Abstract

Introduction

The cannabinoids are a group of terpenophenolic compounds present in the marijuana plant, Cannabis sativa. At present, three general types of cannabinoids have been identified: phytocannabinoids present uniquely in the cannabis plant, endogenous cannabinoids produced in humans and animals, and synthetic cannabinoids generated in a laboratory (Sarfaraz et al., 2008). It is worth noting that Cannabis sativa produces over 80 cannabinoids (Console-Bram et al., 2012). The broader definition of cannabinoids refers to a group of substances that are structurally related to Δ9-tetrahydrocannabinol (Δ9-THC) or that bind to cannabinoid receptors. The chemical definition includes a variety of distinct chemical classes: the classical cannabinoids structurally related to THC, the non-classical cannabinoids, the aminoalkylindoles, the eicosanoids related to the endocannabinoids, quinolines and arylsulphonamides (Woelkart et al., 2008; Console-Bram et al., 2012), and additional compounds that do not fall into these standard classes, but bind to cannabinoid receptors (CB). Multifaceted effects of marijuana can be singled out aiming at evaluation of the potential medical value of marijuana and cannabinoids in specific human diseases, with minimal undesired side effects.

Nomenclature of cannabinoid receptors (CB)

There are two well-characterized CB with distinctly different physiological properties. The psychoactive effects of cannabinoids are associated with the CB1 receptor; the CB2 receptor mainly mediates anti-inflammatory and immunomodulatory actions (Miller and Stella, 2008).

CB2

The second cannabinoid receptor (CB2) was isolated from human myeloid cells in 1992 (Munro et al., 1993). Soon afterwards, CB2 was identified in other species, such as mouse, rat, bovine and zebra fish. The CB2 gene is located on chromosome 1 and 4 in humans and mice, respectively. Unlike the mouse CB2 gene, which is intron-less, the human gene has been reported to have two splice variants (Liu et al., 2009). The longer variant human CB2a, comprised of exon 1a, exon 1b and exon 3, is mostly expressed in testis (about 100-fold more compared to spleen or leukocytes). It is also detected in various regions of the brain (Liu et al., 2009). The shorter variant, human CB2b, consisting of exon 2 and exon 3, is expressed mostly in the spleen and leukocytes, 100-and 30-fold higher, respectively, when compared to CB2b expression in the brain (Liu et al., 2009). Immune cells express high levels of CB2 and there is a hierarchy of CB2 expression within the immune system (B cells > natural killer cells > monocytes > neutrophils > CD8 lymphocytes > CD4 lymphocytes)(Nunez et al., 2004; Yao and Mackie, 2009). The level of expression is dependent on the activation state of the cell and the type of stimuli. Stimulation of splenocytes with LPS leads to CB2 mRNA down- regulation, whereas CD40 co-stimulation results in CB2 mRNA up-regulation (Lee et al., 2001). CB2 receptor sequences are less conserved throughout evolution compared to CB1 receptors. CB2 and CB1 receptors share about 44% identity in humans at the level of amino acids (Liu et al., 2009; Console-Bram et al., 2012). Humans and mice share about 82% identity in amino acid sequence of CB2 (Anday and Mercier, 2005), whereas mice and rat share 93% identity. Human, rat and mouse sequences diverge at the C-terminus. The mouse protein is 13 amino acids shorter, while the rat protein is 50 amino acids longer than the human (Console-Bram et al., 2012). Diversity in CB2 sequences between species can possibly explain differential effects of CB2 agonists in human and rodent models (Figure 1).

Figure 1

Multiple alignment and homology of human and mouse CB1 and CB2 receptors. Sequence alignment was performed using ClustalW tool (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The following symbols denote the degree of conservation observed in each column: …

Medical marijuana

Medical marijuana refers to the parts of the cannabis plant used as a physician-recommended form of herbal therapy or as medicine (synthetic analogs of cannabinoids). In the US recently, use of medical marijuana has surged in the 16 states and the District of Columbia that permit its use. The cannabis plant has a long history of use as a medicine dating back to 2737 B.C. (Ben Amar, 2006). Cannabis sativa contains over 80 different chemical constituents (Molina et al., 2011; Console-Bram et al., 2012). There are three phytocannabinoids (Figure 2): THC, cannabidiol (CBD) and cannabinol (CBN). THC is a primary component of cannabis, responsible for the psychoactive effects of the plant; it has mild analgesic and antioxidant activities (Hampson et al., 1998; Molina et al., 2010). CBD represents up to 40% of the extract of medicinal cannabis. It has been shown to relieve cough, congestion and nausea, convulsion, inflammation and anxiety, and has been shown to be effective in treating multiple sclerosis (MS), anxiety attacks and schizophrenia (Crippa and Zuardi, 2006; Zuardi et al., 2006a; Zuardi et al., 2006b; Kogan et al., 2007; Mechoulam et al., 2007; Lakhan and Rowland, 2009). CBN is a breakdown product of THC and acts as a weak agonist of CB1 and CB2 because of lower affinity than THC toward receptors (Mahadevan et al., 2000).

Figure 2

Prototypical structures of natural and synthetic cannabinoids.

There are several medications (plant extracts or synthetic cannabinoid analogues of THC alone or in combination with CBD) that have been approved for medicinal use. Nabilone® was approved in the US and Canada in 1985 as an anti-nausea treatment for patients undergoing cancer chemotherapy (Todaro, 2012). Sativex® was approved in Canada and the UK for treatment of neuropathic pain in MS and pain associated with cancer (Russo et al., 2007; Sastre-Garriga et al., 2007). Sativex® has been also approved for MS-associated spasticity in many countries, such as New Zealand, UK, Austria, Czech Republic, Denmark, Germany, Sweden, Israel, Italy and Spain (Leussink et al., 2012). In the majority of cases, administration of cannabinoids (natural or synthetic analogs) results in improvement of symptoms of MS, especially spasticity, muscle pain, tremors and bladder control. However, there were differences in the actions of orally administered versus inhaled cannabinoids, probably due to variable absorption of orally administered cannabinoids (Maurer et al., 1990; Russo et al., 2007). Several studies have been done on the pharmacokinetics of cannabinoids during different routes of drug administration. Smoking, the major route, provides a rapid and efficient method for drug delivery from the lungs to the brain, contributing to its abuse potential. The number, duration and spacing of the puffs, as well as holding time and inhalation volumes, greatly affect the degree of exposure (Heishman et al., 1989; Azorlosa et al., 1992). Synthetic THC, Dronabinol (Marinol®), is usually taken orally, but may also be administered rectally. In addition, abuse is being common by the oral route. Absorption is slower when cannabinoids are ingested, with lower and delayed peak THC concentrations (Law et al., 1984; Huestis, 2007). To prevent first-pass metabolism by the liver, Sativex® is administered sublingually, via the oromucosal route. The oromucosal form of administration has the advantage that the first-pass effects are reduced and, compared to smoking, the maximum plasma THC levels are lower and increase more slowly (Karst et al., 2010; Leussink et al., 2012). Because MS is considered to be a relapsing chronic inflammatory disease of the CNS, beneficial anti-inflammatory effects of cannabinoids would provide much needed MS symptom relief. Clinical benefits for the use of cannabinoids in MS patients were also supported by studies in animal models of MS (Baker et al., 2001; Pryce and Baker, 2005; Mestre et al., 2009).

CB1 antagonists (AM251 and SR141716A) lead to changes in efflux of noradrenaline and dopamine in various brain regions, leading to antidepressant effects (Tzavara et al., 2003; Witkin et al., 2005). These studies suggest the possibility of use of cannabinoids in the treatment of mood disorders, such as depression, anxiety, manic depression and posttraumatic stress (Ashton and Moore, 2011).

Marinol® (Dronabinol) was approved by the US and Canada in 1985 for patients undergoing cancer chemotherapy to relieve nausea associated with treatment. In 1992, it was approved for treatment of anorexia associated with AIDS-related weight loss (Dejesus et al., 2007; Todaro, 2012).

Cannabinoids are increasingly being used for treatment of migraine headaches in patients that are not responding to the serotonin 1D agonist, sumatripan (Krymchantowski and Jevoux Cda, 2007). Dronabinol has been shown to inhibit the 5-HT3 receptor involved in emetic and pain responses (Fan, 1995). A patent has been filed for the use of Dronabiniol for treatment of migraines (Barbato, 2007). Canasol® is used for reduction of intraocular pressure in glaucoma (Crandall et al., 2007). Cannabinoid compounds applied topically show hypotensive and neuroprotective effects in mice and monkeys (Williams et al., 2007; Yazulla, 2008). In 1992, the FDA approved the use of Dronabinol to stimulate appetite in AIDS patients suffering from wasting syndrome. This triggered further interest in the development of cannabinoid antagonists that would reduce appetite. Rimonabant has been shown to suppress appetite, producing reduction in body weight (Pi-Sunyer et al., 2006). Dronabinol has been shown to be a potent drug for treatment of HIV-associated sensory neuropathy, the most common peripheral nerve disorder complicating HIV-1 infection (Abrams et al., 2007).

Synthetic cannabinoid agonists

Synthetic cannabinoids are functionally similar to THC, the active part of cannabis. Like THC, they bind to the same cannabinoid receptors in the brain and other organs as the endogenous ligands, anandamide and 2-arachidonylglycerol, which interact with both CB1 and CB2 receptors. More correctly designated as cannabinoid receptor agonists, synthetic cannabinoid agonists were developed over the past forty years as therapeutic agents, often for the treatment of pain. However, it proved difficult to separate their desired properties from unwanted psychoactive effects.

Although often referred to simply as synthetic cannabinoids, many of the substances are not structurally related to the so-called “classical” cannabinoids, i.e., compounds like THC, based on dibenzopyran. The cannabinoid receptor agonists form a diverse group, but most are lipid soluble, non-polar, and consist of 22 to 26 carbon atoms; they would therefore be expected to volatilize readily when smoked. A common structural feature is a side-chain, where optimal activity requires more than four and up to nine saturated carbon atoms. The first figure shows the structure of THC, while the others show examples of synthetic cannabinoid receptor agonists, all of which have been found in “Spice” or other smoking mixtures. The synthetic cannabinoids fall into seven major structural groups (Figure 2):

1. Naphthoylindoles (e.g., JWH-018, JWH-073, JWH-398).
2. Naphthylmethylindoles. (e.g., JWH-175, JWH-195, JWH-197).
3. Naphthoylpyrroles.(e.g., JWH-030, JWH-156, JWH-243).
4. Naphthylmethylindenes (e.g., JWH-176).
5. Phenylacetylindoles (i.e., benzoylindoles, e.g., JWH-250, JWH-253, JWH-313).
6. Cyclohexylphenols (e.g., CP 47,497 and homologs of CP 47,497).
7. Classical cannabinoids (e.g., HU-210).

Compounds in groups 1–5 (JWH compounds) were largely synthesized by Huffman et al. over the past fifteen years (Huffman et al., 1996; Wiley et al., 1998; Huffman et al., 2000; Huffman et al., 2003; Huffman et al., 2005; Huffman et al., 2006; Huffman et al., 2010). The sixth group, cyclohexylphenols, were developed by Pfizer during the 1970’s and 1980’s (Carissimi et al., 1976). The classical cannabinoids (seventh group) is the oldest; synthesis began in the 1960’s following the identification of the chemical structure of THC (Mechoulam and Gaoni, 1965, 1967; Gaoni and Mechoulam, 1971). In general, the agonists show little selectivity between CB1 and CB2, while antagonist compounds are highly selective (>1000 fold selective for CB1 vs. CB2 and vice versa with nanomolar affinity at the relevant receptor)(Console-Bram et al., 2012). The selectivity of these antagonists allows the discrimination of CB1- vs. CB2-mediated effects in vitro and in vivo. Despite the existence of numerous non-selective agonists, there are some that exhibit selectivity. For example, HU-308 is a highly selective CB2 agonist with nanomolar affinity at CB2 and >1000 fold selectivity for CB2 vs. CB1. If a compound shows >100 fold selectivity, it is classified as a selective agonist (Console-Bram et al., 2012).

CB signaling – CB-ligand interactions

CB2, as with the rest of the GPCRs, signals through the three main components of the MAPK pathway, namely, ERK, JNK and p38 (Howlett et al., 2002). However, activation of CB receptor coupling to MAPKs is dependent on cellular content. A wide range of activation and inhibition has been observed dependent on cell type, cell differentiation status and co-modulators of MAPK cascades (Howlett, 2005). CB2 agonism decreased CXCR4-activation mediated G-protein activity and MAPK phosphorylation. Furthermore, CB2 agonists altered cytoskeletal reorganization, by decreasing F-actin levels, impairing productive infection (Costantino et al., 2012). Signaling events associated with inflammatory responses in endothelial cells and monocytes are complex; few studies have addressed potential mechanisms of anti-inflammatory CB2 stimulation. Gertsch et al. investigated intracellular signaling pathways triggered in monocytes by LPS-triggered TNFα and IL-1β production that were suppressed by CB2 agonist. LPS treatment of human monocytes led to a rapid phosphorylation of p38 and JNK1/2 (Gertsch et al., 2008), and CB2 agonist reduced Erk1/2 and JNK1/2 activation (phosphorylation)(Gertsch et al., 2008). Whether or not CB2 modulates ion channels has been controversial. Felder and colleagues (Felder et al., 1995) suggested that CB2 does not modulate potassium or high-voltage calcium channels. More recent work indicates that CB2agonists do modulate these channels and earlier results were due to functional selectivity of the ligands used (Atwood et al., 2012). CB2 affects additional signaling pathways, including activation of phospholipase C, leading to release of calcium (Shoemaker et al., 2005), regulation of small G proteins (such as Rho, Rac and cdc42) (Kurihara et al., 2006), and activation of JNK via the phosphatidyl inositol 3 kinase (PI3K)/Akt pathway (Viscomi et al., 2009; Atwood et al., 2012). In human umbilical vein endothelial cells, cannabidiol could evoke phosphorylation of p44/42 MAPK and PKB/Akt via PI3K pathway (Offertaler et al., 2003).

GPCRs are regulated at many levels. In general, once they are activated by ligand binding, their signaling is attenuated by a desensitization process, followed by internalization. Desensitization involves G protein receptor kinase-mediated phosphorylation of multiple serine or threonine residues of GPCR, followed by binding of the β-arestins. This protein complex is usually localized to clathrin-coated pits or caveoli where it is internalized. The internalized receptor might be transported to endosomes for dephosphorylation and returned to the plasma membrane for the next signaling event or directed to lysosomes for degradation (Atwood et al., 2012). Little is known about the processes involved in CB2 adaption to chronic activation. Most of the knowledge comes primarily from cell lines over expressing CB2. Although the levels of expression in those systems significantly exceed in vivo CB2 levels, the use of expression systems provides very valuable information (Atwood et al., 2012).

CB2 receptor expression in immune and neuroimmune cells

Cannabinoid induced immunomodulation

The mechanism of cannabinoid-induced immunomodulation has been studied in both in vitro and in vivo systems; however, still many questions remain unanswered. It is known that cannabinoids bind to CB1 and CB2 inhibiting adenylate cyclase activity and preventing forskolin-stimulated cAMP activation. This process leads to decreased activity of protein kinase A and subsequently lesser binding of transcription factors to CRE consensus sequences (Condie et al., 1996; Rieder et al., 2010). Cannabinoids clearly modulate immune responses during inflammatory processes and their immunomodulatory effects have been studied in many disease models such as MS, diabetes, septic shock, rheumatoid arthritis, and others (Table 1 and Table 2). Results of animal studies show that cannabinoids exert their immunomodulatory properties in four ways: i) induction of apoptosis, ii) suppression of cell proliferation, iii) inhibition of pro-inflammatory cytokine/chemokine production and increase in anti-inflammatory cytokines, and iv) induction of regulatory T cells (Rieder et al., 2010). It has been shown that THC inhibits proliferation of human lymphocytes in culture (Schwarz et al., 1994) and leads to apoptosis of murine macrophages and T cells (Zhu et al., 1998). McKallip and colleagues showed that THC affects naïve lymphocytes to a greater degree than activated lymphocyte (McKallip et al., 2002b). It was also noted that activated lymphocytes had less expression of CB2, thereby explaining the decreased sensitivity of activated lymphocytes to THC (McKallip et al., 2002b). JWH-015, a CB2 synthetic agonist, inhibited proliferation in T and B cells in a dose-dependent manner and induced apoptosis in splenocytes and thymocytes (Lombard et al., 2007; Rieder et al., 2010). Derocq and colleagues (Derocq et al., 1995) demonstrated that the activity of cannabinoids is not restricted to immunomodulation, and in nanomolar concentrations of synthetic (CP55,940 and WIN55212-2) and natural (THC) cannabinoids increase proliferation of B cells co-stimulated with anti-CD40-antibodies, while at micromolar range (1–100 M) the same cannabinoids inhibited proliferation (Derocq et al., 1995). Interestingly, cannabinol and THC protected human B lymphoblastoid cell line from serum-deprived cell-death and oxidative stress in sub-micromolar concentrations (Chen and Buck, 2000). The fact that activation of CB2 triggers apoptosis in immune cells suggests that targeting CB2 may be a novel approach to the treatment of inflammatory and autoimmune diseases.

Table 1

Anti-inflammatory effects of CB2 activation on immune and neuroimmune cells

Table 2

Anti-inflammatory effects of CB2 activation in animal models

CB receptors in leukocytes and endothelium: relevance to HIV infection

Cannabinoids can affect different pro-inflammatory events associated with HIV infection, such as: i) chemotaxis of immune cells, ii) activation of endothelium and leukocyte infiltration into tissues, iii) injury of endothelial barriers, like the blood brain barrier (BBB), and iv) HIV-1 infection of susceptible cells.

CB2 appears to play a major role in leukocyte migration. Human monocytes treated with the synthetic CB2 agonist, JWH-015, showed diminished migration in response to the chemokines, CCL2 and CCL3, via down-regulation of their receptors and inhibition of IFNγ-induced ICAM-1 expression. Leukocyte migration mediated by RhoA activation was inhibited by CB2 agonists (Kurihara et al., 2006). Cannabinoids could reduce inflammation by interfering with the action of other chemoattractants (Montecucco et al., 2008). Cannabinoids have been reported to inhibit chemokine-induced chemotaxis of various cell types including neutrophils, lymphocytes, macrophages, monocytes and microglia (Miller and Stella, 2008). Recently, it has been shown that CB2 agonist affected dendritic cell migration in vitro and in vivo, primarily through the inhibition of matrix metalloproteinase-9 expression (Adhikary et al., 2012).

In addition to putative effects on leukocytes, anti-inflammatory properties of CB agonists may be related to their actions on the endothelium. CB2 has been found in brain endothelium (Golech et al., 2004; Lu et al., 2008) and in endothelial cells from other organs (Rajesh et al., 2007). Synthetic CB2agonists (JWH-133, HU-308) reduced TNFα-induced activation of human coronary artery endothelial cells in vitro. CB2 agonists reduced secretion of MCP-1 and attenuated monocyte transendothelial migration (Rajesh et al., 2007). Those results are relevant to CB2 anti-inflammatory effects.

Lu and colleagues (Lu et al., 2008) demonstrated that HIV-1 gp120 caused secretion of substance P and dysfunction of brain endothelial cells (Ca2+ influx, decreased barrier tightness, decrease in tight junction (TJ) expression) that was prevented by non-selective CB1/CB2 or CB1 agonists. These compounds diminished monocyte migration across endothelial monolayers pretreated with gp120. The molecular mechanism underlying the beneficial effects of CB agonists was not investigated. Because CB2 agonists modulate immune cell migration, they represent a promising pharmacological approach for development of anti-inflammatory therapeutics. Fraga and colleagues (Fraga et al., 2011) found that CB2 inhibited migration of microglial cells toward HIV Tat protein in a mouse BV-2 microglia-like cell model. In addition, the level of the β-chemokine receptor CCR3 was reduced and its localization was altered.

Our group investigated the effects of CB2 receptor agonists (JWH133 and O1966) on leukocyte-brain endothelial cell interactions. Using primary human brain microvascular endothelial cells and human monocytes, we showed diminished adhesion of leukocytes to activated endothelium and down-regulation of adhesion molecules. In an animal model of systemic inflammation, leukocyte adhesion was significantly attenuated in postcapillary venules in animals treated with CB2 agonists. BBB injury and increased permeability were prevented (Ramirez et al., 2012).

The CB2 agonist, JWH133, was shown to inhibit HIV-1 infection in primary CD4+ T lymphocytes by altering T cell actin dynamics via inactivation of the actin-modulating protein, cofilin. Actin rearrangements are essential for productive infection. CB2 agonist did not alter CXCR4 expression, T cell activation or viral fusion (Costantino et al., 2012). We showed that CB2 agonists diminished HIV replication and HIV LTR activation in monocyte-derived macrophages (personal communication). Cannabimimetic drugs are of particular relevance to HIV-1 associated neurocognitive disorders (HAND) because of their clinical and illicit use in patients with AIDS. The cannabinoid receptor agonist, Win55,212–2, inhibited HIV-1 gp120-induced IL-1β production and synapse loss in a manner reversed by CB2 receptor antagonist. In contrast, Win55,212–2 did not inhibit synapse loss elicited by exposure to the HIV-1 protein Tat. These results indicate that cannabinoids prevent the impairment of network function produced by gp120 and, thus, might have therapeutic potential in HAND (Kim et al., 2011). Molina and colleagues (Molina et al., 2010; Molina et al., 2011) demonstrated that that chronic Δ9-THC treatment decreased plasma and cerebrospinal fluid (CSF) viral load and tissue inflammation significantly reducing morbidity and mortality of simian immunodeficiency virus-infected macaques. THC treatment led to better maintenance of body weight without any alterations in immune responses (Molina et al., 2010). Win55,212–2 has been shown to inhibit HIV-1 expression in CD4+ lymphocytes and microglial cell cultures in a time- and dose-dependant manner (Peterson et al., 2004). Taken together, these studies point to potential therapeutic benefits of CB stimulation in treatment of HIV-1 infection.

Summary

The full potential of CB2 agonists as therapeutic agents remains to be realized. Despite some inadequacies of preclinical models to predict clinical efficacy in humans and differences between the signaling of human and rodent CB2 receptors, the development of selective CB2 agonists may open new avenues in therapeutic intervention. Such interventions would aim at reducing the release of pro-inflammatory mediators particularly in chronic neuropathologic conditions such as HAND or MS. Further studies are needed to delineate the therapeutic effects of CB2 agonists in current efforts to legalize the use of marijuana for medical purposes.

Acknowledgments

References