Università di Pisa
Dipartimento di Farmacia
Corso di Laurea Magistrale in Chimica e Tecnologia Farmaceutiche
Tesi di Laurea:
CANNABINOID CB2 RECEPTOR MODULATORS IN THE TREATMENT
OF RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS
Relatori: Candidata:
Prof.ssa Clementina Manera Caterina Ricardi
Dott.ssa Rebecca Ferrisi
Dott. Torsten Lowin
Anno Accademico 2019-2020
SSD CHIM-08
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Contents
1. GENERAL INTRODUCTION
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1.1. CANNABIS SATIVA AND PHYTOCANNABINOIDS 5 1.2. THE ENDOCANNABINOID SYSTEM 7 1.2.1. The cannabinoid receptors: CB1R and CB2R 7 1.2.2. Endogenous Ligands: Endocannabinoids 11 1.3. ALLOSTERIC MODULATION OF THE CANNABINOID RECEPTORS 15 1.3.1. CB1R allosteric modulators 16 1.3.2. CB2R allosteric modulators 17 1.4 PHYSIOLOGICAL FUNCTIONS OF THE ENDOCANNABINOID SYSTEM AND POTENTIAL THERAPEUTIC APPLICATIONS 19 1.5. RHEUMATOID ARTHRITIS 21 1.5.1. Rheumatoid arthritis treatment 21 1.6. OSTEOARTHRITIS 22 1.6.1. Osteoarthritis treatment 23 1.7. ROLE OF ENDOCANNABINOID SYSTEM IN THE PATHOPHYSIOLOGY OF RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS 24 1.7.1. ECS in SFs 25 1.7.2. ECS in osteoclasts and osteoblasts 25 1.7.3. ECS in chondrocytes 26 1.7.4. ECS in immune cells in arthritic joints 26 1.8. CB2 RECEPTOR AGONISTS IN RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS TREATMENT 322. AIM OF THE THESIS
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2.1. AIM OF THE THESIS 39 2.1.1. Synthesis of EC21a 42 2.1.2. Synthesis of FM6b 45 2.1.3. Synthesis of LV62 473. EXPERIMENTAL PART
49
3
References
63
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1. GENERAL INTRODUCTION
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1.1. CANNABIS SATIVA AND PHYTOCANNABINOIDS
Cannabis sativa L. is an herbaceous annual plant belonging to the family of
Cannabaceae, cultivated mainly in Central Asia (India and China) since ancient times1. Commonly called marijuana, it has been harnessed as a source of fiber, food, psychoactive and recreational compounds. It is the most widely used illegal drug in western countries as it can cause changes in sensory perception along with euphoria and elation. The plant has long been used for medical purposes throughout human history, with first record traced back to ancient China around 5000 years ago. The widely documented uses of marijuana include anti-nociception, anti-inflammation, anticonvulsant, anti-emetic, as well as recreational use, which has largely limited its medical application. Thus, Cannabis has intrigued the scientific community to search for different aspects of its activity and effects in different pathological and physiological conditions2.
Currently, more than 500 natural compounds were identified from C. sativa. Of these, more than 100 are identified as phytocannabinoids (pCBs), which are lipid-soluble chemicals synthesized from fatty acid precursors via a series of transferase and synthase enzymes in female plants of the C. sativa herb3. Their biological effects are mediated by
two members of the G-protein coupled receptor family, cannabinoid receptors 1 and 2 (CB1R, CB2R), localized in the human brain and peripheral tissues. However, they also act
at non-CB receptor sites2.
While more than 100 different cannabinoids can be isolated from C. sativa, trans-Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD) and cannabinol (CBN) are the pCBs that have been investigated to a much greater degree4. The chemical structures of THC, CBD,
and CBN are reported in Figure 1, 2 and 3, respectively.
Δ9-tetrahydrocannabinol (Δ9-THC) is the key compound of the C. sativa with
major psychoactive effects. From a pharmacological point of view, THC is a partial agonist at both CBRs. Anxiety, paranoia, perceptual alterations, and cognitive deficits are the main psychoactive, CB1R- mediated effects of THC. They are caused by the perturbation of
GABA/glutamatergic neurotransmission and dopamine release, and above all, they are generally acute, transient, and self-limited. Moreover, hypo locomotion, hypothermia, catalepsy, analgesia, and increased food intake have been reported after THC administration1.
6 Figure 1. Chemical structure of THC. Cannabidiol (CBD) is a psychotropically inactive component of cannabis accounting for up to 40% of the plant's extract. It was first discovered in 1940, more than 20 years before THC. It has several medicinally relevant properties like inflammatory, anti-oxidative, anti-emetic, anti-psychotic, anticonvulsant, anti-rheumatoid arthritis, and neuroprotective effects. Despite the structural similarity between CBD and D9-THC behaves as an inverse agonist of CBRs5. Figure 2. Chemical structure of CBD. Cannabinol (CBN) is quite similar to THC in terms of structure, yet its affinity for CBRs is relatively low. It is a partial agonist of CB1R while it has a high affinity for CB2R5. Figure 3. Chemical structure of CBN. CBD CBN
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Other pharmacologically important analogues are cannabinoid acids, cannabigerol, and cannabivarins. In addition to the aforementioned pCBs, the pharmacological effects of C. sativa can also be mediated by other components, such as the monoterpenoids
myrcene, limonene, pinene, and the sesquiterpenoid β-caryophyllene6.
1.2. THE ENDOCANNABINOID SYSTEM
The endocannabinoid system (ECS) is a whole signalling system that comprises cannabinoid receptors CBRs, endogenous ligands of cannabinoid receptors (endocannabinoids, eCBs) and the enzymes that regulate the biosynthesis and inactivation of eCBs. This lipid signalling pathway is involved in many important physiological functions in the central and peripheral nervous system and in the immune and endocrine systems6. 1.2.1. The cannabinoid receptors: CB1R and CB2R The discovery in 1990 that an orphan G protein-coupled receptor derived from a rat cerebral cortex mediates pharmacological effects of THC, established the identity of the first cannabinoid receptor, which we now refer to as CB1R. Three years later, a G
protein-coupled receptor expressed in human leukemia cells was identified as a second cannabinoid receptor and named CB2R7.
CBRs are members of the class A G protein-coupled receptor (GPCR) family and share 44% total sequence identity and 68% sequence similarity in the transmembrane regions8. As shown in Figure 4, both receptors share a common feature of class A GPCRs,
possessing a glycosylated extracellular amino-terminal and an intracellular carboxyl-terminal domain connected by seven hydrophobic transmembrane segments, three extracellular and three intracellular loops9.
8 Figure 4. Structure of CB1R and CB2R10. However, they differ in their tissue distribution and exert distinct functions in ECS, where CB1R and CB2R are expressed predominantly in the central nervous system and the
immune system, respectively8.
Specifically, CB1R is mainly expressed pre-synaptically and modulates the release of
neurotransmitters such as serotonin, glutamate, dopamine, noradrenaline and γ-aminobutyric acid (GABA)5. CB
1R is known to mediate the psychotropic effects of
cannabis, which can be ascribed to the wide distribution of CB1R in the brain, with high
concentrations in cerebral cortex, hippocampus, basal ganglia, and cerebellum. Recent evidence has suggested that CB1R are also expressed in a variety of peripheral tissues and
organs, including the spleen, lung, thymus, heart, vasculature, adipose tissue, the gastrointestinal tract, the spinal cord, the adrenal and thyroid glands, liver and reproductive organs, as well as in immune cells6,9.
In comparison, CB2R are mainly expressed in peripheral tissues with immune function,
such as leukocytes, spleen, tonsils and thymus. Moreover, it has been demonstrated the role of the CB2R in a variety of systems including the central nervous systems, as well as
the cardiovascular and respiratory systems, bone, the gastrointestinal tract, liver and the reproductive system9.
As shown in Figure 5 both CB1R and CB2R are coupled through the Gi/o family of
proteins to various signal transduction mechanisms, such as adenylyl cyclase (AC) inhibition, mitogen-activated protein kinase (MAPK) activation, potassium and calcium channel regulation5.
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Typically, CBRs are coupled to Gi or Go proteins and, when activated, inhibit the activity of adenylate cyclase (AC), causing a reduction in cellular cyclic adenosine monophosphate (cAMP) levels and a decrease in protein kinase A (PKA) activity2. However, under certain circumstances, CB1R can switch its coupling of G protein from Gi/o to Gs or Gq. It stimulates AC via Gs proteins and increases intracellular Ca2+ concentration via Gq/11 proteins2. An additional route of CBRs signalling operates via activation of several mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). They are involved in the regulation of cell proliferation, cell cycle control and cell death2. Besides the MAPK signalling, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway is another key regulator of cell growth and death and it is activated by CBRs as well2. Moreover, activation of CB1R induces contrasting effects on different cellular ion
channels. CB1R can inhibit voltage-dependent N-type, P/Q-type, and R-type calcium
channels in various systems and has been reported to stimulate inward-rectifying potassium channels as well2.
Upon G protein activation, specific threonines and serines in the cytoplasmic region of CBRs are susceptible to G protein-coupled receptor kinases (GRKs) phosphorylation, that induces the recruitment of scaffolding protein known as β-arrestin1 and β-arrestin2. Arrestins have been largely known for their role in CBRs desensitization and their major role in CB2R internalization11.
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Other putative cannabinoid receptors
Although only CB1R and CB2R are widely acknowledged as CBRs, several other
receptors, ranging from other G protein-coupled receptors to ion channel receptors, have been recently reported to interact with cannabinoids.
GPR55: G protein-coupled receptor 55 shares numerous cannabinoid ligands with
CBRs, despite its low homology with them. Therefore, some researchers identify GPR55 as a candidate “CB3” receptor. Its broad distribution from central nervous system to
peripheries suggests the importance of GPR55 in various cellular processes and pathologies, proposing it as a potential therapeutic target also in inflammation. The signaling pathways related to GPR55 are not yet fully elucidated, anyway, it is currently found that they differ from the CBRs ones 13.
GPR18: G protein-coupled receptor 18, also known as N-Arachidonyl glycine
receptor (NAGly), is an orphan receptor expressed predominantly in the brain, lungs, thyroid, thymes, testes and ovary, with the highest levels in peripheral blood leukocytes.
Figure 5. Major signalling pathways associated with cannabinoid receptor activation by agonists.
Activation of CBRs is coupled to inhibition of AC with corresponding inactivation of the PKA phosphorylation pathway, or to stimulation of MAPK which events lead to, among other effects, the regulation of expression of several genes. CB1
R stimulation is also coupled to inhibition of voltage-activated Ca2+ channels and stimulation of inwardly rectifying K+ channels.12
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Although GPR18 is less homologous to CB1 and CB2, it is closely related to the ECS. Indeed,
a number of cannabinoid ligands have been decribed to be active as agonists or antagonists, such as THC and NAGly, the endogenous lipid metabolite of anandamide (one of the two main eCBs)14.
TRPV1: Vanilloid receptor type 1 is a ligand-gated cation channel that plays an
important role in pain transmission and modulation. It is located mainly in the nociceptive neurons of the peripheral nervous system. TRPV1 can be activated not only by exogenous agents but also by many endogenous stimuli, including high temperature, low pH, and capsaicin. In addition to direct activation, TRPV1 channel activity is also modulated by inflammatory mediators, such as prostaglandins and bradykinin. The agonistic effect of anandamide, cannabidiol and cannabigerol on this receptor has suggested the association between TRPV1 and the endocannabinoid system15,16. 1.2.2. Endogenous Ligands: Endocannabinoids Endocannabinoids (eCBs) are a family of lipid molecules that act as key regulators of the endocannabinoid system, of which 2-arachidonoylglycerol (2-AG) and anandamide (AEA) (Figure 6) are the best characterized. Figure 6. Chemical structure of AEA and 2-AG. Both eCBs have significant differences in receptor selectivity.
AEA is a high-affinity, partial agonist of CB1R, and almost inactive at CB2R, whereas 2-AG
acts as a full agonist at both CBRs.
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Interestingly AEA has non-cannabinoid sites of action as well. Among those, AEA is able to directly inhibit ion currents mediated by L-type Ca2+ channels and TASK-1 K+ channels. Most notably, AEA is capable to act as a full agonist of TRPV12,17.
ECBs are synthesized, transported and inactivated in respective target tissues differently.
AEA is the is the amide of arachidonic acid (AA) with ethanolamine (EtNH2), and its
formation in neurons is a two-step process. As shown in Figure 7, the first step is the synthesis of the AEA precursor N-arachidonoyl phosphatidylethanolamine (NAPE). It is catalyzed by the enzyme N-acyltransferase (NAT), which transfers the AA residue from a phosphatidylcholine to the amino group of phosphatidylethanolamine (PE). The second step is the cleavage of NAPE to yield AEA, catalyzed by phospholipase D (NAPE-PLD)18. 2-AG is an ester formed from AA and glycerol. As AEA, 2-AG is synthetized in a two-step pathway (Figure 8): in the first step, phospholipase C (PLC) promotes the hydrolysis of membrane phospholipids, such as phosphatidylinositol (PI), and generates 1,2-diacylglycerol (DAG). Then, DAG is converted to 2-AG by the action of diacylglycerol lipase (DAGL)18.
Figure 7. Mechanism of anandamide formation in neurons. First step: synthesis of N-arachidonoyl-
phosphatidylethanolamine (PE), catalysed by acyltransferase. Second step: cleavage of N-arachidonoyl-PE to yield AEA, catalysed by phospholipase D18.
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ECBs are produced on demand and released immediately, since they are highly lipophilic and thus are poorly suited for storage. However, eCBs can be stored by binding to intracellular fatty acid-binding proteins (FABPs), intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). After being released into the intracellular space from postsynaptic neurons, they retrogradely modulate synaptic transmission widely throughout the central nervous system19.
They reach presynaptic CB1Rs and suppress transmitter release, such as glutamate,
acetylcholine and noradrenaline release either transiently (endocannabinoid-mediated short-term depression; eCB-STD) or persistently (endocannabinoid-mediated long-term depression; eCB-LTD). The eCB-STD and eCB-LTD are induced at various types of GABAergic and glutamatergic synapses throughout the brain19.
In most cases, endocannabinoid-mediated retrograde signaling starts with the production of 2-AG, in response to increased intracellular Ca2+ concentration and/or activated G
q/11
-coupled receptors. 2-AG is then released and, traversing the extracellular space, arrives at the presynaptic terminal where it binds to the CB1R. Activated the CB1R, it suppresses the
release of neurotransmitter in two ways: first, by inhibiting voltage-gated Ca2+ channels, which reduce presynaptic Ca2+ influx; second, by inhibiting adenylyl cyclase (AC) and the
subsequent cAMP/PKA pathway2.
Although endocannabinoid retrograde signaling is mainly mediated by 2-AG, AEA can activate presynaptic CB1Rs as well2.
Figure 8. Mechanism of 2-AG formation in neurons. DGL cathalyzes the biosynthesis of 2-AG from
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ECBs are removed from the extracellular space by a two-step process: the transport into cells and the subsequent enzymatic degradation19.
At least four models have been proposed for AEA uptake by cells, and several studies suggest that 2-AG and AEA are transported by the same system. According to the first model, AEA is transported by a carrier protein, which binds and translocates eCBs from one side of the membrane to the other. The second model is that AEA is able to pass through the membrane not by carrier but by simple diffusion. The third model is that AEA undergoes endocytosis through a caveolae-related uptake process19. Finally, it has been found that the AEA uptake in endothelial cells can be mediated by TRPV120.
Endocannabinoids can be then degraded through two different pathways, hydrolysis and oxidation. The enzymes that catalyze the first pathway include FAAH, which degrades AEA into free AA and ethanolamine, and monoacylglycerol lipase (MAGL), which hydrolyzes 2-AG into AA and glycerol. The second pathway involves mainly cyclooxygenases (COX), which induce oxidation of the arachidonic moiety of the endocannabinoids19. In Figure 9 the principal eCBs pathways are illustrated. Figure 9. Anabolic and catabolic pathways of endocannabinoids. Hydrolytic enzymes are involved in both the biosynthesis of eCBs and in their inactivation. The enzymes for 2-AG biosynthesis are PLC and DGL, while NAT and NAPE-PLD are involved in AEA formation. AEA and 2-AG are inactivated by FAAH and MAGL, respectively. Finally, an as yet uncharacterised endocannabinoid membrane transporter (EMT) seems to facilitate both endocannabinoid release and re-uptake12.
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1.3. ALLOSTERIC MODULATION OF THE CANNABINOID RECEPTORS
Besides endogenous cannabinoids and phytocannabinoids, orthosteric ligands at CBRs and ligands able to modulate ECS by binding to allosteric sites of CBRs were developed21.
Allosteric modulators (AMs) of CBRs do not possess intrinsic efficacy but instead enhance or diminish the orthosteric ligand-driven signaling of receptors, binding topographically distinct sites from the orthosteric ones. In this way, AMs modulate receptor activity through conformational changes in the receptor that are transmitted to the orthosteric site, modifying the affinity of an orthosteric ligand for the receptor. This process is described by the “allosteric ternary complex model (TCM)”22.
According to their effects on orthosteric ligand responses, allosteric ligands are divided into Positive Allosteric Modulators (PAMs), Negative Allosteric Modulators (NAMs), and Neutral Allosteric Ligands (NALs). Affinity and/or efficacy of orthosteric ligands are enhanced by PAMs. On the other hand, NAMs decrease receptor function through a reduction of agonist affinity and/or efficacy. Finally, NALs compete with other PAMs or NAMs at the allosteric binding site, without modulating orthosteric ligands activity (Figure 10)22,23.
The ways in which the functions of CBRs can be manipulated for potential therapeutic benefit has been enriched by the existence of allosteric ligands, yet the complexity of their actions provides opportunities for drug screening and development. When comparing with orthosteric ligands, allosteric modulators offer many pharmacological advantages, allowing to overcome side effects such as immunosuppression and psychotropic effects, respectively typical of CB2R and CB1R
orthosteric agonists22,23.
The AMs have a high tissue selectivity, as they act only where ECs (or other orthosteric ligands) are present and do not compete with ligands bound to orthosteric sites, therefore maintaining both temporal and spatial aspects of endogenous physiological signaling; they have a high receptor subtype selectivity essential to reduce side effects, thanks to the higher sequence divergence at allosteric binding sites respect to the orthosteric domains. In addition, since their signal depends on endogenous ligands, an increasing concentration of allosteric modulators beyond the saturation of the allosteric binding site does not enhance the magnitude of the allosteric action. Finally, unique conformations of the
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orthosteric site might be induced and stabilized by allosteric modulators, leading to biased signaling towards the orthosteric ligand (“probe”) that preferentially engages certain signaling interactions rather than other22,23. All these features of allosteric modulation
implicate a reduced side-effect profile. 1.3.1. CB1R allosteric modulators
Allosteric modulators of this class represent interesting prospects for developing ligands, which may reduce the CB1R-related side effects24. The Org compounds (Figure 11), Pregnenolone (Figure 11), and Pepcan-12 (Figure 13) are the main CB1R NAMs developed so far. On the other hand, Lipoxin A4 (Figure 11) is a PAM found to be involved in immune system regulation and in the resolution of inflammation23. PSNCBAM-1 (Figure 11) behaves as a NAM in functional activity and shows a PAM profile in binding25. Finally, ZCZ011 (Figure 11) is characterized by mixed agonist and PAM properties23.
Figure 10. Classification of allosteric modulators and influence on orthosteric ligand function.
Orthosteric agonists bind to the GPCR, inducing a conformational change that lead to the activation of downstream signalling. PAMs, NAMs and NALs bind at distinct sites, enhancing (PAMs), decreasing (NAMs) or not altering (NALs) the affinity (α) and/or the efficacy (β) of the orthosteric ligand. Red arrows show the interaction between the allosteric and the orthosteric ligands. Black arrows denote the orthosteric agonism, that results in downstream activation of signalling pathways22.
17 Figure 11: Chemical structure of CB1R allosteric modulators. 1.3.2. CB2R allosteric modulators
Whereas CB1R AMs are currently playing an increasingly prominent role in the
search for cannabinoid therapeutic agents, few CB2R AMs have been developed so far
Besides its negative allosteric modulation at CB1R, Pepcan-12 (Figure 13) is the first
CB2
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representing a hypothetical target for beneficial effects on many organs23.
Furthermore, Cannabidiol demonstrated to be a NAM at CB2R and a CB1R NAM at low
concentrations. A CBD derivative known as CBD-DMH (Figure 13) has been synthesized as well. CBD-DMH has a NAM/PAM nature that may results in a switch in the response of G-protein signaling or β-arrestin pathway23. N-[5-bromo-1,2-dihydro-1-(4’-fluorobenzyl)-4-methyl-2-oxo-pyridin- 3yl]cycloheptanecarboxamide (EC21a, Figure 13) is the first synthetic allosteric modulator of CB2R reported in literature and has been developed in the lab where I performed my thesis26. Figure 13: Chemical structure of CB2R allosteric modulators.
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1.4 PHYSIOLOGICAL FUNCTIONS OF THE ENDOCANNABINOID SYSTEM AND POTENTIAL THERAPEUTIC APPLICATIONS
Research on the ECS is fervently ongoing with wide-ranging discoveries. The roles of endogenous cannabinoids, phytocannabinoids, and synthetic pharmacological agents acting on the various elements of the ECS have the potential to affect a wide range of pathologies, including emesis, multiple sclerosis, food intake disorders, chronic pain, inflammation, epilepsy, glaucoma, cardiovascular diseases, cancer, Parkinson’s, Alzheimer’s and Huntington’s disease, Tourette’s syndrome, and psychiatric conditions such as depression, autism, and schizophrenia. In addition, the ECS is known to influence neuroplasticity, apoptosis, excitotoxicity, neuroinflammation, and cerebrovascular breakdown associated with stroke and trauma5,27.
The tissue distribution of CB1 and CB2 is shown in Figure 14. CB1R is one of the most
abundant GPCRs in the CNS but is also present in the peripheral nervous system (PNS) and several peripheral organs. CB2Rs, by contrast, are mostly restricted to immune tissues and
cells. However, the concept of CB2R as “the peripheral CBR” has been challenged recently
by several studies demonstrating the expression of the CB2R in the brain. In particular, this
receptor has been also found in the cerebellum, in the brainstem and in the microglia cells, where it is implicated in various processes such as nociception and neuroprotection12.
Figure 14. Distribution of CB1R and CB2R and their associated functions28.
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The preferential distribution of CB1Rs at presynaptic neurons, together with their
coupling to the inhibition of voltage-activated Ca2+ channels, the stimulation of eCBs formation by increased intracellular Ca2+ and activation of other GPCRs, makes the ECS an
ideal natural tool for modulating neurotransmitter release. In particular, eCBs in the CNS intervene in both short-term and long-term forms of synaptic plasticity, regulating cognitive functions and emotions in neuronal circuits of the cortex, hippocampus and amygdala. The abundance of both CB1Rs and eCBs in the basal ganglia and cerebellum
makes targeting this signaling system an ideal way to modulate both movement and posture. Pain perception, cardiovascular, gastrointestinal and respiratory functions also result to be modulated by eCBs, mostly through CB1R. Moreover, their effects on the
release of hypothalamic hormones and peptides, and the regulation of their levels by steroid hormones, lead to modulation of food intake and of the pituitary– hypothalamus– adrenal axis, as well as of both female and male reproduction12. The presence of CB
1Rs on
chondrocytes and osteocytes, as well as evidence of their presence on fibroblast-like synoviocytes, makes CB1Rs particularly interesting in the study of rheumatic diseases6.
The physiological importance of CB2R in cellular and, particularly, humoral immune
responses, has possible implications for inflammation and chronic pain12. CB
2Rs are
expressed in all hematopoietic cells such as lymphocytes, natural killer cells, macrophages, and neutrophils, representing an attractive target for the treatment of the inflammatory status. By modulating the release of anti- or pro-inflammatory cytokines, CB2Rs also
mediate a significant protection in brain microglia against neurotoxicity. In recent years, pharmaceutical companies and academic research laboratories have attempted to identify high-affinity, selective agonists for CB2R, in order to avoid CB1R central effects and
developing molecules with a therapeutic potential in the treatment of different pathologies such as neurodegenerative diseases, pain transduction and perception, ischemic stroke, severe inflammation, autoimmune diseases, osteoporosis, and cancers. Therefore, selective CB2R agonists could represent an attractive target for the
development of neuroprotective therapies and, above all, provide anti-inflammatory effects without psychoactive activities29.
Among all the potential therapeutic applications involving the ECS, I will focus particularly on the involvement of the ECS in the treatment of rheumatoid arthritis (RA) and osteoarthritis (OA).
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1.5. RHEUMATOID ARTHRITIS
Rheumatoid arthritis is a chronic inflammatory autoimmune disease that affects approximately 1% of the adult population. RA is characterized by a persistent joint inflammation leading to cartilage and bone damage, disability and eventually to systemic complications. In addition to a consequent decreased quality of life, life expectancy is reduced, most commonly from accelerated atherosclerosis30. The pathogenesis is the result of a complex interaction between genetic and environmental factors, such as occupational and atmospheric agents and cigarette smoking31.
Chronic inflammation of the synovium, cartilage destruction, and bone loss characterize the pathophysiology of RA. The initial stage of the disease is associated with alteration of the innate and adaptive immune systems with consequent production of autoantibodies. In the following stages of RA, both the innate (neutrophils, macrophages) and adaptive (B and T lymphocytes) immune cells contribute to the amplification and perpetuation of the chronic inflammatory state. Their influx in the synovial cavity promotes inflammation and connective tissue damage by producing cytokines (TNF-α, IL-6, IL-1β), proinflammatory lipids, and metalloproteinases (MMPs), such as MMP-1, -3, and -10. Moreover, osteoclasts become exaggeratedly activated and cause bone resorption31,32. Rheumatoid arthritis synovial fibroblasts (RASFs) also play a key role, being recognized as both engines of joint damage and propagators of the immune response in RA. On one side, they aggressively promote invasion into the extracellular matrix, exacerbating joint damage. On the other one, RASFs produce high amounts of IL-8, MMP-3, and some other chemokines, which perpetuate inflammation and contribute to cartilage destruction. The cascade of events turns the synovial tissue into a hyperplastic and invasive structure teeming with immunocompetent cells, named pannus. The pannus behaves like a locally invasive tumor as it expands causing cartilage destruction and bone erosion, leading to a permanent joint damage32 (Figure 15c). 1.5.1. Rheumatoid arthritis treatment Since there is no cure for RA, the treatment goals are to reduce joint inflammation and pain and prevent or slow down further damage. It is noteworthy to evidence that current RA therapeutics principally interfere with cytokine production or signaling but are
22
often associated with several side effects due to the infection or immune dysfunction33.
The main medications in the management of RA are listed below.
• Disease-modifying antirheumatic drugs (DMARDs) are the mainstay of treatment for RA. Methotrexate is the dominant DMARD. Sulfasalazine, leflunomide, hydroxychloroquine, cyclosporin and gold salts are also widely used33.
• Analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylate, naproxen, ibuprofen and etodolac33.
• Corticosteroid medications (e.g. prednisone) with both anti-inflammatory and immunoregulatory activity33.
• Biological agents such as TNF inhibitors (e.g. infliximab), T-cell costimulatory blockers (e.g. abatacept), B-cell depleting agents (e.g. rituximab) and IL-6 inhibitors (e.g. tocilizumab) are usually taken in combination with DMARDs33. • Janus kinase (JAK) Inhibitors are the latest drug class of disease-modifying drugs to emerge for the treatment of RA. They are small molecule oral treatments, which offer the first truly clinically efficacious long-term oral biologic option in RA34. 1.6. OSTEOARTHRITIS
Osteoarthritis is one of the most common joint diseases and a major cause of disability in the aging population. This chronic degenerative arthropathy affects mainly older people, but in juveniles, young athletes and middle-aged people it can lead to severe pain and physical disability as well. OA most commonly affects the knees, hands, feet, the hips, and the spine and is one of the major reasons for hip and knee replacement surgeries. There are several factors that increase the risk of developing OA, including genetic predisposition, obesity, trauma, gender, muscle weakness, physical activity/inactivity, race, estrogen levels and nutritional status. The most common clinical symptoms are joint pain related to use, stiffness and joint swelling35.
Under normal conditions, cartilage is subjected to a dynamic remodelling process in which low levels of degradative and synthetic enzyme activities are balanced, such that the volume of cartilage is maintained. In OA cartilage, however, this balance is shifted towards degradation, with a resultant fibrillation in the superficial layer of cartilage in the early stage of the disease and, as the pathological process continues, deep fissures
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associated with exfoliation of cartilage fragments develop, resulting eventually in large erosions36,37 (Figure 15b).
Although OA is traditionally described as a prototypical non-inflammatory arthropathy, nowadays there is compelling evidence to suggest that it has an inflammatory component. Many studies have shown the presence of synovitis (inflammation of the synovium) in a large number of patients with OA, demonstrating a direct association between joint inflammation and the progression of the disease. Even though the levels of pro-inflammatory factors in OA synovial fluid are lower than in RA, the OA synovitis is itself cytokine-driven, especially involving tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1. Cytokines stimulate their own production and persuade osteoarthritis synovial fibroblasts (OASFs) and chondrocytes to produce IL-6, IL-8, MMPs, proteases, nitric oxide (NO), prostaglandins and leukotrienes, all of which lead to an increase in cartilage degradation. Latest studies suggest that activated synovial macrophages have a significant impact on the development of OA as well, as they promote the release of both pro-inflammatory cytokines and growth factors35.
1.6.1. Osteoarthritis treatment
At this time, there are no pharmacological agents capable of retarding the progression of OA or preventing the disease, and current treatment for OA is limited to control of
Figure 15. Healthy, OA and RA joint. Comparison between e healthy joint (a), an OA joint (b)
characterized by cartilage destruction and rubbing bones, and a RA joint (c), associated with synovium inflammation and bone erosion38.
24 symptoms and improve functional mobility30. Recommended pharmacological approaches for the management of OA include: • Paracetamol, indicated as the first-line treatment for mild-to-moderate OA36. • Oral and topical NSAIDs37. • COX-2 inhibitors37. • Opioids, such as codeine, oxycodone and tramadol36,37. • Intra-articular injections of corticosteroid or hyaluronic acid derivatives36,37. 1.7. ROLE OF ENDOCANNABINOID SYSTEM IN THE PATHOPHYSIOLOGY OF RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS
Increasing amounts of evidence have confirmed the involvement of ECS in the pathophysiology of OA and RA, and currently patients with rheumatic diseases-associated joint pain are the most prevalent users of therapeutic cannabis products39. Interestingly, the importance of ECS in these disorders was emphasized by the finding of eCBs in the synovial fluid of patients with RA, but not in healthy volunteers; interestingly, CBRs mRNA and proteins have been detected in the synovial tissues of patients with both RA and OA40. Research shows that the ECS, especially activation of CB2R, has a protective function for synovial fibroblasts (SFs), osteoblasts and osteoclasts, T cells, B cells, and macrophages, of which there is compelling evidence to demonstrate its active participation in the development of joint pain40.
In fact, the modulatory role of CB2R in RA is suggested by an increasing number of studies,
one of which revealed the evident increased risk for developing the disease associated with a loss-of function gene polymorphism. In the respective study, the CB2 Q63R gene
polymorphism has been found to have a role in the disease etiology and susceptibility, thus supporting its potential use as a pharmacological target for CB2R selective agonists41.
Indeed, considerable evidence indicates that CB2R is the key target receptor since it
mediates anti-inflammatory effects in RA and OA by decreasing immune cell migration and cytokine production. Specific activation of CB2R may relieve RA and the CB2R
activation-mediated biological functions involve different cell types, as explained in detail below.
25
1.7.1. ECS in SFs
The key role in the pathophysiology of RA is played by SFs, recognized as both engines of joint damage and propagators of the immune response. Previous studies demonstrated that the expression level of CB2R was higher in RASFs than in OASFs and that CB2R
expression in RASFs could be significantly upregulated by proinflammatory factors such as IL-1β, TNF-α, and lipopolysaccharide (LPS). Moreover, it has been found that IL-1β-induced proliferation of RASFs and secretion of IL-6, MMP-3, and MMP-13 in RASFs could be inhibited by the activation of CB2R (Table 1, Figure 16). It has been demonstrated that
the specific CB2R agonist HU-308 significantly decreases in a dose-dependent manner the
IL-1β-induced production of IL-6 from RASFs, and the LPS-induced IL-6 and TNF-α production (Table 1). HU-308 inhibits the inflammatory process by reducing the production of proinflammatory cytokines in RA synovitis via CB2R activation. On the other
hand, MMPs are related to the invasiveness of RASFs and to cartilage erosion, and it has been demonstrated that cartilage could be protected from damage inhibiting MMPs production with HU-308. This CB2R agonist demonstrated to suppress IL-1β-induced
activation of ERK 1/2, and p38 mitogen-activated protein kinase in RASFs as well (Table 1). Another CB2R agonist JWH-133 was also found to inhibit IL-6, MMP-3, and chemokine
ligand 2 production from TNF-α-stimulated RASFs (Table 1).
These findings show that the RA inflammatory conditions lead SFs to express more CB2R
according to an anti-inflammation feedback mechanism, suggesting a complete ECS feedback response in RA joints to counteract synovitis and joint destruction40,42.
1.7.2. ECS in osteoclasts and osteoblasts
Concerning joint damage, considerable evidence indicates that another important role is played by osteoblast (bone-forming cells) and osteoclasts (bone-adsorbing cells). The balance in their number is fundamental to joint health, and overactivation of osteoclasts is a characteristic of RA. Interestingly, both CBRs, 2-AG and AEA have been found in osteoblasts and osteoclasts, as compelling evidence of the involvement of the ECS in the development of the disease. It has been reported that osteoclasts formation is inhibited by pharmacologic antagonism of CB1R, suggesting that this receptor may have a negative
role in joint damage. On the contrary, activation of CB2R has been demonstrated to
26 suppressing bone erosion in RA40 (Table 1, Figure 16).
1.7.3. ECS in chondrocytes
RA is also characterized by irreversible cartilage damage, promoted by chondrocytes apoptosis. Indeed, cell-surface proteins of chondrocytes are the target binding sites of autoantibodies in the synovial fluid of patients with RA. Even if the specific effect of the ECS on chondrocytes and cartilage in RA is yet to be further investigated, evidence shows that activation of CB2R could inhibit IL-1α-induced prostaglandin E2 production, as well as
proteoglycan and collagen degradation (Table 1). CB2R agonism was also reported to
decrease IL-1β-stimulated expression of mRNA and protein of MMP-3 and MMP-13 in human chondrocytes40 (Table 1). 1.7.4. ECS in immune cells in arthritic joints Another important role in the pathophysiology of RA is played by the immune system, and many members of the ECS have been demonstrated to regulate immune cells functions. Indeed, T cells, B cells, and macrophages can be regulated by the activation of CB2R,
thanks to its unique distribution in the peripheral immune system.
The number of macrophages is a sensitive biomarker for response to treatment in RA patients, as it is significantly correlated with joint erosion. They are activated early in response to immune challenge and release pro-inflammatory factors, further augmenting inflammation. In some in vitro experiments, macrophages infiltration and migration in different tissues were found to be inhibited by the activation of CB2R (Table 1, Figure 16).
Moreover, CB2R activation was reported to inhibit pro-inflammatory cytokine production
(in particular, TNF-α and IL-6 production) (Table 1, Figure 16). Although there is no direct data suggesting that the ECS weakens the activity of macrophages in joints, we can hypothesize about their inhibititory potency40.
In addition to macrophages, another component involved in the pathophysiology of rheumatoid synovitis is represented by T cells. They mediate immunoreactions in RA by producing pro-inflammatory cytokine (e.g. TNF-α, IL-1β, IL-6, IL-17), and by promoting cell-cell contact, antigen-specific responses, osteoclast activation, and bone resorption. In T cells, proliferation, differentiation and cytokine release (such as IL-2, IL-17, TNF-α) are
27
inhibited by AEA, whereas 2-AG is able to block T cell chemotaxis (Table 1, Figure 16). Moreover, CB2R activation was reported to reduce T cell differentiation and IL-17 release
(Table 1). The participation of the ECS in T-cell-mediated immune response is also suggested by the change in expression levels of CB1R and CB2R in T cells after activation,
even if the direction of expression change is still controversial40.
CB2R has been also well investigated in terms of regulatory functions also in B cells. B cells
are the source of rheumatoid factor and autoantibodies that contribute to immune complex formation in joints. They act as antigen-presenting cells and contribute to T cell activation. Moreover, B cells produce chemokines and cytokines, both promoting leukocyte infiltration into the joints and synovial hyperplasia. In B cells, the CB2R
expression level varies greatly depending on the stimulus. In general, CB2R activation
modulates B cell migration (Figure 16), cellular energy supply and response to T-cell-independent antigens, suggesting its relevant role in reducing the severity of RA40,41. In Figure 16 the main possible mechanisms responsible for CB2R activation reducing the severity of RA are shown. Figure 16. Possible mechanisms responsible for CB2R activation relieving RA40. As discussed above, the importance of CB2R in relieving RA leads to consider this receptor a potential therapeutic target of RA. In contrast to CB2R, CB1R seems to present
opposite effects, since its activation induces proinflammatory effects by promoting macrophage M1 polarization, generation of reactive oxygen species, fibrosis, and enhancing TLR4 signaling.
28
Therefore, when considering the putative therapeutic implication of cannabinoids in rheumatic diseases, it can be concluded that CB2R agonists might be a good therapeutic
tool that could be also combined with a CB1R antagonist, as long as it is peripherally
restricted in order to avoid their central side effects, to combat RA and OA41.
While the beneficial effects in several arthritis models are limited to CB1R antagonists
providing anti-inflammatory effects, on the other side, the activation of CB1R may lead to
an antinociceptive (Table 1) effect and could reduce the activity of the sympathetic nervous system and its neurotransmitters, thus counteracting the hypertension and metabolic disturbances (Table 1), which often accompany rheumatic diseases41.
In fact, comorbidities such as depression and cardiovascular events are reported in many RA patients, and hypertension and osteoporosis are correlated with the disease. One cause of the development of these conditions is a decreased parasympathetic and an increased sympathetic activity. Cannabinoids might help in this respect, as they counteract neurotransmitter imbalances, modulating the outflow of sympathetic neurotransmitters centrally and peripherally through CB1R. Thus, a CB1R agonist could be useful in the
treatment of hypertension and correlated metabolic disturbances, as CB1R activation leads
to a reduction in noradrenergic tone, reducing blood pressure. Although the reduction of SNS activity via CB1R activation might be beneficial in the early phase of arthritis or to
postpone clinical symptoms, evidence suggests that CB1R antagonist might be better
suited to directly target inflammation in RA41.
Besides the direct effects of cannabinoids on the immune system and inflammation, a significant number of RA patients uses cannabis to treat pain. Although there are only very limited clinical data on the effect of cannabinoids on arthritic pain,
Sativex, a combination of CBD and THC, demonstrated a significant analgesic effect in the
treatment of RA. A reduction in pain on movement and pain on rest, and an improvement in quality of sleep are reported after the administration of the sublingual spray, with only mild or moderate adverse effects, such as transient dizziness, dry mouth and light-headedness41,43.
Pain is modulated by cannabinoids via activating CB1R and CB2R (Table 1), but some
compounds showed to directly modulate the nociceptors TRPV1 and TRPA1. TRPV1 is directly desensitized by CB1R, whereas pain is reduced by CB2R by inhibiting
proinflammatory cytokine production and immune cell infiltration that promote TRPV1 function. CB2
R demonstrated to induce analgesia by stimulating a peripheral release of β- 29
endorphin as well. In addition, an antinociceptive effect might be produced by the interaction of CB2R with peripheral μ-opioid receptors, and additive analgesic effects were
detected in animals treated with a CB2
R agonist along with morphine. Concerning opioid-induced analgesia, it has been demonstrated that a combination of morphine with THC is able to prevent μ-opioid receptor desensitization and to deliver superior pain relief. TRPV1 is a direct target of AEA as well, which is able to desensitize it. Complete FAAH inhibition demonstrated increased pain threshold, and TRPV1 antagonism or desensitizing agonism was reported to decrease proinflammatory cytokine production by macrophages. Besides TRPV1 modulation, AEA is able to promote analgesia via peroxisome-proliferator activated receptor α. Finally, CBD has demonstrated to provide an analgesic effect by activating TRPV1 and serotonin 5-HT1a receptor41.
Whereas a significant amount of research has clarified the contribution of the ECS in the pathophysiology of RA, more studies are needed to elucidate its role in the development and treatment of OA. However, there is a growing body of scientific evidence supporting the anti-inflammatory and antinociceptive role of cannabinoids to treat OA.
The presence of CB1R and CB2R in osteoarthritic synovia and in chondrocytes from
patients with OA is one of the main evidence of the involvement of ECS in the pathophysiology of the disease, together with the fact that 2-AG and AEA have been detected in the synovial fluid derived from patients affected by OA. In addition, OASFs also express AEA and 2-AG catabolic enzymes44,45.
The main feature in OA is the loss of articular cartilage and degradation of cartilage matrix, which is primarily attributed to cartilage breakdown. Cartilage is composed of articular chondrocytes, which maintain its homeostasis, and which are surrounded by an extracellular matrix containing type II collagen, responsible for resistance, and proteoglycans. The contributing main process to cartilage degradation is the proteolysis of collagens and proteoglycans, promoted by proinflammatory cytokines. Cytokines diffuse through the damaged matrix in the chondrocyte environment, inducing MMPs to degrade the cartilage. Evidence has shown that cannabinoids could have a chondroprotective activity, resulting in an inhibition of proteoglycan breakdown and cartilage protection45.
Findings showed that the severity of the disease was reduced in mice treated with
HU-308. Correspondingly, CB2
30
support of this hypothesis, a decrease in proteoglycan production by cultured chondrocytes from CB2R-deficient mice was found. This raises the possibility that
deficiency of CB2R might predispose to OA by altering the proteoglycan content of
cartilage matrix, making it less able to deal with biomechanical stimuli46,47. Additionally, a study found that exposure to WIN-55,212 reduces the activity of MMPs in OA chondrocytes and NO production in bovine articular chondrocytes (Table 1). Moreover, proteoglycan degradation in bovine articular chondrocytes and nasal cartilage explants is inhibited by AEA and WIN-55,212, respectively (Table 1). The results suggest that some cannabinoids may prevent cartilage destruction: on one side, they inhibit cytokine-induced NO production by chondrocytes, preserving them from apoptosis; on the other hand, they inhibit proteoglycan degradation46,48. Recent findings also suggest that AEA
decreases the vitality and mitochondrial activity of murine chondrocytes, contributing to cartilage destruction. Surprisingly, CBRs are not involved in AEA effect46.
Inflammation plays a pivotal role in the aetiology of OA as well, and the cannabinoid receptor system present in the synovium may be an important therapeutic target for the treatment of inflammation and pain associated with OA. Indeed, the most important site of cytokine production is the synovium. In vitro studies have demonstrated that an upregulation of CB1R and a downregulation of CB2R are consequences of exposure of
naive synoviocytes to pro-inflammatory cytokines45. One study showed that WIN-55,212
was able to decrease cytokines and MMP-3 production in RASFs and OASFs, although a CB2R pathway was to exclude. In fact, WIN-55,212 demonstrated to elicit its effect via
TRPV1 and TRPA1 activation or desensitization41. Although the contribution made by the synovium in the development of RA has been demonstrated by a significant amount of research, its contribution in OA has to be further investigated.
The ECS has been shown to be involved in the modulation of OA pain as well. Inflammatory, nociceptive and neuropathic pain demonstrated to be ameliorated by modulating CB1R, CB2R, or FAAH enzymes.
In a monosodium iodoacetate (MIA) model of OA, nociceptive transmission is decreased in knee joint by arachidonyl-2-chloroethylamide (ACEA), a synthetic CB1R agonist. The
efficacy of ACEA is significantly reduced by blocking either CB1R or TRPV1 channel,
suggesting that both receptors are involved in cannabinoid-mediated antinociception (Table 1). In the same model, pain behaviour was decreased by a CB2R agonist, A-796260
31
Local treatment with the FAAH inhibitor URB597 has been found to produce both analgesic and anti-inflammatory effects in the early phases of OA development. This pre-clinical study suggests the utility of FAAH inhibitors as anti- inflammatory agents and demonstrates that early treatment of inflammation can attenuate disease progression44. Anti-nociceptive effect has been discovered with the use of CBD as well. In the end-stage of OA, local administration of CBD attenuates pain and joint inflammation. On the other hand, a prophylactic use of CBD could prevent a subsequent development of chronic pain and nerve damage44.
In addition to their analgesic properties, cannabinoids have an opioid-sparing effect, which would be highly desirable for OA patients. The number of opioids required for pain relief in non-cancer patients using morphine or oxycodone, is significantly decreased by the addition of vaporized cannabis. Similarly, pain scores in patients taking opioids for chronic pain is significantly decreased by the synthetic THC agent dronabinol. This suggests that patients with OA currently taking opioids to manage their chronic pain could gain benefits from cannabinoids44.
Although there is a lack of clinical trials and with ongoing studies exploring their anti- inflammatory effects, it therefore can be concluded that cannabinoids are well placed to enter the field of rheumatic disease treatments.
As concerns OA, current treatment options are not always optimal, and long-term use of some medications leads to major side effects, such as gastrointestinal and cardiovascular events, nausea, constipation, dizziness and headaches. Alleviating pain is one of the main focuses in most of the currently used treatment paradigms in OA which should counteract the reduction in patient mobility. Adverse reactions of the gastrointestinal system can be caused by the administration of NSAIDs, and intraarticular injections of corticosteroids may only temporarily alleviate joint pain. Additionally, the effect of intraarticular injections of hyaluronic acid wears off over time, and they are beneficial only for some patients. The development of disease-modifying drugs for OA is still in the primary phase, and treatment targets could be cytokines, whose inhibition could reduce the inflammatory component of OA. Moreover, preclinical studies have shown the potential of inhibitors of MMPs, but clinical trials in humans were unsatisfactory. There is a lack of success in disease-modifying therapies of OA, and nowadays treatments aim to slow down the progression of the disease before severe pain symptoms occur45.
32 Table 1. CBRs-mediated biological functions related to RA and OA. 1.8. CB2 RECEPTOR AGONISTS IN RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS TREATMENT As it will be discussed below, it is clear that the ECS may counteract RA and OA development and progression, and this could help to overcome several side effects associated with the current treatment options for these rheumatic diseases. The beneficial effects related to ECS are not limited to eCBs, indeed many synthetic and plant-derived cannabinoids have also been reported to be effective to combat pain and inflammation40. As seen in the previous sections, CB1R ligands have limited therapeutic applications, due to their psychotropic effects. On the contrary, CB2R agonists have emerged as a potential CB2R activation CB1R activation effects in RA • Inhibited IL-1β-induced proliferation of RASFs and secretion of IL-6, MMP-3, MMP-1340 • Inhibited LPS-induced production of IL-6 and TNFα40 • Inhibited IL-1β-induced activation of ERK 1/2 and p38 MAPK40 • Inhibited TNFα-induced IL-6 and MMP-3 production40 • Stimulated osteoblasts differentiation40 • Inhibited osteoclasts formation40 • Inhibited IL-1α-induced prostaglandin E2 production40 • Inhibited proteoglycan and collagen degradation40 • Decreased IL-1β-stimulated expression of mRNA and proteins of MMP-3, MMP-1340 • Inhibited macrophages infiltration and degradation40 • Inhibited TNFα and IL-6 production in macrophages40 • Inhibited T cells proliferation, differentiation, chemotaxis and cytokine release (IL-17, IL-2, TNFα)40 • Induced analgesia41 • Reduction of hypertension associated with RA41 • Reduction of metabolic disturbances associated with RA41 • Antinociceptive effect41 effects in OA • Reduced activity of MMPs in chondrocytes46 • Reduced NO production in chondrocytes46 • Reduced proteoglycan degradation in chondrocytes46 • Reduced pain behaviour44 • Decreased nociceptive transmission44
33
anti-inflammatory target, although the development of clinically useful CB2 ligands reveals
to be very challenging. The immunosuppression related with the use of CB2R ligands is
probably one of the reasons that restrain the development of CB2R agonists for human
medicinal use. CB2R agonists induce an increase in the basal level of signaling after binding to the orthosteric site of receptor50 without inducing psychotropic side effects, as CB 2Rs localization is limited to only few areas in the central nervous system. For these reasons, CB2R is a promising target candidate to be considered in many diseases. However, although numerous compounds have been developed and widely used to target
the CB2 receptor, most of the CB2 agonists on the market have several off-targets effects
and are also biased ligands51.
Since I focused my thesis work on the activity of CB2R modulators on the
treatment of RA and OA, in this section only the main CB2R agonists that have proven
potential clinical applications for rheumatic diseases are described. According to their chemical structures, the CB2R agonists taken into account are classified as classical, non-classical and aminoalkylindole agonists7. Classical agonists The classical group consists of dibenzopyran derivatives. It includes THC, HU-210 and JWH-133 (Figure 17). Figure 17. Chemical structures of HU-210 and JWH-133. THC is the constituent of cannabis with the main psychotropic effects and is a CBRs partial agonist. THC anti-inflammatory effects are mainly related to the decrease in IL-1α, IL-1β, IL-6 and TNF-α levels52, and early studies in the 1970s already demonstrated that it could
34
inhibit macrophage function and migration41. Furthermore, THC proved to be effective in
the mouse model of collagen-induced arthritis (CIA). In the respective study, the hypothesis it was demonstrated that THC can elicit antinociception in the paw pressure test, indicating that it can effectively block mechanical nociception in rats. In addition, in the same CIA model, THC showed to have a rapid onset and long duration of action53.
HU-210 is a synthetic analog of THC and displays high affinity for both CB1R and CB2R, due
to its dimethylheptyl side chain7. HU-210 demonstrated to produce phosphorylation of ERK1, ERK2, and p38 MAPK in OASFs and RASFs, providing strong support for functionally coupled cannabinoid receptors in SFs derived from synovia of OA and RA patients. The potential ability of the CB1R and CB2R antagonists SR141716A and SR144528 to block the
effects of the agonist was studied to further investigate the role of CBRs in mediating the effects of HU210. In fact, ERK1- and ERK2- induced phosphorylation in SFs was significantly attenuated by the CB1R antagonist SR141716A. Significant effects were not reached by the
CB2R antagonist SR144528, demonstrating that HU-210 acts via a CB1R- dependent
manner. HU-210- induced phosphorylation of p38 MAPK was not significantly attenuated by the two CBRs antagonists. In another study HU-210 has demonstrated to be helpful in preventing cartilage damage. Chondrocyte cell-surface proteins are the target binding site of autoantibodies from the RA synovial fluid, and chondrocyte apoptosis is involved in the progress of the disease. HU-210 showed to inhibit prostaglandin E2 production in primary cultures of bovine articular chondrocytes. Proteoglycan and collagen degradation in bovine nasal cartilage explant cultures are inhibited as well40,54.
JWH-133 is a CB2R agonist that demonstrated to reduce arthritis severity, inflammatory
cell infiltration and bone destruction, showing promising results in combating chronic inflammation in RA. Indeed, JWH-133 ameliorates pathologic bone destruction in CIA mice via the inhibition of osteoclastogenesis and modulation of inflammatory responses, thereby highlighting its potential as a treatment for human RA. Additionally, JWH-133, but not the CB2R antagonist SR144528, has been found to suppress CIA in mice without toxic
effects. Moreover, osteoclasts formation and osteoclastic bone resorption are attenuated. Infiltration of pro-inflammatory macrophages is decreased as well55. As mentioned in the
previous section, JWH-133 also plays an important role in the inhibition of proinflammatory factor production by RASFs40.
35
Non-classical agonists
The non-classical group contains bicyclic and tricyclic analogs of THC lacking a pyran ring7. HU-308 and CP55,940 (Figure 18) showed promising results in the treatment of rheumatic diseases.
Figure 18. Chemical structures of CP-55940 and HU-308.
In RA, the CB2R selective agonist HU-308 has been reported to effectively suppress
synovitis and alleviate joint destruction by inhibiting the production of autoantibodies and proinflammatory cytokines in CIA models56. An in vitro study demonstrated that the
expression level of CB2R was higher in RASFs than in OASFs, and that CB2R could be
upregulated by proinflammatory factors, such as IL-1β and TNF-α. In the same study they found that IL-1β-induced proliferation of RASFs and secretion of IL-6 and MMPs in RASFs could be inhibited by the administration of HU-308. Moreover, it has been seen that HU-308 suppresses serum levels of anti-collagen II antibodies. Finally, HU-308 demonstrated to stimulate bone formation by promoting osteoblasts differentiation, whereas in vitro it decreased the number of osteoclasts40,42,56.
Concerning OA, findings showed that the severity of the disease was reduced in mice treated with HU-308. Correspondingly, CB2R-deficient mice exhibited a more severe form
of OA than wild-type mice, suggesting that HU-308 protects against OA through a specific CB2R mechanism. In support of this hypothesis, a decrease in proteoglycan production by
cultured chondrocytes from CB2R-deficient mice was found. This raises the possibility that
deficiency of CB2R might predispose to OA by altering the proteoglycan content of
cartilage matrix, making it less able to deal with biomechanical stimuli46,47.
36
Another member of this group is the non-selective CBR agonist CP55,940, whose administration demonstrated to potently reduce inflammatory IL-6 and IL-8 cytokine production by stimulated RASFs, ameliorating inflammation and associated pain in arthritic joints. However, a CBR involvement is probably to exclude. Indeed, although SFs express both CBRs, the CBR antagonists did not significantly modify the inhibition of cytokine secretion induced by CP55,940, leading to conclude that in vitro it exerts its potent anti-inflammatory effect on RASFs via a non-CBR-mediated mechanism57,58.
Furthermore, it has been seen that in vitro CP55,940 decreases macrophage NO production, macrophage migration, and inhibits helper and cytotoxic T cells58. Aminoalkylindole derivate agonists The members of the aminoalkylindole group have structures that differ markedly from both classical and non-classical cannabinoid agonists. The best known is WIN 55,212-2, but JWH-015 demonstrated its potential in the treatment of RA and OA as well7. Both are shown in Figure 19. WIN 55,212-2 (Figure 19) is a very potent CBR agonist that showed to decrease cytokine production of RASFs by activating CB2R and non-cannabinoid receptor targets. Indeed, in a separate study of synovial tissue from patients with RA and OA, production of IL-6, IL-8 and MMPs by stimulated SFs in response to different concentrations of WIN 55,212-2 was investigated, demonstrating its anti-inflammatory effects in low and high concentrations. Low concentrations of WIN 55,212-2 (< 1 µM) decrease cytokine and MMP-3 production in RASFs and OASFs via TRPV1 and TRPA1 activation or desensitization. In higher concentration (4 µM), it has been found that WIN 55,212-2 completely blocks cytokine production, via a TRPA1 dependent pathway as well. Interestingly, the inhibitory effect of
WIN 55,212-2 was partially reversed by CB2R antagonist COR170. In contrast, effects
related to concentrations above 8 µM were no longer inhibited by CB2R antagonist,
suggesting a different cellular target. Furthermore, high concentrations of WIN 55,212-2 diminished SFs adhesion and proliferation, without altering cell viability; in contrast, low concentrations promoted SFs adhesion without influence on cell proliferation. These results, together with the fact that WIN 55,212-2 activates CB2R thereby reducing arthritic
inflammation, underline the potential of WIN 55,212-2 in the treatment of chronic inflammation. Moreover, the identification of TRP channels as low-affinity WIN 55,212-2
37
site of action, comprises an attractive target especially when WIN 55,212-2 is administered directly in the joint to achieve high local concentrations59.
Another study found that exposure to WIN 55,212-2 reduces the activity of MMPs in OA chondrocytes and NO production in bovine articular chondrocytes. Moreover, proteoglycan degradation in nasal cartilage explants was found to be inhibited by WIN
55,212-246,48.
As concerns the selective CB2R agonist JWH-015 (Figure 19), it showed an
anti-inflammatory role in the regulation of the IL-1β-activated anti-inflammatory responses in human RASFs, even though recent evidence suggests that it may exploit non-canonical pathway independent of CB2R to elicit its anti-inflammatory effects. In the respective
study, JWH-015 demonstrated to markedly inhibit the ability of IL-1β to induce production of IL-6 and IL-8, and proinflammatory cyclooxygenase-2 (COX-2) expression. It is been identified that JWH-015 does not utilize CB2R for its anti-inflammatory actions, rather it
has been demonstrated that it interacted with the glucocorticoid receptor (GR)60. Figure 19. Chemical structures of WIN 55212-2 and JWH-015.
38
