Terpenes from Cannabis sativa induce antinociception in a mouse model of chronic neuropathic pain via activation of adenosine A2A receptors.

ABSTRACT: Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including Cannabis sativa. A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, ß-pinene, α-humulene, and ß-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the potential antinociception and mechanism of these Cannabis terpenes in a mouse model of chronic pain. We first tested for antinociception by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with mouse models of chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal antinociception to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs either alone. We then used the adenosine A2A receptor (A2AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A2AR to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A2AR agonists. Together these studies identify Cannabis terpenes as potential therapeutics for chronic neuropathic pain and identify a receptor mechanism for this activity.

Terpenes from Cannabis sativa Induce Antinociception in Mouse Chronic Neuropathic Pain via Activation of Spinal Cord Adenosine A2A Receptors.

Terpenes are small hydrocarbon compounds that impart aroma and taste to many plants, including Cannabis sativa. A number of studies have shown that terpenes can produce pain relief in various pain states in both humans and animals. However, these studies were methodologically limited and few established mechanisms of action. In our previous work, we showed that the terpenes geraniol, linalool, ß-pinene, α-humulene, and ß-caryophyllene produced cannabimimetic behavioral effects via multiple receptor targets. We thus expanded this work to explore the efficacy and mechanism of these Cannabis terpenes in relieving chronic pain. We first tested for antinociceptive efficacy by injecting terpenes (200 mg/kg, IP) into male and female CD-1 mice with chemotherapy-induced peripheral neuropathy (CIPN) or lipopolysaccharide-induced inflammatory pain, finding that the terpenes produced roughly equal efficacy to 10 mg/kg morphine or 3.2 mg/kg WIN55,212. We further found that none of the terpenes produced reward as measured by conditioned place preference, while low doses of terpene (100 mg/kg) combined with morphine (3.2 mg/kg) produced enhanced antinociception vs. either alone. We then used the adenosine A2A receptor (A2AR) selective antagonist istradefylline (3.2 mg/kg, IP) and spinal cord-specific CRISPR knockdown of the A2AR to identify this receptor as the mechanism for terpene antinociception in CIPN. In vitro cAMP and binding studies and in silico modeling studies further suggested that the terpenes act as A2AR agonists. Together these studies identify Cannabis terpenes as potential therapeutics for chronic neuropathic pain, and identify a receptor mechanism in the spinal cord for this activity.

Analgesic Potential of Terpenes Derived from Cannabis sativa.

Pain prevalence among adults in the United States has increased 25% over the past two decades, resulting in high health-care costs and impacts to patient quality of life. In the last 30 years, our understanding of pain circuits and (intra)cellular mechanisms has grown exponentially, but this understanding has not yet resulted in improved therapies. Options for pain management are limited. Many analgesics have poor efficacy and are accompanied by severe side effects such as addiction, resulting in a devastating opioid abuse and overdose epidemic. These problems have encouraged scientists to identify novel molecular targets and develop alternative pain therapeutics. Increasing preclinical and clinical evidence suggests that cannabis has several beneficial pharmacological activities, including pain relief. Cannabis sativa contains more than 500 chemical compounds, with two principle phytocannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). Beyond phytocannabinoids, more than 150 terpenes have been identified in different cannabis chemovars. Although the predominant cannabinoids, Δ9-THC and CBD, are thought to be the primary medicinal compounds, terpenes including the monoterpenes ß-myrcene, α-pinene, limonene, and linalool, as well as the sesquiterpenes ß-caryophyllene and α-humulene may contribute to many pharmacological properties of cannabis, including anti-inflammatory and antinociceptive effects. The aim of this review is to summarize our current knowledge about terpene compounds in cannabis and to analyze the available scientific evidence for a role of cannabis-derived terpenes in modern pain management. SIGNIFICANCE STATEMENT: Decades of research have improved our knowledge of cannabis polypharmacy and contributing phytochemicals, including terpenes. Reform of the legal status for cannabis possession and increased availability (medicinal and recreational) have resulted in cannabis use to combat the increasing prevalence of pain and may help to address the opioid crisis. Better understanding of the pharmacological effects of cannabis and its active components, including terpenes, may assist in identifying new therapeutic approaches and optimizing the use of cannabis and/or terpenes as analgesic agents.

Cannabis sativa terpenes are cannabimimetic and selectively enhance cannabinoid activity.

Limited evidence has suggested that terpenes found in Cannabis sativa are analgesic, and could produce an "entourage effect" whereby they modulate cannabinoids to result in improved outcomes. However this hypothesis is controversial, with limited evidence. We thus investigated Cannabis sativa terpenes alone and with the cannabinoid agonist WIN55,212 using in vitro and in vivo approaches. We found that the terpenes α-humulene, geraniol, linalool, and ß-pinene produced cannabinoid tetrad behaviors in mice, suggesting cannabimimetic activity. Some behaviors could be blocked by cannabinoid or adenosine receptor antagonists, suggesting a mixed mechanism of action. These behavioral effects were selectively additive with WIN55,212, suggesting terpenes can boost cannabinoid activity. In vitro experiments showed that all terpenes activated the CB1R, while some activated other targets. Our findings suggest that these Cannabis terpenes are multifunctional cannabimimetic ligands that provide conceptual support for the entourage effect hypothesis and could be used to enhance the therapeutic properties of cannabinoids.

The endomorphin-1/2 and dynorphin-B peptides display biased agonism at the mu opioid receptor.

BACKGROUND: Opioid agonist activation at the mu opioid receptor (MOR) can lead to a wide variety of physiological responses. Many opioid agonists share the ability to selectively and preferentially activate specific signaling pathways, a term called biased agonism. Biased opioid ligands can theoretically induce specific physiological responses and might enable the generation of drugs with improved side effect profiles. METHODS: Dynorphins, enkephalins, and endomorphins are endogenous opioid agonist peptides that may possess distinct bias profiles; biased agonism of endogenous peptides could explain the selective roles of these ligands in vivo. Our purpose in the present study was to investigate biased signaling and potential underlying molecular mechanisms of bias using 35S-GTPγS and cAMP assays, specifically focusing on the role of adenylyl cyclases (ACs) and regulators of G-protein signaling proteins (RGSs) in CHO, N2a, and SH-SY5Y cell lines, all expressing the human MOR. RESULTS: We found that endomorphin-1/2 preferentially activated cAMP signaling, while dynorphin-B preferentially activated 35S-GTPγS signaling in most cell lines. Experiments carried out in the presence of an isoform selective RGS-4 inhibitor, and siRNA knockdown of AC6 in N2a cells did not significantly affect the bias properties of endomorphins, suggesting that these proteins may not play a role in endomorphin bias. CONCLUSION: We found that endomorphin-1/2 and dynorphin-B displayed contrasting bias profiles at the MOR, and ruled out potential AC6 and RGS4 mechanisms in this bias. This identified signaling bias could be involved in specifying endogenous peptide roles in vivo, where these peptides have low selectivity between opioid receptor family members.

Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for the Mu-Delta Opioid Receptor Heterodimer that Reduces Morphine Withdrawal in Mice.

A major limitation in the study of the mu-delta opioid receptor heterodimer (MDOR) is that few selective pharmacological tools exist and no heteromer-selective antagonists. We thus designed a series of variable-length (15-41 atoms) bivalent linked peptides with selective but moderate/low-affinity pharmacophores for the mu and delta opioid receptors. We observed a U-shaped MDOR potency/affinity profile in vitro, with the 24-atom spacer length (D24M) producing the highest MDOR potency/affinity (<1 nM) and selectivity (≥89-fold). We further evaluated D24M in mice and observed that D24M dose-dependently antagonized tail flick antinociception produced by the MDOR agonists CYM51010 and Deltorphin-II, without antagonizing the monomer agonists DAMGO and DSLET. We also observed that D24M sharply reduced withdrawal behavior in models of acute and chronic morphine dependence. These findings suggest that D24M is a first-in-class high-potency MDOR-selective antagonist both in vitro and in vivo.

Synergistic interaction of the cannabinoid and death receptor systems - a potential target for future cancer therapies?

Cannabinoid receptors have been shown to interact with other receptors, including tumor necrosis factor receptor superfamily (TNFRS) members, to induce cancer cell death. When cannabinoids and death-inducing ligands (including TNF-related apoptosis-inducing ligand) are administered together, they have been shown to synergize and demonstrate enhanced antitumor activity in vitro. Certain cannabinoid ligands have been shown to sensitize cancer cells and synergistically interact with members of the TNFRS, thus suggesting that the combination of cannabinoids with death receptor (DR) ligands induces additive or synergistic tumor cell death. This review summarizes recent findings on the interaction of the cannabinoid and DR systems and suggests possible clinical co-application of cannabinoids and DR ligands in the treatment of various malignancies.

In vitro pharmacological evaluation of the radiolabeled C-terminal substance P analogue Lys-Phe-Phe-Gly-Leu-Met-NH2: Does a specific binding site exist?

In the present paper, we report the synthesis, radiolabeling and comprehensive pharmacological evaluation of a C-terminally truncated tachykinin derivative, 3H-KFFGLM-NH2. The C-terminal fragments of endogenous tachykinins are pharmacophores responsible for interaction with the tachykinin receptors NK1, NK2 and NK3. The N-terminal fragments are responsible for modulation of receptor selectivity and interactions with other receptor systems. To evaluate and separate the function of an NK-pharmacophore from the activity of its parent neurokinin, KFFGLM-NH2 was synthesized in both tritiated and unlabeled forms. It has been proposed that the obtained NK-binding profiles of specific reference ligands and KFFGLM-NH2 differentiate monomeric and dimeric forms of NK receptors. We hypothesize that dimers of NK receptors could be specific receptor(s) for C-terminal fragments of all neurokinins as well as their C-terminal fragments, including H-KFFGLM-NH2. Dissociation of dimers into monomers opens access to additional allosteric binding sites. Fully elongated undecapeptide substance P interacts with both the "tachykinin pocket" and the "allosteric pocket" on the monomeric NK1 receptor, resulting in high and selective activation. However, C-terminal hexapeptide fragment analogues, recognizing only the "tachykinin pocket", may have less specific interactions with all tachykinin receptors in both monomeric and dimeric forms.

Tachykinin NK1 receptor antagonist co-administration attenuates opioid withdrawal-mediated spinal microglia and astrocyte activation.

Prolonged morphine treatment increases pain sensitivity in many patients. Enhanced spinal Substance P release is one of the adaptive changes associated with sustained opioid exposure. In addition to pain transmitting second order neurons, spinal microglia and astrocytes also express functionally active Tachykinin NK1 (Substance P) receptors. In the present work we investigated the role of glial Tachykinin NK1 receptors in morphine withdrawal-mediated spinal microglia and astrocyte activation. Our data indicate that intrathecal co-administration (6 days, twice daily) of a selective Tachykinin NK1 receptor antagonist (N-acetyl-L-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138; 20 µg/injection)) attenuates spinal microglia and astrocyte marker and pro-inflammatory mediator immunoreactivity as well as hyperalgesia in withdrawn rats. Furthermore, covalent linkage of the opioid agonist with a Tachykinin NK1 antagonist pharmacophore yielded a bivalent compound that did not augment spinal microglia or astrocyte marker or pro-inflammatory mediator immunoreactivity and did not cause paradoxical pain sensitization upon drug withdrawal. Thus, bivalent opioid/Tachykinin NK1 receptor antagonists may provide a novel paradigm for long-term pain management.

Repeated morphine treatment-mediated hyperalgesia, allodynia and spinal glial activation are blocked by co-administration of a selective cannabinoid receptor type-2 agonist.

Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine-mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyperalgesia and tactile allodynia in rats. A CB2 (AM 630) but not a CB1 (AM 251) antagonist mitigated this effect. AM 1241 co-treatment also attenuated spinal astrocyte and microglial marker and pro-inflammatory mediator (IL-1ß, TNFα) immunoreactivities in morphine-treated rats, suggesting that CB2 agonists may be useful to prevent the neuroinflammatory consequences of sustained morphine treatment.

Asymmetric synthesis and conformational analysis by NMR spectroscopy and MD of Aba- and α-MeAba-containing dermorphin analogues.

Dermorphin analogues, containing a (S)- and (R)-4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one scaffold (Aba) and the α-methylated analogues as conformationally constrained phenylalanines, were prepared. Asymmetric phase-transfer catalysis was unable to provide the (S)-α-Me-o-cyanophenylalanine precursor for (S)-α-MeAba in acceptable enantiomeric purity. However, by using a Schöllkopf chiral auxiliary, this intermediate was obtained in 88 % ee. [(S)-Aba 3-Gly 4]dermorphin retained µ-opioid affinity but displayed an increased δ-affinity. The corresponding R epimer was considerably less potent. In contrast, the [(R)-α-MeAba 3-Gly 4]dermorphin isomer was more potent than its S epimer. Tar-MD simulations of both non-methylated [Aba 3-Gly 4]dermorphin analogues showed a degree of folding at the C-terminal residues toward the N terminus of the peptide, without however, adopting a stabilized ß-turn conformation. The α-methylated analogues, on the other hand, exhibited a type I/I' ß-turn conformation over the α-MeAba 3 and Gly 4 residues, which was stabilized by a hydrogen bond involving Tyr 5-HN and D-Ala 2-CO.

Recent advances in endomorphin engineering.

Recent statistics from the World Health Organization indicate that a high percentage of people worldwide suffer from a wide variety of acute or cancer-associated chronic pain. At present, with a few exceptions, the treatment of severe pain relies upon oral administration of the mu-opioid receptor-targeting opiate morphine and its surrogates under strict clinical control. In spite of the powerful in vivo efficacy of these drugs, their long-term use is limited by antinociceptive tolerance, physical dependence, and respiratory depression that evolve. As no analgesics with moderate side effect profiles are currently available for the therapy of different types of pain and stages of cancer, considerable efforts must be made in the search for opiate substitutes. Following the recognition that endogenous peptide ligands of the opioid receptors exert striking effects in various pain models, and with the recent advances in chemical synthesis methods, research interest has steadily moved toward peptide-based compounds as potential opioid analgesics. The endomorphins are an attractive set of endogenous opioid peptides that may meet the requirements of opioid-based pain management. By virtue of their excellent mu-opioid receptor labeling and favorable analgesic properties, these tetrapeptides have gained attention in recent years as potential lead compounds. The ever-increasing number of publications in this field strongly suggests that modified analogues of endomorphins could serve as potent substitutes for opiates, with a lower propensity to induce side effects. This review surveys the main results achieved over the past decade regarding the design, radiolabeling, pharmacological characterization, and structure-activity features of a large body of endomorphin derivatives.

Detection of a novel immunoreactive endomorphin 2-like peptide in rat brain extracts.

To pursue further the possible de novo biosynthetic pathway of endomorphins in rat brain we raised antibodies to endomorphin-2 conjugate in rabbits. Antiserum R1 recognized endomorphin-2 with good selectivity as compared to endomorphin-1 with a median detection value of 65.5+/-7.5 pg/tube (n=7), whereas R4 antiserum recognized both endomorphins with similar sensitivity. Neither antisera recognized YP-related di- or tripeptides or YGGF-related opioid sequences (enkephalins, beta-endorphin, dynorphin). Using the same rat brain extraction-RP-HPLC-gradient separation paradigm as previously, antisera detected 144.6+/-40.0 (n=3) pg/g wet brain weight endomorphin-2-like immunoreactivity in the fraction corresponding to standard endomorphin-2 retention time and also in the fraction matching endomorphin-2-OH standard retention time (179.1+/-30.1 pg/g). Since R1 failed to recognize authentic endomorphin-2-OH, the second immunoreactive species must be different from both endomorphin-2 and endomorphin-2-OH. Possible biosynthetic intermediates to endomorphins, synthetic YPFFG and YPWFG had retention times close to the parent endomorphin standards in RP-HPLC gradient separation profile. The former was a mu-opioid receptor agonist of medium potency in the in vitro assays (rat brain RBA>P gamma S binding and mouse vas deferens), whereas the latter was a weak mu-opioid receptor agonist with a significant delta-opioid receptorial action as well and a definite indication of partial agonism.

Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters.

In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [(14)C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K(i) = 24 microm and 3.4 microm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[(3)H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and delta-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K(t)) of 7.6 microm and a maximal velocity (V(max)) of 1.1 nmol x 30 min(-1) x mg of protein(-1). Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[(3)H]Pro is a valuable substrate for both mechanistic and structural studies of H(+)/peptide transporter proteins.