Truncated G protein-coupled mu opioid receptor MOR-1
splice variants are targets for highly potent opioid
analgesics lacking side effects
Susruta Majumdara, Steven Grinnella, Valerie Le Rouzica, Maxim Burgmana, Lisa Polikara, Michael Ansonoffb,
John Pintarb, Ying-Xian Pana, and Gavril W. Pasternaka,1
aMolecular Pharmacology and Chemistry Program and Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065;
andbDepartment of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, NJ 08854
Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 21, 2011 (received for review
September 17, 2011)
Pain remains a pervasive problem throughout medicine, transcend-
ing all specialty boundaries. Despite the extraordinary insights into
pain and its mechanisms over the past few decades, few advances
have been made with analgesics. Most pain remains treated by
opiates, which have significant side effects that limit their utility.
We now describe a potent opiate analgesic lacking the traditional
side effects associated with classical opiates, including respiratory
depression, significant constipation, physical dependence, and,
perhaps most important, reinforcing behavior, demonstrating that
it is possible to dissociate side effects from analgesia. Evidence
indicates that this agent acts through a truncated, six-transmem-
brane variant of the G protein-coupled mu opioid receptor MOR-1.
Although truncated splice variants have been reported for a num-
ber of G protein-coupled receptors, their functional relevance has
been unclear. Our evidence now suggests that truncated variants
when inactive alone, and can comprise new therapeutic targets, as
illustrated by our unique opioid analgesics with a vastly improved
opiate receptor|rewarding behavior|kappa3receptor
relieve pain comes a variety of opioid receptor-mediated side
effects, including respiratory depression, constipation, physical
dependence, and reward behavior felt by many to contribute to
their addictive potential. Most of the clinical opioids act through
mu receptors, which mediate both analgesia and these side
effects. Pharmacological studies have long suggested subtypes of
mu receptors (1) and the possibility of dissociating analgesia
from respiratory depression (2, 3), physical dependence (4), and
the inhibition of gastrointestinal transit (5, 6). However, attempts
to develop opiate analgesics that avoid these side effects have
not been very fruitful. The isolation of a series of splice variants
of the cloned mu opioid receptor from mice (Fig. 1), rats, and
humans with similar splicing patterns (7, 8) reveals a complexity
far exceeding the pharmacological classification of mu receptor
subtypes (1). However, this complexity has yet to be exploited in
generating new classes of opioid analgesics. We now report an
unexpected and unusual target for potent opioid analgesic drugs
that lack respiratory depression, physical dependence, reward
behavior, and significant constipation.
he utility of opioids in the management of pain is not dis-
puted, but they come at a price. Along with their ability to
Recently, we synthesized iodobenzoylnaltrexamide (IBNtxA),
a naltrexone derivative (Fig. 2) (9). In vivo, it is a very potent
analgesic (ED50= 0.48 ± 0.05 mg/kg s.c.) (Fig. 3A and Fig. S1),
∼10-fold more potent than morphine (4.6 ± 0.97 mg/kg s.c.) (10),
with a mechanism of action quite distinct from traditional opi-
ates. It was active s.c. as well as orally (Fig. S1), with a peak effect
after oral administration that was delayed relative to parenteral
administration. In addition to its high potency, IBNtxA also
displayed full efficacy, as indicated by most mice reaching cutoff
values at higher doses. We also assessed IBNtxA analgesia by
using a graded response [percentage maximal possible effect (%
MPE)] (Fig. S2). Its ED50value (0.34 mg/kg; 95% confidence
limits: 0.23, 0.52) was very close to that with quantal responses
(Fig. 3A). Furthermore, IBNtxA analgesia was not limited to the
radiant heat tail-flick assay. IBNtxA was a potent analgesic in the
hot plate assay (ED50= 0.6 mg/kg) as well (Fig. S3).
Opioid receptor KO mice have been valuable in exploring
opioid action. A number of mice targeting MOR-1 have been
are unresponsive to morphine, but one model with a disruption of
exon 1 retained full sensitivity to heroin and morphine-6β-glucu-
loss of all of the variants containing exon 1, this mouse still
expressed a series of MOR-1 variants generated from a second,
upstream promoter associated with exon 11 (Fig. 1). This mouse
of the delta opioid receptor DOR-1 and the kappa1opioid re-
indicating that IBNtxA analgesia did not involve full-length mu
receptors containing exon 1 or delta or kappa1receptors. Its
(ID50= 0.54 ± 0.05 mg/kg s.c.), a well-established opioid antag-
onist (Fig. 3B). Naloxone (ID50= 10.5 ±0.6 mg/kg s.c.) also re-
versed its actions but at doses higher than those needed for
traditional mu opiates such as morphine (ID50= 0.01 mg/kg s.c.)
(15). Its relative insensitivity to a series of selective antagonists,
including β-funaltrexamine (mu), norbinaltorphimine (kappa1),
and naltrindole (delta), further supported a nontraditional opioid
receptor mechanism of action (Fig. S4).
be involved in IBNtxA actions because they are still expressed in
thetriple-KO mice. Lossoftheexon11-associated variants has no
significant effect on morphine or methadone analgesia (16). In
contrast, IBNtxA analgesia was lost in the exon 11 MOR-1 KO
mouse (Fig. 3C), indicating that IBNtxA analgesia involves exon
Author contributions: S.M., S.G., and G.W.P. designed research; S.M., S.G., V.L.R., M.B., L.P.,
M.A., and J.P. performed research; M.A., J.P., and Y.-X.P. contributed new reagents/
analytic tools; S.M., S.G., V.L.R., M.B., L.P., and G.W.P. analyzed data; and Y.-X.P. and
G.W.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| December 6, 2011
| vol. 108
| no. 49www.pnas.org/cgi/doi/10.1073/pnas.1115231108
KO mice, these data suggest that IBNtxA analgesia depends on
exon 11-associated MOR-1 variants lacking exon 1.
The pharmacological profile of IBNtxA also differed from
classical opioids. Respiratory depression is a major issue asso-
ciated with opioids. We therefore examined the effects of
IBNtxA on respiratory rate, a measure of respiratory depression.
Using doses approximately fourfold greater than their respective
analgesic ED50values, we observed the expected decrease in
respiratory rate in mice receiving morphine (20 mg/kg s.c.),
whereas an equivalent dose of IBNtxA (2.5 mg/kg s.c.) did not
change the respiratory rate compared with saline (Fig. 3D).
Inhibition of gastrointestinal transit, manifest clinically as
constipation, is another major clinical concern. Morphine dra-
matically inhibited gastrointestinal transit (Fig. 3E). An equi-
analgesic dose of IBNtxA (0.6 mg/kg s.c.) lowered transit a small
amount, but this effect plateaued, with far greater doses showing
no further inhibition, which clearly distinguished it from the
effects of morphine. However, even this small inhibition of gas-
trointestinal transit appears not to involve the same receptors as
IBNtxA analgesia because the gastrointestinal transit inhibition
persisted in the exon 11 KO mice, which displayed no IBNtxA
analgesia (Fig. S5). Thus, more selective agents might lack even
this small amount of inhibition of gastrointestinal transit seen
Chronic administration of traditional opioids leads to both
tolerance and physical dependence. Daily administration of
morphine (10 mg/kg s.c.) revealed a diminishing response with
no demonstrable analgesia by day 5 (Fig. S6). These chronically
morphine-treated mice were both tolerant and physically de-
pendent, with naloxone precipitating a profound withdrawal
syndrome (Fig. 3F) (4). Chronic IBNtxA dosing also produced
tolerance, although it developed more slowly (Fig. S6). Unlike
morphine, challenging the IBNtxA-tolerant mice with either of
the antagonists naloxone or levallorphan failed to precipitate any
demonstrable withdrawal symptoms, including jumping (Fig. 3F),
implying that chronic IBNtxA does not produce physical de-
pendence. We also observed no cross-tolerance between mor-
phine and IBNtxA analgesia. Implantation of morphine pellets
(75 mg of free base) in mice for 3 d led to marked analgesic
tolerance and physical dependence. After 3 d, IBNtxA (1 mg/kg
s.c.) displayed analgesia in 100% of the pelleted mice (n = 10),
an effect comparable to that seen in naïve mice (93%, n = 15).
Furthermore, IBNtxA did not elicit any signs of withdrawal in
the morphine-dependent mice.
Rewarding behavior is one measure of potential abuse po-
tential. Morphine shows rewarding behavior in the conditioned
place preference assay, whereas the kappa1-selective agonist
U50,488H is aversive. In our studies, IBNtxA was neutral, failing
to show either rewarding or aversive behavior, whereas morphine
was rewarding, as anticipated (Fig. 3G).
The pharmacological profile of IBNtxA suggested that it may
act through a target involving exon 11-associated MOR-1 variants
that lack exon 1. Although
delta, and kappa1receptors in transfected cell lines (9), ∼20% of
total specific125I-BNtxA binding in brain tissue was insensitive to
a combination of high concentrations of selective mu-, delta-, or
kappa1-selective ligands (Table S1). Similar125I-BNtxA-selective
binding levels were present in triple-KO mice (Fig. 4 A and B and
Table S1). Disruption of either exon 11 or exon 2 virtually elim-
inated the binding (Fig. 4A), mimicking in binding studies our
analgesic results in the exon 11 KO model and strongly supporting
a role for exon 11-associated variants in the binding target.
Competition binding studies from triple-KO and WT mouse
brains with traditional mu, delta, and kappa1blockers revealed
profiles quite distinct from traditional binding sites (Table 1), al-
the mu-selective compounds morphine and [D-Ala2,MePhe4,
D-Pen5]enkephalin (DPDPE) and SNC80, the kappa1-selective
drug U50,488H, and orphanin FQ/nociceptin analogs, did not
number of opioids competed binding quite effectively. Martin et al.
initially defined kappa receptors 35 y ago by using the benzomor-
site with high affinity that was comparable to or better than the
traditional kappa1receptor (Ki= 1.8 nM) (17). Other benzomor-
phans considered to have strong kappa actions, as defined by the
criteria of Martin et al., also competed binding very potently, in-
cluding the benzomorphans ethylketocyclazocine, cyclazocine,
and Mr2034. Other structural scaffolds also showed high affinity.
The morphinans levorphanol and butorphanol, and especially the
antagonist levallorphan, were quite potent. Naloxone benzoylhy-
drazone (NalBzoH), which we used to define the kappa3receptor
(17, 18, 22), competed binding effectively, as did the oripavines
etorphine, diprenorphine, and buprenorphine. Naloxone lowered
binding (Ki= 52 nM) more than 10-fold less potently than mu
binding sites (17, 23), possibly explaining its lower potency re-
versing IBNtxA analgesia (ID50= 10 mg/kg s.c.) compared with
125I-BNtxA labels traditional mu,
mouse gene is presented in schematic form and not to scale, with splice
variants shown underneath. There are two major classes of splice variants:
those generated by the promoter associated with exon 1 and an additional
set generated by the exon 11 promoter (designated by the yellow back-
ground). All of the exon 1-associated variants are traditional G protein-
coupled receptors with 7-TM regions. The exon 11-associated variants include
several full-length receptors and both 6-TM and 1-TM variants as indicated.
Schematic of alternative splicing of the mouse Oprm1 gene. The
Fig. 2.Structure of IBNtxA.
Majumdar et al.PNAS
| December 6, 2011
| vol. 108
| no. 49
reversal of morphine analgesia (ID50= 0.01 mg/kg s.c.) (15). This
finding contrasts with levallorphan, which effectively blocked
IBNtxA analgesia at very low doses (ID50= 0.54 mg/kg s.c.),
consistent with its high affinity for the site (Ki= 0.34 nM).
Many of the drugs active at the125I-BNtxA binding site are
potent analgesics in vivo (17–20, 24, 25). To determine whether
they were acting, in part, through mechanisms similar to IBNtxA,
we tested them in the exon 11 KO mouse (Table 2). The anal-
gesic activity of ketocyclazocine, which bound this new site with
very high affinity, was shifted ∼100-fold to the right, with right-
ward shifts for clinical analgesics levorphanol, butorphanol,
and nalbuphine as well. Finally, NalBzoH lost analgesic activity
in the exon 11 KO mice. Doses as high as 100 mg/kg s.c. failed
to reach an ED50comparable with an ED50of 22 ± 5.3 mg/kg s.c.
in WT mice.
The evidence strongly implicated a role for exon 11-associated
variants in IBNtxA actions. However, attempts to demonstrate
125I-BNtxA binding after expression in cells transfected with the
exon 11-associated variants lacking exon 1, such as mMOR-1G,
were unsuccessful. These variants are truncated with only six-
transmembrane (6-TM) domains, lacking the first TM domain of
MOR-1 encoded by exon 1. We therefore examined a possible
role of heterodimerization in the actions of these 6-TM variants.
Heterodimerization of full-length opioid receptors has been
well described, with many showing a pharmacology distinct from
either partner alone, such as the heterodimerization of DOR-1
and KOR-1 to form the kappa2receptor (26). Earlier work from
our laboratory demonstrated that the orphanin FQ receptor
ORL1, which we had initially termed KOR-3 (27–33), dimerized
with the full-length MOR-1 receptor to generate a binding site
with a unique pharmacological profile (34). Furthermore, anti-
sense mapping studies (27, 28) suggested an association of
kappa3analgesia with ORL1. To determine whether ORL1could
partner with a 6-TM MOR-1 variant to generate an125I-BNtxA
binding site, we cotransfected ORL1 with the 6-TM variant
transfected with either MOR-1G or ORL1 alone. However,
coexpression of MOR-1G with ORL1produced a very high-af-
finity125I-BNtxA binding site (KD= 0.24 ± 0.03 nM) (Fig. 4C),
similar in affinity to that seen in brain tissue. This finding implies
that the 6-TM variant alone is insufficient to generate the125I-
BNtxA site in the brain and requires a partner, which may or may
not correspond to ORL1.
125I-BNtxA binding could be detected in cells
IBNtxA is an interesting compound from many perspectives. Its
pharmacological profile in vivo distinguishes it from other opiates
because of its potent analgesia without respiratory depression,
received IBNtxA at the indicated doses and were tested 30 min later at peak
effect to generate the analgesic dose–response curve. ED50values (and 95%
confidence limits) were 0.22 mg/kg (0.13, 0.32) in WT mice and 0.39 mg/kg
(0.15, 0.58) in triple-KO mice by using the radiant heat tail-flick assay. (B)
Reversal of IBNtxA analgesia by levallorphan. Groups of mice (n ≥ 10) re-
ceived IBNtxA (0.75 mg/kg s.c.) and the indicated dose of levallorphan, and
analgesia was assessed 30 min later. (C) IBNtxA analgesia in KO mice. IBNtxA
analgesia (0.5 mg/kg) was determined in groups (n = 10) of WT, triple-KO
(exon 1 MOR-1/DOR-1/KOR-1), and exon 11 MOR-1 KO mice at 30 min. In the
triple-KO mice, analgesia was present and not significantly different from
WT mice. The responses of the exon 11 mice were significantly different
from WT and triple-KO mice. Statistical significance was assessed with the
Fisher exact test. (D) Respiratory rate. Animals were randomly assigned to
receive saline (n = 5), IBNtxA (2.5 mg/kg, n = 5), or morphine (20 mg/kg, n =
5). Each animal’s baseline average breath rate was measured every 5 min for
25 min before drug injection, and breath rates after drug injection are
expressed as a percent of baseline. IBNtxA did not depress respiratory rate
and was not significantly different from saline at any time point, whereas
morphine decreased respiratory depression in comparison with both saline
and IBNtxA (P < 0.001) as determined by repeated-measures ANOVA fol-
lowed by Bonferroni multiple-comparison test. (E) Gastrointestinal transit.
Groups of mice (n = 10) received saline, morphine (5 mg/kg), or IBNtxA (0.3,
0.6, and 1.5 mg/kg) before receiving an oral dose of 0.2 mL of charcoal meal
(2.5% gum tragacanth in 10% activated charcoal in water) by gavage.
Animals were killed 30 min later, and the distance traveled by charcoal was
measured. IBNtxA lowered transit significantly compared with saline (P <
0.05) but less than morphine (P < 0.05) as determined by ANOVA followed
by Tukey’s multiple-comparison test. The inhibition of gastrointestinal
transit seems to plateau even at doses three times higher than the ED50. (F)
Physical dependence. Groups of mice (n ≥ 10) received either morphine (10
mg/kg s.c.) or IBNtxA (1 mg/kg s.c.) for 10 d. They then were challenged with
IBNtxA pharmacology. (A) IBNtxA analgesia. Groups of mice (n = 10)
the indicated antagonist. Naloxone (Nal) precipitated a profound with-
drawal syndrome in the morphine-treated animals, as shown by the number
of jumps per 15 min, which was significantly greater than that in the mor-
phine or IBNtxA controls (i.e., given no antagonist) or in IBNtxA mice given
the indicated antagonist. Mice chronically administered IBNtxA showed no
significant difference from controls when challenged by either naloxone
(Nal, 1 mg/kg s.c.) or levallorphan (Levall, 1 mg/kg s.c.). (G) Conditioned place
preference. Mice randomly were assigned to receive saline (n = 10), IBNtxA
(1 mg/kg, n = 10), or morphine (10 mg/kg, n = 13) in a two-compartment
conditioned place preference assay. The difference score was obtained by
subtracting the amount of time (seconds) spent in the drug-paired com-
partment on test day (Test) from the amount of time (seconds) spent in the
drug-paired compartment on the preconditioning day (Pre). IBNtxA did
not produce place preference and was not significantly different from saline
(P > 0.05), whereas morphine showed preference in comparison with both
saline and IBNtxA (P < 0.05) as determined by ANOVA followed by Tukey’s
| www.pnas.org/cgi/doi/10.1073/pnas.1115231108Majumdar et al.
rewarding nor aversive behavior in conditioned place preference
studies. However, it is possible to further optimize the pharma-
cology. Structure–activity relationships reveal compounds with in-
creased potency and selectivity. Thus, the importance of IBNtxA
comes from opening a unique area of drug development and
a better understanding of receptor targets rather than its own
The mechanism(s) of IBNtxA actions are quite unique and
distinct from any of the traditional opioid receptors. Its potent
reversal by the opiate antagonist levallorphan implies an opiate
mechanism, but its relative insensitivity against several selective
antagonists separates it from the classical drugs. The KO models
give greater insights into the target. The persistence of IBNtxA
analgesia and normal levels of125I-BNtxA binding in the triple-
KO mice rules out a role for delta and kappa1receptors or any of
the full-length MOR-1 splice variants, which all contain exon 1.
On the other hand, the loss of both IBNtxA analgesia and re-
ceptor binding in the exon 11 KO mice clearly implicates a role
for exon 11-associated variants that contain only 6-TM domains.
Heterodimerization has been well demonstrated within the
opioid receptor family, with some complexes showing pharma-
cological profiles unlike either component examined alone (26,
34–39). However, these studies involved dimerization of full-
length receptors. Our studies suggest that heterodimerization of
truncated variants can generate new pharmacological targets.
When examined directly, we failed to see any125I-BNtxA binding
when the 6-TM variant MOR-1G was expressed alone, leading
us to consider whether the target required a partner. We tried
branes from either WT, exon 1, exon 11, or exon 2 KO mice were incubated
with125I-BNtxA (0.3 nM) in the presence of mu (CTAP), kappa1(U50,488H),
or delta (DPDPE) blockers at 1 μM. The triple-KO mice were assayed without
blockers. Only specific binding is reported. The assay was replicated at least
three times. ANOVA revealed no differences for the WT, exon 1, or triple-KO
mice, whereas binding was lost in exon 11 KO mice and markedly reduced in
exon 2 KO mice (P < 0.05). (B) Saturation studies with125I-BNtxA.125I-BNtxA
saturation studies were carried out on mice brain membranes from either
triple-KO or WT mice with blockers. Results are from a representative ex-
periment, and only specific binding is reported. Experiments were replicated
at least three times. KDand Bmaxwere determined by nonlinear regression
analysis (Prism), and the means ± SEM of the replicates were determined. KD
values were best fit with a single site and were as follows: Triple-KO mice,
KD= 0.16 ± 0.04 nM, Bmax= 60.84 ± 1.62 fmol/mg; WT mice, KD= 0.12 ± 0.04 nM,
Bmax= 47 ± 8.4 fmol/mg. There was no significant difference between KDor
Bmaxvalues for the two groups (t test, P > 0.05). (C)125I-BNtxA binding in
MOR-1G- and ORL1-expressing HEK cells. HEK cells were transiently trans-
fected with MOR-1G or ORL1alone or both together. Cell membranes from
all three conditions were incubated with
represent specific binding and were best fit with a single site. The study was
replicated three times, and means ± SEM of the independent replications
revealed KD= 0.24 ± 0.03 nM, Bmax= 18.1 ± 2.21 fmol/mg. No specific
binding was detectible in control cells expressing ORL1or MOR-1G alone or
in nontransfected HEK cells.
125I-BNtxA binding. (A)125I-BNtxA binding in KO mice. Brain mem-
125I-BNtxA (0.08–2 nM). Results
triple-KO and WT mice brain membranes
Competition opioid binding assays with125I-BNtxA in
Morphine, CTAP, DAMGO,
β-endorphin, codeine, meperidine,
DPDPE, SNC80, U50,488H,
dynorphin A, α-neoendorphin,
orphanin FQ/nociceptin (OFQ/N),
OFQ/N(1–11), J-113397, JTC801,
(+)-SKF10,047, (+) pentazocine,
226 ± 40
35.6 ± 5.3
51.9 ± 1.4
20.5 ± 1.8
256 ± 10.2
37.8 ± 10.6
53.2 ± 7.13
32.7 ± 4.1
22.9 ± 3.3
3.3 ± 1.9
29.1 ± 0.02
4.94 ± 0.99
26.3 ± 2.3 28.09 ± 10.96
0.59 ± 0.15
3.71 ± 1.45
8.8 ± 2.5
0.34 ± 0.018
0.04 ± 0.01
13.5 ± 1.6
2.67 ± 0.83
0.21 ± 0.11
1.81 ± 0.67
3.47 ± 1.18
2.92 ± 1.55
2.2 ± 0.71
1.8 ± 0.93
1.0 ± 0.3
7.5 ± 0.6
5 ± 1.5
0.5 ± 0.15
3.14 ± 1.08
22.2 ± 1.9
3.99 ± 1.1
0.2 ± 0.1
4.59 ± 0.58
8.5 ± 5.39
2.75 ± 0.13
3.0 ± 0.86
2.13 ± 0.27
125I-BNtxA competition studies were carried out in mice brain homoge-
nates from triple-KO and WT mice with blockers to prevent binding to
traditional mu, kappa1, and delta opioid receptors as described in Materials
Majumdar et al.PNAS
| December 6, 2011
| vol. 108
| no. 49
coexpression of MOR-1G with ORL1and observed the formation
of a very high-affinity125I-BNtxA binding site (KD= 0.24 nM).
We chose to test ORL1for a number of reasons. First,
BNtxA does not label ORL1when it is expressed alone, making
the detection of a new site simpler. Second, we had already
demonstrated that the full-length MOR-1 will dimerize with
ORL1(34). However, the ability of ORL1to partner with MOR-
1G to form the binding site does not necessarily mean that ORL1
is the endogenous partner. It is entirely possible that the 6-TM
variants can associate with one or more other candidates. Thus,
our findings further illustrate that inactivity of a truncated variant
when expressed alone does not necessarily mean that a truncated
receptor is inconsequential and without relevance, an observation
that may prove important with other truncated G protein-
Classification of the target is not straightforward. Although the
KO studies clearly implicate 6-TM variants generated by the mu
receptor MOR-1 gene Oprm1, its endogenous partner remains
unknown. Pharmacologically, the125I-BNtxA binding site profile
in brain tissue does not correspond to traditional mu, delta, or
kappa1receptors based on its insensitivity toward highly selective
mu, delta, or kappa1agents. However, it has extremely high af-
finity for many of the drugs long considered to be kappa, in-
cluding ketocyclazocine, which was the prototypic drug used by
Martin et al. to define the receptor class (21). It is interesting
that the125I-BNtxA binding profile resembles that of the kappa3
binding site identified with [3H]NalBzoH (17, 18) and that many
of the analgesics proposed to act through that site (17–20, 24, 25)
lost activity in the MOR-1 exon 11 KO mouse. Regardless of the
nomenclature, this target offers a major advance in the design
and development of new highly potent opiates without many of
the drawbacks of the drugs currently available.
Materials and Methods
Male CD-1 and C57 and exon 2 MOR-1 KO mice (25–35 g) were obtained from
Charles River Breeding Laboratories. The exon 1 KO and triple-KO mice came
from J.P.’s laboratory (14, 40), and the exon 11 mice were from our labo-
ratory (16). All mice were maintained on a 12-h light/dark cycle with Purina
rodent chow and water available ad libitum, housed in groups of five until
testing. All animal studies were approved by the Institutional Animal Care
and Use Committee of the Memorial Sloan-Kettering Cancer Center.
Opiates were a gift from the Research Technology Branch of the National
Institute on Drug Abuse (Rockville, MD). IBNtxA and125I-BNtxA were syn-
thesized as previously described (9). Na125I was obtained from Perkin-Elmer.
Miscellaneous chemicals and buffers were purchased from Sigma-Aldrich. All
receptor clones are murine, unless otherwise noted.
125I-BNtxA Binding Assays. Binding assays were carried out in whole-brain
membrane homogenates prepared as previously described (17). Binding in
WT, exon 1, exon 2, and exon 11 knockout mice was carried out in the
presence of mu [D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP)], kappa1
(U50,488H), and delta (DPDPE) blockers at either 1 μM (saturation studies) or
200 nM (competition studies). Binding in triple-KO mice was carried out
in the absence of any blockers. Nonspecific binding was defined by leval-
lorphan (8 μM) and was subtracted from total binding to yield specific
binding. Protein concentrations were determined as described in ref. 41.
KD, Bmax, and Ki values were calculated by nonlinear regression analysis
Analgesia Assays. Tail-flick analgesia was assessed quantally as a doubling or
greater of the baseline tail-flick latency, which ranged from 2 to 3 s, in the
radiant heat tail-flick technique (2, 4, 42, 43), with a maximal 10-s latency to
minimize tissue damage (4, 43). Analysis of the data using graded responses
with %MPE yielded similar responses. The hot plate assay was performed at
55 °C (Ugo Basile) (16). The time(s) elapsing to the first pain response (hind
paw licking or jumping) was scored. A maximal latency of 30 s was used to
minimize any tissue damage. Results were determined as %MPE [(latency
after drug − baseline latency)/(30 − baseline latency)].
Gastrointestinal Motility Assay. Gastrointestinal transit was determined as
described in ref. 5. Animals received the indicated drug followed by a char-
coal meal by gavage. Animals were killed 30 min later, and the distance
traveled by charcoal was measured.
Conditional Place Preference/Aversion. The testing apparatus consisted of two
compartments of equal size separated by a wall with a guillotine-style door
(ENV-512 insert; MedAssociates). One compartment was surrounded by
white walls and had a rod floor, and the other had black walls and a grid
at all times.
Animals were habituated to the environment for 3 h for each of 2 d before
testing and for 1 h on each conditioning session. Baseline preferences were
determined on the preconditioning test day by letting animals explore both
sides freely for 20 min, and the side in which they initially spent more time in
was assigned to saline in the place preference study. Animals were injected on
alternating days for 8 d with either drug or saline and restricted to one
compartment for 20 min. On the postconditioning testing day, animals were
placed in the side paired with saline and allowed to freely explore both
compartments for 20 min. The time spent in each compartment post-
conditioning was calculated and subtracted from the amount of time spent in
each compartment preconditioning to determine the change in each animal’s
preference attributable to conditioning.
Tolerance and Dependence Studies. Tolerance was induced by twice-daily
injections with either morphine (6 mg/kg s.c.) or IBNtxA (1 mg/kg s.c.) or
was determined on day 3 after pellet implantation with either IBNtxA (1 mg/
kg s.c.) ornaloxone (1mg/kgs.c.) to precipitate withdrawal, andanimals were
evaluated for signs of diarrhea and jumping (4, 15).
Respiratory Depression Assessment. Respiratory rate was assessed in awake,
freely moving, adult male CD1 mice with the MouseOx pulse oximeter system
(Starr Life Sciences). Each animal was habituated to the device for 30 min and
for each animal was obtained over a 25-min period before drug injection, and
testing began at 15 min postinjection and continued for a period of 35 min.
Groups of mice (n = 5) were treated s.c. with either morphine (20 mg/kg) or
IBNtxA (2.5 mg/kg) at doses approximately four times their analgesic ED50.
Groups were compared with repeated-measures ANOVA followed by Bonfer-
roni multiple-comparison test.
ACKNOWLEDGMENTS. This work was supported in part by National Institute
on Drug Abuse Grants DA02615, DA06241, DA07242, and DA00220 (to
G.W.P.); DA013997 and DA029244 (to Y.-X.P.); and DA018257 (to J.P.); as
well as the Technology Development Fund of Memorial Sloan-Kettering
Cancer Center (G.W.P.).
Table 2.Opioid analgesia in MOR-1 exon 11 KO mice
ED50, mg/kg s.c.
DrugWTExon 11 KO Shift
0.53 ± 0.07
22 ± 5.3
0.18 ± 0.04
46.96 ± 15.26
0.1 ± 0.01
5.94 ± 3.88
1.58 ± 0.17
1.53 ± 0.06
21.8 ± 10
0.72 ± 0.13
2.58 ± 0.52
1.8 ± 0.14
Dose–response curves were performed for each ligand and replicated at
least three times unless indicated otherwise. All mice were on the C57 back-
ground. Results are the means ± SEM of the independent determinations.
The results for morphine and methadone are from the literature (15).
A number of drugs could not be tested at very high concentrations because
of solubility issues. If the ED50could not be attained, it is stated as greater
than the highest dose tested. The shifts for morphine and methadone are
not significant, but the one for ketocyclazocine is (*P < 0.006).
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| December 6, 2011
| vol. 108
| no. 49