Variation of the net charge, lipophilicity, and side chain flexibility in Dmt1-DALDA: Effect on opioid activity and biodistribution

Article abstract of DOI:10.1021/jm3008079

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-D-Arg-Phe-Lys-NH ₂ ([Dmt¹]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dm

Full text of DOI:10.1021/jm3008079

Article
pubs.acs.org/jmc
Variation of the Net Charge, Lipophilicity, and Side Chain Flexibility
in Dmt1‑DALDA: Effect on Opioid Activity and Biodistribution
Alexandre Novoa,† Sylvia Van Dorpe,‡ Evelien Wynendaele,‡ Mariana Spetea,§ Nathalie Bracke,‡
Sofie Stalmans,‡ Cecilia Betti,† Nga N. Chung,∥ Carole Lemieux,∥ Johannes Zuegg,⊥ Matthew A. Cooper,⊥
Dirk Tourwe,† Bart De Spiegeleer,*,‡ Peter W. Schiller,∥ and Steven Ballet*,†
́
†Department of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
‡Drug Quality and Registration (DruQuaR) Group, Department of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72,
B-9000 Ghent, Belgium
§Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences Innsbruck (CMBI),
University of Innsbruck, Innsbruck, Austria
∥Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal, QC, Canada
⊥Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia
S
* Supporting Information
ABSTRACT: The influence of the side chain charges of the
second and fourth amino acid residues in the peptidic μ opioid
lead agonist Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]-DALDA) was
examined. Additionally, to increase the overall lipophilicity of
[Dmt1]-DALDA and to investigate the Phe3 side chain
flexibility, the final amide bond was N-methylated and Phe3
was replaced by a constrained aminobenzazepine analogue.
The in vitro receptor binding and activity of the peptides, as
well as their in vivo transport (brain in- and efflux and tissue
biodistribution) and antinociceptive properties after peripheral
administration (ip and sc) in mice were determined. The
structural modifications result in significant shifts of receptor
binding, activity, and transport properties. Strikingly, while [Dmt1]-DALDA and its N-methyl analogue, Dmt-D-Arg-Phe-
NMeLys-NH2, showed a long-lasting antinociceptive effect (>7 h), the peptides with D-Cit2 generate potent antinociception
more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt1]-DALDA
and [Dmt1,NMeLys4]-DALDA.
effects between MOR agonists and DOR agonists.1,2 Therefore,
compounds with a mixed μ agonist/δ agonist profile have the
INTRODUCTION
■
Due to the present limitations of the pharmacotherapy of pain,
the development of novel medications for the treatment of
severe and chronic pain represents a high priority goal. To date,
these pathologies rely mostly on opioid analgesics, but the
current available opioid drugs produce a number of side-effects
that limit their clinical use. A considerable amount of work has
been done and is still needed to target more efficacious and
nonaddicting analgesics in order to solve this major health care
problem. One research avenue consists of the discovery and
development of a potent peptide-based analgesic, with reduced
undesired effects, requiring a compound with a high affinity for
μ and/or δ opioid receptors (MOR and DOR, respectively), a
potential to be effective for the treatment of pain with reduced
side effects, as lower doses would be required.3 In parallel to the
development of such dual agonists, mixed MOR agonists/DOR
antagonists were also designed in search of a better pain
management.4−6 Among other beneficial effects, the observa-
tion that the selective blockade of DOR suppressed morphine
tolerance and dependence motivated the development of
compounds with this mixed profile.7
Earlier studies, performed by Schiller and co-workers,8 on the
dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2)-derived
tetrapeptide H-Dmt-D-Arg-Phe-Lys-NH2 (1) ([Dmt1]DALDA,
Figure 1), identified this peptide as a highly potent MOR
potent in vitro agonist activity, a strong antinociceptive effect in
agonist with a high binding affinity at the MOR (Kiμ = 0.143
vivo, an improved resistance to enzymatic degradation, and the
ability to cross the blood−brain barrier (BBB), allowing
systemic administration. More specifically, it was evidenced
nM, 7-fold higher than morphine) and a high metabolic
stability when incubated in sheep blood (t1/2 = 1.8 h).9 The C-
that compounds with a dual MOR/DOR activity present
beneficial pharmacological effects in comparison to highly
Received: June 8, 2012
selective MOR agonists. There have been reports on synergistic
Published: October 29, 2012
© 2012 American Chemical Society
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Figure 1. Structures of Dmt-DALDA (1), Phe, and the conformationally constrained 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba)-
containing dipeptide 2 (with Xxx: an α-amino acid residue).
a
Scheme 1. Synthesis of Fmoc-Aba-Lys(Z)-OH 8a and Fmoc-Aba-Nle-OH 8b Dipeptide Mimetics
a
Reaction conditions: (a) H2 (50 psi), RaNi, H2O/AcOH/Py 1:1:1, 50 °C, 38 h, 78%; (b) 1.1 equiv HCl·H-Lys(Z)-OMe or HCl·H-Nle-OMe, 2.5
equiv NaCNBH3, 20 wt % MgSO4, CH2Cl2, NMM or AcOH, rt, 14 h; (c) 1.1. equiv DCC, Py, CH3CN/H2O 1:1, rt, 14 h; (d) acetone/HCl 1N 1:1,
rt, reflux, 16 h; (e) (i) 4 equiv NH2NH2.H2O, EtOH, reflux, 1.5 h, and (ii) 1.2 equiv Fmoc-OSu, 2.2 equiv NaHCO3, acetone/water 1:2, rt, 2 h.
terminal amidation and insertion of a D-Arg residue in position
2 are likely to assist in the high stability of this peptide.9
Moreover, [Dmt1]DALDA showed extraordinary potency in
the rat tail-flick assay after intrathecal administration (3000
times more potent than morphine),10 and a subcutaneous (sc)
administration also induced a potent analgesic response,11,12
which in turn indicated that this molecule is able to cross the
BBB. Nevertheless, the structural features of this tricharged
peptide responsible for its ability to cross the BBB have not
clearly been determined.
The introduction of a conformational constraint and the N-
methylation of peptidic amide bonds are well-known
procedures to improve the stability and the activity of (opioid)
peptides.13,14 Previously, we have used the 4-amino-1,2,4,5-
tetrahydro-2-benzazepin-3-one (Aba) scaffold 2 (Figure 1) as a
tool for introducing a local conformational constraint in opioid
peptides.15−19 The synthetic Aba residue can be considered as a
constrained phenylalanine where the phenyl group is fixed to
the amine of the next amino acid via a methylene bridge.17
Previous work on opioid peptides indicated that replacement of
Phe by Aba in dermorphin-based sequences improves the DOR
affinity considerably, while preserving the binding to the
MOR.17,18 Additionally, peptides containing this constraint
represent highly active antinociceptive agents when tested in
vivo, as recently reported.19 Similarly, amide N-methylation in
opioid peptide sequences have shown to result in retained
opioid receptor binding and activation,20 as well as improved
BBB passage21 when compared to the nonmethylated
analogues. Therefore, both conformational side chain re-
striction and N-methylation prove to be efficient techniques
in improving the pharmacokinetic and pharmacodynamic
profile of dual MOR/DOR opioid peptides.22
The present work describes the chemical modification of the
MOR lead tetrapeptide [Dmt1]DALDA (Figure 1). The
different modifications that were carried out comprise: (i)
changing the overall net charge in the peptide by replacing the
D-Arg2 residue by a D-Cit2 and/or the Lys4 residue by a Nle4,
(ii) the introduction of a N-methyl group in the amide bond
connecting residues in position 3 and 4 in order to protect the
peptide against enzymatic degradation and to increase the
overall lipophilicity of the peptide, and (iii) introducing a
conformational constraint by means of an Aba3 residue that
replaces Phe3 in the Dmt1[DALDA] sequence. The synthesized
peptides have been investigated for their opioid receptor
binding affinity and agonist potency, BBB permeability, plasma
stability, and antinociceptive activities. Structure−activity
relationship (SAR) studies relating to the substitution pattern
in targeted positions within this series were pursued.
RESULTS AND DISCUSSION
■
Synthesis. All standard and N-methylated amino acids were
obtained from commercial sources, whereas the conformation-
ally constrained aminobenzazepinones of type 2 were prepared
as described below and used in standard solid phase peptide
synthesis.
1. Synthesis of the 4-Amino-2-benzazepin-3-one Scaf-
folds. The preparation of the required Aba-Lys/Nle dipepti-
domimetics 8a,b was performed using a previously reported23
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Table 1. Structures, Receptor MOR/DOR Binding Affinities and in Vitro Potency Data
a
a
a
a
structure
compd MOR Ki (nM) DOR Ki (nM) KOR Ki (nM) Ki ratio μ/δ/κ GPI (μ) IC50 (nM) MVD (δ) IC50 (nM)
b
H-Dmt-D-Arg-Phe-Lys-NH2
1
0.143
0.23
0.57
0.79
0.26
0.26
0.94
2.92
0.61
0.43
2.62
0.93
0.15
2100
15.2
50.0
3.98
309
22.3
16.2
20.9
98.5
56.1
14.4
11.5
>10.000
ND
1/11400/156
1/66/70
1/88/37
1/5/125
1/1190/216
1/62/55
1/42/12
1/2/>3420
1/64/−
1.41
2.2
23.1
6.2
H-Dmt-D-Arg-Phe-Nle-NH2
H-Dmt-D-Cit-Phe-Lys-NH2
H-Dmt-D-Cit-Phe-Nle-NH2
H-Dmt-D-Arg-Phe-NMeLys-NH2
H-Dmt-D-Arg-Phe-NMeNle-NH2
H-Dmt-D-Cit-Phe-NMeLys-NH2
H-Dmt-D-Cit-Phe-NMeNle-NH2
H-Dmt-D-Arg-Aba-Lys-NH2
H-Dmt-D-Arg-Aba-Nle-NH2
H-Dmt-D-Cit-Aba-Lys-NH2
H-Dmt-D-Cit-Aba-Nle-NH2
9
10
11
12
13
14
15
16
17
18
19
20
0.31
1.4
0.89
4.7
8.3
7.0
16.0
39.2
5.03
38.6
7.46
56.9
3.18
0.60
4.35
1.5
1.01
4.4
0.65
1.5
76.0
64 (IC25)
3.1
1.12
ND
1/17/3
1.45
1/22/−
1/3/12
49.0
1000
1.16
0.42
11.1
118
0.78 (IC35)
0.32
c
H-Dmt-D-Arg-Aba-Gly-NH2
1/4/787
a
Values represent means of 3−6 experiments. The GPI functional assay is representative of MOR activation, whereas the MVD is a DOR receptor-
representative assay. Binding affinities of compounds for MOR and DOR opioid receptors were determined by displacement of [3H]DAMGO ([D-
Ala2,NMePhe4,Gly-ol5]enkephalin) and [3H]DSLET ([D-Ser2,Leu5]enkephalin-Thr6) from rat brain membrane binding sites, respectively. KOR
b
receptor binding affinities were determined by displacement of [3H]U69,593 from guinea pig ileum membrane binding sites. Data taken from ref 8.
c
Data taken from ref 15.
and optimized procedure24 (Scheme 1). The phthaloyl-
protected (S)-o-cyano-phenylalanine 3 was obtained by
reaction of methyl 2-[(succinimidooxy)-carbonyl]benzoate25
with the commercially available o-(S)-cyanophenylalanine. The
corresponding o-formyl intermediate 4 was formed by hydro-
genation with Raney nickel in an acetic acid/water/pyridine
mixture. Reductive amination of 4 with lysine and norleucine
amino acid methyl esters resulted in secondary amines 5a/b,
which were subsequently used for the dicyclohexylcarbodii-
mide-induced ring closure to present the desired amino-
benzazepinones 6a/b. Ester hydrolysis, followed by phthaloyl-
removal with hydrazine hydrate and final Fmoc-protection,
gave the required Fmoc-Aba-Lys(Z)/Nle-OH building blocks
8a/b, which were used in the peptide synthesis.
2. Peptide Synthesis. The peptides in this study were
synthesized manually using Nα-Fmoc- or Nα-Boc-protected
amino acids on Rink amide resin (0.31 mmol scale) and MBHA
resin (0.29 mmol scale) using DIC and 1-hydroxybenzotriazole
(HOBt) or O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluro-
nium tetrafluoroborate (TBTU)/N,N-diisopropylethylamine
(DIPEA) as the coupling reagents/mixtures. A 3-fold excess
of the building blocks (Fmoc-Aba-Lys(Z)-OH, Fmoc-Aba-Nle-
OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Z)-OH, Boc-NMeLys(Z)-
OH, Fmoc-Nle-OH, Boc-NMeNle-OH, Fmoc- or Boc-Phe-
OH, Fmoc-D-Arg(Pbf)-OH, Boc-D-Arg(Tos)-OH, Boc-D-Cit-
OH, and Boc-Dmt-OH) and activating agents was applied, and
dry DMF was used as a solvent. Fmoc deprotections were
carried out by treating the resin twice (5 and 15 min) with 20%
piperidine in DMF. Boc-Dmt-OH was used in a final coupling
to directly present the fully unprotected peptide after acidic
cleavage from the resin. Boc-Dmt-OH was coupled with 1,3-
diisopropylcarbodiimide (DIC) and HOBt in order to avoid
side reactions when uronium-based coupling reagents were
used. Final cleavage of the peptide as well as the 2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) side chain
protection group removal was accomplished by treatment
with TFA/CH2Cl2/anisol 90:5:5 for 90 min (Rink resin) or
standard HF/anisol cleavage conditions for 60 min (MBHA
resin). The crude peptides were obtained after evaporation of
the cleavage mixture, precipitation in cold Et2O, dissolving in
glacial acetic acid, and subsequent lyophilization. Final
purification was performed by RP-HPLC on a SUPELCO
DiscoveryBIO wide pore preparative C18 column in 45%
overall yield and was >95% pure as determined by analytical
RP-HPLC. The structures of the compounds were confirmed
by high-resolution electrospray (ESP) ionization mass-
spectrometry.
Biological Evaluation and Structure−Activity Rela-
tionships. In Vitro Opioid Receptor Binding and Activity
(Table 1). The investigated peptides can be subdivided in three
series: (i) peptides 1 and 9−11 in which the side chain charges
were altered, (ii) the analogous series where the α-amine of the
C-terminal residue is methylated (peptides 12−15), and (iii)
the tetrapeptides with restricted side chain flexibility after a
Phe3→Aba3 substitution (compounds 16−19). Peptide 20
(Dmt-D-Arg-Aba-Gly-NH2) was reported recently19 but is
included in this work as a reference compound.
In general, the replacement of D-Arg2 in the H-Dmt-D-Arg-
Phe-Lys-NH2 analogues by D-Cit2 preserves the low- to
subnanomolar MOR binding affinity, produces shifts in κ
opioid receptor (KOR) binding, and enhances the DOR affinity
in most cases (except for 18 vs 16). This MOR/DOR
selectivity shift is in agreement with the in vitro functional
activities at both receptor types, as indicated by the results of
the guinea pig ileum (GPI) and mouse vas deferens (MVD)
bioassays. Structurally, D-Cit resembles D-Arg but it is not
charged at physiological pH. The binding and activation of
MOR and DOR confirms that the charge of the guanidinium
moiety in D-Arg2 is not crucial for receptor recognition, being in
line with earlier findings of other opioid peptides such as, for
example, the dermorphin, endomorphin, and enkephalin
peptides that bear respectively a D-Ala, Pro, or Gly residue in
position 2. Similarly, the side chain charge of the Lys residue in
position 4 was removed via replacement with Nle, a residue that
lacks the terminal amino group in the side chain. As shown in
Table 1, this modification did not markedly affect the
interaction with the MOR receptor but produced a significant
55-fold (1 vs 9), 19-fold (12 vs 13), and 5-fold (16 vs 17)
improvement of both DOR binding and in vitro functional
activity (4-fold (1 vs 9), 5-fold (12 vs 13), and 21- fold (16
(IC25) vs 17)). In this case, the favorable interaction with DOR
may be a result of not only the absence of a side chain charge
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Table 2. Blood−Brain Barrier Transport Data of the [125I]-Peptides (Mean SEM)
peptide
Kin initial (μL/(g × min))
Vbr (μL/g)
capillary fraction (%)
parenchyma fraction (%)
kout (min−1
)
1
9.44 0.41
8.48 1.61
14.91 0.10
18.11 0.78
11.35 0.61
14.89 1.34
19.25 1.02
27.43 4.80
27.12 3.76
21.99 2.81
17.13 2.36
24.01 3.41
23.12 10.26
32.97 9.99
19.39 15.81
25.95 0.95
21.84 1.32
30.95 19.75
76.88 10.26
67.03 9.99
80.61 15.81
74.05 0.95
78.16 1.32
69.05 19.75
0.058 0.11
−0.039 0.11
0.027 0.022
0.028 0.030
0.010 0.038
0.096 0.046
9
10
12
14
20
Figure 2. Mean distribution of the [125I]-peptides into the different tissues.
but also other factors like hydrogen bonding or steric effects
(amine function vs hydrogen) have to be considered. In respect
to the KOR affinity, the Lys4→Nle4 substitution slightly
improves binding when D-Arg2 is preserved (1 vs 9 and 12 vs
13). However, when combined with the D-Arg2→D-Cit2
modification, a significant loss of KOR affinity is observed.
Previously, we have shown that constraining the conforma-
tion of Phe3 in dermorphin-derived tetrapeptides into a 4-
amino-tetrahydro-2-benzazepin-3-one ring 2 leads to highly
potent peptides.18,19 To distinguish the influence of the ring
constraint from that of the N-alkylation of the Gly4 residue, the
“ring opened” analogues containing an amide N-methylation
were prepared and were shown to be of equal potency.21
Therefore, the N-methylated Lys4 analogues of Dmt1-DALDA
were then also investigated. The N-methylation of the Xxx3−
Yyy4 amide bond, on the other hand, influences the cis−trans
isomer equilibrium of the peptide bond between these amino
acids and stabilizes this bond against enzymatic degradation but
also increases the overall lipophilicity of the peptide analogues.
Clearly, when comparing the peptides of the first series
(peptides 1 and 9−11) with the respective peptide analogues of
the second series (12−15), the receptor binding data were very
similar, whereas the functional GPI and MVD assays gave
values slightly in disfavor of MOR activation but in favor of
DOR activity.
activity are obtained, the next analogues including the
conformationally constrained Aba-Lys dipeptide (16 and 17)
presented in vitro activity data with more pronounced shifts.
When Aba3−Lys4 was inserted in the peptide (16), the MOR
affinity was retained, but the DOR affinity increased by a factor
of 23, in agreement with previous findings.18 However, the
ability to activate MOR and DOR decreased substantially.
Because of this drop in activity, we did not perform any further
KOR binding studies for compounds 16 and 18. The ability of
DOR activation was restored in the Nle4 analogue 17 and
concomitantly improved KOR binding considerably (IC50κ 1.12
nM). A combination of D-Cit2 and Aba3−Nle4 substitutions in
19 resulted in the conversion of the potent MOR selective
Dmt1-DALDA 1 into a potent nonselective MOR partial
agonist/DOR full agonist 19.
Previously, the opioid sequence Dmt1-D-Arg2-Aba3-Gly4-NH2
20 was identified as a potent antinociceptive opioid peptide.19
The present data showed that the constrained amino-azepinone
scaffold should not be used in opioid tetrapeptides in
combination with a Lys4 amino acid but preferably in
combination with a Gly4 residue when aiming at combined
MOR/DOR agonists.
Brain Influx and Efflux. Determination of the blood−brain
barrier transport properties (initial influx Kin, Vbr, brain capillary
and parenchyma fraction, and efflux kout) form important
parameters to determine the “druggability” of central nervous
system (CNS) compounds. Therefore, the transport properties
In contrast to the first eight analogues in Table 1, for which
high MOR and DOR receptor binding and potent opioid
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of the peptides 1, 9, 10, 12, 14, and 20 were determined in CD-
1 mice. After labeling with 125I, the radiolabeled peptides were
injected into the jugular vein, and at specified time points after
injection, the mice were decapitated and the brain was isolated
for analysis (Table 2). These data clearly indicate an influx into
the mouse brain for all peptides: peptides 12, 10, 20, and 14
show the highest initial Kin values, indicating a rapid and high
influx of the radiolabeled peptides into the brain, while peptides
9 and 1 demonstrate slower influx properties. Moreover, the
apparent brain distribution volumes, obtained from 15 min
time interval and calculated according to Gjedde,26 indicate
relative high values of 27 μL/g, compared to 15 μL/g for the
negative control bovine serum albumin (BSA). On the basis of
the capillary depletion results, a higher parenchyma versus
capillary distribution for all peptides is observed: the amount of
peptide entering the brain is mostly transferred from the
capillaries to the parenchyma. This is in accordance with
previous distribution results of other opioid peptides.27
However, despite the good brain influx properties of peptide
20, the net effect is expected to be reduced due to the
significant brain to blood efflux transport. Also peptide 1 shows
a non-negligible efflux, while this is less observed for peptides
10 and 12 and not at all for peptides 9 and 14.
Additionally, and in an effort to predict and potentially rank
the ability of the peptides to permeate the BBB via use of an in
vitro test, all compounds were subjected to a Caco-2
permeability assay (Supporting Information). Very low peptide
transport values through the Caco-2 cell monolayers (in both
directions, i.e., apical→basal (A−B) and basal→apical (B−A))
were observed. However, a positive observation that was made
from this study consisted of a low B−A transport, which is
indicative of not hitting p-glycoprotein, an important efflux
transporter that is present at high concentration in the luminal
membrane of the brain endothelia.
In Vivo Tissue Biodistribution. Immediately after brain
dissection, the spleen, liver, lungs, heart, and kidneys were
collected, the tissues were weighed, and the amount of
radioactivity was measured. For each tissue, the percentage of
mass-corrected injected dose (ID) was then calculated as
described in the Experimental Section. The tissue distribution
plots (Figure 2) show a strikingly high liver accumulation for
peptide 9 as a consequence of the Lys4 replacement by Nle4.
Also for peptide 10, a high liver, as well as kidney extraction, is
observed. This higher kidney uptake is also seen for peptides 1
and 20 despite the structural differences between both peptides.
The two peptide analogues containing the amide N-methyl
groups (peptides 12 and 14) present a more balanced
distribution between the different organs. The serum levels
observed 15 min after administration were the highest for
peptide 1, the lowest for peptide 9, and intermediate for
peptides 10, 12, 14, and 20.
In Vitro Metabolic Stability. The in vitro metabolic stability
of unlabeled peptides 1 and 9 was determined in mouse plasma
and brain homogenate using UPLC RP C18-chromatography,
while the other peptides were investigated using the Prevail
organic acid column (HPLC). All peptides could undeniably be
distinguished from tissue components, resulting in a reliable
data evaluation. The peptides were observed to be very stable in
mouse plasma and brain homogenate for at least 2 h (Table 3).
There is also no adsorption nor chemical degradation observed
for the peptides under investigation. The BBB characteristics
can thus be assigned to the native peptides. Comparatively, on
the contrary, endomorphin-1 and endomorphin-2 for example
Table 3. Peptide Recovery after 2 h of in Vitro Incubation
peptide
mean recovery in plasma (%)
mean recovery in brain (%)
1
94.7
90.2
102.6
103.7
96.2
94.0
95.0
99.6
9
10
12
14
20
98.3
112.4
103.9
107.2
show very poor half-lives in mouse plasma: a t1/2 of 4−7 min
indicates a rapid enzymatic degradation of these peptides.28
In Vivo Antinociceptive Properties. Given the superior
potency of peptides 1 and 10 and the absence of promising in
vitro affinity and potency data for the [Aba-Lys]- and [Aba-
Nle]-containing peptides (compounds 16−19), only the
selected [Dmt1]DALDA analogues 1, 9, 10, 12, 14, and 20
were investigated in vivo for their antinociceptive activity using
the tail-flick test in mice after intraperitoneal (ip) and
subcutaneous (sc) administration. The antinociception, result-
ing from ip injection of the compounds, is shown in Figure 3,
with morphine as a positive control. All peptides generated an
antinociceptive effect significantly different from control mice
receiving ip saline. As a reference peptide, the naturally
occurring opioid peptide dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-
Pro-Ser-NH2) was included in the study.29 Figure 3 illustrates
the inability of dermorphin to induce a significant antinoci-
ceptive effect at the applied dose after ip injection.
Peptide 9 had a relatively low antinociceptive activity, similar
to morphine and dermorphin, at 60 min post ip injection. In
contrast, the parent peptide 1 as well as the peptide bearing a D-
Cit residue in position 2 (10) produced potent responses.
Peptide 1 showed a maximum antinociceptive activity at 60 min
post ip injection, while the maximum antinociceptive activity of
peptide 10 was reached at an earlier time point, i.e., at 45 min.
This can be explained by the lower initial Kin value together
with the high serum concentration observed at 15 min post
injection obtained for peptide 1. Peptide 10 showed good initial
brain influx properties, resulting in a relatively rapid onset of
antinociception. The D-Arg2→D-Cit2 substitution changes the
polarity of 1 in such a way that brain influx is accelerated (see
Kin Table 2). The rapid relapse of antinociception, on the other
hand, can be explained by the kidney and liver extraction, seen
at 15 min post injection. Both peptides gave responses that
were superior to the response of peptide 20. Despite the high
initial Kin value of peptide 20, the efflux and kidney uptake
characteristics highly influence its analgesic profile, as a slow
and shallow antinociceptive response is produced by this
peptide. Peptides 12 and 14 showed a rapid increase of
antinociception over time, reaching the highest plateau values at
60−90 min post ip injection. During the first 45 min after
injection, peptide 12 reached potent antinociception at a slower
rate than peptide 14. After the maximal effect, i.e., at the plateau
decline phase, both peptides still showed a high antinociceptive
activity (13−15 times higher than the antinociception of
morphine). These analgesic profiles can be explained by the
high initial brain influx properties (Kin) and insignificant outflux
rate (kout) of both peptides. Taken together, the relatively high
serum concentrations after 15 min ip and the absence of
massive tissue extraction provide an explanation for the
plateauing characteristics. Finally, the disappointing antinoci-
ceptive activity of peptide 9 can be understood by the low
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Figure 3. Antinociceptive characteristics of the [Dmt1]-DALDA analogues after ip administration. (left) Time response curves of the peptides (dose
of 1 μmol/kg, n = 10). Morphine and dermorphin at a dose of 1 μmol/kg (n = 10) were used as positive controls. The error bars represent the
standard error of the mean (SEM). All data were saline-corrected. (right) Area under the curve normalized to a dose of 1 μmol/kg based on
antinociception measured during 120 min postinjection.
initial Kin, indicating a slow onset of antinociception, together
with the excessive liver extraction results.
relative to morphine and a prolonged duration of action (Figure
4A). The high potency that characterizes [Dmt1]DALDA 1 is,
similarly to the ip administration, completely lost upon deletion
of the Lys4 side chain amino group, resulting in analogue 9
(Figure 4B). On the contrary, compound 10 (Figure 4C), as
well as its NMeLys4 analogue 14 (Figure 4E), induce a potent
effect with a peak at 1 h. This observation clearly shows that
only the removal of the Lys4 side chain functionality is
detrimental for antinociception. Following sc administration,
peptide 12, the N-methylated analogue of [Dmt]1DALDA 1,
produced a prolonged antinociception action with a peak effect
around 1−4 h and lasting up to 7 h (Figure 4D), a profile
similar to that of the parent peptide. While it was equipotent to
[Dmt]1DALDA, compound 12 was about 51-fold more potent
than morphine (Table 4). This finding was also true when
comparing peptide 10 and its N-methyl analogue 14, which
shows comparable potencies and similar time course of
antinociceptive action in mice after sc administration and an
augmented potency relative to morphine. On this basis, N-
methylation of the Lys residue in position 4 did not result in
any major alterations in antinociceptive activities when injected
sc, while its replacement with Nle has a detrimental effect.
Compound 20 also produced a significant antinociceptive
action after sc administration with a peak effect at 1 h (Figure
4F) but with a somewhat shorter duration of action than
peptides 1 and 12. It was 3- and 4-fold less potent in eliciting an
antinociceptive effect, respectively. However, compared to
morphine, peptide 20 still shows 14-fold increased analgesic
potency after sc administration. Exchanging the D-Arg2 residue
in peptides 1 and 12 with a D-Cit residue resulted in analogues
10 and 14, respectively, which retained high antinociceptive
potency while having a shorter duration of action (Figure
4C,E).
When analyzing the dose-normalized area under the curve
values (AUCs, 1 μmol/kg) (Figure 3) of the initial 120 min
post injection, we could conclude that peptide 14 (1412%·min)
had the highest antinociceptive effect, followed by peptide 12
(1187%.min), peptide 1 (750%.min), peptide 10 (521%.min),
and peptide 20 (253%·min). The positive control morphine
had a much lower AUC, i.e., 66%·min, similar to peptide 9
(65%·min) and dermorphin (46%·min).
Looking at the structural characteristics of peptides 1−14, it
is clear that the C-terminal amino acid is the most determining
factor for the antinociceptive activity: the presence of NMeLys
(12 and 14) is preferred, followed by Lys (1 and 10), while
other amino acids like Nle (9) decreased its antinociceptive
activity significantly. The effect of varying Arg and Cit at
position 2 is less pronounced but cannot be neglected. It shows
a confounding effect with the amino acid on position 4: if this is
NMeLys, then Cit2 gives a higher activity (14 versus 12), while
if position 4 is Lys, the reverse is true and Arg2 gives the highest
activity (1 versus 10).
In addition to the primary evaluation of antinociceptive
properties of peptides 1, 9, 10, 12, 14, and 20 after ip
administration, a further in vivo study assessing the dose- and
time-dependent antinociceptive effects after sc administration
was performed using the mouse tail-flick test. Antinociceptive
properties of the six peptides, given sc, were compared to those
of morphine and are illustrated in Figure 4. The calculated ED50
values with 95% confidence intervals are shown in Table 4. The
overall profile of antinociceptive effects after sc administration
closely match that determined after ip administration.
Dose-dependent and long duration of action lasting up to 6−
7 h was shown by peptides 1 (Figure 4A), 12 (Figure 4D), and
20 (Figure 4F), while a shorter antinociceptive effect was
produced by peptides 10 (Figure 4C) and 14 (Figure 4E)
lasting about 2−3 h, still being longer than the effect of
morphine. Compound 9 showed no dose-related antinocicep-
tion and failed to reach a 50% effect after sc administration of 6
μmol/kg (Figure 4B). All peptides, except 9, were considerably
more potent in eliciting an analgesic action than morphine
(Figure 4 and Table 4).
Altogether, the new peptidic [Dmt1]DALDA analogues 10,
12, and 14 emerged as potent antinociceptive agents after ip
and sc administration, with several-fold increased potency as
compared to morphine. They differ in kinetic behavior, like
BBB characteristics and tissue distribution, resulting in different
kinetic profiles of the antinociceptive activity.
CONCLUSIONS
■
In line with earlier reports,11,12,30 [Dmt1]DALDA 1 is a
potent opioid antinociceptive agent when administered to mice
sc in the tail-flick test, having a 44-fold increase in potency
From the substitution of the residues of [Dmt1]-DALDA at
position 2 and 4, by structurally related, but uncharged amino
acids (i.e., D-Arg2 by D-Cit2 and Lys4 by Nle4), we conclude that
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Figure 4. Antinociceptive activities of peptides 1, 9, 10, 12, 14, and 20 and morphine after sc administration in the mouse tail-flick test. Dose- and
time-dependent antinociceptive response presented as %MPE. Data are shown as mean SEM.
side chain groups bearing the positive charges in [Dmt1]-
DALDA are not needed for efficient in vitro receptor binding
and activation. However, the removal of either side chain
charges did diminish the well-established MOR subtype
receptor selectivity of [Dmt1]DALDA. Enhanced DOR binding
was observed when one charge and even more when the two
charges were eliminated. This trend was conserved throughout
the “parent” (1 and 9−11), the N-methylated (12−15) and the
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and 14 (Dmt-D-Cit-Phe-(NMe)Lys-NH2), but the latter
peptides produced their peak effect more rapidly (1 h vs 2 h
postinjection for 1/12). The steep increase in potency was also
accompanied by a rapid decrease after reaching the maximum
in antinociceptive effect. These observations are interesting for
the development of peptide-based pharmaceuticals aiming at
different pain types. Peptide 10 could serve as a lead molecule
for the treatment of acute (e.g., postoperative pain), whereas
the long-lasting Dmt-D-Arg-Phe-NMeLys-NH2 12 could be
useful for addressing chronic pain conditions (e.g., neuropathic
pain). As the compounds investigated in this study are potent
dual MOR/DOR (and even KOR, see 17) agonists, they could
potentially be useful for the treatment of pain with reduced side
effects and be effective at low administered doses.
Table 4. Antinociceptive Potencies of Peptides 1, 9, 10, 12,
14, and 20 in Comparison to Morphine after sc
Administration in the Mouse Tail-Flick Test
ED50 μmol/kg (95% confidence
relative potency to
morphine
a
compd
limits)
1
9
0.15 (0.07−0.32)
>6
44
nd
29
51
41
14
1
10
0.23 (0.10−0.55)
0.13 (0.06−0.30)
0.16 (0.07−0.36)
0.47 (0.18−1.2)
6.6 (3.5−12.6)
12
14
20
Morphine
a
ED50 was determined at the time of peak effect (i.e., 2 h for peptide 1
and 12, 1 h for peptides 10, 14, and 20, and 30 min for morphine). nd:
not determined.
EXPERIMENTAL SECTION
■
General. Thin layer chromatography (TLC) was performed on
plastic sheet precoated with silica gel 60F254 (Merck, Darmstadt,
Germany) using specified solvent systems. Mass spectrometry (MS)
was recorded on a Micromass Q-Tof Micro spectrometer using
electrospray (ESP) ionization (positive or negative ion mode). Data
collection was done with Masslynx software. Analytical reversed phase
(RP)-HPLC was performed using an Agilent 1100 series system
(Waldbronn, Germany) with a Supelco Discovery BIO Wide Pore
(Bellefonte, PA, USA) RP C-18 column (25 cm × 4.6 mm, 5 μm)
using UV detection at 215 nm. The mobile phase (water/acetonitrile)
contained 0.1% TFA. The gradient consisted of a 20 min run from 3 to
97% acetonitrile at a flow rate of 1 mL/min. Preparative HPLC was
performed on a Gilson apparatus and controlled with the software
package Unipoint. The RP C18-column (Discovery BIO Wide Pore 25
cm × 21.2 mm, 10 μm) was used under the same conditions as the
analytical RP-HPLC but with a flow rate of 20 mL min−1. A purity of
more than 95% was determined for all compounds by analytical RP-
constrained “Aba” series (16−19). In regard to KOR affinity,
the introduced modifications clearly had an impact on the
binding affinity of the investigated peptides to this opioid
receptor subtype. Low nanomolar KOR binding affinities were
determined for peptide analogues 13, 14, 17, and 19 (with Kiκ
values between 1.12 and 14.4 nM). Because of the liabilities
related to μ opioid analgesics such as morphine, ligands of KOR
have also received considerable attention as potential
analgesics.31 In particular, KOR agonists have therapeutic
potential in pain conditions associated with inflammation.
When looking at the transport properties of the peptide
analogues in this study, all synthesized [Dmt1]-DALDA
peptides show influx into the brain parenchyma. The tissue
distribution, on the other hand, indicated that the charged
aminoalkyl chain of Lys4 and the guanidine moiety of D-Arg2
are of significant importance for the biodistribution of the
peptides. Deletion of these charges, via D-Arg2→D-Cit2 and
Lys4→Nle4 substitutions, decreases the 3+ net charge of the
peptide, resulting in enhanced liver uptake and potentially
influences brain accessibility. As shown in this work, subtle
structural modifications can influence the biodistribution of the
peptide analogues and could lead to a modulated peptide
delivery to the CNS, thus affecting the ratio of centrally vs
peripherally induced analgesia.
The ability of mice to sense a pain stimulus was verified after
peripheral administration of a selection of opioid peptides. In
contrast to the reference peptide [Dmt1]DALDA 1, which
showed a very potent analgesic effect, the low antinociceptive
effect of peptide 9 could be rationalized by a limited brain
intake after massive liver extraction. Peptide 20 showed a rapid
brain influx but also a significant efflux, resulting in lower
antinociception compared to the other peptides such as 1, 12,
and 14.
Peptides 12 and 14 had the highest antinociceptive effect
during the first 2 h of an ip injection, measured as AUC relative
to morphine (normalized at 1 μmol/kg), followed by peptides
1 and 10. The presence of NMeLys4 at the carboxyl terminus of
the peptide sequence is thus preferred for antinociceptive
activity, followed by Lys4, while other amino acids like Gly4 and
Nle4 significantly decreased the antinociception.
1
HPLC using the conditions described above. H NMR and 13C NMR
spectra were recorded at 250 and 63 MHz, respectively, on a Bruker
Avance 250 spectrometer or at 500 and 125 MHz on a Bruker Avance
II 500. Calibration was done with TMS (tetramethylsilane) or residual
solvent signals as an internal standard. The solvent used is mentioned
in all cases and the abbreviations used are: s (singlet), d (doublet), dd
(double doublet), t (triplet), br s (broad singlet), and m (multiplet).
Opioid Receptor Binding Assays. Opioid receptor binding
studies were performed as described in detail elsewhere.35 Binding
affinities for MOR and DOR were determined by displacing,
respectively, [3H]DAMGO (Multiple Peptide Systems, San Diego,
CA) and [3H]DSLET (Multiple Peptide Systems) from rat brain
membrane binding sites, and KOR receptor affinities were measured
by displacement of [3H]U69,593 (Amersham) from guinea pig brain
membrane binding sites. Incubations were performed for 2 h at 0 °C
with [3H]DAMGO, [3H]DSLET, and [3H]U69,593 at respective
concentrations of 0.72, 0.78, and 0.80 nM. IC50 values were
determined form log-dose displacement curves, and Ki values were
calculated from the IC50 values by means of the equation of Cheng and
Prusoff,32 using values of 1.3, 2.6, and 2.9 nM for the dissociation
constants of [3H]DAMGO, [3H]DSLET, and [3H]U69,593, respec-
tively.
Functional GPI and MVD Assays. The guinea pig ileum (GPI)33
and mouse vas deferens (MVD)34 bioassays were carried out as
described in detail elsewhere.35,36 A dose−response curve was
determined with [Leu5]enkephalin as standard for each ileum and
vas preparation, and IC50 values of the compounds being tested were
normalized according to a published procedure.37
Blood−Brain Barrier Transport. Animals. Male Institute for
Cancer Research, caesarean derived-1 (ICR-CD-1) mice (Harlan
Laboratories, Venray, The Netherlands), weighing 25−30 g, were used
according to the Ethical Committee Principles of Laboratory Animal
Welfare as approved by our institute (Ghent University, Faculty of
Veterinary Medicine, 2009−052). Animals were housed under
When the peptide analogues were investigated over a longer
sc postinjection period (up to 7 h) in a dose-dependency study,
it became apparent that two distinct antinociception profiles
were obtained. Comparable ED50 values were measured for
peptides 1 and 12 (Dmt-D-Arg-Phe-(NMe)Lys-NH2) in
comparison with analogues 10 (H-Dmt-D-Cit-Phe-Lys-NH2)
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standard laboratory conditions in a temperature and humidity
regulated environment with food and water ad libitum.
where Am(t) is the amount of radioactivity in brain at time t, Cp(t) the
amount of radioactivity in serum at time t, Kin the brain influx rate
constant, and Vi the initial brain distribution volume.
The exposure time (exp·t) is defined as the integral of the serum
radioactivity over time divided by the radioactivity at time t:
Reagents and Peptides. Bovine serum albumin (BSA), calcium
chloride dehydrate, D-glucose, HEPES, magnesium sulfate heptahy-
drate, potassium chloride, disodium hydrogen phosphate dihydrate,
sodium dihydrogen phosphate monohydrate, sodium chloride, sodium
lactate, urethane, and Krebs−Henseleit buffer were all purchased from
Sigma (St. Louis, MO, USA) or Merck KGaA (Darmstadt, Germany).
Dextran was obtained from AppliChem GmbH (Darmstadt,
Germany), while dermorphin was purchased from Peptide Synthetics
(PPR, Hampshire, United Kingdom). Morphine hydrochloride was
acquired from Belgopia (Louvain-La-Neuve, Belgium).
t Cp(t) dt
∫
o
exp ·t =
Cp(t)
The integral of the radioactivity over time is represented by the area
under the curve (AUC).
Peptides 125I Radiolabeling. The Dmt1-DALDA peptides and
controls (dermorphin and BSA) were labeled by the Iodogen
method.37−39 Briefly, a 1 g/L peptide solution was prepared by
dissolving about 1 mg in phosphate buffer (pH 7.4, 130 mM). A Iodo-
Gen coated tube was previously rinsed with 1 mL of phosphate buffer.
Subsequently, 50 μL of NaI (20 nmol/10 μL) and 10 μL of Na125I
solution (3700 MBq/ml) (Perkin-Elmer, Boston, MA, USA) were
transferred into this Iodo-Gen coated tube. After 6 min of incubation
at room temperature, the oxidation reaction of iodide to iodonium was
stopped by removing the solution from the tube and adding the
iodonium solution to a tube containing 50 μL of peptide solution (1
μg/μL). The iodination reaction of the peptide was allowed to proceed
another 6 min at room temperature, after which the reaction mixture
was purified with an Ag filter. After Ag purification, the radioactivity of
the filtrate was measured using a dose calibrator (Veenstra, The
Netherlands) to dilute the 125I-peptide appropriately.
Finally, the brain/serum ratios (μL/g) were plotted versus exposure
time. The initial slope of the linear portion of this relationship over the
first minutes measured the unidirectional initial influx rate (Kin initial)
from blood to brain. The intercept represents the brain volume of
distribution (Vbr).
As a system suitability controls of this method, BSA, the
impermeable vascular marker, as well as dermorphin, a weak positive
influx control peptide28 were labeled with I-125 and their influx
investigated as described above. In compliance with literature data,40 a
mean influx transfer constant of 0.0849 0.0847 μL/(g × min) was
obtained for BSA, confirming negligible influx. The mean Kin over 15
min for dermorphin was 0.173 0.137 μL/(g × min), while the initial
Kin was 5.52 1.41 μL/(g × min) if the first minutes are considered,
justifying its choice as a weak positive brain influx control.
Capillary Depletion. This method was performed to determine to
what extent the peptides, taken up by the brain, crossed the capillary
wall instead of being trapped in the capillary cells. The method of
Triguero et al.41 as modified by Gutierrez et al.42 was used.
Summarizing, CD-1 mice were anesthetized with urethane (3 g/kg
ip) and the jugular vein was isolated. Then 200 μL of the diluted
peptide solution (10 000 cpm/μL) was injected in the jugular vein. At
specified time points after injection, blood was collected from the
abdominal aorta and the brain was perfused manually with 20 mL of
LR buffer after severing the jugular veins. Subsequently, the brain was
collected, homogenized with 0.7 mL of ice-cold capillary buffer (10
mM HEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1 mM
MgSO4, 1 mM NaH2PO4, and 10 mM D-glucose adjusted to pH 7.4)
in a pyrex glass tube and mixed with 1.7 mL of 26% ice-cold dextran
solution in capillary buffer. The resulting solution was weighed and
centrifuged in a swinging bucket rotor (Eppendorf, Hamburg,
Germany) at 5400g for 30 min at 4 °C. Pellet (capillaries) and
supernatant (parenchyma and fat tissues) were collected, weighed, and
measured in a gamma counter. After centrifuging the obtained blood,
50 μL of serum was collected and measured in a gamma counter.
Compartmental BBB distribution was evaluated from plotting
capillaries/serum and parenchyma/serum activity ratios versus time.
Brain to Blood Transport. This method was performed to quantify
the amount of peptide pumped out of the brain by efflux transport.43
CD-1 mice were anesthetized with urethane (3 g/kg ip), and the skin
of the skull was removed. A hole was then made into the lateral
ventricle, 1 mm lateral and 0.34 mm posterior to the bregma, with a 22
G needle carrying a tubing guard at a constant depth of 2 mm. The
anesthetized mice received an intracerebroventricular (icv) injection of
1 μL of LR solution containing the labeled peptide (25000 cpm/μL)
and 1% BSA at a speed of 360 μL/h for 10 s using a syringe pump
(KDS100, KR analytical, Cheshire, UK). At specified time points after
injection, blood was collected from the abdominal aorta and the mouse
was decapitated. Subsequently, the whole brain was collected, weighed,
and measured in a gamma counter. The efflux rate constant kout
(min−1) was calculated from the linear regression of the natural
logarithm of the residual radioactivity in brain versus time, applying
first-order kinetics.
In addition, a quality control was performed on the filtrated peptide
using a Vydac Everest C18 (250 mm × 4.6 mm ID, 300 Å, 5 μm
particle size) HPLC column (Grace Vydac, Hesperia, CA, USA) in an
oven set at 30 °C, with a mobile phase consisting of (A) 0.1% w/v
formic acid in water and (B) 0.1% w/v formic acid in acetonitrile. A
linear gradient was employed as follows:
Peptide 1: 0−50 min going from 98% to 50% A.
Peptides 9, 12, 14, and 20: 0−40 min going from 98% to 80%
A.
Peptide 10: 0−40 min going from 98% to 60% A.
The flow rate was set at 1.0 mL/min with an injection volume of
100 μL.
The radio-HPLC apparatus consisted of a LaChrom Elite L-2130
pump with degasser, a LaChrom Elite L-2300 column oven, a
LaChrom Elite L-2400 UV detector set at 215 nm (all Hitachi, Tokyo,
Japan), a Rheodyne 7725i manual injector with 100 μL sample loop
(Rheodyne, Rohnert Park, CA, USA), and a Berthold LB500 HERM
radioactivity detector (Berthold Technologies, Bad Wildbad, Ger-
many) equipped with EZChrom Elite version 3.1.7 software for data
acquisition (Scientific Software, Pleasanton, CA, USA).
Multiple Time Regression (MTR). To determine whether the
peptides could enter the brain, multiple time regression analysis was
performed. The CD-1 mice were anesthetized with intraperitoneal (ip)
urethane (3 g/kg), and the jugular vein and carotid artery were
isolated. Then 200 μL of the radiolabeled peptides (30 000 cpm/μL)
in lactated Ringer’s (LR) solution containing 1% BSA were each
injected separately into the jugular vein. At specified time points after
injection, blood was obtained from the carotid artery and the mouse
was decapitated. The brain was removed and weighed, after which
radioactivity was determined in a gamma counter (Wallac Wizard
automatic gamma counter, PerkinElmer, Shelton, CT, USA). The
blood was centrifuged at 10000g for 15 min at 21 °C, and the
radioactivity of the obtained serum was measured.
The theoretical background of the multiple time regression analysis
is based on the Gjedde equation:26
Tissue Distribution. Immediately after brain collection, spleen, liver,
lungs, heart, and kidneys were collected.44 Again, the tissues were
weighed and measured in the gamma counter. For each tissue, the
percentage of time-corrected injected dose (ID) was then calculated:
t Cp(t) dt
∫
A
Cp(t)
m(t)
o
= Kin
+ V
i
Cp(t)
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Atissue
formula: 100(test latency − baseline latency)/(10 − baseline latency).
To avoid tissue damage, a maximum score was assigned (100%) to
animals that failed to respond within 10 s (= cutoff time).
A negative control consisted of a physiological 0.9% saline solution.
Two positive controls were also included, i.e., morphine and
dermorphin, at a dose of 5 mg/kg. All controls were ip administered.
The amount of antinociception was determined using the AUC:
W
tissue
Percentage ID(%) =
× 100
Adose injected
W
animal
where Atissue is the radioactivity of the tissue, Wtissue the weight of the
tissue, Adose injected the amount of radioactivity injected into the vein,
and Wanimal the body weight of the mouse.
In Vitro Metabolic Stability. To obtain information regarding the
metabolic fate of peptides, the nonlabeled peptides were tested in vitro
by incubating in mouse plasma and mouse brain homogenate.45 In
brief, 100 μg of the nonlabeled peptide dissolved in Krebs−Henseleit
buffer (pH 7.4) was incubated in plasma or brain homogenate (250 μL
of mouse plasma or 150 μg of tissue protein, respectively) at 37 °C
while shaking. At suitable time points, aliquots were taken and
immediately transferred into microtubes containing 10% v/v trifluoric
acetic acid (TFA) solution in water to obtain a final concentration of
1:10 v/v solvent/aliquot. The samples were then heated for 5 min at
95 °C, followed by cooling in an ice-bath for 30 min. After
centrifugation of the samples at 15000g for 15 min at 5 °C, the
supernatants were analyzed using UPLC-PDA and HPLC-UV.
Placebos (mouse plasma or tissue homogenate), controls (initially
inactivated tissue proteases), and reference solutions (peptide
dissolved in buffer) were similarly processed. The time-dependent
decrease of the native peptide was quantified and from the linear
n
⎛
⎝
⎞
⎠
(y + y
)
i
i−1
⎜
⎜
⎟
⎟
AUC =
× (xi − xi−1)
∑
2
i=1
within the 120 min time points, y the percentage of antinociception
and x the time after injection (min). Assuming first-order kinetics, the
AUC of the peptides and of morphine is converted to a theoretical
AUCp, respectively AUCm, equivalent to a dose of 1 μmol active
moiety/kg. The morphine normalized AUC for the peptides
(AUCnormalized) is then calculated by:
⎛
⎞
AUCp
AUCnormalized
=
× 100
⎜
⎝
⎟
⎠
AUC
m
B. After sc Administration. Male CD-1 mice (25−35 g) were
purchased from Charles River Laboratories (Sulzfeld, Germany).
Animals were housed under standard laboratory conditions in a
temperature-regulated environment under a controlled 12 h light/dark
cycle with food and water ad libitum. All animal procedures were
approved by the Austrian Ethical Committee on Animal Care and Use
in line with international laws and policies. Morphine hydrochloride
was obtained from Gatt-Koller GmbH (Innsbruck, Austria). All drugs
were dissolved in sterile physiological saline (0.9%) and were
administered sc in a volume of 10 μL per 1 g body weight.
The radiant heat tail-flick test was used to assess antinociceptive
effects of tested drugs after sc administration in mice using an UB
37360 Ugo Basile analgesiometer (Ugo Basile srl, Varese, Italy) as
described.46 The reaction time required by the mouse to move its tail
due to the radiant heat was measured and defined as the tail-flick
latency. A cutoff time of 10 s was also used in order to minimize tissue
damage, and the % antinociception or MPE for each dose
administered was calculated as above. At least three doses were
tested, and five to six animals per dose were used. Antinociceptive
ED50 values and 95% confidence limits were calculated by the method
of Litchfield and Wilcoxon (1949).47
Compound Synthesis and Characterization. Both Fmoc-Aba-
Lys-OH 8a and Fmoc-Aba-Nle-OH 8b were prepared according to
reported procedures (see Supporting Information).23,24 Subsequently,
all peptides were assembled as mentioned before by standard SPPS
protocols employing Fmoc- or Boc-protected amino acid residues or
dipeptides (8a and 8b).
Peptide Characterization. H-Dmt-D-Arg-Phe-Lys-NH2 (1).
HPLC (standard gradient): tret = 8.46 min. TLC Rf (EBAW), 0.50;
TLC Rf (BAW), 0.24. ESI-HRMS [M + H+]: m/z = 640.3950
(calculated for C32H48H+N9O5: 640.3929).
regression between the quantified decrease and time, the half-life (t1/2
)
was determined as:
ln(2)
k
t1/2
=
where k is the first-order rate constant.
If the calculated half-life was more than two times the experimental
time, i.e., more than 4 h, no quantitative half-life was given and the
results expressed as “stable”.
UPLC-PDA analyses were performed using Waters Acquity H-Class
Biosamples FTN, a Waters Acquity H-Class BioQuaternary solvent
manager, a Waters Acquity H-Class column module, and a Waters
Acquity H-Class photodiode array detector (UV spectrum was
recorded between 190 and 400 nm) equipped with Waters Empower
Pro software version 2 (all Waters, Milford, MA, USA). Analysis was
performed using a Waters Acquity UPLC BEH300 C18 column (100
mm × 2.1 mm ID, 300 Å, 1.7 μm particle size) in an oven set at 35 °C.
The following solvent system was used: (A) 10 mM ammonium
carbonate and (B) acetonitrile. The gradient method consisted of a
linear increase from 5% B to 60% B in 20 min, followed by 10 min
reconditioning with initial composition 95/5 v/v A/B. The flow rate
was set at 0.58 mL/min, and the injection volume was 5 μL.
The degradation of the peptides was also studied by use of an
HPLC system consisting of a Waters Alliance 2695 separations
module, a Waters column heater, and a Waters 2487 dual wavelength
absorbance detector, fitted with Empower Pro software version 2 (all
Waters, Milford, MA, USA). Quantification of the degradation was
performed at 210 nm. Analysis was achieved using a Grace Alltech
Prevail organic acid column (250 mm × 4.6 mm I.D., 110 Å, 5 μm
particle size) in an oven set at 30 °C, using (A) 25 mM phosphate
buffer pH 8.0 and (B) acetonitrile. The following linear gradient
method was used: from 10% B to 70% B in 45 min, followed by 15
min with the initial composition (90/10 v/v A/B) to recondition the
system. The flow rate was set at 1.0 mL/min with an injection volume
of 20 μL.
H-Dmt-D-Arg-Phe-Nle-NH2 (9). HPLC (standard gradient): tret
=
10.57 min. TLC Rf (EBAW), 0.62; TLC Rf (BAW), 0.70. ESI-HRMS
[M + H+]: m/z = 625.3793 (calculated for C32H47H+N8O5: 625.3820).
H-Dmt-D-Cit-Phe-Lys-NH2 (10). HPLC (standard gradient): tret
=
8.68 min. TLC Rf (EBAW), 0.52; TLC Rf (BAW), 0.35. ESI-HRMS
[M + H+]: m/z = 641.3759 (calculated for C32H47H+N8O6: 641.3770).
H-Dmt-D-Cit-Phe-Nle-NH2 (11). HPLC (standard gradient): tret
=
Antinociceptive Properties. A. After ip Administration. The
same type of mice as used for the BBB transport studies were used in
this assay. Antinociception was assessed using the 55 °C tail
withdrawal method in CD-1 mice. Each mouse (10 mice per group)
was first tested for consistency in baseline latency by immersing its tail
in the water and recording the time to response. Mice were then
administered the controls as well as the test compounds at a dose of 5
mg/kg body weight and tested for antinociception at various time
points afterward. Antinociception was calculated as % antinociception
or % MPE (percent maximum possible effect) using the following
11.06 min. TLC Rf (EBAW), 0.72; TLC Rf (BAW), 0.74. ESI-HRMS
[M + H+]: m/z = 626.3651 (calculated for C32H46H+N7O6: 626.3661).
H-Dmt-D-Arg-Phe-NMeLys-NH2 (12). HPLC (standard gradient):
tret = 8.43 min. TLC Rf (EBAW), 0.52; TLC Rf (BAW), 0.24. ESI-
HRMS [M + H+]: m/z = 654.4064 (calculated for C33H50H+N9O5:
654.4086).
H-Dmt-D-Arg-Phe-NMeNle-NH2 (13). HPLC (standard gradient):
tret = 10.67 min. TLC Rf (EBAW), 0.62; TLC Rf (BAW), 0.70. ESI-
HRMS [M + H+]: m/z = 639.3983 (calculated for C33H49H+N8O5:
639.3977).
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H-Dmt-D-Cit-Phe-NMeLys-NH2 (14). HPLC (standard gradient): tret
= 8.67 min. TLC Rf (EBAW), 0.55; TLC Rf (BAW), 0.37. ESI-HRMS
[M + H+]: m/z = 655.3915 (calculated for C33H49H+N8O6: 655.3926).
H-Dmt-D-Cit-Phe-NMeNle-NH2 (15). HPLC (standard gradient):
tret = 11.81 min. TLC Rf (EBAW), 0.72; TLC Rf (BAW), 0.74. ESI-
HRMS [M + H+]: m/z = 640.3823 (calculated for C33H48H+N7O6:
640.3817).
(solution); kout, brain efflux rate coefficient; MOR, μ-opioid
receptor; MPE, maximal possible effect; MTR, multiple time
regression; MVD, mouse vas deferens; Nle, norleucine; NMM,
N-methyl morpholine; Kin, unidirectional brain influx or uptake
transfer constant; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofur-
an-5-sulfonyl; Phth, phthaloyl; RaNi, Raney nickel; SAR,
structure−activity relationship; sc, subcutaneous; SEM, stand-
ard error of the mean; TBTU, O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate; Vbr, brain
distribution volume; Z, benzyloxycarbonyl
H-Dmt-D-Arg-Aba-Lys-NH2 (16). HPLC (standard gradient): tret
=
8.21 min. TLC Rf (EBAW), 0.52; TLC Rf (BAW), 0.24. ESI-HRMS
[M + H+]: m/z = 653.3913 (calculated for C33H48H+N9O5: 652.3929).
H-Dmt-D-Arg-Aba-Nle-NH2 (17). HPLC (standard gradient): tret
=
11.25 min. TLC Rf (EBAW), 0.75; TLC Rf (BAW), 0.74. ESI-HRMS
[M + H+]: m/z = 637.3826 (calculated for C33H47H+N8O5: 637.3820).
REFERENCES
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=
■
8.43 min. TLC Rf (EBAW), 0.55; TLC Rf (BAW), 0.32. ESI-HRMS
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ASSOCIATED CONTENT
* Supporting Information
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■
S
Characterization of nonkey compounds, ED50 values of selected
peptides and morphine at different time points after sc
administration, and Caco-2 cell permeability data. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For S.B.: phone, +32 2 6293292; E-mail: sballet@vub.ac.be.
For B.D.S.: phone, +32 9 2648100; E-mail, bart.despiegeleer@
ugent.be.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The work of S.B., D.T., and P.W.S. was supported by a
collaboration convention between the Minister
■
̀
e du Dev
́
eloppe-
ment Economique, de l'Innovation et de l'Exportation du
Queb
́
ec, and the Fund for Scientific ResearchFlanders (FWO
Vlaanderen) (PSR-SIIRI-417). S.V.D. thanks the Institute for
the Promotion of Innovation through Science and Technology
in Flanders (IWTVlaanderen) for a Ph.D. grant (no. 73402),
and a Special Research Fund from the Ghent University (BOF)
grant and mandate (no. 01J22510 and 01D38811) (E.W.,
B.D.S., S.S.). The research of P.W.S. was also supported by
grants CIHR (MOP-89716), and the NIH (DA-004443).
M.A.C. is supported by a NHMRC Australia Fellowship
AF511105.
ABBREVIATIONS USED
■
Aba, 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one; AUC,
area under the curve value; BBB, blood−brain barrier; BSA,
bovine serum albumin; Cit, citrulline; CNS, central nervous
system; DAMGO, [D-Ala2,NMePhe4,Gly-ol5]enkephalin; Dmt,
2′,6′-dimethyl-(S)-tyrosine; DOR, δ-opioid receptor; DSLET,
[D-Ser2,Leu5]enkephalin-Thr6; ESP, electrospray ionization
mass-spectrometry; GPI, guinea pig ileum; HEPES, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid; HOBt, 1-hy-
droxybenzotriazole; ID, injected dose; ip, intraperitoneal; iv,
intravenous; KOR, κ-opioid receptor; LR, lactated Ringer’s
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Shenderovich, M. Design of peptides, proteins and peptidomimetics in
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