Decoding the Postulated Entourage Effect of Medicinal Cannabis: What It Is and What It Isn’t

The ‘entourage effect’ term was originally coined in a pre-clinical study observing endogenous bio-inactive metabolites potentiating the activity of a bioactive endocannabinoid. As a hypothetical afterthought, this was proposed to hold general relevance to the usage of products based on Cannabis sativa L. The term was later juxtaposed to polypharmacy pertaining to full-spectrum medicinal Cannabis products exerting an overall higher effect than the single compounds. Since the emergence of the term, a discussion of its pharmacological foundation and relevance has been ongoing. Advocates suggest that the ‘entourage effect’ is the reason many patients experience an overall better effect from full-spectrum products. Critics state that the term is unfounded and used primarily for marketing purposes in the Cannabis industry. This scoping review aims to segregate the primary research claiming as well as disputing the existence of the ‘entourage effect’ from a pharmacological perspective. The literature on this topic is in its infancy. Existing pre-clinical and clinical studies are in general based on simplistic methodologies and show contradictory findings, with the clinical data mostly relying on anecdotal and real-world evidence. We propose that the ‘entourage effect’ is explained by traditional pharmacological terms pertaining to other plant-based medicinal products and polypharmacy in general (e.g., synergistic interactions and bioenhancement).


Introduction-The Emergence of the 'Entourage Effect' Term
Ever since the discovery of the endocannabinoid system (ECS) in 1988, the scientific community has had a strong interest in exploring the therapeutic potential of Cannabis sativa L. (hereafter referred to as "Cannabis").The ECS is almost ubiquitously distributed throughout the body and as such is implicated in maintaining homeostasis across multiple physiological functions.It is often observed as being either up-or downregulated in different disease states.Targeting this system with ECS modulatory compounds, e.g., exogenous Cannabis-derived cannabinoids (e.g., ∆ 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD)) can aid in rebalancing the system to homeostasis, resulting in therapeutic effects.It is generally recognized that the system is composed of cannabinoid receptors (e.g., cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2)); endocannabinoid signaling molecules (e.g., anandamide (AEA) and 2-arachidonoylglycerol (2-AG)); and enzymes responsible for the metabolism and availability of endocannabinoids.These ECS components are regulated in response to disturbances in the homeostasis of various body systems at any given time [1].
To the best of our knowledge, the 'entourage effect' term was used for the first time in a pre-clinical study performed by Ben-Shabat et al. in 1998 [2].They found that endogenous metabolites (i.e., fatty acid glycerol esters), which are otherwise individually pharmacologically inactive, potentiated the activity of the endocannabinoid 2-AG when tested collectively in different in vitro and in vivo studies.The potentiated effect was only observed at specific metabolite concentration ranges.This was as a hypothetical afterthought described as the 'entourage effect' and proposed to potentially be of broad relevance to the medicinal use of Cannabis-based products.The authors referred to bioactive compounds derived from plants as being accompanied by chemically related compounds (i.e., 'entourage compounds'), the latter being bio-inactive when administered individually.The observations by Ben-Shabat et al. could therefore potentially lead to findings suggesting that plant products exert effects that resemble those found in nature more than isolated single compounds from the plant [2,3].
The application of the 'entourage effect' term in the literature was later on juxtaposed with polypharmacy more broadly, pertaining to full-spectrum medicinal Cannabis products, exerting an overall higher effect compared to single compounds (e.g., THC and CBD) isolated from the plant or their synthetic analogues.Advocates of the term suggest the 'entourage effect' mechanism to be the underlying reason that many patients claim to experience an overall better effect from full-spectrum Cannabis products [4,5].This, however, relies mostly on anecdotal and real-world evidence from observational studies.Critics, on the contrary, state that the 'entourage effect' is unsupported by sound evidence and that the term primarily is used for marketing purposes to promote sales in the currently blooming medicinal Cannabis industry [6,7].
The aim of this scoping review is to discuss the main literature claiming the existence of the 'entourage effect' and to decode the referenced evidence from a pharmacological angle.Research disputing the existence of the 'entourage effect' is also included to provide an objective point of view on the matter.The overall aim is to clarify if evidence-based rationales exist that justify the usage of the 'entourage effect' term when referring to a superior therapeutic effect of full-spectrum medicinal Cannabis products compared to single compound analogues.The review is based on literature searches performed on PubMed until April 2023, with the following combination of search terms: "entourage effect", "synergy", "full-spectrum", and "medicinal cannabis"; or "medical cannabis", "cannabinoid", and "terpene"; or "terpenoid", "herbal bioenhancer", "polypharmacy", and "polypharmacology".Reference lists of key reviews and articles were assessed for additional articles of importance to the topic.Reviews, perspectives, and original research of both pre-clinical and clinical origin have been included.

Possible Pharmacological Mechanisms Involved in the 'Entourage Effect'
To progress Cannabis-based products from their current status as medicinal Cannabis under national control to a conventional drug supported by clinical evidence and with centralized approval by, e.g., the EMA and FDA, it is necessary to adhere to scientific pharmacological terminology.A misleading tendency-describing the 'entourage effect' as synergistic effects arising between the Cannabis-derived cannabinoids and terpenes resulting in a collective potentiated beneficial therapeutic effect-is evident.This implies that the compounds act at the same receptor targets while avoiding the fact that antagonistic interactions might also arise.Decoding the proclaimed entourage effect with a pharmacological lens therefore seems warranted.

Pharmacokinetic and Pharmacodynamic Interactions
In recent decades, research has focused highly on discovering and determining the pharmacological effects involved in the 'entourage effect' [8].The exact mechanisms of actions are still unknown; however, in general, they are believed to involve pharmacokinetic and pharmacodynamic interactions between the compounds of the winterized extract, that is, with plant waxes removed but cannabinoids and auxiliary compounds like terpenes and flavonoids preserved.Interactions occur when one compound's activity is affected by other compounds either negatively or positively.Drug interactions can be of a pharmacokinetic and pharmacodynamic nature, which collectively can exert both beneficial and adverse clinical outcomes.Pharmacokinetic interactions affect the involved compounds' absorption, distribution, metabolism, and excretion (ADME), ultimately impacting their bioavailability.Pharmacodynamic interactions between compounds of the full-spectrum extract affect the efficacy of the dosed medicine.These interactions happen due to differences in receptor/enzyme targets and/or binding affinities that either enhance or suppress the bioactivity of other involved compounds [9][10][11].

Additive, Synergistic, and Antagonistic Effects
Multiple types of combinatory effects are possible between the Cannabis compounds, including additive effects and synergistic as well as antagonistic interactions [9].Additive effects between compounds are a pure summation of the individual compounds' effects.Antagonistic interactions result in a combinatory effect lower than the sum of individual effects.This is often considered negative in the context of therapeutic effect but can be beneficial if the dampened antagonized effect reduces unwanted adverse effects.Synergistic interactions are the result of two or more compounds working in concert to cause a potentiated effect greater than the sum of their individual effects [12].The 'entourage effect' is often described as being caused by beneficial synergistic effects, while antagonistic effects or additive adverse effects often are avoided in the discussion [7].Underlying mechanisms of actions resulting in such synergistic effects, in addition to the elimination of adverse effects, can, as mentioned above, be of pharmacokinetic and pharmacodynamic origin [12-14].

Bioenhancers
Bioavailability refers to the fraction of a drug that reaches the systemic circulation and ultimately its therapeutic target(s).Bioenhancers increase bioavailability, thus enabling the drug to reach its therapeutic response at a lower dose, carrying the additional benefit of reducing the likelihood of adverse effects [15].For a bioactive compound to exert its full therapeutic potential, its absorption and resultant bioavailability is paramount.Depending on the route of administration, the oral route being the most restrictive, limitations in permeability, water solubility, and first-pass metabolism in the liver can decrease the compound's bioavailability.Cannabinoids are intrinsically prone to poor bioavailability as a result of their lipophilic chemical nature and thus poor water solubility as well as the reported first-pass metabolism of both CBD and THC [16].Bioenhancer mechanisms of actions can therefore involve, e.g., the enhancement of absorption, inhibition of drug efflux membrane transporters, and inhibition of cytochrome P-450 (CYP450) liver enzymes.Examples of bioenhancers of natural origin are, e.g., grapefruit juice, citric acid, aloe vera, flavonoid curcumin, menthol, and eicosapentaenoic fatty acid.These are, among many others, applied as excipients in different final drug products in traditional pharmacology [17,18].
These pharmacological mechanisms of action ultimately impact the compounds' individual and collective effects and the resultant clinical outcome experienced by the patient.

Proclaimed Entourage Compounds
Following the original findings by Ben-Shabat et al., other studies have investigated different endocannabinoid-like compounds and referred to them as 'entourage compounds' when they were observed to potentiate the activity of the endocannabinoids 2-AG and AEA (e.g., [19][20][21][22]).
Two types of 'entourage effects' have been defined in relation to Cannabis-derived compounds: 'intra-entourage' and 'inter-entourage'.The former refers to either cannabinoidto-cannabinoid or terpene-to-terpene interactions, and the latter refers to cannabinoid-toterpene interactions [23].
THC itself has been reported to be a partial agonist of both CB1 and CB2, in addition to possessing affinity and exerting effects at other targets, observed across numerous pre-clinical studies (Table A1).THC can exert mixed agonistic and antagonistic effects depending on different factors such as cell type, receptor expression state, and the presence of other ligands with affinity for the same targets as THC (e.g., endocannabinoids or other cannabinoids derived from the Cannabis plant).The concentration of THC in relation to other potentially co-administered compounds (i.e., 'entourage compounds') also impacts the pharmacological effect [24].
CBD binds to a multitude of targets (summarized in Table A1) and as such possesses a promiscuous and complex pharmacological profile [25].The polypharmacology of CBD is under extensive investigation for a variety of postulated pharmacological effects across multiple pathologies (e.g., neurological, neuropsychiatric, and inflammatory disorders), explaining why more data are available on this compound [26,27].Some of CBD's mechanisms of action, such as binding to CB1 as an allosteric negative modulator [28], can impact the bioactivity of THC.This has led to the proposal that CBD is an 'entourage compound' [4,5,29].CBD additionally can impact the pharmacokinetics of THC by, e.g., inhibiting some hepatic CYP enzymes.As such, the metabolism of THC into its more potent psychoactive metabolite (i.e., 11-OH-THC) will be delayed [30,31].CBD furthermore can modulate endocannabinoid pharmacokinetics by inhibiting fatty acid amide hydrolase (FAAH), thus inhibiting the degradation of AEA [32].
Terpenes have also been referred to as 'entourage compounds' due to their ability to, e.g., increase blood-brain barrier permeability, thus increasing the pharmacokinetic properties of, e.g., THC [30,33].A non-exhaustive list of pharmacological effects pertaining to terpenes is provided in Table A2.
Flavonoids are another group of compounds present in Cannabis, and more than 20 different flavonoids have been identified [34].The bioactivities and therapeutic potentials of these compounds have not yet been studied in depth.However, Cannaflavins A-C have been reported to possess anti-inflammatory, neuroprotective, anti-cancer, and anti-viral effects.These compounds are therefore likely to contribute to the collective therapeutic effect exerted by the administration of a Cannabis-derived extract and as such could be perceived as 'entourage compounds' too.
The abovementioned observations, among others, have led to the proposal that THC can be perceived as a 'silver bullet', while the additional compounds derived from Cannabis can be perceived as a collective 'synergistic shotgun' [4,5,30].

Evidence Perceived as Supporting the 'Entourage Effect'
In line with the original definition of the 'entourage effect', several endocannabinoidlike compounds have been investigated for their 'entourage effect' in relation to the endocannabinoids 2-AG and AEA.An 'entourage effect' of 2-AG and related endogenous compounds (2-linoleoylglycerol (2-LG) and 2-palmitoylglycerol (2-PG)) with an analgesic effect in cultured neurons was observed.A delay in transient receptor potential vanilloid 1 (TRPV1) signaling was seen when the compounds were administered in combination [19].Another group of researchers [20] observed the same tendency, with AEA congeners (palmitoylethanolamide (PEA) and oleamide (OEA)) potentiating the vasorelaxant effect of AEA via TRPV1 targeting in rats.PEA and analogue compounds have been reported to act as 'entourage compounds' by preventing AEA inactivation, thereby potentiating its activity [21].
Several pre-clinical studies covering different disease areas have shown an increased efficacy from full-spectrum Cannabis extracts or combinations of CBD and THC, with or without additional compounds, compared to single compounds (e.g., THC or CBD) [4,[35][36][37][38][39][40].As an example, concerning the anti-cancer potential of Cannabis, Blasco-Benito et al. [36] reported a more potent anti-tumor response from a plant-based extract compared to pure THC.This enhanced therapeutic efficacy was not caused by the presence of five of the most common terpenes.It was rather believed to be a result of multiple targets and mechanisms of action affected by an array of compounds present in the extract.LaVigne et al. [41] reported that Cannabis-derived terpenes (i.e., α-humulene, geraniol, linalool, and β-pinene) are cannabimimetic (i.e., exerting cannabinoid-like effects) and selectively enhance the activity of cannabinoids.All terpenes activated CB1 in vitro and exerted selective additive effects when co-administered with a CB1 agonist.The authors considered these observations as evidence of the 'entourage effect'.The terpenes β-caryophyllene and αhumulene were reported to contribute to the cytotoxic effect together with low levels of cannabichromene (CBC).CBD was, however, reported as the main cytotoxic compound in 32 different hemp inflorescences.These findings were stated to illustrate 'inter-entourage effects' between the terpenes and cannabinoids and 'intra-entourage effects' between CBC and CBD.However, antagonistic interactions from unidentified compounds in the extracts were also reported [42].Yekhtin et al. [40] observed differential specific anti-inflammatory effects of THC-and CBD-based extract products, with a higher activity of Cannabis extracts compared to pure cannabinoids.However, similarities in anti-inflammatory effects exerted by the extracts with differing THC and CBD content suggest that these major cannabinoids do not solely determine the final anti-inflammatory potential related to a certain Cannabis extract.
Corroborating these findings, Baram et al. [37] reported that different extracts with equivalent doses of THC affected apoptosis-inducing efficacy differently across different cancer cell lines.The fact that cancer cell lines express cannabinoid receptors differently might explain the heterogenous effects exerted by different extracts on different cell lines, even though the mechanism cannot be explained.The authors stated that matching synergistic compound compositions with specific cancer cell lines might hold the potential to optimize cancer treatment.This was supported by Li et al. [43], who observed differing anti-cancer and anti-inflammatory properties across high-THC Cannabis extracts from 25 different chemovars.It was concluded that the presence of certain terpenes and other cannabinoids (e.g., CBD and cannabigerol (CBG)) exerted modulating effects on THC's anti-cancer properties.It was additionally proposed that the observed effects might be explained by the presence of compounds such as flavonoids.Namdar et al. [38] observed that only terpenes and cannabinoids co-produced in Cannabis chemovars exert specific interactions and induce enhanced cell cytotoxicity.This was most apparent at natural compound ratios present in Cannabis inflorescence (i.e., flowers)-derived extracts.These observations were concluded to be a proof of concept of the 'inter-entourage effect' existence.Supporting these findings, Raz et al. published a study [44] showing synergistic effects between specific terpenes and THC in CB1 activation, which was most apparent at natural ratios present in Cannabis.Another study [45] observed that several compounds (e.g., ∆ 8 -THC, ∆ 6a,10a -THC, 11-OH-∆ 9 -THC, cannabinol (CBN), and PEA) possessed partial agonistic binding affinities at CB1 and modulated CB1 signaling in the presence of THC.
Gallily et al. [46] found that the bell-shaped biphasic dose-response associated with the administration of purified CBD (THC Pharm.GmbH, Germany) could be overcome by the administration of a Cannabis-derived standardized extract highly enriched with CBD (Avidekel, i.e., clone 202, Tikun Olam, Israel).This was proposed to be caused by synergistic effects occurring between CBD and additional compounds present in the extract.It was concluded that a standardized Cannabis extract was recommended for the management of various inflammatory conditions.Another group of researchers [47] reported a 14-fold higher plasma concentration of cannabidiolic acid (CBDA) when administered as part of a Cannabis extract as opposed to as a single compound.This was explained to be the result of CBG and THC interactions with a specific drug efflux pump, causing the inhibition of the pump.As CBDA is a substrate of this pump, the inhibition results in the prevention of CBDA efflux from the blood into the intestinal lumen, which caused the enhanced plasma concentrations.It was suggested that the extract could be perceived as a natural vehicle enhancer of CBDA.
Dahlgren et al. [48] published data derived from an open-label phase II clinical trial showing improvements in primary outcomes of anxiety-related symptoms from a fullspectrum high-CBD sublingual solution administered for four weeks.The enrolled patients (N = 14) achieved and maintained symptom reductions while experiencing few side effects.Additionally, secondary outcomes such as mood, sleep, and quality of life were observed to be improved.This was achieved by the administration of a smaller dose (approx.30 mg/day) compared to another clinical trial testing isolated CBD from extract (approx.300 mg/day) for its anxiolytic effects [49].These preliminary data will be further assessed in the ongoing RCT.Similar findings have been reported by other research groups, such as Pamplona et al., who observed benefits of CBD-rich extracts in contrast to purified CBD in relation to treatment-resistant epilepsy [50].
Several reviews and perspectives claiming the therapeutic potential of the 'entourage effect' and elucidating it in a primarily optimistic perspective have been published [4,5,29,51].These papers have in common that they refer to the same few pre-clinical original research papers presented above (e.g., [2,[36][37][38]).

THC and CBD Combinatory Effects
Research showing beneficial therapeutical effects from co-administering THC and CBD, mainly in the form of the Cannabis-based medicinal product Sativex ® (i.e., nabiximol), has by some been interpreted as supporting evidence of the 'entourage effect'.Sativex ® contains an almost equal ratio of THC and CBD, in addition to other minor compounds present in trace amounts.One heavily referenced example is the randomized controlled trial (RCT) by Johnson et al. [52] assessing the analgesic effect of Sativex ® compared to a THCpredominant extract and placebo in cancer patients.Sativex ® exerted significant analgesic effects compared to both the THC extract and placebo.As the only salient difference was the presence of CBD in Sativex ® , it was proposed that the enhanced analgesic effect was a result of synergistic effects between THC and CBD.Sepulveda et al. [53] reported differential effects of CBD and THC regarding chemotherapy-induced neuropathic pain reduction.In general, the administration of pure THC or a high-THC extract was most effective.Pure CBD had little effect, whereas a high-CBD extract was more effective; however, it was not as potent as the high-THC extract.These differential effects might be a result of additional bioactive compounds present in the extract.
Niesink et al. [54] performed a critical scrutiny of the scientific literature to assess the claim that CBD can protect against THC-associated adverse effects.Only a few studies supported this, however, with inconsistent results.An RCT study performed by Englund et al. [55] investigated four different THC:CBD ratios (i.e., 10 mg THC and 0, 10, 20, or 30 mg CBD) to determine if the presence of CBD in different doses improves the safety of Cannabis consumption.Forty-six (46) healthy infrequent Cannabis users reported negative outcomes from inhaling vaporized 10:0 THC:CBD mg Cannabis.CBD did not significantly alter this outcome at any co-administered dose.It was therefore concluded that THC:CBD co-administration in common therapeutically relevant ratios does not protect against THCassociated adverse effect acute occurrences.
CBD's modulation of the THC response allows for the administration of higher THC doses, potentially enhancing the clinical efficacy and creating a better safety profile.This is of particular importance to patients whose condition requires the presence of THC for therapeutic efficacy [29].Boggs et al. [31] reviewed the pre-clinical and clinical evidence showing functional interactions between CBD and THC.The clinical data show mixed results, with some showing CBD's potential to attenuate THC-associated adverse effects and others showing exacerbation or no impact.The authors found the scientific consensus to be limited by a scarcity of articles as well as confounding factors (e.g., self-reporting bias, variability in administration patterns, methodologies, etc.).An increasing interest in CBD's alleviating and modulatory effects on THC's adverse effects is evident, which has led to the development of Sativex ® [30].
The abovementioned key articles have in general been perceived as evidence supporting the 'entourage effect' existence, even though most of these are either pre-clinical, observational, or review articles and do not follow double-blinded randomized trials.

Evidence Perceived as Disputing the 'Entourage Effect'
Murataeva et al. [56] stated that different 2-AG congeners (2-LG, 2-oleoylglycerol (2-OG) and 2-PG) did not bind as orthosteric ligands to cannabinoid receptors.The congeners, however, have been suggested to potentiate the activity of 2-AG, reportedly by inhibiting its degradation.Murataeva et al. observed that the congeners did not inhibit CB1-dependent neurotransmission and failed to potentiate the 2-AG-mediated depolarization-induced suppression of excitation neuron signaling.The authors concluded that these 2-AG congeners acted as antagonists, involving neuron calcium channel inhibition and to some extent CB1 internalization.This suggests a more nuanced relationship than previously believed for 'entourage compounds'.Another group of researchers [22] investigated the neuroprotective effects of 2-AG and PEA.When administered individually, both compounds exerted neuroprotective effects.However, when used in combination, they canceled each other's effects.This was suggested to be caused by counteracting effects on microglial cells and possibly allosteric receptor modulation.
Research groups [57,58] have concluded the absence of an 'entourage effect' involving terpenes commonly found in Cannabis.The terpenes did not modulate the functional activity of THC at CB1 or CB2 nor did they modulate the actions of Cannabis-derived cannabinoids or endocannabinoids at other non-cannabinoid receptors, i.e., transient receptor potential ankyrin 1 (TRPA1) or TRPV1 channels [59].This might, however, differ depending on the experimental system used to assess the compounds.
In a recently published perspective paper by Cogan et al. [6], the literature perceived as supporting the 'entourage effect' was critically analyzed.In general, the pre-clinical and clinical studies in the field were found to be inconclusive, even contradictory, and limited in scope as they were based on only a few Cannabis-derived compounds.
Reviews by, among others, Russo et al. [4,5] claiming the therapeutic potential of the 'entourage effect' have been criticized for misinterpreting the original observations [2,3] in an attempt to scientifically back the use of full-spectrum Cannabis extract products with the hypothesized entourage effect term [6].The reviews place the 'entourage effect' in a mostly optimistic context, with claims of almost only beneficial effects, while generally avoiding an assessment of potentially undesired adverse effects.In fact, one of these reviews [4] is among the most heavily referenced articles used to support the existence of the 'entourage effect'.
Cogan et al. [6] concluded that the 'entourage effect' term is unfounded by current research and that perspectives in favor of the 'entourage effect' represent a misrepresentation and abuse of the research for the benefit of marketing purposes in a currently poorly regulated medicinal Cannabis industry.An editorial paper titled "Waiting for the entourage" supported these claims [60].The commercialization of Cannabis-based products has indeed grown rapidly and as a result seems to have outpaced the research field [61].
According to some researchers, there is no basis for expecting net beneficial effects of the 'entourage effect'.Increased risks of adverse effects may just as well arise between the Cannabis compounds (e.g., cannabinoids and/or terpenes), either by augmenting negative effects or diminishing beneficial effects, which is also referred to as the 'contra-entourage effect' [6,7].A study supported this [62], showing significant synergistic cytotoxic activity exerted by specific fractions of a full-spectrum Cannabis extract compared to the whole full-spectrum extract.Two significant effective synergistic interacting extract fractions contained the following: (1) mainly CBD (98.3%) and low concentrations of THC (0.3%), CBG (0.2%), and a trace amount of cannabidivarin (CBDV) (0.09%) and (2) mainly CBG (58.8%) and CBD (38.2%) as well as low concentrations of THC (0.7%) and CBC (0.4%), respectively.The synergistic effects were dependent on the relative ratios between the cannabinoids.These findings were proposed by the authors to not only be caused by additive effects of CBD in both fractions but also potential synergistic interactions between CBD and CBG, which has been reported by other research groups [63].The synergy might also be a result of the activation of multiple targets and pathways besides CB1 and CB2 receptors.The depletion of non-active and antagonistic compounds from the full-spectrum extract might cause higher specific cytotoxic efficacy while reducing the concentration, and dose, of the fractions needed to exert a significant effect (i.e., a 'contra-entourage effect').Raup-Konsavage et al. [64] reported that CBD did not display an 'entourage effect' in regard to anti-cancer effects.It was observed that pure CBD exerted an equally or more potent anti-cancer effect compared to several CBD oil extracts that additionally contained minor amounts of other cannabinoids (e.g., CBC, CBG, CBN, and THC).The authors did, however, state that these in vitro observations did not rule out potential synergistic effects arising between extract compounds.Supporting these findings, Crippa et al. [65] published data on two case studies showing worsening seizures from CBD-enriched Cannabis-derived extract administration in two children with treatment-refractory epilepsy.Initially, symptom improvement occurred, but it was followed by worsening symptoms, with typical signs of THC intoxication reported.Seizure remission and improvement in signs of intoxication were obtained when the children were switched to the same dose of a purified CBD product.However, these observations need to be assessed in high-quality RCTs and in the context of the administered doses.A CBD-enriched extract containing a lower THC percentage may not lead to intoxication and potentially carry therapeutic value.
In summary, when collectively assessing the data behind the proclaimed entourage effect, it is evident that the pre-clinical research primarily is based on single-compound combinations or extracts of highly unknown molecular composition.This also applies to the clinical studies in the field, where a lack of knowledge of the extract composition challenges the interpretation of the pharmacological mechanisms of action of a specific Cannabis extract.As illustrated in Tables A1 and A2, the multi-targeting potential of selected Cannabis-derived compounds, results in complex combinatory effects, which are as of yet poorly understood [24].
Although evidence exists that indicates the enhanced therapeutic efficacy of extracts compared to single compounds, the question remains whether this proves the existence of the 'entourage effect' or if it is merely a sign of specific compound combinations exerting enhanced therapeutic efficacy (as, e.g., shown in the study by Mazuz et al. [62]).Traditional pharmacology describes this as polypharmacy, and it is the basis for active pharmaceutical ingredient (API) combinations exerting combinatory beneficial pharmacological effects.In this context, existing pharmacological terms, like additive effects, synergistic interactions, and bioenhancers, seem to be perfectly applicable in explaining the pharmacological effects underlying the proclaimed entourage effect term.For that reason, it appears unnecessary to introduce a term such as 'entourage effect' exclusively to Cannabis-based products.

"Dirty Drugs" and Drug-Drug Interactions
In pharmacology, the informal term "dirty drug" refers to a drug that targets multiple targets within the body and exerts a wide range of effects, both desired and undesired.An example is the atypical, or second generation, antipsychotic medicinal product olanzapine, which acts as an antagonist at both dopamine (i.e., D2) and serotonin (i.e., 5-HT-2A) receptors.This exerts beneficial effects on positive as well as negative symptoms related to schizophrenic pathophysiology [66].Olanzapine has, however, also been observed to result in adverse effects such as weight gain [67].
"Dirty drugs" additionally raise the risk of drug-drug interactions when co-administered with other drugs.This is particularly critical, when administering drugs with a narrow therapeutic index.Several cannabinoids are known to be metabolized by hepatic CYP enzymes, which can either inhibit or enhance other compounds', or drugs', metabolism and vice versa.One of the most commonly administered cannabinoids, CBD, is an inhibitor of a CYP subtype responsible for the metabolism of several antidepressants and opioids.Consequently, the hypothetical scenario of CBD co-administration with other active compounds (e.g., antidepressants or opioids) may result in an increase in API serum concentrations, ultimately affecting the resultant pharmacological response [7].For that reason, pharmaceutical companies aim to avoid having these so-called "dirty" drugs in their pipeline and instead focus on designing drugs as selectively as possible.On the contrary, opioids and cannabinoids have been observed to exert synergistic effects in the case of, e.g., pain management, when co-administered.Thus, knowledge of specific active compound combinations exerting beneficial combinatory effects might provide alternative treatment options for patients with unmet medical needs.This might even lead to lower drug doses needed to provide a therapeutic effect, as, e.g., observed by the opioid-sparing effect of cannabinoids [68].
Many diseases are caused by a multifactorial causality, where single-compound medicinal products often fail to target all the disease-affected targets and as a result fail to manage the disease effectively.It has therefore been proposed that multi-compound products, such as Cannabis extracts, can provide beneficial effects in the management of such multifactorial diseases, being perceived as a more holistic treatment approach (e.g., [12,69]).As an example, Lehar et al. [70] observed that combinations of selected compounds resulted in synergistic effects and improved therapeutic selectivity as a result of multi-target effects.Consequently, the therapeutic dose could be reduced, additionally minimizing occurrences of adverse effects often associated with the administration of high drug doses.In fact, treatment with Cannabis extracts has been proposed to reduce polypharmacy, which is the realistic scenario for many patients living with multiple diseases and related symptoms.

Factors Affecting Assessment of Potential 'Entourage Effects' 7.1. Chemovar Compound Variability and Product Heterogeneity
Chemovar genotype, growth conditions, manufacturing processes, and storage conditions are all factors affecting the full-spectrum profile of a specific chemovar.The drug delivery system and route of administration of the resultant chemovar extract, or single compounds isolated from the extract, impact the final bioavailability and pharmacological effect exerted in the body [35].The extraction method, e.g., solvent-based or supercritical CO 2 , impacts the chemical profile of the extract, highlighting the importance of improved transparency and regulation in the labeling of Cannabis extract products [61].A complete profiling of the Cannabis product is necessary to provide proof of batch-to-batch consistency and is key to developing a truly standardized Cannabis-based product for medicinal purposes [71].
Hundreds of different Cannabis chemovars exist, and more than 500 compounds related to chemical classes like cannabinoids, terpenes, and flavonoids have been reported [35,72,73].Many of these compounds possess individual bioactivities affected by the compounds they are co-administered with.This can somewhat be regarded as supporting the existence of an 'entourage effect', or at least combinatory effects, which, however, can be both beneficial and unfavorable.Cannabis has therefore been proposed to be perceived as a versatile plant and treasure trove, rather than just a single drug [35,74].The primary focus within the research field has been on the cannabinoids THC and CBD, but these only constitute 2 out of more than 140 different cannabinoids discovered to date.Collectively, these factors contribute to the high heterogeneity of Cannabis-based medicinal products or derived extracts, which might partly explain the contradicting findings across studies, both pre-clinical and clinical [35].

Relative THC:CBD Ratio, Dose, and Administration Route
Across studies in the field of medicinal Cannabis, there are marked variabilities in dose, the route of administration, and THC:CBD ratios, in addition to relative ratios of other compounds present in different Cannabis extracts.These factors collectively impact pharmacological effects and clinical outcomes.Many observational studies are scarce in information on the investigated Cannabis product types and doses administered, which is often compared across different diseases and Cannabis products sourced from different suppliers.This challenges the comparison of study outcomes, resulting in questionable conclusions across reviews and meta-analyses, whenever study data are pooled [75].
The relative THC:CBD ratio has been observed to impact the therapeutic effect of Cannabis-based medicines.For example, high-THC products are administered for indications such as nausea and vomiting, whereas an equal THC:CBD ratio is beneficial in, e.g., pain and multiple sclerosis, while high-CBD products can be used for the treatment of, e.g., depression and epilepsy [76,77].However, it is important to state that these reported effects and underlying mechanisms of actions are dose-dependent.Depending on the severity and progression of a disease, varying the dosing scheme for a specific patient is necessary.The inherent polypharmacy pertaining to multi-compound Cannabis extract products, in addition to the polypharmacology of the individual compounds, as illustrated in Tables A1 and A2, will define the product's impact on a certain pathophysiological disease state.
The final pharmacological outcome depends on the combined effect of the co-administered compounds as they impact each other's ability to bind to their respective targets.Furthermore, biphasic (i.e., when low and high doses exert opposite effects) dose-responses have been reported as, e.g., THC is demonstrated to be anxiolytic at low doses and anxiogenic at higher doses [78].The biphasic effect is additionally influenced by potentially co-administered compounds.This was observed in a pre-clinical study in which the bell-shaped curve, associated with CBD administered in its pure form, was counteracted by the administration of a high-CBD extract with a full-spectrum profile [46].A higher dose increases the risk of cannabinoid off-target binding to targets with lower affinity and selectivity [79].THC is a partial agonist of CB1 and CB2 but can exert a mixed agonist-antagonist profile depending on the cell type and its expression of receptors, the experimental settings (e.g., in vitro or in vivo), and the co-presence of phytocannabinoids or endocannabinoids with stronger affinity to the target(s) [24,80].
The high lipophilicity of cannabinoids restricts their bioavailability when administered orally via conventional drug delivery strategies.Studies on ingested THC report bioavailability ranging from 4-20% [81,82], with approx.6% reported for CBD in humans [83,84].Many in vitro and in vivo studies in the field assess pharmacological effects in a scenario in which the compound of interest is injected directly into the target site, thus providing 100% bioavailability.This is an unrealistic pharmacological scenario in a clinical setting where the oral administration route often is preferred [79].
Thus, simple mixtures with carrier oils, like medium-chain triglycerides (MCT), being the most readily available product type on the market, lose most of their cannabinoid dose in the gastrointestinal tract following oral administration.This may partly explain the non-optimal and non-significant clinical outcomes reported across several studies in the field [35].Therefore, more sophisticated formulations, so-called enabling technologies, are needed to increase the bioavailability and lower the dose needed to exert a therapeutic effect.

Conclusions
Based on the published literature included within this scoping review, it is evident that there is a lack of sound evidence supporting the existence of the proclaimed Cannabis-related entourage effect.The literature shows contradictory, equivocal, and inconclusive findings, with both advocates and critics of the 'entourage effect' expressing their observations and opinions in several reviews, based primarily on the same existing original articles, of which there are relatively few.Certainly, indications exist of multi-compound and full-spectrum Cannabis extract products exerting enhanced efficacy and a broader therapeutic index.However, the pharmacological underlying mechanisms of action are currently based on contradictory and simplistic pre-clinical and clinical data and as such currently remain scientifically unfounded.As concluded in a recently performed review [85], focusing on potential synergistic and 'entourage' effects of Cannabis-based medicines as analgesic, further clinical trials and in vitro studies are necessary to definitively demonstrate their analgesic effects and therapeutic potential in general, both as single compounds and in various combinations.
The 'entourage effect' term was originally coined as a hypothetical afterthought in a pre-clinical study to describe bio-inactive compounds potentiating a bioactive compound's activity.The term was then used in the context of polypharmacy to refer to an enhanced therapeutic effect arising from administration of multi-compound Cannabis-based products, without clear justifications [6].The 'entourage effect' is frequently connected to the designation of synergistic effects.This is misleading from a pharmacological point of view as it implies that the Cannabis compounds target the same receptor system, where an actual synergistic interaction and pharmacological amplifying effect can arise.The general reference to the 'entourage effect' as an enhanced therapeutic effect omits the fact that compounds present in the Cannabis-based product might also exert antagonistic interactions.Potentially, this can result in an unwanted adverse effect, referred to as the 'contra-entourage effect' [7], causing confusion when used in the context of medicinal products.Furthermore, the referral to the 'entourage effect' between cannabinoids and/or terpenes conflicts with the original definition of the term as most of these compounds are themselves bioactive, as illustrated in Tables A1 and A2.
Other natural plant-based products composed of multiple compounds have found their way into the pharmaceutical research field.Interestingly, their collective and interacting effects have not been explained as an 'entourage effect' but rather by the usage of traditional pharmacological terms such as synergistic/antagonistic interactions, additive effects, and bioenhancement [86].It can therefore be speculated if the 'entourage effect' term is scientifically valid or if it is in fact at the borderline of pseudoscience.The problematic misuse of the term by the Cannabis industry to justify unique selling propositions of their Cannabis chemovars ultimately risks affecting the patients negatively.In line with Cogan et al. [6], we therefore propose that the 'entourage effect' is explained using established pharmacological terminology pertaining to polypharmacy in general (e.g., synergistic interactions and biohancement).

Future Perspectives
Sometimes less is more-a statement that most often applies to traditional pharmaceutical drug development, predominantly following the single-compound approach [87].Standardized product formulations of exact compositions (i.e., APIs, e.g., cannabinoids, terpenes, and auxiliary components) with known individual and collective mechanisms of action are essential.A focus on Cannabis-derived ECS modulatory compounds beyond THC and CBD should be prioritized as they hold the potential for unique combinatory pharmacological effects.This should be explained by traditional pharmacological terms and verified in pre-clinical and clinical studies [23,88], resulting in less contradictory clinical outcomes, as opposed to by using "drug cocktails" with an unknown compound content and resultant unpredictable interaction effects [8,12].A possible approach is to utilize analytical chemistry tools (e.g., high-performance liquid chromatography (HPLC)) to characterize the fingerprint of a Cannabis product and to understand the chemical foundation of the 'entourage effect'.This should lead to less ambiguity in the identification of specific chemovars for certain disease treatments [89].
Availability of evidence-based high-quality data, in line with best practice procedures, are more likely to provide doctors with the decision basis needed to prescribe Cannabisbased medicine to their patients, many suffering from unmet medical needs by conventional medicinal products.Certainly, specific cannabinoid and terpene combinations hold the potential to develop design-specific Cannabis-based combination drugs with unique therapeutic potentials [44,90].It has, however, been advocated that extracts can also be too pure, causing the loss of synergistic therapeutic potential [5,87].It is required by regulatory laws to demonstrate superior clinical efficacy or the improved safety of a drug combination product composed of several APIs compared to a single API-based product [7,14,91,92].These aspects need to be established and tackled before Cannabis-based products can develop into conventional medicinal products, i.e., with a solid clinical package to support doctors in their preparation of an effective treatment plan for their patients.Appendix B  For a more extensive review on terpene mechanisms of actions and pharmacological effects, see these extensive reviews on the subject: Goncalves et al. [225], Liktor-Busa et al. [226], and Odieka et al. [33,71].Abbreviations: 5-hydroxytryptamine receptor 1A (5-HT-1A); adrenergic receptor alpha-1 (A1A); adrenergic receptor alpha-2 (A2A); cannabinoid receptor 2 (CB2); Extracellular-regulated kinase/serine/threonine kinase (ERK/AKT); Farnesyltransferase (FTase); gamma-aminobutyric acid type A receptor(GABA-A); mitogen-activated protein kinase (MAPK); Nuclear factor kappa B (NFκB); peroxisome proliferator-activated receptor alpha/gamma (PPARα/γ); Toll-like receptor 4 (TLR4); transient receptor potential cation channel type A1 (TRPA1); transient receptor potential vanilloid type 1 (TRPV1).

Table A1 .
[71]]: Pre-clinical in vitro study; **: pre-clinical in vivo study; ***: clinical study; N.B.: This table is non-exhaustive, broadly elucidating selected compounds and some of their potential pharmacological effects currently present in the pre-clinical literature.Depending on study parameters, the compounds show differing, sometimes biphasic, affinities and effects at different targets, thus highlighting the contradictory and equivocal evidence state.For a more extensive review on cannabinoid mechanisms of action and pharmacological effects, see these extensive reviews on the subject: Morales et al.[24], Stasiulewicz et al.[196], Almeida et al.[197], Oultram et al.[198], Vitale et al.[25], Peng et al.[199], Matheson et al.[200], Odieka et al.[71], and Castillo-Arellano et al. *

Table A2 .
Selected Cannabis sativa L.-derived terpenes, their targets, mechanisms of action, and potential resultant pharmacological effects.

Table A2 .
Cont.Pre-clinical in vitro study.** Pre-clinical in vivo study.*** Clinical study.N.B.: This table is non-exhaustive, broadly elucidating selected compounds and some of their potential pharmacological effects currently present in the pre-clinical literature.Depending on study parameters, the compounds show differing, sometimes biphasic, affinities and effects at different targets, thus highlighting the contradictory and equivocal evidence state. *