Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8-13) fragment as a potential first-in-class analgesic

Article abstract of DOI:10.1021/jm100092s

The neurotensin hexapapetide fragment NT(8-13) is a potent analgesic when administered directly to the central nervous system but does not cross the blood-brain barrier. A total of 43 novel derivatives of NT(8-13) were evaluated, with one, ABS212 (1), being most active in four rat models of pain when administered peripherally. Compound 1 binds to human neurotensin receptors 1 and 2 with IC₅₀ of 10.6 and 54.2 nM, respectively, and tolerance to the compound in a rat pain model did not develop after 12 days of daily administration. When it was administered peripherally, serum levels and neurotensin receptor binding potency of 1 peaked within 5 min and returned to baseline within 90-120 min; however, analgesic activity remained near maximum for >240 min. This could be due to its metabolism into an active fragment; however, all 4- and 5-mer hydrolysis products were inactive. This pharmacokinetic/pharmacodynamic dichotomy is discussed. Compound 1 is a candidate for development as a first-in-class analgesic.

Full text of DOI:10.1021/jm100092s

J. Med. Chem. 2010, 53, 4623–4632 4623
DOI: 10.1021/jm100092s
Identification and Functional Characterization of a Stable, Centrally Active Derivative of the
Neurotensin (8-13) Fragment as a Potential First-in-Class Analgesic
Francis M. Hughes Jr.,†,‡ Brooke E. Shaner,† Lisa A. May,† Lyndsay Zotian,† Justin O. Brower,‡ R. Jeremy Woods,‡
Michael Cash,‡ Dustin Morrow,† Fabienne Massa,§ Jean Mazella,§ and Thomas A. Dix*,†,‡
†Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, Medical University of South Carolina Campus,
280 Calhoun Street, P.O. Box 250140, Charleston, South Carolina 29425-2303, ‡Argolyn Bioscience, Inc., 2530 Meridian Parkway, Suite 200,
Durham, North Carolina 27713, and §Institut de Parmacologie Moleculaire and Cellulaire, UMR 6097, Universiteꢀ de Nice-Sophia Antipolis,
Centre National de la Recherche Scientifique, 660 Route des Lucioles, 06560, Valbonne, France
Received January 21, 2010
The neurotensin hexapapetide fragment NT(8-13) is a potent analgesic when administered directly to
the central nervous system but does not cross the blood-brain barrier. A total of 43 novel derivatives of
NT(8-13) were evaluated, with one, ABS212 (1), being most active in four rat models of pain when
administered peripherally. Compound 1 binds to human neurotensin receptors 1 and 2 with IC50 of 10.6
and 54.2 nM, respectively, and tolerance to the compound in a rat pain model did not develop after 12
days of daily administration. When it was administered peripherally, serum levels and neurotensin
receptor binding potency of 1 peaked within 5 min and returned to baseline within 90-120 min;
however, analgesic activity remained near maximum for >240 min. This could be due to its metabolism
into an active fragment; however, all 4- and 5-mer hydrolysis products were inactive. This pharmaco-
kinetic/pharmacodynamic dichotomy is discussed. Compound 1 is a candidate for development as a
first-in-class analgesic.
Introduction
BBB would have great potential for development as pharma-
ceutical agents.
Neurotensin (NTa) is a tridecapeptide first isolated from
bovine hypothalamus over 30 years ago.1 The hexapeptide
C-terminal fragment NT(8-13) [Arg8-Arg9-Pro10-Tyr11-
Ile12-Leu13] was shown to retain the full activity of the parent
compound,2 defining it as the active component of the mole-
cule and the logical lead for development of NT-based
therapeutics. Although its effects are not limited to the
nervous system,3,4 NT is known to act as a neurotransmit-
ter/neuromodulator where it induces several important phy-
siological effects in mammals including analgesia5 and
hypothermia.6 It also has been shown to have potential as
an antipsychotic compound based on its blockade of amphe-
tamine-induced locomotor hyperactivity.7,8 NT exerts its
effects through binding of two brain receptors, NTR-1 (also
referred to as NTS-1) and NTR-2 (NTS-2), although the
relative roles of each receptor in the physiological responses
are not well-understood.9-11 Numerous experiments with
both NT and NT(8-13) suggest that all central activities
attributable to NT and derivatives only manifest following
direct application into the CNS12 while peripheral adminis-
tration is ineffectual. This probably is a result of the com-
pounds’ polarity and short half-lives in plasma.13 Hence, NT
derivatives that could be delivered peripherally and cross the
Two previously described NT(8-13) analogues (NT1 and
NT69L) displayed significant analgesic and antipsychotic
effects when injected ip.14,15 However, various aspects limited
their usefulness as a drug including the induction of significant
tolerance after a single injection.16 Research in this laboratory
and others has shown that minor changes to the structure of
NT(8-13) can effect extreme changes in hypothermic, anti-
psychotic, or analgesic responses.12,13,15,17-19 Specifically,
modification of the N-terminus of NT(8-13) and substitution
of tLeu at position 12 produce compounds exhibiting in-
creased stability in blood and antipsychotic activity in rats19,20
(alsounpublisheddata);the most activeof thesecompounds is
ABS201 (2)21 (compound 11 in earlier manuscript).
Compound 2 also exhibits minor analgesic activity. In this
paper, structural modification of 2 and subsequent evaluation
in various rat models have been used to identify new NT(8-13)
derivatives possessing dramatically improved analgesic proper-
ties. A library of 44 proprietary modifications to the NT(8-13)
parent compound have been screened, with ABS212 (1) chosen
as the lead compound based on its superior activity in the
hot plate model. Compound 1 was further screened in a battery
of analgesic models and its pharmaceutical properties were
explored in order to evaluate its potential for further develop-
ment as a first-in-class analgesic.
*To whom correspondence should be addressed. Address: Department
of Pharmaceutical and Biomedical Sciences, Medical University of South
Carolina, 280 Calhoun Street, QF304, Charleston, South Carolina 29403.
Phone: 843-876-5092. Fax: 843-792-1617. E-mail: dixta@musc.edu.
a Abbreviations: NT, neurotensin; BBB, blood-brain barrier; NTS,
neurotensin receptor; CNS, central nervous system; MPE, maximum
possible effect; PK, pharmacokinetic; PD, pharmacodynamic.
Results
Synthesis of Analogues. NT and NT(8-13) are pluripotent
peptides that have activity as analgesics and antipsychotic
r
2010 American Chemical Society
Published on Web 05/19/2010
pubs.acs.org/jmc

4624 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12
Hughes et al.
Figure 1. Structures of cationic natural and non-natural amino acids substituted for Arg8 in NT(8-13).
and hypothermic agents only when administered directly
into the CNS. Previously, we developed a derivative of
NT(8-13), 2, that has potent antipsychotic but weak an-
algesic properties when dosed ip or iv.13 This analogue
possesses several significant alterations from the parent
compound including a modified N-terminus and Arg8 resi-
due, and substitution of tLeu for Ile.12 The primary result of
these alterations was to block peptidase-catalyzed hydrolysis
at Arg8-Arg9 and Tyr11-Ile12, respectively.22 To expand
upon previous findings, a series of 43 additional analogues
were synthesized that feature further modification of the
N-terminus and Arg residue with the anticipation that we
would uncover compounds featuring changes/improvements
in their relative antipsychotic/analgesic profiles. The struc-
tures of the new peptides are given in Figure 1.
Analysis of Analogues in the Hot Plate Model. As an initial
screen for analgesic activity, all peptides were evaluated
in the hot plate analgesic model using a standard dosing of
10 mg/kg ip. This value was an approximate molar equiva-
lent dose to 5 mg/kg morphine to provide a potency bench-
mark. Figure 2 shows a typical pharmacodynamic (PD)
response curve comparing 1 to morphine and saline. For
Figure 2. Compound 1 benchmarked with morphine in the hot
plate model. Rats were dosed ip with 1 at 10 mg/kg, morphine at
5 mg/kg, or sterile saline and analyzed at the indicated time points in
the hot plate model. Individual points indicate the mean ( SEM;
n g 6 for all treatments.
morphine the response reaches 100% of the maximal possible
effect (MPE) after 15-30 min and then decreases in a time-
dependent manner, returning to baseline by 200 min. When
dosed with 1, the time at 100% MPE was greatly prolonged
and did not begin to diminish significantly for 180 min.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4625
Table 1. Comparison of Peptides in the Hot Plate Assaya
Effect of 1 in Other Models of Pain. To evaluate the
potential usefulness of 1 as a general analgesic, its effectiveness
in four additional rat models of pain (acetic acid writhing, tail
flick, formalin, and Chung models) was determined. As shown
in Figure 4, 1 was quite potent in the acetic acid writhing assay
with an EC50 of 0.16 mg/kg (approximately 10-fold more
potent than the hot plate). This increase in potency relative
to the hot plate may be a result of the high sensitivity of this
assay.23,24 In the tail flick assay (Figure 5A), 1 significantly
increased the latency of tail withdrawal (indicated at the %
MPE) in a manner similar to morphine. A dose response
analysis revealed an EC50 of 1.84 mg/kg, very similar to what
was seen in the hot plate assay (Figure 5B). Figure 6A presents
a typical PD response curve in the formalin model. Compound
1 again was significantly more potent than morphine in this
model. The dose response curve (Figure 6B) indicated an EC50
of 2.05 mg/kg, again similar to the hot plate model. In the
Chung model of neuropathic pain (Figure 7, performed at a
single dose of 10 mg/kg) 1 elicits a maximal response within 15
min which is maintained for over 1 h before beginning to fade.
Effect of 1 on Hypothermia. A well-known central effect of
NT and its active derivatives is the reduction of core body
temperature (hypothermia). Figure 8A is a typical PD
response curve obtained with 1 that demonstrates a signifi-
cant and sustained (>4 h) drop in temperature. Interest-
ingly, the dose response curve of the area under the PD
curves (Figure 8B) gave an EC50 of 0.78 mg/kg, significantly
lower than the EC50 associated with central pain mediation.
This is likely due to the hypothermic and analgesic effects
being modified through different NTRs.
Lack of Induction of Tolerance by 1. Previously reported
NT analogues have been shown to induce tolerance after as
little as a single injection.16 In order to assess potential
development of tolerance to 1, rats were injected with 10
mg/kg daily for 12 consecutive days. On the indicated days
(Figure 9) rats were analyzed in the hot plate analgesia model
immediately following injections. As seen in Figure 2, initial
injections resulted in a strong and sustained increase in
response latency in this assay, again demonstrating the
significant analgesic activity of this compound. However,
even after 12 daily injections, the animals still displayed a
similar PD response, clearly showing that tolerance is not
developing to 1 over the time course of the experiments.
Binding to NTRs. To ensure that 1 is binding to appro-
priate receptors with sufficient affinity to generate the PD
responses, binding assays were performed with rat NTR-1.
As shown in Figure 10A there was dose-dependent binding
of 1 to rat NTR-1 with a KD of 23.31 nM. Equivalent
experiments could not be performed with cloned rat NTR-
2, as various investigators have not been able to obtain stable
constructs (E. Richelson, personal communication). How-
ever, the binding to human NTR-1 and NTR-2 was assessed
and demonstrated EC50 of 10.6 and 54.2 nM, respectively.
For virtually all NT derivatives, their relative affinities for
human versus rat NTRs remain consistent.25,26
time at
MPE (min)
compd
m/e (obsd/calcd)
MPE
AUC
201 (2)
202
203
204
205
206
207
208
209
210
211
212 (1)
213
214
215
216
217
218
219
220
221
222
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
802.60/802.02
816.63/816.04
830.65/830.07
844.72/845.10
788.60/787.99
802.64/802.02
816.65/816.04
830.68/831.08
774.60/773.96
788.60/787.99
nd
100.00
100.00
60.94
19.90
86.00
55.80
84.90
67.20
89.40
82.40
nd
45
75
0
9928
12182
4281
1898
11015
6001
7004
8788
9507
12105
nd
0
0
0
0
15
0
0
nd
135
0
816.59/817.05
816.56/816.00
830.60/830.03
844.69/844.06
844.63/844.06
872.72/872.11
842.65/842.04
nd
100.00
48.52
100.00
63.30
74.48
10.00
79.31
nd
18084
3515
8874
6106
12400
781
75
0
0
0
0
5793
nd
nd
0
nd
856.71/856.07
nd
55.00
76.07
nd
3569
7462
nd
0
nd
*60
0
788.67/787.99
802.65/802.02
816.75/816.04
830.79/831.08
774.61/773.96
788.64/787.99
nd
100.00
59.32
29.00
55.70
48.00
100.00
nd
8416
5345
3985
3853
3520
5733
nd
0
0
0
30
nd
0
816.69/817.05
760.62/759.94
774.33/773.96
788.40/787.99
802.42/803.02
802.44/801.98
816.48/816.00
830.51/830.03
830.55/830.03
858.68/858.08
828.57/828.01
842.63/842.04
828.69/828.01
nd
84.00
51.69
100.00
100.00
100.00
50.86
100.00
92.28
37.36
95.68
76.40
79.62
82.80
nd
11109
3585
17320
11180
7022
4039
8396
5152
2148
7425
7506
6637
6110
nd
0
120
90
60
0
75
0
0
15
0
0
0
nd
0
856.69/856.07
80.48
7119
a Three major end points are compared: (1) the maximal possible
effect (MPE) that was obtained with that drug, (2) the time that the
maximal effect was maintained, and (3) the area underneath the phar-
macodynamic response curve.
The remaining compounds in Figure 1 also were tested in this
model.
In order to facilitate a meaningful comparison of relative
potency of all compounds tested, Table 1 reports three major
end points of the model: (1) the maximal effect (as a
percentage of the MPE) obtained with the given compound,
(2) the time that 100% MPE was maintained, and (3) the area
underneath the PD response curve. Analysis of that table
indicates that 1 was the most efficacious of the analogues and
thus was chosen for further study.
Potency of 1 with Various Routes of Administration. Po-
tency studies were undertaken to determine the EC50 of 1 and
to evaluate whether it differed as a function of the route
of administration. As shown in Figure 3, the EC50 of 1 was
2.64 mg/kg when delivered iv. Significant differences were
not found when the drug was administered ip, sc, or im.
Calcium Mobilization. To ensure that binding to receptor
actually results in activation, the flux of calcium (a well
established second messenger response) was measured in the
rat NTR-1 expressing cells in response to increasing con-
centrations of 1. As shown in Figure 10B 1 activated calcium
flux with an EC50 of 27.9 nM, a concentration very similar to
its binding affinity.
PK Analysis. As a further step toward understanding the
mechanism of action of 1, a PK analysis was performed.

4626 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12
Hughes et al.
Figure 3. Effect of route of administration of 1 in the hot plate model. Rats were injected ip, iv, sc, or im with various concentrations of 1 and
then assessed in the hot plate assay after 60 min. EC50 values are as follows: ip = 2.00 mg/kg (95% confidence levels of 1.543 and 2.583 mg/kg);
iv = 2.64 mg/kg (95% confidence levels of 1.261 and 5.546 mg/kg); sc = 3.84 mg/kg (95% confidence levels of 1.567 and 9.392 mg/kg); im =
2.45 mg/kg (95% confidence levels of 1.376 and 4.369 mg/kg). n = 3 for all doses. Individual points indicate the mean ( SEM.
To test this possibility, the kinetics of NTR-1 binding in the
plasma of rats after injection of 1 were examined and are
overlaid on the data in Figure 11. As shown, very quickly
(1 min) after iv injection there is a large increase in NTR-1
binding that begins to rapidly decrease by 15 min. After
60 min, levels have returned to concentrations not significantly
different from nontreated animals. Notably, the kinetic profile
of receptor binding mirrored almost exactly the level of parent
compound measured by mass spectrometry. Indeed, calcu-
lated half-lives (T1/2) were 0.407 and 0.438 h, respectively.
To further ensure that metabolites of 1 were not respon-
sible for the demonstrated PD response, the various possible
hydrolytic metabolites of this compound were synthesized
and the 5-mers and 4-mers assayed in the hot plate model.
Table 2 lists these possible metabolites, while Figure 12
shows the results of that analysis. In response to treatment
Figure 4. Dose response of 1 in the writhing model. Rats received
with 1 response latency rapidly increased to maximum (30 s)
and remained high during the course of the experiment. In
contrast, no increase in response latency was seen with any of
the possible metabolites. There was an early increase with 4
that did not approach maximal and rapidly returned to
baseline; this is probably a spurious result. Because removal
of one or two amino acids from either end completely
abolished activity, further analysis of the 3- and 2-mers
was deemed unnecessary. These results strongly argue that
hydrolytic metabolites are not responsible for the long
lasting PD effect observed with the parent compound.
varying concentrations of 1 iv before analysis in the writhing model.
EC50 = 0.16 mg/kg, with 95% confidence levels of 0.072 69 and
0.3669 mg/kg; n = 3 for all doses. Individual points indicate the
mean ( SEM.
After injection of rats with 1 mg/kg 1, blood levels were
assessed at increasing time points by mass spectrometry. As
shown in Figure 11, the initial spike in concentration ex-
pected immediately following injection was observed. Sur-
prisingly, however, these levels rapidly decreased and
returned to baseline by 60 min despite the fact that the PD
analgesic response remained near maximal for at least 180 min.
One possible explanation for the discrepancy between the
PK and PD is that a hydrolysis product or an unidentified
metabolite of 1 passes through the BBB to activate the NTRs
and maintain the sustained analgesia well after 1 is degraded.
Discussion
Neurotensin (NT) has been shown to be an effective
endogenous pain modulator27 and as such is an excellent

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4627
Figure 6. Effect of 1 in the formalin model. (A) Typical PD response
elicited by 1, morphine, and saline. Rats were dosed ip with 1 at
10 mg/kg, morphine at 5 mg/kg, or sterile saline and analyzed in the
formalin assay for 30 min; n = 6 for each treatment. Individual points
indicate the mean ( SEM. (B) Dose response of 1. Rats were injected
ip with various doses of 1 and assessed in the tail flick model 30 min
later. The EC50 was 2.06 mg/kg, with 95% confidence levels of 1.27
and 3.30 mg/kg; n = 6 for all doses. Individual points indicate the
mean ( SEM.
Figure 5. Effects of 1 in the tail flick model. (A) Typical PD response
elicited by 1, morphine, and saline. Rats were dosed ip with 1 at
10 mg/kg, morphine at 5 mg/kg, or sterile saline and analyzed in the
tail flick assay at 30 and 60 min; n = 6 for each treatment. Individual
points indicate the mean ( SEM. Single asterisks denote values
significantly different from saline, with p < 0.05, whereas double
asterisks indicate p < 0.01. (B) Dose response of 1. Rats were injected
ip with various doses of 1 and assessed in the tail flick model 30 min
later. The EC50 was 1.57 mg/kg, with 95% confidence levels of 0.967
and 2.552 mg/kg; n = 6 for all doses. Individual points indicate the
mean ( SEM.
candidate for pharmaceutical applications. However, this
peptide and its fully active fragment NT(8-13) suffer from
several druggability issues including deliverability, stability,
selectivity, and efficacy. When derivatives are designed
to address these issues, several elements of the peptide must
be retained in order not to adversely degrade its activity.
In particular, the positively charged side chains at the
N-terminus2,28 and the free carboxylate at position 13 are
necessary for significant activity. Loss of potency also is noted
when Arg8 is changed from L- to D-.28,29
We have previously developed a derivative of NT(8-13), 2,
that retained the structural features necessary for activity but
included a modified N-terminus and Arg8 residue and sub-
stituted tLeu for Ile.12 This compound had strong antipsy-
chotic but weak analgesic activity. In this study a series of 43
additional analogues were synthesized that feature further
modification of the N-terminus and Arg residue (Table 1). As
anticipated, this resulted in compounds with a wide variety of
activities in the hot plate model of acute pain.
Figure 7. Effect of 1 in the Chung model. Rats were given an ip dose
of 10 mg/kg 1 or saline and analyzed in the Chung assay for 30 min;
n = 5 for both treatments. Individual points indicate the mean ( SEM.
for a class of NT(8-13)-based antipsychotic drugs.20 In
addition, analogues containing Arg8 plus a methyl addition
(ABS214 and ABS238) or hLys alone (2 and ABS225) per-
formed quite well. In these cases activity was not affected by a
change in the N-terminus group. These results indicate that
smaller side chains at Arg8 are more effective analgesics.
Overall, however, it was surprising that broader structure-
activity relationships did not result from the data, which may
indicate a complex cross-talk between binding and activating
NTR-1 and NTR-2 to promote the observed physiological
responses.
A few generalizations can be made on the structure-func-
tion relationships of the current series of NT(8-13) deriva-
tives. In the Arg8 position, nearly all Orn substituted
derivatives (ABS209-ABS212 (1) and ABS234-ABS236)
possessed significantly increased activity (five of eight analo-
gues reached 100% MPE, while two others were over 80%).
The effect of the Orn substitution has been similarly reported
Data from the hot plate model enabled identification of 1 as
the best lead candidate for further characterization. This
molecule demonstrated a potent antinociceptive response in

4628 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12
Hughes et al.
Figure 8. Effect of 1 in the hypothermia assay. (A) Typical PD res-
ponse elicited by 1 and saline. Rats were dosed iv with 1 at 10 mg/kg
or sterile saline and analyzed in the hypothermia assay for 4 h; n g 4
for each treatment. Individual points indicate the mean ( SEM. (B)
Dose response of 1. Rats were injected iv with various doses of 1 and
assessed in the hypothermia assay. The total area under the various
PD curves was calculated and used to generate the dose-response
curve. EC50 = 0.78 mg/kg with 95% confidence levels of 0.190 and
3.237 mg/kg; n = 3 for all doses. Individual points indicate the mean
( SEM.
Figure 10. Evaluation of 1 binding and activation of rat NTR-1.
(A) Radioligand competition assays with [125I]NT were performed
and the data normalized to percent of maximal binding. Individual
experiments were measured in triplicate, and each experiment was
performed a minimum of three independent times. The data points
represent the mean ( SEM of all experiments. The Ki was 23.31 nM
with 95% confidence levels of 14.01 and 38.79 nM. (B) Calcium
release assays were performed on the fluorometric imaging plate
reader (FLIPR, Molecular Devices Corp.). Individual points indicate
the mean (SEM of at least three independent measurements. The EC50
was 27.88 nM with 95% confidence levels of 17.62 and 44.10 nM.
Figure 9. Evaluation of tolerance development in response to
repeated daily doses of 1. Six rats were separated into two groups
of three, and both groups were injected iv with 10 mg/kg 1 per day
for 12 days consecutively. Each set was allowed at least 48 h between
consecutive evaluations in the hot plate model. Thus, any given data
point represents the mean ( SEM of three rats (one set), although
overall a total of six rats were analyzed for the development of
tolerance.
Figure 11. PK analysis of 1 levels and receptor binding activity
overlaid on PD information from the hot plate model. Rats were
injected with 1 mg/kg 1. At each time point of 1 (or 5), 15, 30, 60, 120,
180, and 240 min, three rats were assessed using the hot plate model.
Thus, individual points represent the mean ( SEM of three in-
dependently assessed rats. Animals were immediately sacrificed
after analysis, and plasma was isolated and analyzed in the NTR-1
radiolabel competition assay. Thus, individual points represent the
mean ( SEM of three independently assessed rats. Plasma concen-
trations were calculated from a standard curve prepared in plasma
from untreated rats. Plasma levels of 1 were also assessed by MS in a
separate experiment performed by Covance, Inc. Individual points
represent the mean ( SEM of at least three independently assessed
rats.
four additional rat analgesic models. The EC50 values for
three of the four iv analgesic tests were quite similar
(approximately 2.0 mg/kg), validating the use of the hot plate
model as an initial screening tool. A significantly lower EC50
was detected in the acetic acid writhing assay that may be a
result of the enhanced sensitivity of this assay relative to the
others.23,24
This EC50 did not differ because of route of administration,
suggesting it is freely permeable throughout the periphery.
Repeated administration of 1 for 12 straight days did not

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4629
result in the development of significant tolerance to the drugs
even though previous studies with other NT(8-13) analogues
found tolerance after a single dose.16
uptake and distribution between the blood and the brain and/
or compound binding to the central NTRs manifesting long-
lived molecular changes that sustain the analgesic activity.
The discovery and development of peptide drugs provide
the most direct route for exploiting genomic and proteomic
insights into the treatment of disease. Peptides can act as
agonists to positively affect biological processes, with the
potential to repair damaged, injured, or undeveloped tissue.
Peptide therapeutics represent an extremely powerful ap-
proach that can reduce the time and risk of bringing a drug
to market. Compared to synthetic small molecules, drugs
based on native peptides have the potential for shorter and
more reliable development cycles because their physiological
activity is well-defined prior to development and they are
much less likely to cause unexpected toxicities. We conclude
that 1 is an exciting new candidate for potential development
as a broad-spectrum, first-in class analgesic.
Analysis of the PK of this drug surprisingly revealed that it
is rapidly cleared from the circulation (<60 min), despite the
fact that the analgesic response is maintained for nearly 4 h.
This possibly explains the absence of tolerance that is gen-
erally due to desensitization following long-term exposure of
drugs. The observed dichotomy between the PK and PD
cannot be explained by a previously unrecognized metabolite
of the analogue because NTR-1 binding decreased at an
identical rate to levels of parent compound measured by
LC/MS. Moreover, none of the possible 5-mer or 4-mer
hydrolytic metabolites of 1 possessed significant endogenous
activity. Thus, the mechanisms of this PK/PD dichotomy
remain undefined although it could be explained by selective
Table 2. The 5-mer and 4-mer Hydrolytic Fragments of 1
Experimental Section
structure
m/e (obsd/calcd)
Peptide Synthesis. Appropriately protected non-natural ami-
no acid analogues of Arg and Lys were synthesized as described
previously by our laboratory30-35 and others36 for incorpora-
tion at the 8 position of NT(8-13) via Merrifield solid-phase
synthesis (Figure 1). All analogues were determined to be greater
than 95% enantiomerically pure. Peptides were prepared by
manual solid-phase peptide synthesis on Wang resin (Fmoc
chemistry), purified by reverse-phase HPLC, and isolated and
studied as the trifluoroacetate salts.19 Peptide identity was
determined by MALDI-TOF MS on a Voyager DE-STR system
compd
Length: 5-mers
3
4
Me3-Orn Arg Pro Tyr tLeu
Arg Pro Tyr tLeu Leu
703.52/704.88
661.47/660.80
Length: 4-mers
Me3-Orn Arg Pro Tyr
Arg Pro Tyr tLeu
5
6
7
590.38/5912.70
548.30/547.61
nd
Pro Tyr tLeu Leu
Figure 12. Evaluation of all potential 5- and 4-mer hydrolysis products of 1 in the hot plate model. All possible 5- and 4-mer products that
might result from proteolysis from the N-terminal, the C-terminal, or both were synthesized and injected into rats iv at 12.5 mmol/kg. The
change in concentration from mg/kg was necessary to ensure equimolar levels, given the differences in molecular weights. Each graph is
the individual PD response curve for that hydrolysis product, and each point on the graphs represents the mean ( SEM of at least three rats
independently assessed at that time point.

4630 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12
Hughes et al.
4117 mass spectrometer (Applied Biosystems, Foster City, CA),
and the peptides were determined to be greater than 95% pure
(MS data provided in Table 1).
not supporting rat, 2 = paw raised, and 3 = paw being licked,
nibbled, or shaken) for a period of 30 min (60 min after drug
application). An average pain response was calculated for a
minimum of six animals and plotted over time. The dose-
response curves and ED50 values were generated using Graph-
Pad Prism.
Animals. All animal work was reviewed and approved by the
Institutional Animal Care and Use Committee (IACUC) at the
Medical University of South Carolina. Animal work at the
University of California and Covance were reviewed and ap-
proved by their institutions’ IACUCs. All experimental proto-
cols were performed in accordance with the guidelines set forth
in the NIH Guide for the Care and Use of Laboratory Animals,
published by the Public Health Service. All protocols were
performed on male Sprague-Dawley rats (Harlan, Prattville
AL, 240-280 g), which were housed in an AAALAC-approved
colony room maintained at a constant temperature and humid-
ity. Animals (two per cage) were kept on a 12 h light-dark cycle
with ad libitum access to food and water. Because many of the
assays are susceptible to learning phenomena, which results in
progressive decrease of the physiological response or behavior
to be monitored, rats were used no more than three times per
analgesic assay. Only experimentally naive rats were used in the
formalin test.
Pain Models. Hot Plate Model. The hot plate model evalu-
ates central pain attenuation in a rodent using an acute thermal
stimulus known to be mediated through central processing
pathways.37-39 In this assay the rat is treated with compound
and, at a series of time points thereafter, assessed on a hot plate
analgesia meter (Columbus Instruments, Columbus, OH), es-
sentially a flat surface maintained at 53.0 ( 0.2 ꢀC. After the rat
is placed on the hot surface, the time until the rat lifts, nibbles, or
shakes one of its hind paws is recorded, which is known as the
response latency. To avoid tissue damage, animals were not
allowed to remain on the hot plate longer than 30 s. The
individual experiments were scored as the percent of maximal
possible effect (%MPE) calculated using the following equa-
tion: %MPE = [(postdrug latency - predrug latency)/(cutoff
predrug latency)] ꢀ 100. The dose-response curves and ED50
values were generated using GraphPad Prism.
Chung Model. The Chung model for neuropathic pain was
performed in the laboratories of Tony Yaksh (University of
California, San Diego). To assess tactile thresholds, rats are
placed in a clear plastic, wire mesh-bottomed cage divided into
individual compartments. Animals are allowed to acclimate,
and then baseline thresholds are assessed prior to drug treat-
ment. To assess the 50% mechanical threshold for paw with-
drawal, von Frey hairs are applied to the plantar midhind paw,
avoiding the tori (footpads). The eight von Frey hairs used are
designated by log(10 ꢀ force required to bend hair, mg) and
range from 0.4 to 15.1 g (numbers 3.61-5.18). Each hair is
pressed perpendicularly against the paw with sufficient force to
cause slight bending and held for approximately 6-8 s. A
positive response is noted if the paw is sharply withdrawn.
Flinching immediately upon removal of the hair is also con-
sidered a positive response. Absence of a response (“-”) is cause
to present the next consecutive stronger stimulus. A positive
response (“þ”) is cause to present the next weaker stimulus.
Stimuli are presented successively until either six data points are
collected or the maximum or minimum stimulus is reached. If a
minimum stimulus is reached and positive responses still oc-
curred, the threshold is assigned an arbitrary minimum value of
0.25 g. If a maximum stimulus is presented and no response
occurred, a maximum threshold value of 15 g is assigned. If a
change in response occurs, either “-” to “þ” or “þ” to “-”,
causing a change in the direction of stimulus presentation from
descending to ascending or vice versa, four additional data
points are collected subsequent to the change. The resulting
pattern of responses are tabulated and the 50% response thresh-
old is computed using the formula
logðthreshold; mg ꢀ 10Þ ¼ Xf þ kr
Acetic Acid Writhing Model. This protocol is a well-estab-
lished model of analgesia.40-42 Rats are administered drug iv
and then rested for 20 min before ip injection with 2 mL/kg of
3% acetic acid. Animals were then placed in a 10 in. by 10 in.
chamber and allowed to rest an additional 10 min. For the next
20 min, rats are observed every 20 s. At each point individual rats
are scored on whether they are writhing or not. Writhing is
defined as a constriction of the abdominal area, often with
extension of the hind legs. At the end of the observation period
the percent of the time the animal was observed to be writhing
was calculated. The dose-response curves and ED50 values were
generated using GraphPad Prism.
Tail Flick Model. This model evaluates phasic pain, involving
less surface area stimulation than the hot plate model.43 This
protocol was performed by placing rats in individual Plexiglas
containment and immersing the distal 3 cm of the rat’s tail in a
hot (49 ( 1 ꢀC) water bath. The elapsed time was measured from
insertion in the water to tail withdrawal. The cutoff latency for
tail withdrawal was 10 s, at which time the animal’s tail was
removed from the water bath. Assays were scored as percent
MPEs over time as discussed above (hot plate model). The
dose-response curves and ED50 values were generated using
GraphPad Prism.
Formalin Model. The formalin model was performed accord-
ing to the methods of Dubuisson and Dennis.44 As opposed to
the procedures described above, this evaluates behavioral
changes rather than changes in response latency and is consid-
ered a model of chronic pain. The rats were administered either
control or test compound. Thirty minutes later, 50 μL of a 5%
formalin solution in sterile saline was injected intradermally into
the right forepaw. The animal was placed in a clear Plexiglas
cage, and the animal’s posture was graded at 1 min intervals on a
scale from 0 to 3 (0 = normal posture, 1 = paw on ground but
where Xf is the value of the last von Frey hair applied, k is the
correction factor based on pattern of responses (from calibra-
tion table), and r is the mean distance in log units between
stimuli. On the basis of observations on normal, unoperated rats
and sham-operated rats, the cutoff of a 15.1 g hair is selected as
the upper limit for testing.45
Hypothermia Assay. Hypothermia assays were performed as
previously described.46 Briefly, rats were placed in Plas-Labs
plastic restraining apparatuses (250-500 g model) and rectal
probes (Physitemp, RET-2) lubricated with mineral oil and
inserted in the animals’ rectums. Probes were connected to a
microprobe thermometer (Physitemp, BAT-12) equipped with a
switchbox (Physitemp, SWT-5). Rats were allowed to equili-
brate in the restraining apparatus for 15 min before initial
temperatures were recorded. For injections, peptides were dis-
solved in saline and injected into the tail vein. Subsequent
temperature measurements were taken every 15 min for 4 h.
The area under the curve, the resulting dose-response curve,
and the EC50 values were generated using GraphPad Prism
software.
Tolerance to the Hot Plate Model. For tolerance experiments,
six rats were separated into two groups of three. Both groups
were injected iv with 10 mg/kg 1 per day for 12 days consecu-
tively. To avoid learning behaviors, each set was allowed at least
48 h between consecutive evaluations in the hot plate model as
described above. Thus, any given data point represents the mean
( SEM of three rats (one set), although overall a total of six rats
were analyzed for the development of tolerance.
NTR-1 and NTR-2 Binding Assays. Binding to Rat NTR-1.
NTR binding assays were performed as previously described.19
LTK cells expressing rat NTR were propagated in using stan-
dard cell culture techniques. For the assay, cells were scraped off

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4631
the plates, washed, and resuspended in binding buffer (20 mM
Hepes (pH 7.4), 50 mM NaCl, 1.5 mM CaCl2, 0.1 mg/mL
L-lysine, 1% BSA, 0.01% NaN3) at a concentration of 2 ꢀ
106 cells/mL. Samples for the determination of KD were pre-
pared by resuspending compound in binding buffer at various
concentrations. For PK analysis, animals were sacrificed by
CO2 asphyxiation at the appropriate times and blood was
harvested into a prechilled heparin-coated tube by heart punc-
ture. Tubes were then centrifuged (500g) to pellet cells, and the
resulting plasma was immediately aliquoted and frozen at
-70 ꢀC for subsequent analysis. For generation of a standard
curve, compound was resuspended and diluted in plasma iso-
lated from untreated rats.
To perform the assay, 50 μL of sample was combined in a
1.5 mL microcentrifuge tube with 50 μL of cells (100 000 cells)
and 50 μL containing 0.0375 pmol of [125I]NT (approximately
100 000 CPM, diluted in binding buffer). The mixture was
allowed to incubate for 60 min at room temperature to come
to equilibrium. Following incubation, 1 mL of ice cold binding
buffer was added, the cells were pelleted, and the supernatants
were aspirated. Cells were washed again in 1 mL of ice cold
binding buffer, and the tip of the tubes containing the pelleted
cells were cut off and counted in a γ counter.
visual inspection of the raw data. PK parameters calculated
included half-life (t1/2), area under the concentration-time
curve from time 0 to the last time point (AUC0-t), and area under
the concentration-time curve from 0 to infinity (AUC0-¥). PK
parameters were calculated by using WinNonlin Professional
edition (Pharsight Corp., version 5.2).
Acknowledgment. The authors thank Dr. Tony Yaksh and
colleagues at the University of California at San Diego for
performing the Chung assay. This work was supported by
Grant GM-079044 (T.A.D.) from the National Institutes of
Health, National Institute of General Medical Sciences. This
research was conducted in a facility constructed with support
from the National Institutes of Health, Grant C06 RR015455
from the Extramural Research Facilities Program of the
National Center for Research Resources.
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