Non-cannabinoid CB1, non-cannabinoid CB2 antinociceptive effects of several novel compounds in the PPQ stretch test in mice
Introduction
The study of cannabinoids as analgesics stems from findings that compounds like delta9-tetrahydrocannabinol (Δ9-THC), the major psychoactive component of marijuana, can produce antinociception without the respiratory depression associated with opioid analgesics (Gaoni and Mechoulam, 1964). In animal models, a “tetrad” of behavioral effects, including antinociception in the tail-flick test, has proved to be a reliable indicator of cannabimimetic activity (Martin et al., 1991). However, cannabinoids such as Δ9-THC are also active in other antinociceptive tests, including the phenyl-p-quinone (PPQ) stretch test (Pearl et al., 1968).
The central effects of cannabinoids are mediated by the cannabinoid CB1 receptor, a G protein-coupled membrane receptor localized in brain regions associated with pharmacological actions such as analgesia (Herkenham et al., 1990, Matsuda et al., 1990). An endogenous ligand for the cannabinoid CB1 receptor, arachidonylethanolamide (anandamide), also exhibits cannabinoid-like pharmacology and dose-dependent analgesia (Devane et al., 1992). Anandamide is also a full agonist at the TRPV1 vanilloid receptor, a member of the large family of transient receptor potential (TRP) channels comprised of a diverse group of non-voltage-gated cation channels involved in sensory signaling and is found throughout the whole neuroaxis, plus a variety of nonneural tissues (Szallasi, 2002, Szallasi and Appendino, 2004).
®-(+)-arachidonyl-1′-hydroxy-2′-propylamide (®-methanandamide) is a chiral, metabolically stable congener of anandamide with higher binding preference for the cannabinoid CB1 receptor. Studies have shown that chronic exposure to ®-methanandamide in rats result in changes in cannabinoid CB1 receptor binding and mRNA expression in the basal ganglia, cerebellum and the hippocampus (Romero et al., 1999). Studies have also shown that ®-methanandamide possesses cannabimimetic properties (Abadji et al., 1994) by the mouse “tetrad” of hypothermia, hypokinesia, ring immobility and antinociception (Martin et al., 1991).
Palmitoylethanolamide, an endogenous cannabimimetic fatty acid derivative (Devane et al., 1992), binds to CB2 cannabinoid receptors on mast cells and down-modulates mast cell activation in vitro (Mazzari et al., 1996). Like anandamide, palmitoylethanolamide is anti-hyperalgesic in both visceral and somatic pain models and acts synergistically with anandamide in reducing pain responses in the formalin test (Calignano et al., 1998, Jaggar et al., 1998). However, palmitoylethanolamide binds with low affinity to both cannabinoid receptor subtypes (Di Marzo, 1998, Lambert and Di Marzo, 1999) and may behave as an “enhancer” of endogenous anandamide (Di Marzo et al., 2000c). Palmitoylethanolamide has been shown to reduce formalin-induced, dextran-induced and carrageenan-induced edema and hyperalgesia when administered orally to rats (Mazzari et al., 1996), as well as inhibiting hyperalgesia after sciatic nerve ligation in rats (Helyes et al., 2003).
N-vanillyl-arachidonyl-amide (arvanil) is a chemical “hybrid” between anandamide and capsaicin (Di Marzo et al., 2000a). It is among the most potent inhibitors of the anandamide membrane transporter (Szallasi and Di Marzo, 2000) and is 4-fold more potent than anandamide at cannabinoid CB1 receptors (Melck et al., 1999). Arvanil also activates TRPV1 receptors with higher potency than either anandamide or capsaicin (De Petrocellis et al., 2000). The effect of i.t. administration of arvanil are not blocked by i.t. administration of antagonists to the cannabinoid CB1 or TRPV1 receptors (Di Marzo et al., 2000a), nor did these receptor antagonists reverse the analgesic effect of arvanil in the rat formalin test (Brooks et al., 2002). Nevertheless, arvanil is a potent in vivo analgesic, vasodilator and anti-inflammatory compound (Szallasi and Di Marzo, 2000).
Cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB597) is a selective fatty acid amide hydrolase (FAAH) inhibitor (Kathuria et al., 2003). It has been shown to increase brain anandamide levels and magnify anandamide responses by inhibiting intracellular FAAH activity in wild-type and FAAH−/− mice (Fegley et al., 2005). It has also been demonstrated in FAAH−/− mice that a reduction of FAAH activity results in the reduction of pain sensation and enhancement of endocannabinoid signaling (Cravatt et al., 2001).
Acetaminophen is a well-known antipyretic analgesic, often classified as a nonsteriodal anti-inflammatory drug (NSAID), but different because of failure to reduce the inflammation of rheumatoid arthritis. Though a weak inhibitor of the synthesis of prostaglandins of COX-1 and COX-2 in broken cell systems, therapeutic concentrations of acetaminophen inhibit prostaglandin synthesis in intact cells in vitro involving the COX-2 pathway when levels of arachidonic acid and peroxide tone are low (Graham and Scott, 2005). N-(4-Hydroxyphenyl)-arachidonamide (AM404), a potent activator of TRPV1, as well as a ligand at cannabinoid CB1 receptors and an inhibitor of cellular anandamide uptake, has been recently identified as a novel metabolite of acetaminophen. This provides a link between acetaminophen and the cannabinoid/vanilloid receptor system (Högestätt et al., 2005).
Due to the increasing evidence that cannabinoid and TRPV1 systems influence each other in the transmission and/or blockade of pain perception, it is beneficial to evaluate these classes of compounds in antinociceptive paradigms. In this study, we examined the analgesic and anti-hyperalgesic effects of cannabinoid- and vanilloid-like compounds as well as acetaminophen, in mice by the PPQ method (a visceral pain model), using different routes of administration and potential receptor antagonists. PPQ has become a standard test agent for preclinical screening of potential analgesic compounds in mice because it correlates well with similar analgesic activity in humans (Dubinsky et al., 1987). We also conducted molecular studies with short-term administration of Δ9-THC, ®-methanadamide and arvanil to assess the possibility of any receptor protein changes via cannabinoid, TRPV1 and opioid receptors.
Section snippets
Animals
All animal studies were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No.80-23, 1996). Male ICR mice (Harlan Laboratories, Indianapolis, IN) weighing 16 to 20 g were housed 4 per cage in an animal care facility maintained at 22 ± 2 °C on a 12 h light/dark cycle. Food and water were given ad libitum. Animals were brought to the
Results
These studies examined the antinociceptive efficacy of various cannabinoid and vanilloid-like compounds in a novel paradigm, the PPQ test. All drugs were tested via several routes of administration to determine by which route each drug was most efficacious. We were also testing for oral efficacy due to the possible clinical relevance, given that oral administration of cannabinoids yields high analgesic effects (Cichewicz et al., 1999). The PPQ test, a visceral pain model, is an effective test
Discussion
Many researchers have studied Δ9-THC and other cannabinoids as important analgesic compounds. The typical tests used to assess pain-relieving abilities in rodents include the tail-flick and the hot-plate tests, based on supraspinal and spinal reflexes that mediate the signaling of pain stimuli. However, little information exists about cannabinoid or vanilloid drugs in the PPQ test, a visceral pain model mediated by the prostaglandin system. The studies presented here suggest a non-cannabinoid CB
Acknowledgement
This work was supported by NIDA Grants DA-09789, DA-05274 and K02-DA-00186.
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