Deuteration: Versus ethylation-strategies to improve the metabolic fate of an 18F-labeled celecoxib derivative

Article abstract of DOI:10.1039/d0ra04494f

The inducible isoenzyme cyclooxygenase-2 (COX-2) is closely associated with chemo-/radioresistance and poor prognosis of solid tumors. Therefore, COX-2 represents an attractive target for functional characterization of tumors by positron emission tomography (PET). In this study, the celecoxib derivative 3-([18F]fluoromethyl)-1-[4-(methylsulfonyl)phenyl]-5-(p-tolyl)-1H-pyrazole ([18F]5a) was chosen as a lead compound having a reported high COX-2 inhibitory potency and a potentially low carbonic anhydrase binding tendency. The respective deuterated analog [D2,18F]5a and the fluoroethyl-substituted derivative [18F]5b were selected to study the influence of these modifications with respect to COX inhibition potency in vitro and metabolic stability of the radiolabeled tracers in vivo. COX-2 inhibitory potency was found to be influenced by elongation of the side chain but, as expected, not by deuteration. An automated radiosynthesis comprising 18F-fluorination and purification under comparable conditions provided the radiotracers [18F]5a,b and [D2,18F]5a in good radiochemical yields (RCY) and high radiochemical purity (RCP). Biodistribution and PET studies comparing all three compounds revealed bone accumulation of 18F-activity to be lowest for the ethyl derivative [18F]5b. However, the deuterated analog [D2,18F]5a turned out to be the most stable compound of the three derivatives studied here. Time-dependent degradation of [18F]5a,b and [D2,18F]5a after incubation in murine liver microsomes was in accordance with the data on metabolism in vivo. Furthermore, metabolites were identified based on UPLC-MS/MS. This journal is

Full text of DOI:10.1039/d0ra04494f

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Deuteration versus ethylation – strategies to
18
improve the metabolic fate of an F-labeled
Cite this: RSC Adv., 2020, 10, 38601
celecoxib derivative†
a
a
*
Markus Laube,
Martin Ullrich,
Klaus Kopka,
‡
Cemena Gassner,‡ab Christin Neuber, ‡a Robert Wodtke,
a
a
a
c
Cathleen Haase-Kohn,
Torsten Kniess,
Reik Loser,
Evamarie Hey-Hawkins
Martin Kockerling,
¨
¨
a
a
d
ab
*
and Jens Pietzsch
The inducible isoenzyme cyclooxygenase-2 (COX-2) is closely associated with chemo-/radioresistance and
poor prognosis of solid tumors. Therefore, COX-2 represents an attractive target for functional
characterization of tumors by positron emission tomography (PET). In this study, the celecoxib derivative
3-([18F]fluoromethyl)-1-[4-(methylsulfonyl)phenyl]-5-(p-tolyl)-1H-pyrazole ([18F]5a) was chosen as a lead
compound having a reported high COX-2 inhibitory potency and a potentially low carbonic anhydrase
binding tendency. The respective deuterated analog [D2,18F]5a and the fluoroethyl-substituted derivative
[
18F]5b were selected to study the influence of these modifications with respect to COX inhibition
potency in vitro and metabolic stability of the radiolabeled tracers in vivo. COX-2 inhibitory potency was
found to be influenced by elongation of the side chain but, as expected, not by deuteration. An
automated radiosynthesis comprising 18F-fluorination and purification under comparable conditions
provided the radiotracers
[
18F]5a,b and [D2,18F]5a in good radiochemical yields (RCY) and high
radiochemical purity (RCP). Biodistribution and PET studies comparing all three compounds revealed
bone accumulation of 18F-activity to be lowest for the ethyl derivative [18F]5b. However, the deuterated
analog [D2,18F]5a turned out to be the most stable compound of the three derivatives studied here.
Time-dependent degradation of [18F]5a,b and [D2,18F]5a after incubation in murine liver microsomes was
in accordance with the data on metabolism in vivo. Furthermore, metabolites were identified based on
UPLC-MS/MS.
Received 20th May 2020
Accepted 11th October 2020
DOI: 10.1039/d0ra04494f
rsc.li/rsc-advances
of anti-inammatory drugs taking into account that the long-
term gastrointestinal toxicity of common non-steroidal anti-
1 Introduction
inammatory drugs (NSAIDs) like ibuprofen could be circum-
vented with this new class of COX-2-selective inhibitors (Cox-
ibs). Today however, only a few Coxibs such as celecoxib,
etoricoxib, and the prodrug parecoxib are in clinical use
because of general concerns about the cardiovascular safety in
long-term applications of this inhibitor class.1,2 The mode of
action is based on the inhibition pattern for cyclooxygenase
(COX), an enzyme which converts arachidonic acid to prosta-
glandin H2. Thereby, COX catalyzes a key step in the synthesis of
autocrine and paracrine acting prostaglandins and throm-
boxane A2 that are involved in the regulation of a multitude of
physiological and pathophysiological processes. Two isoforms
of cyclooxygenases are known: the constitutively and
throughout the whole body expressed isoform COX-1 which
regulates mainly homeostatic processes and its counterpart
COX-2 that is nearly absent in the body with exception of some
organs, such as kidney and brain. However, COX-2 is induced in
inammatory conditions and neoplasia. The COX-2 pathway
hence provides prostaglandins at inamed sites and,
Celecoxib (tradename Celebrex®) is an FDA-approved anti-
inammatory, analgesic, and antipyretic drug which belongs
to the class of selective cyclooxygenase-2 (COX-2) inhibitors and
can be prescribed for the treatment of rheumatoid arthritis,
osteoarthritis, and acute pain. The development of this drug in
the late 1990s represented a breakthrough in the development
aHelmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer
Research, Bautzner Landstrasse 400, 01328 Dresden, Germany. E-mail: m.laube@
hzdr.de; j.pietzsch@hzdr.de
b
¨
Faculty of Chemistry and Food Chemistry, School of Science, Technische Universitat
Dresden, Mommsenstrasse 4, D-01062 Dresden, Germany
cUniversity of Rostock, Institute of Chemistry, Department of Inorganic Solid State
Chemistry, Albert-Einstein-Str. 3a, D-18059 Rostock, Germany
dLeipzig University, Faculty of Chemistry and Mineralogy, Institute of Inorganic
Chemistry, Johannisallee 29, D-04103 Leipzig, Germany
† Electronic supplementary information (ESI) available. CCDC 1428720 and
1433314. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/d0ra04494f
‡ M. L., C. G., and C. N. contributed equally to this manuscript.
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furthermore, in tumors which makes it a potential target for
Based on our interest in the development of radiolabeled
diagnosis and treatment of a variety of disorders. COX-2 is COX-2 inhibitors34–37 and to minimize non-specic binding, we
overexpressed in chronic inammatory diseases such as rheu- aimed to synthesize the respective methylsulfonyl-substituted
matoid arthritis or in neurodegenerative diseases such as Par- celecoxib derivatives and thus we herein describe two
kinson's and Alzheimer's. In addition, as a key player in tumor- attempts to increase their metabolic stability. In general,
associated inammation, COX-2 modulates radiation sensi- possibilities to increase the metabolic stability of a radiotracer
tivity, progression, and metastasis in a variety of cancers and containing 18F bonded to an sp3-hybridized carbon atom
leads to a reduction in overall survival.1–8
include (i) the deuteration vicinal to the uorine substituent38–41
A high ‘molecular contrast’ between healthy tissue and or at other parts of the molecule,41–43 (ii) the replacement of an
18
pathological lesions also forms the basis for successful alkyl chain by a cycloalkyl ring,44 (iii) the use of CF2 F groups,20
radionuclide-based approaches. From the perspective of the and (iv) the application of uorophenyl-instead of uoroalkyl-
oncologically oriented radiochemist/radiopharmacologist, substituted analogs wherever possible.45–47 As an early
COX-2 is a highly promising drugable target because (i) under example, Zhang et al. demonstrated that utilization of a [18F]
physiological conditions it is nearly absent in tissues except uoromethoxy-d2 group in
a peripheral benzodiazepine
kidneys, heart, and brain, (ii) its expression is inducible and receptor ligand decreased its metabolization and, hence, 18F-
COX-2 is overexpressed at inammatory sites and in (inamed) deuorination in the brain of rats but not in primates.39
tumor tissue, and (iii) the availability of clinically approved Accordingly, the use of [18F]uoroethoxy-d4 group was found to
drugs would allow the clinician to intervene in a personalized increase the metabolic stability of verapamil analogs.41 In the
manner in, e.g., radioresistant tumors with high COX-2 present study, we focus on the development of a deuterated and
expression. Among a multitude of labeled COX-2 inhibi- a uoroethyl-substituted celecoxib analog (Fig. 1). We present
tors,9–11 a wide variety of celecoxib-based radiotracer approaches the synthesis, COX inhibition studies, radiolabeling with
have been described in the literature (Fig. 1) for studies on uorine-18 as well as radiopharmacological evaluation in vitro
absorption, distribution, metabolism, and excretion (carbon-14 and in vivo for three methylsulfonyl-substituted celecoxib
(ref. 12)) as well as for positron emission tomography (PET; derivatives to identify potent and metabolically stable
carbon-11,13–18 uorine-18 (ref. 19–24)) or single photon emis- radiotracers.
sion computed tomography (SPECT; iodine-123/125,25–29, tech-
netium-99m30,31). However, up to now no radiotracer could be
transferred to clinical application because of high non-specic
2 Results and discussion
2.1. Synthesis
binding, metabolic instability, inability to demonstrate COX-2-
specic binding in vivo, and/or lack of data from primates or
rst-in-patient studies. As one example, the [18F]uoromethyl-
substituted celecoxib derivative was synthesized by Uddin
et al.21 and evaluated in an inammation model as well as in
tumor xenogras of COX-2-negative human colorectal carci-
noma (HCT116) and COX-2-expressing human head and neck
squamous cell carcinoma (1483 HNSCC) providing evidence for
COX-2-specic uptake of this radiotracer in vivo. Unfortunately,
no further studies on this radiotracer have been presented up to
now. One reason might be, as shown by Uddin et al.,21 that the
[18F]uoromethyl-substituted radiotracer is prone to 18F-
deuorination in vivo and, presumably, binds to carbonic
anhydrase in the erythrocytes as it is known for celecoxib and
other sulfonamide-substituted Coxibs.28,32,33
The synthetic route for the labeling precursors 4a,b and [D2]4a
as well as the uorine-19-substituted reference compounds 5a–
d and [D2]5a comprises a three-step synthesis starting from the
diketoacid esters 1a–c (Scheme 1).
At rst, Knorr pyrazole synthesis by cyclocondensation of the
b-dicarbonyl compounds 1a–c with methylsulfonylphenylhy-
drazine hydrochloride in methanol furnished the acid ester-
substituted pyrazoles 2a–e in 15–96% yield. As commonly
utilized for the synthesis of celecoxib, the reaction of the a,g-
diketoacid ester 1a with the respective phenylhydrazine leads
predetermined by the ꢀI and ꢀM effect of the neighboring ester
group to the initial condensation at the a-keto group and,
hence, regioselectively to the 1,5-diphenyl-substituted pyrazole
2a. In comparison, in the b,d-diketoacid ester 1b the
Fig. 1 Schematic presentation of selected radiolabeled celecoxib derivatives reported in the literature12–31 and the aim of this work. The
generalized structure shows the vicinal diphenyl-substituted pyrazole as the core structure of celecoxib derivatives. The black dots indicate only
the position of the radiolabel in the respective study. Beside the radiolabel, author and year are given as a reference.
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Scheme 1 Synthesis of labeling precursors and reference compounds. Reagents and conditions: (a) 4-methylsulfonylphenylhydrazine hydro-
chloride, MeOH, 70 ꢁC, 21 h; (b) LiAlH4, THF, 70 ꢁC, 1.5 h or r.t., 0.5 h; (c) LiAlD4, THF, 70 ꢁC, 1.5 h; (d) (Ts)2O, DMAP, pyridine, DCM, ꢀ10 ꢁC / r.t.,
1–2 d; (e) DAST, DCM, r.t., 2 d.
corresponding keto group is adjacent to a methylene group inhibition assay. In the next step, the four pyrazole derivatives
exerting a +I-effect instead which disfavors the initial conden- 2a–d were reduced using lithium aluminum hydride in THF to
sation at this site and therefore results in the non-regioselective form the alcohols 3a–d, and in case of 2a also with lithium
reaction towards the 1,5-diphenyl- (2b,c: 47–69% yield) and 1,3- aluminum deuteride in THF to form the deuterated alcohol [D2]
diphenyl-substituted (2d,e: 15–17% yield) products. The struc- 3a, in 61–96% yield. The radiolabeling precursors 4a,b and [D2]
tural identity of the regioisomers was conrmed by NMR 4a, bearing a tosyl leaving group, were synthesized in 48–76%
spectroscopy, and X-ray crystal structure analysis in case of 2e yield by conversion of the respective alcohols with tosyl anhy-
(Fig. 2, le). Exemplarily, 2e showed coupling in NOESY and dride in DCM utilizing 4-(dimethylamino)pyridine (DMAP) and
ROESY spectra for the signal of the methylene group (d ¼ 3.91 pyridine as bases. The uoro-substituted reference compounds
parts per million (ppm)) to the pyrazole proton (6.85 ppm) and 5a–d and [D2]5a were obtained by uorination of the alcohols
to the aromatic protons of the para-methylsulfonyl-substituted 3a–d and [D2]3a, respectively, with (diethylamino)sulfur tri-
phenyl ring (7.79 ppm). In contrast, in NOESY and ROESY uoride (DAST) in DCM in 47–90% yield. The X-ray crystal
spectra of 2c only the coupling of the methylene group (3.76 structure obtained for the 1,5-diphenyl-3-uoroethyl-
ppm) to the pyrazole proton (6.57 ppm) was observed (for substituted reference compound 5c (Fig. 2, right) conrmed
selected expansions of NOESY spectra of 2c and 2e see ESI, the structural identity of this regioisomer. Crystals of 5c con-
Fig. S1 and S2†). The asymmetric unit of crystals of 2e contains tained two symmetry-independent molecules in the asymmetric
only the regioisomer 2e, not 2c (see ESI, Fig. S3†). Although the unit (ESI, Fig. S4†). Both have the same group connectivity, such
pyrazoles 2d,e do not exhibit the vicinal diaryl heterocyclic that they are the same regioisomers. They only differ in dihedral
structure of Coxibs we selected 2d and further processed this angles around single bonds (i.e. C1–C4, C3–C13, C9–S1) as
compound to generate 5d as a negative control for our COX a result of crystal packing effects.
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Fig. 2 Molecular structures of compounds 2e (left) and 5c (right) in the crystal (ORTEP plot with atom labeling scheme, displacement thermal
ellipsoids are drawn at 50% probability level. Only one of the two independent molecules of the asymmetric unit of 5c is shown).
2.2. COX inhibition
2.3. Radiosynthesis
COX inhibition potency was determined in vitro for the refer- Radiolabeling with [18F]uoride was achieved starting from the
ence compounds 5a–d and [D2]5a (Table 1) using puried ovine tosylated precursors 4a,b and [D2]4a under standard conditions
COX-1 and recombinant human COX-2 enzyme in the (Scheme 2 and Table 2) in a synthesis sequence using an
commercially available ‘COX Fluorescent Inhibitor Screening automated radiosynthesizer (TRACERlab FX-N). Aer azeo-
Assay KIT’ (Cayman Chemical, Ann Arbor, MI). The data ob- tropic drying of [18F]uoride, labeling was performed at 80 ꢁC in
tained for the COX-2-selective inhibitor 5a are in accordance acetonitrile for 15 min followed by dilution of the mixture with
with the inhibition pattern previously determined by Uddin eluent, ltration, and purication by semi-preparative HPLC. In
et al. with a 14C-based COX assay.21 As expected, deuteration did order to obtain the radiotracer in high chemical purity, we
not markedly change COX-2 inhibitory potency of [D2]5a, while adjusted the eluent to 40% MeCN/60% water with 0.1% tri-
both prolongation to the uoroethyl chain resulting in the tolyl uoroacetic acid (TFA) and allowed the tracers to be eluted from
derivative 5b and the lack of a methyl substituent in derivative the HPLC column aer a separation time of approximately
5c weakened the inhibitory potency and selectivity for COX-2. 50 min. Aer a C18-based solid phase extraction to remove the
The regioisomer 5d, which does not contain the typical vicinal eluent, the nal tracers [18F]5a,b and [D2,18F]5a were isolated in
diarylheterocyclic structure of Coxibs, showed neither COX-2 radiochemical yields (RCY) between 33 and 38% and high
nor COX-1 inhibition as anticipated.
radiochemical purity (RCP > 99%) as well as chemical purity (CP
Table 1 COX inhibition data for compounds 5a–d and [D2]5a
R1
R2
IC50 (COX-1) [mM]
IC50 (COX-2) [mM]
Selectivity indexc
5aa
5a
[D2]5a
5b
CH3
CH3
CH3
CH3
H
CH2F
CH2F
CD2F
CH2CH2F
CH2CH2F
>4
0.60
0.51
0.97
2.53
4.97
>7
>100
>100
38.51
>100
>196
>103
15
5c
>20
5d
—
—
>100
>100
—
Celecoxib
>100
0.04 ꢂ 0.02b
a
b
c
Previously reported by Uddin et al.21 Mean ꢂ SD of six independent experiments. Selectivity index ¼ IC50 (COX-1)/IC50 (COX-2).
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group which is a major determinant of substrate and CA
inhibitor specicity57 and hence are not likely potent CA
inhibitors. However, it is possible that other interactions play
a role in cellular binding effects observed in this investigation
because in both cell lines a COX-independent blocking effect
was observed at a concentration of 100 mM celecoxib which is
higher than the IC50 value of the interactions with moderate
affinity described above. Because of that and insufficient COX
inhibition potencies, compounds 5a,b and [D2]5a did not meet
the requirements for in vivo studies in tumor xenogra-bearing
mice. Instead, we focused on biodistribution studies in healthy
rats to evaluate the inuence of deuteration or elongation on
metabolic stability for the celecoxib-based radiotracers.
Scheme 2 Radiosynthesis of the 18F-labeled celecoxib derivatives [18F]
5a,b and [D2,18F]5a. Reagents and conditions: (a) [18F]fluoride/K2CO3/
Kryptofix® 222 (K222), MeCN, 80 ꢁC, 15 min.
> 92%) as an ethanolic solution with high activity concentration
suitable for further in vitro and in vivo applications (for radio-
HPLC chromatograms and radio-TLC data of nal radio-
tracers, see ESI, Fig. S5†). The log D value (pH 7.4) was deter-
mined to be 2.72 for [18F]5a and [D2,18F]5a, and 2.65 for [18F]5b
by an HPLC-based method.35,48
2.5. Metabolic stability in vivo
To assess the in vivo behavior of [18F]5a,b and [D2,18F]5a in
healthy rats with focus on metabolic stability, (i) ex vivo bio-
distribution at 5 and 60 min post injection (p.i.) was analyzed,
(ii) small animal PET imaging was performed up to 90 min p.i.,
and (iii) blood samples were analyzed for metabolites at 20 and
60 min p.i.
2.4. Cellular uptake
For evaluation of COX-2 selectivity in vitro, cell uptake was
Biodistribution experiments (Fig. 4, see ESI, Tables S5 and
studied in two melanoma cell lines with known COX expression S6†) showed a similar pharmacokinetic pattern for [18F]5a,b and
pattern:35 the COX-2-negative Mel-Juso and COX-2-positive [D2,18F]5a. At 5 min p.i., the highest initial radioactivity
A2058 cell line, both expressing COX-1 at low but constitutive concentration was found in liver, adrenals, and brown as well as
levels. Cellular binding and uptake of [18F]5a,b and [D2,18F]5a white adipose tissue (BAT/WAT). Whereas radioactivity
was found to be independent of the COX-2 expression since concentration considerably decreased in almost all organs from
cellular uptake by COX-2-negative Mel-Juso cells was slightly 5 to 60 min p.i., radioactivity concentration in intestine, urine,
higher compared to uptake by COX-2-positive A2058 cells and femur increased over time. At 60 min p.i., the hepatobiliary
(Fig. 3). Furthermore, inhibition with the known COX-2- and renal excretion was found to increase within the row [18F]5a
selective inhibitor celecoxib resulted in a reduction of cell < [D2,18F]5a < [18F]5b resulting in liver-to-bladder ratios of 2.3,
uptake, however, in both cell lines in a comparable manner 3.2, and 2.6. By contrast to excretion, 18F-deuorination
pointing to a saturation of other binding sites instead of COX-2. tendency clearly decreased in the row [18F]5a > [D2,18F]5a >
For celecoxib it is known that it can also bind and interact with [18F]5b resulting in femur uptakes (standardized uptake value,
other targets which contributes to its anticarcinogenic SUV) of 4.1 ꢂ 0.6, 2.6 ꢂ 0.3, and 1.0 ꢂ 0.2, respectively.
effects.49–51 As examples, interactions with moderate affinity like
the inhibition of 3-phosphoinositide-dependent kinase
Small animal PET imaging was in accordance with the bio-
1
distribution experiments showing a rapid clearance of [18F]5a,
(PDK1, IC50 ¼ 3.5 mM,52 IC50 ¼ 48 mM (ref. 53)), protein kinase B [D2,18F]5a, and [18F]5b from the blood and a fast excretion of
(PKB, IC50 ¼ 28 mM (ref. 50 and 53)), phosphodiesterase-5 radioactivity into the intestine and the bladder (Fig. 5). In line
(PDE51A, IC50 ¼ 16 mM (ref. 54)), or sarcoplasmic/ER Ca2+ with the lipophilicity of the compounds, hepatobiliary excretion
ATPase (SERCA, IC50 ¼ 35 mM (ref. 49 and 55)) and the high predominated renal excretion for all three tracers. From the
affinity interaction with carbonic anhydrases (CA, hCA-IX IC50
¼
dynamic PET study it appears that [18F]5b gets eliminated even
16 nM, hCA-XII IC50 ¼ 18 nM (ref. 49 and 56)) have been faster than [18F]5a or [D2,18F]5a. However, with regard to 18F-
described. Compounds 5a,b and [D2]5a lack the sulfonamide deuorination, [18F]5b was clearly superior to [18F]5a or
Table 2 Results of radiosyntheses for the 18F-labeled celecoxib derivatives [18F]5a,b and [D2,18F]5a
[
18F]5a
[D2,18F]5a
[
18F]5b
Isolated RCYa
Am [GBq mmolꢀ1
RCP
35 ꢂ 9% (n ¼ 8)
7–54
>99%
33 ꢂ 6% (n ¼ 4)
13–44
>99%
38 ꢂ 8% (n ¼ 6)
4–40
>99%
]
CP
>94%
>92%
>94%
Synthesis timeb
log D7.4,HPLC
116 min (n ¼ 8)
129 min (n ¼ 4)
121 min (n ¼ 6)
2.72
2.72
2.65
a
Mean ꢂ SD of n independent experiments. b Mean of n independent experiments.
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Fig. 3 Representative cellular binding and uptake of [18F]5a,b and [D2,18F]5a in COX-2-negative Mel-Juso and COX-2-positive A2058 cells at
37 ꢁC. Blocking was performed by preincubation with 100 mM celecoxib. Results are shown as mean ꢂ SD of one representative experiment
performed in quadruplicate.
[D2,18F]5a, which means that prolongation of the alkyl chain is trichloroacetic acid (TCA) revealed that about 25–29% of activity
benecial resulting in signicantly decreased radioactivity in blood was bound to plasma proteins. Low binding of [18F]
concentration in epiphyses (Fig. 5 red circle) and skeleton. 5a,b and [D2,18F]5a to erythrocytes is in line with the absence of
These results are in accordance with previous reports showing a sulfonamide group typically interacting with carbonic anhy-
that deuteration38–41 and prolongation of uoro-substituted drase. With regard to metabolization, the formation of free [18F]
alkyl chains58 decreased the 18F-deuorination rate in vivo. Of uoride and more hydrophilic metabolites was observed for all
note, prolongation of the alkyl chain does not necessarily three radiotracers (Fig. 6C). However, radiotracers showed
inuence the stability in blood plasma as shown for O-[18F]u- a different number of metabolites and different amounts of
oromethyl-, O-[18F]uoroethyl, and O-[18F]uoropropyl tyrosine intact compound. For all three radiotracers, no intact parent
derivatives58 or even increased 18F-deuorination as shown for compound was excreted into the urinary bladder or the intes-
5-[18F]uoroalkyl pyrimidine nucleosides59 in a comparison tine so that only metabolites and [18F]uoride could be
between propyl-, butyl-, and pentyl-substituted derivatives.
observed there.
With the aim to elucidate the differences in the metabolic
At 60 min p.i., 28% of intact [18F]5a was found in blood
fate of [18F]5a, [D2,18F]5a, and [18F]5b, metabolites of the three (Fig. 6D) together with two more hydrophilic metabolites (S ¼
radiotracers were analyzed in samples of blood as well as of 28%). Both metabolites were found to be excreted into the urine
liver, urine, and intestinal content by radio-HPLC (Fig. 6, see (ESI, Fig. S6 and S9†). In comparison, 52% of the deuterated
ESI, Fig. S6–S8†) and radio-TLC (see ESI, Fig. S9†) aer i.v. analog [D2,18F]5a was intact at 60 min p.i. and only one radio-
injection in healthy rats. Analysis of blood samples (Fig. 6A) active metabolite (14%) was present (Fig. 6D). However, two
conrmed the rapid blood clearance of [18F]5a, [D2,18F]5a, and metabolites were observed in intestinal content and urine (ESI,
[18F]5b resulting from very fast tracer distribution (t1/2 ¼ 0.6, Fig. S7 and S9†).
1.3, and 1.0 min, respectively) followed by a considerably slower
For the [18F]uoroethyl derivative [18F]5b, 30% of the parent
tracer elimination (t1/2 ¼ 19.5, 17.0, and 20.7 min). At 60 min compound was intact at 60 min p.i (Fig. 6D). showing the
p.i., for the three radiotracers 16–23% of activity in blood was presence of three metabolites in blood (S ¼ 43%). For this
found in erythrocytes, whereas about 70% of activity in blood tracer, excretion into the intestine via four metabolites and into
was retrieved in blood plasma (Fig. 6B). Precipitation with the urinary bladder via two metabolites was observed (ESI,
Fig. 4 Biodistribution of [18F]5a,b and [D2,18F]5a in healthy rats at 5 min (A) and 60 min p.i. (B). Results are shown as mean ꢂ SD of two inde-
pendent experiments each performed in quadruplicate (8 animals for each time point and tracer).
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Fig. 5 Maximum intensity projections of small animal PET imaging after i.v. injection of [18F]5a, [D2,18F]5a, and [18F]5b in healthy rats. Tracer
distribution was investigated for 60 min p.i. in dynamic mode (A) and, afterwards, the whole animal was scanned in static mode (B). Different
organs are marked as H-heart, V-vein, K-kidney, L-liver, I-intestine, and B-bladder. For better visualization, PET images (scaled to SUVmax ¼ 7.0)
are overlayed with CT images (scaled between 400 and 6000 Houndsfield Units (HU)).
Fig. S8 and S9†). Hence, excretion into the urinary bladder and carboxyl derivative by cytosolic alcohol dehydrogenases ADH1
the intestine are dominant factors determining the metabolic and ADH2, and nally converted to a minor extent to the
fate of [18F]5a,b and [D2,18F]5a which is in accordance with O-glucuronide by UDP glucuronosyltransferases.12,51 Also
other results from the literature.
Takashima-Hirano et al.13,14 showed that isotopically labeled
The lead celecoxib is metabolized by CYP2C9 and to lower [11C]celecoxib is oxidized via the hydroxymethyl form to the
extent (<25%) by CYP3A4 to the hydroxyl form, oxidized to the carboxylic acid derivative. While in blood and liver [11C]
Fig. 6 (A) Blood clearance rate (% ID per mL of intact compound) after i.v. injection of [18F]5a,b and [D2,18F]5a. (B) Radioactivity distribution in
blood components with and without TCA precipitation. (C) Analytical radio-HPLC chromatograms of rat blood plasma at 60 min p.i. (D) Ratio of
[
18F]fluoride, intact compound, and radiometabolites at 20 and 60 min p.i. determined by radio-TLC (*) and radio-HPLC (**).
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Fig. 7 (A) Analytical radio-HPLC chromatograms of [18F]5a, [D2,18F]5a, and [18F]5b incubated for 60 min with murine liver microsomes and
structure of metabolites determined by UPLC-MS/MS; (B) time dependency of metabolism of intact tracer and formation of [18F]fluoride and 18F-
metabolites based on radio-TLC.
celecoxib was found to be mostly intact, the carboxylic acid was oxidation to the hydroxylated metabolites as rate limiting steps
the major radioactive component in the bile suggesting domi- in radiotracer metabolization. Moreover, liver microsomes
nant excretion of this radiometabolite into the intestine. Simi- contain avin monooxygenases and uridine glucuronide
larly, for
[
18F]5a,b and [D2,18F]5a only more polar transferase, which allow further phase I and II transformations.
radiometabolites were found to be excreted into the urinary Enzymes like aldehyde oxidase, glutathione transferase, sulfo-
bladder and the intestine while both the intact compound and transferase, or other cytosolic cofactors are not present in this
radiometabolites were present in blood plasma. The deuterated subcellular fraction.60
analog [D2,18F]5a showed faster blood clearance compared to
For the experiment, [18F]5a, [D2,18F]5a, and [18F]5b were
[18F]5a (Fig. 6A) presumably caused by both tissue tracer prepared at the same day to ensure comparable activity of the
distribution of intact compound, e.g., to white adipose tissue liver microsomes. All three radiotracers were subjected to
(Fig. 4) as well as a metabolic shi towards the more extensive oxidative conditions in the murine liver microsome assay for 10,
oxidation to the carboxylic acid and excretion in this form. Of 30, 60, and 120 min and samples were analyzed by radio-HPLC
note, a fast metabolization of [11C]celecoxib was also observed and radio-TLC (Fig. 7). Furthermore, carrier-added samples
in male baboon, i.e. only 17% of intact radiotracer was found at were subjected to the same conditions and analyzed aer
90 min p.i. in blood.18
radioactive decay by UPLC-MS/MS to identify the structure of
the metabolites. While for [18F]5a and [D2,18F]5a the formation
of one major metabolite was observed, two metabolites were
formed from [18F]5b (Fig. 7A and ESI, Fig. S13–S24†). Of note,
glucuronidation was exemplarily investigated for [18F]5b by
applying oxidative conditions as described in the experimental
section with addition of MgCl2 (5 mM), alamethicin (50 mg
mLꢀ1), and uridine diphosphate glucuronic acid (UDPGA, 5
mM) but glucuronidation was not observed in this experimental
set-up (data not shown). UPLC-MS/MS experiments veried the
2.6. Metabolic stability in vitro
Murine liver microsome assay was performed to investigate the
time-dependent formation of 18F-bearing metabolites and their
structure in more detail. Different liver subcellular fractions
such as liver microsomes, liver S9 fractions, and liver cytosol are
principally available differing in their capability to catalyze
phase I and phase II reactions. We decided to use liver micro-
somes to follow cytochrome P450 (CYP)-mediated phase I
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Fig. 8 Comparison of 18F-defluorination and 18F-bearing metabolite formation tendency obtained for [18F]5a, [D2,18F]5a, and [18F]5b in vitro and
in vivo based on selected results of this study. Formation of [18F]fluoride in vivo (left, orange bars) refers to SUV values for femur obtained in
biodistribution studies 60 min p.i. Formation of 18F-bearing metabolites in vivo (right, orange bars) refers to % of activity in blood plasma samples
as analyzed by radio-TLC and radio-HPLC 60 min p.i. Formation of [18F]fluoride and 18F-bearing metabolites in vitro (blue bars) refers to % of
activity as analyzed by radio-TLC after 60 min incubation with murine liver microsomes.
formation of the respective hydroxylated 18F-bearing metabo- other celecoxib derivatives, only non-specic17,19,24,26,28 or
lites for all three compounds as well as the formation of no16,20,25,27,29–31 information about the detailed metabolism is
a deuorinated and hydroxyl-substituted metabolite in case of currently available.
[18F]5a and [D2,18F]5a. The time-dependent formation of [18F]
uoride and other 18F-bearing metabolites was found to follow
3 Conclusion
different trends. While 18F-deuorination decreased in the
order [18F]5a > [18F]5b > [D2,18F]5a, the amount of metabolites
bearing covalently bound 18F increased in the order [D2,18F]5a <
[18F]5a < [18F]5b (Fig. 7B and ESI, Fig. S10–S12†). Hence,
deuteration effectively enhanced the metabolic stability as
observed in vitro and in vivo.
In this study, alterations in the substitution pattern of a cele-
coxib derivative were studied with respect to deuteration and
elongation of the side chain in position 3 of the pyrazole ring.
The radiotracers [18F]5a,b and [D2,18F]5a were examined within
detailed in vitro and in vivo studies. COX inhibitory potency of
[D2]5a was found to be comparable to the potent and selective
COX-2 inhibitor 5a, while the ethyl derivative 5b turned out to
be slightly less potent. However, all three radiotracers [18F]5a,b
and [D2,18F]5a did not show COX-2 specic binding in vitro so
that radiopharmacological evaluation in vivo focused on meta-
bolic stability in healthy rats. Our results demonstrate marked
effects of both deuteration and elongation on 18F-deuorination
and formation of 18F-bearing metabolites in vitro and in vivo
(Fig. 8).
In principle, oxidative metabolism and excretion to urinary
bladder and intestine were found to be dominant factors
determining the metabolic fate of all three investigated radio-
tracers which is in accordance with other results from the
literature. As a main result, deuteration was shown to be most
benecial as it effectively decelerates metabolic transformation
without compromising the biological activity of the molecule. In
this sense, it is part of ongoing research to envisage the addi-
tional exchange of the CH3 with a CD3 group at the tolyl ring to
result in a radiotracer candidate with highest possible meta-
bolic stability for this kind of substance class. Other COX-2-
targeting radiotracers, e.g. the [18F]uoromethyl-substituted
valdecoxib derivative61 for which the evaluation in monkey
was substantially hampered by 18F-deuorination, might
accordingly benet from similar modications.
Our results showed that oxidative metabolism represents
a dominant factor determining the metabolic fate of [18F]5a,b
and [D2,18F]5a which is in accordance with previous reports. For
example, aer administration of
a
non-radioactive
uoromethyl-substituted celecoxib derivative Uddin et al.21
also identied the oxidatively deuorinated metabolite bearing
a hydroxymethyl group in the inamed footpad. In accordance,
[11C]celecoxib is oxidized to the carboxylic acid derivative,13,14
a fact that also causes fast metabolization of [11C]celecoxib in
male baboon18 which shows the need for metabolic stabilization
also in primates. Deuteration as shown for [D2,18F]5a in this
study effectively suppressed metabolization in vitro and in vivo
suggesting that this might be a useful strategy for further
developments. Interestingly, other studies indicated that the
metabolic degradation at the 5-phenyl ring can be lowered as
well. For example, a 18F-labeled celecoxib derivative having a 4-
[18F]uoro-5-phenyl-pyrazole22 as structural key feature but no
oxidizable methyl group has been reported to be metabolically
stable and was found to be intact in brain, liver, intestine, and
blood plasma of healthy mice. Also, only a small amount of
metabolites has been observed for the ortho-18F-celecoxib
derivative23 which has a uorine in ortho-position to the
methyl group indicating that steric hindrance or substitution
nearby can also lower the oxidative metabolism at this site. For
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Ethical statement
All animal experiments were carried out according to the
guidelines of the German Regulations for Animal Welfare. The
protocols were approved by the local Ethical Committee for
Animal Experiments (Landesdirektion Sachsen; AZ 24-9168.21-
4/2004-1).
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Author contributions
Conceptualization and design: M. L., T. K., J. P.; methodology
and validation: M. L., J. P.; investigation (acquisition, analysis
and interpretation of data): M. L., C. G., C. N., R. W., R. L., M. K.,
E. H.-H.; writing – original dra: M. L., C. G., C. N., R. W., R. L.;
writing – review and editing: M. L., M. U., C. H.-K., K. K., E. H.-
H., J. P.; administrative, technical, or material support: K. K., J.
P.; study supervision: J. P.
Conflicts of interest
14 M.
Takashima-Hirano,
Y.
Cui,
T.
Takashima,
E. Hayashinaka, Y. Wada, Y. Watanabe, H. Doi and
M. Suzuki, J. Labelled Compd. Radiopharm., 2009, 52, S435.
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There are no conicts to declare.
Acknowledgements
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The excellent technical assistance of Peggy Nehring, Kay
¨
Fischer, Uta Lenkeit, Mareike Barth, Catharina Knofel, Johanna
Wodtke, Regina Herrlich, and Andrea Suhr is greatly acknowl-
edged. We thank Ralf Bergmann, PhD, for supporting the PET
experiments. We also thank Friedrich-Alexander Ludwig, PhD,
for his support in establishing the liver microsome assay as well
as stimulating and fruitful discussions. The authors thank
Stephan Preusche, Martin Kreller, PhD, and the cyclotron team
for providing [18F]uoride. The authors thank the Helmholtz
Association for supporting this work through the Helmholtz
Cross-Programme Initiative “Technology and Medicine—
Adaptive Systems”. The authors are also thankful to the Deut-
sche Forschungsgemeinscha (DFG) for supporting this work
within the Collaborative Research Centers Transregio 67
“Functional Biomaterials for Controlling Healing Processes in
Bone and Skin—From Material Science to Clinical Application”
(59397982-CRC/TRR 67/3, B5) and Transregio 205 “The Adrenal:
Central Relay in Health and Disease” (314061271-CRC/TRR 205/
1, B10). This work also is part of the research initiative
“Radiation-Induced Vascular Dysfunction (RIVAD)”.
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