Radiofluorinated histamine H3 receptor antagonist as a potential probe for in vivo PET imaging: Radiosynthesis and pharmacological evaluation

Article abstract of DOI:10.1016/j.bmc.2012.03.024

The histamine H₃ receptor (H₃R) plays a role in cognition and memory processes and is implicated in different neurological disorders, including Alzheimer's disease, schizophrenia, and narcolepsy. In vivo studies of the H₃R occupancy using a radiolabeled PET tracer would be very useful for CNS drug discovery and development. We report here the radiosynthesis, in vitro and in vivo evaluation of a novel ¹⁸F- labeled high-affinity H₃R antagonist ¹⁸F-ST889. The radiosynthesis was accomplished via nucleophilic substitution of the mesylate leaving group with a radiochemical yield of 8-20%, radiochemical purity >99%, and specific radioactivity >65 GBq/μmol. ¹⁸F-ST889 exhibited high in vivo stability and rather low lipophilicity (log D_7.4 = 0.35 ± 0.09). In vitro autoradiography showed specific binding in H ₃R-rich brain regions such as striatum and cortex. However, in vivo PET imaging of the rat brain with ¹⁸F-ST889 was not successful. Possible reasons are discussed.

Full text of DOI:10.1016/j.bmc.2012.03.024

Bioorganic & Medicinal Chemistry 20 (2012) 2889–2896
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Radiofluorinated histamine H3 receptor antagonist as a potential probe
for in vivo PET imaging: Radiosynthesis and pharmacological evaluation
Svetlana V. Selivanova a, , Michael Honer a, Francine Combe a, Kathleen Isensee b, Holger Stark b,
⇑
Stefanie D. Krämer a, P. August Schubiger a, Simon M. Ametamey a
a Center for Radiopharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich (Swiss Federal Institute of Technology), Wolfgang-Pauli-Strasse 10,
CH-8093 Zurich, Switzerland
b Institute of Pharmaceutical Chemistry, ZAFES/LiFF/OSF, Johann Wolfgang Goethe University, Biozentrum Max-von-Laue-Strasse 9, 60348 Frankfurt-am-Main, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The histamine H3 receptor (H3R) plays a role in cognition and memory processes and is implicated in dif-
ferent neurological disorders, including Alzheimer’s disease, schizophrenia, and narcolepsy. In vivo stud-
ies of the H3R occupancy using a radiolabeled PET tracer would be very useful for CNS drug discovery and
development. We report here the radiosynthesis, in vitro and in vivo evaluation of a novel 18F-labeled
high-affinity H3R antagonist 18F-ST889. The radiosynthesis was accomplished via nucleophilic substitu-
tion of the mesylate leaving group with a radiochemical yield of 8–20%, radiochemical purity >99%,
Received 16 December 2011
Revised 5 March 2012
Accepted 9 March 2012
Available online 17 March 2012
Keywords:
Histamine H3 receptor
H3R
PET imaging
Neurological disorders
CNS
and specific radioactivity >65 GBq/l
mol. 18F-ST889 exhibited high in vivo stability and rather low lipo-
philicity (logD7.4 = 0.35 0.09). In vitro autoradiography showed specific binding in H3R-rich brain
regions such as striatum and cortex. However, in vivo PET imaging of the rat brain with 18F-ST889 was
not successful. Possible reasons are discussed.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
located mainly in the central nervous system (CNS) and is scarce
in the periphery. It is distributed in many brain areas, namely the
Histamine receptors are G-protein coupled receptors (GPCRs).
They exist in four different subtypes named H1R to H4R. H1R and
H2R are targets for anti-allergy and anti-ulcer medications, respec-
tively.1 H4R is claimed to be acting in inflammation and immune
response.2,3 H3R plays an important role in neuronal activities such
as cognition and memory.4 It is thought to be involved in different
neurological disorders, including Alzheimer’s disease,5 attention-
deficit/hyperactivity disorder,5 narcolepsy,6,7 epilepsy,6 and
schizophrenia8 among others.1,9 H3R was first identified pharmaco-
logically in 1983 as a mediator of histamine release.10 It is accepted
now that among other physiological functions H3R regulates excre-
tion of histamine and other important neurotransmitters, such as
acetylcholine, noradrenalin, dopamine, and serotonin.11–13 H3R is
cerebral cortex, striatum, hypothalamus, substantia nigra,
accumbens nucleus, olfactory tubercle, caudate, and amygdala, as
identified by autoradiography in rodents.8,14,15
In vivo studies of the distribution and density of H3R in humans
and quantification of the receptor occupancy would be useful for
CNS drug discovery and development. Positron-emission tomogra-
phy (PET) can offer such an opportunity. For this, special probes la-
beled with positron-emitting radionuclides have to be developed.
These radiolabeled compounds must possess very high affinity
(low nanomolar to subnanomolar range), high target selectivity
and appropriate pharmacokinetics, including the capability to
cross the blood–brain barrier (BBB). The main radioisotopes used
in PET are carbon-11 (11C) and fluorine-18 (18F). 18F has excellent
physical characteristics, including a longer half-life, allowing for
lengthier synthetic procedures and the possibility to deliver to
centers, which do not have their own dedicated radiochemistry
laboratories and a cyclotron.
Attempts to prepare PET tracers for H3R started back in the
1990’s (Fig. 1). Two imidazole derivatives were radiolabeled with
18F and 11C, giving 18F-FUB-272 and 11C-UCL-1829, respectively.
Biodistribution studies with 18F-FUB-272 showed poor brain
uptake and high non-specific binding. Similarly, 11C-UCL-1829
revealed low brain penetration and very high uptake in the lungs.16
Radiofluorinated VUF 5000 was considered as a potential probe but
Abbreviations: H3R, histamine H3 receptor; CNS, central nervous system; GPCRs,
G-protein coupled receptors; BBB, blood–brain barrier; CYP, cytochrome-P450; P-
gp, P-glycoprotein; PET, positron emission tomography; EOB, end of bombardment;
EOS, end of synthesis; HPLC, high-performance liquid chromatography; UPLC, ultra
performance liquid chromatography; TLC, thin-layer chromatography; DMF,
dimethylformamide; DMSO, dimethylsulfoxide; PBS, phosphate buffered saline;
TFA, trifluoroacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonate;
DMEM, Dulbecco’s modified Eagle medium; MW, molar weight; MDR1, multidrug
resistance protein 1; MDCK, Madin–Darby canine kidney.
⇑
Corresponding author. Tel.: +41 44 633 60 94; fax: +41 44 633 13 67.
E-mail address: svetlana.selivanova@pharma.ethz.ch (S.V. Selivanova).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.024

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S. V. Selivanova et al. / Bioorg. Med. Chem. 20 (2012) 2889–2896
the reaction is multistep and involves distillation of a radiolabeled
intermediate, which is not optimal for an eventual automation.
11C-GSK189254 progressed to the first human PET study in healthy
volunteers and was found to be promising in quantifying H3R in
humans.25,26 Despite recent developments and progress of 11C-
labeled radioligands into clinical trials, there is still a need for
potent and specific H3R tracers labeled with a longer-lived 18F.
Recently, a series of novel non-imidazole histamine H3R antago-
nists were described,27 based on the lead structure of JNJ-
520785228,29 and FUB 88030 (Fig. 2). These compounds possess high
affinity and selectivity to H3R in vitro. In vivo potency was found to
be high after oral administration to mice. Compound ST889 (Fig. 2)
was selected as a potential PET tracer candidate due to its high
binding affinity (hH3R Ki = 0.094 nM)27 and amenability to 18F-
radiolabeling.
This paper describes the radiosynthesis, in vitro and in vivo
evaluation of 18F-ST889 as a potential PET probe for imaging H3R.
2. Results and discussion
2.1. Chemistry and radiochemistry
The precursor for the radiolabeling and cold reference com-
pound were synthesized as described previously.27
Figure 1. Previously attempted H3R PET tracers.
Radiosynthesis (Scheme 1) was performed in one step and opti-
mized for solvent (acetonitrile, DMF, DMSO), reaction time (5–
30 min), and temperature (90–140 °C). Acetonitrile was the pre-
ferred solvent giving higher yields. Heating up to 140 °C in DMF
or DMSO did not offer any improvement. Precursor concentration
showed low brain uptake in rats.17,18 Synthetic steps were de-
scribed for 18F-fluoroproxyfan,19 which shows saturable binding
in vitro.20 To our knowledge, no in vivo imaging was reported for
this compound to date. The failure of the imidazole-containing
compounds in general might be explained by their interaction with
cytochrome-P450 (CYP) enzymes.21 Ligands without imidazole
moiety were also described. Selective H3R ligand JNJ-10181457 la-
beled with 11C had promising in vitro properties but failed in vivo
due to high non-specific binding, which could not be blocked with
H3R antagonists.22 More recently, a short report from Merck Labo-
ratories described a carboxamide, which was labeled with either
11C or 18F and showed good in vivo binding and specificity.23 In
vivo PET studies in rhesus monkeys using either of these tracers
proved that they could be used to measure receptor occupancy
by H3R inverse agonists.24 However, the radiofluorinated carbox-
amide analogue was obtained in rather modest yields. Moreover,
in a range of 17–35
noticeably, thus lower concentration was used. It should be noted
that precursor concentration of 10–12 mol/mL gave considerably
lmol/mL did not influence the reaction yield
l
lower yields. Purification was accomplished by semi-preparative
HPLC and the product was collected in a volume of 3–4 mL. The final
formulation was performed using solid phase extraction: the prod-
uct was concentrated on a Sep-Pak Light C18 cartridge and eluted
with 0.5 mL ethanol. Dilution with 9.5 mL of phosphate buffered
saline (PBS) resulted in a solution acceptable for in vivo biological
applications. Calculated radiochemical yields based on the isolated
product were 8–20% (decay-corrected to the end of bombardment
(EOB), n = 12). Typically, 4.1–9.5 GBq (110–257 mCi) of 18F-ST889
could be obtained within 100–110 min (including HPLC purifica-
tion) starting from ꢀ80–100 GBq (2.1–2.7 Ci) of 18F-fluoride.
To prepare an ethanol-free formulation, the eluate from the
Sep-Pak cartridge was evaporated at room temperature under
reduced pressure and a stream of nitrogen and the residue was
reconstituted in PBS. Analysis of the formulated solution showed
that 18F-ST889 partially decomposed during the evaporation proce-
dure, most probably due to self-radiolysis. Two polar products of
the decomposition were detected in addition to the parent com-
pound. One, likely 18F-fluoride, was eluting with the solvent front,
followed by a second unidentified compound, which was present
only in selected batches. The extent of radiolysis was dependent
on the shelf time. Radiochemical purity of the formulated in PBS
18F-ST889 decreased to 89% 30 min after formulation and declined
further to 73% after 2 h. Radiolysis is a known phenomenon for
many compounds with high specific radioactivity and activity
concentration.31 It can be often avoided by addition of ascorbic acid
or sodium ascorbate to the ethanolic solution of the radioactive
Figure 2. Non-imidazole H3R antagonists.
Scheme 1. Optimized radiolabeling conditions.

S. V. Selivanova et al. / Bioorg. Med. Chem. 20 (2012) 2889–2896
2891
compound before evaporation. Addition of ascorbic acid (2 mg/mL,
2.4. Lipophilicity (logD7.4)
calculated for the final formulated solution volume) suppressed
radiolysis and the final product was obtained in high activity
concentration in ethanol-free aqueous solution. However, the
ethanol containing formulation was used for biological studies
described herein.
In vivo H3R targeting in the CNS requires good BBB passage of
the ligand. The partition coefficient logP is often used to evaluate
the ability of a drug to cross BBB. 18F-ST889 is a base with two pro-
ton-accepting nitrogen atoms, which are protonated at physiolog-
ical pH. For easily ionizable compounds the distribution coefficient
logD at physiological pH 7.4 (logD7.4) is used instead.32
2.2. Quality control
Partitioning of 18F-ST889 between octanol and phosphate buffer
(pH 7.4) was determined using the shake-flask method.33 The
experimental logD7.4 = 0.35 0.09 was calculated from three inde-
pendent determinations. This is a relatively low value but may still
allow significant tracer concentration in the brain. Significant BBB
permeation by passive diffusion is expected for compounds with
logD7.4 between 0 and 3.32,34 However, many exceptions exist to
this rule, including effective brain radiotracers.35 There is also clear
evidence that structurally similar JNJ-5207852 (Fig. 2) reaches high
brain levels 60 min after subcutaneous administration in mice.29
The addition of a fluoro-substituent to one of the piperidine rings
may result in a minor loss in lipophilicity but is not expected to les-
sen brain accessibility drastically.36,37
Quality control of 18F-ST889 was performed on an aliquot of the
formulated radiopharmaceutical ready for injection using an ana-
lytical HPLC system. The pH of the clear colorless radiotracer solu-
tion was between 7 and 8. The product was >98% chemically and
radiochemically pure and the formulation contained no precursor.
The apparent specific activities were 65–100 GBq/
lmol (1.7–
2.7 Ci/
l
mol) at the end of synthesis (EOS). 18F-ST889 was radio-
chemically stable in the solution for at least 4 h (confirmed by
UPLC).
2.3. Binding affinity to rat H3R
The Ki of the non-radioactive ST889 to human recombinant H3R
was reported to be 0.094 nM (pKd 10.03).27 To exclude significant
species differences in the binding affinity of 18F-ST889 between
human and rat H3R we determined the equilibrium dissociation
constant Kd of 18F-ST889 to native rat brain H3R in a pilot experi-
ment using rat whole brain homogenate. The estimated Kd value
2.5. In vitro metabolic stability in plasma and liver microsomes
The stability of 18F-ST889 in plasma and liver microsomes was
tested in vitro by incubating the radioligand with human or rodent
plasma and liver microsomes at 37 °C. 18F-ST889 showed good
in vitro plasma stability without marked decomposition during
the first 60 min. Minor defluorination occurred in human plasma
after 90 min when 18F-ST889 accounted for 98% of the total radio-
activity in the sample with a decomposition degree still under 3%
after 120 min.
was 0.34 nM (pKd 9.47) with maximum binding density (Bmax
)
930 fmol/mg protein. The Bmax for the structurally similar JNJ-
5207852 was reported to be 57 fmol/mg protein determined in
rat cerebral cortex membranes.29 Non-linear curve fitting and Scat-
chard plot analysis (Fig. 3) revealed one high affinity binding site.
The estimated Kd of a low affinity interaction was P3
lM at Bmax
18F-ST889 was stable in human and rodent liver microsomes up
to 120 min. The oxidative activity of the enzymes was confirmed
with testosterone as positive control. After 90 min 60% of the initial
testosterone was oxidized by human microsomes, while it was
completely metabolized by rat microsomes.
220 pmol/mg protein. The high Bmax value excludes specific bind-
ing to GPCRs in case of the low affinity interaction site. The binding
potential (Bmax/Kd) is about 40-fold higher for the high affinity
binding, ruling out significant non-specific accumulation of the
tracer in brain due to the low affinity binding. Only 55% of
radioactivity was displaced by 10
l
M (R)-
a
-methylhistamine
2.6. In vitro autoradiography
while 10
l
M cold ST889 displaced the 18F-analogue (0.06 nM)
completely (99.8%). The determined subnanomolar affinity to
rat H3R, although a slightly lower than to human H3R, is suitable
for PET imaging. A similar species-dependence was described
for JNJ-5207852 with a pKi of 9.24 to human H3R and 8.90 to
rat H3R.29
In vitro autoradiography was carried out on horizontal rat brain
slices with 18F-ST889 alone or in the presence of 100
lM hista-
mine. Tracer binding showed a heterogeneous distribution with
the highest density in the striatum and cerebral cortex (Fig. 4).
Moderate 18F-ST889 binding was identified in hippocampus and
Figure 3. Binding affinity of ST889 to rat brain crude membranes. Rat brain crude membranes were incubated with 0.06 nM 18F-ST889 and various concentrations of cold
ST889. Bound ST889 (parameter B) and the molar concentration of free (unbound) ST889 were calculated from bound 18F-ST889 and total ST889. A high and a low affinity
binding site were identified by non-linear curve fitting (A). Continuous line: fit function shown in (Eq. 1); dashed line: contribution of high affinity binding site (fitted Kd
0.34 nM); dash-dot line: contribution of low affinity binding site (fitted Kd P3 lM). The results are displayed as a Scatchard plot with the fitted parameters (B). Expanded x-
axis scale is in the insert.

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S. V. Selivanova et al. / Bioorg. Med. Chem. 20 (2012) 2889–2896
thalamus, whereas binding in the cerebellum was clearly lower.
This distribution of ligand binding is consistent with previously
published data on the localization of H3R in the rat brain.9 More-
over, the tracer binding to striatum, cortex, hippocampus and thal-
2.8. PET imaging
Based on the in vitro characterization described above, 18F-
ST889 was further evaluated as a potential radiotracer for CNS
H3R imaging in vivo. A whole body scan at 70–100 min post injec-
tion (p.i.) revealed that 18F-ST889 or its radiometabolites are
cleared mainly via the renal pathway (Fig. 5). Highest activity con-
centrations were found in the urinary bladder and the kidney pel-
vis. Hepatobiliary elimination of the tracer was also apparent by
activity accumulation in the liver and intestine. Furthermore, the
whole body PET image of 18F-ST889 suggested minor defluorina-
tion of the parent compound at this late time point since some
radioactivity uptake was observed in joints and the skeleton, in
particular the spinal column. High 18F-ST889 accumulation was
identified in various glandular tissues such as thyroid, adrenals
and salivary glands. Noteworthy, high levels of thyroid accumula-
tion were observed with other potential H3R probes, such as 123I-
iodoproxyfan and 125I-WYE-230949, and was attributed to
in vivo deiodination of the radiotracer and accumulation of free io-
dine in the thyroid, which is a known phenomenon.41,42 This expla-
nation is not applicable in our case. There must be another
mechanism of non-specific glandular accumulation of the tracer
or its metabolite(s).
amus was displaceable by co-incubation of 18F-ST889 with 100
lM
histamine. These results demonstrate the specificity of 18F-ST889
for targeting H3R in the rat brain in vitro.
2.7. Transport in P-glycoprotein-transfected cells in vitro
Besides low passive permeation across the brain capillary endo-
thelium, efflux by P-glycoprotein (P-gp) in the luminal plasma
membrane of the brain endothelial cells is another major obstacle
for many compounds, in particular at tracer concentration.
In vivo P-gp susceptibility of bases with MW >250 and number
of N and O atoms <4, to which category 18F-ST889 belongs, is
hardly predictable.38 We investigated the passive permeation
across a tight cell layer and possible efflux of 18F-ST889 by P-gp
with MDR1-transfected MDCK cells (n = 6 from 2 independent
experiments). In this assay, P-gp transport of a substrate would re-
sult in a higher apparent permeability (Papp) coefficient from basal
to apical (B?A) than from apical to basal (A?B).39 The Papp A?B of
18F-ST889 was (1.6 0.7) ꢁ 10ꢂ7 cm/s and Papp B?A was
(1.2 0.7) ꢁ 10ꢂ7 cm/s, resulting in a ratio B?A/A?B of 0.8, indi-
cating that 18F-ST889 is not a substrate for human P-gp. Similar
No appreciable accumulation of 18F-ST889 could be observed in
the H3R-expressing forebrain areas (striatum and cerebral cortex)
(Fig. 6). Only one hot spot was observed within the brain and co-
registered CT images suggested that this basally located hot spot
most probably corresponds to the pituitary gland. High accumula-
tion of radioactivity was also visible in other secretory tissues such
as the Harderian and intraorbital lacrimal glands and salivary
gland. Co-administration of the blocking compound, ST900
(Ki = 0.068 nM to recombinant human H3R)27 (1 mg per kg body
weight) or ciproxifan (Ki = 0.654 nM to rat cortex H3R)43 (2 mg
per kg body weight) with 18F-ST889 did not block the signal
(Fig. 7) suggesting nonspecific tracer accumulation. Interestingly,
radioactivity in other brain regions such as cerebral cortex and cer-
ebellum was slightly elevated under both blocking conditions.
Such a phenomenon could result from blocking of peripheral bind-
ing sites or metabolizing enzymes resulting in a greater availability
values were obtained in the presence of 10 lM elacridar, a specific
P-gp inhibitor, and with the parent cell line MDCK (data not
shown). Therefore, no efflux of 18F-ST889 by P-gp is expected at
the BBB of humans. However, differences in P-gp-efflux may exist
between species, which was shown previously by transport exper-
iments with MDR1 (human) and mdr1a (mouse) transfected epi-
thelial cells.40 Papp values of the P-gp substrate 3H-vinblastine in
the parallel experiment using the same MDCK-MDR1 cultures were
(2.2 1.4) ꢁ 10ꢂ7 cm/s A?B and (32.4 9.6) ꢁ 10ꢂ7 cm/s B?A,
with a ratio B?A/A?B of 15, confirming P-gp functionality.
When 100 lM verapamil was added to the apical compartment,
Papp A?B for 18F-ST889 increased to (30.3 10.8) ꢁ 10ꢂ7 cm/s,
while Papp B?A was hardly affected (1.9 0.7) ꢁ 10ꢂ7 cm/s. The ef-
fect on 18F-ST889 permeation was not seen at 10
lM verapamil,
excluding any specific effect as well as an increase in barrier per-
meation under non-toxic verapamil doses to rats in vivo.
Although 18F-ST889 is not a substrate for P-gp transport, BBB
permeation may be at the limit for significant brain uptake as
concluded from the relatively low Papp A?B and logD7.4 values.
The extent of plasma protein binding and receptor density in the
brain may have high impact on the final radiotracer accumulation
level.
Figure 5. Series of horizontal whole body slices (ventral to dorsal) through a rat
injected with 18F-ST889. The images were obtained by reconstructing data from 70
to 100 min after injection (b–urinary bladder, t–thyroid, j–joint, l–liver, i–intestine,
k–kidney, a–adrenal, s–spinal column).
Figure 4. In vitro 18F-ST889 autoradiography of horizontal sections of a rat brain
under baseline (A) and blocking (B) conditions (co-incubation with 100
histamine).
lM

S. V. Selivanova et al. / Bioorg. Med. Chem. 20 (2012) 2889–2896
2893
Figure 6. Representative horizontal PET/CT slices through a rat’s head injected with 18F-ST889. The images were obtained by reconstructing data from 0 to 60 min after
injection (averaged). CT data are given in grey values whereas PET data are shown in color (highest activity concentration: yellow, moderate activity concentration: red).
Figure 7. Series of horizontal slices through a rat’s head (ventral to dorsal). The animal was injected with 18F-ST889 alone (A, baseline) or co-injected with 2 mg/kg ciproxifan
(B). The images were obtained by reconstructing data from 0 to 60 min after injection (averaged).
of the radioligand in blood.44 Evaluation of the dynamic PET data
demonstrated that brain signals reached a plateau within 10 min
after tracer injection and were persistent over the 60 min acquisi-
tion time without any significant washout. Furthermore, time-
activity curves for other organs, in particular glandular tissue,
showed stable radioactivity level or even slight increase (data not
shown). Taken together, our in vivo data indicate that 18F-ST889
does not bind selectively to H3R in the rat brain.
suitable trial of the tracer utility for imaging humans. It is worth
to mention that three successful imaging studies of H3R reported
previously were performed in rhesus monkeys,24 pigs,45 and
humans.25,26
4. Experimental
All commercial reagents and solvents were used as supplied by
Sigma–Aldrich or Merck unless stated otherwise. Reactions were
monitored by UPLC. The system was Acquity UPLC from Waters,
equipped in addition with a FlowStar LB 513 radiodetector (Bert-
hold Technologies) and a UPLC dedicated reversed-phase Acquity
2.9. In vivo metabolism
Only intact radiotracer was detected in plasma samples taken at
5, 15, and 30 min after injection. Urine sample taken at 30 min
after injection displayed three radioactive compounds, one of them
being 18F-ST889, which accounted for 90% of the total radioactivity.
Therefore, the tracer stability may not be the reason for the low
brain uptake. The radiometabolites (7% and 3%) were more hydro-
philic. The radiometabolite eluting with solvent front (7%) may
suggest partial defluorination in vivo, which results in visible bone
uptake at late imaging time (Fig. 5).
BEH C18, particle size 1.7
l
m, 100 ꢁ 2.1 mm column. The mobile
phase was a gradient of acetonitrile in water with 0.1% trifluoro-
acetic acid (TFA) from 0% to 100% over 2 min, flow rate of
0.7 mL/min. Analytical HPLC was performed on an Agilent 1100
series system equipped with a Raytest Gabi Star radiodetector.
Analytical reversed phase column was Phenomenex Gemini C18,
particle size 5
l
m, 250 ꢁ 4.6 mm. A gradient of acetonitrile in
water with 0.1% TFA from 10% to 25% over 12 min, flow rate of
1 mL/min was used. Semipreparative HPLC was carried out on an
HPLC system equipped with a Merck-Hitachi L-6200A Intelligent
pump, a 5 mL injection loop, a Knauer Variable Wavelength
Monitor UV-detector, and an Eberline RM-14 radiodetector on a re-
3. Conclusion
We conclude that despite proven specificity of 18F-ST889 in vi-
tro, the compound is not suitable as a PET tracer for brain imaging
of H3R in rats. Most probably it is related to restricted BBB passage
due to the moderate lipophilicity and low permeation coefficient.
Brain imaging in (non-human) primates could provide for a more
versed-phase Phenomenex Gemini C18 column, particle size 5 lm,
250 ꢁ 10 mm. The mobile phase consisted of isocratic 10% acetoni-
trile/90% water with 0.1% TFA v/v, eluted at 4 mL/min flow rate.
UV-absorbance was detected at 226 nm. TLC plates were

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S. V. Selivanova et al. / Bioorg. Med. Chem. 20 (2012) 2889–2896
Macherey-Nagel SIL G-25 UV254 normal phase silica. The mobile
phase consisted of acetonitrile/50 mM ammonium bicarbonate
buffer pH 8 (3:1). Protein concentrations were determined using
Genesys 10 UV Scanning spectrophotometer (Thermo Fischer
Scientific).
4.4. Preparation of crude rat brain membranes
The rats (Sprague–Dawley, 7–8 weeks old, mixed gender) were
sacrificed by decapitation. The brains were removed, separated
from the cerebellum and homogenized (Polytron PT-2100, Kinem-
atica) in 10 volumes of ice-cold 10 mM Tris/acetate buffer pH 7.4
containing sucrose (0.32 M) for 1 min at setting 4. After centrifuga-
tion (1000ꢁg, 15 min, 4 °C) the supernatant containing the mem-
branes was collected and kept aside. The pellet was resuspended
in five volumes of Tris buffer (as above) and the homogenizing pro-
cedure was repeated. The pellet was discarded and the superna-
tants were combined and centrifuged at 17000ꢁg for 20 min at
4 °C to precipitate the membranes. The supernatant was decanted;
the pellet was resuspended in 5 mM Tris/acetate buffer pH 7 and
centrifuged again (17000ꢁg, 20 min, 4 °C). This last procedure
was repeated three times. The final pellet was reconstituted in
5 mM Tris/acetate buffer pH 7.4, stored in aliquots at ꢂ80 °C, and
thawed on the day of the experiment. Protein content was deter-
mined by Bradford assay using bovine serum albumin as standard
protein.46
4.1. 18F-fluoride production
No-carrier-added (n.c.a) 18F-fluoride was produced via the
18O(p,n) 18F nuclear reaction in a fixed-energy Cyclone 18/9 cyclo-
tron (IBA, Belgium). For this >98% isotopically enriched 18O-water
(Nukem GmbH, Germany) was irradiated by 18 MeV proton beam.
Small volume liquid target (0.5 mL) was used to produce radioac-
tivity for reaction optimization experiments. Large volume liquid
target (2.0 mL) was used when labeled compound was produced
for biological experiments. Produced 18F-fluoride/18O-water solu-
tion was transferred using a helium stream from the target to a
shielded hot cell equipped with a manipulator, where radiosynthe-
sis was performed. Typical production of 18F-fluoride at EOB of the
2 mL target for 35 mAh (45 min) was 80–100 GBq.
4.2. Radiosynthesis
4.5. Binding affinity to rat H3R
The binding assay was performed with 6 ꢁ 10ꢂ11
M
18F-ST889
Precursor for radiolabeling and cold reference compound were
synthesized as described previously.27
(specific radioactivity 52 GBq/lmol at EOS) alone or in combination
18F-fluoride (ꢀ80–100 GBq) was trapped on an anion exchange
Sep-Pak Light Accell Plus QMA cartridge (Waters) preconditioned
with 5 mL 0.5 M potassium carbonate solution followed by 10 mL
water and flushed with 10 mL air. It was eluted with 1.5 mL solu-
with cold ST889 at 10 different concentrations, ranging from
3 ꢁ 10ꢂ11 M to 10ꢂ5 M in a final volume of 0.2 mL (3 samples per
concentration). Nonspecific binding was estimated in the presence
of 10
l
M (R)-a-methylhistamine, an established selective H3R
tion
of
Kryptofix
2.2.2
(4,7,13,16,21,24-hexaoxa-1,10-
ligand. The membranes (75
lg/mL protein) were incubated to-
diazabicyclo[8.8.8]-hexacosane) (5 mg) and potassium carbonate
(1 mg) in 80% acetonitrile in water. Effluent was collected to a
tightly closed 10 mL Reacti-Vial (Thermo Fisher Scientific) and sol-
vent was evaporated under stream of nitrogen and reduced pres-
sure (50–80 mbar) at 100 °C. The residue was azeotropically
dried by addition of 3 ꢁ 1 mL of dry acetonitrile. Precursor ST-
gether with above mentioned compounds in HEPES buffer (30 mM
sodium 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (Na-HEPES),
110 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, and 1.3 mM MgCl2, pH 7.4)
for 1 h at room temperature. The incubation was terminated by
addition of ice cold HEPES buffer and rapid filtration through glass
microfiber GF/B grade filters (Whatman) presoaked in 0.05% poly-
ethylenimine (Fluka). The filters were washed three times with
4 mL ice-cold HEPES buffer and radioactivity trapped on the filters
was counted in an automatic gamma counter (Wallac 1480 Wizard
300, Perkin Elmer). Kd and Bmax values were estimated using (Eq. 1) by
non-linear curve fitting in Excel (MS Office 2007, Microsoft):
928 (1.97–2.35 mg, 4.8–5.7 lmol) predissolved in 300 ll acetoni-
trile was added to the dry 18F-Kryptofix complex and the reaction
mixture was heated at 100 °C for 30 min. After 5 min of cooling at
room temperature 4 mL of 0.1% TFA solution in water (v/v) was
added to the vial and injected into semi-preparative HPLC at once
(for conditions see Section 4). The radioactive fraction eluting at
tR = 17–18 min was collected and radio-fluorinated product was
concentrated using solid phase extraction. For this, the collected
solution was diluted with 10 mL water for injection and passed
through Sep-Pak Light C18 cartridge (preconditioned in advance
with 5 mL ethanol followed by 10 mL H2O). Cartridge was washed
with additional 10 mL water for injection and 18F-ST889 was
eluted with 0.5 mL ethanol to a penicillin vial. Ethanol fraction
was diluted with 9.5 mL PBS and passed through a 13 mm Nalgene
Bmax;1 ꢁ Ka;1 ꢁ ð½ST889totalꢃ ꢂ ½ST889boundꢃÞ
B ¼
1 þ Ka;1 ꢁ ð½ST889totalꢃ ꢂ ½ST889boundꢃÞ
Bmax;2 ꢁ Ka;2 ꢁ ð½ST889totalꢃ ꢂ ½ST889boundꢃÞ
þ
ð1Þ
1 þ Ka;2 ꢁ ð½ST889totalꢃ ꢂ ½ST889boundꢃÞ
The parameter B indicates the quantity of ST889 bound in mol/
mg protein; Bmax is the maximal binding capacity in mol/mg pro-
tein. Ka is the association constant, that is, 1/Kd, in Mꢂ1. [ST889total
]
and [ST889bound] are the total and bound molar concentrations of
ST889 in the incubation mixture, respectively. The bound concen-
tration was calculated from the amount trapped on the filter and
the sample volume. The estimated binding parameters were con-
firmed by Scatchard plot, that is, B/([ST889total]ꢂ[ST889bound]) ver-
sus B.
0.2 lm sterile filter (Thermo Fisher Scientific) to a sterile vial, giv-
ing an injectable sterile solution containing 5% ethanol by volume,
which was used for biological experiments.
4.3. Quality control
An aliquot of known volume and radioactivity of the final for-
mulated solution was injected into analytical HPLC for quality con-
trol, which required approximately 15 min (for conditions see
Section 4). The retention time of the radiolabeled product was
10.7 min. and the formulation contained no precursor (expected
tR = 11.5 min). The specific radioactivity was determined from the
quality control run matching the area of the UV absorbance peak
at 226 nm, which co-eluted with the radiolabeled product, to a
standard calibration curve calculated using known concentrations
of the non-radioactive reference compound.
4.6. Lipophilicity determination (logD7.4
)
Lipophilicity was determined using the shake-flask method33 by
partitioning the radiolabeled compound between 1-octanol and
aqueous phosphate buffer solution at pH 7.4. Both solvents were
pre-saturated with each other. First, octanol (2 mL), saturated with
buffer, aqueous phosphate buffer (2 mL), saturated with octanol,
and formulated 18F-ST889 solution (50
ll) were mixed in a separa-
tion funnel. After phase separation the aqueous phase was

S. V. Selivanova et al. / Bioorg. Med. Chem. 20 (2012) 2889–2896
2895
discarded to remove any possible polar impurities. Next, the
purified octanol phase (300 l), containing the radiotracer
l), satu-
rated with octanol, were mixed in three Eppendorf microcentrifuge
tubes. The Eppendorf tubes were tightly closed and rotated on an
upside-down shaker for 30 min. The phases were separated by
The sections were placed in a light-tight cassette and exposed to
BAS-TR 2025 phosphor imaging plate (RaytestFuji). After
15 min exposure time the imaging plates were scanned in a BAS-
5000 bio-imaging analyzer with a resolution of 50 m (Fujifilm).
Data were analyzed and processed with the AIDA 2.31 software
(RaytestFuji).
l
a
(ꢀ700 kBq), and fresh phosphate buffer at pH 7.4 (300
l
l
20 min centrifugation at 6700ꢁg. An aliquot (30
ll) of both phases
from each Eppendorf tube was removed and the radioactivity of
these samples was counted in a gamma counter. The distribution
coefficient D7.4 was calculated dividing the averaged radioactivity
concentration in the octanol phase by the averaged radioactivity
concentration in the aqueous phase. The reported logD7.4 is the
mean of three independent determinations.
4.10. In vitro P-gp transport
Madin–Darby canine kidney cells strain II transfected with hu-
man P-glycoprotein (MDCKII-hMDR1) were provided by the Neth-
erlands Cancer Institute (Amsterdam, Netherlands). Cells were
cultured in Dulbecco’s modified Eagle medium (DMEM) containing
GlutaMAX (Gibco, Invitrogen), 100 U/mL penicillin/100 lg/mL
4.7. In vitro plasma stability
streptomycin mixture (Invitrogen), and 10% fetal bovine serum
(Invitrogen) and were split 1:5 twice weekly. For transport studies
Human or rodent plasma was centrifuged at room temperature
the cells were seeded into 4.2 cm2, 0.4
lm BD Falcon polyethylene
at 270ꢁg for 10 min. An aliquot of 50
l
l (ꢀ60 MBq) of radiolabeled
terephthalate culture inserts (BD Biosciences) at a density of
500,000 cells/cm2. Culture medium (3 mL) on the apical (insert)
and basal (well) sides was replaced daily. Transport experiments
were performed 3 days after the seeding. The medium was re-
placed by DMEM containing 10 mM HEPES and 1% fetal calf serum,
and 400 kBq 18F-ST889 (formulated in PBS containing 5% ethanol)
or 20 kBq 3H-vinblastine (2.96 GBq/mmol, Perkin Elmer), a known
P-glycoprotein substrate, were added either to the apical or the ba-
sal compartment (donor compartments). The cultures were incu-
compound solution was added to the plasma supernatant
(ꢀ0.5 mL) and vortexed. The resulting plasma containing 18F-
ST889 was distributed into six Eppendorf microcentrifuge tubes,
70
point, already contained 70
incubated at 37 °C in an Eppendorf Thermomixer Compact (Vau-
dauxEppendorf). The samples were quenched with 70 l methanol,
l
l (ꢀ7 MBq) to each. The first tube, which represented zero time
l
l methanol. Five other tubes were
l
one by one, after 5, 30, 60, 90, and 120 min. Precipitated plasma
proteins were removed by centrifugation at 6700ꢁg for 10 min.
The supernatants were injected into UPLC for analysis.
bated at 37 °C on a rocking plate and 100 ll samples were
withdrawn from the acceptor compartments after 20, 40, 60 and
from both the acceptor and donor compartments after 80 min.
18F-ST889 and 3H-vinblastine were quantified with a gamma coun-
ter and a LS6500 liquid scintillation counter (Beckman Coulter),
respectively. The experiments were repeated in the presence of
4.8. In vitro stability in liver microsomes
Human and rat liver microsomes were purchased from BD Bio-
sciences as 20 mg protein/mL solution and stored at ꢂ80 °C. They
were thawed before use and stored on ice. The general assay pro-
cedure suggested by the supplier was optimized for our use. For-
verapamil hydrochloride (10
(10 M) to inhibit P-gp function. Permeation coefficients (Papp
were calculated according to (Eq. 2):
lM and 100 lM) and elacridar
l
)
mulated 18F-ST889 (100
adenine dinucleotide phosphate (NADPH) (100
tion), and PBS (775 l of 0.15 M solution) were added to an Eppen-
dorf microcentrifuge tube. The mixture was incubated at 37 °C for
5 min. The enzymatic reaction was initiated by addition of 25 l of
human or rat liver microsomes. Hundred microliter of this solution
was withdrawn right away and quenched with 100 l of ice-cold
acetonitrile to represent zero time point. The mixture was then
incubated for additional 120 min. Samples (100 l) of the incubat-
ing solution were transferred into a different Eppendorf tube and
quenched with 100 l of ice-cold acetonitrile at 15, 30, 60, and
120 min. The samples were vortexed and centrifuged at 6700ꢁg
for 3 min. The supernatants (50 l) were transferred into HPLC
vials and analyzed by UPLC. Testosterone (100 l of 0.5 mM solu-
tion, final concentration 50
M) was used instead of 18F-ST889 as
a positive control. A mixture of testosterone (100 l of 0.5 mM
solution) and PBS (900 l of 0.15 M solution) without microsomes
l
l, ꢀ5 MBq), reduced nicotinamide
1
DQr
Dt
Papp
¼
ꢁ
ð2Þ
l
l of 10 mM solu-
A ꢁ C0
l
where A is the area of the cell layer (4.2 cm2), C0 is the initial com-
pound concentration (mol/cm3) in the donor compartment, and
is the quantity (mol) of compound transported to the acceptor com-
partment over the period of time t (sec). A ratio Papp (B?A)/Papp
DQr
l
D
l
(A?B) >1 in the absence of a P-gp inhibitor indicates that com-
pound is a P-gp substrate.
l
4.11. In vivo PET imaging
l
Animal care and all experimental procedures were approved by
the Veterinary Office of the Canton Zurich. Adult male Sprague–
Dawley rats were obtained from Charles River (Germany) and were
allowed free access to food and water. For PET scanning rats were
immobilized by an isoflurane inhalation anesthesia using an oxy-
gen/air mixture as carrier gas. Monitoring of anesthesia was per-
formed according to protocols published previously.47 In the first
PET experiment, a rat was scanned in a whole body configuration
to obtain data on whole body distribution of 18F-ST889 and the
main elimination pathways. For this purpose, a rat (475 g) was in-
jected with 29.1 MBq (0.33 nmol) of the tracer and scanned in the
quad-HIDAC PET tomograph (Oxford Positron Systems, UK) during
70–100 min post injection in a single bed position. Acquired data
were reconstructed in a single time frame with a bin size of
1.0 mm and a matrix size of 250 ꢁ 100 ꢁ 100 mm. In a second
set of PET experiments, the heads of four rats were scanned in
the GE VISTA PET/CT tomograph, which is characterized by high
sensitivity but a limited axial field-of-view of 4.8 cm.48 Animals
were anesthetized and fixed on the bed of the tomograph before
l
l
l
l
l
was incubated along with other samples and served as a negative
control and to determine initial area under UV peak for testoster-
one. UV absorption for the testosterone was measured at 245 nm.
4.9. In vitro autoradiography
Whole brain horizontal slices (20 lm) of a male Sprague
Dawley rat mounted on SuperFrost microscope adhesion slides
(MenzelThermo Fisher Scientific) were thawed for 30 min and
incubated for 45 min with a 0.01 nM solution of 18F-ST889 in PBS
containing 5% ethanol (v/v) at room temperature. Nonspecific
binding was determined in the presence of 100 lM histamine.
The slices were washed three times for 3 min in PBS at 4 °C and
twice for 5 s in distilled water and then air dried for 20 min at rt.

2896
S. V. Selivanova et al. / Bioorg. Med. Chem. 20 (2012) 2889–2896
abstract, XII International Symposium on Radiopharmaceutical Chemistry,
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tracer injection. Two animals were injected into a tail vein with
18F-ST889 alone (injected dose 29–44 MBq; injected mass 0.27–
0.33 nmol), one animal was co-injected with the blocker ST900
(1 mg per kg body weight), and another animal was co-injected
with the blocker ciproxifan (2 mg per kg body weight). All animals
were scanned for 0–60 min after tracer injection in a dynamic PET
acquisition mode. After the PET scan one animal was subjected to a
CT scan to obtain fused PET/CT images for a better anatomical
assignment of radioactive signals. Raw data were acquired in list-
mode and reconstructed in user-defined time frames (dynamic:
5 ꢁ 2, 6 ꢁ 5, 2 ꢁ 10 min; static: 1 ꢁ 60 min) with a voxel size of
0.3875 ꢁ 0.3875 ꢁ 0.775 mm and a matrix size of 175 ꢁ 175 ꢁ
61. Image files were evaluated by ROI analysis using the dedicated
PMOD software.49 Time-activity curves were normalized to the in-
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4.12. In vivo metabolism
A rat was injected with ꢀ250 MBq (ꢀ7 nmol, 300
lL) of the tra-
cer via a lateral tail vein. Blood samples were withdrawn at 5 and
15 min after injection. The animal was sacrificed at 30 min after
injection and blood and urine was collected. The samples were
placed into 1.5 ml Eppendorf microcentrifuge tubes and centri-
fuged (5000ꢁg, 5 min, 4 °C). The supernatants were decanted into
separate Eppendorf tubes and the proteins were precipitated by
addition of an equal volume of ice-cold methanol. The tubes were
centrifuged (5000ꢁg, 5 min, 4 °C). The supernatants were trans-
ferred into HPLC vials and analyzed by UPLC and TLC (see condi-
tions in Section 4).
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