L. species contain hundreds of phytocompounds of which about 550 to 600 have been identified [1,2]. Although the intoxicating delta-9-Tetrahydrocannabinol (THC) is the primary cannabinoid found in “drug-type” cannabis
(marijuana, an obsolete and pejorative slang term for drug-type Cannabis
), Cannabidiol (CBD) is the main cannabinoid in hemp (“fibre-type” Cannabis
). The term “hemp” is therefore used for those Cannabis
varieties that are low in THC (<0.2% by European law), “drug-type Cannabis
” for those that are rich in THC, and “Cannabis
” as overall term.
cultivars are often characterised by their THC/CBD ratio but a variety of terpenes have also been described as characteristic, additional markers among a number of other phytocompounds that vary considerably between chemical varieties or “chemovars” [3,4].
The term “strain” is commonly misapplied to chemovars of Cannabis
in common parlance, but is properly pertinent to bacteria and viruses, but not plants [5-8]. In fact, neither CBD nor THC is formed enzymatically by the plant. Both substances are the decarboxylated form of Cannabidiolic Acid (CBDA) and delta-9-Tetrahydrocannabinolic Acid (THCA) respectively, induced in nature by slowly aging (mainly by light), or in post-harvest processing e.g., by heating. About 20 to 50-times higher concentrations of acid cannabinoid (THCA) are found in dried but otherwise unheated Cannabis
Cannabigerolic Acid (CBGA) is the precursor of CBDA, THCA and cannabichromenic acid, CBCA. CBGA is rapidly converted to the a.m. acids CBDA, CBCA, THCA or decarboxylated to Cannabigerol (CBG). Unsurprisingly, whilst CBD is usually the primary constituent, CBG predominant fibre hemp varieties also exist (e.g., the French Santhica-23, Santhica-27
) without notable concentrations of THC (THCA) and CBD (CBDA) [5,10].
Each of these phytocannabinoids has its own individual pharmacologic effects and interacts with various targets, including but not limited to the so called Endocannabinoid System (ECS) of humans as will be discussed in this text.
Since its discovery in the 1990s, the ECS emerged as one of the largest signalling and regulatory network of the organism, consisting basically of ligands, namely endocannabinoids, receptors and enzymes which are responsible for their formation and deactivation. The ECS owes his discovery largely to the historic research on the mechanisms of the psychotropic THC. After THC, CBD is the next most widely investigated phytocannabinoid.
CBD, isolated first in 1940, was given to human subjects already in 1972 to study antiepileptic effects [7,11]. From 1998 onwards, CBD was subject of an intensive development program of GW Pharmaceuticals, together with its combination with THC (“nabiximols”, US adopted name). Public interest increased tremendously in 2013/2014, when several media (e.g., CNN,https://edition.cnn.com/2013/08/07/health/charlotte-child-medical-marijuana/index.html
) reported the case of a little girl suffering from Dravet syndrome, a type of rare, in-born form of epilepsy, that was successfully treated with a whole plant extract high in CBD (~ 17% CBD and 0.3% to 0.5% THC). The reduction of seizures from over 40 per day to 2-4 per month was so spectacular that many other families with similar cases as well as physicians became highly interested in CBD; at this time, anti-epileptic properties of CBD had already been reported, and larger prospective clinical trials were underway [7,12-14]. In June 2018 purified CBD (>98%) derived from Cannabis
(Epidiolex®, GW Pharmaceuticals) received marketing authorisation as orphan drug by the US Food and Drug Authority for treatment of Dravet- and Lennox Gastaut - syndrome.
Cannabidiol (CBD) is a major non-intoxicating component of hemp. Reports have suggested that CBD possesses anti-epileptic, anxiolytic, anti-inflammatory, anti-psychotic and anti-hyperalgesic properties [8,15,16].
CBD acts on the canonical G-protein coupled receptor CB1 as a negative allosteric modulator; this alters the signalling of agonists such as delta-9-Tetrahydrocannabinol (THC) but also of antagonists such as rimonabant . At CB2 receptor, CBD is a partial agonist [18,19]. Therefore, treatment with therapeutic doses of CBD does not cause the common side effects of CB1-agonists, such as psychotomimetic effects, increase of appetite and anxiety, nor those of antagonists, such as weight loss and depression. Instead, CBD targets a number of other receptors and ion-channels, as well as enzymes and other constituents within the endocannabinoid system as will be described later. However, the mechanism of action of CBD in producing such effects is still insufficiently known.
In this article, selected phytocompounds of hemp (Cannabis
) are briefly discussed as well as their putative targets, in order to give an overview on the extremely complex interaction commonly called as “entourage effect”; lacking more systematic studies particularly in man, a number of mechanisms must remain assumptions. It is beyond the scope of this article to summarise all experimental work that has been published on specific ligands, receptors and ion channels. Primacy is given to most recent articles and reviews rather than to original papers.
The Composition of Phytocompounds of Hemp (Cannabis) Demonstrates a Large Variability
Different hemp (Cannabis
) varieties differ not only in their ratio of the two major Cannabinoids, CBD (CBDA) and THC (THCA), but even one and the same cultivar can demonstrate a considerable year to year variability of the profile of components when grown as outdoor culture; the variability of Glass-house cultures is much less as light, temperature and humidity are more stable. In addition to the almost 200 cannabinoids (including synthetic products) that are known today, terpenes and phenolic compounds (particularly flavonoids) may contribute to overall effects in a way that is still insufficiently known.
Terpenes, nature’s largest group of phytocompounds, are important components also in Cannabis
and can represent about 3% to 10% of the trichome content by weight [20,21]. More than 150 were identified in Cannabis
; their profile contributes to the characteristic of a specific cultivar. Only a few terpenes are more prominent and found in a majority of cultivars; monoterpenes such as myrcene (prominent also in hop), alpha-pinene, D-limonene, trans-ocimene and alpha-terpinolene make up to 28.3 mg/g or 0.7% of flower dry weight [3,4,22-26]. Limonene and perillyl alcohol, a precursor of limonene, demonstrated anticancer effects in phase I/II clinical studies [27,28]. Other major terpenes are sesquiterpenes, particularly ß-caryophyllene that occurs also in rosemary, or alpha-humulene, occurring in hops. Some terpenes are reported to interact directly or indirectly with the ECS as will be outlined later. Their use is considered as safe; numerous of those found in Cannabis
are listed in the database on “food flavourings” of the EU (https://webgate.ec.europa.eu/foods_system/main/
) and/or the US “GRAS-List” (https://www.femaflavor.org
). Myrcene and caryophyllene, the main terpenes in essential oil from Cannabis
, demonstrated measurable effects on autonomic nervous system parameters with beneficial subjective effects in volunteers . Terpenes’ medicinal properties are supported by numerous in vitro
and in vivo
studies that used human cells, and show anti-inflammatory, antioxidant, analgesic, anticonvulsive, antidepressant, anxiolytic, anticancer, neuroprotective, anti-mutagenic, anti-allergic, antibiotic and anti-diabetic effects [21,30,31].
Flavonoids are less numerous in Cannabis
than terpenes. Among the over 6,000 flavonoids (a subgroup of phenolics) known, 26 have been identified in Cannabis
so far. The content of flavonoids in Cannabis
is highly variable and seems to be influenced more by growth conditions than by genetics . The major flavonoids present in the leaves and flowers of the hemp cultivars Felina and Futura given as examples are orientin, vitexin, luteolin-7-O-beta-D-glucuronide, apigenin-7-O-beta-D-glucuronide  and cannflavin A; cannflavins are unique for Cannabis
and can make up to over 400 mcg/g hemp inflorescence . Flavonoids have their own pharmacological effects. A meta-analysis suggested that consumption of dietary flavonoids and subtypes (isoflavones, flavonols) has protective effects such as against ovarian and colorectal cancer [34,35]; luteolin as example, demonstrated excellent anti-inflammatory properties in man [36,37]. Although phenolics are considered as valuable components in maintaining health, little is actually known about contributions to the “entourage effect”.
The most important (agroclimatic) factors influencing the profile of phytocompounds are summarised in table 1 below, although differences between cultivars exist.
In a field experiment that examined eight industrial hemp varieties over six years, the THC content in Futura 77 varied by a factor of twenty two (between 0.045 % and 1.000 %), the CBD content varied by a factor of three (between 1.011 % and 3.261 %). Overall, agroclimatic conditions seem to affect in general the content of THC more than of CBD, as the variability was distinctly higher in six of eight cultivars (variation of max. /min. concentration: THC between 4.2 and 25, CBD between 2.5 and 5.7 . Unsurprisingly, a marked regional influence has been observed as well. When cultures of Futura 75, another hemp variety authorized by the European Community, was grown in parallel in four different regions of Morocco, cultures of the region of Agadir had 50% higher concentrations of THC than those of Beni-Mellal (0.035% vs 0.021%. .
The profile of phytochemicals originally present in the plant is considerably modified by processes after harvest as summarised below (Table 2), particularly by drying and extraction .
As can be seen, a number of pre-harvest factors, post-harvest conditions and processes influence the final profile of phytocompounds, among which the selection of the strain, the conditions of growth and harvest of the herbal material, drying and extraction is likely to have the greatest influence. Drying at temperatures below 50°C yielded the highest amount of total phenolics ; temperatures above 100°C decrease not only the content of phenolics and volatile terpenes but also of cannabinoid acids such as CBDA and THCA. The nature of the solvent distorts the genuine profile further; already small changes of the solvent polarity influence the extraction efficiency, e.g., ethanol (marginally less polar than methanol) extracts the cannabinoids slightly better than methanol (THC>THCA>CBDA>CBD . When investigating supercritical CO2-extracts, cannabinoid potency increased by factors of 3.2 for THC but 4.0 for CBD in concentrates compared to flowers, sesquiterpenes up to a factor of 8.9 .
In summary, a large number of factors, many of which are not usually standardised, influence the final content of cannabinoids and other phytosubstances in dried flowers and leaves. Processing differences from manufacturer to manufacturer, changes the profile even further. Products may thus be very different in composition, from batch to batch and from different suppliers.
Different Cannabis Cultivars Have Different Effects
Cannabis is a genetically highly diverse plant, and confusion arises from the hundreds of fantasy names and cultivars, often very similar, and that are the results of crossing, breeding, and selection mechanisms. This makes comparisons between cultivars even more challenging. Nowadays there are attempts to characterise the huge number of cultivars at their DNA-level, a project which is shared between Phylos, an agricultural genomics company (https://phylos.bio/), and the American Museum of Natural History. As an example, the OG Kush clone group-GAL 303 represents a sample that is widely regarded to be the original; it contains 63 genetically-identical samples submitted from different people of the “Cannabis community”.
Cannabis consumer claim that cultivars not only differ in their “characters” in terms of smell and flavour but also in terms of biological effects, despite that the “potency” (THC-content) cannot be perceived by smelling . It is not fully clear yet how such popular claims can be linked to differences in chemical composition and therapeutic effects, but volatile terpenes likely play a role.
Cannabis consists only of a single species Cannabis sativa L.; the ratio between CBD and THC is often taken as a further characteristic [38,63]. Notable differences in effects related to large differences concerning the THC/CBD ratio are summarised in table 3, although it has for many years been argued that effects cannot be accounted for in terms of the THC and/or CBD content alone [64,65].
Compiled from [67-71].
*CBD may potentiate some of the THC side effects when the CBD:THC ratio is around 1.8:1; a much higher ratio of 20:1 blocks THC effects [71,72]; psychoactive effects are mediated via CB1 receptors which are dose- and time-dependently down regulated by THC .
According to a meta-analysis, no significant differences have been observed in outcomes on patients treated for chronic pain, either with THC or CBD; both treatments reduced pain significantly when compared to placebo but were less effective than a THC: CBD buccal spray .
Effects related to other phytocompounds than CBD and THC are therefore of interest. In a study in which seven different Cannabis cultivars were administered to 442 participants, two cultivars, Bubba Kush and Chocolope, both with approximately the same content of THC (17-22%) and CBD (0.1%), rated at the extremes concerning their influence on anxiety; Bubba Kush was the most effective and Chocolope the least . After quantification of 29 terpenes and four Cannabinoids (CBD, THC, CBG, CBC), most striking differences were found between the contents of trans-nerodiol (already known to reduce anxiety as well as to enhance bioavailability) and guaiol, gamma-terpinene and sabinene hydrate (having the least effect on anxiety). According to other investigations, eucalyptol (1,8-cineole) has also been reported to reduce anxiety . Further examples, linked to improvement of sleeping disorders and social anxiety, are caryophyllene, linalool and myrcene which are terpenes that widely occur in Cannabis and hemp .
Phenolic compounds are likely contributing as well. They have been isolated and detected from flowers, leaves, stem, roots, seeds, twigs and pollen and can be found in hemp-flower tee . They are not only potent antioxidants; many of them, including apigenin, kaempferol, luteolin, naringin and quercetin have anxiolytic properties that may have influenced the results of the study mentioned above [33,76].
Effects of CBD are Complex and Modified by a Large Number of Concomitant Phytocompounds
All phyto substances have not only their own pharmacologic activity but interact directly or indirectly with a large number of targets that are affected by CBD and/or other cannabinoids, directly or indirectly. This can significantly modify the overall clinical effect. Although it is commonly believed that these mixtures achieve their function in humans in a synergistic manner, individual components of an extract may also have opposite effects. Further, in addition to their pharmacological effects, some substances are well known to enhance bioavailability. CBDA, as an example, has been described to increase the bioavailability of CBD by a factor of two . In addition, some flavonoids, a subgroup of phenolics that occur in Cannabis, such as quercetin, genistein, and naringin (naringenin is the aglycone of naringin) also enhance the bioavailability of other substances ; some of these flavonoids are already used in novel drug delivery formulations . It is assumed that such bioenhancers modulate membrane dynamics and lipid environment, thus increasing the permeability at the site of absorption .
Inhibition of metabolising liver enzymes such as CYP3A4 (by terpenes or sesquiterpenes e.g., ß-caryophyllene) or CYP3A5 (by luteolin), CYP2D6 (by naringenin) or CYP2C9, CYP2C19 (by quercetin), all of which are found in Cannabis, is another mechanism that increases the bioavailability of various substances, particularly of CBD . Such very complex mixtures in extracts have thus a broad range of overlapping effects that may vary with their composition and concentration. Unfortunately, the composition of Cannabis products included in clinical studies and for products available on the market is insufficiently characterised; systematic investigations are missing. Overall effects of extracts rich in CBD thus differ from those observed with pure CBD .
Out of the numerous and varying phytocompounds of Cannabis, CBD is not only considered as the main cannabinoid of hemp and primary substance responsible for the overall effects but it has also been investigated more thoroughly than the large number of non-cannabinoids and minor cannabinoids, except THC. Studies on isolated substances, although necessary, cannot foresee effects of natural extracts where many compounds interact. Of approximately 65 targets that have been reported for CBD , many of them function like in a network; an overview on selected targets that may be relevant for effects as well as potential interactions are given in figure 1.
As many conditions are multifactorial in nature, multitarget treatments such as with CBD should therefore have therapeutic advantages. Interactions may occur through various mechanisms including:
• Bioavailability (example: inhibition/induction of metabolism)
• Interference with cellular transport processes (example: inhibition of anandamide uptake/transport/degradation by CBD)
• Activation of pro-drugs or deactivation of active compounds to inactive metabolites; alteration of phytocompounds via bacterial fermentation in the intestine
• Action of synergistic partners at different points of the same signalling cascade (multi-target effects)
• Inhibition of binding to target proteins (example: reduction of psychotropic effects of THC by CBD that acts as a negative allosteric modulator of CB1 receptors)
• Indirectly increasing/decreasing ligands such as Anandamide (AEA) or 2-Arachidonoylglycerol (2-AG) (example: modulation of the endocannabinoid tone by nutritional supply of omega-3 / omega-6 fatty acids and/or inhibiting the deactivation and/or modulating the synthesis of endocannabinoids)
When interactions (synergism) between a number of phyto substances such as in extracts is possible, effective concentrations of substances can be much lower, but one can only speculate as respective systematic studies are missing.
The theoretical mechanisms of action of CBD and of other ligands seem to be very complex whereby the overwhelming amount of data still comes from experimental studies. Although it is often argued that effects in vivo need concentrations in a magnitude as observed under experimental conditions, correlation between binding studies with pure substances on one hand and the situation in the human organism, particularly in diseased conditions on the other, is often poor. As an example, experimental Inhibition (IC50) of various cytochrome P450 liver enzymes (CYP2C19, 3A4, 2D6, 2C9) by CBD is in the range between 0.8 mcM for CYP2C19 and 5.6 mcM for CYP2C9 , concentrations that are too high to be achieved via oral route. Nonetheless, CBD can increase the plasma levels of drugs administered concomitantly via inhibition of their metabolisation. Examples are warfarin S (metabolised by CYP 2C9) or N-desmethylclobazam (metabolised by CYP 2C19) where CBD increases the respective plasma levels despite that repeated doses of 700mg CBD result in average plasma concentrations of only 6 to 11 ng/ml (0.019 to 0.035 nM) which are roughly 100.000 times lower than the IC50 values .
CBD treatment (800mg CBD per day) was also accompanied by a significant increase in serum AEA as well as of other substrates of Fatty Acid Amid Hydrolase (FAAH), the enzyme responsible for the degradation of AEA in man, suggestive for an inhibition of the uptake/transport or of the enzyme , despite that CBD does not inhibit human FAAH in vitro in ligand assays . Similar, effects of terpenes (citrus fragrance) have been observed at very low, nearly undetectable serum concentrations in animals and depressive subjects [31,88]. Consequently, components in concentrations as low as 0.05% have been considered of pharmacological interest .
Whilst preclinical studies, including in rodents, can provide valuable insight into possible performance in humans, caution must always be shown in extrapolation of such data to humans.
Experience has demonstrated that the predictability of rodent models for toxicity in man fails in 57% . This is even more important for extracts where individual substances do not act in isolation but together with many other compounds.
In view of these limitations of any extrapolation of preclinical effects to effects in man, comparisons between Cannabis chemovars, between genuine extracts and between well defined extracts and pure substances would be of particular interest. Not only such studies are almost missing, extracts lack unfortunately characterisation beyond the content of CBD and THC and therefore also comparability between extracts.
In the following, effects of selected hemp (Cannabis) components on the endocannabinoid system and related targets are summarised as an illustration of the complexity of the numerous mechanisms and interactions that, in theory, are possible; many signalling systems seem to be “redundant” and act in parallel. To note, observations mainly result from preclinical studies. In the absence of systematic studies in man, the contribution of individual compounds to overall effects (“entourage effect”) must remain assumptions.
The Canonical Endocannabinoid Receptors CB1, CB2 and GPR55
All three receptors are members of the large family of G-Protein Coupled Receptors (GPCRs). CBD is a negative allosteric modulator of CB1 and possibly also of CB2; this interferes with the binding of both, agonists and antagonists [17-19]. On the “orphan” receptor” GPR55, CBD acts as an antagonist . THC is a well-known partial agonist of CB1 and CB2, whereas its role on GPR55 is still unclear, as results are conflicting. A dose- and time-dependent desensitization / down regulation of CB1 receptors by agonists has been reported . CB1 availability is on average 41% higher in males than female subjects and could explain sex differences .
Little is known about the respective acids, CBDA and THCA which are the main cannabinoids in unheated flowers and extracts. CBDA has weak activity at CB1 and CB2 receptors; THCA is thermal unstable which complicates investigations about its binding to cannabinoid receptors [93,94]. As THCA-A (and also CBDA) shows anticonvulsant activities in vivo they likely passed the blood-brain barrier in case of disease-related impairment . CBG binds to CB1 (antagonist) and CB2 and is an antagonist to GPR55 .
CB1 and CB2 are targeted also by some flavonoids, the largest group of phenolics, such as quercetin, and terpenes notably the sesquiterpene ß-caryophyllene which is a selective agonist of CB2; it is widely found in hemp [97-99].
The only naturally occurring CB1- / CB2-antagonist detected so far is THCV, a D9-THC homologue with a shorter side-chain . It is a neutral antagonist at CB1, in contrast to rimonabant (SR141716A), but an agonist at high doses [101,102].
The CB1 receptor influences (among other) appetite, memory and learning, addiction disorders, motor dysfunction, schizophrenia, depression, bipolar- and anxiety disorders. Beyond the brain, CB1 receptors also function in liver and adipose tissues, vascular as well as cardiac tissue, reproductive tissues and bone; its expression is increased in most tumours including leukaemia, probably as a protective mechanism as loss of CB1 accelerates tumour growth [103,104].
CB2 is involved in immune regulation; in the brain, CB2 receptors are found on microglial cells and are transiently elevated in traumatic brain injury . CBD at nanomolar concentrations was able to significantly reduce the effect of a selective CB2R agonist, whereas it is also described as partial agonist . Activation of CB2 stimulates bone formation, represses bone resorption and mediates renal fibrosis [106,107].
GPR55 is widely expressed in the central nervous system, glial cells, caudate, putamen, frontal cortex, striatum, hypothalamus, as well as in dorsal root ganglia neurons and adipocytes, chondrocytes and the gastrointestinal tract. It is upregulated in cancer, inflammation, pain and possibly regulates bone formation, blood pressure and insulin secretion [108,109]. At present, little is known about functional ligands other than the endogenous Lysophosphatidyl-inositol (LPI).
Other receptors such as GPR3, GPR6 and GPR12 have also been described as being activated by CBD as inverse agonist but research is still at the beginning .
Acetylcholine Receptors (AChRs)
Nicotinic- (n-AChRs) as do the muscarinic- (m-type receptors) respond to the neurotransmitter Acetylcholine (ACh) which is deactivated by Acetylcholinesterase (AChE). Inactivation leads to acetylcholine accumulation and hyperstimulation, a common strategy to treat Alzheimner’s disease. Apart from its role in neurotransmission, AChE is involved in the regulation of cell proliferation, differentiation, apoptosis and cell-cell interaction.
CBD is a reversible inhibitor of n-AChRs and has been shown to increase dose-dependently the extracellular levels of ACh and wakefulness in rats . In addition, terpenes such as 1,8-cineole, ß-caryophyllene, linalool, (+), (-)-alpha-pinene, ß-myrcene and (+)-3-carene that occur in hemp are also known as effective inhibitors of AChE, in vitro and in vivo, increasing the concentration of this neurotransmitter [112,113]; synergistic inhibition has been described between caryophyllene oxide and 1,8-cineole . Phenolics such as luteolin, naringenin or quercetin are active as well although less . Endogenous inhibitors of n-AChRs are AEA, 2-Arachidonoylglycerol (2-AG) and Arachidonic Acid (AA) which is a metabolite of AEA and 2-AG, but also of omega-6-phospholipids . AEA inhibits the function of n-AChRs in thalamic synaptosomes via a non-competitive, cannabinoid-independent mechanism.
n-AChR receptors are found throughout the peripheral and central nervous system but also on cancer cells and are involved in reward-circuits, appetite, stress-related behaviours, neuronal development and neurodegenerative disorders. In muscle, receptors mediate the neuromuscular transmission ; this may play a role in motor neuron diseases such as Amyotrophic Lateral Sclerosis (ALS).
5-Hydroxytryptamine Receptors (5-HT, Serotonin Receptors)
5-HT receptors belong to the group of G protein-coupled receptors with serotonin as the primary endogenous ligand. Various types and subtypes are known that modulate the release of excitatory and inhibitory neurotransmitters, such as glutamate, Gamma Amino Butyric Acid (GABA), dopamine, acetylcholine but also a wide range of hormones and other substances. As 5-HT receptors cross-talk with other receptors (e.g., synergistic effects of 5-HT3 with CB1), in vivo effects are very complex; they may play also a role in longevity.
CBD is an agonist of 5-HT1A and increased 5-HT firing via desensitization of 5-HT1A receptors in rats in doses of 5mg/kg/day [118,119]. It is an agonist of 5-HT2A, and an antagonist of 5-HT3A; CBG is an antagonist to 5-HT1A .
Therefore, moderate doses of CBG and CBD may oppose one another at the 5-HT1A receptor, at least in the regulation of nausea and vomiting . Other agonists on 5-HT1A are CBDA (110-times more potent), and THCV [90,122]. Agonists of 5-HT1A and 5-HT2A are anxiolytic, antidepressant, attenuate nausea, vomiting, motor- and cognitive impairment, cerebral infarction/ hypoxic brain damage (stroke), stress and mechanical allodynia. Serotonin agonists are effective in treatment of migraine and cluster headache.
On 5-HT3A, CBD acts, similar to THC and AEA, as non-competitive allosteric inhibitor [123-126]. 5-HT3 receptors are further inhibited by eucalyptol (1,8-cineole) and linalool, terpenes that occur also in hemp .
CBDA binds weakly to 5-HT5A that mediates psychiatric effects .
Adenosine, the endogenous ligand, is, similar to serotonin, an autacoid with rapid generation and destruction. The most important receptor types are A1, A2A, A2B, and A3. Adenosine mediates a multitude of physio-pathological effects on central nervous-, cardiovascular-, peripheral- and immune-systems. Receptors are members of the G-Protein Coupled Receptor (GPCR) family. A well known antagonist is caffeine.
Adenosine A1 receptor (A1A)
CBD and CBC are indirect agonists acting via inhibition of transport and/or uptake . Significant preventive effects on ventricular tachycardia/ arrhythmias have been observed in rats with a CBD dose as low as 0.05mg/kg .
Agonists protect against reperfusion injury, are neuro- and cardioprotective/ anti-arrhythmic, protect against ischemia/ reperfusion-induced ventricular arrhythmias following coronary artery occlusion and decrease hypoxic brain damage after occlusion (stroke). Further on, activation reduces glutamate release (as in seizures, excitotoxicity and oxidative stress). A1A is involved in wound healing, anti-nociception and fibrosis [101,131]. Both, A1A and A2A, cross-talk with CB2.
Adenosine A2 receptor (A2A)
CBD is an agonist; limonene, a monoterpene, exhibits selective affinity, likely responsible for its sedative effect .
A2A receptors mediate glutamate uptake and neuropathic pain; they are neuroprotective, antinociceptive/ anti-inflammatory; reduce glutamate-, IL-6, TNFa-, COX-2, iNOS-release and attenuate brain damage in neurodegenerative disorders, inflammation of lung and retina, in vitro and in vivo. Agonists promote also angiogenesis, macrophage expression, collagen production and downregulate matrix metalloproteinase (MMP 9, 2, 14), which are involved in collagen breakdown (important in cancer), and increase Hepatic Stellate Cell (HSC) proliferation [132-135].
Gamma Amino Butyric Acid Receptors (GABARs)
Different GABA receptor subtypes exist; one of the most important is GABAA; CBD, AEA and 2-AG are positive allosteric modulators at GABAA [124,136,137]. Already low doses of CBD, known to be effective in Dravet syndrome, increase GABAA currents comparable to that induced by a classical benzodiazepine, flunitrazepam . Terpenes such as linalool, and some flavonoids such as apigenin or luteolin also activate GABAA. GABAA receptors mediate fast inhibitory neurotransmission in the mammalian central nervous system which plays a role in the treatment of stress, anxiety, cognitive disorders, insomnia, depression, schizophrenia, epilepsy, tuberous sclerosis and pain.
The level of enhancement seen with either CBD or 2-AG was maximal on α2-containing GABAA receptor subtypes.
Glycine Receptors (GlyRs)
Similar to GABA receptors, glycine receptors are chloride-selective ion channels that mediate rapid synaptic inhibitory neurotransmission in the brain stem and spinal cord . Presynaptic GlyRs are believed to regulate releases of major neurotransmitters including GABA, dopamine, and glutamate. GlyRs such as alpha-1 GlyR form homo- and heteromers with other receptors. CBD enhances allosterically the activity of the alpha-1 and alpha-3 glycine receptors in low concentrations (12.8 to 18.1 mcM), which is likely to contribute to its anti-inflammatory and analgesic effects . GlyRs are activated also by THC [90,125]. In contrast, 2-AG is an antagonist; results for AEA are conflicting.
Drugs that activate glycinergic signalling by potentiating glycine receptor activity or inhibiting transporter activity are anti-inflammatory, neuroprotective and contribute to analgesia [141,142].
Transient Receptor Potential Cation Channels (TRPs)
TRP cation channels are ancient, evolutionarily conserved trans-membrane proteins that are found in all organisms and almost all organs. TRP ion channels not only act as ‘polymodal cellular sensors’ on sensory neurons but are also functionally expressed by a multitude of non-neuronal cell types. TRP channels display an extraordinary diversity of roles as sensors of changes of the local environment and homeostasis. These changes can be of chemical (e.g., pH, osmolarity, taste) or of physical nature (mechanical pressure, stretch, thermal, light). Lipids that change the physico-chemical properties of the cellular membrane influence therefore indirectly proper functioning of TRPs and other trans-membrane proteins. Unsurprisingly, ion channels are important nociceptors that play a role in many fundamental cellular processes and are involved in a number of hereditary as well as acquired diseases such as Duchenne muscular myopathy, Olmsted syndrome, cancer or diabetes . Current evidence supports that TRP channels are altered in inflammation and cancer, including proliferation, differentiation, apoptosis, migration and angiogenesis. They can cross-talk with other receptors. Some confusion may arise from the possibility that both, agonists as well as antagonists, can serve for the same therapeutic indication. This comes from the fact that these channels do not have two discrete states only, open (conducting) and closed (non-conducting), but also an inactivated/desensitized state, which after more or less long exposure to agonists or continuous stimulation are not able to conduct ions across any longer because the channel gate closes .
Ankrin type (TRPA1)
TRPA1 is the only member of TRPA channel subfamily in mammals. It is involved in cold hypersensitivity (< 17°C), acidic pH, secondary hyperalgesia (tactile allodynia and mechanical hyperalgesia in the area surrounding an injury) and neuropathy induced by diabetes and chemotherapy, but also in visceral pain, inflammatory bowel diseases, allergic contact dermatitis; TRPA1 channels are upregulated in nasopharyngeal carcinoma as well as in migraine and are mainly expressed in human fibroblasts, keratinocytes and megakaryocytes [145-147].
TRPA1 is activated and desensitized by a number of cannabinoids (rank order of potency: CBC > CBD > CBN > CBDV > CBG > THCV > CBGV >THCA > CBDA > CBGA > THCVA, but also by Arachidonic Acid (AA), as well as by hemp flavonoids such as quercetin, gingerol and eugenol [125,148,149]. The presence of such phytocompounds, in addition to cannabinoids, enhances their effects on TRPA1 channels . Examples for antagonists are camphor, thymol, menthol, 1,8-cineole, which can attenuate nociception, neurogenic inflammation in skin, non-histaminergic pruritus, colitis and release of vasoactive substances [151-153].
Melastatin type (TRPM8, “menthol receptor”)
TRPM8 is sensitive to temperatures between 10°C to 28°C. Activation is inhibited by CBD (rank: CBD > CBG > THCA > CBN > THCV > CBDV > CBGA > THCVA > CBGV > CBDA > THCv, but also by AEA and omega-3 and omega-6 fatty acids, including AA [90,125,144,148]. Inhibition reduces the response to menthol. Further to menthol; and camphor, TRPM8 is activated by terpenes that have been found in Cannabis such as linalool, eugenol or eucalyptol (1,8-cineole); naringenin blocks the activation on the level of the translation of the TRPM8-gene . A widespread over-expression is found in prostate carcinoma, particularly in androgen receptor-dependent carcinoma, breast, colon, pancreas, lung and skin tumours, in opposition to low or undetectable levels in the corresponding normal tissues [144,155]. Inhibition has pro-apoptotic effects. TRPM8 plays also a role in migraine pathogenesis and the control of bladder function .
Vanilloid type (TRPV), TRPV1 (Type 1, “capsaicin receptor”)
- is activated (opened) by mechanical, thermal (T >43°C), and chemical (e.g., low pH) stimuli. TRPV1 channels were found colocalised with CB2 receptors. CBD, CBG, CBN, CBDV, CBGV, and THCV are agonists that rapidly desensitise the ion channel making them insensitive; the most potent compound being again is CBD which decreased pain behaviour in an animal osteoarthritis model . CBDA is a partial agonist of TRPV1.
Endogenous activators are 2-AG, Palmitoylethanolamid (PEA), Oleoylethanolamide (OEA), virodhamine and high concentrations of AEA. Non-cannabinoid agonists found in Cannabis are the terpenes thymol and eugenol; others are capsaicin, piperine and the widely used OTC substance N-acetylcysteine. TRPV1 is involved in inflammatory osteoarthritis pain, neurogenic inflammation, neuronal over-excitability, neurotoxicity, anxiety, emesis, epilepsy, metabolic disorders and hyperkeratosis and induce apoptosis in cancer cells [147,153,158,159].
TRPV2 is activated at temperatures over 52°C. A number of cannabinoids show agonist activity (rank order of potency THC > CBD > CBGV > CBG > THCV > CBDV > CBN . TRPV2 is also activated by THCA [90,94]. Similar to TRPM8, TRPV2 was found over-expressed in cancer cell lines .
TRPV3 is activated by temperature of 33°C to 39°C. It is activated / rapidly desensitized by CBD, but also by THC, THCV and CBC [90,100,160,161]. CBDA is a very weak activator. A number of monoterpenes also can activate TRPV3 such as (+) borneol, thymol, camphor, eugenol, menthol and carvacrol [151,162]. TRPV3 is predominantly expressed in skin and can form hetero-tetrameric channels with TRPV1; it is upregulated in the skin of patients with atopic dermatitis, chronic itch (e.g., Olmsted syndrome) and hair loss .
TRPV4 is activated at temperatures between 27°C and 42°C, mechanical forces (e.g., cell swelling), but also by THCV (THCV> CBD), CBG, CBGA, CBGV, THC and CBN, by AA, some endocannabinoids (AEA, 2-AG) and by apigenin [100,151]. It is a crucial regulator in the progression of fibrosis including myocardial fibrosis, cystic-, pulmonary-, hepatic- and pancreatic fibrosis . Further on, it plays a role in tumour angiogenesis, inflammation of airways and vascular relaxation induced by P450 epoxygenase-dependent metabolites of endocannabinoids [165,166].
Interestingly, TRPV2, TRPV3 and TRPV4 are so far the only TRP channels that THC is able to activate.
Nuclear Transcription Factor kappa B (NF-kB), Nuclear Factor Erythroid-Derived 2 (Nrf2)
In contrast to other cannabinoids, CBD has profound gene-regulating properties. Well known examples are the nuclear transcription factor kappa B (NF-kB) and Nrf2. Both are governed by the Peroxisome Proliferator Activated Receptor gamma (PPARg) which is a widely expressed ligand-modulated nuclear hormone receptor; both transcription factors play a key role in tissue protection and repair, either by activation of Nrf2 and/or by inhibition of NF-kB . Studies revealed that CBD is a transcriptional repressor that can control cell proliferation and differentiation through DNA methylation which is an important step in epigenetic regulation .
Concentrations of PPARg agonists such as CBD matters: low concentrations promote cell survival, whereas high increase cell loss. Beneficial effects have been reported in a number of animal brain-inflammation models, including ischemic stroke [169,170].
In these respects, CBD differs from THC: in vitro, CBD altered the expression of over ten times more genes than THC. From the 1298 transcripts found to be differentially regulated by the treatments, 680 gene probe sets were up-regulated by CBD, only 58 by THC. On the other hand, 524 gene products were down-regulated by CBD but only 36 by THC. CBD-specific gene expression profile showed changes associated particularly with oxidative stress .
NF-κB regulates the transcription of genes involved in inflammation and pain (expression of genes of pro-inflammatory cytokines such as IL-1b, IL-6, TNFa, expression of inflammatory mediators such as COX-2, inducible nitric oxide synthase (iNOS) but also in lipid transport/storage and cell proliferation [172,173]. NF-κB is induced by harmful stimuli including high glucose levels, and is commonly elevated in older subjects. Suppression has therefore a high potential for the prevention and management of chronic diseases.
NF-κB is inhibited, directly or indirectly via agonists of PPARg, such as CBD, CBC, CBG, THCA, THC (but not THCV), endogenous compounds such as AEA, 2-AG, PEA, AA, as well as by a number of terpenes that occur in hemp such as a/ß-amyrin, ß-caryophyllene, a-pinene, borneol, eugenol, a-humulene, D-limonene, ß-myrcene, nerolidol, and hemp-flavonoids such as apigenin, genistein, kaempferol, luteolin, myricetin, naringenin, orientin, rutin, vitexin and quercetin [90,98,173,174]. Other activation-inhibitors are vitamin E and linoleic acid. Quercetin has specific protective effect on Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS), a common disease of urology, which is mediated by anti-inflammation, anti-oxidation, and at least partly through NF-κB and MAPK signalling pathways in vivo .
Nrf2 is another important transcription factor that is targeted by CBD and other hemp components; it controls genes encoding proteins formed in response to oxidative stress such as in stroke, cardio-vascular disorders, chronic liver disease or excito-neurotoxicity, in contrast to NF-kB that controls mainly inflammatory proteins. Nrf-2 and PPARg regulate each other but also the nuclear factors, NF-kB and Nrf2, seem to interact . Nrf2 controls phase II detoxifying enzymes and transporters . Nrf2 is over-expressed in several types of human cancer, notably of the lung, breast, ovary, prostate, pancreas, colon and skin .
Agonists such as CBD enhance the expression of Nrf2 target genes. A number of polyphenols/ flavonoids that occur in hemp are also agonists, e.g., apigenin, genistein, kaempferol, luteolin, lutein, naringenin, quercetin .
The most important endogenous ligands activating CB1, CB2 and GPR55 are AEA, 2-AG and noladin ether; virodhamine is an antagonist of CB1 but an agonist of CB2 . Endocannabinoids are also ligands for other targets such as AChR, 5-HT3, GABAR, TRPM8, TRPV1, CPR119 and PPARg. Their level is influenced by sex, age and conditions such as obesity and dysmetabolism. AEA levels positively correlate with the Body Mass Index (BMI) and waist circumference irrespective of age and sex, total cholesterol with N-Palmitoylethanolamide (PEA) and N-Oleoylethanolamide (OEA) levels . As CBD and THC influence the availability of endocannabinoids indirectly via competition to the Fatty Acid Binding Proteins (FABP), affecting metabolisation and/or cellular uptake, they modulate the endocannabinoid tonus and signalling.
Fatty Acid Amid Hydrolase (FAAH)
FAAH is the enzyme responsible for the breakdown of AEA into Arachidonic Acid (AA) + ethanolamine; it degrades also OEA, 2-OG and PEA which are known as competitive substrates and endogenous agonists of GPR55, GPR119, PPARalpha and TRPV1 [100,181-183]. Further on, FAAH plays a role in the uptake of AEA into the cell whereby cells display different sensitivities to FAAH inhibition. This mechanism modulates the availability of free AEA further .
FAAH inhibition increases the basal levels of AEA and of other ligands and therefore an interaction with targets such as CB1, GABAA, TRPV1 and PPARgamma; it also antagonises the function of AChRs and 5-HT1A. It may be speculated that this adds to restore indirectly a dysregulated endocannabinoid system, thereby enhancing hippocampal neurogenesis, possibly also learning and memory . In addition, inhibition of FAAH has anxiolytic and neuroprotective effects; it is anti-convulsant, anti-inflammatory and anti-nociceptive (through interaction of a.m. ligands with CB1 and PPARs). Interestingly, FAAH levels are altered in obese subjects .
Further to FAAH, Lipoxygenases (LOX) are able to degrade AEA as well. Their inhibition (e.g., by CBD, THC) also enhances the interaction of AEA and of other endocannabinoids with a large number of targets. Lipoxygenases (5-LOX, 12-LOX, 15-LOX) are known to be crucial enzymes in the biosynthesis of pro-inflammatory prostaglandin E2 and leukotrienes using not only AEA and 2-AG but also AA as substrate . Some of the degradation products of AEA have biological activities of their own, but their impact on biological processes within the ECS is actually not sufficiently known.
Other enzymes that degrade AEA are Cyclooxygenase-2 (COX-2) and some Cytochrome P450 (Cyt P450) enzymes (CYP3A4). COX-2 is inhibited by Cannabidiolic Acid (CBDA), the parent compound of CBD, but not by CBD itself; MAGL inactivates AEA also to some extent .
CBD is a moderate inhibitor of FAAH but more potent than other phytocannabinoids. Of 11 pure cannabinoids tested (CBC, CBD, CBG, CBN, CBDA, CBGA, CBDV, CBGV, THCA, THCV, THCVA) CBD was relatively the most potent inhibitor [101,150]. It is also an inhibitor of AEA-uptake/transport. A number of flavonoids that occur in hemp, also inhibit FAAH (genistein, kaempferol > apigenin ≈ quercetin > luteolin) .
Monoacyl Glycerollipase (MAGL)
MAGL is the main enzyme responsible for breakdown of 2-Arachidonoylglycerol (2-AG). To a minor extent of less than 10%, the degradation of 2-AG is attributed to two serine hydrolases, α/β-hydrolase 6 and 12 (ABHD6 and -12) . 2-AG is the main endocannabinoid in brain with levels that are up to 800-times higher than those of AEA, and targets CB1 and CB2 receptors as well as GABA-, Glycine receptors, TRPV1 and NF-kB; it is a key regulator of neurotransmitter release. 2-AG is dramatically increased under acute brain ischemia, is raised by menopause and obesity, and correlates with triglyceride and cholesterol levels [188,189]. MAGL inactivates also AEA. Other enzymes that degrade 2-AG, although to a much lesser extent, are Lipoxygenases (LOX-12, 5, 15), COX-2, possibly FAAH and cytochrom P450 enzymes (CYP2J2) [190-193].
So far, of the cannabinoids tested, only THCV inhibits 2-AG degradation by MAGL. CBD inhibits 2-AG degradation via LOX but not via COX or MAGL. Other Cannabinoids (CBG, CBC and THCA) exert effects only at high concentrations [100,101,125].
Among terpenes, only the triterpenes pristimerin, a-amyrin, b-amyrin and euphol have been reported to inhibit 2-AG hydrolysis by MAGL; in addition, ß-amyrin can inhibit ABHD6 and ABHD12 [97,183].
Inhibition of MAGL is neuroprotective, attenuates neuro-inflammation, lowers amyloid-β levels and plaques (with a potential use in Alzheimer’s disease); this results in reduced activity in the prostaglandin-mediated pro-inflammatory signalling cascades, and improves learning and memory deficits. MAGL is over-expressed in aggressive human cancer cells. MAGL levels decrease with age and are, similar to FAAH, down regulated in obese subjects [194,195].
In conclusion, a countless number of interactions probably occur after administration of Cannabis (hemp) products. Some but not all phytocompounds may act synergistically; the overall effect on the organism depends on the profile of these compounds and their concentrations but also on the health state of the organism. Likely the “entourage effect” is therefore not always the same and depends primarily on the profile of phytocompounds. Whereas the pharmacological effects of pure CBD have been extensively studied and are well described with generally consistent results, effects observed with genuine hemp (Cannabis) extracts vary and lack systematic investigations. In consequence, pharmacological and clinical effects are not predictable as the profile as well as the concentration of phytocompounds markedly differs and is rarely sufficiently characterised. In fact, each hemp (Cannabis) product must be considered to be unique. Research so far fails in most cases adequately to characterise the study material(s) beyond the content of the decarboxylated main cannabinoids CBD and THC. Unfortunately, investigations on well characterised cultivars as well as genuine extracts are still in their infancy, such that systematic comparisons between different, well characterised products and pure cannabinoids are missing. For research purposes it is absolutely necessary to more fully characterise the phytocompound constituents of formulations and extracts, rather than simply list their CBD- and THC-concentrations. As a minimum, the percentages of the respective cannabinoid acids, eventually including the content of Cannabigerol (CBG) as the precursor substance of CBD and THC, as well as an analytical profile of the most prominent terpenes and phenolic compounds should be included in order to enable comparisons and improve further research. With a better understanding it might be possible to select cultivars and/or extracts for specific clinical indications or health benefits.