Development of a peptidomimetic antagonist of neuropeptide FF receptors for the prevention of opioid-induced hyperalgesia

Article abstract of DOI:10.1021/cn500219h

Through the development of a new class of unnatural ornithine derivatives as bioisosteres of arginine, we have designed an orally active peptidomimetic antagonist of neuropeptide FF receptors (NPFFR). Systemic low-dose administration of this compound to rats blocked opioid-induced hyperalgesia, without any apparent side-effects. Interestingly, we also observed that this compound potentiated opioid-induced analgesia. This unnatural ornithine derivative provides a novel therapeutic approach for both improving analgesia and reducing hyperalgesia induced by opioids in patients being treated for chronic pain.

Full text of DOI:10.1021/cn500219h

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Article
Development of a peptidomimetic antagonist of neuropeptide
FF receptors for the prevention of opioid-induced hyperalgesia
Frédéric Bihel, Jean-Paul Humbert, Séverine Schneider, Isabelle Bertin, Patrick Wagner,
Martine Schmitt, Emilie Laboureyras, Benoit Petit-Demoulière, Elodie Schneider,
Catherine Mollereau, Guy Simonnet, Frédéric Simonin, and Jean-Jacques Bourguignon
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/cn500219h • Publication Date (Web): 14 Jan 2015
Downloaded from http://pubs.acs.org on January 26, 2015
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Development of a peptidomimetic antagonist of
neuropeptide FF receptors for the prevention of
opioidꢀinduced hyperalgesia.
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Frédéric Bihel,a‡ Jean-Paul Humbert,b Séverine Schneider,a Isabelle Bertin,b Patrick Wagner,a
Martine Schmitt,a Emilie Laboureyras,c Benoît Petit-Demoulière,d Elodie Schneider,e Catherine
Mollereau,f Guy Simonnet,c Frédéric Simonin,b&* and Jean-Jacques Bourguignon a*
aUniversity of Strasbourg, CNRS, UMR7200, Faculty of pharmacy, 67400 Illkirch
Graffenstaden, France
bUniversity of Strasbourg, CNRS, UMR7242, ESBS, 67412 Illkirch Graffenstaden, France
cUniversity of Bordeaux Ségalen, INCIA, CNRS UMR 5287, 33076 Bordeaux, France
dInst. Clinique de la Souris, Strasbourg Univ., Illkirch, France
ePhenopro, Illkirch, France
fUniversity of Toulouse, CNRS, UMR5089, IPBS, 31077 Toulouse, France
KEYWORDS. NPFF; GPCR; GPR147; GPR74; peptidomimetic; unnatural amino acid;
ornithine; arginine; bioisoster; Opioidꢀinduced hyperalgesia.
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ABSTRACT
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Through the development of a new class of unnatural ornithine derivatives as bioisosteres of
arginine, we have designed an orally active peptidomimetic antagonist of neuropeptide FF
receptors (NPFFR). Systemic lowꢀdose administration of this compound to rats blocked opioidꢀ
induced hyperalgesia, without any apparent sideꢀeffects. Interestingly, we also observed that this
compound potentiated opioidꢀinduced analgesia. This unnatural ornithine derivative provides a
novel therapeutic approach for both improving analgesia and reducing hyperalgesia induced by
opioids in patients being treated for chronic pain.
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INTRODUCTION
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Development of drugs that can effectively and safely treat both acute and chronic pain
(resulting from postoperative surgery, cancer, neuropathies, etc.) remains a major unmet
challenge of pharmacotherapy. Opiate analgesics, such as morphine and fentanyl, continue to be
the cornerstones for treating moderate to severe pain. However, with chronic administration,
their usefulness is limited by prominent sideꢀeffects such as tolerance and dependence. These
side effects have been proposed to result from adaptive modifications in cellular responsiveness,
primarily involving desensitization and downꢀregulation of opioid receptors.1
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An intriguing hypothesis is that the decreased effectiveness of opioids with longꢀterm use may,
in part, result from activation of antiꢀopioid systems that produce hyperalgesia, thus diminishing
2ꢀ4
the net analgesic effect of opioid agonists.
This phenomenon is termed opioidꢀinduced
hyperalgesia (OIH). In rats, both acute and prolonged opioid treatment induces a long lasting
OIH that persists for several days after opioid administration is stopped.5ꢀ8 In humans, several
reports indicate that both acute and chronic opioid treatments may be associated with paradoxical
hyperalgesia and/or allodynia.9ꢀ11 Enhancements in pain sensitivity have been also reported in
heroin addicts.12 Several neuromodulators have antiꢀopioid properties, including NMDA,
cholecystokinin (CCK), nociceptin/orphanin FQ and neuropeptide FF (NPFF).3,13ꢀ17
The NPFF system is composed of two GPCR subtypes, NPFFR1 (GPR147) that binds the
mammalian neuropeptides VF (NPVF: VPNLPQRFꢀNH2) whereas NPFFR2 (GPR74) has a
greater affinity for neuropeptide FF (FLFQPQRFꢀNH2). These two receptors belong to the
GPCR subfamily called the RFꢀamide receptors, which also includes GPR10, GPR54 and
GPR103. This subfamily is known to bind endogenous neuropeptides that have the Cꢀterminal
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sequence ArgꢀPheꢀNH2. Originally identified as an inducer of hyperalgesia and a modulator of
opiate analgesia,18 subsequent in vitro and in vivo studies have implicated NPFF in the regulation
of the cardiovascular system,19ꢀ21 body temperature,22ꢀ23 feeding behavior,24 metabolism,25 and
water balance.26ꢀ27
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Based on the observation that the Cꢀterminal sequence ArgꢀPheꢀNH2 is common to all of the
neuropeptide ligands of the RFꢀamide GPCR family,28ꢀ31 we previously investigated the Nꢀ
acylated dipeptide RF9 as a nonꢀselective antagonist of both hNPFFR1 and hNPFFR2 (Table
1).32 Stable enough to be subcutaneously coꢀadministrated with opiates, RF9 completely blocked
the delayed and long lasting paradoxical hyperalgesia induced by chronic opiate treatment, and
prevented the development of associated tolerance.16,32 Moreover, in agreement with its
antagonist function at both NPFF receptor subtypes, RF9 was shown to prevent hypothermia and
hypertension induced by NPFF or NPVF.23,32ꢀ33 Starting from the Nꢀacylated dipeptide ArgꢀPheꢀ
NH2, here we describe the design of an NPFF antagonist derived from an unnatural ornithine
amino acid that is able to prevent opioidꢀinduced hyperalgesia after oral administration in rats.
RESULTS AND DISCUSSION
Structure-activity relationships (SAR) of the dipeptide RF-NH2. In a recent study, we
reported on an extensive optimization of the Nꢀterminal region of the amidated dipeptide Argꢀ
PheꢀNH2, which allowed for the identification of compound 2 bearing the 2ꢀphenylbenzoyl
moiety at the Nꢀterminus as a selective subnanomolar ligand of NPFFR1 (Table 1).34 Starting
with RF9 and compound 2 as compounds of reference, we performed a structureꢀactivity
relationship analysis of the dipeptide series. All dipeptide compounds were synthesized by
standard Fmocꢀbased solidꢀphase peptide synthesis (FmocꢀSPPS) on Rink amide resin.
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Table 1. Binding affinity of Nꢀacylated dipeptides towards NPFF1 & 2 receptorsa
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Ki hNPFF1
(nM)b
Ki hNPFF2
(nM)b
Cpd
R
X1ꢀX2
RF9
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ArgꢀPhe
CitꢀPhe
22 ± 5
43 ± 1
880 ± 120
> 13 µM
2
ArgꢀPhe
0.32 ± 0.02
920 ± 50
3
ArgꢀAla
2 400 ± 440
> 10 µM
[3H]ꢀFFRFꢀNH2 was used as radioligand for competition assays with both
hNPFF1 & 2 receptors. b Values are expressed as the mean ± SEM
a
As reported previously, we have shown that the natural amino acid configuration of the
sequence LꢀArgꢀLꢀPhe of RF9 was optimal for both receptors. We then evaluated the mode of
interaction of the arginine residue with NPFF receptors. The guanidine moiety can establish two
types of interaction within the active site. The first one is electrostatic through its ability to be
protonated at physiological pH and the second one involves a set of Hꢀbond acceptorꢀdonor
interactions through the various amino groups. To address the importance of the electrostatic
interaction, we replaced the arginine amino acid of RF9 by a citruline residue (1), which bears a
urea moiety in place of a guanidine on the side chain. Like guanidine, urea can establish Hꢀbond
acceptorꢀdonor interactions, but lacks electrostatic interactions. This change led to a drastic loss
in affinity towards both NPFF receptors, confirming the requirement for an electrostatic
interaction at this position. Indeed, replacement of arginine by citruline in the NPFF sequence
was recently reported as leading to a dramatic loss in potency for both NPFF receptors.28 To
determine the importance of the aromatic ring close to the Cꢀterminus, the phenylalanine in
compound 2 was replaced by an alanine (3). This resulted in a significant loss of affinity,
emphasizing the requirement of an aromatic ring close to the Cꢀterminal end. This result is
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consistent with the fact that [Alaꢀ8]NPFF was reported as not active towards both NPFF
receptors.28
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SAR of the arginine derivatives. In 1989, Allard et al. reported that replacing the Cꢀterminal
carboxamide by a carboxylic acid led to a complete loss of activity.35ꢀ36 However, more recently,
it has been reported that the NPY antagonist BIBP3226, based on a single arginine core resulting
from the deletion of the Cꢀterminal carboxamide of the ArgꢀTyr sequence, was a potent
antagonist of both NPFF receptors.30 Based on this latter result, the Cꢀterminal carboxamide
function was deleted from dipeptide 2, leading to arginine derivative 4a (Table 2). The deletion
of the Cꢀterminal amide (4a) led to a twoꢀorder of magnitude loss in affinity toward hNPFFR1
(Ki = 58 nM) and a significant gain in affinity toward hNPFFR2 (Ki = 250 nM), in comparison
with the dipeptide 2. This result highlights the critical role played by the Cꢀterminal carboxamide
as a Hꢀbond acceptor/donor system in the binding to hNPFFR1 but not to hNPFFR2. The
corresponding Dꢀenantiomer 4b led to slight decrease of affinity for hNPFFR1, but interestingly,
a gain of affinity for hNPFFR2 was observed, suggesting that the Dꢀconfiguration would be
more favorable to hNPFFR2. However, these negative or positive effects of the configuration on
the affinity towards hNPFFR1 or hNPFFR2 respectively, are still moderate, maybe because of
the spatial flexibility of the arginine side chain which compensates the configuration effect. By
increasing the length of the alkyl chain (4d) or by adding a chlorine atom in the paraꢀposition
(4f), the affinity for hNPFFR1 was further improved. These results suggest the presence of a
hydrophobic pocket. The introduction of a Nꢀmethyl group at the Cꢀterminal part of 4a led to 4h,
which showed a potent affinity towards both receptors, similar to that observed with the
reference compound RF9. This result highlights the fact this Cꢀterminal amide is not involved in
a Hꢀbond donor interaction with either of the receptors. Based on this result, we applied a
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rigidification of the Cꢀterminal part through the introduction of cyclic molecules such as
tetrahydroisoquinoline (4i) or piperidine (4j, 4k) moieties, resulting in a drop of affinity for
hNPFFR1 and a slight gain of affinity for hNPFFR2 with compounds 4i and 4j. 4k, bearing a 4ꢀ
benzylpiperidine moiety, showed a similar affinity for hNPFFR1 than 4a. At the difference of 4i
and 4j, the phenyl group at the Cꢀterminus of 4k is in free rotation around a methylene moiety.
This last result indicates that a flexible aromatic group is preferred for occupying the
hydrophobic pocket.
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Table 2. Binding affinity of arginine derivatives towards NPFF1 & 2 receptorsa
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Conditions: (a) amine (1.1 eq.), BOP (1.1 eq.), NMM (2
eq.) DMF, rt; (b) DBU (2 eq.), DCM, rt; (c) 2ꢀ
Phenylbenzoic acid (1.1 eq.), BOP (1.1 eq.), NMM (2 eq.),
DCM, rt; (d) TFA, DCM, rt.
Ki NPFF1
Ki NPFF2
Cpd
C*
R
(nM)b
(nM)b
RF9
2
22 ± 5
0.32 ± 0.02
58 ± 18
43 ± 1
920 ± 50
250 ± 43
97 ± 5
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D
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74 ± 4
15 ± 3
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53 ± 12
21 ± 1
69 ± 2
84 ± 19
nd
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320 ± 11
190 ± 19
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53 ± 13
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nd
4k
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C*: amino acid configuration; [3H]ꢀFFRFꢀNH2 was used as radioligand for
competition assays with both hNPFF1 & 2 receptors. b Values are expressed as the
mean ± SEM; c nd : not determined.
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We next explored the side chain of the arginine moiety, by introducing on the guanidine
moiety an aromatic moiety using several alkyl spacers (Table 3). The Nγꢀsubstituted
argininamide derivatives 7b-d were prepared by guanidinylation of the corresponding
ornithinamide precursor 6 bearing a diphenylmethyl group on the terminal part. The
guanidinylation reagents 5a-c were synthesized by alkylation of N,N’ꢀbis(tertꢀ
butoxycarbonyl)ꢀSꢀmethyl isothiourea under standard Mitsunobu conditions (Table 3). Taken
as a reference, compound 7a (no substituent on the guanidine) exhibited an affinity towards
both NPFF receptors similar to that of 4a. The introduction of an aromatic moiety on the
guanidine group (compounds 7b to 7d) did not improve their affinities towards both hNPFFR1
and hNPFFR2. Indeed, there seemed to be neither steric hindrance, nor additional interaction,
so we can hypothesize that the guanidine moiety does not interact with a residue buried into
the transmembrane domain, but more likely with an acidic residue located at the surface of the
receptor. This result is consistent with the model of binding proposed by BeckꢀSickinger et al.,
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who hypothesized an interaction between the guanidine moiety of Arg with an anionic
residue on a loop of the GPCR. Using siteꢀdirected mutagenesis, two anionic residues
(Asp6.59 located near the extracellular end of the TMD6, and Glu5.27 located on the TMD5
of both NPFF receptors) were postulated to form the negatively charged pocket responsible for
the binding of the guanidinium moiety of the endogenous ligands.
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Table 3. Synthesis and Binding affinity of arginine derivatives towards NPFF1 & 2 receptorsa
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Conditions: (a) ROH (2 eq.), DIAD (2.2 eq.), PPh3 (2.2
eq.), THF; (b) 5a-c (1.2 eq.), TEA (3 eq.), DMF; (c) TFA,
DCM.
Ki NPFF1
Ki NPFF2
Cpd
R
(nM)b
(nM)b
RF9
2
7a
7b
7c
22 ± 5
0.32 ± 0.02
53 ± 4
43 ± 1
920 ± 50
284 ± 40
180 ± 46
165 ± 52
162 ± 49
H
ꢀCH2ꢀPh
ꢀ(CH2)2ꢀPh
ꢀ(CH2)3ꢀPh
43 ± 33
49 ± 12
27 ± 4
7d
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[3H]ꢀFFRFꢀNH2 was used as radioligand for competition assays with both
hNPFF1 & 2 receptors. b Values are expressed as the mean ± SEM
Identification and SAR of ornithine derivatives. The presence of a salt bridge led us to
investigate the effect of replacing the guanidine moiety by other cationic species, such as
secondary or tertiary amines, that exist as salts at physiological pH. Primary amine 8 was easily
obtained starting from the commercially available ornithine (Scheme 1). Reductive amination
between the ornithine derivative 8 and benzaldehyde using NaBH4 led to the corresponding
secondary amine 9. To introduce a tertiary amine, we developed an efficient strategy starting
from the commercially available FmocꢀLꢀGlu(OtBu)ꢀOH (Scheme 2). After introducing the Cꢀ
and Nꢀterminus using the classical Fmocꢀstrategy, the tertbutyl ester was cleaved with
trifluoroacetic acid in DCM, affording compound 10. The resulting carboxylic acid was activated
with isobutylchloroformiate, and then reduced by NaBH4 to the corresponding alcohol derivative
11. After a mesylation step, a series of ornithine derivatives bearing tertiary amines were
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obtained by nucleophilic substitution with various secondary amines. Using piperidine in the last
step, this sevenꢀstep synthesis was performed at a gramꢀscale, with up to 40% overall yield.
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Scheme 1 Synthesis of an ornithine derivative bearing a benzylamine on the side chain.
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Conditions: (a) Phenethylamine (1.1 eq.), BOP (1.1 eq.), NMM (2 eq.), DMF, rt; (b) DBU (2 eq.), DCM, rt; (c) 2ꢀ
Phenylbenzoic acid (1.1 eq), BOP (1.1 eq.), NMM (2 eq.), DCM, rt; (d) TFA, DCM, rt; (e) PhCHO (2 eq.), TEA (4
eq.), DCM/MeOH, then NaBH4 (3 eq.).
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Scheme 2 General synthesis of ornithine derivatives bearing tertiary amines on the side chain.
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Conditions: (a) Phenethylamine (1.1 eq.), BOP (1.1 eq.),
NMM (2 eq.), DMF, rt; (b) DBU (2 eq.), DCM, rt; (c) 2ꢀ
Phenylbenzoic acid (1.1 eq.), BOP (1.1 eq.), NMM (2 eq.),
DCM, rt; (d) TFA, DCM, rt; (e) iBuCOCl (2.1 eq.), NaBH4
(5 eq.), NMM (2.1 eq.), THF, ꢀ20°C; (f) MsCl (2.5 eq.),
TEA (2.5 eq.), DCM, rt; (g) amines (3.5 eq.), NaI (0.1 eq.),
MeCN, 40°C.
As shown in Table 4, replacement of arginine by ornithine led to a full loss of affinity towards
both NPFF receptors. However, with the introduction of a benzyl group on the ornithine side
chain (9) some affinity was recovered, especially towards NPFF1 (Ki = 400 nM). Similar results
were obtained for the tertiary amine 12a, and with the other moieties bearing a benzene ring
(12b, 12c, and 12d). While as constrained tertiary amine derivatives 12b, 12c and 12d did not
improve the affinity for hNPFFR1 in comparison with the more flexible 12a, their affinity
towards hNPFFR2 was enhanced. 12d showed a 30ꢀfold improvement of affinity for hNPFFR2
in comparison with 12a. Interestingly, 12d appeared to be slightly more selective towards
NPFF2 (Ki = 34 nM) vs NPFF1 (Ki = 400 nM). This result is remarkable as 12d constitutes the
first example of a reversed selectivity profile, and is likely due to the establishment of an
hNPFFR2ꢀspecific interaction between the benzyl moiety and an aromatic amino acid located at
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proximity of the anionic pocket formed by Asp6.59 and Glu5.27. In contrast, the introduction of
a piperidine moiety on the side chain led to compound 12e, which exhibited a slight increase in
affinity and a preference for hNPFFR1 (Ki = 170 nM). The presence of a second protonatable
nitrogen was well tolerated (12f) for hNPFFR1, but did not improve the affinity. A similar result
was obtained with the Hꢀbond acceptor group of the piperazine derivative 12g. In contrast, both
compounds (12f, 12g) shown no affinity for hNPFFR2 (Ki > 10 µM), highlighting the hypothesis
of an anionic pocket less lipophilic for hNPFFR1 than for hNPFFR2, which is consistent with the
better affinity of 12d toward hNPFFR2.
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Table 4. Binding affinity of ornithine derivatives towards NPFF1 & 2 receptorsa
Ki NPFF1
(nM)b
Ki NPFF2
(nM)b
Cpd
R
RF9
22 ± 5
43 ± 1
2
8
9
0.32 ± 0.02
> 10 µM
400 ± 38
920 ± 50
> 10 µM
2300 ± 66
NH2
NHꢀBn
12a
12b
680 ± 160
500 ± 99
1100 ± 510
320 ± 99
12c
485 ± 35
ndc
12d
12e
12f
400 ± 71
172 ± 13
232 ± 17
34 ± 12
563 ± 164
> 10 µM
12g
319 ± 13
> 10 µM
a
[3H]ꢀFFRFꢀNH2 was used as radioligand for competition assays with both
b
c
hNPFF1 & 2 receptors. Values are expressed as the mean ± SEM; nd : not
determined.
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Functional activities towards both NPFF receptors. We examined the activity of a selection
of compounds representative of each chemical series through in vitro functional experiments
performed on CHO cells expressing NPFF receptors. Given the preferential coupling of
hNPFFR1 and hNPFFR2 to Gi/o proteins,38ꢀ39 the agonist activity of the selected compounds was
inferred from their ability to inhibit forskolinꢀstimulated cAMP accumulation in NPFFRꢀ
expressing cells. In agreement with the already reported antagonist properties of RF9,32 no
agonistic activity was found for either the arginine derivatives (4d, 4h, 7a, 7b) or the unnatural
ornithine derivatives (12b, 12e) at concentrations up to 10 µM.
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In vivo activity on fentanyl-induced hyperalgesia. As previously described, fentanyl
administration in rats induces a shortꢀlasting analgesia (few hours), followed by a hyperalgesic
state that remains for several days.6 The coꢀadministration of an NPFF antagonist (e.g. RF9, 2)
with an opiate (e.g. fentanyl, morphine) was found to prolong analgesia, while strongly reducing
hyperalgesia (Fig. 1).16,32,34 Using the RandallꢀSelitto test (paw pressure) in rats, the ornithine
derivatives 12b and 12e were selected along with the arginine derivative 4h to be coꢀ
administrated (0.5 mg/kg, s.c.) with fentanyl and evaluated for their ability to inhibit fentanylꢀ
induced hyperalgesia (Fig. 1).
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Figure 1 Inhibition of fentanylꢀinduced hyperalgesia by selected compounds in rats. On day 0
(D0), fentanyl injections (4 × 80 µg/kg, s.c.) were performed every 15 min for 1 h, resulting in a
total dose of 320 µg/kg. NPFF antagonists (0.5 mg/kg, s.c.) or saline injections were performed
30 min before the first administration of fentanyl. Nociceptive threshold was measured by the
pawꢀpressure test. Hyperalgesia inhibition % was evaluated on day two (D2). Values are
expressed as the mean ± SEM. *Dunnett test P < 0.001 for comparison between saline group and
tested compounds.
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*
*
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*
The dipeptides RF9 and 2 were previously reported to be strongly active in this model.34 In
contrast, 4h was surprisingly found to have little effect in vivo, despite its high affinities towards
both NPFF1 & 2 receptors (Table 2, Ki = 11 and 25 nM, respectively). Although less potent in
terms of affinity for both NPFFRs, the ornithine derivatives 12b and 12e strongly counteracted
fentanylꢀinduced hyperalgesia at low doses.
Metabolic stability and physicochemical properties. The lack of in vivo activity of 4h may be
due to rapid metabolic degradation. To test this possibility, intrinsic clearance in rat liver
microsomes and halfꢀlife time in rat serum were evaluated for compounds 2, 4h, and 12e (Table
5). Dipeptide 2 was quite stable in liver microsomes (Clint = 8 µL/min/mg protein), but was
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hydrolyzed in rat serum to the corresponding Cꢀterminal carboxylic acid (detected by LCꢀMSꢀ
MS, t1/2 = 1 h 46 min). In contrast, arginine and ornithine derivatives 4h and 12e showed good
stability in rat serum, but a significant clearance in rat liver microsomes (Table 5). Using LCꢀ
MSꢀMS, we were able to detect an oxidation at the Cꢀterminal phenethyl moiety. As 4h and 12e
exhibit the same metabolic profile, the difference of in vivo efficacy may be explained by their
ability to cross the bloodꢀbrain barrier (BBB) after systemic administration. Many reports have
highlighted the critical role of a small subset of physicochemical data on BBB permeability.40ꢀ43
According to S. Hitchcock, the application of four simple physicochemical rules may help to
identify the compounds bearing favorable BBB permeability: (a) PSA < 90 Å2; (b) HBD < 3; (c)
2 < LogD7.4 < 5; (d) MW < 450 Da.43 Table 5 shows a comparison of these four physicochemical
properties applied to the five compounds tested in vivo. First, in spite of their in vivo efficacy,
both dipeptide ArgꢀPheꢀNH2 derivatives (RF9 and 2) do not respect any of the four rules. One
way to explain the central efficacy of these compounds would be to consider a specific transport
across the BBB through a carrierꢀmediated transport system. Recently, dipeptides such as Argꢀ
Leu were shown to be substrates of the transporter Pept1, which is known to play an important
role in the active transport of dipeptides through the intestinal epithelium.44 In contrast, the
arginine derivative 4h exhibits slightly better physicochemical properties than RF9 and 2, but
only one rule is fulfilled. In vivo, this compound demonstrated poor activity. Following our
hypothesis of an active transport of dipeptide ligands, we may consider that 4h is not recognized
by any specific transporter, impairing its ability to reach the CNS. Finally, the switch from a
guanidine group (4h) to tertiary amines (12b and 12e) led to a great improvement of the
probability for these compounds to cross the BBB through passive diffusion. Indeed, 12e follows
most of the Hitchcock rules, with a molecular weight just above the threshold of 450 Da.
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Notably, there is a great improvement of the polar surface area, which is reported as a critical
property for passive BBB diffusion.45 Therefore, 12e appears in vivo as efficient as RF9, in spite
of a lower affinity towards both NPFF receptors. A low polar surface area is also a dominating
determinant for oral absorption,45 making of 12e a good candidate to be an orallyꢀavailable
NPFF antagonist able to block opioidꢀinduced hyperalgesia.
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Table 5. Metabolic stability and physicochemical properties of a set of NPFFR antagonists
RF9
2
4h
12e
12b
MW
(g/mol)
482
500
11.90
0.02
471
11.97
1.40
483
8.94
3.43
531
8.32
5.05
pKaa
11.87
ꢀ0.87
Loga
D7.4
PSAa
(Å2)
163
165
113
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HBD
Clint
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NTe
NTe
46
51
t1/2
(min)
a
NTe
stabled
stabled
NTe
106
Calculator Plugins (Marvin 6.0.2, 2013, http://www.chemaxon.com) were used for structure property prediction and
calculation; b Intrinsic clearance in rat liver microsomes as a measure of metabolic stability. Values are expressed in µL/min/mg
of protein; c Halfꢀlife time in rat serum as a measure of metabolic stability; d No degradation detected after 2 h in serum. e NT =
Not tested.
Preventive effect of 12e on fentanyl-induced hyperalgesia after oral administration. With an
aqueous solubility greater than 24 mM, 12e was easily solubilized in physiological solution
before being administered per os to rats. Figure 2 shows that the preventive effect of 12e on
fentanylꢀinduced hyperalgesia is doseꢀdependent, with a complete reversal at a dose of 3 mg/kg.
Interestingly, 12eꢀfentanyl treated rats still exhibited analgesia after 6 h, which was not the case
for the salineꢀfentanyl group. Thus, 12e seems to prolong the opioidꢀinduced analgesia, similar to
what was observed with RF9.16 These results are in good agreement with the fact that NPFF
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receptors are known to reduce the analgesic effect of exogenous opioids, while promoting
tolerance to analgesics. Although it has been proposed that the NPFFR1 receptor subtype is
mainly involved,46 the respective roles of both NPFFR1 and NPFFR2 receptor subtypes in these
functions is still unclear. Indeed, both NPFFR1 and NPFFR2 endogenous agonists, RFRP3 and
NPFF (respectively), have been shown to display hyperalgesic or antiꢀmorphine properties and
localization in the CNS of both receptors is consistent with a potential role in the modulation of
nociception and antiꢀopioid effects.17 As 12e displays a low selectivity between these two NPFF
receptor subtypes, it is therefore difficult to say from our data whether one or both subtypes are
responsible for the antiꢀhyperalgesic activity of this compound. In the future, development of
highly selective compounds (antagonists and agonists) for NPFFR1 and NPFFR2 receptors
should greatly help us to decipher the respective role of each NPFF receptor subtype in the
modulation of the effects associated with acute and chronic opiate treatments.
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Figure 2. Preventive effect of 12e on fentanylꢀinduced hyperalgesia. On day 0 (D0), four
successive injections of fentanyl (80 µg/kg, s.c.) were performed every 15 min for 1 hour,
resulting in a total dose of 320 µg/kg. 12e (0.3 (ꢀ), 1 (ꢀ) and 3 (ꢁ) mg/kg) or saline (ꢂ) was
orally administered 30 min before the first administration of fentanyl. Nociceptive threshold was
measured by the pawꢀpressure test. Inset: Hyperalgesic index (HI) is the area between the
baseline and the curve for nociceptive threshold on the days after fentanyl administration,
normalized to control. Values are expressed as the mean ± SEM. *Dunnett’s test P < 0.001 for
comparison between saline group and Compound 12e group.
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Selectivity of 12e towards a set of protein-targets. We next evaluated the ability of 12e to bind
to a set of 45 proteinꢀtargets, including GPCRs (opioid, dopamine, muscarinic receptors, etc.),
amine transporters (SERT, NET, DAT), and ion channels (e.g., 5HT3, NMDA, and hERG)
(Table S1 in Supp. Information). Tested at 1 µM, 12e exhibited no or very low affinity for any of
these targets, except for the µꢀopioid receptor (MOR). 12e had a significant affinity for MOR (Ki
= 1020 ± 50 nM SEM), but no agonistic effect of 12e was observed on CHO cells expressing
MOR (cAMP assay). In vivo, 12e did not block the analgesia induced by fentanyl (Fig. 2) and
when administered alone did not lead to any analgesic or hyperalgesic effect (not shown),
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indicating that at the doses used this compound did not display any agonist or antagonist activity
at MOR. More investigations are required to see if this modest affinity towards MOR is
correlated with the potentiation of the opioidꢀinduced analgesia observed in our experiments.
Finally, chronic administration of 12e to mice (up to 30 mg/kg, i.p., once per week for 6 weeks)
did not exhibit any visible toxicity, side effects, or any negative impact on muscular strength,
locomotor activity or body mass evolution.
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In conclusion, using drug design and bioisosterism concepts applied to the dipeptide RF9, we
have developed unnatural analogs of ornithine as antagonists of NPFF receptors. Tested in vivo,
compound 12e is the first orally active NPFF antagonist capable of preventing opioidꢀinduced
hyperalgesia at low doses. Although we cannot exclude that the development of highly selective
NPFFR1 or NPFFR2 antagonists could be an interesting strategy for the treatment of chronic
pain, the lack of selectivity of 12e, as well as its the good pharmacological profile in terms of
antiꢀOIH efficacy, selectivity, and preꢀclinical observations, suggest that 12e is a potential lead
for preclinical studies for the treatment of acute and chronic pain. A lead optimization program
for the further development of this compound is now in progress.
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Materials and Methods
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Compound synthesis
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See supporting informations
Binding assays
hNPFFR1 and hNPFFR2 receptors : Membranes from CHO cells stably expressing hNPFFR1 or
hNPFFR2 Nꢀterminally fused to a Flag sequence (SFꢀhNPFFR1 and SFꢀhNPFFR2, respectively)
were prepared as described.47 SFꢀhNPFFR1 or SFꢀhNPFFR2 membranes (10 µg) were incubated
for 30 min at 25 °C in a final volume of 0.5 ml containing 50 mM HEPES pH 7.4, 1 mM CaCl2,
1 mM MgCl2, 0.1% bovine serum albumin, 10 nM and 3 nM [3H]ꢀFFRFamide (CEA, Paris;
specific activity, 13,6 Ci/mmol) for SFꢀhNPFFR1 and SFꢀhNPFFR2, respectively and the ligands
to be tested. Nonꢀspecific binding was determined in the presence of 1 µM neuropeptide FF for
SFꢀhNPFFR2 and 1 µM neuropeptide VF for SFꢀhNPFFR1. Typical total and nonspecific
binding were 1200 and 200 dpm, respectively for SFꢀhNPFFR1 and SFꢀhNPFFR2. Incubation
mixtures were rapidly filtered and washed with 50 mM HEPES pH 7.4, 1 mM CaCl2, 1 mM
MgCl2, on 96 wells GF/B unifilter (Perkin Elmer). Unifilter plates where then dried for 1 h at
65°C. Bound radioactivity was determined by scintillation counting with 30 µL of scintillation
cocktail (Oꢀscint, Perkin Elmer) per well on a Topcount scintillation apparatus (Perkin Elmer).
MOR receptor: Membrane preparations from HEK293 cells expressing human MOR and
receptor binding assay were performed as described by Becker et al.47
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cAMP measurements in cells expressing hNPFFR1 or hNPFFR2.
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The inhibition by compounds of forskolinꢀstimulated cAMP accumulation in CHO cells
expressing hNPFFR1 and hNPFFR2 was assessed according to Gicquiaux et al.48 with minor
modifications. CHO cells stably expressing SFꢀhNPFFR1 or SFꢀhNPFFR2 were harvested,
seeded in 96ꢀwell plates (10,000 cells per well) in DMEM/Ham’s F12 (1/1) supplemented with
5% FCS, penicillin (100 U/mL) and streptomycin (100 µg/mL), and grown at 37 °C in a
humidified atmosphere with 5% CO2 for 1ꢀ2 days until 90% confluency. Cells were washed with
0.2 mL PBS, then incubated for 10 min at 37°C with 0.2 mL assay buffer (150 mM NaCl, 5 mM
KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 10 mM HEPES, 10
mg/ml BSA, pH 7.4). The buffer was then replaced for 10 min with 0.06 mL of fresh buffer
containing 1 mM IBMX before cell exposure to 10 mM forskolin, without or with compounds to
be tested (10ꢀ10 to 10ꢀ5 M), for 10 min. The incubation medium (0.1 mL final volume) was
maintained at 37°C during the whole procedure. The reaction was stopped by adding 40 mL iceꢀ
cold 0.5 M HCl and freezing at ꢀ80°C. After 1 h, cells were disrupted by sonication and
centrifuged at 2000 g for 15 min. The resulting supernatants were stored at ꢀ20°C until
quantitation of cAMP levels by radioimmunoassay.
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In vitro metabolic stability in rat liver microsomes
All incubations were performed individually for each test compound. Compounds (50 µM) were
incubated at 37°C under standard incubation conditions : phosphate buffer (pH 7.4), NADPH (1
mM), rat liver microsomes (0.1 mg protein). Final concentration of DMSO was 0.5%. At
different times (0, 5, 15, 30, and 45 minutes), aliquots (35 µL) of the reaction mixture were
stopped with 100 µL of cold acetonitrile. Upon centrifugation of the resultant mixture,
supernatants were analyzed by a generic HPLC method performed on a Dionex Ultimate 3000
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(Waters XSelect CSH C18 column – 5 µm – 4.6 x 50 mm) with the following parameters : Flow
rate of 1 mL/min, column temperature: 30°C, solvent system: A (MeOH) and B (0.05% TFA in
H2O), t = 0 min to 12 min: 20 to 100% of A then t = 12 min to t= 15 min: 100% of A. The ratio
of product was determined by integration of UV spectra recorded at 202 nm. Metabolic stability
was determined by the disappearance of the tested compound over time. The Lnꢀlinear plots of
the % of compound remaining based on chromatographic peak area versus time were plotted,
and the slope was calculated by linear fitting of the curve. The microsomal intrinsic clearance
(Clint, expressed in µL/min/mg protein) was calculated using the equation Clint = 0.693/(t1/2) (mg
of microsomal protein / volume of incubation).49
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In vitro metabolic stability in rat serum
All incubations were performed individually for each test compound. Compounds (50 µM) were
incubated at 37°C in rat serum (200 µL). Final concentration of DMSO was 0.5%. At different
times (0, 15, 30, 60, and 120 minutes), aliquots (35 µL) of the reaction mixture were stopped
with 100 µL of cold acetonitrile. Upon centrifugation of the resultant mixture, supernatants were
analyzed by a generic HPLC method performed on a Dionex Ultimate 3000 (Waters XSelect
CSH C18 column – 5 µm – 4.6 x 50 mm) with the following parameters : Flow rate of 1
mL/min, column temperature: 30°C, solvent system: A (MeOH) and B (0.05% TFA in H2O), t =
0 min to 12 min: 20 to 100% of A then t = 12 min to t = 15min: 100% of A. The ratio of product
was determined by integration of UV spectra recorded at 202 nm. Metabolic stability was
determined by the disappearance of the tested compound over time. The Lnꢀlinear plots of the %
of compound remaining based on chromatographic peak area versus time were plotted, and the
slope was calculated by linear fitting of the curve. The in vitro halfꢀlife time (t1/2, expressed in
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minutes) was estimated by using 0.693/k where k is the biotransformation rate constant and
corresponds to the slope of the Lnꢀlinear curve.
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Solubility in HEPES pH 7.4
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3 mg of compound 12e was added to 200 µL of buffer (HEPES pH 7.4), and the heterogeneous
mixture was agitated for 24 h at 20°C. After centrifugation (15 000 g – 10 min), supernatant was
diluted with buffer and analyzed by HPLC (Gilson) on a Kinetex C18 column (100 A, 50 x 4.6
mm) using the following parameters : Flow rate of 2 mL/min, solvent system: A (MeCN) and B
(0.1% TFA in H2O), t= 0.2 min to 3 min: 5% to 95% of A. The ratio of product was determined
by integration of UV spectra recorded at 235 nm.
Animals
Experiments were performed on male SpragueꢀDawley rats (Charles River, France), weighing
250 ꢀ 350 g, housed five per cage and maintained under a 12:12 light:dark cycle (lights on at
08:00 am) at a constant room temperature of 23 ± 2 °C. The animals had free access to food and
water. Pharmacological tests and care of the animals were performed in accordance with the
Guide for Animal Care and Use (NIH, 2011).
Drug administration
Fentanyl citrate was obtained from SigmaꢀAldrich (Saint Quentin Fallavier, France). All drugs
were dissolved in physiological saline (NaCl 0.9%) and injected subcutaneously or by oral route.
Control animals were administered with an equal volume of saline.
Measurement of the nociceptive threshold
Nociceptive thresholds in rats were determined by a modification of the RandallꢀSelitto method,
the pawꢀpressure vocalization test in which a constantly increasing pressure is applied to the hind
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paw until the rat squeaks. The Basile analgesimeter (Apelex, Massy, France; stylus tip diameter:
1 mm) was used. A 600 g cutoff value was employed for preventing tissue damage.
Experiments were performed essentially as described.6 Briefly, the effect of fentanyl on
nociceptive threshold was studied using an injection procedure designed to partly mimic its use
in surgery. Fentanyl (or saline) was injected 4 times at 15 min intervals (80 µg/kg, s.c. per
injection resulting in a total dose of 320 µg/kg). On day 0, nociceptive threshold was measured
every 2 h for a period of 6 h after the first fentanyl injection and once daily during the 5
following days (D1 to D5). Pharmacological compounds (RF9, 2, 4h, 12b and 12e) were
administered (30 min before the first fentanyl injection.
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Data and statistical analyses
Data were expressed as the mean nociceptive threshold ± SEM. Student's paired tꢀtest was used
to assess paired comparisons of nociceptive threshold values to evaluate changes of the
nociceptive threshold before the beginning of experiments (Dꢀ1 and D0 basal values). To evaluate
the timeꢀcourse effects of treatments on nociception, ANOVAs were performed on the
nociceptive threshold values and followed by post hoc analysis using Dunnett’s test. A difference
was considered as significant for p < 0.05. Hyperalgesia index (HI), represented by the area
above the curve for the days following fentanyl treatment, were calculated for each rat by the
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trapezoidal method and were expressed as a mean percentage ± SEM of the reference index
(100%: HI associated with hyperalgesia observed in the control group). Ki values are expressed
as means ± SEM from at least two separate experiments performed in duplicate.
Supporting Information
Compound synthesis and analytical characterization; Selectivity of 12e against 45 protein targets.
This information is available free of charge via the internet at http://pubs.acs.org/.
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Author Information
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Corresponding Authors
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‡ Corresponding author concerning medicinal chemistry: E-mail: fbihel@unistra.fr
& Corresponding author concerning pharmacological evaluation: E-mail: simonin@unistra.fr
Author Contributions : * These authors contributed equally to this work
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Funding
This work was supported by grants from Agence National de la Recherche (ANRꢀ06ꢀNeuroꢀ041ꢀ
01, ANRꢀ08ꢀEBIOꢀ014ꢀ01), Alsace BioValley (Conectus, Pharmadol), Conseil Régional
d’Alsace (Pharmadol), Communauté Urbaine de Strasbourg (Pharmadol), ICFRC (Pharmadol),
OSEO (Pharmadol), Direction Générale des Entreprises (Pharmadol). This work has been
published within the LABEX ANRꢀ10ꢀLABXꢀ0034_Medalis and received financial support
from the French government managed by "Agence National de la Recherche" under "Programme
d'investissement d'avenir.
Notes
The authors declare no competing financial interest
Uncategorized References
(1) Kieffer, B. L. and Evans, C. J. (2002) Opioid tolerance ꢀ In search of the holy grail. Cell 108,
587ꢀ590
(2) Ossipov, M. H., Lai, J., Vanderah, T. W. and Porreca, F. (2003) Induction of pain facilitation
by sustained opioid exposure: relationship to opioid antinociceptive tolerance. Life Sci 73, 783ꢀ
800
(3) Rothman, R. B. (1992) A Review of the Role of AntiꢀOpioid Peptides in MorphineꢀTolerance
and Dependence. Synapse 12, 129ꢀ138
(4) Simonnet, G. and Rivat, C. (2003) Opioidꢀinduced hyperalgesia: abnormal or normal pain?
Neuroreport 14, 1ꢀ7
ACS Paragon Plus Environment
26

Page 27 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
(5) Celerier, E., Laulin, J. P., Corcuff, J. B., Le Moal, M. and Simonnet, G. (2001) Progressive
enhancement of delayed hyperalgesia induced by repeated heroin administration: A sensitization
process. J Neurosci 21, 4074ꢀ4080
7
8
9
(6) Celerier, E., Rivat, C., Jun, Y., Laulin, J. P., Larcher, A., Reynier, P. and Simonnet, G. (2000)
Longꢀlasting hyperalgesia induced by fentanyl in rats ꢀ Preventive effect of ketamine.
Anesthesiology 92, 465ꢀ472
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(7) Larcher, A., Laulin, J. P., Celerier, E., Le Moal, M. and Simonnet, G. (1998) Acute tolerance
associated with a single opiate administration: Involvement of NꢀmethylꢀDꢀaspartateꢀdependent
pain facilitatory systems. Neuroscience 84, 583ꢀ589
(8) Mao, J. R., Price, D. D. and Mayer, D. J. (1995) Mechanisms of Hyperalgesia and Morphineꢀ
Tolerance ꢀ a Current View of Their Possible Interactions. Pain 62, 259ꢀ274
(9) Crofford, L. J. (2010) Adverse effects of chronic opioid therapy for chronic musculoskeletal
pain. Nat Rev Rheumatol 6, 191ꢀ197
(10) Angst, M. S. and Clark, J. D. (2006) Opioidꢀinduced hyperalgesia ꢀ A qualitative systematic
review. Anesthesiology 104, 570ꢀ587
(11) Mao, J. R. (2002) Opioidꢀinduced abnormal pain sensitivity: implications in clinical opioid
therapy. Pain 100, 213ꢀ217
(12) White, J. M. (2004) Pleasure into pain: The consequences of longꢀterm opioid use. Addict
Behav 29, 1311ꢀ1324
(13) Ueda, H. and Ueda, M. (2009) Mechanisms underlying morphine analgesic tolerance and
dependence. Front Biosci 14, 5260ꢀ5272
(14) Mollereau, C., Mazarguil, H., Zajac, J. M. and Roumy, M. (2005) Neuropeptide FF (NPFF)
analogs functionally antagonize opioid activities in NPFF2 receptorꢀtransfected SHꢀSY5Y
neuroblastoma cells. Mol Pharmacol 67, 965ꢀ975
(15) McNally, G. P. (1999) Pain facilitatory circuits in the mammalian central nervous system:
their behavioral significance and role in morphine analgesic tolerance. Neurosci Biobehav R 23,
1059ꢀ1078
(16) Elhabazi, K., Trigo, J. M., Mollereau, C., Mouledous, L., Zajac, J. M., Bihel, F., Schmitt,
M., Bourguignon, J. J., Meziane, H., Petitꢀdemouliere, B., Bockel, F., Maldonado, R. and
Simonin, F. (2012) Involvement of neuropeptide FF receptors in neuroadaptive responses to
acute and chronic opiate treatments. Brit J Pharmacol 165, 424ꢀ435
(17) Ayachi, S. and Simonin, F. (2014) Involvement of Mammalian RFꢀAmide Peptides and
Their Receptors in the Modulation of Nociception in Rodents. Frontiers in endocrinology 5, 158
(18) Yang, H. Y. T., Fratta, W., Majane, E. A. and Costa, E. (1985) Isolation, Sequencing,
Synthesis, and Pharmacological Characterization of 2 Brain Neuropeptides That Modulate the
Action of Morphine. P Natl Acad Sci USA 82, 7757ꢀ7761
(19) Laguzzi, R., Nosjean, A., Mazarguil, H. and Allard, M. (1996) Cardiovascular effects
induced by the stimulation of neuropeptide FF receptors in the dorsal vagal complex: An
autoradiographic and pharmacological study in the rat. Brain Res 711, 193ꢀ202
(20) Prokai, L., Zharikova, A. D., Juhasz, A. and ProkaiꢀTatrai, K. (2006) Cardiovascular effects
of neuropeptide FF antagonists. Peptides 27, 1015ꢀ1019
(21) Roth, B. L., Disimone, J., Majane, E. A. and Yang, H. Y. T. (1987) Elevation of Arterialꢀ
Pressure in Rats by 2 New Vertebrate Peptides FlfqpqrfꢀNh2 and AgeglsspfwslaapqrfꢀNh2
Which Are Immunoreactive to FmrfꢀNh2 Antiserum. Neuropeptides 10, 37ꢀ42
ACS Paragon Plus Environment
27

ACS Chemical Neuroscience
Page 28 of 30
1
2
3
4
5
6
(22) Fang, Q., Wang, Y. Q., He, F., Guo, J., Guo, J., Chen, Q. and Wang, R. (2008) Inhibition of
neuropeptide FF (NPFF)ꢀinduced hypothermia and antiꢀmorphine analgesia by RF9, a new
selective NPFF receptors antagonist. Regul Peptides 147, 45ꢀ51
7
8
9
(23) Mouledous, L., Barthas, F. and Zajac, J. M. (2010) Opposite control of body temperature by
NPFF1 and NPFF2 receptors in mice. Neuropeptides 44, 453ꢀ456
(24) Murase, T., Arima, H., Kondo, K. and Oiso, Y. (1996) Neuropeptide FF reduces food intake
in rats. Peptides 17, 353ꢀ354
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(25) Lefrere, I., de Coppet, P., Camelin, J. C., Le Lay, S., Mercier, N., Elshourbagy, N., Bril, A.,
BerrebiꢀBertrand, I., Feve, B. and Krief, S. (2002) Neuropeptide AF and FF modulation of
adipocyte metabolism ꢀ Primary insights from functional genomics and effects on betaꢀ
adrenergic responsiveness. J Biol Chem 277, 39169ꢀ39178
(26) Kalliomaki, M. L. and Panula, P. (2004) Neuropeptide FF, but not prolactinꢀreleasing
peptide, mRNA is differentially regulated in the hypothalamic and medullary neurons after salt
loading. Neuroscience 124, 81ꢀ87
(27) Majane, E. A. and Yang, H. Y. T. (1991) Mammalian FmrfꢀNh2ꢀLike Peptide in Rat
Pituitary ꢀ Decrease by Osmotic Stimulus. Peptides 12, 1303ꢀ1308
(28) Findeisen, M., Rathmann, D. and BeckꢀSickinger, A. G. (2011) StructureꢀActivity Studies
of RFamide Peptides Reveal SubtypeꢀSelective Activation of Neuropeptide FF1 and FF2
Receptors. Chemmedchem 6, 1081ꢀ1093
(29) Mazarguil, H., Gouarderes, C., Tafani, J. A. M., Marcus, D., Kotani, M., Mollereau, C.,
Roumy, M. and Zajac, J. M. (2001) Structureꢀactivity relationships of neuropeptide FF: role of
Cꢀterminal regions. Peptides 22, 1471ꢀ1478
(30) Mollereau, C., Mazarguil, H., Marcus, D., Quelven, I., Kotani, M., Lannoy, V., Dumont, Y.,
Quirion, R., Detheux, M., Parmentier, M. and Zajac, J. M. (2002) Pharmacological
characterization of human NPFF1 and NPFF2 receptors expressed in CHO cells by using
NPYY1 receptor antagonists. Eur J Pharmacol 451, 245ꢀ256
(31) Vyas, N., Mollereau, C., Cheve, G. and McCurdy, C. R. (2006) Structureꢀactivity
relationships of neuropeptide FF and related peptidic and nonꢀpeptidic derivatives. Peptides 27,
990ꢀ996
(32) Simonin, F., Schmitt, M., Laulin, J. P., Laboureyras, E., Jhamandas, J. H., MacTavish, D.,
Matifas, A., Mollereau, C., Laurent, P., Parmentier, M., Kieffer, B. L., Bourguignon, J. J. and
Simonnet, G. (2006) RF9, a potent and selective neuropeptide FF receptor antagonist, prevents
opioidꢀinduced tolerance associated with hyperalgesia. P Natl Acad Sci USA 103, 466ꢀ471
(33) Wang, Y. Q., Guo, J., Wang, S. B., Fang, Q., He, F. and Wang, R. (2008) Neuropeptide FF
receptors antagonist, RF9, attenuates opioidꢀevoked hypothermia in mice. Peptides 29, 1183ꢀ
1190
(34) Gealageas, R., Schneider, S., Humbert, J. P., Bertin, I., Schmitt, M., Laboureyras, E.,
Dugave, C., Mollereau, C., Simonnet, G., Bourguignon, J. J., Simonin, F. and Bihel, F. (2012)
Development of subꢀnanomolar dipeptidic ligands of neuropeptide FF receptors. Bioorg Med
Chem Lett 22, 7471ꢀ7474
(35) Allard, M., Geoffre, S., Legendre, P., Vincent, J. D. and Simonnet, G. (1989)
Characterization of Rat SpinalꢀCord Receptors to Flfqpqrfamide, a Mammalian Morphine
Modulating Peptide ꢀ a Binding Study. Brain Res 500, 169ꢀ176
(36) Payza, K., Akar, C. A. and Yang, H. Y. T. (1993) Neuropeptide Ff Receptors ꢀ Structureꢀ
Activity Relationship and Effect of Morphine. J Pharmacol Exp Ther 267, 88ꢀ94
ACS Paragon Plus Environment
28

Page 29 of 30
ACS Chemical Neuroscience
1
2
3
4
5
6
(37) Findeisen, M., Wurker, C., Rathmann, D., Meier, R., Meiler, J., Olsson, R. and Beckꢀ
Sickinger, A. G. (2012) Selective Mode of Action of GuanidineꢀContaining NonꢀPeptides at
Human NPFF Receptors. J Med Chem 55, 6124ꢀ6136
7
8
9
(38) Elshourbagy, N. A., Ames, R. S., Fitzgerald, L. R., Foley, J. J., Chambers, J. K., Szekeres,
P. G., Evans, N. A., Schmidt, D. B., Buckley, P. T., Dytko, G. M., Murdock, P. R., Milligan, G.,
Groarke, D. A., Tan, K. B., Shabon, U., Nuthulaganti, P., Wang, D. Y., Wilson, S., Bergsma, D.
J. and Sarau, H. M. (2000) Receptor for the pain modulatory neuropeptides FF and AF is an
orphan G proteinꢀcoupled receptor. J Biol Chem 275, 25965ꢀ25971
(39) Hinuma, S., Shintani, Y., Fukusumi, S., Iijima, N., Matsumoto, Y., Hosoya, M., Fujii, R.,
Watanabe, T., Kikuchi, K., Terao, Y., Yano, T., Yamamoto, T., Kawamata, Y., Habata, Y.,
Asada, M., Kitada, C., Kurokawa, T., Onda, H., Nishimura, O., Tanaka, M., Ibata, Y. and Fujino,
M. (2000) New neuropeptides containing carboxyꢀterminal RFamide and their receptor in
mammals. Nat Cell Biol 2, 703ꢀ708
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(40) Abraham, M. H. (2004) The factors that influence permeation across the bloodꢀbrain barrier.
Eur J Med Chem 39, 235ꢀ240
(41) Goodnow, R. A., Guba, W. and Haap, W. (2003) Library design practices for success in
lead generation with small molecule libraries. Comb Chem High T Scr 6, 649ꢀ660
(42) Gratton, J. A., Abraham, M. H., Bradbury, M. W. and Chadha, H. S. (1997) Molecular
factors influencing drug transfer across the bloodꢀbrain barrier. J Pharm Pharmacol 49, 1211ꢀ
1216
(43) Hitchcock, S. A. (2008) Bloodꢀbrain barrier permeability considerations for CNSꢀtargeted
compound library design. Curr Opin Chem Biol 12, 318ꢀ323
(44) Zhang, L. H., Zhang, L., Luo, T., Zhou, J., Sun, L. Y. and Xu, Y. H. (2012) Synthesis and
Evaluation of a DipeptideꢀDrug Conjugate Library As Substrates for PEPT1. Acs Comb Sci 14,
108ꢀ114
(45) Kelder, J., Grootenhuis, P. D. J., Bayada, D. M., Delbressine, L. P. C. and Ploemen, J. P.
(1999) Polar molecular surface as a dominating determinant for oral absorption and brain
penetration of drugs. Pharmaceut Res 16, 1514ꢀ1519
(46) Lameh, J., Bertozzi, F., Kelly, N., Jacobi, P. M., Nguyen, D., Bajpai, A., Gaubert, G.,
Olsson, R. and Gardell, L. R. (2010) Neuropeptide FF Receptors Have Opposing Modulatory
Effects on Nociception. J Pharmacol Exp Ther 334, 244ꢀ254
(47) Becker, J. A. J., Wallace, A., Garzon, A., Ingallinella, P., Bianchi, E., Cortese, R., Simonin,
F., Kieffer, B. L. and Pessi, A. (1999) Ligands for kappaꢀopioid and ORL1 receptors identified
from a conformationally constrained peptide combinatorial library. J Biol Chem 274, 27513ꢀ
27522
(48) Gicquiaux, H., Lecat, S., Gaire, M., Dieterlen, A., Mely, Y., Takeda, K., Bucher, B. and
Galzi, J. L. (2002) Rapid internalization and recycling of the human neuropeptide YY1 receptor.
J Biol Chem 277, 6645ꢀ6655
(49) Obach, R. S., Baxter, J. G., Liston, T. E., Silber, B. M., Jones, B. C., MacIntyre, F., Rance,
D. J. and Wastall, P. (1997) The prediction of human pharmacokinetic parameters from
preclinical and in vitro metabolism data. J Pharmacol Exp Ther 283, 46ꢀ58
ACS Paragon Plus Environment
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