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Radiosynthesis and in vivo Evaluation of Carbon-11 (2S)-3-(1H-Indol-3-yl)-2-{[(4-methoxyphenyl)carbamoyl]amino}-N-{[1-(5-methoxypyridin-2-yl)cyclohexyl]methyl}propanamide: An Attempt to Visualize Brain Formyl Peptide Receptors in Mouse Models of Neuroinflammation.

TL;DR: The very first attempt to visualize in vivo formyl peptide receptors (FPRs) in mouse brain by positron emission tomography (PET) is described and useful information is provided for the design and characterization of future potential PET radioligands for visualization of brain FPRs by PET.
Abstract: Here, we describe the very first attempt to visualize in vivo formyl peptide receptors (FPRs) in mouse brain by positron emission tomography (PET). FPRs are expressed in microglial cells where they mediate chemotactic activity of β-amyloid peptide in Alzheimer disease and, thus, are involved in neuroinflammatory processes. To this purpose, we have selected (2S)-3-(1H-Indol-3-yl)-2-{[(4-methoxyphenyl)carbamoyl]amino}-N-{[1-(5-methoxypyridin-2-yl)cyclohexyl]methyl}propanamide ((S)-1), that we have previously identified as a potent non-peptidic FPR agonist. (S)-[(11) C]-1 has been prepared in high radiochemical yield. (S)-[(11) C]-1 showed very low penetration of blood-brain barrier and, thus, was unable to accumulate into the brain. In addition, (S)-[(11) C]-1 was not able to label FPRs receptors in brain slices of PS19 and APP23 mice, two animal models of Alzheimer disease. Although (S)-[(11) C]-1 was not suitable to visualize FPRs in the brain, this study provides useful information for the design and characterization of future potential PET radioligands for visualization of brain FPRs by PET.

Summary (2 min read)

Introduction

  • Neuroinflammation is a complex, dynamic, multicellular process that plays a central role in a variety of neurological diseases including neurodegenerative disorders.
  • In particular, in vivo imaging of microglia can offer a measure of the inflammatory process and a means of tracking the progression of those pathologies that trigger an immune activation in the brain and the efficacy of therapeutic treatments over time [12].
  • FPRs are expressed in several immune cells including leukocytes, monocytes/macrophages, and microglia, and are considered to play relevant roles in innate immunity and host defense mechanisms and chemotaxis [14].
  • Among FPRs, the FPRL-1, and its murine homologue FPR2, appear to be relevant to the proinflammatory aspects of AD, because FPRL-1 is a chemotactic receptor for Ab42, the 42 amino acid form of Ab, which induces monocytes migration and activation [15].
  • Conversely, non-peptidic small molecules may have some advantages because they can be suitably designed to modulate properties, such as potency, selectivity, lipophilicity, and cell permeability, that are pivotal for a potential radiotracer [23].

Lipophilicity

  • Lipophilicity indices were measured by a reversed-phase HPLC method consisting in a PerkinElmer Series 200 LC apparatus equipped with a PerkinElmer 785A UV/VIS detector set at 254 nm.
  • UV signals were monitored and obtained peaks integrated using a personal computer running PerkinElmer Turbochrom Software.
  • This mobile phase composition was chosen for the analysis due to reasonable retention times for the compounds analyzed.
  • The compounds were dissolved in MeOH and the measurements were made at a flow rate of 1 ml/min.

Serum Stability and Permeability Evaluation

  • Before embarking in the radiolabeling of (S)-1, the authors evaluated two additional features that are of importance for brain PET radiotracers.
  • First, the authors examined if (S)-1 was a P-glycoprotein (P-gp) substrate, because PET tracers that are not substrates of the P-gp efflux pump have higher chances for crossing the BBB.
  • Therefore, the authors evaluated the efflux ratio between basal-to-apical (BA) and apicalto-basal (AB) fluxes in Caco-2 cells monolayer (BA/AB) of compound (S)-1.
  • Second, the authors evaluated if (S)-1 was stable in serum for, at least, the timeframe of a PET scan.
  • (S)-1 showed the desired stability with degradation half-life (t1/2) longer than 4 h .

PET Studies

  • While these studies can offer important information on the interaction between the radiotracer and the target, they cannot predict radiotracer brain uptake or other biodistribution issues.
  • PET images were generated by averaging dynamic data at 0 – 90 min after injection of (S)-[11C]-1, and indicated minimal radioactivity in the brain (top panel in Fig. 1).
  • To this end, the authors selected the APP23 mice model, a transgenic mouse strain showing a robust formation of amyloid plaques, and the PS19 mouse model that is characterized by progressive accumulation of fibrillary tau tangles.
  • These data indicated that the occupancy of FPRs by (S)-[11C]-1 at 1 nM was very low and was not sufficient to autoradiographically label FPRs receptors in the brain.
  • High © 2016 Wiley-VHCA AG, Z€urich www.cb.wiley.com radioactivity levels were observed in the liver of control and LPS-treated rats, suggesting that (S)-1 undergoes rapid and massive hepatic clearance (Fig. 3).

Conclusions

  • In conclusion, the authors have reported here the very first attempt to visualize in vivo brain FPRs that are expressed in microglial cells where they mediate chemotactic activity of Ab peptide in AD.
  • These results could be due to possible low affinity of (S)-[11C]-1 for the target receptor in vivo, concomitant with the rapid in vivo clearance of the radioligand.
  • In addition, most of the FPRL-1 agonists reported, if not all, have been assessed solely for their ability to activate (EC50) and not for their affinity (KD or Ki) for the receptor.
  • The results presented here might suggest it is not the case.
  • This work was supported in part by Grant-in-Aid for Scientific Research on Innovative Areas (‘Brain Environment’) 23111009 (M. H.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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FULL PAPER
Radiosynthesis and in vivo Evaluation of Carbon-11 (2S)-3-(1H-Indol-3-yl)-2-{[(4-
methoxyphenyl)carbamoyl]amino}-N-{[1-(5-methoxypyridin-2-yl)cyclohexyl]methyl}
propanamide: An Attempt to Visualize Brain Formyl Peptide Receptors in Mouse
Models of Neuroinflammation
by Enza Lacivita*
a
), Madia Letizia Stama
a
), Jun Maeda
b
), Masayuki Fujinaga
b
), Akiko Hatori
b
), Ming-Rong Zhang
b
),
Nicola A. Colabufo
a
)
c
), Roberto Perrone
a
), Makoto Higuchi
b
), Tetsuya Suhara
b
), and Marcello Leopoldo
a
)
c
)
a
) Dipartimento di Farmacia Scienze del Farmaco, Universit
a degli Studi di Bari ‘Aldo Moro’, via Orabona, 4, IT-70125,
Bari (phone: +39-080-5442750, fax: +39-080-5442231, e-mail: enza.lacivita@uniba.it)
b
) National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-9-
1 Anagawa, Inage-ku, Chiba, Chiba 263-8555, Japan
c
) BIOFORDRUG s.r.l., Spin-off, Universit
a degli Studi di Bari ‘Aldo Moro’, via Orabona, 4, IT-70125, Bari
Here, we describe the very first attempt to visualize in vivo formyl peptide receptors (FPRs) in mouse brain by positron
emission tomography (PET). FPRs are expressed in microglial cells where they mediate chemotactic activity of b-amyloid
peptide in Alzheimer disease and, thus, are involved in neuroinflammatory processes. To this purpose, we have selected (2S)-
3-(1H-Indol-3-yl)-2-{[(4-methoxyphenyl)carbamoyl]amino}-N-{[1-(5-methoxypyridin-2-yl)cyclohexyl]methyl}propanamide ((S)-
1), that we have previously identified as a potent non-peptidic FPR agonist. (S)-[
11
C]-1 has been prepared in high
radiochemical yield. ( S)-[
11
C]-1 showed very low penetration of bloodbrain barrier and, thus, was unable to accumulate into
the brain. In addition, (S)-[
11
C]-1 was not able to label FPRs receptors in brain slices of PS19 and APP23 mice, two animal
models of Alzheimer disease. Although (S)-[
11
C]-1 was not suitable to visualize FPRs in the brain, this study provides useful
information for the design and characterization of future potential PET radioligands for visualization of brain FPRs by PET.
Keywords: Formyl peptide receptors, Neuroinflammation, PET, Radiosynthesis, Ureidopropanamide.
Introduction
Neuroinflammation is a complex, dynamic, multicellular
process that plays a central role in a variety of neurological
diseases including neurodegenerative disorders. Activation
of microglia, the innate immune system of the central ner-
vous system (CNS), represents one of the hallmarks of neu-
roinflammation [1]. In normal condition, activation of
microglia exerts a protective function, providing tissue
repair by releasing anti-inflammatory cytokines and neu-
rotrophic factors [2][3]. Upon neuronal injury or infection,
microglia becomes overactivated leading to the release of
neurotoxic and proinflammatory factors [4][5]. Recent liter-
ature evidences also indicate that microglial activation is a
phenotypically and functionally diverse process, which may
change depending on aging, stage of disease, or presence of
other inflammatory events [2][6].
Several studies indicate that neuroinflammation is an
early and continuous feature of Alzheimer disease (AD).
Amyloid-b (Ab), which is considered central to the patho-
genesis of this disease, provokes the recruitment of micro-
glia and astrocytes to the sites in close proximity to Ab
deposits. Ab is able to interact with numerous surface
receptors that are expressed by microglial cells such as
formyl peptide receptors (FPRs) [7], Toll-like receptors
[8], scavenger receptors [9], and receptors for advanced
glycation end products (RAGE) [10]. Binding of Ab to
these receptors induces proinflammatory gene expression
and subsequent production of cytokines and chemokines
[11].
Molecular imaging techniques, such as positron emis-
sion tomography (PET) and single-photon emission com-
puted tomography (SPECT), can noninvasively visualize
specific targets of the inflammation cascade through speci-
fic and sensitive probes and, as such, can be powerful
tools to track the progression of neuroinflammatory pro-
cesses including those in AD. In particular, in vivo imag-
ing of microglia can offer a measure of the inflammatory
process and a means of tracking the progression of those
pathologies that trigger an immune activation in the brain
and the efficacy of therapeutic treatments over time [12].
Over the past decade, several targets for molecular imag-
ing of neuroinflammation have been studied, including
translocator protein 18 kDa (TSPO), that is considered a
hallmark of activated microglia [13], and endothelial
adhesion molecules, that are involved in the recruitment
DOI: 10.1002/cbdv.201500281 © 2016 Wiley-VHCA AG, Z
urich
Chem. Biodiversity 2016, 13, 875 883 875

of circulating leukocytes [12]. However, none of these
molecular targets has been fully validated as a biomarker
for the visualization of microglial activation.
The human FPR family belongs to the class A of G-pro-
tein coupled receptors and includes three subtypes, termed
FPR, FPR-like type-1 (FPRL-1), and type-2 (FPRL-2).
FPRs are expressed in several immune cells including
leukocytes, monocytes/macrophages, and microglia, and
are considered to play relevant roles in innate immunity
and host defense mechanisms and chemotaxis [14]. Among
FPRs, the FPRL-1, and its murine homologue FPR2,
appear to be relevant to the proinflammatory aspects of
AD, because FPRL-1 is a chemotactic receptor for Ab
42
,
the 42 amino acid form of Ab, which induces monocytes
migration and activation [15]. FPRL-1 also is involved in
the uptake and fibrillary aggregation of Ab
42
in mononu-
clear phagocytes by rapidly internalizing the Ab
42
-FPRL-1
complexes into cytoplasmic region [16]. FPRL-1 mediates
Ab
42
-induced senescence in neural stem/progenitor cells in
the hippocampus of APP/PS1 mice, an animal model of
AD [17]. Recently, it has been demonstrated that the
expression levels of FPRL-1 in primary microglial cells
increase after exposure to Ab and the recognition of Ab by
the FPRL-1 seems to be the starting point of the signaling
cascade inducing the inflammatory state [18]. Slowick et al.
has evidenced that APP/PS1 mice show higher expression
level of FPRs as compared to wild-type littermates. In par-
ticular, a significant increase in expression of FPR and
FPRL-1 in glial cells was observed in the cortex and hip-
pocampus through immunofluorescence and real-time PCR
analyses [19]. Therefore, FPRL1 could represent an inter-
esting target for monitoring in vivo the onset and progres-
sion of neuroinflammation in AD through molecular
imaging techniques.
To the best of our knowledge, only two studies have
been reported to date dealing with in vivo imaging of
FPRs. Locke et al. [20] have reported the radiosynthesis
and in vivo evaluation of cFLFLFK-PEG-
64
Cu, a peptide
analog of formyl peptide. The radiolabeled peptide was
able to bind to neutrophils in vitro and to accumulate
peripherally at sites of inflammation in vivo. Zhang et al.
[21] have evaluated the ability of cFLFLF-
64
Cu in the
detection of macrophages in pancreatic islets and in
the aorta in comparison with [
18
F]-fluorodesoxyglucose.
The ability of these radiotracers to penetrate and dis-
tribute into the brain was not studied. In general, the
in vivo use of peptides is hampered by short half-life due
to rapid proteolysis in plasma. Moreover, peptides cannot
cross bloodbrain barrier (BBB) unless they interact with
specific transport systems [22]. Conversely, non-peptidic
small molecules may have some advantages because they
can be suitably designed to modulate properties, such as
potency, selectivity, lipophilicity, and cell permeability,
that are pivotal for a potential radiotracer [23].
Recently, we have reported on the identification of a
series of non-peptidic agonists for FPRs with 3-(1H-indol-
3-yl)-2-[3-(4-nitrophenyl)ureido]propanamide structure
[24]. Among the studied compounds, we have focused our
attention on (S)-1 (Table) as a PET radiotracer candidate,
because (S)-1 demonstrated potent interaction with FPRs
by inducing Ca
2+
mobilization in transfected HL-60 cells
and in human neutrophils. (S)-1 was also able to potently
induce chemotaxis in human neutrophils (Table) [24].
Importantly, (S)-1 showed a MeO group amenable of easy
labeling of the molecule with
11
C. Therefore, on the basis
of such considerations, we have selected (S)-1 as a candi-
date for in vivo PET imaging of FPRs. To the best of our
knowledge, no study aimed at the visualization of FPRs in
activated microglial cells has been reported to date.
Results and Discussion
Lipophilicity
Lipophilicity is a major factor influencing passive brain
entry and can be determined in various theoretical and
Table. Chemical structure and biological properties of (S)-1
Ca
2+
Mobilization
in transfected HL-
60 cells EC
50
[lM]
a
)
Neutrophils EC
50
[lM] Log k
0
Plasma stability t
1/2
[h] BA/AB
FPR FPRL-1 Chemotaxis Ca
2+
mobilization w/o inhibitor With inhibitor
0.26 0.19 0.030 0.086 0.31 > 4 3.5 3.2
a
) Data taken from [24].
876 Chem. Biodiversity 2016, 13, 875 883
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urich

experimental ways [25]. For this purpose, we assessed the
retention factor of (S)-1, log k
0
as lipophilicity index,
using a reversed-phase HPLC method (Table) [26]. We
determined also the retention factor of PD-176252, a
bombesin antagonist that is structurally related to (S)-1
and that is known to be active in vivo in various behav-
ioral tests after i.p. administration in the rat and guinea
pig [27], suggesting that it is able to accumulate into the
brain. The log k
0
for (S)-1 and PD-176252 were 0.31 and
0.43, respectively, indicating that the two compounds have
similar lipophilic properties and, thus, it is likely that (S)-1
is able to enter into the brain.
Serum Stability and Permeability Evaluation
Before embarking in the radiolabeling of (S)-1, we evalu-
ated two additional features that are of importance for
brain PET radiotracers. First, we examined if (S)-1 was a
P-glycoprotein (P-gp) substrate, because PET tracers that
are not substrates of the P-gp efflux pump have higher
chances for crossing the BBB. Therefore, we evaluated
the efflux ratio between basal-to-apical (BA) and apical-
to-basal (AB) fluxes in Caco-2 cells monolayer (BA/AB)
of compound (S)-1. Generally, a cutoff value of 3 is used
to distinguish P-gp substrate from nonsubstrate [28]. The
efflux ratio was evaluated in the presence or in the
absence of a P-gp inhibitor in order to assess the magni-
tude of the interaction with P-gp interaction. (S)-1
showed an efflux ratio of 3.5 and 3.2 in the absence or in
the presence of a P-gp inhibitor, respectively (Table).
These data indicated that (S)-1 can cross biological mem-
branes and has very weak interaction with P-gp. Second,
we evaluated if (S)-1 was stable in serum for, at least, the
timeframe of a PET scan. (S)-1 showed the desired stabil-
ity with degradation half-life (t
1/2
) longer than 4 h
(Table).
Radiochemistry
As stated above, we selected (S)-1 as a candidate for the
preparation of a PET radiotracer, because it presents a
MeO group that can be easily radiolabeled with a posi-
tron emitter
11
C. We chose to prepare the desmethyl
derivative (S)-5, which bears the OH group on the pheny-
lureidic moiety of the molecule, because of its chemical
accessibility.
The desmethyl precursor for
11
C-radiolabeling (S)-5
was easily prepared as shown in Scheme 1.(S)-Boc-tryp-
tophan was activated with N,N
0
-carbonyldiimidazole and
condensed with the amine 2, prepared as previously
reported [29], to give the Boc-protected derivative (S)-3.
Subsequently, the latter compound was deprotected with
trifluoroacetic acid to give the amine (S)-4, which was
condensed with 4-aminophenol in the presence of N,N
0
-
carbonyldiimidazole to give the ureido derivative (S)-5 in
good yield.
The radiosynthesis of (S)-[
11
C]-1 was successfully per-
formed in high yield using a home-made automated syn-
thesis system [30]. (S)-[
11
C]-1 was prepared by reacting
the precursor (S)-5 with [
11
C]-MeI. [
11
C]-MeI was pre-
pared by reducing cyclotron-produced [
11
C]-CO
2
with
LiAlH
4
, followed by iodination with 57% HI. After distil-
lation and drying, [
11
C]-MeI was transferred into a DMF
solution of (S)-5 and NaOH. The [
11
C]-methylation
Scheme 1
(S)-Boc-tryptophan activated with N,N
0
-carbonyldiimidazole. b) Trifluoroacetic acid. c) 4-Aminophenol, N,N
0
-carbonyldiimidazole. d)[
11
C]MeI.
Chem. Biodiversity 2016, 13, 875 883 877
© 2016 Wiley-VHCA AG, Z
urich www.cb.wiley.com

proceeded efficiently in 5 min at 70°. Semipreparative
RP-HPLC purification of the mixture gave (S)-[
11
C]-1 in
15 3(n = 5) radiochemical yield (decay-corrected)
based on [
11
C]-CO
2
. Starting from 12.2 to 18.5 GBq of
[
11
C]-CO
2
, 0.73 1.41 GBq of (S)-[
11
C]-1 were produced
(31 min of synthesis time from the end of bombardment
(EOB)).
The identity of (S)-[
11
C]-1 was confirmed by coinjec-
tion with unlabeled (S)-1 on analytical RP-HPLC. In the
final product solutions, the radiochemical purity was
higher than 99% at EOS. No significant peak correspond-
ing to unreacted (S)-5 was observed in the final product.
Moreover, the radioligand did not show radiolysis at
room temperature after 180 min from formulation,
showing adequate radiochemical stability within the time-
frame of one PET scan. The analytical results were in
compliance with our in-house quality control/assurance
specifications for radiopharmaceuticals.
PET Studies
Fol low ing a heuristic approach, we decided to evaluate
the ability of (S )-[
11
C]-1 to acc umulate in mouse brain
bypassing in vitro autoradiography studies. While these
studie s can offer important in formati on on the interac-
tion bet ween the radiotracer and the target, they cannot
predict radiotracer brain uptake or other biodistribution
issues. In vivo dynamic P ET scans of normal rat brains
were conducted immediately after bolus intravenous
injection of the radioligand. PET images were generated
by averaging dynamic data at 0 90 min after injection
of (S)-[
11
C]-1, and indicated minimal radioactivity in the
brain (top panel in Fig. 1). Timeradioactivity curve for
the whole brain also demonstrated an initial spike cor-
responding to radioactivity in cerebral vessels, followed
by very low retention of radioactivity in the b rain (bot-
tom panel in Fig. 1).Thesedataindicatedthat
(S)-[
11
C]-1 was not able to accumulate into the brain
and, therefore, it is not suitable for imaging of FPRL-1
in living brain. The lack of brain uptake was quite
unexpected based on the permeability study in Caco-2
cell monolayer.
Subsequently, we tested the ability of (S)-[
11
C]-1 to
label FPRs in mouse brain in autoradiography studies. To
this end, we selected the APP23 mice model, a transgenic
mouse strain showing a robust formation of amyloid pla-
ques, and the PS19 mouse model that is characterized by
progressive accumulation of fibrillary tau tangles. Both
animal models are characterized by abundant accumula-
tion of activated microglia in tight association with tau
and amyloid deposits that can be visualized through an
in vitro autoradiography or an in vivo PET with suitable
radioligands [31 33]. The brain tissue slices from aged
PS19 and APP23 mice and from their wild-type litter-
mates were incubated for 1 h with (S)-[
11
C]-1 solution at
1n
M concentration (2 mCi/l). Total binding (TB) and
nonspecific binding (NSB) of (S)-[
11
C]-1 were assessed by
challenging the radioligand in the absence or in the pres-
ence of 10 l
M of fMLF. No specific radioligand binding
was observed in the sections from any of the mouse
strains, despite massive gliosis in PS19 and APP23 mouse
brains (Fig. 2). These data indicated that the occupancy
of FPRs by (S)-[
11
C]-1 at 1 nM was very low and was not
sufficient to autoradiographically label FPRs receptors in
the brain.
In parallel, we evaluated the ability of (S)-[
11
C]-1 to
label FPRs in peripheral inflammation. To this end, pneu-
monia was induced in rats by administration of
lipopolysaccharide (LPS). LPS is a bacterial endotoxin
that is used to stimulate the immune system through acti-
vation of macrophage-like cells in peripheral tissues.
Moreover, it has been reported that LPS is able to upreg-
ulate expression of FPRs in both peripheral tissues and
microglial cells [34]. According to established experimen-
tal procedures [35], the animals were administered intra-
tracheally with LPS (1.25 mg, 1 d before the scan) to
induce the inflammatory response. Pulmonary radioactiv-
ity retention in the LPS-treated rats was very low and
Fig. 1. (top) Sagittal PET images showing distribution of radioacticity
in the rat brain and surrounding tissues after intravenous administra-
tion of (S)-[
11
C]-1. PET data were generated by summation of dynamic
data at 0 90 min after radioligand injection, and were merged onto
the MRI anatomical template. (bottom) Time-radioactivity curve in
the whole brain of a rat after intravenous administration of (S)-[
11
C]-1.
878 Chem. Biodiversity 2016, 13, 875 883
www.cb.wiley.com © 2016 Wiley-VHCA AG, Z
urich

equivalent to control levels (Fig. 3). These data indicated
that (S)-[
11
C]-1 was not able to label FPRs in this animal
model. post mortem Examinations of LPS-treated animals
proved profound infiltration of neutrophils in the lungs of
LPS-treated rats. Therefore, it is possible that in vivo (S)-
[
11
C]-1 has low affinity for FPRs. However, high
Fig. 2. In vitro autoradiographic labeling of coronal mouse brain slices with (S)-[
11
C]-1. Brain samples were derived from PS19 and APP23 trans-
genics and their wild-type littermates. The images demonstrate radioligand binding without blockade (total binding, TB) and with 10 l
M fFML
(nonspecific binding, NSB).
Fig. 3. Horizontal (top) and sagittal (bottom) PET images showing distribution of radioactivity in the lungs and liver at 20 90 min after intra-
venous administration of (S)-[
11
C]-1 in normal control rat (right) and rat with LPS-induced lung inflammation (left).
Chem. Biodiversity 2016, 13, 875 883 879
© 2016 Wiley-VHCA AG, Z
urich www.cb.wiley.com

Citations
More filters
Journal ArticleDOI
TL;DR: A new series of ureidopropanamide derivatives were designed with the goal of converting functional activity from agonism into antagonism and to develop new FPR2 antagonists, and showed that they decreased the production of reactive oxygen species in mouse microglial N9 cells after stimulation with lipopolysaccharide.
Abstract: Formyl peptide receptor-2 (FPR2) is a G protein-coupled receptor belonging to the N-formyl peptide receptor (FPR) family that plays critical roles in peripheral and brain inflammatory responses. FPR2 has been proposed as a target for the development of drugs that could facilitate the resolution of chronic inflammatory reactions by enhancing endogenous anti-inflammation systems. Starting from the structure of the FPR2 agonists (R)- and (S)-4 and 2, we designed a new series of ureidopropanamide derivatives with the goal of converting functional activity from agonism to antagonism and to develop new FPR2 antagonists. Although none of the compounds behaved as antagonist, some of the compounds were able to induce receptor desensitization, thus functionally behaving as antagonists. Evaluation of these compounds in an in vitro model of neuroinflammation showed that they reduced reactive oxygen species (ROS) production in mouse microglial N9 cells after stimulation with lipopolysaccharide (LPS). These FPR2 ligands may protect cells from damage due to inflammation-associated oxidative stress.

8 citations

Journal ArticleDOI
TL;DR: In this paper, the role of FPRs in the development and prognosis of neurological diseases is discussed, and it is shown that FPR2 acts as a key molecule that mediates the active resolution of inflammation, which binds with corresponding receptors to reduce the expression and activation of pro-inflammatory composition.
Abstract: Formyl peptide receptors (FPRs) are a group of G protein-coupled cell surface receptors that play important roles in host defense and inflammation. Owing to the ubiquitous expression of FPRs throughout different cell types and since they interact with structurally diverse chemotactic agonists, they have a dual function in inflammatory processes, depending on binding with different ligands so that accelerate or inhibit key intracellular kinase-based regulatory pathways. Neuroinflammation is closely associated with the pathogenesis of neurodegenerative diseases, neurogenic tumors and cerebrovascular diseases. From recent studies, it is clear that FPRs are important biomarkers for neurological diseases as they regulate inflammatory responses by monitoring glial activation, accelerating neural differentiation, regulating angiogenesis, and controlling blood brain barrier (BBB) permeability, thereby affecting neurological disease progression. Given the complex mechanisms of neurological diseases and the difficulty of healing, we are eager to find new and effective therapeutic targets. Here, we review recent research about various mechanisms of the effects generated after FPR binding to different ligands, role of FPRs in neuroinflammation as well as the development and prognosis of neurological diseases. We summarize that the FPR family has dual inflammatory functional properties in central nervous system. Emphasizing that FPR2 acts as a key molecule that mediates the active resolution of inflammation, which binds with corresponding receptors to reduce the expression and activation of pro-inflammatory composition, govern the transport of immune cells to inflammatory tissues, and restore the integrity of the BBB. Concurrently, FPR1 is essentially related to angiogenesis, cell proliferation and neurogenesis. Thus, treatment with FPRs-modulation may be effective for neurological diseases.

8 citations

Journal ArticleDOI
01 Aug 2017
TL;DR: The review summarizes the developments of various radiolabeled ligands for PET imaging of neuroinflammation, based on detection of isotope labeled tracers, which emit positrons.
Abstract: Non-invasive molecular imaging techniques can enhance diagnosis of neurological diseases to achieve their successful treatment. Positron emission tomography (PET) imaging can identify activated microglia and provide detailed functional information based on molecular biology. This imaging modality is based on detection of isotope labeled tracers, which emit positrons. The review summarizes the developments of various radiolabeled ligands for PET imaging of neuroinflammation.

1 citations

References
More filters
Journal ArticleDOI
TL;DR: A functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement is shown and suggests both formyl peptide receptors play an essential role in Aβ1-42-induced signal transduction in glial cells.
Abstract: Recent studies suggest that the chemotactic G-protein-coupled-receptor (GPCR) formyl-peptide-receptor-like-1 (FPRL1) and the receptor-for-advanced-glycation-end-products (RAGE) play an important role in the inflammatory response involved in neurodegenerative disorders such as Alzheimer’s disease (AD). Therefore, the expression and co-localisation of mouse formyl peptide receptor (mFPR) 1 and 2 as well as RAGE in an APP/PS1 transgenic mouse model using immunofluorescence and real-time RT-PCR were analysed. The involvement of rat or human FPR1/FPRL1 (corresponds to mFPR1/2) and RAGE in amyloid-β 1–42 (Aβ1-42)-induced signalling were investigated by extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, the cAMP level in primary rat glial cells (microglia and astrocytes) and transfected HEK 293 cells was measured. Formyl peptide receptors and RAGE were inhibited by a small synthetic antagonist WRW4 and an inactive receptor variant delta-RAGE, lacking the intracytoplasmatic domains. We demonstrated a strong increase of mFPR1/2 and RAGE expression in the cortex and hippocampus of APP/PS1 transgenic mice co-localised to the glial cells. In addition, the Aβ1-42-induced signal transduction is dependant on FPRL1, but also on FPR1. For the first time, we have shown a functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition a possible physical interaction between FPRL1 as well as FPR1 and RAGE was shown with co-immunoprecipitation and fluorescence microscopy. The results suggest that both formyl peptide receptors play an essential role in Aβ1-42-induced signal transduction in glial cells. The interaction with RAGE could explain the broad ligand spectrum of formyl peptide receptors and their important role for inflammation and the host defence against infections.

77 citations

01 Jan 2009
TL;DR: This new radiolabeled peptide targeting the FPR binds to neutrophils in vitro and accumulates at sites of inflammation in vivo and may prove to be a useful tool to probe inflammation or injury.
Abstract: Thesynthesisandvalidationofanew,highlypotent 64Cu-labeled peptide, cFLFLFK-PEG- 64 Cu, that targets the formyl peptide receptor (FPR) on leukocytes is described. The peptide ligand is an antagonist of the FPR, designed not to elicit a chemotactic response resulting in neutropenia. Evidence for the selective binding of this synthesized ligand to neutrophils is provided. PET properties of the compound were evaluated in a mouse model of lung inflammation. Methods: The FPR-specific peptide, cinnamoyl-F-(D)L-F-(D)L-FK (cFLFLF), was sequentially conjugated with a bifunctional polyethylene glycol moiety (PEG, 3.4 kD) and a 2,29,2$,2$9-(1,4,7,10-tetraazacyclododecane1,4,7,10-tetrayl)tetraacetic acid (DOTA) through a lysine (K) spacer and finally labeled with 64 Cu-CuCl2 to form cFLFLFKPEG-64Cu.The bindingaffinity andstimulation potency of the ligand toward human neutrophils were assessed in vitro. Blood kinetic and organ biodistribution properties of the peptide were studied in the mouse. Ten male C57BL/6 mice were used in this study; 4 control mice and 6 administeredKlebsiella pneumonia. PET/CT scans were performed to assess the localization properties of the labeled peptide in lungs 18 h after tracer administration. Lung standardized uptake values (SUVs) were correlated with lung neutrophil activity as measured by myeloperoxidase assays. Immunohistochemistry was performed to confirm that neutrophils constitute the majority of infiltrating leukocytes in lung tissue 24 h after Klebsiella exposure. Results: In vitro binding assays of the compound cFLFLFKPEG-64Cu to the neutrophil FPR yielded a dissociation constant of 17.7 nM. The functional superoxide stimulation assay exhibited negligible agonist activity of the ligand with respect to neutrophil superoxide production. The pegylated peptide ligand exhibited a blood clearance half-life of 55 6 8 min. PET 18 h after tracer administration revealed mean lung SUVs and lung myeloperoxidase activities for Klebsiella-infected mice that were 5- and 6-fold higher, respectively, than those for control mice. Immunohistochemistry staining confirmed that the cellular infiltrate in lungs of Klebsiella-infected mice was almost exclusively neutrophils at the time of imaging. Conclusion: This new radiolabeled peptide targeting the FPR binds to neutrophils in vitro and accumulates at sites of inflammation in vivo. This modified peptide may prove to be a useful tool to probe inflammation or injury.

76 citations

Journal ArticleDOI
TL;DR: It is suggested that endogenous BB-like peptides at the DRN evoke the release of 5-HT from the limbic nerve terminals originating from the raphé, specifically at the ventral hippocampus, resulting in anxiogenesis.
Abstract: The effects of PD 176252 [3-(1H-indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]-2-methyl-2-[3-(nitro-phenyl)ureido]propionamide], a nonpeptide bombesin (BB) BB1/BB2 receptor antagonist, were assessed in rats using several ethologically relevant tests of anxiety. Consistent with a role for the bombesin family of peptides in subserving anxiety behaviors, the antagonist increased social interaction (3.75 and 7.5 mg/kg, i.p.), dose-dependently attenuated the number of vocalizations emitted by guinea pig pups separated from their mother (1-30 mg/kg, i.p.), reduced latency to approach a palatable snack in an anxiogenic (unfamiliar) environment, and reduced the fear-potentiated startle response (5 and 10 mg/kg, i.p., and 100-200 ng per rat, i.c.v.). When administered directly to the dorsal raphe nucleus (DRN), PD 176252 (20-500 ng) increased social interaction under aversive conditions, as did the 5-HT1A receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (50 ng). Furthermore, intra-DRN microinfusion of the peptide antagonist (PD 176252) suppressed, whereas its agonist [neuromedin B (NMB)-30] promoted, the in vivo release of 5-HT in the ventral hippocampus. In parallel, the suppressed social interaction elicited by intra-DRN administration of NMB was attenuated by a systemically administered 5-HT2C (but not 5-HT1A) receptor antagonist. Together, these findings suggest that endogenous BB-like peptides at the DRN evoke the release of 5-HT from the limbic nerve terminals originating from the raphe, specifically at the ventral hippocampus, resulting in anxiogenesis. The finding that this action was attenuated by BB receptor (BB1 and/or BB2) antagonists suggests that these compounds may represent a novel class of anxiolytic agents.

71 citations

Journal ArticleDOI
12 Sep 2012-PLOS ONE
TL;DR: It is concluded that PET with [18F]FEDAC may be a useful tool for imaging TSPO expression and evaluating progress of lung inflammation.
Abstract: Purpose The translocator protein (18 kDa) (TSPO) is highly expressed on the bronchial and bronchiole epithelium, submucosal glands in intrapulmonary bronchi, pneumocytes and alveolar macrophages in human lung. This study aimed to perform positron emission tomography (PET) imaging of lung inflammation with [(18)F]FEDAC, a specific TSPO radioligand, and to determine cellular sources enriching TSPO expression in the lung. Methods An acute lung injury model was prepared by intratracheal administration of lipopolysaccharide (LPS) to rat. Uptake of radioactivity in the rat lungs was measured with small-animal PET after injection of [(18)F]FEDAC. Presence of TSPO was examined in the lung tissue using Western blot and immunohistochemical assays. Results The uptake of [(18)F]FEDAC increased in the lung with the progress of inflammation by treatment with LPS. Pretreatment with a TSPO-selective ligand PK11195 showed a significant decrease in the lung uptake of [(18)F]FEDAC due to competitive binding to TSPO. TSPO expression was elevated in the inflamed lung section and its level responded to the [(18)F]FEDAC uptake and severity of inflammation. Increase of TSPO expression was mainly found in the neutrophils and macrophages of inflamed lungs. Conclusion From this study we conclude that PET with [(18)F]FEDAC may be a useful tool for imaging TSPO expression and evaluating progress of lung inflammation. Study on human lung using [(18)F]FEDAC-PET is promising.

70 citations

Journal ArticleDOI
TL;DR: Specific biological assays have been carried out to establish the P-gp interacting mechanism of tested compounds discriminating between substrates and inhibitors and both the basicity and the presence of H-bond donor or acceptor groups seem to be negligible.

56 citations

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Frequently Asked Questions (1)
Q1. What are the contributions mentioned in the paper "Full paper radiosynthesis and in vivo evaluation of carbon-11 (2s)-3-(1h-indol-3-yl)-2-{[(4- methoxyphenyl)carbamoyl]amino}-n-{[1-(5-methoxypyridin-2-yl)cyclohexyl]methyl} propanamide: an attempt to visualize brain formyl peptide receptors in mouse models of neuroinflammation" ?

Lacivita et al. this paper presented an attempt to visualize brain formyl peptide receptors ( FPRs ) in mouse models of neuroinflammation.