Involvement of the cannabimimetic compound, N-palmitoyl-ethanolamine, in inflammatory and neuropathic conditions: Review of the available pre-clinical data, and first human studies

The endogenous cannabimimetic compound, and anandamide analogue, N-palmitoyl-ethanolamine (PEA), was shown to exert potent anti-inflammatory and analgesic effects in experimental models of visceral, neuropathic and inflammatory pain by acting via several possible mechanisms. However, only scant data have been reported on the regulation of PEA levels during pathological conditions in animals or, particularly, humans. We review the current literature on PEA and report the results of three separate studies indicating that its concentrations are significantly increased during three different inflammatory and neuropathic conditions, two of which have been assessed in humans, and one in a mouse model. In patients affected with chronic low back pain, blood PEA levels were not significantly different from those of healthy volunteers, but were significantly and differentially increased (1.6-fold, P<0.01, N = 10 per group) 30 min following an osteopathic manipulative treatment. In the second study, the paw skin levels of PEA in mice with streptozotocin-induced diabetic neuropathic pain were found to be significantly higher (1.5-fold, P<0.005, N = 5) than those of control mice. In the third study, colonic PEA levels in biopsies from patients with ulcerative colitis were found to be 1.8-fold higher (P<0.05, N = 8–10) than those in healthy subjects. These heterogeneous data, together with previous findings reviewed here, substantiate the hypothesis that PEA is an endogenous mediator whose levels are increased following neuroinflammatory or neuropathic conditions in both animals and humans, possibly to exert a local anti-inflammatory and analgesic action.     

Candidate genes for cannabis use disorders: findings, challenges and directions

Aim   Twin studies have shown that cannabis use disorders (abuse/dependence) are highly heritable. This review aims to: (i) review existing linkage studies of cannabis use disorders and (ii) review gene association studies, to identify potential candidate genes, including those that have been tested for composite substance use disorders and (iii) to highlight challenges in the genomic study of cannabis use disorders.
Methods   Peer-reviewed linkage and candidate gene association studies are reviewed.
Results   Four linkage studies are reviewed: results from these have homed in on regions on chromosomes 1, 3, 4, 9, 14, 17 and 18, which harbor candidates of predicted biological relevance, such as monoglyceride lipase ( MGLL ) on chromosome 3, but also novel genes, including ELTD1 [epidermal growth factor (EGF), latrophilin and seven transmembrane domain containing 1] on chromosome 1. Gene association studies are presented for (a) genes posited to have specific influences on cannabis use disorders: CNR1 , CB2 , FAAH , MGLL , TRPV1 and GPR55 and (b) genes from various neurotransmitter systems that are likely to exert a non-specific influence on risk of cannabis use disorders, e.g. GABRA2 , DRD2 and OPRM1 .
Conclusions   There are challenges associated with (i) understanding biological complexity underlying cannabis use disorders (including the need to study gene–gene and gene–environment interactions), (ii) using diagnostic versus quantitative phenotypes, (iii) delineating which stage of cannabis involvement (e.g. use versus misuse) genes influence and (iv) problems of sample ascertainment.     

Regulation of anandamide tissue levels by N-arachidonylglycine

N-arachidonylglycine (NAGly), the carboxylic analog of the endocannabinoid anandamide, occurs in rat and bovine brain as well as in peripheral sites and shows activity against tonic, formalin-induced pain. It was also observed, using cell membrane preparations, that it inhibits the hydrolytic activity of fatty acid amide hydrolase (FAAH) on anandamide (N-arachidonylethanolamide). These data suggested that it may serve as an endogenous regulator of tissue anandamide concentrations. In this report, we show findings derived from mass spectrometric analyses, indicating that blood levels of anandamide in rats given 10 mg/kg p.o. of NAGly were increased significantly by more than 9-fold when compared with vehicle-treated controls. In vitro evidence in RAW 264.7 cells using a deuterium-labeled NAGly demonstrated that it was not a precursor or source of arachidonic acid for the observed 50% rise in anandamide levels, suggesting that the increase was due to some effect other than increased biosynthesis of anandamide. Moreover, the findings presented here suggest that NAGly can serve as a model for the design of agents to provide pharmacological control of tissue anandamide concentrations.     

More Aroused, Less Fatigued: Fatty Acid Amide Hydrolase Gene Polymorphisms Influence Acute Response to Amphetamine

Amphetamine is a stimulant drug that enhances attention and feelings of alertness. Amphetamine''s effects are known to be modulated by endogenous cannabinoids, which are degraded by the enzyme fatty acid amide hydrolase (FAAH). In this study we investigated inter-individual differences in mood response to amphetamine in relation to four polymorphisms in the FAAH gene, including the FAAH missense variant rs324420C → A (Pro129Thr), which was previously found to be associated with street drug use and addictive traits. One hundred and fifty-nine healthy Caucasian volunteers participated in a three-session, double-blind crossover study receiving either placebo or oral d-amphetamine (10 and 20 mg). Associations between individual genotypes and levels of self-reported Arousal (Profile of Mood States) after d-amphetamine ingestion were investigated using two-way ANOVAs/ANCOVAs. Association analyses for haplotypes were performed using the adaptive permutation approach implemented in PLINK. Genotypes at rs3766246 and rs2295633 were significantly associated with increased ratings of Arousal (p<0.05) and Fatigue (p<0.01) after the 10-mg dose. Fatigue levels were also found to be associated with the haplotypes CCC and TAT formed from rs3766246, rs324420, and rs2295633 (p<0.05). These data suggest that the endocannabinoid system influences variation in subjective response to amphetamine. This has important implications for understanding the role of endogenous cannabinoids in response to amphetamine, studies of poly-substance abuse, and understanding the genetic determinants of inter-individual differences in stimulant effects and risk of abuse.     

Stress-induced analgesia

For over 30 years, scientists have been investigating the phenomenon of pain suppression upon exposure to unconditioned or conditioned stressful stimuli, commonly known as stress-induced analgesia. These studies have revealed that individual sensitivity to stress-induced analgesia can vary greatly and that this sensitivity is coupled to many different phenotypes including the degree of opioid sensitivity and startle response. Furthermore, stress-induced analgesia is influenced by age, gender, and prior experience to stressful, painful, or other environmental stimuli. Stress-induced analgesia is mediated by activation of the descending inhibitory pain pathway. Pharmacological and neurochemical studies have demonstrated involvement of a large number of neurotransmitters and neuropeptides. In particular, there are key roles for the endogenous opioid, monoamine, cannabinoid, γ-aminobutyric acid and glutamate systems. The study of stress-induced analgesia has enhanced our understanding of the fundamental physiology of pain and stress and can be a useful approach for uncovering new therapeutic targets for the treatment of pain and stress-related disorders.     

Effects of cocaine in 5-lipoxygenase-deficient mice

Summary. 5-Lipoxygenase (5-LOX), along with 12-lipoxygenase and cyclooxygenases, metabolizes arachidonic acid into eicosanoids. In rodents, 12-lipoxygenase deficiency alters behavioral responses to cocaine. We used 5-LOX-deficient mice and their controls to investigate cocaine’s actions. After repeated cocaine injections, the increase in locomotor activity was greater in 5-LOX-deficient mice. Since the 5-LOX pathway may regulate the levels/metabolism of arachidonoylethanolamide (AEA) we assayed the AEA levels in the striatum, the binding of the endogenous AEA to the cannabinoid receptor CB1R, and anandamide hydrolase (FAAH) activity in the striatum, hippocampus, and cortex. Striatal AEA levels decreased after repeated cocaine injections. Cocaine also decreased CB1R binding in all brain regions studied and the only significant differences between 5-LOX-deficient and control mice was the greater hippocampal FAAH activity in 5-LOX-deficient mice. Our results demonstrated that a 5-LOX deficiency alters sensitivity to repeated cocaine. It should be investigated whether a human 5-LOX gene polymorphism affects cocaine’s actions. M. Maccarrone, H. Manev: These authors contributed equally as the senior authors. Correspondence: Hari Manev, Department of Psychiatry, The Psychiatric Institute, University of Illinois, 1601 West Taylor Street, MC912 Chicago, IL 60612, USA     

CNR1 and FAAH variation and affective states induced by marijuana smoking

Background: Polymorphisms in cannabinoid receptor type 1 (encoded by CNR1) and fatty acid amide hydrolase (encoded by FAAH) have been associated with cannabis dependence, but it remains unknown whether variation within these genes influences cannabis’ acute effects on affect.

Objective: Conduct a secondary data analysis study to determine whether previously observed acute effects of tetrahydrocannabinol (THC) on mood was dependent upon variation in CNR1 and FAAH.

Methods: A balanced placebo design was used crossing marijuana administration (i.e., 0% THC vs. 2.8% THC) with stimulus expectancy. Participants (N = 118; 64% male) provided DNA and completed the Profile of Mood States questionnaire prior to and after smoking. Haplotypes were constructed from genotyped single nucleotide polymorphisms for CNR1 (rs1049353 and rs806368) and FAAH (rs4141964, rs324420, and rs11576941); rs2023239 (CNR1) and rs6703669 (FAAH) were not part of a phased haplotype block. Analyses tested both main and interaction effects for genotype across CNR1 and FAAH, and drug, and expectancy effects.

Results: THC increased levels of POMS Tension-Anxiety and Confusion-Bewilderment over and above the effects of variation in CNR1 and FAAH. Significant drug X genotype/haplotype and expectancy X genotype/haplotype interaction effects were observed for some but not all mood states [e.g., ‘C’ allele carriers of rs2023239 who received THC had higher levels of Anger-Hostility (β= 0.29 (0.12), p= .02) compared to those who received placebo].

Conclusion: These preliminary findings suggest individual differences in mood states after using marijuana depend on genetic variation. Such information might be useful in understanding either motivation for use of marijuana and/or risk for associated behaviors.     

A role for vanilloid receptor 1 (TRPV1) and endocannabinnoid signalling in the regulation of spontaneous and L-DOPA induced locomotion in normal and reserpine-treated rats

Although most commonly associated with actions at cannabinoid CB1 receptors on the extracellular surface of the plasma membrane, the endocannabinoid anandamide (AEA) is also transported into the cell, by the putative anandamide membrane transporter (AMT), and activates the vanilloid receptor 1 (TRPV1) at an intracellular site. AEA is then inactivated by fatty acid amide hydrolase (FAAH). As systemic administration of TRPV1 ligands reduces locomotor activity in normal rodents, we hypothesised that activation of TRPV1 by endocannabinoids could play a role in the control of voluntary movement and that such actions could be regulated by AMT and FAAH. Motor activity was assessed in normal, in reserpine-treated, and in reserpine-treated rats treated with L-DOPA. In normal rats, the TRPV1 agonist capsaicin (1 mg/kg) or the FAAH inhibitor URB597 (10 mg/kg) caused a significant reduction in movement in both the horizontal (locomotion) and vertical (rearing) planes (-45% and -53% respectively with capsaicin; -33% and -37% for URB597). Capsaicin-induced hypolocomotion was attenuated by the TRPV1 antagonist, capsazepine. There was no effect of capsaicin, URB597 or the AMT inhibitor OMDM-2 on motor activity in reserpine-treated rats. L-DOPA treatment of reserpine-treated rats elicited high levels of motor activity in both the horizontal and vertical planes. Horizontal activity was attenuated by capsaicin (1 mg/kg, -60%), but not by URB597 (10 mg/kg) or OMDM-2 (5 mg/kg). Vertical activity was attenuated by capsaicin (1 mg/kg, -61%) and by URB597 (10 mg/kg, -54%), but not by OMDM-2. These data suggest that activation of the TRPV1 system can suppress spontaneous locomotion in normal animals and modulates several L-DOPA-induced behaviours in reserpine-treated rats.     

Lack of effect of chronic pre-treatment with the FAAH inhibitor URB597 on inflammatory pain behaviour: evidence for plastic changes in the endocannabinoid system

BACKGROUND AND PURPOSE

Elevating levels of endocannabinoids with inhibitors of fatty acid amide hydrolase (FAAH) is a major focus of pain research, purported to be a safer approach devoid of cannabinoid receptor-mediated side effects. Here, we have determined the effects of sustained pharmacological inhibition of FAAH on inflammatory pain behaviour and if pharmacological inhibition of FAAH was as effective as genetic deletion of FAAH on pain behaviour.

EXPERIMENTAL APPROACH

Effects of pre-treatment with a single dose, versus 4 day repeated dosing with the selective FAAH inhibitor, URB597 (i.p. 0.3 mg·kg⁻¹), on carrageenan-induced inflammatory pain behaviour and spinal pro-inflammatory gene induction were determined in rats. Effects of pain induction and of the drug treatments on levels of arachidonoyl ethanolamide (AEA), palmitoyl ethanolamide (PEA) and oleolyl ethanolamide (OEA) in the spinal cord were determined.

KEY RESULTS

Single, but not repeated, URB597 treatment significantly attenuated the development of inflammatory hyperalgesia (P < 0.001, vs. vehicle-treated animals). Neither mode of URB597 treatment altered levels of AEA, PEA and OEA in the hind paw, or carrageenan-induced paw oedema. Single URB597 treatment produced larger increases in AEA, PEA and OEA in the spinal cord, compared with those after repeated administration. Single and repeated URB597 treatment decreased levels of immunoreactive N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) in the spinal cord and attenuated carrageenan-induced spinal pro-inflammatory gene induction.

CONCLUSION AND IMPLICATIONS

Changes in the endocannabinoid system may contribute to the loss of analgesic effects following repeated administration of low dose URB597 in this model of inflammatory pain.     

Cannabinoid receptor type 1 expression during postnatal development of the rat retina

Cannabinoid receptor type 1 (CB1R) participates in developmental processes in the central nervous system (CNS). The rodent retina represents an interesting and valuable model for studying CNS development, because it contains well-identified cell types with clearly established and distinct developmental timelines. Very little is known about the distribution or function of CB1R in the developing retina. In this study, we investigated the expression pattern of CB1R in the rat retina during all stages of postnatal development. Western blots were performed on retinal tissue at different time points between P1 and adulthood. In order to identify the cells expressing the receptor and the age at which this expression started, immunohistochemical co-staining was carried out for CB1R and markers of the different cell types comprising the retina. CB1R was already present at P1 in various cell types, i.e., ganglion, amacrine, horizontal, and mitotic cells. In the course of development, it appeared in cone photoreceptors and bipolar cells. For some cell types (bipolar, Müller, and some amacrine cells), CB1R was transiently expressed, suggesting a potential role of this receptor in developmental processes, such as migration, morphological changes, sub-identity acquisition, and patterned retinal spontaneous activity. Our results also indicated that CB1R is largely expressed in the adult retina (cone photoreceptors and horizontal, most amacrine, and retinal ganglion cells), and may therefore contribute to retinal functions. Overall these results indicate that, as shown in other structures of the brain, CB1R could play an instrumental role in the development and function of the retina.