scheme 1. Structure of anle138b (1), and reaction scheme for
C-
radiomethylation of
3 using either nucleophilic substitution or reductive
methylation selectively yielding pyrazole-methylated
4 and 5 or aniline-
methylated 2, respectively.
Figure 2. Exemplary results from substitutive (A) and reductive (B)
11
radiomethylation of 3. A: [ C]MeI was trapped in a solution of 1 mg 3 and 5
First, we characterized the fibril binding of 2 using a competition
assay with tritiated 1 on recombinant human αSyn fibrils. This
assay showed high-affinity binding of 2 to αSyn fibrils with an
IC50 value of 1.6 nM, making this compound a promising
candidate for imaging synucleinopathies (Figure 1).
11
2 3
mg Cs CO in 500 µl DMSO and heated to 110 °C for 5 min. [ C]4 and 5 are
11 11
the main products while no [ C]2 is formed. B: [ C]MeI was trapped in a
solution of 5 mg TMAO and 1 mg 3 in 350 µl DEF cooled to -20 °C. After
incubation at 60 °C for 3 min, 7.6 mg NaBH
3
CN in 60 µl DEF and 1.2 mL 100
mM citrate phosphate buffer pH 5 were added and the resulting mixture was
11
heated to 100 °C for 5 min. [ C]2 was the main radioactive product. The
precursor 3 is visible in both UV chromatograms.
When we applied these conditions to radiolabeling, we
immediately observed formation of the desired product in
reasonable yields (>300 MBq), without generation of the
previously observed side products. This indicates that canonical
and reductive radiomethylation can be used as orthogonal
strategies for radiolabeling of the aniline-containing branch of
the anle138b (1) family (Scheme 1), enabling selective
production of three different radiotracers from a single precursor.
Figure 2B shows
a typical chromatogram from such an
3
Figure 1. Competitive binding of 2 and [ H]1 to αSyn fibrils. Fibrils were
experiment. After some experiments with separate additions of 3
and the reducing agent after the TMAO reaction we found that 3
can already be added to the TMAO solution without negatively
affecting the outcome of the reaction. Omitting TMAO from this
solution massively reduced the formation of 2 from 47 % to only
5 % (radiochemical conversion), demonstrating that the reaction
is indeed mainly mediated by TMAO-dependent formaldehyde
formation. Using this synthesis route we reliably obtained yields
probed with tritiated 1 (1 nM) in the presence of a dilution series of 2. Mean
values ±SD (n=3) are presented.
11
We then set out to directly methylate 3 with [ C]MeI. We
expected the reaction to concurrently occur both at the aniline
nitrogen and the pyrazole nitrogen atoms, with a tendency for
aniline methylations with weaker bases or with no additional
2
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1
1
11
of 600-2000 MBq [ C]2 after 30 min (35 µAh) target irradiation.
Although this methylation approach enabled high radiochemical
yields, the obtained molar radioactivities were comparatively low
plate with different oligomerization states, [ C]2 showed
preferential binding to αSyn fibrils (Figure 3B) compared to
oligomers (not significant, Dunnett’s test) and monomers (p =
0.0341, Dunnett’s test). The low signal intensity in this assay
only allowed for qualitative verification of [ C]2 fibril binding
within the scope of this work, while quantification of binding data
(in addition to abovementioned IC50) will be established in future
using our tritium-based assay.
(
15.1 ± 3.4 GBq/µmol at end of synthesis. For other tracers we
1
1
typically achieve 60-200 GBq/µmol) and the synthesis thus
requires further optimization for successful imaging of low-
abundant targets such as αSyn aggregates. Regardless of this
remaining
challenge,
the
reliable
and
high-yielding
radiosynthesis approach allowed us to analyze the in vitro and in
vivo characteristics of 2.
When designing PET tracers for brain imaging, not only affinity
and selectivity but also suitable physicochemical properties like
the logP value and molecular weight are crucial constraints.
LogP values between 1 and 4 are considered as optimal,
Table 1. Conditions for direct radiomethylation of 3 with MeI. Reaction time for
all reactions was set to 5 min. Radiochemical conversions (%) were calculated
from semipreparative HPLC chromatograms.
[
16]
depending on the author, although the meaningfulness of logP
[
17]
is under discussion. With its (calculated) logP of 5.21, 2 is not
considered optimal in this regard and might show a tendency for
unspecific lipid binding but seemed lipophilic enough to cross
the blood-brain barrier. We thus analyzed the in vivo
biodistribution after tail vein injection in healthy rats. During the
Solvent
DMSO
Base
Temperature
°C)
2 (%)
4 (%)
5 (%)
(
NaOH,
110
0
28.2
58.5
11
dynamic PET scans the tracer [ C]2 showed a clear penetration
of the blood brain barrier in all animals. It is homogenously
distributed in the complete brain with a higher midbrain uptake
5
µl 5 M
DMSO
DMSO
DMSO
DMF
Cs CO
2
3
110
110
110
110
0
18.4
7.2
0
45.4
14.6
0
(
Figure 4A). This is also observed in the kinetics of the tracer
NaHCO
3
2.1
9.3
0
1
1
where the lowest uptake of [ C]2 is seen in the cortex. Figure
4B shows the uptake kinetics (%ID/cm ) for the cerebellum,
3
-
-
brainstem, cortex and striatum with an initial perfusion peak after
the injection. After a drop, a slow increase can be observed until
0
0
2
5 minutes after injection, followed by a slow washout of the
1
1
To assess serum stability of [ C]2, rat serum was incubated with
the tracer at 37 °C for 1.5 hours and analyzed by radio-HPLC.
The chromatograms (Suppl. Fig. 2) did not show any signs of
activity.
Ex vivo biodistribution was carried out directly after PET
1
1
measurements with [ C]2. The data are presented in Figure 4C.
The highest activity concentrations (in %ID/g) are observed in
the left and right kidney (0.52±0.30, 0.69±0.17 respectively) as
well as in the liver (0.79±0.22) which might represent excretion
of the tracer after 75 minutes of PET measurement. In the brain,
the cortex and the cerebellum show the lowest radioactivity
concentration (0.25±0.05, 0.24±0.05 %ID/g) whereas the
brainstem shows the highest uptake values (0.30±0.05 %ID/g).
In vivo stability was analyzed by injection of the radiotracer into
healthy rats (178.5±35.8 MBq) and HPLC analysis of cleared
brain lysates and plasma samples after 7 and 15 minutes (see
Suppl. Fig. 5). Despite the low sensitivity of our radio-HPLC
detector we were able to verify that the majority of radioactivity
1
1
metabolism or degradation of [ C]2 in serum over 90 minutes.
1
1
in the brain was indeed [ C]2 at 7 min and 15 min post injection.
The overall distribution and in particular the pattern within the
brain seems to be suited for application in relevant animal
models, although the time-activity curves showed atypical brain
uptake kinetics after the typical drop following the perfusion peak.
This might be caused by the rather high logP value and will
require deeper kinetic analysis using a blood sampling approach
to record the arterial input function. To exclude suboptimal
serum stability of the tracer as an alternative explanation of the
unexpected time-activity curve, we analyzed the stability of
[ C]2 by co-incubation with rat serum. Since the tracer was
completely stable within the time of incubation, metabolism (e.g.
cytochrome P450-mediated demethylation) in a different organ
with efficient release of metabolites back into the serum and
accumulation of the metabolite might cause atypical brain
uptake. This, however, was not supported by ex vivo HPLC of
brain lysates where the main radioactivity 15 min post injection
was found in the form of the parent compound.
Figure 3. Binding assays using αSyn fibrils. A: αSyn fibrils were incubated
11
with 3 nM [ C]2 in the presence (blocking) or absence (binding) of 1 µM of the
non-radioactive compound. Wells containing no αSyn fibrils (“uncoated”) were
used as negative control. Binding to αSyn fibrils was statistically significant
(
p=0.0167) and completely blocked (p=0.0057) by addition of excess 2. B:
11
Three different states of αSyn were used for coating of the plate. [ C]2
preferentially bound to fibrillar synuclein (p=0.0341). Binding was not
statistically significant (n.s.) for monomeric and oligomeric αSyn.
1
1
After immobilizing αSyn in microtiter plates and probing it with
1
1
[
C]2 we quantified the bound activity using autoradiography.
We were able to detect statistically significant (p = 0.0167,
Tukey’s test) fibril binding of the tracer that was blockable (p =
0.0057, Tukey’s test) to baseline levels by excess (1 µM) of the
non-radioactive compound (Figure 3A). When we coated the
3
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11
Figure 4. Pharmacokinetic characteristics of [ C]2 in healthy rats (n=4, mean values ± SD). A: Dynamic PET scan (coronal plane) of all 39 frames of the brain
11
11
demonstrates brain uptake and distribution suited for imaging with even distribution of [ C]2 in the whole brain. B: Brain uptake of [ C]2 drops after the initial
perfusion peak and then slowly increases over time to a maximum uptake after 25 min, after which the activity slowly washes out. C: Ex vivo biodistribution after
75 min uptake time shows median uptake values in the brain between 0.24±0.05 (cerebellum) and 0.30±0.05 %ID/g (brainstem). Organ uptake was highest in the
liver with 0.79±0.22%ID/g.
To use the brain time-activity curves for calculation of binding
potential maps, we are now applying more sophisticated
approaches involving the arterial input function, and in parallel
we are following up on potential tissue metabolism. This will
allow us to further improve the tracer regarding both
pharmacokinetics and metabolic stability, as well as
quantification of αSyn in disease models, going beyond this
study in healthy animals where we would not expect αSyn
binding.
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1
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[
[
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1
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in healthy rats in vivo. This is the first report of
C
radiosynthesis and biodistribution analysis of this compound
class. Next steps will focus on improvement of the clogP value,
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between αSyn and other types of aggregates. Improved next-
generation candidates will be evaluated in animal models of
disease and on human pathological tissues. Although further
work is needed, these compounds might bear the potential for
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models, and in patients suffering from synucleinopathies.
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The research leading to these results has received funding from
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2013) under REA grant agreement n°602646. We thank Kerstin
Overkamp and Gerhard Wolf, MPIBPC, for performing the HPLC
and ESI-MS analyses, and Ramona Stumm and Elena Kimmerle,
WSIC, for performing automated radiosyntheses.
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Keywords: α-synuclein • radiotracer • Parkinson’s disease •
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5
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Entry for the Table of Contents
Development of radiotracers for alpha-synuclein has proven challenging. Here we describe a radiolabeling strategy for the
aggregation modulator anle253b (2) and demonstrate its successful penetration of the blood-brain barrier in healthy rats as well as its
high-affinity binding to aggregated alpha-synuclein in vitro.
6
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