targeting cannabinoid receptors,possible methods?

Medicinal & health benefits of cannabis
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duke
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targeting cannabinoid receptors,possible methods?

Post by duke »

hi all wouldnt it be good if when using cannabis for a cancer or other treatment but being able to target more precisely the area requiring treatment? the first few chapters are mostly understandable to me and delving deeper i find interesting but my lack of biology degree dont help,what do you reckon?
i found and read and shared this (minus the images)full text from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404216/ so not my work! :idn:
Abstract
Cannabinoid receptors (CB1 and CB2), as part of the endocannabinoid system, play a critical role in numerous human physiological and pathological conditions. Thus, considerable efforts have been made to develop ligands for CB1 and CB2, resulting in hundreds of phyto- and synthetic cannabinoids which have shown varying affinities relevant for the treatment of various diseases. However, only a few of these ligands are clinically used. Recently, more detailed structural information for cannabinoid receptors was revealed thanks to the powerfulness of cryo-electron microscopy, which now can accelerate structure-based drug discovery. At the same time, novel peptide-type cannabinoids from animal sources have arrived at the scene, with their potential in vivo therapeutic effects in relation to cannabinoid receptors. From a natural products perspective, it is expected that more novel cannabinoids will be discovered and forecasted as promising drug leads from diverse natural sources and species, such as animal venoms which constitute a true pharmacopeia of toxins modulating diverse targets, including voltage- and ligand-gated ion channels, G protein-coupled receptors such as CB1 and CB2, with astonishing affinity and selectivity. Therefore, it is believed that discovering novel cannabinoids starting from studying the biodiversity of the species living on planet earth is an uncharted territory.

Keywords: cannabinoid receptor type 1 (CB1) and type 2 (CB2), phytocannabinoids, synthetic cannabinoids, structural analysis, animal venoms
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1. Introduction
Cannabis sativa, commonly known as marijuana, is a plant which has been used throughout human history to treat a wide variety of ailments, such as pain and anxiety, and for recreational purposes. The first natural product isolated from the cannabis plant and then characterized was the phytocannabinoid cannabinol (CBN) [1], followed by the isolation and pharmacological elucidation of the psychoactive Δ9-tetrahydrocannabinol (Δ9-THC) and the non-euphoric cannabidiol (CBD) [1,2]. The knowledge about the structure and pharmacology of CBN, Δ9-THC and CBD has led to major breakthroughs in our understanding of the effects of this plant. Insights into the mechanism of the action of phytocannabinoids led to the identification of two G protein-coupled receptors (GPCRs), cannabinoid receptors type 1 (CB1) and type 2 (CB2) [3,4]. Consequently, endogenous ligands of cannabinoid receptors, also known as endogenous cannabinoids or endocannabinoids, were identified [2,5]. Amid the recognized endocannabinoids, anandamide (synonym for N-arachidonoylethanolamine or AEA) and 2-arachidonoyl glycerol (2-AG) were discovered first [6,7,8]. Thereafter, it became clear that endocannabinoids and cannabinoid receptors are pleiotropic signaling molecules belonging to the endocannabinoid system, which also involves the enzymes that catabolize these compounds [9,10,11,12]. This signaling system has been shown to contribute to re-establishing homeostasis after insults, which highlights the therapeutic opportunities for multiple pathologies, such as pain, inflammation, cardiovascular regulation, metabolic disorders, cancer and neurodegenerative disorders [2,13]. In addition, CB1 and CB2 have been proven to play a crucial role in various bioactivities of phytocannabinoids [14], indicating the significance of cannabinoid receptors for the therapeutic effects of the cannabis plant. These discoveries subsequently inspired the constant generation of a wide variety of synthetic cannabinoids with similar or distinct structures as compared with endo- and phyto-cannabinoids. Simultaneously with the progress made in the medical field to develop selective CB1 or CB2 ligands that can modulate biological functions and treat associated diseases, some synthetic cannabinoids have become problematic in the field of recreational use, such as SPICE and K2 [15].

As a therapeutic target, CB2 has a remarkable advantage over CB1 regarding its expression pattern. CB1 is mainly expressed in the human central nervous system (CNS) (Figure 1), and is the main receptor responsible for the psychotropic effects of Δ9-THC as well as the deleterious psychiatric side effects of drugs targeting CB1 [2,16]. The CB1 inverse agonists rimonabant (SR141716) and taranabant (MK-0364) were developed as anti-obesity drugs, but both produce crippling CNS side effects, such as anxiety, depression, and suicidal ideation [17,18,19]. As a consequence, they were either withdrawn from the market or dropped in clinical trials. In contrast, CB2 is predominantly expressed in peripheral tissues, such as the immune system, where it modulates immunological function, cell migration and cytokine release [16,20] (Figure 1). CB2 expression has also been detected in the brain, albeit to a much lower extent in comparison to the immune system or the level of CB1 expression [16] (Figure 1). Notwithstanding a rather limited expression of CB2 in the peripheral nervous system and the CNS, it is undeniable that the CB2 plays an active role in neurological activities, including nociception and neuroinflammation [21,22]. Some CB2-selective agonists have been developed, showing significant efficacy in in vitro assays and in animal models without displaying unwanted psychoactive effects. Examples of such CB2-selective agonists are JWH-015, HU-308 and GW-405833 [23,24,25,26,27]. So far, besides a few phytocannabinoids and their analogs, no other CB targeting drugs have reached the market yet for clinical use. Thus, it is believed that selectively targeting CB2 provides a promising pathway of new drug discovery in the area of natural products for the treatment of a number of disorders while avoiding the severe psychiatric side effects associated with CB1.

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Figure 1
Major localization of CB1 and CB2 and their associated physiology in the human body. CB1: the majority of CB1 was found to be expressed in the brain, where it modulates various neurological activities. CB1 is located in the peripheral tissues, and, although to a lesser extent, also participates in the modulations of local tissue functions. CB2: the predominant expression of CB2 was revealed to be in the immune system (such as the spleen), where it exhibits the effects of immune modulation, and other peripheral tissues.

Recently, structural determination of CB1/CB2 coupled to the Gi protein has revolutionized our understanding of their structures and functions [28,29], alongside a parallel revolution in the methods for determining structures of cannabinoid receptors [30]. For the past few years, X-ray crystallography has been the method of choice for elucidating CB1 and CB2 structures [30,31,32,33]. Thanks to the advent of higher resolution cryo-electron microscopy (cryo-EM) structures that eliminate the problem of crystal-packing artifacts and generate an ensemble of structures [34], cryo-EM has now become the primary method in order to obtain ligand-bound CB1 or CB2 in the active state coupled to the heterotrimeric G protein complex [28,29]. Therefore, the activation mechanisms of CB1 and CB2 have been revealed [28,29]. This is expected to facilitate the rational structure-based design/discovery of drugs selectively targeting cannabinoid receptors.

In the meantime, some efforts have been made to discover novel cannabinoids over the past few years, leading to the emergence of peptide-type ligands of CB1 and/or CB2 from natural sources, other than the cannabis plant. Examples hereof are hemopressin (Hp) and the related peptides VD-Hpα and RVD-Hpα found in mice, rats or humans, as well as Pep19 derived from peptidyl-prolyl cis-trans isomerase A in humans, showing a variety of in vivo pharmacological effects depending on CB1, e.g., antinociception and neuromodulation [35,36]. Moreover, Pep19 did not exhibit CNS side effects in rats [35]. These peptides represent valuable starting points for the development of peptide drugs targeting cannabinoid receptors.

Based on the available literature on cannabinoids, it is evident that natural products have been an important source of CB1 and CB2 ligands. Cannabinoid receptors are one of the primary targets of natural products, since over 600 natural GPCR ligands have been isolated from plants, animals, fungi, and bacteria; they predominantly target aminergic, opioid, cannabinoid, and taste 2 receptors [37]. Among the diverse natural GPCR ligands, nature-derived peptides isolated from bacteria, fungi, plants, and venomous animals are an emerging compound class for GPCR ligand discovery according to published data [37]. Over 50% of nature-derived peptides targeting GPCRs discovered so far originate from animal venoms [37]. Animal venoms contain a true pharmacopeia of peptides acting on molecular targets, e.g., GPCRs, often with impressive affinity and selectivity [38]. Examples of the value of venom peptides in guiding the development of human therapeutics targeting GPCRs include the antidiabetic exenatide (Byetta®) from the venomous Gila monster (Heloderma suspectum) [38] and the analgesic cobratide (also known as cobratoxin) from the Chinese cobra (Naja atra). None of the known venom peptides have been described as ligands of cannabinoid receptors to the best of our knowledge. Therefore, for future perspective, animal venoms can be seen as a promising and yet untapped source to find selective and potent ligands of CB1 and/or CB2.

In this review, we first briefly introduce the cannabinoid receptors CB1 and CB2, and then discuss the key similarities and diversities of their activation mechanisms based on the structural information obtained by cryo-EM. Furthermore, we provide an overview of the current research status of phytocannabinoids and synthetic cannabinoids that have been shown to be ligands of CB1 and/or CB2. In the light of a natural products perspective, recently emerged novel cannabinoids, i.e., peptide-type ligands of cannabinoid receptors from animal sources, are summarized and the potential of animal venoms as a source of novel cannabinoids is demonstrated. In addition, CB1 or CB2 expression systems that can be used to rapidly screen unlabeled natural products in vitro are described in the final section.

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2. Cannabinoid Receptors
CB1 is encoded by the gene CNR1 and consists of 472 amino acids in humans (473 amino acids in rats and mice) [16]. The amino acid sequence identity among these species is 97–99% [16]. CB2 is encoded by the gene CNR2, which consists of 360 amino acid in humans. It shares only 44% sequence homology with CB1 at the protein level [16]. Additionally, CB2 has greater species differences between humans and rodents, compared with CB1, as its amino acid sequence identity among humans and rodents is slightly higher than 80% [16].

CB1 and CB2 are both class A (rhodopsin-like) GPCRs. Generally, the structure of cannabinoid receptors contains seven transmembrane alpha helices (TMHs) arranged to form a closed bundle and loops connecting TMHs that extend intra- and extracellularly [34]. In addition, it contains an extracellular N terminus and an intracellular C terminus that begins with a short helical segment (Helix 8)-oriented parallel to the membrane surface [34]. CB1 primarily couples to Gi/o protein and, under certain conditions, couples to Gs and Gq, while CB2 only couples to Gi/o [34], to trigger the further activation and downstream signaling.

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3. Ligand-Bound CB1/CB2-Gi Complex
3.1. Activation Mechanism of CB1 and CB2
Ligand-bound cryo-EM structures of the active cannabinoid receptors in complex with Gi (Figure 2A,C) have recently been built and utilized to reveal activation mechanisms of CB1 and CB2. The overall structures of the active CB1-Gi and CB2-Gi complexes are alike [29]. The binding poses of agonists in CB1 and CB2 are superimposable [28,29]. Moreover, the agonist-binding pockets and conformations of critical residues for the receptor activation are almost identical between the active conformations of CB1 and CB2 [29].

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Figure 2
(A) Binding pocket of human CB1-Gi complex bound to agonist AM841 and human CB2-Gi complex bound to agonist AM12033. Orange cartoon, CB1 structure; green cartoon, CB2 structure; yellow sticks, AM841; orange sticks, AM12033. (B) Comparison of the “toggle switch” residue conformation in human CB1, orange cartoon; and in human CB2, green cartoon. (C) Binding pocket of human CB1-Gi complex bound to agonist FUB and human CB2-Gi complex bound to agonist WIN 55,212-2. Marine cartoon, CB1 structure; purple cartoon, CB2 structure; green sticks, FUB; cyan sticks, WIN 55,212-2. (D) Comparison of the “toggle switch” residue conformation in human CB1, marine cartoon; and in human CB2, purple cartoon. (A) and (B) are derived from Hua et al. [29], (C) and (D) are derived from Xing et al. [28].

On the other hand, intriguing differences in the activation process of both cannabinoid receptors have also been revealed by structural analysis. Firstly, in the case of agonist-bound cannabinoid receptor-Gi complexes, the cytoplasmic region of transmembrane region 5 (TM5) in CB1 is simply extended and moves inward during activation, resulting in more polar and hydrophobic interactions with α5 of the Gαi protein [29]. In contrast, the cytoplasmic portion of the TM5 in CB2 extends and moves outward to form extensive interactions with the α5 helix of Gαi [29]. Secondly, in CB1, TM6 in the intracellular region moves inward to interact with α5 of the Gαi protein [29]. However, a large outward movement of the intracellular part of TM6 in CB2 occurs to accommodate the mounting of α5 from the Gαi protein [29]. In addition, the residues on the cytoplasmic ends of TM5 and TM6 in CB2 shift modestly upward, relative to those of CB1 [28]. The movements of TM5 and TM6 have a certain significance in the activation processes of cannabinoid receptors: an outward movement of TM6 is suggested as a characteristic of cannabinoid receptor activation and an extension of TM5 can result in new interactions with Gαi [29]. Notably, the critical so-called ‘‘toggle switch’’ residues of cannabinoid receptors show differences for both receptors. In CB1, the “twin toggle switch”, F200 and W356 (Figure 2B), experiences synergistic conformation changes, while in CB2, the “single toggle switch’’, W258 (corresponding to W356 in CB1) (Figure 2B), triggers the activation and the downstream signaling [29]. Alternatively, when taking into consideration residue F117 in CB2 (corresponding to F200 in CB1), another way to think about the differences of the toggle switch in the cannabinoid receptors is the following: the distance between F200 and W356 in CB1 is longer than that between F117 and W258 in CB2 (Figure 2D) [28]. This is a result of the upward position of W356 and the rotation of F200 in CB1 compared with the analogous residues in CB2 [28]. The different arrangement of the toggle switch in CB2 causes a rotation of F202 in its TM5 in comparison with the corresponding L287 in CB1 [28]. In general, CB2 only experiences minor conformational changes upon agonist binding, while CB1 is exceptional and displays larger conformational changes when modulated by agonists [29]. Furthermore, the high plasticity of CB1 during the transitions between different states facilitates its inherent ability to respond to a diverse array of ligands compared to CB2 [29].

3.2. Implications for CB1 and CB2 Ligand Selectivity
Structure determination of cannabinoid receptors coupled to Gi indicates that discovering a selective agonist may be a huge challenge due to similar binding pockets in both cannabinoid receptors. Nevertheless, there is clear-cut evidence that some highly selective CB1 or CB2 agonists can be obtained. For example, arachidonyl-2’-chloroethylamide (ACEA) and arachidonylcyclopropylamide (ACPA) are potent and selective CB1 agonists, as described further in “4.2.2. CB1-selective agonists”; JWH-133, JWH-015 and PM-226 are potent and selective CB2 agonists, as described further in “4.2.3. CB2-selective agonists”. This strongly suggests that the assumed critical differences of activation processes between CB1 and CB2 might be a good starting point for the design of cannabinoid receptor-selective drugs. As we emphasized earlier, the difference of agonist-binding activation modes between CB1-Gi and CB2-Gi complexes may find its basis in the “toggle switch”. Although the agonist-bound CB1-Gi and CB2-Gi overlap very well, the “toggle switch” concept in cannabinoid receptors is regarded as a crucial role in determining efficacy of a ligand [28,29]. When the notorious switch is constrained by binding of an antagonist or inverse agonist, the activation of cannabinoid receptors can be blocked or reversed [28].

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4. Phytocannabinoids and Synthetic Cannabinoids
4.1. Phytocannabinoids
To date, over 100 unique phytocannabinoids have been identified [14,39]. These phytocannabinoids can be classified into several subclasses, including the Δ9-THC type, the Δ8-THC type, the CBD type, the CBN type, and several others [14]. Among these, Δ9-THC and CBD are the compounds that have been investigated the most [40]. They have been shown to bind to cannabinoid receptors and elicit the characteristic beneficial or psychoactive effects associated with cannabis. The beneficial effects of the phytocannabinoids are mediated by multiple targets, thus not solely the cannabinoid receptors, CB1 and CB2, as many people believe [14]. Two kinds of novel phytocannabinoids have recently emerged (Δ9-THCP and CBDP) [39] (see further). Here, we provide an overview on the recently reported novel phytocannabinoids and the well-known/typical phytocannabinoids that are the most thoroughly studied to date.

4.1.1. Δ9-Tetrahydrocannabinol (Δ9-THC)
The psychotropic effects of cannabis are considered to be produced essentially by Δ9-THC (Table 1a); for example, acute psychotic reactions and a temporary decline in cognitive functioning in human [1,2,40,41]. The psychoactive effects of cannabis are predominantly attributed to partial agonist activity of Δ9-THC at CB1 [14,40] (Table 1a). Moreover, Δ9-THC is also characterized as a partial agonist of CB2 [14,42] (Table 1a). As a typical partial agonist, Δ9-THC has a mixed agonist–antagonist effect which is presumably dependent on the cell type, the expression of receptors, and the presence of endocannabinoids or other full agonists [14]. Regarding the unwanted side effects, the safety concerns raised in connection with Δ9-THC as a psychoactive agent preclude its widespread use in the clinic. Δ9-THC undoubtedly has a range of important therapeutic benefits, such as appetite stimulation, analgesia, and anti-emetic effects, mediated by either CB1 and CB2 or non-cannabinoid targets [40,43]. This drove further large-scale investigations, leading to the approval of nabiximol (Sativex®), a combination of THC and CBD, for the treatment of pain and/or spasticity in multiple sclerosis which was a milestone in cannabis research [44]. Sativex® is a preparation administered in the form of an orally mucosal spray and licensed in more than 27 countries as a formulation delivering a consistent concentration at a one-to-one ratio of Δ9-THC:CBD [45]. After the optimization of preparation and delivery methods, another product, called Cannador®, came to the market, delivering Δ9-THC:CBD within a narrow concentration range and at a two-to-one ratio, in the form of an orally administered capsule [45]. In relation to this, many of the recent studies on medical cannabis have focused on various forms of Δ9-THC + CBD administration [46,47,48,49] and co-administration of Δ9-THC with first-line neurotherapeutic drugs [50]. On the other hand, the synthesis of Δ9-THC analogs is another effective way to avoid or reduce its severe side effects. Details of synthetic cannabinoids are summarized further in the “Synthetic cannabinoids” section.

Table 1
Structures, binding type and bioactivities of Δ9-THC, CBD, Δ9-THCP and CBDP.

Phytocannabinoids Binding Type/CB Ki (nM)/CB EC50/IC50 (nM)/CB Bioactivity
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(a)
Δ9-THC Partial agonist/CB1, CB2 5.00~80.0/CB1
1.70~75.0/CB2 [14] 13.0~87.0/CB1
41.8, 61.0/CB2 [14] Analgesic,
antiemetic,
orexigenic [53];
relief from muscle spasms/spasticity in multiple sclerosis [61]
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(b)
CBD Antagonist/inverse agonist, negative allosteric modulator/CB1
Partial agonist/CB2 73.0~>10,000/CB1
370~>10,000/CB2 [14] 3860/CB1
503, 2270/CB2 [14] Anti-inflammatory,
anti-nociceptive,
anti-oxidant,
anti-ischemic, neuroprotective, immunosuppressive [62];
anxiolytic [43,62]
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(c)
Δ9-THCP Agonist/CB1, CB2 1.20/CB1
6.20/CB2 [39] NA Analgesic [39]
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(d)
CBDP NA NA NA NA
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Ki: binding constant; EC50: half-maximal effective concentration; IC50: half-maximal inhibitory concentration; NA: not available.

4.1.2. Cannabidiol (CBD)
Unlike Δ9-THC, CBD (Table 1b) is regarded as a clinically interesting compound for its therapeutic potential in several disorders, including anti-inflammatory, analgesic, anti-anxiety, and antitumor properties [40,43,51,52]. Moreover, it has low addictive, hallucinogenic, and toxic side effects [40,43,51]. A number of studies have investigated CBD to determine its activity at cannabinoid receptors and shown very low affinity for CB1 and CB2 [14] (Table 1b). It was reported that CBD can act as an antagonist/inverse agonist at certain concentrations below which it binds to both CB1 and CB2 orthosteric sites [53]. Recently, several studies demonstrated that CBD can act as a negative allosteric modulator of CB1, which alters the potency and efficacy of the orthosteric ligands but does not activate the receptor itself [54,55,56]. For CB2, the study showed that CBD can act as a partial agonist [54]. These results could explain the reported ability of CBD to functionally antagonize some undesirable effects of Δ9-THC in animal studies and clinical studies in humans without attenuating positive effects, thus increasing the therapeutic index of Δ9-THC. In addition, CBD exerts analgesic effects in rats by interacting with several target proteins including CB1 which involves in nociceptive control [57]. In addition, the potential immunological or anti-inflammatory effects of CBD are likely mediated via CB2 [43]. In other words, the mechanistic bases of the effects of the phytocannabinoid CBD are still not fully explained. CBD is also reported to be a potent ligand of transient receptor potential vanilloid 1 (TRPV1) and TRP melastatin 8 (TRPM8) channels [58]. As CBD shows significant efficacy as a therapeutic agent with broad safety, the U.S. Food and Drug Administration (FDA), in June 2018, approved the first drug Epidiolex®. This is an oral solution composed of the active ingredient CBD, derived from marijuana, to treat rare and severe forms of epilepsy. The proof of concept delivered by Epidiolex® drives further research formulating CBD in order to apply CBD to other various diseases or to improve the efficacy of other medical drugs in co-administration [59,60].

4.1.3. Δ9-Tetrahydrocannabiphorol (Δ9-THCP) and Cannabidiphorol (CBDP)
At the end of last year, two novel phytocannabinoids, Δ9-tetrahydrocannabiphorol (Δ9-THCP) and cannabidiphorol (CBDP), were isolated from Cannabis sativa [39]. These common names were derived from the traditional naming of phytocannabinoids based on the resorcinyl residue, in this case corresponding to sphaerophorol [39]. Δ9-THCP is a Δ9-THC homolog with a seven-term side alkyl chain (Table 1c) which is longer than the five-term side alkyl chain of Δ9-THC (Table 1a). Δ9-THCP can bind with high affinity to both CB1 and CB2 in a radioligand binding assay [39] (Table 1c). Its affinity for CB1 is significantly higher compared to the reported data of Δ9-THC, as shown in Table 1a. Further in vivo evaluation of Δ9-THCP confirmed its cannabimimetic activity of decreasing locomotor activity and rectal temperature, inducing catalepsy and producing analgesia, thereby mimicking the properties of a full CB1 receptor agonist [39]. The cannabimimetic activity of Δ9-THCP is several times higher than that of Δ9-THC [39]. As the pharmacological activity of Δ9-THC is particularly ascribed to its affinity for CB1 receptor, it is suggested that this affinity can be increased by elongating the alkyl side chain [63]. Thus, the in vivo results of Δ9-THCP show the significance of the length of the side alkyl chain on the resorcinyl moiety in modulating the ligand affinity at CB1 [39]. Another novel phytocannabinoid was named cannabidiphorol (CBDP), which is a CBD homolog with a seven-term side alkyl chain (Table 1d). At present, no data on the pharmacological effects of CBDP are available [39].

4.2. Synthetic Cannabinoids
Synthetic cannabinoids constitute the most diverse group of cannabinoids in regard to functional profile and chemical structure [40]. Originally, the synthetic cannabinoids were used as pharmacological tools to delineate the cannabinoid receptor-mediated activity [21]. Thus, their structural characteristics allow them to bind to one of the known cannabinoid receptors present in human cells, CB1 and/or CB2 [15]. After decades, some synthetic cannabinoids emerged on the market as alternatives to phytocannabinoids for recreational drug use. Several hundreds of different synthetic cannabinoids have been produced up to date, sometimes with subtle structural changes [15,22]. These synthetic cannabinoids can be divided into classical, nonclassical, amino-alkylindoles, eicosanoids and others in terms of chemical structure [53]. Many of these synthetic cannabinoids are used in pharmacological studies involving structure–activity relationships, receptor binding studies and detailed mechanisms of action of these drugs. The FDA has approved three synthetic cannabis-related drug products: Marinol® (dronabinol), Syndros® (dronabinol), and Cesamet® (nabilone) [64]. Marinol® and Syndros® include the active ingredient dronabinol, a synthetic Δ9-THC which is considered the psychoactive intoxicating component of cannabis (i.e., the component responsible for the “high” people may experience from using cannabis). Their therapeutic uses in the United States include the treatment of nausea associated with cancer chemotherapy and the treatment of anorexia associated with weight loss in AIDS patients [64]. Another FDA-approved drug, Cesamet®, contains the active ingredient nabilone, which has a chemical structure similar to THC and is synthetically derived. Cesamet®, similarly to dronabinol-containing products, is indicated for nausea associated with cancer chemotherapy and neuropathic pain [64].

4.2.1. Mixed CB1/CB2 Agonists
Most known synthetic agonists of cannabinoid receptors show little selectivity between CB1 and CB2 [21], but exhibit stronger affinity for cannabinoid receptors compared to endo- and phytocannabinoids [65]. The synthetic cannabinoids that are most commonly used in the laboratory as CB1 and CB2 receptor agonists fall essentially into three chemical groups: classical, nonclassical and amino-alkylindole. Three notable examples of such compounds are 11-hydroxy-∆8-THC-dimethylheptyl (HU-210), CP-55,940 and WIN-55,212-2. HU-210 (Table 2a), an example of a classical synthetic cannabinoid, is a highly potent cannabinoid receptor agonist, and its potency and affinity at cannabinoid receptors exceed those of many other cannabinoids [66]. The high potency and affinity of HU-210 are believed to result from replacing the pentyl side chain on Δ9-THC with a dimethylheptyl group [53]. Furthermore, pharmacological effects of HU-210 in vivo are exceptionally long-lasting [66]. The non-classical synthetic cannabinoid, CP-55,940 (Table 2b), is a cannabinoid receptor full agonist that is considerably more potent than Δ9-THC [67,68]. Moreover, it has comparable affinity for both CB1 and CB2 receptors in the low nanomolar range and it is highly potent in vivo [69]. Like CP-55,940, the amino-alkylindole synthetic cannabinoid WIN-55,212 (Table 2c) exhibits relatively high potency for both CB1 and CB2, and possesses CB1 and CB2 affinities in the low nanomolar range. However, in contrast to CP-55,940, it has slightly greater affinity for CB2 than for CB1 [66] (Table 2c). Overall, agonists of the cannabinoid receptors are involved in cognition, memory, anxiety, control of appetite, emesis, motor behavior, sensory, autonomic and neuroendocrine responses, immune responses and inflammatory effects, liver injury and hepatocellular carcinoma [53]. Currently, among synthetic CB1/CB2 mixed agonists, only nabilone (Table 1d) is in the phase III of the clinical trial for non-motor symptoms in Parkinson’s disease from ClinicalTrials.gov [70].

Table 2
Structures, chemical type and bioactivities of mixed CB1/CB2 agonists.

Synthetic Cannabinoids Chemical Type Ki (nM)/CB EC50 (nM)/CB Bioactivity
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(a)
HU-210 Classical 0.0608~0.730/CB1
0.170~0.524/CB2 [66] 0.0702/CB1 [71]
NA/CB2 Analgesic [53];
neuroprotective [72,73]
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(b)
CP-55,940 Nonclassical 0.500~5.00/CB1
0.690~2.80/CB2 [66] 0.0462~31.0/CB1 [24,71,74]
NA/CB2 Anti-nociceptive, anti-emetic [53]
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(c)
WIN-55,212-2 Amino-alkylindole 1.89~123/CB1
0.280~16.2/CB2 [66] 5.50~3000/CB1 [71,74,75,76,77,78]
NA/CB2 Analgesic, anti-inflammatory [53];
neuroprotective [79]
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(d)
Nabilone Classical 1.84/CB1
2.19/CB2 [66] NA Analgesic, antiemetic, anti-inflammatory [53];
neuroprotective [80,81]
Ki: binding constant; EC50: half-maximal effective concentration; NA: not available.

4.2.2. CB1-Selective Agonists
The starting point for the development of a CB1-selective agonist was the AEA molecule. Through changing the atom on the 1′, 2′ or 2 carbon of AEA, its CB1 selectivity can be enhanced, leading to synthesis of CB1-selective agonists, such as methanandamide (Table 3c) and O-1812 (Table 3d) [66]. So far, the most potent CB1-selective agonists developed are arachidonyl-2´-chloroethylamide (ACEA) (Table 3a) and arachidonylcyclopropylamide (ACPA) (Table 3b), both of which exhibit reasonably high CB1 potency. ACEA displays nanomolar affinity at CB1 and >1000-fold selectivity over CB2 [21], while ACPA displays >300-fold selectivity over CB2 [74]. In general, compounds with an agonistic effect and sufficient affinity to CB1 have a potential for abuse as cannabis substitutes. However, considering the medical potency of CB1-selective agonists, there is still an attractive interest in those compounds exploring different pharmacological strategies to minimize the unwanted CNS side effects and maximize the beneficial therapeutic effects. For example, ACEA and ACPA have both been studied and have been shown to have anti-depressant [82,83] and anti-nociceptive effects [84,85]. Furthermore, ACEA has been receiving considerable attention in terms of co-administration with different antiepileptic drugs. It potentiated the anticonvulsant activity of antiepileptic drugs in various animal models of epilepsy and stimulated neurogenesis in the brain of mice, showing no possible acute adverse effects [86,87,88,89,90]. This combinational activity could be beneficial to avoid the severe side effects of CB1-selective agonists. However, no CB1-selective agonist is currently in the stage of the clinical trial according to ClinicalTrials.gov [70].

Table 3
Structures, chemical type and bioactivities of CB1-selective agonists.

Synthetic Cannabinoids Chemical Type Ki (nM)/CB EC50 (nM)/CB1 Bioactivity
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(a)
ACEA Eicosanoid 1.40, 5.29/CB1
195, >2000/CB2 [66] 0.0317, 51.0 [74] Anti-depressant [86];
anti-nociceptive [53];
anti-ulcer [84];
neuroprotective [91,92];
potentiating activity of antiepileptic drugs [87];
reducing cognitive impairment [91]
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(b)
ACPA Eicosanoid 2.20/CB1
715/CB2 [66] 0.0551, 37.0 [74] Anti-depressant, anxiolytic [83];
anti-nociceptive [53];
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(c)
Methanandamide Eicosanoid 17.9~28.3/CB1
815~868/CB2 [66] 1000 [77] Analgesic, anti-emetic, orexigenic, anti-proliferation, anti-migration [53]
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(d)
O-1812 Eicosanoid 3.40/CB1
3870/CB2 [66] NA Anti-nociceptive, suppressing spontaneous activity and catalepsy [93];
anti-convulsant [94]
Ki: binding constant; EC50: half-maximal effective concentration; NA: not available.

4.2.3. CB2-Selective Agonists
Concerning CB2-selective agonists, the most widely used experimental tool is the classical cannabinoid, JWH-133 (Table 4a), and the less selective amino-alkylindole, JWH-015 (Table 4b), developed by Dr John Huffman [66]. Both compounds not only bind with higher affinity to CB2 than to CB1, but also behave as a potent CB2-selective agonist in functional assays [66]. Other notable CB2-selective agonists include PM-226 (Table 4c), HU-308 (Table 4d), the GlaxoSmithKline compound GW-405833 (Table 4e), Merck Frosst (now known as Merck Canada) compounds L-759,633 (Table 4f) and L-759,656 (Table 4g). CB2-selective agonists have undoubtedly been the focus in the field of therapeutic uses, because modulation of the CB2 is an interesting approach avoiding CNS related side effects, to treat pain, inflammation, arthritis, addictions, neuroprotection, and cancer, among other possible therapeutic applications [24,27,95,96,97,98,99,100]. Interestingly, the use of known CB2-selective agonists (i.e., JWH-015 and L-759,656) for treating or preventing a disease associated with immune dysfunction such as HIV disease was proposed in an US patent published in 2012 [22]. Over the past decade, published patents have claimed >150 synthetic selective agonists of CB2 [22]. Nowadays, new ligands designed to interact with CB2 as selective agonists are currently the subject of research both by academia and industry. Furthermore, a number of reports dealing with in vivo and in vitro models have shown positive and very interesting results (as summarized in Table 4). Nevertheless, there has still been limited success in clinical trials, partly due to the lack of translation from preclinical models and also due to the differences across species [22,101,102]. At present, at least three unique synthetic CB2 agonists have reached clinic trials, including GW842166X, S-777469 and JBT-101 from ClinicalTrials.gov [22,70,103].

Table 4
Structures, chemical type and bioactivities of CB2-selective agonists.

Synthetic Cannabinoids Chemical Type Ki (nM)/CB EC50 (nM)/CB2 Bioactivity
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(a)
JWH-133 Classical 677/CB1
3.40/CB2 [104] 63.0 [105] Attenuating neurodegenerative and spatial memory impairment [27]; improving cerebral infarction [106];
anti-inflammatory,
ameliorating sepsis [107];
anti-cancer [53];
anti-nociceptive [108];
protective effects on renal ischemia-reperfusion injury [109] and against cardiotoxicity [110]
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(b)
JWH-015 Amino-alkylindole 383/CB1
13.8/CB2 [104] NA Attenuating neurodegenerative,
neuroprotective [106]; immunomodulatory,
anti-inflammatory [53];
anti-nociceptive [111];
anti-cancer [112];
anti-obesity [25]
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(c)
PM-226 Classical >40,000/CB1
12.8/CB2 [95] 38.7 [95] Neuroprotective [95];
anti-neuroinflammatory [78]
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HU-308 Nonclassical >10,000/CB1
22.7/CB2 [66] 5.57 [23] Anti-convulsant, neuroprotective [106]; anti-inflammatory [96,97];
anti-nociceptive [96];
anti-dyskinesia [98];
osteoprotective [97];
alleviating septic lung injury [113]
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(e)
GW-405833 Amino-alkylindole 4772/CB1
3.92/CB2 [24] 0.650 [24] Anti-nociceptive,
anti-inflammatory [99];
protective effects on acute liver injury [114]
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(f)
L-759,633 Classical 1043, 15850/CB1
6.40, 20.0/CB2 [66] 8.10 [115] Analgesic [53]
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(g)
L-759,656 Classical 529~>20000/CB1
11.8~57.0 [66] 3.10 [115] Analgesic [53]
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Ki: binding constant; EC50: half-maximal effective concentration; NA: not available.

4.2.4. CB1-Selective Antagonists/Inverse Agonists
Since the discovery of CB1 and the subsequent development of the CB1-selective and potent antagonist SR141716 (also called Rimonabant) (Table 5a) by Sanofi-Aventis [19,116], there has also been considerable interest in the therapeutic potential of CB1-selective antagonists. Researchers found their therapeutic potential in the treatment of disorders in which the endocannabinoid system appears to induce undesirable symptoms following its upregulation [1]. Other notable CB1 selective antagonists include analogs of rimonabant, AM-251 (Table 5b) and AM-281 (Table 5c) [117]. Rimonabant, AM-251 and AM-281 not only act as antagonists attenuating effects of CB1 agonists, but act as inverse agonists which can by themselves elicit responses in some CB1-containing tissues that are opposite in direction from those elicited by CB1 agonists [117]. More specifically, they appear to produce inverse cannabimimetic effects in at least some tissues by somehow reducing the constitutive activity of CB1. The constitutive activity is understood as the coupling of CB1 to its effector mechanisms that, it is thought, can occur in the absence of exogenously added or endogenously released CB1 agonists [117]. Since the withdrawal of rimonabant from the market in 2008, due to its severe psychiatric side effects, research on CB1-selective antagonists’ potential pharmacological effects has continued. For instance, a recent study showed that rimonabant protects against light-induced retinal degeneration in vitro and in vivo via regulating CB1 [118]. More recently, it was shown to exhibit neuroprotective effects in a retinal degeneration model by blocking CB1 [119]. At the same time, as a selective antagonist/inverse agonist of CB1, implications of rimonabant in weight loss, anti-diabetes and reduced drug dependency have been established [53]. However, although research on the development of synthetic CB1-selective antagonists sounded very promising, it remains associated with unideal convoys. Several compounds have been withdrawn from commercial markets and clinical trials [120]. Besides rimonabant, taranabant (MK-0364) (Table 5d) and otenabant (CP-945,598) (Table 5e) were both discontinued in phase III clinical trials for treating obesity due to the risk/reward ratio [17,121] and surinabant (SR147778) (Table 5f) was discontinued from clinical trials for smoking cessation [53]. Moreover, there is no CB1-selective antagonist which is now in the stage of the clinical trial according to ClinicalTrials.gov [70]. Therefore, the current strategy towards tackling these adverse effects may be to restrict binding of CB1 antagonists to CB1 in CNS, limit their crossing of the blood brain barrier, or co-administer CB1 antagonists with drugs blocking side effects.

Table 5
Structures, chemical type and bioactivities of CB1-selective antagonists/inverse agonists.

Synthetic Cannabinoids Chemical Type Ki (nM)/CB IC50 (nM)/CB1 Bioactivity
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(a)
Rimonabant (SR141716) Others 1.80~12.3/CB1
702~13200/CB2 [66] 5.60~48.0 [116,122] Anti-obesity,
smoking cessation [53];
protective effects of retinal degeneration [118];
neuroprotective [119]
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(b)
AM-251 Others 7.49/CB1
2290/CB2 [66] 3.00 [122] Anti-obesity; anti-depressant [53]; potentiating activity of antidepressant drugs [123];
improving albuminuria and renal tubular structure [124] as well as recognition memory [125]
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(c)
AM-281 Others 12.0/CB1
4200/CB2 [66] 9.91 [122] Improving cognitive deficits [53]; facilitatory effect on recognition memory [126];
ameliorating spatial learning and memory impairment [127]; protective effects against cardiotoxicity [110]
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(d)
Taranabant (MK-0364) Others 0.130, 0.270/CB1
170, 310/CB2 [104] 0.290 [128] Anti-obesity [129];
smoking cessation [130];
anti-nociceptive [131];
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(e)
Otenabant (CP-945,598) Others 0.120~0.700/CB1 [132,133]
7663/CB2 [132] 13.1 [122] Anti-obesity [120]
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(f)
Surinabant (SR147778) Others 3.50/CB1
442/CB2 [134] 9.60 [134] Anti-obesity, smoking cessation, suppressing alcohol preference [53]
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Ki: binding constant; IC50: half-maximal inhibitory concentration; NA: not available.

4.2.5. CB2-Selective Antagonists/Inverse Agonists
The most notable CB2-selective antagonists/inverse agonists are the Sanofi-Aventis diarylpyrazole, SR144528 [135] (Table 6a) and 6-iodopravadoline (AM-630) [115] (Table 6b). Both compounds bind with much higher affinity to CB2 than to CB1, exhibit marked potency as CB2-selective antagonists and behave as inverse agonists that can produce inverse cannabimimetic effects at CB2 by themselves [66]. In fact, less attention has been paid to CB2-selective antagonists/inverse agonists compared to agonists, as indicated by a small number of patents and pharmacological studies over the past few years. For example, the US patent for AM-630 describes this compound as a CB2-selective antagonist and proposes the use of AM-630 for treating or preventing a disease associated with immune dysfunction such as HIV disease [22]. In addition, AM-630 has been shown to effectively inhibit inflammatory osteolysis in the differentiation medium system [136] and to potentiate the activity of conventional antidepressant drugs in vivo [26]. In addition, in contrast to CB2-selective agonists, no CB2-selective antagonist has been in the stage of clinical trial so far from ClinicalTrials.gov [70].

Table 6
Structures, chemical type and bioactivities of CB2-selective antagonists/inverse agonists.

Synthetic Cannabinoids Chemical Type Ki (nM)/CB IC50 (nM)/CB2 Bioactivity
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(a)
SR144528 Others 50.3~>10,000/CB1
0.280~5.60/CB2 [66] 39.0 [135] Anti-nociceptive [53]
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(b)
AM-630 Amino-alkylindole 5152/CB1
31.2/CB2 [66] 12.3 [122] Inhibiting inflammatory osteolysis [136]; potentiating activity of antidepressant drugs [26]; improving memory, anti-oxidant [137]
Ki: binding constant; IC50: half-maximal inhibitory concentration; NA: not available.
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