Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the brain

Article abstract of DOI:10.1016/j.nucmedbio.2020.03.003

Introduction: Since the approval of three ¹⁸F labeled β-amyloid-targeting PET imaging agents, Amyvid (florbetapir f18, AV-45), Neuraceq (florbetaben f18, AV-1) and Vizamyl (flutemetamol f18, F-PIB), they have increasingly been employed to assis

Full text of DOI:10.1016/j.nucmedbio.2020.03.003

NMB-08133; No of Pages 10
Nuclear Medicine and Biology xxx (xxxx) xxx
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Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio
Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of
β-amyloid in the brain
Zhihao Zha a,b, Karl Ploessl a, Seok Rye Choi a, David Alexoff a, Hank F. Kung a,b,
⁎
a
Five Eleven Pharma Inc., Philadelphia, PA 19104, USA
b
Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Introduction: Since the approval of three 18F labeled β-amyloid-targeting PET imaging agents, Amyvid
(florbetapir f18, AV-45), Neuraceq (florbetaben f18, AV-1) and Vizamyl (flutemetamol f18, F-PIB), they have in-
creasingly been employed to assist differential diagnosis of Alzheimer's disease in patients with dementia. Also,
they are frequently used in selecting patients participating drug trials aiming to reduce β-amyloid (Aβ) plaques
in the brain. The first approved tracer in this class was [18F]AV-45, which is metabolized rapidly in blood and
some of its N-demethylated metabolites cross the blood brain barrier and resulted in lowering the image con-
trast. To improve metabolic stability of [18F]AV-45, we hypothesized that substituting N-CH3 with N-CD3 at the
metabolically labile position, creating [18F]D3FSP, may reduce in vivo N-demethylation. We report the preclinical
evaluation of [18F]D3FSP as an Aβ imaging agent.
Received 25 February 2020
Received in revised form 13 March 2020
Accepted 18 March 2020
Available online xxxx
Keywords:
Alzheimer's disease
Amyvid
Deuterium
In vivo metabolism
β-Amyloid
Methods: Preclinical pharmacology of [18F]D3FSP was evaluated using in vitro autoradiography and competitive
binding assay. Biodistribution of [18F]D3FSP was evaluated in wild-type CD-1 mice. In vivo metabolism in mice
and in vitro microsomal metabolism were analyzed by HPLC. Single dose acute toxicity of D3FSP was also per-
formed in rats.
Results: [18F]D3FSP showed high binding affinity to β-amyloid plaques (Ki = 3.44 1.22 nM, a value similar as
AV-45 (Ki = 4.02 0.22 nM)), and displayed excellent β-amyloid binding in AD brain sections consistent with
known Aβ regional distribution. After an iv injection it exhibited good initial brain uptake and fast washout in
wild-type CD-1 mice. In vitro microsomal metabolism and in vivo metabolism in mice did not result in any signif-
icant differences between [18F]D3FSP and [18F]AV-45. No treatment-related mortality or any adverse effects were
observed in single dose acute toxicity studies administered at hundred-folds of maximum human dose.
Conclusion: A new small molecule, [18F]D3FSP, was prepared and tested as an alternative to [18F]AV-45 to reduce
N-demethylation in vivo. This strategy did not lead to better in vivo stability. However, [18F]D3FSP displayed very
similar Aβ targeting property comparable to [18F]AV-45. Preclinical studies suggest that [18F]D3FSP is useful as a
β-amyloid-targeting PET imaging agent.
© 2020 Published by Elsevier Inc.
1. Introduction
indicated for positron emission tomographic (PET) imaging of the
brain in cognitively impaired adults undergoing evaluation for
Alzheimer's disease (AD) is the most common type of dementia. It is
projected that 14 million people will live with AD in the USA by 2060 [1]
. AD is a progressive and fatal neurodegenerative disorder. Accumula-
tion of β-amyloid plaques (Aβ) and intracellular neurofibrillary tangles
(Tau) in the brain are two neuropathologic hallmarks of AD. The detec-
tion of these hallmarks has shown diagnostic and prognostic value in
the management of Alzheimer's disease [2–5].
In 2012, the Food and Drug Administration (FDA) has approved a
new radiopharmaceutical agent, Florbetapir F18 (Amyvid, AV-45), to
assist clinicians in detecting causes of cognitive impairment other than
Alzheimer's disease. Florbetapir F18 injection (Amyvid™, Eli Lilly) is
Alzheimer's disease and other causes of cognitive decline [6]. The devel-
opment of β-amyloid plaque-targeting radiotracers has enabled non-
invasive PET imaging to quantitate β-amyloid deposition in a living
human brain [7–12]. As shown in Fig. 1, three PET tracers for in vivo vi-
sualization of brain β-amyloid plaques have been approved for clinical
use by the US Food and Drug Administration (FDA) and European Med-
icines Agency (EMA): [18F]AV-45 ([18F]florbetapir; Amyvid™) [13],
[
18F]AV-1 ([18F]florbetaben; Neuraceq™) and [18F]3′-F-PIB ([18F]
flutemetamol; Vizamyl™).
Recently, β-amyloid PET imaging has become an important tool in
the transformation of AD research, which could potentially accelerate
therapeutic drug development [14]. β-Amyloid PET is likely to contrib-
ute to better patient care as well [15]. Recent changes in using bio-
markers (Aβ deposition in the brain or Aβ in CSF) as a biological
⁎
Corresponding author at: Five Eleven Pharma Inc., Philadelphia, PA 19104, USA.
E-mail address: kunghf@sunmac.spect.upenn.edu (H.F. Kung).
https://doi.org/10.1016/j.nucmedbio.2020.03.003
0969-8051/© 2020 Published by Elsevier Inc.
Please cite this article as: Z. Zha, K. Ploessl, S.R. Choi, et al., Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the
brain, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2020.03.003

2
Z. Zha et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
Fig. 1. Three FDA approved PET tracers, Amyvid, Neuraceq and Vizamyl, for imaging β-amyloid plaques in assisting diagnosis of Alzheimer's disease (AD).
definition of AD was announced by the National Institute on Aging and
Alzheimer's Association [2,16]. New diagnostic recommendations for
the preclinical, mild cognitive impairment, and dementia stages of
Alzheimer's disease defined by its underlying pathologic processes can
be documented by in vivo biomarkers, i.e. using β-amyloid PET imaging
to measure Aβ load in patients [16]. Accessing the status of β-amyloid
plaque accumulation in the brain by non-invasive PET imaging is critical
for accurate diagnosis of AD, and also beneficial to selecting patients for
developing therapy for AD. Since the first approval of [18F]AV-45
(florbetapir f18, Amyvid) in 2012 [13], β-amyloid PET imaging agents
are being utilized in a clinical setting [17,18]. They improve subject se-
lection and treatment response monitoring for anti-amyloid therapies
during early phases of the disease [19,20].
Because [18F]AV-45 and [18F]AV-1 share close similarity in their
chemical structures, they showed as expected similar pharmacoki-
netic and metabolic fates [23]. The preclinical and clinical PK data
of both tracers showed that the radiolabeled metabolites, N-
demethylated metabolites, may contribute significantly to the total
radioactivity, and the N-demethylated metabolites displayed a 20
to 30-fold lower binding affinity [12,21,24] to the β-amyloid (Aβ)
plaques in the brain (Fig. 2). Preclinical experiments suggest that
these metabolites penetrate intact BBB and distribute into the
brain. Slowing down N-demethylation could lower the metabolites
entering the brain, which could contribute to an increase of specific
signal measured by PET imaging.
Deuterated medications are an active area in drug development
[25–27]. Various strategies to enhance metabolic stability might be
used and the deuterated drug approach is one of them [28]. Deuterium
(D) is a naturally-occurring, nonradioactive isotope of hydrogen (H). D
has a 2-fold higher mass than H, leading to a reduced vibrational
stretching frequency of the C–D bond. Also, C–D bonds are shorter
than C\\H bonds by 0.005 Å [27]. Therefore, the activation energy re-
quired for reaching the transition state for bond cleavage is greater for
C–D than C–H, and it is harder to break C-D bonds than C\\H bonds.
The difference in the stability of isotopically substituted molecules is re-
ferred to as the primary kinetic isotopic effect (KIE) [29]. To observe
high KIE by deuterium substitution, it is necessary that the C–H cleavage
step is at least partially rate-limiting among the multistep enzyme-
catalyzed metabolism. The efforts to exploit isotope effects in the design
of radiotracers resulted in a mixed outcome [30–33]. Deuterium substi-
tution in small molecules often does not result in a change of the shape,
As shown in Fig. 1, all three tracers contain an N-methyl group which
is crucial for high binding affinity to β-amyloid plaques. It was also
confirmed that N-demethylation is one of the major pathway of metab-
olism in
[ [
18F]AV-45 [12,21,22] and 18F]AV-1 [23]. Besides N-
demethylated derivatives, the other major radioactive metabolites
were identified as the N-acetylated derivative [18F]AV-267 (Fig. 2)
[22]. The same metabolites were identified in human plasma [21].
Both metabolites - derived from N-demethylation - exhibited lower
binding affinity to β-amyloid plaques, but they are capable of penetrat-
ing blood brain barrier (BBB). Therefore, the 18F-metabolites in the
brain could contribute to an increased nonspecific binding and lowering
the specificity for detecting β-amyloid plaques in the brain. It was hy-
pothesized that slowing down the N-demethylation by deuterium sub-
stitution of hydrogen might increase the brain uptake of the parent
radiotracer and reduce non-specific binding by the metabolites.
Fig. 2. Chemical structures of [18F]AV-45, its known radioactive metabolites and [18F]D3FSP.
Please cite this article as: Z. Zha, K. Ploessl, S.R. Choi, et al., Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the
brain, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2020.03.003

Z. Zha et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
3
size, charge or target pharmacology. However, because a carbon-
2. Results
deuterium chemical bond is stronger than a carbon-hydrogen bond,
deuterium substitution may slow down drug metabolism. This attenua-
tion of metabolism can lower the injection dose, lower radiation expo-
sure and sometimes improve image contrast [27]. In 2017, FDA
approved deuterotetrabenazine for the treatment of choreas associated
with Huntington's disease [34], the same indications of the parent drug,
tetrabenazine. This announcement provided a stamp of approval and
gave an endorsement for the pharmaceutical industry to develop addi-
tional deuterated derivatives of existing drugs as new chemical entities
[35].
Synthesis of D3FSP, D3AV-1 and related precursors and stan-
dards are included in the supplemental information. Results of
preparation and quality controls of [18F]D3FSP and [18F]AV-45 fol-
lowing established procedures are also included in the supplemen-
tal information.
Radiolabeling procedures (Fig. 3) were similar to published
methods for [18F]AV-45 [22]. Precursor (4) was radiolabeled with ac-
tivated [18F]fluoride with K222/K2CO3 in DMSO for 15 min at 110 °C.
The solution was allowed to cool and the protection group was
cleaved with 10% HCl solution at 100 °C for 10 min. The product,
Among many metabolic reactions catalyzed by cytochrome P450
(CYP450), amine N-dealkylation can be slowed down by deuterium
substitution when N-dealkylation is the rate-limiting step in drug
metabolism [36]. Mechanistic studies of model P450 enzyme cata-
lyzed N-demethylation reactions have shown that the first step pro-
ceeds via a hydrogen atom extraction (and thus susceptible to effects
of deuterium substitution), followed by oxidation reactions leading
to the removal of the N-methyl group [37]. Deuterium substitution
would in theory show a slower kinetics in breaking of C-D bond. It
was reported that [18F]AV-1 was metabolized by several CYP en-
zymes. Enzyme CYP4F2 predominantly mediates N-demethylation,
whereas CYP2J2 and CYP3A4 contribute predominantly to the for-
mation of polar metabolites. Among those, CYP2J2 and CYP4F2
were identified as the main enzymes involved in the metabolism of
[
18F]D3FSP, was then purified by semi-preparative HPLC. Activities
used ranged from 442 MBq to 4.1 GBq (n = 8), with radiochemical
purities of 98.5 1.3%. Radiochemical yields (decay corrected)
were 55 11% with molar activities (Am) of 22.8 12.4 GBq/μmol.
Similarly, [18F]AV-45 was prepared and tested for comparison with
[
18F]D3FSP as described in this paper.
2.1. In vitro competitive binding assay
In vitro binding studies, using either [18F]D3FSP or [18F]AV-45 as the
“hot ligand”, showed that hydrogen to deuterium substituted analog
(“cold” AV-45 vs D3FSP) exhibited the same excellent binding affinity
to β-amyloid plaques from AD human brain (Ki = 3 to 4 nM). No effect
was observed for deuterium substitution on binding affinity; all deuter-
ated agents, D3FSP and D3AV-1 displayed the same binding affinity as
their hydrogen analogs (Table 1).
[
18F]AV-1 [38]. From the results of [18F]AV-1, it is reasonable to
infer that the same CYP enzymes might also be involved in the me-
tabolism of [18F]AV-45.
Hydrogen–deuterium substitutions have been adopted in direct
proximity to [18F]fluoride to avoid 18F-defluorination [39]. A suc-
cessful example of the stabilization by means of deuteration consists
of the preparation of [18F]FE-(+)-DTBZ-D4 [40]. In vivo studies with
2.2. In vitro autoradiography of AD brain sections with [18F]D3FSP
Various postmortem AD brain sections were incubated in vitro with
[
18F]FE-(+)-DTBZ showed high accumulation of radioactivity in
[
18F]AV-45 or [18F]D3FSP to directly compare the tracer binding to β-
joints and bones. To improve the metabolic stability, [18F]FE-(+)-
DTBZ-D4 was developed and showed a six fold increase in half-life
stability. The main improvement was the considerably reduced
bone uptake when comparing with the non-deuterated tracer. Be-
cause [18F]AV-45 did not show significant defluorination, we have
selected not to substitute fluoroalkyl chains with deuterium. The hy-
drogens in the N-methyl group, which are known to metabolic deg-
radation, were chosen to substitute hydrogen on the N-methyl
group with deuterium.
amyloid plaques. The images of autoradiography (Fig. 4) showed a
broad spectrum of varying signal intensities obtained over the tissue
samples from different AD patients reflecting different levels of β-
amyloid plaques. The darkly speckled band around the edge of the pos-
itive tissue sections reflects [18F]D3FSP or [18F]AV-45 labeling of β-
amyloid plaques present in the gray matter, while the light central
area of the tissue reflects white matter that is not specifically labeled
by [18F]D3FSP or [18F]AV-45 (Fig. 4).
Blocking of [18F]D3FSP or [18F]AV-45 binding to β-amyloid plaques
in these brain sections by several known β-amyloid binding ligands or
self-blocking resulted in complete blocking of β-amyloid binding of
both tracers (Fig. 5). Blocking studies demonstrate that [18F]D3FSP
and [18F]AV-45 bind to the same sites, and the “cold” ligands - AV-45,
AV-1, PIB and IMPY compete with [18F]D3FSP and [18F]AV-45 for the
same binding sites.
To address the metabolic instability of [18F]AV-45, we have synthe-
sized a deuterated derivative of [18F]AV-45, [18F]D3FSP, in which N-
methyl group that plays a key role in its in vivo metabolism was replaced
with a N-trideuteromethyl group (–NCD3) (Fig. 2). Replacement of hy-
drogen with deuterium could improve the in vivo stability, which
could lead to better in vivo signal to noise ratios in the brain. Therefore,
[
18F]D3FSP and the ‘cold’ D3FSP were prepared and characterized with
respect to their in vitro binding to Aβ plaques in postmortem AD brain
tissue homogenates, in vivo biodistribution, in vitro microsomal and
in vivo metabolism in mice, and acute toxicity in rats. We compared
data of [18F]D3FSP with those of [18F]AV-45 which is an FDA approved
drug with proven safety and effectiveness.
2.3. In vitro liver microsomal metabolism
In vitro microsomal metabolism were conducted by incubation of
[
18F]D3FSP or [18F]AV-45 using the same batch of mouse and human
liver microsomes. The metabolites of [18F]D3FSP or [18F]AV-45 were
Fig. 3. Preparation of [18F]D3FSP, [18F]AV-45, [18F]AV-1 and [18F]D3AV-1.
Please cite this article as: Z. Zha, K. Ploessl, S.R. Choi, et al., Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the
brain, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2020.03.003

4
Z. Zha et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
Table 1
In vitro binding affinity to β-amyloid plaques of human AD brain homogenates (Ki, nM, Avg SD, n = 3).
measured using human and mouse liver microsomes in the presence of
an NADPH-generating system at 37 °C for 1, 5, 15, 30, 60 and 120 min.
The radioactive incubation mixtures were extracted and assayed using
HPLC, and the profiles of parent compounds and metabolites were de-
termined by reverse-phase HPLC. Incubation with mouse liver micro-
somes of [18F]D3FSP and [18F]AV-45 led to four metabolites, and five
metabolites were found after incubation with human liver microsomes.
The rate of formation of various metabolites for [18F]D3FSP and [18F]AV-
45 was analyzed by plotting the amount of the parent and each of the
metabolites against incubation time (Fig. 6).
metabolized fractions in plasma increased over time (Fig. 7. And
Fig. 8). Within 30 min after injection, only 21% of the parent [18F]
D3FSP was present in plasma. The profiling and identification of the me-
tabolites was performed by HPLC with radioactive detection and com-
parison with the data of [18F]AV-45. [18F]F-N-demethylated product
([18F]AV-160) and [18F]AV-267 were identified with cold standard com-
pounds previously [22], but other hydrophilic metabolites were not
characterized. Results suggested that both [18F]D3FSP and [18F]AV-45
displayed very similar in vivo metabolism profiles in the brain of mice.
The deuterium substitution apparently did not lead to any significant
changes in the metabolism in the brain of mice.
The study shows that both [18F]AV-45 and [18F]D3FSP were rapidly
metabolized by human and mouse liver microsomes (Fig. 6A and B).
After 30 min incubation with the mouse microsomes, only 1.5 to 3% of
the parent ligands remained and the dominant metabolite was the N-
demethylated product (M1) accounting for about 50% of the original ac-
tivity. The biological half-life of [18F]AV-45 and [18F]D3FSP was esti-
mated to be b5 min in this study. The metabolites detected after
incubation of [18F]AV-45 and [18F]D3FSP in liver microsomes from
mouse and human were the N-demethylated products, lipophilic me-
tabolites and hydrophilic metabolites. However, the results suggest
that there are no differences between [18F]D3FSP and [18F]AV-45. Both
agents showed very similar pattern of metabolites qualitatively and
quantitatively (Fig. 6C and D).
Analysis of plasma and brain extracts following iv administration
of [18F]D3FSP or [18F]AV-45 in normal mice were performed. Radio-
HPLC profiles of extracts from the brain showed three radioactive
metabolite peaks along with [18F]D3FSP with retention times at 2,
9.7, 10.9, and 13.4 min, respectively (HPLC profiles see SI Fig. S4a).
The pattern was again very similar to that of [18F]AV-45 (Fig. 7a
and b).
On the other hand, plasma showed two more radioactive metabolite
peaks with retention times at 4.3 and 16.6 min (HPLC profiles see SI
Fig. S4b). The main radioactive metabolite peak eluted at 2 min was
highly polar and remained to be identified. These findings are in accor-
dance with the in vitro metabolites observed after incubation with
mouse and human microsomes. The percentage of parent [18F]D3FSP
exponentially decreased as a function of time representing 74.2
2.4. Biodistribution
7.8% at 2 min and 20.8
2.3% at 30 min post-injection, respectively.
Following an iv injection into the tail vein of normal mice, [18F]D3FSP
distributed primarily to the liver and kidneys. Within 2 min post-
injection, there was 6.3% injected dose per gram (% dose/g) in the
brain. However, the activity cleared rapidly from the brain dropping to
1.7% dose/g in 30 min as shown in Table 2. Biodistribution studies of
During the same time, the percentage of the main polar 18F-
metabolite increased and reached 20.9 2.3% of plasma radioactivity.
Again a very similar pattern of metabolites were observed for [18F]AV-
45 (Fig. 8a and b). These radioactivity peaks associated with metabolites
may be due to the presence of [18F]fluoroacetate (or [18F]fluoroacetic
acid) generated by O-dealkylation of the tracer. The major radioactive
metabolite, N-demethylated product in plasma reached 50.8 4.0% at
60 min post-injection. Since there is no target binding site in the brain
of normal mouse, the percentage of [18F]D3FSP in the brain was very
low. In vitro microsomal metabolism and in vivo metabolism in mice
were not significantly different between the two radiotracers, [18F]AV-
45 and [18F]D3FSP; the deuterium substitution apparently did not
show modification of in vivo metabolism.
[
18F]D3FSP in normal mice have shown that, as expected, it is rapidly
distributed to the brain but not retained in the absence of β-amyloid de-
posits. Distribution and elimination from other tissues were also rapid.
Excretion was mainly via the gastrointestinal route. The biodistribution
of [18F]D3FSP (Table 2) and [18F]AV-45 (Table 3) in wild type CD-1 mice
showed very similar results. Both the [18F]D3FSP and [18F]AV-45
showed comparable brain penetration at 2 min and similar brain reten-
tion at later time points (60 and 120 min). The rapid clearance from the
brain without β-amyloid plaques would likely reduce non-specific
binding to the brain tissue leading to less non-specific binding in imag-
ing. Ratios of brain and blood uptake and clearance in mice of [18F]
D3FSP showed an excellent initial uptake in the brain and was followed
by a rapid clearance from the brain (Table 4).
It was stated in the package insert of Amyvid that the maximum
chemical dose per injection would be 50 μg [13]. Acute pharmaco-
logical toxicity test was performed in rats using one hundred times
of estimated maximum human dose. Rats received either vehicle
or 0.52 mg/kg D3FSP once on Day 1 via iv injection at a dose volume
of 4 mL/kg. On Day 2 and Day 15 after drug administration, clinical
observations, body weight measurements, blood samples for the
evaluation of hematology, clinical chemistry, and coagulation end-
points and histological examinations of organs were performed.
No drug related pathological findings by macroscopic or microscopic
examinations in any of the animals at either sacrifice intervals were
observed.
2.5. In vivo metabolism in normal mouse
The parent activity of [18F]D3FSP and [18F]AV-45, and their radioac-
tive metabolites from plasma and brain after iv injection in normal mice
were analyzed using HPLC. Both [18F]D3FSP and [18F]AV-45 were rap-
idly metabolized into various radioactive metabolites, and the
Please cite this article as: Z. Zha, K. Ploessl, S.R. Choi, et al., Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the
brain, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2020.03.003

Z. Zha et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
5
Fig. 4. Comparison of β-amyloid plaques binding between (a) [18F]AV-45 and (b) [18F]D3FSP and in vitro autoradiograms of frozen brain tissue sections from postmortem AD patients. Both
tracers showed consistent and comparable β-amyloid plaque binding.
3. Discussion
and hydrophobicity, it is possible that deuterated drugs bind more
tightly to their biological targets. However, we found no change of bind-
ing affinity with deuterium substitution, and the effect, if any, was not
measurable. Autoradiography of postmortem brain tissue sections
with [18F]D3FSP and [18F]AV-45 was performed to support selective
and specific binding of [18F]D3FSP to Aβ plaques. [18F]D3FSP showed
high specificity in binding to β-amyloid plaques by in vitro homogenate
binding assay and brain section autoradiography.
Generally, deuterated drugs retain their original biochemical po-
tency and selectivity, but deuteration can produce substantial benefits
to the overall pharmacological profile of the resulting compounds if cor-
responding C\\H bonds are involved in critical steps in pharmacokinet-
ics of drugs. Typically, cleavage of C\\H bond is the starting point of N-
demethylation and limiting N-demethylation might enhance the brain
delivery. Biodistribution of [18F]D3FSP in normal mice showed the abil-
ity of the tracer to rapidly cross BBB and rapid clearance from normal
mice brain. Results of biodistribution of [18F]D3FSP were comparable
with those of [18F]AV-45. Possible kinetic isotope effect in the
metabolism of [18F]D3FSP was limited to N-demethylation, which did
not induce a pronounced overall deuterium kinetic isotope effect. Deu-
terium substitution of N-methyl group of AV-45 might not produce
large enough effects in pharmacokinetics to modify the ultimate out-
come in imaging β-amyloid plaques in the brain. Although [18F]D3FSP
did not yield a positive kinetic isotope effect, we have successfully cre-
ated a new chemical entity which shows comparable preclinical proper-
ties to the FDA approved drug, [18F]AV-45.
Significant amount of information on in vivo metabolism of [18F]AV-
45 in humans as well as in mice has been reported in the literature
(Fig. 9) [21,22]. In vivo metabolism of [18F]AV-45 in mice showed that,
at 30 min after an intravenous injection, only 30% of the parent [18F]
AV-45 remained in the plasma. The biological half-life of [18F]AV-45 in
mouse plasma was estimated to be b30 min. Metabolite profiling and
identification of the metabolites were done by HPLC with radioactive
detection and liquid chromatography/mass spectroscopy analysis. Sim-
ilar to that reported previously in mice and in humans several metabo-
lites of [18F]AV-45 were identified [21,22]. One of the major plasma
metabolites was N-demethylated [18F]AV-45 (designated as [18F]AV-
160), which constituted about 48% of the metabolites at 30 min after in-
jection. The brain uptake of [18F]AV-160 at 2 min after injection was
4.5%ID/g of tissue, and decreased to 1.8%ID/g at 60 min. The initial up-
take of the parent [18F]AV-45 was 1.5-fold higher than this metabolite.
Also, a lipophilic metabolite, [18F]AV-267, was identified. No significant
binding to Aβ plaques was observed with either of the metabolites using
AD brain-section autoradiography and the in vitro AD brain homogenate
binding assay. The inhibition constants of AV-160 and AV-267 (Ki = 54
and 398 nM, respectively) indicate at least a 20–100 fold reduction of
binding affinity to Aβ plaques in AD brain tissue homogenates, as com-
pared with that of AV-45 (Ki = 2.87 nM). N-demethylation was also
identified as main metabolic pathway in [18F]AV-1 [42,43].
In vitro binding assay and in vitro autoradiography studies were used
to measure [18F]D3FSP affinity to β-amyloid plaques in the AD brain tis-
sue homogenates. Because deuterium and hydrogen differ both in size
One other purpose of this research is to register a new β-amyloid
plaque imaging agent for AD patients in China and other Asian countries
(a)
(b)
Control AV-45
D3FSP
AV-1
PIB
IMPY
Fig. 5. Autoradiography images of postmortem AD brain slices by in vitro section labeling with (a) [18F]AV-45 and (b) [18F]D3FSP: control and blocking of β-amyloid plaque binding by
various competing drugs. Concentrations of competing drugs were 1–5 μM.
Please cite this article as: Z. Zha, K. Ploessl, S.R. Choi, et al., Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the
brain, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2020.03.003

6
Z. Zha et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
Fig. 6. Time-course of in vitro microsomal metabolism of (a) [18F]AV-45 with mouse microsomes (b) [18F]D3FSP with mouse microsomes (c) [18F]AV-45 with human microsomes (d) [18F]
D3FSP with human microsomes. M1 is the corresponding N-demethylated [18F]AV-45 or [18F]D3FSP tracer. [18F]AV-45 and [18F]D3FSP displayed very similar in vitro metabolism profiles.
currently not served by the current FDA approved agents. Although,
there are already three FDA approved β-amyloid plaque imaging agents
available in the US, Japan and European countries, none of them has
been successfully introduced in other markets. There is an enormous
demand for β-amyloid plaque imaging in China and other Asian coun-
tries, because the current AD population is over ten million, which is
likely more than the AD patients in the US and Europe combined.
There is clearly an unmet clinical need in Asian countries and it would
be entirely fair to develop one additional β-amyloid plaque imaging
agent just for these countries. The new agent, [18F]D3FSP, might serve
as a clinical tool for assisting diagnosis and benefiting AD patients in
these countries currently not being served by the approved β-amyloid
plaque imaging tracers.
[
18F]D3FSP displayed high specificity in binding to β-amyloid
plaques similar to that observed for [18F]AV-45 (Amyvid). In vitro and
in vivo metabolism in mice suggested that both agents showed very
similar pattern of metabolites, but deuterium substitution did not
change in vivo metabolism. In vivo biodistribution in normal mice also
exhibited very similar high brain penetration and rapid wash-out, a nec-
essary property for effective brain imaging agents. It is reasonable to
conclude that the hypothesis of improving the in vivo properties by
using deuterated [18F]D3FSP to reduce the N-demethylation did not
Table 2
Table 3
Biodistribution of [18F]D3FSP in wild type CD-1 mice (% dose/g, n = 3).
Biodistribution of [18F]AV-45 in wild type CD-1 mice (% dose/g, n = 3).
Organ
2 min
30 min
60 min
120 min
Organ
2 min
30 min
60 min
120 min
Blood
Heart
Muscle
Lung
Kidney
Spleen
Liver
2.16
5.08
3.21
4.88
9.51
2.50
12.00
6.26
1.74
0.05
0.39
0.14
0.16
0.80
0.12
0.70
0.23
0.18
2.93
2.40
1.79
2.90
6.41
1.52
11.99
1.71
2.24
0.27
0.39
0.06
0.47
1.86
0.21
0.71
0.37
0.44
2.08
1.72
1.20
1.97
3.74
1.22
8.15
1.53
2.76
0.07
0.19
0.04
0.25
0.47
0.12
0.75
0.20
0.37
1.36
1.18
0.75
1.31
2.09
0.83
5.47
1.26
4.41
0.12
Blood
Heart
Muscle
Lung
Kidney
Spleen
Liver
2.28
4.22
2.57
4.04
9.60
1.60
11.08
7.33
1.62
0.15
0.58
0.08
0.71
0.96
0.78
2.83
1.54
0.26
3.27
2.54
1.59
1.98
8.52
1.61
12.72
1.66
2.34
0.27
0.31
0.22
0.33
1.17
0.22
0.61
0.15
0.18
2.39
2.03
1.31
2.32
5.79
2.36
9.66
1.49
3.21
0.46
0.13
0.05
0.15
1.53
0.74
0.16
0.16
0.54
1.66
1.51
1.01
1.54
2.67
1.07
6.01
1.29
2.81
0.24
0.03
0.07
0.03
0.02
0.07
0.29
0.04
0.77
0.24
0.23
0.23
0.49
0.20
1.03
0.06
0.87
Brain
Bone
Brain
Bone
Please cite this article as: Z. Zha, K. Ploessl, S.R. Choi, et al., Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the
brain, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2020.03.003

Z. Zha et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
7
Table 4
0.7 mL K222/K2CO3 solution (40 mg K2CO3, 22 mg K222, 18.4 mL ACN,
3.6 mL water) into a 1 mL conical vial. The solution was dried
azeotropically twice with 1 mL acetonitrile at 80 °C. Ligand 4 (1.2 mg)
was dissolved in 0.5 mL DMSO and added to the dried [18F]fluoride
and vial was crimped. The mixture was heated for 15 min at 110 °C.
The mixture was cooled at RT for 1 min; the vial was then opened and
250 μL HCl (10%) was added and heated at 100 °C for 10 min. The reac-
tion medium was added to 8 mL ice cold water, then 0.85 mL 1 N NaOH
was added (final pH ~6). The mixture was loaded on an activated Oasis
HLB 3 cc (60 mg) extraction cartridge. The cartridge was rinsed with
2 × 3 mL water and the product was eluted with 1 mL ACN. The eluted
acetonitrile solution was diluted with 1 mL water and injected into prep
HPLC: Phenomenex Gemini C18, 250 × 10 mm, ACN/water 60/40, 4 mL/
min, 350 nm. The fraction at 11–12 min was collected. The fraction was
diluted with 30 mL water and pushed through an activated Oasis HLB
3 cc (60 mg) extraction cartridge; the cartridge was washed with
3 mL water. The product was eluted with 1 mL EtOH (containing ascor-
bic acid). The ethanolic solution was concentrated under argon to about
100 μL volume and diluted with 900 μL saline. The ligand was evaluated
by radio HPLC (Supelco Ascentis C18 150 × 4.6, ACN/10 mM AFB (am-
monium formate buffer) 50/50, 1 mL/min, 350 nm) ([18F]D3FSP: RCP:
98.5 1.3% (n = 8), RCY: 54 11% (decay corrected), molar activity
Ratios of brain and blood uptake and clearance in normal mice.
Brain
2 min/60 min
Blood
2 min/60 min
Brain/blood
2 min
Brain/blood
60 min
[
[
18F]D3FSP
18F]AV-45
4.1
4.9
1.0
1.0
2.9
3.2
0.7
0.6
work as originally envisioned. However, the new deuterium substituted
agent, [18F]D3FSP, might be useful for imaging β-amyloid plaques in the
brain. Recently, results of a phase I clinical study in humans (Dr. Dean
Wong, Johns Hopkins University, un-published data, IND #137713,
SNMMI abstract 2019) suggested that in the same AD patient [18F]
D3FSP and [18F]AV-45 exhibited very similar patterns of brain distribu-
tion reflecting the β-amyloid plaque specific binding. However, there
was no measurable delayed metabolic N-demethylation in vivo between
[
18F]D3FSP and [18F]AV-45 in AD patients suggesting that the original
hypothesis could not be realized.
4. Conclusion
A new β-amyloid plaque targeting PET imaging agent, [18F]D3FSP - a
N-trideuteromethyl (–NCD3) analog of [18F]AV-45, was prepared and
tested. In vivo and in vitro studies suggest that [18F]D3FSP binds to β-
amyloid plaques with high affinity and displays a suitable pharmacoki-
netic characteristics comparable to [18F]AV-45. But the deuterium sub-
stitution did not lead to significant beneficial kinetic effects on slowing
in vivo N-demethylation of [18F]D3FSP. However, results of preclinical
studies suggest that [18F]D3FSP might be a suitable candidate as a new
Aβ imaging agent to assist diagnosis of AD for a large patient population
in countries currently not being served by FDA approved agents.
(Am): 22.8
were prepared with RCP: 98.4% (n = 6), RCY 46 8 (decay corrected),
m = 22 14 GBq/μmol.
12.4 GBq/μmol). Similarly, [18F]AV-45 and [18F]AV-1
A
5.3. In vitro competitive binding assay
Competitive binding assays were performed in 12 × 75 mm
borosilicate glass tubes. The reaction mixture contained 100 μL of AD
brain homogenates (20–25 μg), 100 μL of [18F]AV-45 or [18F]D3FSP
(~150,000 cpm), and 100 μL of competing compounds (10−5 to
10−10 M diluted serially in PBS containing 0.1% bovine serum albumin)
in a final volume of 250 μL. Nonspecific binding was defined in the pres-
ence of 1 μM of IMPY (6-iodo-2-(4′-dimethylamino-)phenyl-imidazo
[1,2-a]pyridine) in the same assay tubes [41]. The mixture was incu-
bated for 60 min at room temperature, and the bound and the free ra-
dioactivity were separated by vacuum filtration through Whatman GF/
B filters using a Brandel M-24R cell harvester, followed by washing
with PBS buffer three times. The radioactivity on the filters was counted
with a gamma counter (Wizard2, Perkin-Elmer). Data were analyzed
using the nonlinear least-square curve fitting program LIGAND to deter-
mine IC50. Ki was calculated by Cheng-Prusoff equation using 3.51 nM as
Kd of AV-45 and D3FSP.
5. Materials and methods
Synthesis of D3FSP and related agents see Supplemental
information.
5.1. Material
All reagents and solvents were purchased commercially (Aldrich,
Acros, or Alfa Inc.) and were used without further purification, unless
otherwise indicated. Solvents were dried through a molecular sieve sys-
tem (Pure Solve Solvent Purification System; Innovative Technology,
Inc.).
5.2. Radiosynthesis of [18F]D3FSP and [18F]AV-45
5.4. In vitro autoradiography AD brain section labeling of [18F]D3FSP
An activated Sep-Pak Light (Waters, Accell Plus QMA Carbonate)
Frozen brains from AD and control subjects were cut into 20 μm sec-
was loaded with
[
18F]fluoride (0.4–4.1 GBq) and eluted with
tions. The sections were incubated with [18F]D3FSP in 40% ethanol at a
Fig. 7. Time-course of in vivo metabolism profiles of (a) [18F]AV-45 (b) [18F]D3FSP in the brain of normal mice: M1: Hydrophilic metabolite, M2: N-Demethylated AV-45 ([18F]AV-160), M3:
Lipophilic metabolite ([18F]AV-267).
Please cite this article as: Z. Zha, K. Ploessl, S.R. Choi, et al., Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the
brain, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2020.03.003

8
Z. Zha et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
Fig. 8. Time-course of in vivo metabolism of (a) [18F]AV-45 (b) [18F]D3FSP in the plasma of normal mice: M1: Hydrophilic metabolite, M2: Hydrophilic metabolite, M3: N-Demethylated
AV-45 ([18F]AV-160), M4: Lipophilic metabolite, ([18F]AV-267), M5: Lipophilic metabolite.
concentration of 0.3 nM for 1 h. The sections were then washed with
40% ethanol (2-min wash three times), followed by rinsing with water
for 30 s. After drying, the sections were exposed to image plate for
30 min. Digitized images were acquired with Typhoon FLA 7000 (GE
Healthcare).
5.6. Biodistribution in CD-1 mice
Animal studies were undertaken in compliance with University of
Pennsylvania IACUC guidelines related to the conduct of animal experi-
ments. CD-1 mice (20–26 g, male) were injected with [18F]D3FSP or
[
18F]AV-45 (720–925 kBq/mouse), 150 μL formulated in 10% ethanol
and 90% saline directly into the tail vein. The mice were sacrificed by
cardiac puncture under isoflurane anesthesia at various time points (2,
30, 60, and 120 min) after the injection. Organs of interest were re-
moved and weighed, and the radioactivity was counted with a γ-
counter. The injected percentage dose per organ was calculated by a
comparison of the tissue counts to suitably diluted aliquots of the
injected material. Each time point consisted of a group of 3 animals.
5.5. In vitro liver microsomal metabolism
RapidStart™ NADPH regenerating system, human liver microsomes
(Pool of 50, mixed gender, Lot No 1610016) and CD1 mouse liver micro-
somes (Pool of 1200, male, Lot No 1610148) were purchased from
Sekisui XenoTech (Lenexa, KS). RapidStart™ NADPH regenerating sys-
tem was reconstituted with 4 mL water. 25 μL of the NADPH-
generating system, 2.22 MBq of [18F]D3FSP or [18F]AV-45 in 100 μL
phosphate buffer solution, and 15 μL of human or mouse liver micro-
somes were employed in the study. The control vials were prepared
by the same way without microsomes. The capped vials were gently
shaken and placed in an incubator at 37 °C. At 1, 5, 15, 30, 60 and
120 min, 20 μL of the mixtures were quenched with 100 μL of acetoni-
trile, shaken vigorously for 10 s and centrifuged for 3 min at 5000 ×g.
The acetonitrile layer was removed shortly before HPLC analysis. The
RP-HPLC was performed using Agilent 1100 Series HPLC equipped
with an Agilent XDB C18 column and the radiometric peaks were de-
tected using a Bioscan flow detector.
5.7. In vivo metabolites analysis by HPLC
Between 37 and 74 MBq of [18F]D3FSP or [18F]AV-45 was injected
into the tail vein of CD-1 mice (three mice per each group). The mice
were sacrificed at 2, 30 or 60 min after the injection. The blood samples
were centrifuged at 2000 ×g for 3 min to separate plasma. Plasma sam-
ples were mixed with equal volumes of acetonitrile followed by centri-
fugation at 5000 ×g for 5 min to remove the denatured proteins. The
supernatant was then analyzed directly by HPLC. The HPLC analysis
was performed on an Agilent XDB-C18 column (150 × 4.6 mm) with
0.1% trifluoroacetic acid in water and acetonitrile gradient at a flow
Fig. 9. Proposed metabolic pathways in humans of (a) [18F]AV-45 (florbetapir f18, Amylvid) by AVID Radiopharmaceuticals [44] and (b) [18F]AV-1(florbetaben f18, Neuraceq) by Bayer
Healthcare [45]. As indicated, one of the major metabolites were the N-demethylated derivatives for [18F]AV-45 and [18F]AV-1. The metabolites showed lower binding affinities to Aβ
plaques in the brain.
Please cite this article as: Z. Zha, K. Ploessl, S.R. Choi, et al., Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the
brain, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2020.03.003

Z. Zha et al. / Nuclear Medicine and Biology xxx (xxxx) xxx
9
rate of 1 mL/min. For the determination of metabolites in the brain,
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HPLC. The retention times under these conditions were approximately
2.0 and 4.3 min for polar metabolites, 9.7 min for N-demethylated prod-
uct, 10.9 min for parent ligand and 13.4 min and 16.6 min for lipophilic
metabolites.
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Please cite this article as: Z. Zha, K. Ploessl, S.R. Choi, et al., Preclinical evaluation of [18F]D3FSP, deuterated AV-45, for imaging of β-amyloid in the
brain, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2020.03.003