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Orally Active Opioid mu/delta Dual Agonist MGM-16, a Derivative of the

Indole Alkaloid Mitragynine, Exhibits Potent Antiallodynic Effect on
Neuropathic Pain in Mice

Article in Journal of Pharmacology and Experimental Therapeutics · December 2013

DOI: 10.1124/jpet.113.208108 · Source: PubMed

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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 348:383–392, March 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics

Orally Active Opioid m/d Dual Agonist MGM-16, a Derivative of

the Indole Alkaloid Mitragynine, Exhibits Potent Antiallodynic
Effect on Neuropathic Pain in Mice s

Kenjiro Matsumoto, Minoru Narita, Naotaka Muramatsu, Terumi Nakayama, Kaori Misawa,
Mariko Kitajima, Kimihito Tashima, Lakshmi A. Devi, Tsutomu Suzuki, Hiromitsu Takayama,

Downloaded from jpet.aspetjournals.org at Josai Kokusai UnivLib Yakugaku-Bu(KIN) on February 6, 2014

and Syunji Horie
Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International University, Chiba, Japan (K.Ma., N.M., K.T.,
S.H.); Department of Toxicology (K.Ma., M.N., T.S.) and Department of Pharmacology (K.Ma., M.N.), Hoshi University School of
Pharmacy and Pharmaceutical Sciences, Tokyo, Japan; Department of Molecular Structure and Biological Function, Graduate
School of Pharmaceutical Sciences, Chiba University, Chiba, Japan (T.N., K.Mi., M.K., H.T.); and Department of Pharmacology
and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York (L.A.D.)
Received September 26, 2013; accepted December 9, 2013

ABSTRACT
(E)-Methyl 2-((2S,3S,7aS,12bS)-3-ethyl-7a-hydroxy-8-methoxy- deferens contractions. Systemic administration of MGM-16
1,2,3,4,6,7,7a,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3- produced antinociceptive effects in a mouse acute pain model
methoxyacrylate (7-hydroxymitragynine), a main active constituent and antiallodynic effects in a chronic pain model. The anti-
of the traditional herbal medicine Mitragyna speciosa, is nociceptive effect of MGM-16 was approximately 240 times
an indole alkaloid that is structurally different from morphine. more potent than that of morphine in a mouse tail-flick test, and
7-Hydroxymitragynine induces a potent antinociceptive effect its antiallodynic effect was approximately 100 times more potent
on mouse acute pain through m-opioid receptors. In this study, than that of gabapentin in partial sciatic nerve-ligated mice,
we developed dual-acting m- and d-opioid agonists MGM-15 especially with oral administration. The antinociceptive effect
and MGM-16 from 7-hydroxymitragynine for the treatment of of MGM-16 was completely and partially blocked by the
acute and chronic pain. MGM-16 showed a higher potency than m-selective antagonist b-funaltrexamine hydrochloride (b-FNA)
that of 7-hydroxymitragynine and MGM-15 in in vitro and in vivo and by the d-selective antagonist naltrindole, respectively, in a
assays. MGM-16 exhibited a high affinity for m- and d-opioid tail-flick test. The antiallodynic effect of MGM-16 was completely
receptors, with Ki values of 2.1 and 7.0 nM, respectively. MGM- blocked by b-FNA and naltrindole in a neuropathic pain model.
16 showed m- and d-opioid full agonistic effects in a guanosine These findings suggest that MGM-16 could become a class of a
59-O-(3-[35S]thiotriphosphate) binding assay and in a functional compound with potential therapeutic utility for treating neuro-
test using electrically elicited guinea pig ileum and mouse vas pathic pain.

Introduction
Morphine has been proposed for the treatment of neuro-
pathic pain, but it has incomplete efficacy and dose-limiting
adverse effects (Gilron et al., 2005). To develop additional
This work was supported in part by Grants-in-Aid for Scientific Research
from the Ministry of Culture, Sports, Science, and Technology of Japan;
analgesics, derivatives have been synthesized by simplification
a Grant-in-Aid for Scientific Research from the Japan Society for the and introduction of substituents into morphine’s chemical
Promotion of Science; Special Funds for Education and Research (Development
of SPECT Probes for Pharmaceutical Innovation) from the Ministry of
structure (Corbett et al., 2006). Thus, most morphine-derived
Education, Culture, Sports, Science and Technology of Japan; and Iodine opioid analgesics used clinically have m-receptor agonist pro-
Research Project at Chiba University.
dx.doi.org/10.1124/jpet.113.208108.
files. Although all three major types of opioid receptors (m, d,
s This article has supplemental material available at jpet.aspetjournals.org. and k) are able to mediate analgesia and antinociception, they

ABBREVIATIONS: CHO, Chinese hamster ovary; DAMGO, [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin; DPDPE, [D-Pen2, D-Pen5]-enkephalin; b-FNA,
b-funaltrexamine hydrochloride; GIT, gastrointestinal transit; 7-hydroxymitragynine, (E)-methyl 2-((2S,3S,7aS,12bS)-3-ethyl-7a-hydroxy-8-
methoxy-1,2,3,4,6,7,7a,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate; IGIT, inhibitory effects on gastrointestinal transit; MGM-9,
(E)-methyl 2-(3-ethyl-7a,12a-(epoxyethanoxy)-9-fluoro-1,2,3,4,6,7,12,12b-octahydro-8-methoxyindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate; MGM-15, (E)-
methyl 2-((2S,3S,7aS,12aR,12bS)-3-ethyl-7a-hydroxy-8-methoxy-1,2,3,4,6,7,7a,12,12a,12b-decahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate;
MGM-16, (E)-methyl 2-((2S,3S,7aS,12aR,12bS)-3-ethyl-9-fluoro-7a-hydroxy-8-methoxy-1,2,3,4,6,7,7a,12,12a,12b-decahydroindolo[2,3-a]quinolizin-2-
yl)-3-methoxyacrylate; norBNI, nor-binaltorphimine dihydrochloride; NTI, naltrindole; U69593, (5a,7a,8b)-(1)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro
[4.5]dec-8-yl]-benzeneacetamide.

383
384 Matsumoto et al.

have different pharmacological activities. Recently, a prom- physiologic conditions. Furthermore, we examined the anti-
inent role of d-opioid receptors in chronic pain such as allodynic effects of MGM-16 in a mouse sciatic nerve ligation
neuropathic pain and inflammatory pain was reported from model. We found that the orally active m- and d-opioid receptor
studies using mutant animals and selective agonists (Nadal agonist MGM-16 produces potent effects against acute and
et al., 2006; Nozaki et al., 2012). In a neuropathic pain rat neuropathic pain.
model, d-opioid receptor protein expression was increased
compared with a control rat in the dorsal root ganglion (Kabli
and Cahill, 2007). d-Opioid receptor activation leads to de- Materials and Methods
creased chronic pain but weakly influences acute pain, in Preparation of MGM-15. NaBH4 (4.3 mg, 0.11 mmol) was added
contrast to m-opioid receptor activation (Gaveriaux-Ruff et al., to a stirred solution of 7-hydroxymitragynine (37.5 mg, 0.091 mmol) in
2011). A novel strategy for pain management is to use dual- dry MeOH (1.1 ml) at 0°C under an argon atmosphere. After 30
acting and/or mixed opioid agonists (Matsumoto et al., 2008; minutes, H2O was added to the reaction mixture. The mixture was
Cremeans et al., 2012). Therefore, opioid agonists, which act on concentrated under reduced pressure and poured into saturated
not only d- but also m-opioid receptor subtypes, might be broad- aqueous NaHCO3 solution. The whole mixture was extracted with 5%
spectrum analgesics useful to treat a variety of painful MeOH/CHCl3 three times. The combined extract was washed with
conditions. brine, dried over MgSO4, and evaporated to give a residue that was pu-
rified by silica gel column chromatography (ethyl acetate/n-hexane 5
The traditional herbal medicine Mitragyna speciosa has
35:65). The purified compound was then crystallized from ethyl
long been used in Thailand for its opioid-like effects (Burkill, acetate to give 26.3 mg of MGM-15 (y. 70%).
1935) and as a replacement for opium (Suwanlert, 1975). This MGM-15; m.p.: 219–223°C (ethyl acetate). UV (MeOH) lmax nm (log
medicinal herb contains many indole alkaloids (Takayama, e): 288 (3.13), 277 (3.06), 239 (4.16), 232 (4.14), 214 (4.50). Infrared
2004). (E)-Methyl 2-((2S,3S,7aS,12bS)-3-ethyl-7a-hydroxy-8- spectroscopy (KBr) nmax cm21: 3341, 2954, 1698, 1614, 1467, 1283. 1H
methoxy-1,2,3,4,6,7,7a,12b-octahydroindolo[2,3-a]quinolizin- NMR (500 MHz, CDCl3) d ppm: 7.40 (1H, s, H-17), 7.00 (1H, dd, J 5
2-yl)-3-methoxyacrylate (7-hydroxymitragynine), a main active 8.1, 8.1 Hz, H-11), 6.35 (1H, d, J 5 7.9 Hz, H-12), 6.31 (1H, d, J 5 8.2
constituent of this plant, is an indole alkaloid that is struc- Hz, H-10), 3.87 (1H, br.s, Na-H), 3.83 (3H, s, 9-OCH3), 3.80 (3H, s, 17-
turally different from morphine (Horie et al., 2005). We have OCH3), 3.70 (3H, s, 22-OCH3), 3.47 (1H, br.s, H-2), 2.95 (2H, m, H-15
reported that 7-hydroxymitragynine induces a potent anti- and H-21), 2.94 (1H, s, 7-OH), 2.53 (2H, m, H-5 and H-14), 2.25 (1H,
ddd, J 5 12.3, 12.3, 2.3 Hz, H-5), 2.16 (2H, m, H-3 and H-21), 2.03 (1H,
nociceptive effect on mouse acute pain through the activation
d, J 5 14.3 Hz, H-6), 1.92 (1H, ddd, J 5 13.6, 13.6, 4.3 Hz, H-6), 1.79
of m-opioid receptors. It is approximately 14 times more (1H, m, H-19), 1.58 (1H, br.d, J 5 11.3 Hz, H-20), 1.39 (1H, d, J 5 12.8
potent than morphine when orally administered (Matsumoto Hz, H-14), 1.23 (1H, m, H-19), 0.85 (3H, dd, J 5 7.3, 7.3 Hz, H3-18). 13C
et al., 2008). Furthermore, 7-hydroxymitragynine inhibited NMR (125 MHz, CDCl3) d ppm: 169.1 (C-22), 160.3 (C-17), 155.9 (C-9),
gastrointestinal transit (GIT) less potently than morphine at 149.9 (C-13), 129.3 (C-11), 121.3 (C-8), 111.8 (C-16), 105.2 (C-12),
each equally antinociceptive dose in mice (Matsumoto et al., 101.9 (C-10), 77.1 (C-7), 69.8 (C-2), 61.6 (17-OCH3), 61.6 (C-3), 58.5 (C-
2006a). We investigated the structural similarities between 21), 55.1 (9-OCH3), 51.2 (22-OCH3), 50.7 (C-5), 40.7 (C-20), 40.2 (C-15),
morphine and 7-hydroxymitragynine using molecular model- 35.2 (C-6), 28.5 (C-14), 19.1 (C-19), 13.1 (C-18). Electron ionization
ing techniques. However, we could not superimpose all three mass spectrometry (%) m/z: 416 (M1, 61), 400 (96), 399 (100), 398 (97),
functional groups, i.e., a nitrogen atom, a benzene residue, 397 (78), 383 (41), 256 (64), 214 (84). Anal. Calcd for C23H32O5N2: C,
66.32; H, 7.74; N, 6.73. Found: C, 66.08; H, 7.77; N, 6.71. CD (c 5 0.29
and an oxygen atom on the benzene ring in their structures
mM, MeOH, 24°C), Δ« (l nm): 0 (318), 11.4 (293), 0 (257), 21.0 (248),
(Matsumoto et al., 2005a). Therefore, we developed novel 0 (233), 10.2 (231), 0 (227), 27.7 (215), 10.1 (208).
opioid analgesics derived from 7-hydroxymitragynine that have Preparation of MGM-16. NaBH4 (2.1 mg, 0.056 mmol) was
different pharmacophore groups from morphine that bind to added to a stirred solution of 10-fluoro-7-hydroxymitragynine (23.7
m-opioid receptors. We found a novel dual-acting m- and k-opioid mg, 0.055 mmol) in dry MeOH (0.5 ml) at 0°C under an argon
agonist, (E)-methyl 2-(3-ethyl-7a,12a-(epoxyethanoxy)-9- atmosphere. The reaction mixture was stirred for 30 minutes. After
fluoro-1,2,3,4,6,7,12,12b-octahydro-8-methoxyindolo[2,3-a] adding H2O, the reaction mixture was poured into saturated aqueous
quinolizin-2-yl)-3-methoxyacrylate (MGM-9), which has stronger NaHCO3 solution. The whole mixture was extracted with 5% MeOH/
antinociceptive and weaker rewarding effects than morphine CHCl3 three times. The combined extract was washed with brine,
(Matsumoto et al., 2008). 7-Hydroxymitragynine-related dried over MgSO4, and evaporated to give a residue that was purified
by amino-silica gel column chromatography (ethyl acetate/n-hexane 5
indole alkaloids have interesting pharmacological charac-
1:1) to give 22.6 mg of MGM-16 (y. 95%).
teristics such as high oral potency and few side effects. MGM-16; UV (MeOH) lmax nm (log e): 297 (3.13), 276 (3.10), 240
Therefore, further investigation of the development of novel (4.22), 226 (4.11), 205 (4.60). Infrared spectroscopy (KBr) nmax cm21:
analgesics against acute and chronic pain is warranted. 3364, 2947, 1701, 1626, 1490, 1284, 1240. 1H NMR (500 MHz, CDCl3)
In this study, we hypothesized that a dual-acting m- and d ppm: 7.41 (1H, s, H-17), 6.76 (1H, dd, J 5 12.8, 8.2 Hz, H-11), 6.28
d-opioid agonist derived from 7-hydroxymitragynine could not (1H, dd, J 5 8.5, 3.1 Hz, H-12), 4.01 (3H, d, J 5 2.7 Hz, 9-OCH3), 3.79
only induce potent antinociceptive effects against acute pain (3H, s, 17-OCH3), 3.69 (3H, s, 22-OCH3), 3.46 (1H, d, J 5 3.1 Hz, H-2),
but also induce an antiallodynic effect against neuropathic 2.95 (2H, m, H-15 and H-21), 2.77 (1H, br.s, 7-OH), 2.52 (2H, m, H-5
pain. We synthesized the novel m/d-opioid dual agonists and H-14), 2.25 (1H, m, H-5), 2.15 (2H, m, H-3 and H-21), 1.95 (2H, m,
(E)-methyl 2-((2S,3S,7aS,12aR,12bS)-3-ethyl-7a-hydroxy-8-methoxy- H-6), 1.76 (1H, m, H-19), 1.58 (1H, br.d, J 5 11.3 Hz, H-20), 1.37 (1H,
d, J 5 12.8 Hz, H-14), 1.23 (1H, m, H-19), 0.84 (3H, dd, J 5 7.3, 7.3 Hz,
1,2,3,4,6,7,7a,12,12a,12b-decahydroindolo[2,3-a]quinolizin-2-yl)-
H3-18). 13C NMR (125 MHz, CDCl3) d ppm: 169.0 (C-22), 160.3 (C-17),
3-methoxyacrylate (MGM-15) and (E)-methyl 2-((2S,3S,7aS, 148.4 (d, J 5 237.4 Hz, C-10), 145.1 (C-13), 143.2 (d, J 5 11.9 Hz, C-9),
12aR,12bS)-3-ethyl-9-fluoro-7a-hydroxy-8-methoxy-1,2,3,4, 126.8 (C-8), 116.4 (d, J 5 21.5 Hz, C-11), 111.7 (C-16), 105.8 (d, J 5 6.9
6,7,7a,12,12a,12b-decahydroindolo[2,3-a]quinolizin-2-yl)-3- Hz, C-12), 77.4 (C-7), 70.0 (C-2), 61.6 (17-OCH3), 61.4 (C-3), 61.2 (d,
methoxyacrylate (MGM-16) and clarified their pharmacolog- J 5 7.8 Hz, 9-OCH3), 58.4 (C-21), 51.2 (22-OCH3), 50.5 (C-5), 40.7
ical profiles using in vitro and in vivo experiments under (C-20), 40.1 (C-15), 35.2 (C-6), 28.4 (C-14), 19.0 (C-19), 13.1 (C-18).
 MGM-16, a Novel Dual-Acting m- and d-Opioid Agonist 385
CD (c 5 0.26 mM, MeOH, 24°C), Δ« (l nm): 0 (337), 12.7 (297), 0 (255), equilibration, the tissues were transmurally stimulated with a train
24.2 (236), 23.8 (227), 28.4 (214), 10.1 (207). Fast atom bombardment of 10 pulses of 0.5-ms duration with 2-ms intervals every 1 minute by
(nitrobenzyl alcohol) mass spectrometry m/z: 435 [M1H]1. High- a stimulator (SEN-7203). Contractions were isometrically recorded by
resolution fast atom bombardment (nitrobenzyl alcohol/polyethylene using a displacement transducer (TB-651T; Nihon Kohden). The
glycol) mass spectrometry: calcd. for C23H32O5N2F: 435.2295, found: effects of drug treatments on the twitch contractions evoked by
435.2301. transmural stimulation from the ring electrodes were examined. The
Drugs. The drugs used in this study were morphine hydrochloride height of the twitch response to transmural stimulation was
(Takeda Chemical Industries, Osaka, Japan), naloxone hydrochloride measured before and after the drug challenge. The responses were
(MP Biomedicals, Irvine, CA), gabapentin (Cayman Chemical Com- expressed as contraction percentage of the twitch response to the
pany, Ann Arbor, MI), naltrindole hydrochloride, nor-binaltorphimine transmural stimulation before the drug challenge as 100%.
dihydrochloride (norBNI), [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin Receptor Binding. Guinea pig brain membrane protein (without
(DAMGO), [D-Pen2, D-Pen5]-enkephalin (DPDPE), (5a,7a,8b)-(1)-N- cerebellum, 500 mg) fractions were incubated with 1 nM [3H]DAMGO
methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (50.0 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) or
(U69593), cyprodime hydrobromide, and [Met]-enkephalin (Sigma- 1 nM [3H]U69593 (55.0 Ci/mmol; GE Healthcare, Little Chalfont,
Aldrich, St. Louis, MO) and b-funaltrexamine hydrochloride (b-FNA; Buckinghamshire, UK) in a final volume of 1 ml of 50 mM Tris-HCl
Tocris-Cookson, Bristol, UK). buffer. Rat brain membrane protein fractions were incubated with
For experiments with guinea pig ileum and mouse vas deferens, 2 nM [3H]DPDPE (25.2 Ci/mmol; PerkinElmer Life and Analytical
7-hydroxymitragynine-related indole alkaloids and cyprodime hydro- Sciences, Boston, MA) in a final volume of 1 ml of 50 mM Tris-HCl
bromide were first dissolved in 100% dimethylsulfoxide to yield a buffer. Nonspecific binding was measured by the inclusion of 10 mM
5 mM solution, and then subsequently diluted with distilled water. naloxone. The incubation periods were 1, 2.5, and 1 hour for
The other drugs were dissolved in distilled water. For subcutaneous [3H]DAMGO, [3H]DPDPE, and [3H]U69593, respectively, at 25°C.
administration, 7-hydroxymitragynine-related indole alkaloids were The reaction was terminated by rapid filtration under reduced
dissolved in 0.01 N hydrochloric acid. Other drugs were dissolved in pressure through glass fiber filters (Whatman GF/C, presoaked in
saline. For oral administration and receptor-binding assays, MGM- 0.25% polyethyleneimine; Whatman, Clifton, NJ). After filtration,
15, MGM-16, 7-hydroxymitragynine, and gabapentin were dissolved filters were washed three times with 3 ml of cold 50 mM Tris-HCl and
in 0.01 N hydrochloric acid. counted in 4 ml of Ultima Gold scintillation cocktail (PerkinElmer
In the antinociceptive test, naloxone hydrochloride (2 mg/kg), Life and Analytical Sciences). The ability of unlabeled drugs to inhibit
naltrindole (3 mg/kg), norBNI (20 mg/kg), or b-FNA (40 mg/kg) was specific radioligand binding was expressed as the IC50 value, which is
administered subcutaneously 15 minutes, 30 minutes, 24 hours, or 24 the molar concentration of the unlabeled drug necessary to displace
hours before drug injection, respectively. These protocols were 50% of the specific binding. Inhibition constants (Ki) of unlabeled
described by Matsumoto et al. (2005a). compounds were calculated as described by Cheng and Prusoff (1973).
Animals. Male ddY strain mice (Japan SLC, Hamamatsu, Japan), Guanosine 59-O-(3-[35S]Thiotriphosphate) Binding Assay.
male Hartley strain guinea pigs (Japan SLC), and male SD rats The agonist activities of the test compounds (MGM-15 and MGM-
(Japan SLC) were used. Animals were housed in a temperature- 16) and DAMGO for m-opioid receptor– or Met-enkephalin for d-opioid
controlled room at 24°C with lights on from 7:00 AM to 5:00 PM and receptor–positive controls were measured by radioactivities of
free access to food and water. All experiments were performed in guanosine 59-O-(3-[35S]thiotriphosphate) ([35S]GTPgS). m-Opioid re-
compliance with the Guiding Principles for the Care and Use of ceptor and d-opioid receptor agonist activities were performed using
Laboratory Animals approved by the Japanese Pharmacological a membrane fraction prepared from Chinese hamster ovary (CHO-
Society and the guidelines approved by the Ethics Committee on K1) K1 cells expressing recombinant m- and d-opioid receptors. The
Animal Care and Animal Experimentation of Josai International membranes of cells stably expressing rat m- and d-opioid receptors
University. The number of animals used was kept to the minimum were suspended with binding assay buffer [50 mM Tris-HCl (pH 7.4)
necessary for a meaningful interpretation of the data, and animal containing EDTA (1 mM), MgCl2 (12.5 mM), NaCl (100 mM), bovine
discomfort was kept to a minimum. serum albumin (0.5%), and GDP (3 mM)]. The membranes (10 mg)
Electrical Stimulation of Guinea Pig Ileum. Guinea pig ileum were incubated at 25°C for 1 hour in 200 ml of binding assay buffer
was dissected and placed in Krebs-Henseleit solution (in mM: NaCl, with various concentrations of the test compound or positive control
112.08; KCl, 5.90; CaCl2, 1.97; MgCl2, 1.18; NaH2PO4, 1.22; NaHCO3, (0.1–100,000 nM) and 50 pM [35S]GTPgS (specific activity, 1000 Ci/
25.00; and glucose, 11.49). The ileum was placed under 1g tension in mmol; PerkinElmer Life and Analytical Sciences) in a 96-well plate.
a 10-ml organ bath containing the nutrient solution. The bath was The reaction was terminated by filtration, and MicroScint-20
maintained at 37°C and continuously bubbled with a mixture of 95% (PerkinElmer Life and Analytical Sciences) was added to the plate.
O2 and 5% CO2. Tissues were stimulated by a platinum needle-ring Radioactivity in the samples was determined using a liquid scintil-
electrode (Iwashiya Kishimoto Medical Instruments, Kyoto, Japan). lation analyzer. Nonspecific binding was measured in the presence of
After equilibration, the ileum was transmurally stimulated with 10 mM unlabeled GTPgS. Agonist activation by each compound was
monophasic pulses (0.2 Hz and 0.1-ms duration) by a stimulator expressed as the percentage stimulation, which was calculated as (T1 –
(SEN-7203; Nihon Kohden, Tokyo, Japan). Contractions were iso- T0)/(T2 – T0)  100, where T0 is the nonspecific activity, T1 is the
tonically recorded using a displacement transducer (TD-112S; Nihon [35S]GTPgS activity in the presence of various concentrations of test
Kohden). The effects of drug treatments on the twitch contractions compounds or positive control, and T2 is the maximal stimulation of
evoked by transmural stimulation from the ring electrodes were [35S]GTPgS binding by the positive control.
examined. The height of the twitch response to transmural stimula- All measurements were performed in duplicate. Three independent
tion was measured before and after the drug challenge. Responses experiments were conducted in this study, and data are expressed as
were expressed as contraction % of the twitch response to the mean 6 S.E.M. The EC50 values (a concentration that produces 50% of
transmural stimulation before the drug challenge as 100%. maximal stimulation of the [35S]GTPgS binding) and Emax (percent-
Electrical Stimulation of Mouse Vas Deferens. Mouse vas age of maximal stimulation in the [35S]GTPgS binding) were
deferens was dissected and placed in Krebs-Henseleit solution without estimated from saturation analysis of agonist-stimulated [35S]GTPgS
MgCl2. The tissues were placed under 0.2g tension in a 10-ml organ binding using program XLfit (Microsoft, Redmond, WA).
bath containing the nutrient solution. The bath was maintained at Tail-Flick Test. Mice respond to a focused heat stimulus by
37°C and continuously bubbled with a mixture of 95% O2 and 5% CO2. flicking or moving their tail from the path of the stimulus, thereby
Tissues were stimulated by a platinum needle-ring (the ring was exposing a photocell located in the tail-flick analgesia meter (Ugo
placed 20 mm above the base of a 5-mm-long needle) electrode. After Basile Tail-flick Unit 7360; Ugo Basile, Comerio, Italy) immediately
386 Matsumoto et al.

below the tail. The reaction time is automatically recorded. Before 7-hydroxymitragynine and semisynthetic compounds derived
treatment with drugs, vehicle, or saline, the nociceptive threshold was from 7-hydroxymitragynine (Fig. 1) were evaluated by mea-
measured three times, and the mean of the reaction time was used as suring inhibition of the twitch contraction induced by electri-
the predrug latency for each mouse. A cutoff time of 10 seconds was cal stimulation in the guinea pig ileum preparation (Table 1).
used to prevent tissue damage.
MGM-15 and MGM-16 were prepared by NaBH4 reduction of
Antinociception in the tail-flick test was quantified using the per-
centage of maximum possible effect and calculated as: % maximum
7-hydroxymitragynine and 10-fluoro-7-hydroxymitragynine,
possible effect 5 [(test latency – predrug latency)/(cutoff time – respectively. MGM-15 and MGM-16 showed a higher potency
predrug latency)]  100. than 7-hydroxymitragynine, based on their pD2 values. The
GIT. Mice were fasted, with water available ad libitum, for 18 introduction of a fluoro group onto the C-10 position (MGM-
hours before the experiments. Fifteen minutes after subcutaneous 16) led to a higher potency than MGM-15. MGM-16 showed an
administration of 7-hydroxymitragynine, MGM-15, MGM-16, mor- inhibitory effect as potent as that of the m-selective agonist
phine, vehicle, or saline, a 0.25-ml charcoal meal (an aqueous suspen- DAMGO.
sion of 10% charcoal and 5% gum arabic) was orally administered. Effects of MGM-15 and MGM-16 on Electrically
Fifteen minutes after oral administration of 7-hydroxymitragynine, Stimulated Twitch Contraction in Mouse Vas Defer-
MGM-15, MGM-16, or vehicle, and 30 minutes after oral administra-
ens. To clarify the effect of MGM-15 and MGM-16 on d-opioid
tion of morphine or distilled water, a charcoal meal was orally ad-
ministered. These time points were established by the time of peak
receptors, we investigated the effect of MGM-15 and MGM-16
effect for each drug and by the protocol as previously reported using mouse vas deferens (Table 2). MGM-15 and MGM-16
(Matsumoto et al., 2006a). Thirty minutes after administration of the inhibited the electrically elicited mouse vas deferens contrac-
charcoal meal, the animal was killed by cervical dislocation, and the tion in a dose-dependent manner, as did 7-hydroxymitragy-
small intestine from the pylorus to the ileocecal junction was carefully nine and the d-selective agonist DPDPE. MGM-16 showed
removed. Both the length of the small intestine from the pylorus to the a higher potency than MGM-15. MGM-16 showed an in-
ileocecal junction and the farthest distance to which the charcoal meal hibitory effect as potent as that of DPDPE. The concentration-
had traveled were measured. For each animal, the GIT was calculated response curves for MGM-15 and MGM-16 were shifted to the
as the percentage of the distance traveled by the charcoal meal right in the presence of the m-opioid receptor antagonist
relative to the total length of the small intestine. The inhibition of GIT
cyprodime and k-opioid antagonist norBNI (Fig. 2). The
(%) was calculated as: inhibition of GIT (%) 5 [(saline or vehicle GIT –
drug GIT)/(saline or vehicle GIT)]  100.
curves of MGM-15 and MGM-16 were further shifted to the
Neuropathic Pain Model. The mice were anesthetized with 3% right in the presence of cyprodime, norBNI, and the d-selective
isoflurane. We produced a partial sciatic nerve injury by tying a tight antagonist naltrindole (Table 3).
ligature with an 8-0 silk suture around approximately one-third to Receptor Binding. The affinities of MGM-15 and MGM-
one-half the diameter of the sciatic nerve on the right side (ipsilateral 16 for the three opioid receptor types were determined by
side) under a light microscope (SD30; Olympus, Tokyo, Japan), as evaluating the inhibition of radioligand binding to m-, d-, and
described previously (Seltzer et al., 1990; Malmberg and Basbaum, k-opioid receptors (Table 3). MGM-15 and MGM-16 had
1998). In sham-operated mice, the nerve was exposed without a relatively high affinity for m- and d-opioid sites. The
ligation. The sciatic nerve–ligated mice exhibit thermal hyperalgesia [3H]DAMGO and [3H]DPDPE binding was displaced by
and mechanical hyperalgesia on the ipsilateral side, indicating the
MGM-15 and MGM-16 in a concentration-dependent manner
state of neuropathic pain hypersensitivity (Narita et al., 2008). In the
present study, the antinociceptive assay was performed 7 days after
in the membrane samples from guinea pig and rat brain,
partial sciatic nerve ligation. respectively (data not shown).The Ki value for MGM-15
Measurement of Mechanical Allodynia. The paw withdrawal
threshold was determined as described by Chaplan et al. (1994). The
mice were individually placed in suspended cages with wire mesh
bottoms and allowed to acclimate to their environment for at least 30
minutes. The hind paw was stimulated with a series of calibrated
(0.02–1.48 g) von Frey filaments applied to the plantar surface. The
50% paw withdrawal threshold that is nonparametrically distributed
was determined using the up-down paradigm. Testing was initiated
with 0.17-g hair, and a positive response was indicated by a withdrawal
of the paw. The paw withdrawal threshold was measured repeatedly at
the multiple time points indicated after administration of each drug.
Statistical Analysis. The data are expressed as the mean 6 S.E.M.
Statistical analyses were performed with the two-tailed Student’s t
test for comparison of two groups and by a one-way analysis of
variance followed by a Bonferroni multiple-comparisons test for compar-
ison of more than two groups. A P value , 0.05 was considered sta-
tistically significant. ED50 values and 95% confidence limits were
determined using the Litchfield-Wilcoxon method (Litchfield and
Wilcoxon, 1949). The statistical significance of differences between MGM-
15 and MGM-16 in [35S]GTPgS binding assay was assessed using two-way
analysis of variance followed by Bonferroni multiple-comparison tests.

Results
Effects of 7-Hydroxymitragynine-Related Indole
Alkaloids on Electrically Stimulated Twitch Contrac- Fig. 1. Chemical structure and conformation of 7-hydroxymitragynine,
tion in Guinea Pig Ileum. The opioid agonistic activities of MGM-15, and MGM-16.
 MGM-16, a Novel Dual-Acting m- and d-Opioid Agonist 387
TABLE 1
pD2 values and maximum percentage inhibition of mitragynine
derivatives in guinea pig ileum
Each value represents mean 6 S.E.M. of data obtained from four to six animals.

Compound pD2 Value Maximum Inhibition

%
Morphine 7.16 6 0.05 79.4 6 5.7
MGM-15 8.26 6 0.05 78.2 6 3.4
MGM-16 8.81 6 0.09 89.0 6 2.3
7-Hydroxymitragynine 7.58 6 0.12 79.6 6 3.0
DAMGO 8.46 6 0.06 87.2 6 1.8

displacement of [3H]DAMGO and [3H]DPDPE binding to m-

and d-opioid sites was 6.4 6 0.30 and 16 6 1.0 nM,
respectively. The Ki value for MGM-16 displacement of
[3H]DAMGO and [3H]DPDPE was 2.1 6 0.028 and 7.0 6
0.23 nM, respectively. MGM-15 and MGM-16 weakly displaced
[3H]U69593 binding to k-opioid sites.
[35S]GTPgS Binding Assay. We next investigated the
ability of MGM-15 and MGM-16 to activate G-proteins in
CHO-K1 cells expressing recombinant m- and d-opioid
receptors (Fig. 3; Table 4). The m-opioid agonist DAMGO or
d-opioid agonist [Met]-enkephalin produced a concentration-
dependent increase in [35S]GTPgS binding to the CHO-K1 cell
membrane. MGM-16 also showed a concentration-dependent
increase in [35S]GTPgS binding to CHO-K1 cell membranes
expressing recombinant m- and d-opioid receptors. In contrast, Fig. 2. Concentration-response curves of MGM-15 (A) and MGM-16 (B)
MGM-15 showed a smaller increase in the binding of for electrical stimulation–induced contraction in mouse vas deferens in
the absence or presence of cyprodime (Cyp) and norBNI or Cyp, norBNI,
[35S]GTPgS than that of MGM-16. and naltrindole (NTI). Responses are expressed as contraction % of the
Antinociception of MGM-15 and MGM-16 in Mouse twitch contraction before agonist addition. Data represent mean 6 S.E.M.
Tail-Flick Test by Subcutaneous and Oral Administra- of five animals.
tion. Antinociceptive effects of MGM-15 and MGM-16 were
investigated in acute thermal pain tests in mice. MGM-15 and
MGM-16 induced dose-related antinociceptive responses in their corresponding antinociceptive ED50 values. In the case
tail-flick tests after subcutaneous and oral administration (Fig. of morphine, the IGIT ED50 value was much smaller than its
4). The effect peaked at 15–30 minutes after injection. The antinociceptive ED50 value.
antinociceptive effect of MGM-15 was approximately 15 and 50 Effects of Selective Antagonists on MGM-15 and
times more potent than that of morphine after subcutaneous MGM-16 Antinociception in Mouse Tail-Flick Test. To
and oral administration, respectively. The antinociceptive determine the opioid receptor type selectivity of MGM-15 and
effect of MGM-16 was approximately 71 and 240 times more MGM-16 antinociception, mice were pretreated with selective
potent than that of morphine after subcutaneous and oral opioid receptor antagonists in the tail-flick test (Fig. 5).
administration, respectively (Tables 5 and 6). The antinociceptive effect of MGM-15 and MGM-16 was
Next we investigated the effects of MGM-15 and MGM-16 completely blocked by the irreversible m-opioid receptor se-
on the inhibition of GIT (IGIT) and their antinociceptive lective antagonist b-FNA. These effects were partially and
effects in comparison with morphine and 7-hydroxymitragy- significantly blocked by the d-opioid receptor selective antag-
nine (Tables 5 and 6). The IGIT ED50 values of 7-hydroxymi- onist naltrindole. The selective k-opioid antagonist norBNI
tragynine, MGM-15, and MGM-16 were slightly larger than was ineffective against MGM-15- and MGM-16-induced

TABLE 2
pD2 values and maximum inhibitory percent of DPDPE, MGM-15, MGM-16, and 7-hydroxymitragynine on electrical
stimulation–induced contraction in mouse vas deferens in the absence or the presence of cyprodime (Cyp) and nor-BNI or Cyp,
nor-BNI, and NTI
Each value represents mean 6 S.E.M. of pD2 value (maximum inhibitory percent) obtained from four or five animals.

pD2 Value of:

Compound
Compound Alone Compound + Cyp and norBNI Compound + Cyp, norBNI, and NTI

DPDPE 8.16 6 0.20 (89.3 6 1.1) 7.80 6 0.17 (90.7 6 2.2) 6.29 6 0.10 (90.5 6 1.6)
MGM-15 7.56 6 0.06 (85.7 6 2.3) 7.10 6 0.11 (82.3 6 5.2) 6.30 6 0.08 (78.8 6 3.3)
MGM-16 8.08 6 0.11 (92.9 6 1.8) 7.50 6 0.11 (95.8 6 1.1) 6.52 6 0.22 (91.4 6 3.0)
7-Hydroxymitragynine 7.22 6 0.13 (82.7 6 3.9) 5.98 6 0.16 (80.2 6 3.3) 5.55 6 0.12 (78.2 6 4.8)
388 Matsumoto et al.

TABLE 3 TABLE 4
Ki values for the inhibition of m- and k-opioid binding to guinea pig brain [35S]GTPgS intrinsic efficacies and EC50 values of MGM-15, MGM-16,
membranes and d-opioid binding to rat brain by test compounds DAMGO, and [Met]-enkephalin in CHO-K1 cells expressing recombinant
The m-binding sites were labeled with [3H]DAMGO (1 nM), d-binding sites with [3H] m- and d-opioid receptors
DPDPE (2 nM), and k-binding sites with [3H]U69593 (1 nM). Data are expressed as Each value represents the mean 6 S.E.M. of three independent samples.
the mean Ki value 6 S.E.M. for three or four determinations performed in
duplicate. m-Opioid Receptor d-Opioid Receptor
Compound
Ki of: EC50 Emax EC50 Emax
Compound
3 3 3
[ H]DAMGO (m) [ H]DPDPE (d) [ H]U69593 (k) nM nM

nM
MGM-15 250 6 61 72 6 4.7 1492 6 425 70 6 4.9
MGM-16 18 6 0.055 94 6 1.8 61 6 3.2 105 6 8.1
Morphine 2.7 6 0.24 40 6 6.0 55 6 4.1 DAMGO 8.0 6 0.69 100 N.D. N.D.
MGM-15 6.4 6 0.30 16 6 1.0 55 6 4.0 [Met]-enkephalin N.D. N.D. 1.3 6 0.26 100
MGM-16 2.1 6 0.028 7.0 6 0.23 29 6 1.5
DAMGO 1.2 6 0.092 N.D. N.D. N.D., not determined.
DPDPE N.D. 1.2 6 0.13 N.D.
U69593 N.D. N.D. 0.66 6 0.073
N.D., not determined.
demonstrated higher potency than MGM-15 in vitro and in
an in vivo acute pain model. Thus, we investigated the
antinociception. When these opioid antagonists were adminis- antiallodynic effect of MGM-16 in a neuropathic pain model.
tered subcutaneously alone at the doses used in the present Mice with partial sciatic nerve ligation exhibited marked
study, they did not produce any effects in the tail-flick test neuropathic pain-like behavior on the ipsilateral side 7 days
(data not shown). after the nerve ligation. We evaluated the antiallodynic
Effect of MGM-16 on Thermal Hyperalgesia Induced effects induced by subcutaneous and oral administration of
by Sciatic Nerve Ligation in Mice. MGM-16 showed MGM-16 in sciatic nerve–ligated mice using von Frey
potent m/d-opioid dual agonistic activities and further filaments (Fig. 6). MGM-16 (0.1, 0.2, and 0.4 mg/kg) dose-
dependently increased the ipsilateral paw withdrawal threshold
in sciatic nerve–ligated mice, and maximal antihyperalgesic
responses were seen at 15 or 30 minutes after subcutaneous
administration of MGM-16 (Fig. 6A). MGM-16 (0.2 mg/kg)
reversed the threshold to control level in sciatic nerve–ligated
mice. With oral administration, MGM-16 (0.5, 1, and 2 mg/kg)
dose-dependently increased the ipsilateral paw withdrawal
threshold in sciatic nerve–ligated mice, and maximal anti-
hyperalgesic responses were seen 30 minutes after admin-
istration (Fig. 6B). MGM-16 at 1 mg/kg and gabapentin at
100 mg/kg reversed the threshold to control level in sciatic
nerve–ligated mice.
To investigate the contribution of the opioid receptor sub-
types in the antiallodynic effect of MGM-16, sciatic nerve–
ligated mice were pretreated with selective opioid receptor
antagonists (Fig. 7). The antiallodynic effect of MGM-16 was
completely blocked by b-FNA and naltrindole in the chronic
pain model. The selective k-opioid antagonist norBNI was
ineffective against MGM-16-mediated antinociception.

Discussion
In the present study, we synthesized the dual-acting m- and
d-opioid agonists MGM-15 and MGM-16 from 7-hydroxymi-
tragynine for the treatment of acute and chronic pain. MGM-
16 showed higher opioid agonist potency than that of
7-hydroxymitragynine and MGM-15 in vitro and in vivo. When
administered orally, MGM-16 showed 100-fold-higher analge-
sic potency than that of gabapentin in sciatic nerve–ligated
mice. Therefore, MGM-16 could be useful for the treatment of
chronic pain such as mechanical allodynia. To our knowledge,
Fig. 3. (A) Concentration-response curves of MGM-15, MGM-16, and this is the first study to investigate the antiallodynic effect of
DAMGO for [35S]GTPgS binding to CHO-K1 cells expressing recombinant
m-opioid receptors. (B) Concentration-response curves of MGM-15, MGM-
mitragynine-related indole alkaloids using a neuropathic
16, and [Met]-enkephalin for [35S]GTPgS binding to CHO-K1 cells mouse pain model.
expressing recombinant d-opioid receptors. The membrane-bound The alkaloid 7-hydroxymitragynine was found to possess
[35S]GTPgS was measured and expressed as % stimulation relative to the most potent opioid agonistic effects among the constitu-
the basal level. Each symbol represents the mean 6 S.E.M. of three
independent samples. *P , 0.05; **P , 0.01; ***P , 0.001 vs. MGM-16 ents isolated from the Thai herbal medicine M. speciosa
group by Bonferroni correction. (Horie et al., 2005). We have previously synthesized opioid
 MGM-16, a Novel Dual-Acting m- and d-Opioid Agonist 389

Fig. 4. Time course of the antinociceptive effects produced by subcutaneous (A and B) or oral (C and D) administration of MGM-15 (A and C) and MGM-
16 (B and D) in the tail-flick test in mice. Each value represents mean 6 S.E.M. of data obtained from eight mice.

analgesics from the indole alkaloid mitragynine (Takayama Takayama et al. (2006) reported that the dimension or
et al., 2002, 2006; Matsumoto et al., 2004, 2008; Takayama, electronegativity of the functional group at the C10 position of
2004). We have surveyed these compounds for their opioid mitragynine derivatives is important in eliciting opioid ago-
agonistic activities in vitro to elucidate the specific structure nistic effects. Among those derivatives, the C10-fluorinated
necessary for its pharmacophore to bind to opioid receptors. A derivative, MGM-9, showed the highest potency (Takayama
nitrogen atom, a benzene residue, and an oxygen function et al., 2006). MGM-16, the C10-fluorinated derivative of
play a significant role in producing opioid agonistic activity MGM-15, showed the most potent opioid agonistic effects
(Matsumoto et al., 2005a). The conversion of an indolenine among the derivatives tested previously (Takayama et al.,
moiety in 7-hydroxymitragynine (MGM-15) and 10-fluoro-7- 2002, 2006; Matsumoto et al., 2008). MGM-16 showed full
hydroxymitragynine into an indoline derivative (MGM-16) led agonistic properties on m- and d-opioid receptors. MGM-16
to an increase in agonistic potency. This indicates that the sp3 also showed higher affinities and intrinsic efficacies than
carbon at the C2 position (Fig. 1), which spatially configured MGM-15 on m- and d-opioid receptors in vitro. A benzene
the benzene ring in the ligands, was more efficient at exerting residue of 7-hydroxymitragynine-related indole alkaloids
the opioid activity. plays an essential role in producing opioid agonistic activity

TABLE 5
ED50 values of antinociceptive effects and IGIT produced by subcutaneous administration of morphine,
7-hydroxymitragynine (7-OHMG), MGM-15, and MGM-16 in mice
ED50 represents the median effective dose (mg/kg) (95% confidence limits).

ED50 of:
Test
Morphine 7-OHMG MGM-15 MGM-16

mg/kg
Tail-flick 4.57 (3.12–6.69) 0.80 (0.48–1.33) 0.30 (0.18–0.50) 0.064 (0.040–0.10)
IGIT 1.07 (0.40–2.86) 1.19 (0.54–2.63) 0.35 (0.18–0.70) 0.089 (0.040–0.188)
390 Matsumoto et al.

TABLE 6
ED50 values of antinociceptive effects and IGIT produced by oral administration of morphine,
7-hydroxymitragynine (7-OHMG), MGM-15, and MGM-16 in mice
ED50 represents the median effective dose (mg/kg) (95% confidence limits).

ED50 of:
Test
Morphine 7-OHMG MGM-15 MGM-16

mg/kg
Tail-flick 63.0 (37.2–106.8) 4.43 (1.57–6.93) 1.26 (0.84–1.88) 0.263 (0.165–0.420)
IGIT 11.7 (5.6–24.6) 7.50 (3.95–14.2) 2.20 (0.18–0.70) 0.451 (0.230–0.882)

for the specific structure necessary for its pharmacophore to tests using selective antagonists, MGM-15 and MGM-16
bind to opioid receptors (Matsumoto et al., 2005a). It is showed similar opioid receptor type selectivity. Taken together,
speculated that the fluorine group at the C10 position on the the findings show that fluorination at the C10 position
benzene residue strengthens pharmacophore binding to increased the affinity to m- and d-opioid receptors but did not
opioid receptors because the lone fluorine pair easily forms change the selectivity to the opioid receptor subtypes.
hydrogen bonds with the receptor molecule (Takayama Subcutaneously and orally administered MGM-16 showed
et al., 2006). Based on receptor-binding studies and tail-flick dose-dependent and antiallodynic effects in sciatic nerve–ligated
mice. The antinociceptive effect of MGM-16 was completely
and partially blocked by the m-selective antagonist b-FNA
and by the d-selective antagonist naltrindole, respectively, in
an acute pain model. The antiallodynic effect of MGM-16 was
completely blocked by b-FNA and naltrindole in a chronic
pain model. The contribution of d-opioid receptors to the
antiallodynic effect of MGM-16 in the neuropathic pain model
was higher than in the acute pain test. As previously reported,
upregulation of d-opioid receptors is induced in a rat model of
peripheral nerve injury to the dorsal root ganglion (Kabli and
Cahill, 2007). d-Opioid receptor knockout mice showed an
increase in mechanical allodynia under neuropathic pain
induced by partial sciatic nerve ligation (Nadal et al., 2006;
Gaveriaux-Ruff et al., 2011). However, d-opioid receptor
knockout mice showed normal pain responses to acute pain.
Furthermore, d-opioid selective agonists induce clear anti-
allodynic effects in various animal models of neuropathic
pain but do not show clear antinociceptive effects on acute
nociception (Gaveriaux-Ruff et al., 2011). Thus, d-opioid
receptor activation leads to controlled mechanical allodynia.
This indicates that the d-opioid receptor agonistic properties
of MGM-16 are involved in its antiallodynic effects in sciatic
nerve–ligated mice.
Several studies showed the effectiveness of m-opioid re-
ceptor agonists and demonstrated that m-opioid receptors
contribute to the control of mechanical allodynia in neuro-
pathic pain (Mansikka et al., 2004; Finnerup et al., 2010).
Indeed, m-opioid receptor knockout mice showed an increase
in mechanical allodynia under neuropathic pain induced by
partial sciatic nerve ligation (Nadal et al., 2006; Gaveriaux-
Ruff et al., 2011). Furthermore, m-opioid receptor agonists
attenuate mechanical allodynia in sciatic nerve–ligated mice
(Mansikka et al., 2004). This suggests that the m-opioid
receptor contributes to alleviating mechanical allodynia. In
this study, we found that the antiallodynic effect of MGM-16
Fig. 5. Effects of opioid receptor antagonists on antinociception by was significantly blocked by a m-opioid receptor antagonist.
subcutaneous administration of MGM-15 (A) and MGM-16 (B). The Therefore, the m-opioid receptor agonistic activities of MGM-
antinociceptive effects of MGM-15 and MGM-16 were determined in mice
by the tail-flick test after subcutaneous administration of the following
16 also contribute to its antiallodynic effect in partial sciatic
antagonists: b-FNA (40 mg/kg), naloxonazine (NLZ; 35 mg/kg), NTI (3 mg/ nerve–ligated mice.
kg), and norBNI (20 mg/kg). Measurements were performed 15 minutes The traditional Thai herbal medicine M. speciosa has long
after subcutaneous administration of MGM-15 (0.5 mg/kg) or MGM-16 (0.1 been used in Thailand for its opioid-like effects (Burkill, 1935)
mg/kg) in the tail-flick test. Each value represents mean 6 S.E.M. of eight
or nine mice. ###P , 0.001 vs. vehicle-treated mice by Student’s t test. and as a replacement for opium (Suwanlert, 1975). We have
**P , 0.01; ***P , 0.001 vs. MGM-15 or MGM-16 by Bonferroni correction. studied the pharmacological activities of mitragynine, a major
 MGM-16, a Novel Dual-Acting m- and d-Opioid Agonist 391

Fig. 6. Effects of MGM-16 on mechanical allodynia in partial sciatic nerve–ligated mice. MGM-16 was administered subcutaneously (A) and orally (B),
and the paw withdrawal thresholds on the ipsilateral side were determined repeatedly. Gabapentin (Gab; 100 mg/kg) was administered orally as
a positive control. Several doses of MGM-16 and 100 mg/kg gabapentin are presented at 0.5 and 1 hour after administration, respectively. Each value
represents mean 6 S.E.M. of six or seven mice. **P , 0.01; ***P , 0.001 vs. vehicle by Bonferroni correction.

alkaloid of this herb (Watanabe et al., 1997; Matsumoto et al., dose-dependently increases GTPgS binding to CHO-K1 cells
2005b), and 7-hydroxymitragynine, a main active alkaloid of expressing recombinant m- and d-opioid receptors. We in-
this herb (Matsumoto et al., 2004, 2005a, 2006a). Recently, vestigated the antinociceptive effects of MGM-16 ingested
additional research has been performed on the opium-like orally, based on the traditional usage of M. speciosa and the
effects of M. speciosa and mitragynine and the unique relevance of this route for clinical administration. Following
chemical and pharmacological properties of mitragynine- oral administration, the antinociceptive effect of MGM-16 was
related indole alkaloids (Adkins et al., 2011; Raffa et al., about 240 times more potent than that of morphine in the
2013). We synthesized indole alkaloids such as the partial mouse tail-flick test. Gabapentin, a synthetic analog of
opioid agonist 9-hydroxycorynantheidine, the opioid antago- g-aminobutyric acid, was initially developed as an anticon-
nist corynantheidine, and the m/k-opioid dual agonist MGM-9. vulsant and later licensed for the management of postherpetic
These compounds showed less rewarding effects, based on neuralgia (Gilron, 2007). In our results, gabapentin is
the structure-activity relationship study (Takayama, 2004; effective for mechanical allodynia but very high doses (100
Matsumoto et al., 2006b, 2008). In this study, we synthesized mg/kg p.o.) were needed to alleviate it, as reported previously
a novel m/d-opioid dual agonist, MGM-16. MGM-16 showed (Kiso et al., 2008). Under this experimental condition, the
opioid agonistic effects as potent as those of DAMGO and antiallodynic effect of MGM-16 is approximately 100 times
DPDPE in a functional assay using isolated guinea pig ileum more potent than that of gabapentin in partial sciatic
and mouse vas deferens, respectively. Previously, there were nerve–ligated mice. MGM-16 exerts a potential ability to
no reports on G-protein action following the binding of treat chronic pain more effectively than existing drugs.
mitragynine-related indole alkaloids to opioid receptors In conclusion, the present study demonstrated that MGM-
(Raffa et al., 2013). In this study, we show that MGM-16 16 derived from Mitragyna alkaloid 7-hydroxymitragynine
392 Matsumoto et al.

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of six mice. ###P , 0.001 vs. vehicle by Student’s t test. ***P , 0.001 vs. orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa.
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Matsumoto K, Takayama H, Narita M, Nakamura A, Suzuki M, Suzuki T,
first study to report the antiallodynic effects of the alkaloids Murayama T, Wongseripipatana S, Misawa K, and Kitajima M, et al. (2008) MGM-
derived from mitragynine. MGM-16 may become a new class 9 [(E)-methyl 2-(3-ethyl-7a,12a-(epoxyethanoxy)-9-fluoro-1,2,3,4,6,7,12,12b-
octahydro-8-methoxyindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate], a derivative
of seed compound with potential therapeutic utility for of the indole alkaloid mitragynine: a novel dual-acting mu- and kappa-opioid ag-
treating neuropathic pain. onist with potent antinociceptive and weak rewarding effects in mice. Neuro-
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Matsumoto K, Yamamoto LT, Watanabe K, Yano S, Shan J, Pang PK, Ponglux D,
Acknowledgments
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Authorship Contributions Nozaki C, Le Bourdonnec B, Reiss D, Windh RT, Little PJ, Dolle RE, Kieffer BL,
Participated in research design: Matsumoto, Narita, Horie, Suzuki, and Gavériaux-Ruff C (2012) d-Opioid mechanisms for ADL5747 and ADL5859
effects in mice: analgesia, locomotion, and receptor internalization. J Pharmacol
Takayama. Exp Ther 342:799–807.
Conducted experiments: Matsumoto, Muramatsu, Nakayama. Raffa RB, Beckett JR, Brahmbhatt VN, Ebinger TM, Fabian CA, Nixon JR, Orlando
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from a non-poppy source. J Med Chem 56:4840–4848.
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