Synthesis and biological evaluation of an orally active glycosylated endomorphin-1

Article abstract of DOI:10.1021/jm300418d

The endogenous opioid peptide endomorphin-1 (1) was modified by attachment of lactose to the N-terminus via a succinamic acid spacer to produce compound 2. The carbohydrate modification significantly improved the metabolic stability and membrane permeability of 2 while retaining μ-opioid receptor binding affinity and agonist activity. Analogue 2 produced dose-dependent antinociceptive activity following intravenous administration in a chronic constriction injury (CCI) rat model of neuropathic pain with an ED₅₀ of 8.3 (±0.8) μmol/kg. The corresponding ED₅₀ for morphine was 2.6 (±1.4) ±mol/kg. Importantly, compound 2 produced dose-dependent pain relief after oral administration in CCI rats (ED ₅₀ = 19.6 (±1.2) ±mol/kg), which was comparable with that of morphine (ED₅₀ = 20.7 (±3.6) μmol/kg). Antineuropathic effects of analogue 2 were significantly attenuated by pretreatment of animals with the opioid antagonist naloxone, confirming opioid receptor-mediated analgesia. In contrast to morphine, no significant constipation was produced by compound 2 after oral administration.

Full text of DOI:10.1021/jm300418d

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of an Orally Active Glycosylated
Endomorphin-1
Pegah Varamini,† Friederike M. Mansfeld,† Joanne T. Blanchfield,† Bruce D. Wyse,‡,§ Maree T. Smith,‡,§
and Istvan Toth*,†,§
‡
†School of Chemistry and Molecular Biosciences, and Centre for Integrated Preclinical Drug Development, The University of
Queensland, Brisbane, QLD 4072, Australia
§School of Pharmacy, The University of Queensland, Brisbane, QLD 4102, Australia
S
* Supporting Information
ABSTRACT: The endogenous opioid peptide endomorphin-1 (1) was modified by attachment of lactose to the N-terminus via
a succinamic acid spacer to produce compound 2. The carbohydrate modification significantly improved the metabolic stability
and membrane permeability of 2 while retaining μ-opioid receptor binding affinity and agonist activity. Analogue 2 produced
dose-dependent antinociceptive activity following intravenous administration in a chronic constriction injury (CCI) rat model of
neuropathic pain with an ED50 of 8.3 ( 0.8) μmol/kg. The corresponding ED50 for morphine was 2.6 ( 1.4) μmol/kg.
Importantly, compound 2 produced dose-dependent pain relief after oral administration in CCI rats (ED50 = 19.6 ( 1.2) μmol/
kg), which was comparable with that of morphine (ED50 = 20.7 ( 3.6) μmol/kg). Antineuropathic effects of analogue 2 were
significantly attenuated by pretreatment of animals with the opioid antagonist naloxone, confirming opioid receptor-mediated
analgesia. In contrast to morphine, no significant constipation was produced by compound 2 after oral administration.
models of acute5 and neuropathic pain6 with less cardiorespir-
INTRODUCTION
Neuropathic pain is a complex, chronic pain state that may
■
atory depression than opioid alkaloids.7 In spite of the relatively
high stability of endomorphins when compared with other
develop secondary to damage to, or dysfunction of, the
peripheral or central nervous system.1 Pharmacological
opioid peptides, the rapid degradation of endomorphins by
membrane-bound exo- and endopeptidases in the blood limits
management of neuropathic pain is challenging for clinicians
due to its heterogeneous nature. Currently available drug
their usefulness as analgesics.8,9 Furthermore, the blood−brain
barrier (BBB) contains several enzymes that can degrade
treatments, including anticonvulsants (e.g., gabapentin),
peptides and inhibit their penetration into the brain.10 Because
antidepressants (e.g., amitriptyline), and antiarrhythmics, only
cause pain relief in up to 50% of patients2 and often produce
of their hydrophilicity, endomorphins, like all small peptides,
are not able to cross the BBB by transmembrane diffusion.11
Previous studies demonstrated that endomorphins do not elicit
antinociceptive activity following peripheral administration in
rodents.12 Even after delivering the peptides directly into the
central nervous system in animal models, endomorphins had a
short duration of action (<30 min).13,14 As a result,
modification of the structure of endomorphins presents a
simple and viable method to enhance their bioavailability to the
brain.
dose-limiting side effects.2,3 Additionally, morphine, the
prototypic strong opioid analgesic, has reduced potency in
neuropathic pain and produces a range of adverse effects
including nausea, vomiting, constipation, sedation, analgesic
tolerance, and cardiorespiratory depression.3 Hence, consid-
erable research attention has been directed toward investigation
of endogenous opioid peptides as possible novel analgesic
agents for producing potent analgesia with minimal opioid-
related adverse effects. Two important members of this group
are endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, 1) and endomor-
phin-2 (Tyr-Pro-Phe-Phe-NH2). They exhibit high selectivity
and affinity for μ-opioid receptors (MOR),4 the main opioid
receptor type that mediates analgesia. Centrally administered
endomorphins produce potent antinociception in rodent
Oral administration is the most convenient and favorable
route for delivering drugs; nevertheless, it has proven to be
Received: February 29, 2012
Published: June 8, 2012
© 2012 American Chemical Society
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extremely challenging for peptides and proteins.15 In an
attempt to overcome the shortcomings associated with oral
bioavailability of peptides, a diverse array of chemical
modifications have been explored including esterification,16
amidation17 (dermorphin analogues), and cyclization (α-
conotoxin Vc1.1).18 A well-studied approach to produce
systemically active peptide analogues is conjugation to
saccharide units.19 Peptide glycosylation can promote biological
stability as well as increase permeability across biological
barriers via active or facilitated transport.20,21 Previously, we
showed that incorporation of sugars into the structure of short
peptides can improve their oral bioavailability.22 In the present
study, we applied this approach to stabilize the structure of
parent peptide 1, reduce its vulnerability to proteolysis, and
confer oral activity for the relief of neuropathic pain.
monolayers with Papp 1.1 ( 0.5) × 10−7 cm/s, whereas
compound 2 exhibited significant permeability with Papp 7.6
( 0.2) × 10−5 cm/s, which was a remarkable 700-fold increase
from that of the parent peptide (p < 0.001).
The possible role of a lactose receptor and lactose selective
transporter28,29 was examined by assessing the Caco-2 cell
permeability of analogue 2 in the presence of 20 mM lactose,
glucose, or galactose. The addition of lactose inhibited the
absorption of analogue 2, suggesting receptor-mediated
absorption. Galactose, the terminal monosaccharide of lactose,
caused less inhibition of the uptake of compound 2. The
presence of glucose had no effect on the permeability of
compound 2, suggesting that the absorption did not utilize any
of the known glucose transporters (see Supporting Information,
Figure 1).
Previous work by our group showed that attachment of
glucose succinamic acid to the N-terminus of peptide 1,
reduced MOR binding affinity considerably.23 However, lactose
modification has been reported to promote the uptake and
activity of CNS peptides.24,25 To investigate this, we modified
the N-terminus of peptide 1 using lactose with a succinamic
acid spacer. The resulting compound 2 was studied for its in
vitro biological activity including plasma stability, membrane
permeability, MOR, δ-opioid receptor (DOR), and κ-opioid
receptor (KOR) binding affinity as well as MOR agonist
activity. Additionally, the in vivo analgesic activity of derivative
2 was assessed in a rat model of neuropathic pain. The
nonselective opioid receptor antagonist, naloxone hydro-
chloride was used to confirm the role of opioid receptors in
mediating pain relief. In vivo pharmacological characterization
of analogue 2 was also extended to evaluate its potency relative
to morphine to induce constipation in rats.
Receptor Binding and Functional Activity. Receptor-
binding scintillation proximity assay (SPA) was performed to
investigate the affinity of compounds 1 and 2 at MORs, DORs,
and KORs using membranes derived from stably transfected
CHO-K1 cells which express the corresponding receptors.
Confirmation of the agonist behavior of the peptide derivatives
at MORs was attained by measuring the accumulation of cyclic
adenosine monophosphate (cAMP) upon stimulation of SH-
SY5Y cells with forskolin. SH-SY5Y is a human neuroblastoma
cell line which endogenously expresses MORs and DORs.
Treatment with MOR agonists has an inhibitory effect on the
cAMP pathway, resulting in a reduction of intracellular cAMP
formation.30
Compound 2 exhibited a decrease in binding affinity for the
MORs compared with the parent peptide 1 at Kiμ 14.9 ( 0.65)
and 0.29 ( 0.019) nM, respectively (Figure 2, Table 1). Like
RESULTS
■
Compounds 1 and 2 (Figure 1) were synthesized via solid
phase peptide synthesis (see Supporting Information, Scheme
Figure 1. Structure of compounds 1 and 2.
1) using the Fmoc in situ neutralization protocol26 and purified
to a single peak (>95% purity) by analytical reverse phase high
performance liquid chromatography (RP-HPLC) and charac-
terized using electrospray ionization mass spectrometry (ESI-
MS). Purified yields of the compounds varied from 84% for
peptide 1 to 24% for analogue 2. In vitro assays were used to
demonstrate the stability, membrane permeability, and func-
tional activity of peptide 2. Incubation of compound 2 with
human plasma showed an increase in the stability. The half-life
of the parent peptide 1 was 2.7 min, while the half-life of its
Figure 2. Binding affinities for compounds 1, 2, and morphine
determined by competitive displacement of [3H]DAMGO. Parent
peptide 1 and morphine exhibited a higher binding affinity at MOR
compared with compound 2. Data presented are mean SEM from
three separate experiments, each performed in triplicate.
parent peptide 1, compound 2 exhibited weak receptor binding
affinities at DOR and KOR with Kiδ and Kiκ values above 1000
nM (Table 1). The binding affinity of morphine was also tested
at MOR, DOR, and KOR for comparison with Kiμ, Kiδ, and Kiκ
values 0.14 ( 0.032), 295 ( 19.8), and 13.3 ( 1.7) nM,
respectively.
Compounds 1 and 2 inhibited forskolin-stimulated cAMP
formation in a concentration-dependent manner, with the
carbohydrate derivative displaying a 9-fold lower potency than
analogue 2 was 57.3 min. The apparent permeability (Papp
)
values were evaluated using Caco-2 cell monolayers.
Propranolol was used as a positive control, with Papp of 1.5
( 0.03) × 10−4 cm/s, because it is recognized as being
completely absorbed from the gastrointestinal tract (GI).27
Peptide 1 displayed low permeability through Caco-2 cell
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Table 1. Summary of in Vitro Results
a
b
c
d
e
compd
plasma stability,t1/2 (min) Papp ( × 10−7, cm/s)
Kiμ (nM)
Kiδ (nM)
Kiκ (nM)
Kiδ/μ
Kiκ/μ
cAMP, IC50 (nM)
1
2
2.70 1.9
57.3 6.6*
ND
1.1 0.52
760.0 19.9***
ND
0.29 0.019
14.9 0.65
0.14 0.032
2765 176
4751 354
295 19.8
2048 424
1566 165
13.3 1.7
9534
319
7062
105
95
65.4 1.13
590.2 11.9
ND
morphine
2107
a
The apparent permeability (Papp, cm/s) through Caco-2 cell monolayers. The data were quantified by LC/MS and each point is expressed as mean
b
c
d
SD (n = 4). Kiμ versus [3H]DAMGO. Kiδ versus [3H]DPDPE. Kiκ versus [3H]diprenorphine. Binding affinity values were determined using
eight concentrations of each compound in the range 10 pM to 1 μM and performed in three independent experiments, each in triplicate. Data are
e
expressed as mean SEM. The IC50 values (nM) were estimated from concentration−response curves using nonlinear regression for inhibition of
forskolin-stimulated cAMP formation. *p < 0.05, ***p < 0.001 compared with compound 1 in the same experiment.
the parent peptide at MORs. IC50 values increased from 65.4
( 1.13) nM for compound 1 to 590.2 ( 11.9) nM for
compound 2 (Figure 3, Table 1).
Figure 4. Plasma concentration versus time curves for compounds 1
and 2 following oral administration of single doses at 10 μmol/kg. The
plasma concentrations were quantified by LC/MS, and each
concentration is expressed as mean SEM (n = 4).
of compound 2 was evaluated in CCI rats after oral
administration at the doses that produced pain relief following
Figure 3. Concentration−response curves for the inhibitory effects of
iv administration. Oral doses of compound 2 produced dose-
dependent relief of mechanical allodynia in the ipsilateral
(injured side) hindpaw of CCI rats. Compound 2 produced
significant pain relief at 8 and 16 μmol/kg with the peak effect
at 0.75−1 h postdosing and was active for up to 2 h (Figure
6a). Compound 2 at 8 μmol/kg was equi-effective, with
morphine at the same dose after oral administration (Figure
6b) with ED50 ( SEM) values of 19.6 ( 1.2) and 20.7 ( 3.6)
μmol/kg respectively.
Bolus iv and oral doses of compound 2 at 5 and 8 μmol/kg,
respectively, produced insignificant antinociception in the
contralateral (uninjured) hindpaw in contrast to morphine
which produced significant antinociception in the contralateral
hindpaw at corresponding doses (see Supporting Information,
Figure 2, p < 0.001).
Naloxone Hydrochloride Pretreatment. Pretreatment of
CCI rats with a single bolus subcutaneous (sc) dose of
naloxone at 10 mg/kg abolished the analgesic effects of
compound 2 at 5 μmol/kg iv and morphine at 2 μmol/kg iv
(Figure 7, p < 0.001).
Constipation-Inducing Properties. The constipating
potency of compound 2 relative to morphine was investigated
using the castor oil-induced diarrhea assay and the charcoal test
in order to assess the effect on stool hydration and GI motility,
respectively. Single oral doses of compound 2 up to 16 μmol/
kg (p > 0.05) did not significantly inhibit castor oil-induced
diarrhea or GI transit of a charcoal meal. The mean ( SEM)
GI propulsion (%) was 58.5 ( 2.2) % for rats treated with
compound 2, which was not significantly different (p > 0.05)
from that observed for vehicle-treated animals (66.5 3.8%).
By contrast, single oral doses of morphine at 8 μmol/kg
significantly inhibited castor oil-induced diarrhea (p < 0.001)
compounds 1 and 2 on forskolin-stimulated cAMP formation in SH-
SY5Y cells. Data presented are mean
SEM from three separate
experiments, each performed in triplicate. Nonlinear regression as
implemented in GraphPad Prism was used to estimate IC50 values.
Oral Absorption. To examine whether the high in vitro
permeability of compound 2 translated into oral absorption of
the peptide, 10 μmol/kg of compound 2 was administered to
rats via oral gavage and the plasma concentrations of the
peptide were measured at 30, 60, and 90 min postdosing. The
peak plasma concentration of 22 ( 5.1) nmol/mL was
observed at 60 min postdosing for compound 2 (Figure 4).
In contrast, the parent peptide 1 was not detected in plasma at
any time following oral administration of the same dose.
Antinociceptive Activity. To investigate the impact of
lactose conjugation on the pain-relieving effects of parent
peptide 1 following systemic administration, the CCI rat model
of neuropathic pain was used. Single doses of compounds 1 and
2 from 0.16 to 16 μmol/kg were administered intravenously
(iv) and orally (po) to CCI rats. Morphine was used at 2 (iv)
and 8 (po) μmol/kg p.o. Compound 2 produced significant
analgesic activity at 5 and 16 μmol/kg, with a rapid onset of
action at 15 min postdosing and a duration of action of 1.5 h
following iv bolus administration (Figure 5a,b). The ED50
( SEM) values for compound 2 and morphine were
determined at 8.3 ( 0.8) and 2.6 ( 1.4) μmol/kg, respectively,
after iv administration. Compound 1 did not produce
significant analgesia after iv administration even at the highest
dose tested (Figure 5a). The apparent membrane permeability
coefficient for analogue 2 (Table 1), as well as its presence in
plasma following oral administration (Figure 4), suggested its
possible oral absorption. On the basis of these data, the efficacy
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Figure 5. Intravenous dosing of compound 2 produced significant pain relief in the ipsilateral hindpaw of the CCI rat model of neuropathic pain. (a)
Normalized (Δ) paw withdrawal thresholds (PWT) versus time curves for single iv bolus doses of compounds 1 and 2, morphine, and vehicle in
CCI rats. (b) Extent and duration of analgesia (areas under the curve (AUC) versus time, ΔPWT AUC values) shows that single iv bolus doses of
compound 2 evoked significant analgesia at 5 and 16 μmol/kg, relative to vehicle. The ΔPWT AUC at 5 μmol/kg is comparable with that for
morphine at 2 μmol/kg. Each value represents mean SEM (n = 6). ***p < 0.001 compounds 1 and 2 vs vehicle treated CCI rats.
Figure 6. Dose-dependent analgesic activity was produced by single oral doses of compound 2 in the ipsilateral hindpaw of the CCI rat model of
neuropathic pain. (a) The time course for the antinociceptive effects of compound 2, morphine, and vehicle in CCI rats after oral gavage. (b) ΔPWT
AUC values showing significant relief of mechanical allodynia produced by oral gavage of compound 2 at 8 and 16 μmol/kg relative to the vehicle.
The effect at a dose of 8 μmol/kg is comparable with the effect of morphine at the equivalent dose. Each value represents mean SEM (n = 6−8).
**p < 0.01, ***p < 0.001 compounds 2 vs vehicle treated CCI rats.
(Figure 8a) and markedly decreased GI transit of a charcoal
meal (36.7 2.9%, p < 0.001) (Figure 8b).
degrading endomorphins.33 Endomorphins are also the
substrates of the proline-specific aminopeptidases, such as
aminopeptidase P (APP; EC 3.4.11.9) and aminopeptidase M
(APM, EC 3.4.11.2).34 Carboxypeptidase Y (CPY, EC 3.4.16.1)
can hydrolyze peptide amides such as endomorphins35 and
unlike many carboxypeptidases does not require a free C-
terminal carboxyl group.36
The MOR, DOR, and KOR binding affinities of the
synthesized peptides were determined by competitive displace-
ment assay using [3H]DAMGO, [3H]DPDPE, and [3H]-
diprenorphine to label MOR, DOR, and KOR, respectively. In
line with previous reports,4 peptide 1 showed a high affinity and
selectivity for MORs. While the lactose modification in
compound 2 led to a drop in receptor binding affinity and
agonist activity at MORs, it was still in the nanomolar range.
DISCUSSION
■
We showed that modification of peptide 1 with a lactose
residue in position 1 (2) resulted in significant pain relief in
CCI rats after both iv and oral administration.
The in vitro data demonstrated very low stability for peptide
1, which is consistent with literature reports.31,32 Conversely,
compound 2 was significantly more stable in human plasma
than the parent peptide 1. There are a group of enzymes in
plasma that can specifically hydrolyze peptide bonds.
Dipeptidyl peptidase IV (DPP IV, EC.3.4.14.5) is a
membrane-bound serine protease, which is the main enzyme
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values) and fractional absorption values (Fa) in humans. In this
comparison, it was shown that for compounds with high Fa
(%), the corresponding Papp values are in the range, 10−5−10−6
cm/s.38 On the basis of this classification, compound 2 is within
the range of highly permeable compounds across barriers like
the BBB. The Caco-2 cell model has been shown to be a
reliable model when studying CNS targeted compounds, for
either passive or active transport across the BBB.39,40
Previous studies demonstrated that glycosylation improved
the brain delivery and intestinal absorption of some other
peptides such as somatostatin,41 met-enkephalin,42 dermorphin,
and deltorphin analogues.16,43 These studies postulated that the
improved bioavailability of the glycosylated peptide analogues
stemmed from improved diffusion from peripheral sites into the
bloodstream due to increased water solubility and active
transport via glucose transporter 1 (GLUT-1), resulting in a
higher rate of influx of the compounds from the blood to the
brain.44
Figure 7. Pretreatment with the opioid receptor antagonist, naloxone
hydrochloride (10 mg/kg sc), abolished the antinociception produced
by a single iv bolus dose of compound 2 in the ipsilateral hindpaws of
opioid-naive CCI rats (5 μmol/kg, n = 6) in a manner similar to
morphine (2 μmol/kg, n = 6) ***p < 0.001, vs vehicle pretreated rats.
Following oral administration to CCI rats, compound 2
evoked significant analgesia. N-terminal conjugation of peptide
1 with lactose succinamic acid produced a derivative with
significantly improved oral activity and comparable potency
with morphine, for which various oral dosage forms are
currently in clinical use. The pain-relieving activity of
compound 2 in CCI rats was naloxone-sensitive, which is
consistent with previous reports on the key role of opioid
receptors in mediating the analgesic activity of peptide 1.14
Structure−activity studies of enkephalin-based opioid glyco-
peptides revealed that disaccharide derivatives were significantly
more potent than any of the monosaccharides after peripheral
administration.24 In line with these findings, conjugation of
peptide 1 with glucose residues resulted in over a 100-fold
decrease in MOR binding.23 The exact mechanism by which
glycosylation improves BBB or GI transport has yet to be
elucidated.42 However, it is believed that glycopeptides
promote negative membrane curvature on the surface of
endothelial cells, which may increase endocytosis and result in
enhanced transport through membranes via transcytosis.32,45,46
Conjugation of peptide 1 with a lactose residue caused
alterations in the excretion profile of the peptide and
subsequently led to a significant systemic exposure of
compound 2 after oral dosing in rats. Opioid peptides are
predominantly cleared from the body via fecal−oral routes.47,48
It has previously been shown that shifting from hepatic to renal
No increase in DOR and KOR binding affinities was observed
for analogue 2, demonstrating that it maintained selectivity for
MORs. Morphine produced profound affinity for MORs,
however, it showed a lack of selectivity for these receptors,
which was consistent with previous studies.30,37
Despite the decrease in binding affinity and agonist activity of
analogue 2 at the MORs, an unprecedented 700-fold increase in
membrane permeability relative to the native peptide was
observed. Lactose conjugation not only increased the stability
and permeability of the peptide in vitro but also enhanced
absorption across the GI mucosa in vivo. Hence, significant
systemic exposure of compound 2 was observed after oral
dosing in rats in contrast to the parent peptide 1. Lactose
significantly inhibited the membrane permeability of compound
2, while galactose and glucose showed low or no inhibitory
effect in the Caco-2 cell monolayer assay. These findings
suggested receptor-mediated or lactose-selective transporter-
mediated absorption of compound 2 as previously re-
ported.28,29 Furthermore, glycopeptides are believed to interact
reversibly with the membrane and subsequently promote
transport through brain capillaries by transcytosis.32 According
to the Biopharmaceutics Classification System, there is a high
correlation between Caco-2 cell permeability coefficients (Papp
Figure 8. Assessment of the constipation-inducing activity of compound 2 relative to morphine in drug-naive rats following oral administration. (a)
Lack of inhibitory effect of compound 2 in doses up to 16 μmol/kg on induction of diarrhea following oral administration of castor oil in contrast to
significant inhibitory effects of morphine at 8 μmol/kg. (b) Lack of effect of compound 2 (16 μmol/kg) and vehicle on the gastrointestinal transit of
a charcoal meal in contrast to the inhibitory effect of morphine (8 μmol/kg) in rats. Data are the mean SEM (n = 8). ***p < 0.001, vs vehicle.
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chemicals were purchased from Sigma Aldrich (VIC, Australia). All cell
culture reagents were purchased either from Sigma Aldrich or Gibco
(VIC, Australia).
clearance can increase plasma stability and antinociceptive
effects of opioid peptides.48 Because hydrophilicity of the
glycopeptides is increased compared with the parent peptides, it
is plausible that glycosylation shifts elimination to the renal
pathway and this may contribute to the higher stability of these
conjugates.
The pain-relieving effects of compound 2 were selective to
the injured hindpaw with insignificant effects produced in the
contralateral hindpaw. In contrast, morphine produces pain
relief affecting both the injured and uninjured hindpaws of CCI
rats. It is plausible that derivative 2 produces an antineuropathic
effect by a different mechanism to that of morphine. These
observations are consistent with a previous report which
showed selective antihyperalgesic responses in the inflamed
hindpaw following continuous central (intrathecal) admin-
istration of peptide 1 in carrageenan-induced inflammatory pain
in rats.49
Hydrogenation was performed using a H-Cube continuous-flow
hydrogenation reactor from ThalesNano Nanotechnology Inc.,
Hungary. Absorbance measurements were performed on a Varian
Cary 50 Bio UV/vis spectrophotometer (λ = 570 nm). Electrospray
ionization mass spectrometry (ESI-MS) was carried out on a PE Sciex
API3000 triple quadrupole mass spectrometer using a mixture of
solvent A (0.1% formic acid in water) and B (0.1% formic acid in 9:1
acetonitrile/water) at 0.05 mL/min. LC-MS was carried out using
solvents A and B at a flow rate of 0.3 mL/min, and a splitter after the
Phenomenex Luna C18 column (5 μm, 50 mm × 2.0 mm) was used to
achieve a flow rate of 0.05 mL/min into the ion source of the mass
spectrometer. Preparative HPLC was carried out on a Waters 600
controller and pump with a 490E programmable multiwavelength UV/
visible detector, and analytical HPLC was performed on an Agilent
1100 system fitted with a binary pump, autosampler, and a UV
detector set to a wavelength of 214 nm.
For new opioid analgesics to be considered for clinical
applications, it is essential that they have less opioid-related
adverse effects than morphine. A common side effect produced
by MOR agonists is constipation, comprising a reduction in
stool hydration as well as inhibition of GI motility, resulting in
delayed GI transit.50 Two methods were applied to evaluate the
constipation-inducing propensity of derivative 2. These
included assessment of inhibition of castor oil-induced diarrhea
and reduced gastrointestinal transit of a charcoal meal. No
significant constipation was observed in either assay following
oral administration of compound 2 at doses higher than those
required to fully reverse allodynia. In contrast, morphine
induced significant constipation in both tests at a dose equal to
the effective analgesic dose in CCI rats.
Chemistry. 2,3,6-Tetra-O-acetyl-4-O-(2′,3′,4′,6′-tetra-O-acetyl-
β-D-galactopyranosyl)-β-D-glucopyranosylbromide (3). A mixture
of β-D-lactose (15 mmol, 5.1 g), lithium perchlorate (15 mmol, 1.6 g),
and acetic anhydride (150 mmol, 14 mL) was stirred at 40 °C under
an argon atmosphere for 16 h. Then the reaction mixture was cooled
on an ice bath to 4 °C, and 32% hydrogen bromide in acetic acid (80
mmol, 14.3 mL) was added dropwise. Once the addition was
complete, the solution was stirred for 5 h and subsequently diluted
with 50 mL of dry dichloromethane (DCM). This was washed with
saturated bicarbonate (5 × 30 mL) and brine (1 × 30 mL), dried over
magnesium sulfate, and then filtered. Upon evaporation of the solvent,
the product (6.7 g, 64%) spontaneously crystallized and was used
without further purification for the next step.
2,3,6-Tetra-O-acetyl-4-O-(2′,3′,4′,6′-tetra-O-acetyl-β-D-galacto-
pyranosyl)-β-D-glucopyranosylazide (4). Compound 3 (9.6 mmol,
6.7 g) was dissolved in 40 mL of acetone and a solution of sodium
azide (11.5 mmol, 0.75 g) in 10 mL of water was added carefully. After
the mixture had been stirred at room temperature for 17 h, it was
concentrated under reduced pressure and the resulting white crystals
were washed with ice−water and dried under vacuum (6.2 g, 98%).
1H NMR (300 MHz, CDCl3): δ = 5.35 (dd, 1H, H-4′), 5.10 (m, 2H,
H-2′, H-3), 4.95 (m, 2H, H-3′, H-2), 4.64 (d, 1H, H-1), 4.48 (d, 1H,
H-1′), 4.11 (m, 3H, H-6a′, H-6b′, H-6a), 3.87 (m, 1H, H-5′), 3.80 (m,
2H, H-4, H-6b), 3.71 (m, 1H, H-5), 2.15 (s, 3H, OAc), 2.10 (s, 3H,
OAc), 2.08 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.03
(s, 3H, OAc), 2.01 (s, 3H, OAc). Melting point: 83−85 °C.
4-Oxo-4-[(2,3,6-tetra-O-acetyl-4-O-{2′,3′,4′,6′-tetra-O-acetyl-β-D-
galactopyranosyl}-β-D-glucopyranosyl)amino]butanoic Acid (5).
Compound 4 (0.4 mmol, 260 mg) was dissolved in 20 mL of dry
DCM and then pumped through an H-Cube continuous-flow
hydrogenation reactor, which was operating with the following
settings: T 35 °C, PH2 40 bar, flow rate 2 mL/min. The reaction
was complete after 1 h, and the solvent was evaporated and replaced
with fresh dry DCM for the next reaction step. Pyridine (1.5 mL) and
4-dimethylaminopyridine (0.04 mmol, 5 mg) were added, and the
solution was cooled in an ice bath. Succinic anhydride (0.8 mmol, 80
mg) was added in small portions, and the mixture was stirred for 24 h,
letting it warm to room temperature slowly. The solution was diluted
with DCM (20 mL) and washed with 5% hydrochloric acid (3 × 30
mL). The organic phase was dried over magnesium sulfate and filtered,
and the solvent was removed under reduced pressure. The residue was
purified using gradient flash column chromatography (2:1 ethyl
acetate/hexane to 100% ethyl acetate, 0.5% acetic acid) to yield a
white powder (165 mg, 56%).
CONCLUSION
■
We have successfully designed and developed an orally active
sugar-modified derivative of native peptide 1 with high
potential for the treatment of neuropathic pain. Analogue 2 is
the first reported orally active derivative of this opioid peptide.
Its modification with a lactose moiety facilitates GI uptake
possibly by providing a substrate to enhance transporter-
mediated uptake associated with oral delivery. Remarkably, this
compound exhibited strong antineuropathic activity without
producing constipation, a major side effect of existing opioid
analgesics. This is a highly novel finding in the opioid analgesic
field and will be further investigated to reveal the underlying
mechanisms. Our present findings suggest that analogue 2 can
be developed as a novel opioid analgesic agent to treat
neuropathic pain.
EXPERIMENTAL SECTION
■
General. Peptide synthesis grade dimethylformamide (DMF),
trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and piperidine
were purchased from Merck Biosciences (Kilsyth, VIC, Australia).
HPLC-grade acetonitrile was purchased from RCI Labscan Ltd.
(Bangkok, Thailand). Fmoc-protected amino acids and Rink amide
MBHA resin (100−200 mesh, 0.4−0.8 mmol/g loading) were
obtained from Novabiochem (Melbourne, VIC, Australia) or
Mimotopes (Clayton, VIC, Australia). Frozen membranes providing
human MOR, KOR, and DOR derived from stably transfected CHO-
K1 cells (1 × 400 units/μL), wheatgerm agglutinin (WGA) coated
poly vinyl toluene SPA beads, [3H]DAMGO (49.6 Ci/mmol,
[Tyrosyl-3,5-3H(N)]−), [3H]DPDPE (25.2 Ci/mmol, [Tyrosyl-
2,6-3H(N)]−), [3H]diprenorphine (50 Ci/mmol, [15,16-3H]−),
384-well OptiPlates and ProxiPlates, and clear adhesive Topseal-A
were purchased from Perkin-Elmer (Massachusetts, USA). All other
1H NMR (300 MHz, CDCl3): δ = 6.44 (d, 1H, JNH,1 = 9.3 Hz, NH),
5.35 (dd, 1H, J4′,5′ = 1.0 Hz, J3′,4′ = 3.5 Hz, H-4′), 5.29 (dd, 1H, J3,4
=
8.9 Hz, J2,3 = 9.6 Hz, H-3), 5.22 (dd, 1H, JNH,1 = 9.3 Hz, J1,2 = 9.3 Hz,
H-1), 5.10 (dd, 1H, J1′,2′ = 7.9 Hz, J2′,3′ = 10.4 Hz, H-2′), 4.95 (dd, 1H,
J3′,4′ = 3.5 Hz, J2′,3′ = 10.4 Hz, H-3′), 4.84 (dd, 1H, J1,2 = 9.6 Hz, J2,3
=
9.6 Hz, H-2), 4.47 (d, 1H, J1′,2′ = 7.9 Hz, H-1′), 4.43 (m, 1H, H-6a),
4.14 (m, 2H, H-6b,H-6a′), 4.07 (m, 1H, H-6b′), 3.87 (m, 1H, H-5′),
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3.76 (dd, 1H, J3,4 = 8.8 Hz, J4,5 = 8.8 Hz, H-4), 3.73 (m, 1H, H-5), 2.72
(m, 1H, CH2CO), 2.63 (m, 1H, CH2CO), 2.47 (m, 2H, CH2CO),
2.15 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.05 (s, 3H,
OAc), 2.05 (s, 3H, OAc), 2.04 (s, 3H, OAc), 1.96 (s, 3H, OAc).
Melting point: 97−98 °C.
surgery with regard to posture of the affected hindpaw, exploring
behavior, body weight, and signs of autotomy.
Assessment of Von Frey Paw Withdrawal Thresholds. Paw
withdrawal thresholds (PWTs) were assessed in the rat hindpaws
using a set of calibrated Von Frey filaments. For each testing session,
rats were acclimatized for at least 15 min in wire mesh metabolic cages.
Von Frey filaments which delivered forces in the range 2−20 at 2 g
increments were used to quantify the threshold for hindpaw
withdrawal in response to application of a graded non-noxious
stimulus for each hindpaw. Commencing with 6 g, the Von Frey
filament was applied to the plantar surface of the footpad of the
hindpaw so that the filament bent slightly for 3 s. Lifting the hindpaw
or avoidance of the filament was considered a positive response. If the
rat responded, the next filament in a descending sequence was tested.
If no response was observed, the next filament in an ascending
sequence was applied. Mechanical allodynia was fully developed in the
ipsilateral (injured side) hindpaw by 14 days post-CCI surgery (PWTs
≤ 6 g). Rats not responding to any of the filaments were arbitrarily
assigned a PWT value of 20 g.
Evaluation of Oral Absorption. Male Sprague−Dawley rats,
weighing 280−310 g, were gavaged with 500 μL of a 20 mM solution
of compound 1 or 2 in water. Blood samples were taken from the tail
vein after 30, 60, and 90 min and collected in heparin-coated tubes.
The blood was centrifuged to remove red blood cells, and 100 μL of
plasma was added to 300 μL of acetonitrile to precipitate proteins. The
suspension was centrifuged a second time, and the supernatant was
analyzed by LC-MS as described in the analysis of the Caco-2
monolayer permeability assay analysis. Ethics approval was obtained
from The University of Queensland Animal Ethics Committee for
these experiments.
Solid-Phase Peptide Synthesis. The peptide was assembled on
Rink amide MBHA resin using HBTU (4 equiv)/DIPEA (5 equiv)
activation and the in situ neutralization protocol for Fmoc chemistry.51
The following protected amino acids were used and double coupled:
Fmoc-Phe, Fmoc-Trp(Boc), Fmoc-Pro, and Fmoc-Tyr(tBu). Com-
pound 5 (1.3 equiv) was activated with HBTU (1.25 equiv)/DIPEA
(2 equiv) and coupled overnight. The resin was then washed with
DMF, DCM, and MeOH and treated with 75% hydrazine hydrate in
MeOH twice for 30 min. After washing with MeOH, the resin was
dried under vacuum overnight. Cleavage of the peptide was achieved
by treatment of the resin with TFA/triisopropylsilane/water
(95:2.5:2.5) for 2 h. The solvent was removed under a stream of
air, the crude peptide precipitated by addition of cold diethyl ether and
then dissolved in acetonitrile/water/TFA (50:50:0.1) and lyophilized.
Purification. The crude peptide was purified by preparative RP-
HPLC using a Vydac C18 column (22 mm × 250 mm) with a gradient
of 20% to 60% solvent B (solvent A, 0.1% TFA in water; solvent B,
90% acetonitrile and 0.1% TFA in water) over 70 min at a flow rate of
10 mL/min. The purity of peptides in the collected fractions was
confirmed by ESI-MS and analytical RP-HPLC using a Vydac C18
column (5 μm, 4.6 mm × 250 mm) and a gradient of 100% A to 100%
B over 30 min at a flow rate of 1 mL/min. Fractions containing pure
peptides were combined and lyophilized.
Compound 1 Yield: 108 mg, 84%. Exact mass calculated, 611.2976;
found, 611.2987 [M + H+].
Naloxone-Sensitivity of Analgesia. Groups of CCI rats (n = 6)
were pretreated with single subcutaneous doses of either vehicle or
naloxone hydrochloride (10 mg/kg) at 5 min prior to iv administration
of single bolus doses of compound 2 (5 μmol/kg), morphine (2
μmol/kg), or vehicle. PWTs were determined just prior to vehicle or
naloxone administration and at the following times after test item
administration: 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, and 3 h.
Compound 2 Yield: 452 mg, 24%. Exact mass calculated,
1056.4173, found, 1056.4152 [M + Na+].
In Vitro Experiments. Cell Culture. The Caco-2 human epithelial
colorectal adenocarcinoma cell line was obtained from the American
Type Culture Collection (Rockville, USA). Caco-2 cells were
maintained in T-75 flasks (Nunc, Australia) with Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 1% L-glutamine, and 1% nonessential amino acids at 95%
humidity at 37 °C in an atmosphere of 5% CO2. The culture medium
was changed every second day. When the cells reached 80%
confluence, they were subcloned using 0.25% trypsin−EDTA. Passage
numbers 55−75 were used in the assays. SH-SY5Y cells were
maintained in DMEM:Hams F12 medium supplemented with 10%
FBS, 1% glutamine, 1% nonessential amino acids, and 1000 U/mL
streptomycin/penicillin. Cells were subcloned in the same manner as
Caco-2 cells. Passage numbers 10−20 were used in the assays.
In Vivo Experiments. Animals. Male specific pathogen free (SPF)
Sprague−Dawley (SD) rats weighing between 160 and 200 g were
purchased from The University of Queensland Biological Resources
breeding facility and kept in groups of three for at least a 3 day
acclimatization period prior to initiation of experimental procedures.
Rats were housed in an artificially lit room on a 12 h light/12 h dark
cycle at controlled temperature (22.2 0.2 °C; mean SEM) and
humidity (51.6−65.2%) with food and water available ad libitum. The
animal experimentation protocols were approved by The University of
Queensland Animal Ethics Committee (AEC no. CIPDD/366/09/
NHMRC).
Surgery Procedure. Chronic constriction injury (CCI) of the sciatic
nerve was performed according to the method of Bennett and Xie52 in
adult male SD rats. Briefly, while rats were deeply anaesthetized with
3% isoflurane delivered in oxygen, the left common sciatic nerve was
exposed at midthigh level by blunt dissection through the biceps
femoris. Proximal to the trifurcation, ≈10 mm of nerve was freed of
adhering tissue, four loose ligatures (3.0 silk) were tied around the
sciatic nerve (≈1 mm apart), and the incision was closed in layers.
After surgery, topical antibiotic powder (benzylpenicillin) was applied
to the wound to prevent infection and rats were kept warm during
surgical recovery. Rats were housed individually and inspected from
the time of CCI surgery until experimentation on day 14 post-CCI
Assessment of Constipation Inducing Properties. Castor Oil-
Induced Diarrhea Test in Rats. The constipating capacity of 2 was
compared to that of morphine using the castor oil-induced diarrhea
method. Drug-naive rats weighing 200−250 g were fasted for
approximately 16 h before the start of experimentation but were
permitted free access to water. Rats were then placed into individual
wire-mesh cages without access to food or water for a 1 h
acclimatization period prior to drug or vehicle administration.
At completion of the acclimatization period, each rat received
compound 2 (16 μmol/kg), morphine (8 μmol/kg), or vehicle via oral
administration. Five minutes after test item or vehicle administration, 1
mL of castor oil was administered by oral gavage. Rats were observed
hourly up to 8 h after the administration of castor oil. At each
observation time, the presence or absence of diarrhea was recorded. A
scoring system was used based on the weight of feces boli and the
feces consistency. Weight was scored from 0 (no feces) to 3 (>3 g of
feces), and consistency was scored from 0 (normal-shape feces) to 3
(shapeless feces with large amounts of liquid). Diarrhea was
considered to be present when both weight of feces and the feces
consistency were scored at ≥2.
Charcoal Test. GI transit was assessed using the charcoal meal test
in groups of drug-naive rats. Animals (200−250 g) were fasted for
approximately 16 h before experimentation but had free access to
water. Groups of rats (n = 8 per group) received oral doses of
compound 2 at 16 μmol/kg, morphine at 8 μmol/kg, and vehicle just
prior to oral gavage of an aqueous activated charcoal suspension (1 mL
of 10% activated charcoal and 5% gum Arabic). One hour later, rats
were euthanized with intraperitoneal (ip) sodium pentobarbital (65
mg per rat) followed by cervical dislocation. The intestine was
carefully removed and separated from the omentum while avoiding
stretching. The total length of the small intestine was measured from
the pylorus to the ileocecal junction as well as the most distal point of
migration of the charcoal meal. GI propulsion was calculated as the
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percentage of the distance traveled by the charcoal meal relative to the
total length of the small intestine. GI propulsion <55% indicated the
inhibition of bowel propulsion.
Data Analysis. For in vivo studies, the PWT versus time curve data
were normalized by subtracting predosing baseline PWT values from
the PWT of each time point for each individual rat to obtain ΔPWT
values as follows:
brain barrier; Boc, tert-butoxycarbonyl; cAMP, 3',5'-cyclic
adenosine monophosphate; DCM, dichloromethane; DMF,
dimethylformamide; ED50, dose effective in 50% of test
subjects; EDTA, ethylenediaminetetraacetic acid; ESI, electro-
spray ionization; Fmoc, 9-fluorenylmethoxycarbonyl; GI,
gastrointestinal; GLUT-1, glucose transporter 1; HPLC, high-
performance liquid chromatography, high-pressure liquid
chromatography; IC50, half-maximum inhibitory concentration;
iv, intravenous; Ki, , inhibition constant; LC-MS, liquid
chromatography-mass spectrometry, ; m, multiplet (spectral),
meter(s), milli, isotopic mass, magnetic quantum number (ESR
normalized (Δ) PWT value
= postdosing PWT−predosing baseline PWT
ΔPWT versus time curves were then constructed. The extent and
duration of analgesia (area under the curves (AUC) versus time for
ΔPWT; ΔPWT AUC values) was estimated using trapezoidal
integration for individual CCI rats. Mean ( SEM) ΔPWT AUC
versus log dose curves were constructed and the AUC values were
normalized relative to the maximum possible ΔPWT AUC value to
give % maximum possible effect (% MPE). Nonlinear regression as
implemented in the GraphPad Prism (v5.0) software program
(GraphPad Software) was used to estimate the ∼ED50 values.
Statistical Analysis. During all animal experiments, the inves-
tigator was blinded to the type and dose of the administered
compounds. All data are expressed as mean ( SEM). One-way
ANOVA and Dunnett’s post hoc test were used to compare stability
and permeability results, normalized ΔPWT AUC between the
ipsilateral and contralateral hindpaws, in addition to inhibition of GI
propulsion and castor-oil induced diarrhea produced by compound 2
and morphine relative to the vehicle. All comparisons between two
treatment groups, including comparisons between ΔPWT AUC values
of naloxone and vehicle pretreated CCI rats and the ED50 value for
compound 2, were performed using the nonparametric Mann−
Whitney test as implemented in the GraphPad Prism (v5.0) software
package. The statistical significance criterion was P < 0.05.
and NMR spectroscopy); sc, subcutaneous; t-Bu, tert-butyl; t1/2
,
half-time
REFERENCES
■
(1) Truini, A.; Cruccu, G. Pathophysiological mechanisms of
neuropathic pain. Neurol. Sci. 2006, 27, S179−S182.
(2) Dworkin, R. H.; O’Connor, A. B.; Backonja, M.; Farrar, J. T.;
Finnerup, N. B.; Jensen, T. S.; Kalso, E. A.; Loeser, J. D.; Miaskowski,
C.; Nurmikko, T. J.; Portenoy, R. K.; Rice, A. S. C.; Stacey, B. R.;
Treede, R.-D.; Turk, D. C.; Wallace, M. S. Pharmacologic management
of neuropathic pain: evidence-based recommendations. Pain 2007,
132, 237−251.
(3) Gentilucci, L. New trends in the development of opioid peptide
analogues as advanced remedies for pain relief. Curr. Top. Med. Chem.
2004, 4, 19−38.
(4) Zadina, J. E.; Hackler, L.; Ge, L.-J.; Kastin, A. J. A potent and
selective endogenous agonist for the μ-opiate receptor. Nature 1997,
386, 499−502.
(5) Horvath, G. Endomorphin-1 and endomorphin-2: pharmacology
of the selective endogenous μ-opioid receptor agonists. Pharmacol.
Ther. 2000, 88, 437−463.
(6) Przewlocki, R.; Przewlocka, B. Opioids in chronic pain. Eur. J.
Pharmacol. 2001, 429, 79−91.
(7) Czapla, M. A.; Gozal, D.; Alea, O. A.; Beckerman, R. C.; Zadina, J.
E. Differential cardiorespiratory effects of endomorphin 1, endomor-
phin 2, DAMGO, and morphine. Am. J. Respir. Crit. Care Med. 2000,
162, 994−999.
(8) Sugimoto-Watanabe, A.; Kubota, K.; Fujibayashi, K.; Saito, K.
Antinociceptive effect and enzymatic degradation of endomorphin-1 in
newborn rat spinal cord. Jpn. J. Pharmacol. 1999, 81, 264−270.
(9) Tomboly, C.; Peter, A.; Toth, G. In vitro quantitative study of the
degradation of endomorphins. Peptides 2002, 23, 1573−1580.
(10) Allt, G.; Lawrenson, J. G. The blood−nerve barrier: enzymes,
transporters and receptorsa comparison with the blood−brain
barrier. Brain Res. Bull. 2000, 52, 1−12.
(11) Egleton, R. D.; Abbruscato, T. J.; Thomas, S. A.; Davis, T. P.
Transport of opioid peptides into the central nervous system. J. Pharm.
Sci. 1998, 87, 1433−1439.
(12) Perlikowska, R.; Gach, K.; Fichna, J.; Toth, G.; Walkowiak, B.;
do-Rego, J. C.; Janecka, A. Biological activity of endomorphin and
[Dmt1] endomorphin analogs with six-membered proline surrogates in
position 2. Bioorg. Med. Chem. 2009, 17, 3789−3794.
ASSOCIATED CONTENT
* Supporting Information
■
S
Scheme for synthesis of compounds 1 and 2 and additional
figures. Full experimental details of plasma stability assay, Caco-
2 cell permeability assay, receptor-binding scintillation prox-
imity assay (SPA), inhibition of cAMP accumulation, and
description of the test and control item administration for in
vivo pharmacology experiments. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: (+61) 7 3346 9892. Fax: (+61) 7 3365 4273. E-mail: i.
toth@uq.edu.au. Address: School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane, QLD
4072, Australia.
Notes
The authors declare no competing financial interest.
(13) Soignier, R. D.; Vaccarino, A. L.; Brennan, A. M.; Kastin, A. J.;
Zadina, J. E. Analgesic effects of endomorphin-1 and endomorphin-2
in the formalin test in mice. Life Sci. 2000, 67, 907−912.
(14) Goldberg, I. E.; Rossi, G. C.; Letchworth, S. R.; Mathis, J. P.;
Ryan-Moro, J.; Leventhal, L.; Su, W.; Emmel, D.; Bolan, E. A.;
Pasternak, G. W. Pharmacological characterization of endomorphin-1
and endomorphin-2 in mouse brain. J. Pharmacol. Exp. Ther. 1998,
286, 1007−1013.
ACKNOWLEDGMENTS
■
This work was supported by infrastructure purchased with
funds from Pegah Varamini’s International Postgraduate
Research Scholarship (IPRS), the Queensland Government
Smart State Research Facilities Fund, and by research funds
from the National Health and Medical Research Council
Development Grant 569855. We thank Julie Bergeon for her
assistance in the synthesis of lactose succinamic acid.
(15) Soltero, R., Oral Protein and Peptide Drug Delivery; John Wiley &
Sons, Inc.: New York, 2005; pp 189−200.
(16) Ogawa, T.; Araki, M.; Miyamae, T.; Okayama, T.; Hagiwara, M.;
Sakurada, S.; Morikawa, T. Synthesis and antinociceptive activity of
orally active opioid peptides: improvement of oral bioavailability by
esterification. Chem. Pharm. Bull. (Tokyo) 2003, 51, 759−771.
ABBREVIATIONS USED
δ, chemical shift in parts per million downfield from
tetramethylsilane; AUC, area under the curve; BBB, blood−
■
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Article
(17) Ogawa, T.; Miyamae, T.; Okayama, T.; Hagiwara, M.; Sakurada,
S.; Morikawa, T. Structure−activity relationships (SAR) of [D-
Arg2]dermorphin(1−4) analogues, N(alpha)-amidino-Tyr-D-Arg-Phe-
X. Chem. Pharm. Bull. (Tokyo) 2002, 50, 771−780.
(18) Clark, R. J.; Jensen, J.; Nevin, S. T.; Callaghan, B. P.; Adams, D.
J.; Craik, D. J. The engineering of an orally active conotoxin for the
treatment of neuropathic pain. Angew. Chem., Int. Ed. Engl. 2010, 49,
6545−6548.
(19) Muthu, D.; Polt, R. New Prospects for Glycopeptide Based
Analgesia: Glycoside-Induced Penetration of the Blood−Brain Barrier.
Curr. Drug Delivery 2005, 2, 59−73.
(20) Polt, R.; Palian, M. M. Glycopeptide analgesics. Drugs Future
2001, 26, 561−576.
(21) Simerska, P.; Moyle, P. M.; Toth, I. Modern lipid-,
carbohydrate-, and peptide-based delivery systems for peptide, vaccine,
and gene products. Med. Res. Rev. 2009, 31, 520−547.
(22) Toth, I.; Malkinson, J. P.; Flinn, N. S.; Drouillat, B.; Horvath, A.;
Erchegyi, J.; Idei, M.; Venetianer, A.; Artursson, P.; Lazorova, L.;
Szende, B.; Keri, G. Novel lipoamino acid- and liposaccharide-based
system for peptide delivery: application for oral administration of
tumor-selective somatostatin analogues. J. Med. Chem. 1999, 42,
4010−4013.
(23) Koda, Y.; Del Borgo, M.; Wessling, S. T.; Lazarus, L. H.; Okada,
Y.; Toth, I.; Blanchfield, J. T. Synthesis and in vitro evaluation of a
library of modified endomorphin 1 peptides. Bioorg. Med. Chem. 2008,
16, 6286−6296.
(24) Elmagbari, N. O.; Egleton, R. D.; Palian, M. M.; Lowery, J. J.;
Schmid, W. R.; Davis, P.; Navratilova, E.; Dhanasekaran, M.; Keyari, C.
M.; Yamamura, H. I.; Porreca, F.; Hruby, V. J.; Polt, R.; Bilsky, E. J.
Antinociceptive structure−activity studies with enkephalin-based
opioid glycopeptides. J. Pharmacol. Exp. Ther. 2004, 311, 290−297.
(25) Do Carmo, G. P.; Polt, R.; Bilsky, E. J.; Rice, K. C.; Negus, S. S.
Behavioral pharmacology of the mu/delta opioid glycopeptide
MMP2200 in rhesus monkeys. J. Pharmacol. Exp. Ther. 2008, 326,
939−948.
(26) Alewood, P.; Alewood, D.; Miranda, L.; Love, S.; Meutermans,
W.; Wilson, D., Rapid in situ neutralization protocols for Boc and
Fmoc solid-phase chemistries. In Methods in Enzymology; Gregg, B. F.,
Ed.; Academic Press: New York, 1997; Vol. 289, pp 14−29.
(27) Johnsson, G.; Regardh, C. G. Clinical pharmacokinetics of beta-
adrenoreceptor blocking drugs. Clin. Pharmacokinet. 1976, 1, 233−263.
(28) Klink, D.; Yu, Q. C.; Glick, M. C.; Scanlin, T. Lactosylated poly-
L-lysine targets a potential lactose receptor in cystic fibrosis and non-
cystic fibrosis airway epithelial cells. Mol. Ther. 2003, 7, 73−80.
(29) Kato, Y.; Onishi, H.; Machida, Y. Biological characteristics of
lactosaminated N-succinyl-chitosan as a liver-specific drug carrier in
mice. J. Controlled Release 2001, 70, 295−307.
(30) Harrison, C.; McNulty, S.; Smart, D.; Rowbotham, D. J.;
Grandy, D. K.; Devi, L. A.; Lambert, D. G. The effects of
endomorphin-1 and endomorphin-2 in CHO cells expressing
recombinant μ-opioid receptors and SH-SY5Y cells. Br. J. Pharmacol.
1999, 128, 472−478.
(31) Liu, H. M.; Zhang, B. Z.; Liu, X. F.; Wang, C. L.; Ni, J. M.;
Wang, R. Endomorphin-1 analogs with enhanced metabolic stability
and systemic analgesic activity: Design, synthesis, and pharmacological
characterization. Bioorg. Med. Chem. 2007, 15, 1694−1702.
(32) Egleton, R. D.; Mitchell, S. A.; Huber, J. D.; Palian, M. M.; Polt,
R.; Davis, T. P. Improved blood−brain barrier penetration and
enhanced analgesia of an opioid peptide by glycosylation. J. Pharmacol.
Exp. Ther. 2001, 299, 967−972.
(33) Janecka, A.; Staniszewska, R.; Gach, K.; Fichna, J. Enzymatic
degradation of endomorphins. Peptides 2008, 29, 2066−2073.
(34) Harbeck, H. T.; Mentlein, R. Aminopeptidase P from rat brain.
Purification and action on bioactive peptides. Eur. J. Biochem. 1991,
198, 451−8.
(36) Berne, P. F.; Blanquet, S.; Schmitter, J. M. Carboxypeptidase Y-
catalyzed transpeptidation of esterified oligo- and polypeptides and its
use for the specific carboxyl-terminal labeling of proteins. J. Am. Chem.
Soc. 1992, 114, 2603−2610.
(37) Mignat, C.; Wille, U.; Ziegler, A. Affinity profiles of morphine,
codeine, dihydrocodeine and their glucuronides at opioid receptor
subtypes. Life Sci. 1995, 56, 793−799.
(38) Smetanova, L.; Stetinova, V.; Svoboda, Z.; Kvetina, J. Caco-2
cells, biopharmaceutics classification system (BCS) and biowaiver.
Acta Med. (Hradec Kralove, Czech Republic) 2011, 54, 3−8.
(39) Lundquist, S.; Renftel, M.; Brillault, J.; Fenart, L.; Cecchelli, R.;
Dehouck, M. P. Prediction of drug transport through the blood−brain
barrier in vivo: a comparison between two in vitro cell models. Pharm.
Res. 2002, 19, 976−981.
(40) Garberg, P.; Ball, M.; Borg, N.; Cecchelli, R.; Fenart, L.; Hurst,
R. D.; Lindmark, T.; Mabondzo, A.; Nilsson, J. E.; Raub, T. J.;
Stanimirovic, D.; Terasaki, T.; Oberg, J. O.; Osterberg, T. In vitro
models for the blood−brain barrier. Toxicol. In Vitro 2005, 19, 299−
334.
(41) Albert, R.; Marbach, P.; Bauer, W.; Briner, U.; Fricker, G.;
Bruns, C.; Pless, J. SDZ CO 611: a highly potent glycated analog of
somatostatin with improved oral activity. Life Sci. 1993, 53, 517−525.
(42) Egleton, R. D.; Mitchell, S. A.; Huber, J. D.; Janders, J.;
Stropova, D.; Polt, R.; Yamamura, H. I.; Hruby, V. J.; Davis, T. P.
Improved bioavailability to the brain of glycosylated Met-enkephalin
analogs. Brain Res. 2000, 881, 37−46.
(43) Negri, L.; Lattanzi, R.; Tabacco, F.; Orru, L.; Severini, C.;
Scolaro, B.; Rocchi, R. Dermorphin and Deltorphin Glycosylated
Analogues: Synthesis and Antinociceptive Activity after Systemic
Administration. J. Med. Chem. 1999, 42, 400−404.
(44) Polt, R.; Porreca, F.; Szabo, L. Z.; Bilsky, E. J.; Davis, P.;
Abbruscato, T. J.; Davis, T. P.; Harvath, R.; Yamamura, H. I.; Hruby,
V. J. Glycopeptide enkephalin analogues produce analgesia in mice:
evidence for penetration of the blood−brain barrier. Proc. Natl. Acad.
Sci. U.S.A. 1994, 91, 7114−7118.
(45) Broadwell, R. D.; Balin, B. J.; Salcman, M. Transcytotic pathway
for blood-borne protein through the blood−brain barrier. Proc. Natl.
Acad. Sci. U.S.A. 1988, 85, 632−636.
(46) Polt, R.; Dhanasekaran, M.; Keyari, C. M. Glycosylated
neuropeptides: A new vista for neuropsychopharmacology? Med. Res.
Rev. 2005, 25, 557−585.
(47) Weber, S. J.; Greene, D. L.; Hruby, V. J.; Yamamura, H. I.;
Porreca, F.; Davis, T. P. Whole body and brain distribution of
[3H]cyclic [D-Pen2, D-Pen5] enkephalin after intraperitoneal, intra-
venous, oral and subcutaneous administration. J. Pharmacol. Exp. Ther.
1992, 263, 1308−1316.
(48) Witt, K. A.; Huber, J. D.; Egleton, R. D.; Roberts, M. J.; Bentley,
M. D.; Guo, L.; Wei, H.; Yamamura, H. I.; Davis, T. P.
Pharmacodynamic and pharmacokinetic characterization of poly-
(ethylene glycol) conjugation to met-enkephalin analog [D-Pen2, D-
Pen5]-enkephalin (DPDPE). J. Pharmacol. Exp. Ther. 2001, 298, 848−
856.
(49) Emese, C.; Gabriella, J.; Geza, T.; Ildiko, D.; Gyorgy, B.;
Gyongyi, H. Antinociceptive effect of continuous intrathecal
administration of endomorphin-1. Pain 2001, 94, 31−38.
(50) Holzer, P. Opioid receptors in the gastrointestinal tract. Regul.
Pept. 2009, 155, 11−17.
(51) Alewood, P.; Alewood, D.; Miranda, L.; Love, S.; Meutermans,
W.; Wilson, D. Rapid in situ neutralization protocols for Boc and
Fmoc solid-phase chemistries. In Methods in Enzymology; Gregg, B. F.,
Ed.; Academic Press: New York, 1997; Vol. 289, 14−29
(52) Bennett, G. J.; Chung, J. M.; Honore, M.; Seltzer, Z., Models of
neuropathic pain in the rat. Curr. Protoc. Neurosci. 2003, Unit 9.14.
(35) Peter, A.; Toth, G.; Tomboly, C.; Laus, G.; Tourwe, D. Liquid
chromatographic study of the enzymatic degradation of endomor-
phins, with identification by electrospray ionization mass spectrometry.
J. Chromatogr., A 1999, 846, 39−48.
5867
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