Orally Active Opioid Compounds from a Non-Poppy Source

psychoactive properties,(1) primarily by laborers as a stimulant to counteract work fatigue. ...... With Psychoactive Effects From Salvia divinoru...
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Orally Active Opioid Compounds from a Non-Poppy Source Robert B. Raffa,* Jaclyn R. Beckett, Vivek N. Brahmbhatt, Theresa M. Ebinger, Chrisjon A. Fabian, Justin R. Nixon, Steven T. Orlando, Chintan A. Rana, Ali H. Tejani, and Robert J. Tomazic Temple University School of Pharmacy, 3307 N. Broad Street, Philadelphia, Pennsylvania 19140, United States ABSTRACT: The basic science and clinical use of morphine and other “opioid” drugs are based almost exclusively on the extracts or analogues of compounds isolated from a single source, the opium poppy (Papaver somniferum). However, it now appears that biological diversity has evolved an alternative source. Specifically, at least two alkaloids isolated from the plant Mitragyna speciosa, mitragynine ((E)-2-[(2S,3S)-3-ethyl-8-methoxy-1,2,3,4,6,7,12,12b-octahydroindolo[3,2-h]quinolizin-2yl]-3-methoxyprop-2-enoic acid methyl ester; 9-methoxy coryantheidine; MG) and 7-hydroxymitragynine (7-OH-MG), and several synthetic analogues of these natural products display centrally mediated (supraspinal and spinal) antinociceptive (analgesic) activity in various pain models. Several characteristics of these compounds suggest a classic “opioid” mechanism of action: nanomolar affinity for opioid receptors, competitive interaction with the opioid receptor antagonist naloxone, and twoway analgesic cross-tolerance with morphine. However, other characteristics of the compounds suggest novelty, particularly chemical structure and possible greater separation from side effects. We review the chemical and pharmacological properties of these compounds.

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INTRODUCTION Morphine and other opiates derived from a poppy source (Papaver somniferum) are effective pain relievers. However, the constipation, respiratory depression, and concerns of abuse with these compounds place certain restrictions on their clinical use. In addition, analgesic tolerance develops, as does crosstolerance to related compounds, limiting substitution options when tolerance or dependence develops. Alternatives might provide valuable clinical options. The Mitragyna speciosa plant is known as “kratom” (Thailand) or “biak-biak” (Malaysia) in Southeast Asia1 (Figure 1). The plant has been used for its distinctive psychoactive properties,1 primarily by laborers as a stimulant to counteract work fatigue.2,3 At higher doses, opium-like effects prevail.2,3 It has also been chewed or dissolved in teas and used as treatment for opiate withdrawal, fever reduction, analgesia, diarrhea, coughing, hypertension, and depression.2−4 More than 20 alkaloids, several of which are biologically active, have been isolated from the M. speciosa plant,5 with mitragynine (Figure 2a) being the major one, accounting for 66.2% of the crude base1 and 6% by weight of the dried plant. Paynantheine, speciogynine, 7α-hydroxy-7H-mitragynine (first isolated in 20046), and speciociliatine comprise 8.6%, 6.6%, 2% (0.04% of the dried plant), and 0.8%, respectively, of the leaves of a Thai M. speciosa.1 Mitragynaline, mitragynalinic acid, corynantheidaline, and corynantheidalinic acid also have been isolated from M. speciosa.1 Ajmalicine and mitraphylline are additional indole alkaloids structurally related to mitragynine that have been isolated from M. speciosa. Many of the early studies on M. speciosa focused on isolating the alkaloids found in the leaf extracts. Mitragynine is a minor constituent in young plants, and it is the dominant indole alkaloid in older plants.5,7 The distribution of alkaloids varies from plant to plant and from young plants to older ones, and a likely pathway for the © XXXX American Chemical Society

biogenesis of alkaloid compounds within the plant has been established.7 The M. speciosa alkaloids mitragynine (MG), 7α-hydroxy7H-mitragynine (7-OH-MG), and mitragynine pseudoindoxyl were discovered to have high affinity (nanomolar range) for opioid receptors.1 Whereas MG is present in much higher concentration in the Thai plant (66%) than the Malaysian plant (12%),1 7-OH-MG occurs in about equal quantities in both plants. MG and 7-OH-MG also exhibit opioid-mediated (naloxone-sensitive) antinociceptive activity, 7-OH-MG more than 40-fold more potent than mitragynine and 10-fold more potent than morphine.6 Tolerance and cross-tolerance to morphine, withdrawal signs (sign of physical dependence), and conditioned place-preference have also been demonstrated. On the basis of in vitro radioligand receptor binding assays and in vivo behavioral characterizations, MG, 7-OH-MG, and several analogues appear to have features similar to classic opioids. However, their non-Papaver source and nonmorphine-like chemical structures suggest a unique class of opioid analgesics. We review the evidence for opioid classification, novel structural features, and greater separation between antinociception (analgesia) and some classic opioid side effects.

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DO MG AND 7-OH-MG DISPLAY OPIOID-LIKE PHARMACOLOGY? Substances are typically classified as “opioid” based on generally accepted criteria, which include binding affinity for one or more of the major 7TM-GPCR (seven-transmembrane G-proteincoupled receptor) subtypes of opioid receptor (μ, δ, κ, and ORL-1), morphine-like in vivo effects such as pain relief Received: January 29, 2013

A

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(manifested by abstinence-induced and/or antagonist-induced withdrawal signs), and (least definitively) by some similarity of chemical structure to morphine or another known opioid. Opioid Pharmacology: In Vitro. Two studies have reported the affinities of MG and/or 7-OH-MG for opioid receptors in homogenates of guinea pig brain. The subtypeselective ligands DAMGO ( D -Ala 2 ,N-MePhe 4 ,Gly-ol]enkephalin), DPDPE (D-Pen2-D-Pen5-enkephalin), and U69,593 (N-methyl-2-phenyl-N-[(5R,7S,8S)-7-(pyrrolidin-1-yl)1-oxaspiro[4.5]dec-8-yl]acetamide) were used to assess affinity at the μ, δ, and κ opioid receptor subtypes, respectively. The affinity of MG is 7.2 nM at μ sites, 60 nM at δ sites, and >1000 nM at κ sites (nearly 10-fold selectivity for μ over δ sites and greater than 1000-fold selectivity for μ over κ sites).8 In comparison, the affinity of morphine in the same study was 3.5 nM at μ sites, 417 nM at δ sites, and 468 nM at κ sites (selectivity of >100-fold for μ over δ sites and >100-fold for μ over κ sites). The affinity of 7-OH-MG in the same study was 13 nM at μ sites (about 1/2 that of MG), 155 nM at δ sites, and 123 nM at κ sites (selectivity of >10-fold for μ over δ sites and nearly 10-fold for μ over κ sites). Thus, both MG and 7-OHMG have affinity comparable with that of morphine for μ opioid receptors. Morphine displays >100-fold μ site selectivity over δ and κ sites. MG displays about 10-fold μ site selectivity over δ and >1000-fold selectivity over κ sites, and 7-OH-MG displays >10-fold μ site selectivity over δ and κ sites. Therefore, the relative receptor binding profile of MG is μ > δ ≫ κ, and that of 7-OH-MG is μ > δ ≅ κ (compared to morphine’s profile of μ ≫ δ ≅ κ). A subsequent study6 reported similar results for 7-OH-MG (9.8 nM at μ sites, 145 nM at δ sites, and 195 nM at κ sites). We are not aware of binding studies at the ORL-1 (opioid-receptor-like, nociceptin, orphanin FQ) receptor (but see inactivity in isolated tissue assay below). Opioid Pharmacology: Isolated Tissue. As a measure of intrinsic activity (efficacy) at opioid receptors, the relative potency of morphine and MG was measured in the classic electrically stimulated guinea pig ileum isolated tissue preparation.8 Electrically stimulated contraction of guinea pig ileum smooth muscle is inhibited by opioids.9 MG was found to be about 1/4 as potent as morphine but had nearly the same intrinsic activity and thus is a full agonist in this test. 7-OH-MG exhibited 10-fold greater potency than morphine and essentially the same intrinsic activity as morphine and thus is also a full agonist in this test.10 Since the inhibitory effects of MG on electrically stimulated guinea pig ileum are not affected by CompB (a nonpeptidic ORL-1 receptor antagonist), MG does not act on ORL-1 receptors.11 Related compounds in M. speciosa, such as MG-pseudoinoxyl (Figure 2b), speciogynine, speciociliatine, 7-hydroxyspeciocillatine, and paynantheine, also exhibit agonist action in electrically stimulated guienea-pig ilium,1,8 and an antagonist, corynantheidaline (Figure 2c), reverses morphine-inhibited twitch contraction in guinea pig ileum.1 Opioid Pharmacology: In Vivo. Antinociception: Mice, Rats, and Dogs. Subcutaneous (sc) administration of 7-OHMG (5 mg/kg) produces a full antinociceptive effect in the mouse tail-flick test.12 The effect was significantly different from saline for more than 60 min. In the same study, oral 7-OH-MG (10 mg/kg), but not oral morphine (20 mg/kg), produced a full antinociceptive effect. A central site of action was demonstrated by dose-related (30−100 nmol) antinociception following intracerebroventricular administration of MG (about 10-fold less potent than morphine) and MG-pseudoindoxyl

Figure 1. M. speciosa: (top) leaves (courtesy of Uomo Vitruviano from the Wikipedia Web site, http://en.wikipedia.org/wiki/File:Mitragyna_ speciosa111.JPG); (bottom) powdered form (courtesy of Ingenium f r o m W i k i m e d i a , h t t p : / / c o m mons .wikimedia.org /wi k i/ File:Powdered_kratom.jpg).

Figure 2. (a) Octahydroindoloquinazoline scaffold. Shown is m i t r a g y n i n e ( M G ) ( ( E ) - 2 - [ ( 2S ,3 S ) -3 -e t h y l - 8 - m e t h ox y 1,2,3,4,6,7,12,12b-octahydroindolo[3,2-h]quinolizin-2-yl]-3-methoxyprop-2-enoic acid methyl ester). (b) MG-pseudoindoxyl. (c) Corynantheidaline (morphine-antagonist).

(analgesia in humans, antinociception in animals), miosis, constipation, respiratory depression, tolerance, cross-tolerance to known opioid, development of physical dependence B

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these results,4 but one is that MG (consistent with a μ opioid receptor agonist action) substitutes for morphine in this test. Antinociception in Mice: Tolerance. Tolerance to (diminution of) the antinociceptive effect of opioids develops over the course of repeated administrations. For example, the antinociceptive effect of 10 mg/kg morphine administered sc twice daily declines from nearly 100% on day 1 in the mouse tail-flick test to nearly no effect on day 5.19 A similar pattern of development of tolerance was observed for the same dose of 7OH-MG. In addition, two-way cross-tolerance between morphine and 7-OH-MG was observed (a diminished 7-OHMG effect in morphine-tolerant mice and a diminished morphine effect in 7-OH-MG-tolerant mice). The development of antinociceptive tolerance is not indicative of opioid activity, but two-way cross-tolerance to morphine is. Classic Opioid Side Effects. In addition to therapeutic pain relief, opioids produce characteristic side effects. In particular, these include emesis, inhibition of cough (antitussive action), constipation (or antidiarrheal action), respiratory depression, and Straub tail in rodents (vertical or nearly vertical position of the tail, sometimes with a slight forward pointing of the tip). In summary, below are the results of a study in which several of these effects were examined:13 • Orally administered MG dose-dependently inhibited a model of cough reflex in unanesthetized dogs. • MG did not produce emesis in dogs at doses that produced full antinociceptive effect orally (in contrast, codeine produced emesis within the antinociceptive dose range). • Although depression of respiration was not tested directly, it was observed while conducting related tests that “... codeine is more active than [MG] in depressing respiration following intravenous injection in anesthetized dogs13”. • There was no indication that MG induced Straub tail. • Oral administration of MG at the high end of its antinociceptive dose−response curve (55.2 mg/kg) produced 18% inhibition of gastrointestinal (GI) transit of a charcoal meal in rats (compared to 70−75% inhibition of transit by morphine and codeine); intraperitoneally administered 7-OH-MG (36.8 mg/kg) produced no significant effect. • To our knowledge, diuresis (a characteristic effect of several κ-opioid agonists) has not been reported for MG or 7-OH-MG Inhibition of Gastrointestinal Transit. Morphine inhibits GI transit in animals, and clinically, the dose of morphine required for analgesic effect often exceeds that which causes constipation. The relative separation between MG-induced antinociception and constipation (i.e., inhibition of GI transit) noted above was examined further in a more recent study18 using the charcoal meal test in mice. 7-OH-MG inhibited transit in a dose-dependent manner, but the study concluded “Thus, it is estimated [7-OH-MG] is 4.9−6.4 times less constipating than morphine at antinociceptive doses”. Taken together, the two studies13,18 suggest that MG and 7-OH-MG might benefit from a nonopioid component to their in vivo action or they might bind in some unique way or have advantageous opioid receptor (sub)type relative affinities, since pharmacological effects of κ agonism are somewhat counter to μ-elicited adverse effects (e.g., GI protection instead of GI transit inhibition).

(approximately 1/2 as potent as morphine). The maximum effect of both compounds by this route was greater than 50%, but less than the full effect produced by morphine (1−10 nmol). In the mouse hot-plate test (54.5 °C), MG (92 mg/kg) produces 100% antinociception by oral administration, but the same dose was ineffective by sc administration.13 MG is also active, with oral potency (16−21 mg/kg) about 1 /2 that of codeine (7−11 mg/kg), in the rat tail-flick test and Randall and Selitto rat paw pressure test.13 It is also active in an antinociceptive test in dogs (hind leg flick), with oral potency about equal to that of codeine (2−8 mg/kg).13 Antinociception: In Vivo Receptor Profile in Mice. The antinociceptive effects produced by the peripheral (intraperitoneal) or central (intracerebroventricular) administeration of MG to mice is antagonized by central (intracerebroventricular) administration of the reversible opioid receptor antagonist naloxone.14 The opioid receptor subtype selectivity of MG-induced intracerebroventricular antinociception has been assessed using the receptor-selective compounds cyprodime (μ), naltrindole (δ), and nor-binaltorphimine (nor-BNI) (κ).12 Consistent with the binding studies in vitro, morphine- and 7-OH-MG-induced antinociception in the mouse tail-flick and hot-plate (55 °C) was antagonized by the nonselective opioid receptor antagonist naloxone and the μselective antagonist cyprodime but less so or not at all by the δselective antagonist naltrindole or the κ-selective antagonist nor-BNI. MG-induced antinociception in the rat tail-flick test was not antagonized by the mixed opioid agonist/antagonist nalorphine in an early study.13 The reason for this difference is not known, but given the now-known receptor binding profile, the more recent studies are likely to be the more instructive. Naloxone-Precipitated Withdrawal in Mice. Morphine or 7-OH-MG was injected sc to mice twice daily (09:00 and 19:00) following standard protocols.15−17 The dose progressively increased over a period of 5 days according to (morphine, 7-OH-MG) (mg/kg): first day (8, 15), second day (20, 25), third day (30, 35), fourth day (40, 45), and fifth day (45 at 09:00 only). Naloxone (3 mg/kg, sc), at a dose that inhibits antinociception,18 was injected 2 h after the final dose, and the mice were immediately placed on a circular cylinder (30 cm in diameter × 70 cm height) and observed for withdrawal signs for 60 min.19 The 7-OH-MG-treated mice displayed classic opioid withdrawal signs (jumping, rearing, urination, and forepaw tremor) when challenged by naloxone. This finding is consistent with the in vitro and in vivo properties of the compound. Naloxone-challenge did not induce as much diarrhea in 7-OH-MG-treated mice as it did in morphinetreated mice. This is consistent with a smaller constipatory effect or might be due to testing less than an equiantinociceptive dose of 7-OH-MG compared to morphine. Naloxone-Precipitated Withdrawal in Zebrafish. Zebrafish have dopaminergic projections in forebrain regions analogous to the mammalian mesolimbic system and display behavioral signs and biochemical changes during withdrawal from psychotropic drugs, including morphine.4 In a recent study,4 zebrafish were exposed to morphine (1.5 mg/L) for 2 weeks, then tested for signs of abstinence-induced withdrawal, either alone or in the presence of MG (2 mg/L). In the absence of MG, withdrawal from morphine elicited significant alterations in a variety of measures of normal swimming. In contrast, in the presence of MG, morphine-withdrawn fish displayed swimming behaviors comparable to those of control or to those that were morphine-treated. There are several possible interpretations of C

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Conditioned Place-Preference in Mice. In this test, mice learn to associate one discernible location (“place”) with a drug being administered in that place (established during a “conditioning” phase) and an identifiable different “place” with no drug or control being administered. At a preset time after the final conditioning session, the animals are allowed to choose which location they prefer, i.e., to display a “placepreference”, indicated by the difference between the amount of time spent in the drug-administered place and the drug-neutral place. Mice display a preference for opioid-administered places in this type of test. In such a test, 7-OH-MG (0.5, 1, or 2 mg/ kg, sc) induced significant and dose-related place-preference compared to vehicle.20 At similar antinociceptive doses, 7-OHMG and morphine exhibited about equal conditioned placepreference. While such results are not exclusive for an opioid mechanism of action, they are consistent with the in vitro and other in vivo results with 7-OH-MG.

Figure 4. Poor overlap of the low-energy conformations of MG and morphine.19

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RECEPTOR SUBTYPE SELECTIVITY Opioid μ-selective structures display a large degree of diversity. Opioid δ-selective compounds typically contain a hydrophobic region (such as an indole or spiroindane) that binds in a hydrophobic pocket of the receptor and might also contribute to selectivity by providing added bulk that inhibits binding to other sites. For example, a tryptophan within the binding cavity of the μ receptor subtype appears to provide steric hindrance for the added bulk associated with δ-selective compounds. When tryptophan is switched to alanine, leucine, or lysine (smaller amino acids than tryptophan), δ-selective compounds can bind.22 7-OH-MG does not contain this hydrophobic region, so it is not unexpected that it binds with greater affinity to μ sites than δ sites. κ-Selective compounds typically contain a basic moiety that binds to a glutamate within the κ binding pocket.22 Structure−Activity Relationship (SAR). Since the selectivity of a compound for an opioid receptor subtype cannot be determined simply by the presence or absence of one or more functional groups,22 selected modifications have been made to the structure of 7-OH-MG in order to identify the most important molecular groups and understand how these groups affect the compound’s activity. The results identified the C9 position, C7 position, Nb lone pair, and β-methoxyacrylate moiety as the four major sites1,18 (Table 1). (Note: In the IUPAC nomenclature the methoxy group is attached at the C8 carbon atom of the octahydroindoloquinazoline scaffold, whereas the same carbon is referred to as C9 position in the coryantheidine scaffold.) • The C7 position is important for binding affinity. For example, the addition of OH (7-OH-MG) decreases the binding affinity for μ and κ receptor subtypes by about 1 /2 but increases in vivo antinociceptive potency (previously discussed). The addition of OCH3 (7methoxy-MG), OCH 2 CH 3 (7-ethoxy-MG), or OCOCH3 (7-acetoxy-MG) group in the C7 position leads to reduced potency and intrinsic activity. • The C9 position on the corynantheidine scaffold is important for the intrinsic activity of these compounds. For example, when the methoxy group at the C9 position of the corynantheidine nucleus is sequentially replaced by hydroxyl (9-hydroxycorynantheidine) to demethoxy (corynantheidine), the intrinsic activity evolves from full agonist to partial agonist to antagonist. • A C10-fluorinated, ethylene glycol-bridged derivative of MG (MGM-9) displays nanomolar binding affinity (7− 18 nM) at μ- and κ-opioid receptors, oral antinociceptive potency greater than morphine, and weaker inhibition of

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DO MG AND 7-OH-MG HAVE OPIOID-LIKE CHEMICAL STRUCTURE? Comparison to the Structure of Morphine. A cursory comparison suggests some similarities in the chemical structures of MG, 7-OH-MG, and morphine. Each has the three functional groups traditionally deemed important for opioid receptor binding: a tertiary nitrogen atom, a benzene residue, and a phenolic hydroxyl (Figure 3).21 However, the

Figure 3. Comparison of the 2D chemical structures of the morphinederived opiate “template” (top) and of mitragynine (bottom).

groups do not overlay well in three-dimensional space8 (Figure 4), and it is believed that they bind to opioid receptors differently than does morphine.19 7-OH-MG differs from MG by a hydroxyl group at the C7 position. The hydroxyl increases potency significantly, resulting in 7-OH-MG being nearly 50fold more potent than MG and more than 10-fold more potent than morphine in the guinea pig ileum preparation.1 This is possibly due18 to greater lipophilicity than morphine and faster distribution across the blood−brain barrier. D

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Table 1. Structure−Activity Relationship of the Agonist Action of Mitragynine (MG) and Analogues in the Isolated Guinea-Pig Ileum Preparation (Tested at 100 pM to 30 μM)1,a

a

Efficacy and potency are expressed as inhibition of electrically induced twitch compared to morphine (%). NI = inactive or naloxone-insensitive.

some affinity for α2-adrenoceptors.25 However, a study of the effect of MG on cAMP formation suggested that MG does not act directly on these receptors to produce antinociceptive activity.26 MG also displays some affinity (details not reported) for 5-HT2C, 5-HT7, and dopamine D2 receptors.25 Other Characteristics: In Vivo. 5-HT, Norepinephrine. A possible in vivo action of MG on 5-HT systems was investigated. Similar to morphine and tramadol, MG was found to suppress 5-HT-induced mouse head-twitch response.27 This might represent an action on 5-HT2A receptors, α2-adrenoceptors, or a combination of both.27 Given a possible action of MG at 5-HT2A receptors, MG was tested for antidepressant activity using the mouse forced-swim and tailsuspension tests, two behavioral models commonly used to screen for antidepressant activity. MG was moderately active in these tests, suggesting that it might have behaviorally relevant effect on 5-HT systems.28 This is consistent with a previous study in which an aqueous extract of M. speciosa demonstrated an antidepressant activity in a withdrawal model (mice), leading the authors to speculate that the effect was mediated through 5HT or noradrenergic systems.29 It is also consistent with the suggestion that the chemical structure of 7-OH-MG has characteristic features that resemble those of 4-hydroxytryptophan, which is a 5-HT2A ligand. Indeed, a role for descending noradrenergic and 5-HT systems in MG-induced antinociception has been postulated.30 A recent study appears to have ruled out a role for cannabinoid CB1 receptors in the antinociceptive action of MG.31

gastrointestinal transit than morphine at equianalgesic doses.20 • Modification of the β-methoxyacrylate residue in MG (−C−CO2−CH3) (e.g., by substitution with −CO2−H or −CH2−OH) results in compounds that exhibit no or only very weak opioid agonistic activity, indicating that this structural feature is needed for opioid receptor activity. • The Nb lone pair appears to be essential for opioid agonist activity, in agreement with modifications of opioids that reduce the basicity of the nitrogen atom, which is reported to be an essential feature of the opioid pharmacophore.21 MG-pseudoindoxyl. MG-pseudoindoxyl has high affinities for μ- and δ-opioid receptors (0.01 and 3.0 nM, respectively) and negligible affinity for κ receptors (79 nM).1 Its naloxonesensitive inhibitory effect on guinea pig ileum preparation is more potent than that of mitragynine or morphine (100-fold and 20-fold, respectively), but it has much less antinociceptive activity than does morphine, which is speculated to be due to instability of the compound in the brain.18,19

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DO MG AND 7-OH-MG DISPLAY NON-OPIOID PROPERTIES? Other Characteristics: In Vitro. In addition to the opioidergic receptor system, noradrenergic and 5-HT (5hydroxytryptamine, serotoninergic) systems are involved in analgesic pathways of the central nervous system.23,24 These pathways are believed to contribute to significant descending modulatory effects on the afferent transmission of pain signals from neurons in the dorsal horn of the spinal cord. MG displays

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METABOLISM In rodents and humans, MG undergoes phase I and phase II hepatic metabolism and is excreted in the urine. The E

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Figure 5. Postulated metabolic pathways of MG in humans.32

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ABUSE POTENTIAL The opioid-like receptor binding and in vivo activity of extracts of M. speciosa raise the question of abuse potential. For many decades, laborers in Southeast Asia have used mitragynine in low doses as a stimulant and energy supplement. In higher doses, it has been used as an opium substitute or to manage opium withdrawal symptoms.34 Abuse has led to the drug being made illegal in Thailand, Malaysia, Myanmar, and Australia.5 It is still a popular street drug in these areas, and reports of abuse in other countries have appeared (street names such as “thang”, “kakuam”, “thom”, “ketum”, and “biak” in the U.S.).35 Mitragynine is widely available on the Internet, and several Web sites sell it in the form of leaves (whole or crushed), as an extract, powder or encapsulated powder, or extract-resin “pies”. Seeds and whole plants for cultivation use are also available. Several Web sites provide information about mitragynine and its use. Many of these sites include users’ experiences with it. Mitragynine is not currently controlled in the United States under the Controlled Substances Act, but the United States DEA (Drug Enforcement Administration) has placed it on their list of drugs and chemicals of concern.35

metabolism of MG has been studied using solid-phase extraction from human urine and analysis by liquid chromatography−linear ion trap mass spectrometry.32 A number of phase I metabolites have been identified in addition to at least three glucuronides (Figure 5). The extent to which they contribute to biological effects is currently unknown. Some contribution is suggested by the greater oral vs sc activity of MG in some antinociceptive tests.13

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TOXICOLOGY Single- and multiple-dosing of mitragynine has been studied in rats and dogs.13 In rats, single oral doses of as high as 806 mg/ kg produced no toxicity and 30 multiple oral doses of up to 50 mg/kg/day produced no side effects. In dogs, five daily oral doses of 16 mg kg−1 day−1 and two additional days of oral 32 mg kg−1 day−1 produced no side effects. At higher doses and longer exposures, transient clinical findings (primarily blood dyscrasias) were observed. To our knowledge, there are no well-defined human studies of toxicity of MG, and in the single case report of a death in the United States, the medical examiner did not include mitragynine toxicity in the cause of death, which was ruled accidental toxicity due to propylhexedine (a potent α2adrenoceptor agonist found in some nasal decongestant inhalers).33

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DISCUSSION The Mitragyna speciosa plant, which is indigenous to southeastern Asia, particularly Thailand and Malaysia, contains more F

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GI transit. Therefore, 7-OH-MG had a 10-fold greater separation between antinociception and constipation than did morphine. There are several possibilities (in decreasing order of opioid distinction): the compounds bind to opioid receptors in a novel or distinct manner; they activate different (or different combinations of) G proteins or other downstream secondmessenger transduction pathways; a nonopioid component of its pharmacology either contributes to antinociception or counters side effects; or a metabolite contributes to antinociception or counters side effects. The potentially important clinical advantages of such a profile warrant additional study of the exact mechanism. The possibility of differential (“biased”) intracellular signaling has not yet been reported. The metabolism and toxicity of MG have been studied extensively, and the byproducts of hepatic metabolism have been determined. MG undergoes phase I and phase II metabolism. The metabolites are renally eliminated. To our knowledge, the detailed pharmacology or side effect profiles of the metabolites have not been described. Similar to classic opioids, M. speciosa compounds have been subject to abuse. They are widely available on the Internet in various forms, along with information about their use and psychotropic effects. They are not currently regulated under the United States Controlled Substances Act, but they are listed as chemicals of concern by the U.S. Drug Enforcement Administration. In summary, “opioid” pharmacology and medicinal chemistry, as the name implies, have been almost exclusively based on the natural and synthetic analogues of compounds from a single source: opium. It appears that biodiversity has provided an alternative natural source of “opioid” compounds: M. speciosa. They meet the criteria of high affinity for known opioid receptors and centrally mediated, opioid-receptor antagonist-sensitive antinociception in several animal models. However, their structural distinctiveness is mirrored in their apparently reduced side effect profile. Taken together, the data appear to be sufficient to justify the claim that compounds from M. speciosa, such as MG and 7-OH-MG, and synthetic analogues of these naturally occurring alkaloids are a novel (or at least distinct) class of opioids.

than 20 alkaloids, at least 3 of which display significant affinity (nanomolar) for opioid receptors. Two of them, mitragynine and 7-hydroxymitragynine, have been characterized as agonists based on their in vivo activity (no reports of interaction with GTPγS binding were found). One of them, corynantheidine, has been characterized as an antagonist (with nanomolar receptor binding affinity selectivity of μ ≫ δ ≅ κ). Thus, the compounds satisfy the most important modern in vitro criteria for classification as an opioid, namely, significant affinity for one or more of the known opioid receptors. The binding assays suggest relatively high selectivity for μ-opioid receptors, with some δ- and κ-opioid receptor activity. The compounds also display opioid-like activity in a classic isolated tissue preparation: the guinea pig ileum. MG inhibits electrically-induced guinea pig ileum contraction. Compared with morphine, its relative inhibitory activity is 95%. More importantly, the inhibition is reversed by the opioid receptor antagonist naloxone. The compounds also exhibit classic opioid-receptor-mediated antinociceptive effect in several animal models (no human studies were identified) and in several species (including oral activity in dogs). A central site of action has been demonstrated, since (i) antinociception was produced by direct injection (intracerebroventricular) into the brain and (ii) peripheral or central administration was antagonized by the central (intracerebroventricular) administration of naloxone, the latter confirming that the antinociception was produced via opioid receptors. Therefore, that these compounds produce centrally mediated antinociception through opioid receptors has been firmly established. The elucidation of a structure−activity relationship for the natural products and their synthetic analogues lends further support for a receptor-based mechanism of action. However, it is here that the M. speciosa compounds and their analogues begin to differentiate from classic opioids. Despite some important superficial similarities to classic opioid chemical structures (tertiary nitrogen, benzene ring, and phenolic hydroxyl group), molecular modeling, to date, indicates that they do not overlay in the classic opioid template. It has been concluded, therefore, that MG, 7-OH-MG, and related compounds represent a class of opioid compounds structurally distinct from morphine. It is hypothesized, based on this structural distinctness, that these compounds bind to opioid receptors in a fashion different from morphine. The actual details are still a matter of study. Nevertheless, such an in vitro differentiation might account for the observed differences in the in vivo profile of these compounds compared to classic opioids. The evidence clearly establishes that MG, 7-OH-MG, and their agonist analogues produce antinociception via opioid receptors. However, in terms of side effects, the in vivo profile of these compounds does not entirely parallel the classic opioid profile, giving credence to their status as a novel class of opioids. For example, on the basis of limited observations, the compounds appear to produce less emesis and less respiratory depression than does codeine. More detailed investigation of these end points is still needed. The evidence regarding constipation as a side effect is more compelling (assessed by inhibition of GI transit). Full dose−response curves have been obtained for morphine and 7-OH-MG in mouse tail-flick and hot-plate (55 °C) tests and for inhibition of GI transit, all in mice, all by sc administration, and all in the same study. In both antinociception tests, 7-OH-MG was approximately 10-fold more potent than morphine but was equally potent in inhibing

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AUTHOR INFORMATION

Corresponding Author

*Phone: +1 215 707-4976. E-mail: robert.raff[email protected]. Notes

The authors declare no competing financial interest. Biographies Robert B. Raffa earned Bachelor’s degrees in Chemical Engineering and Physiological Psychology (University of Delaware), Master’s degrees in Biomedical Engineering (Drexel University, PA) and Toxicology (Thomas Jefferson University, PA), and a Ph.D. in Pharmacology under the direction of Prof. Ronald J. Tallarida (Temple University School of Medicine, PA). He was co-team-leader for CNS Analgesics Drug Discovery at Johnson & Johnson and past president of the Mid-Atlantic Pharmacology Society. He is currently Professor of Pharmacology in the Department of Pharmaceutical Sciences at Temple University School of Pharmacy and Research Professor in the Department of Pharmacology of Temple University School of Medicine. Jaclyn R. Beckett, Vivek N. Brahmbhatt, Theresa M. Ebinger, Chrisjon A. Fabian, Justin R. Nixon, Steven T. Orlando, Chintan A. G

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Rana, Ali H. Tejani, and Robert J. Tomazic are Pharm.D. students at Temple University School of Pharmacy, PA.

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ACKNOWLEDGMENTS The authors thank Wayne Childers, Ph.D., and Michael R. Borenstein, Ph.D., Temple University School of Pharmacy, Philadelphia, PA, for helpful discussions and suggestions.

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ABBREVIATIONS USED 5-HT, 5-hydroxytryptamine (serotonin); 7-OH-MG, 7-hydroxymitragynine (7α-hydroxy-7H-mitragynine); 7TM-GPCR, seven-transmembrane G-protein-coupled receptor; DAMGO, 2 4 2 5 D-Ala ,N-MePhe ,Gly-ol]enkephalin; DPDPE, D-Pen -D-Pen enkephalin; MG, mitragynine

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