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Russo The case for the entourage effect and conventional breeding of clinical cannabis No strain no gain Front Plant Sci 2019

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The topic of Cannabis curries controversy in every sphere of influence, whether politics, pharmacology, applied therapeutics or even botanical taxonomy. Debate as to the speciation of Cannabis, or a lack thereof, has swirled for more than 250 years. Because all Cannabis types are eminently capable of cross-breeding to produce fertile progeny, it is unlikely that any clear winner will emerge between the “lumpers” vs. “splitters” in this taxonomical debate. This is compounded by the profusion of Cannabis varieties available through the black market and even the developing legal market. While labeled “strains” in common parlance, this term is acceptable with respect to bacteria and viruses, but not among Plantae. Given that such factors as plant height and leaflet width do not distinguish one Cannabis plant from another and similar difficulties in defining terms in Cannabis, the only reasonable solution is to characterize them by their biochemical/pharmacological characteristics. Thus, it is best to refer to Cannabis types as chemical varieties, or “chemovars.” The current wave of excitement in Cannabis commerce has translated into a flurry of research on alternative sources, particularly yeasts, and complex systems for laboratory production have emerged, but these presuppose that single compounds are a desirable goal. Rather, the case for Cannabis synergy via the “entourage effect” is currently sufficiently strong as to suggest that one molecule is unlikely to match the therapeutic and even industrial potential of Cannabis itself as a phytochemical factory. The astounding plasticity of the Cannabis genome additionally obviates the need for genetic modification techniques.
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fpls-09-01969 January 8, 2019 Time: 15:54 # 1
published: 09 January 2019
doi: 10.3389/fpls.2018.01969
Edited by:
Giuseppe Mandolino,
Council for Agricultural and
Economics Research, Bologna, Italy
Reviewed by:
Gianpaolo Grassi,
CREA-CIN Rovigo, Italy
Raffaella Pergamo,
Council for Agricultural and
Economics Research, Rome, Italy
Ethan B. Russo;
Specialty section:
This article was submitted to
Plant Breeding,
a section of the journal
Frontiers in Plant Science
Received: 31 October 2018
Accepted: 19 December 2018
Published: 09 January 2019
Russo EB (2019) The Case
for the Entourage Effect
and Conventional Breeding of Clinical
Cannabis: No “Strain,” No Gain.
Front. Plant Sci. 9:1969.
doi: 10.3389/fpls.2018.01969
The Case for the Entourage Effect
and Conventional Breeding of
Clinical Cannabis: No “Strain,” No
Ethan B. Russo*
International Cannabis and Cannabinoids Institute, Prague, Czechia
The topic of Cannabis curries controversy in every sphere of influence, whether politics,
pharmacology, applied therapeutics or even botanical taxonomy. Debate as to the
speciation of Cannabis, or a lack thereof, has swirled for more than 250 years. Because
all Cannabis types are eminently capable of cross-breeding to produce fertile progeny,
it is unlikely that any clear winner will emerge between the “lumpers” vs. “splitters” in
this taxonomical debate. This is compounded by the profusion of Cannabis varieties
available through the black market and even the developing legal market. While labeled
“strains” in common parlance, this term is acceptable with respect to bacteria and
viruses, but not among Plantae. Given that such factors as plant height and leaflet
width do not distinguish one Cannabis plant from another and similar difficulties in
defining terms in Cannabis, the only reasonable solution is to characterize them by their
biochemical/pharmacological characteristics. Thus, it is best to refer to Cannabis types
as chemical varieties, or “chemovars.” The current wave of excitement in Cannabis
commerce has translated into a flurry of research on alternative sources, particularly
yeasts, and complex systems for laboratory production have emerged, but these
presuppose that single compounds are a desirable goal. Rather, the case for Cannabis
synergy via the “entourage effect” is currently sufficiently strong as to suggest that one
molecule is unlikely to match the therapeutic and even industrial potential of Cannabis
itself as a phytochemical factory. The astounding plasticity of the Cannabis genome
additionally obviates the need for genetic modification techniques.
Keywords: cannabis, cannabinoid, marijuana, hemp, genomics, genetically modified organism,
tetrahydrocannabinol, cannabidiol
Earlier data on taxonomy of Cannabis was previously reviewed (Russo, 2007), which will be
herein summarized and supplemented. Cannabis is a dioecious annual of the Cannabaceae
family which traditionally includes hops, Humulus spp. Alternatively, Cannabis has also been
assigned to Moraceae, Urticaceae, or even in the Celtidaceae families on the basis of chloroplast
restriction site maps (Weigreffe et al., 1998), and chloroplast mat K gene sequences (Song
et al., 2001). More recently, the Cannabaceae have subsumed eight genera: Celetis, Pteroceltis,
Aphananthe, Chaetachme, Gironniera, Lozanella, Trema, and Parasponia, comprising 170 odd
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Russo Conventional Breeding of Cannabis
species (McPartland, 2018), a finding supported by genetic
analysis of four plastid loci (Yang et al., 2013). Current
research on fossil pollen samples associated with the ecological
associations of Cannabis with steppe companion species
(Poaceae, Artemisia, Chenopodiaceae), and Humulus (hops) with
forest genera (Alnus, Salix, Populus), have established that
although Cannabis seems to have originated in the Tibetan
Plateau at least 19.6 million years ago, it has also been indigenous
to Europe for at least a million years (McPartland et al., 2018),
and refuted the conventional wisdom that this “camp follower”
was brought there by man.
The species assignation of Cannabis itself is fraught with
great debate. Cannabis sativa, meaning “cultivated Cannabis,
was so named by Fuchs, among others, in 1542 (Fuchs,
1999), an assignation 211 years before the systematization of
botanical binomials Linnaeus in his Species Plantarum (Linnaeus,
1753). Lamarck subsequently suggested Cannabis indica, a more
diminutive intoxicating Indian plant from India, as a separate
species (Lamarck, 1783). The issue has remained unresolved
in the subsequent centuries with two opposing philosophies.
Ernest Small has championed the single species concept (Small
and Cronquist, 1976). Polytypic treatments of Cannabis also
gained adherents (Schultes et al., 1974;Anderson, 1980)
on morphological criteria suggesting separation of Cannabis
sativa L. Cannabis indica Lam. and Cannabis ruderalis Jan.,
a scheme supported by systematic chemotaxonomy. Principal
component analysis (PCA) of 157 Cannabis accessions from
around the world assessed allozyme frequencies at 17 gene
loci suggested a split (Hillig, 2005b). “Sativa” gene pools from
eastern European ruderal samples were linked to narrow-
leaflet European and Central Asian fiber and seed plants,
while an “indicagrouping encompassed Far Eastern seed
and fiber plants and drug plants with broad-leaflets from
most of the rest of the world, along with wild accessions
from the Indian subcontinent. Central Asian roadside samples
(Cannabis ruderalis) were thought to represent a third group.
Gas chromatography (GC) and starch-gel electrophoresis studies
also suggested species separation of sativa and indica (Hillig and
Mahlberg, 2004).
Agronomic factors in 69 samples suggested inclusion of
eastern hemp and drug plants in Cannabis indica (Hillig,
2005a), a division supported by fragment length polymorphisms
(Datwyler and Weiblen, 2006).
More recently, PCA seemed to point to terpenoid content
as the most convincing distinguishing chemotaxonomic markers
between putative sativa and indica species (Elzinga et al., 2015).
Similarly, PCA was felt to separate drug Cannabis from hemp
(Sawler et al., 2015). A recent study demonstrated demarcation
of Cannabis drug from hemp accessions via genotyping of
13 microsatellite loci across the genome, not merely genes
affecting cannabinoid or fiber production (Dufresnes et al.,
2017). Professor Giovanni Appendino has reported the presence
of the cis-19-THC stereo-isomer only in the hemp accessions
(Giovanni Appendino, personal communication). However,
these distinctions may well pass by the wayside given the current
trend to crossbreed hemp with drug cultivars to avoid legislative
restrictions on THC content.
The Cannabis species controversy, Cannabis sativa vs. indica
vs. afghanica, has continued unabated to the current day
with impassioned arguments advanced by the protagonists
(Clarke and Merlin, 2013, 2016;Small, 2015;McPartland
and Guy, 2017;Small, 2017). This author, having been
on every side of the issue at one time or another, has
chosen to eschew the irreconcilable taxonomic debate as
an unnecessary distraction (Piomelli and Russo, 2016), and
rather emphasize that only biochemical and pharmacological
distinctions between Cannabis accessions are relevant. In
his recent seminal review, McPartland agreed, “Categorizing
Cannabis as either ‘Sativa’ and ‘Indica’ has become an exercise in
futility. Ubiquitous interbreeding and hybridization renders their
distinction meaningless.” (McPartland, 2018) (p. 210).
An additional non-sensical nomenclature controversy
pertains in common parlance to Cannabis “strains,” an
appellation that is appropriate to bacteria and viruses, but
not plants (Bailey and Bailey, 1976;Usher, 1996;Brickell et al.,
2009), especially so with Cannabis where the chemical variety,
abbreviated “chemovar” is the most appropriate appellation
(Lewis et al., 2018).
2011 was a landmark year for Cannabis genomics, as Medical
Genomics and Nimbus Informatics issued an online report on
the complete 400 million base-pair genomic sequence, which was
shortly joined by a draft genome and transcriptome (van Bakel
et al., 2011).
This development sparked prominent publicity and
controversy as to what it might portend. Whereas, the human
genome was analyzed some 20 years earlier, the implications for
Cannabis were subject to great speculation.
The news catalyzed a flurry of new research, but considerable
progress had already been achieved in applied Cannabis genetics.
The identification and synthesis of 19-tetrahydrocannabinol
(THC) was accomplished in Israel 1964 (Gaoni and Mechoulam,
1964), but it was not until much later before successful cloning
of its biosynthetic enzyme, tetrahydrocannabinolic acid synthase
(THCA synthase) (Sirikantaramas et al., 2004;Figure 1). Enzyme
crystallization followed (Shoyama et al., 2005). Cannabidiolic
acid synthase, which catalyzes cannabidiolic acid (CBDA), the
precursor of cannabidiol (CBD), had been previously identified
and produced in pure form (Taura et al., 1996;Figure 1).
These developments stimulated additional findings, including the
archeological phytochemical discovery of THCA synthase in a
2700 year old Cannabis cache from a tomb in Central Asia along
with two previously unreported single nucleotide polymorphisms
(SNPs) in the enzyme’s gene sequence (Russo et al., 2008).
By 2011, the enzymes for the production of the major
phytocannabinoids had been identified. Similarly, selective
advanced Mendelian breeding yielded Cannabis varieties rich
in specific single components. Thus, high-THC and high-
CBD plants were produced for pharmaceutical development
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FIGURE 1 | Biosynthetic pathways and enzymes (red) of Cannabis sativa, indication the natural species Helichrysum umbraculigerum, and alternative species
(in color) that have been genetically modified to produce subsequent products [redrawn and updated from (Russo, 2011) using ACD/ChemSketch 2017.2.1].
(de Meijer et al., 2003;de Meijer, 2004), with analogous breeding
of high-cannabigerol (CBG) (de Meijer and Hammond, 2005)
and cannabichromene (CBC) lines (de Meijer et al., 2009a). The
selective breeding also extended to propyl phytocannabinoid
analogs, tetrahydrocannabivarin (THCV), cannabidivarin
(CBDV), cannabigerivarin (CBGV), and cannabichromivarin
(CBCV) (de Meijer, 2004). The availability of plants with high
titers of these “minor cannabinoids” portend interesting new
pharmaceutical applications (Russo, 2011;Russo and Marcu,
Access to the Cannabis genome might simplify production
of THC-knockout plants via CRISPR technology (clustered
regularly-interspaced short palindromic repeats). While this
could be attractive for industrial hemp breeding, a prior
generation of plant husbandry has already yielded hemp cultivars
that easily fulfill international restrictions that require 0.1% or
less THC content (Wirtshafter, 1997;McPartland et al., 2000;
Small and Marcus, 2003). In fact, cannabinoid-free Cannabis
with no functional cannabigerolic acid synthase (Figure 1) has
also been produced conventionally (de Meijer et al., 2009b).
Thus, it remains unclear that genetic engineering of Cannabis
is even necessary for this plant whose incredible plasticity
already displays bountiful biochemical diversity. Introduction
of genetically modified organism (GMO) Cannabis would
incite considerable controversy among certain segments of the
population, and likely provoke a flurry of legal entanglements
over patent and breeding rights.
One may easily imagine a variety of additional science fiction
scenarios. In the 1990s an Internet hoax spread the rumor that
an apocryphal Professor Nanofsky had introduced genes for
THC production into oranges (Citrus x. sinensis (L.) Osbeck).
Although this could be technologically achievable, such an effort
would be no more than a laboratory carnival act in light of
the prodigious cannabinoid production from Cannabis itself.
A stealthy peppermint chemovar (Mentha xpiperita Lamiaceae)
sporting illicit phytocannabinoids in the glandular trichomes of
its leaves might be more logical choice for such underground
subversive daydreams and send rhizomes and runners along
watercourses worldwide.
Prior claims of production of cannabidiol from hops
(Humulus lupulus L. Cannabaceae) and flax (Linum
usitatissimum L. Linaceae) are unsubstantiated, but
cannabigerolic acid and cannabigerol were detected in
South African Helichrysum umbraculigerum Less. Asteraceae
(Bohlmann and Hoffmann, 1979;Appendino et al., 2015;Russo,
2016;Figure 1), but without reference to its concentration.
This claim was confirmed recently with trace amounts observed
from dried samples of aerial parts (Mark Lewis, personal
Because the complexity of purely de novo biochemical
synthesis of cannabinoids has been deemed non-cost effective
(Carvalho et al., 2017), alternative microbial hosts have been
suggested (Zirpel et al., 2017). In 2004, cDNA cloning of THCA
synthase was achieved, allowing conversion of cannabigerolic
acid (CBGA) to THC (Sirikantaramas et al., 2004), and an 8%
THCA production in tobacco hairy roots (Nicotiana tabacum
cv.Xanthi Solanaceae) was demonstrated on CBGA feeding
(Figure 1). The enzyme was also expressed in the insect,
Spodoptera frugiperda (J.E. Smith) Noctuidae (fall armyworm)
via a recombinant baculovirus. Subsequently, this research group
turned to yeasts, Pichia pastoris (now Komagataella phaffii Phaff
Saccharomycetaceae) (Taura et al., 2007;Figure 1), and achieved
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a CBGA to THCA conversion of 98% over 24 h, with yield of
32.6 mg/L of medium. A recombinant form of THCA synthase
proved 4.5X more efficient than in Cannabis and 12X that in
S. frugiperda. This process was subsequently optimized with a
64.5-fold improvement in activity (Zirpel et al., 2018), with a
reported production in K. phaffii of 3.05 g/L of THCA after 8 h of
incubation at 37C. A simple calculation provides that this yield
could also be achieved from extraction of just 15 g of 20% THCA
herbal Cannabis.
Cannabis terpenoid production is similarly possible in
alternative hosts. Saccharomyces cerevisiae Meyen ex E.C. Hansen
Saccharomycetaceae mutants deficient in farnesyl diphosphate
synthase enzyme accumulate geranyl pyrophosphate instead,
which is shunted into the production of medically useful
terpenoid, linalool (Oswald et al., 2007;Figure 1). Similarly,
other researchers have harnessed the biosynthetic capabilities of
mitochondria in S. cerevisiae to increase farnesyl diphosphate
production of sesquiterpenoids (Farhi et al., 2011), although not
ones common to Cannabis.
At present, the existing Cannabis genomic sequences are
not fully annotated. Consequently, applied foreknowledge and
detective work will be necessary to acquire practical data on
genetic function in Cannabis. The greatest potential in such
investigation will lie in the realm of epigenetics, underlying
hereditable changes in gene expression or phenotype of the
plant. The most salient deficiency is a lack of knowledge
regarding regulation of cannabinoid production. Understanding
the biosynthetic pathways and regulation of terpene synthases
producing the Cannabis terpenoids has barely been initiated
(Booth et al., 2017) and remain ripe targets of additional research
(Russo, 2011).
An additional problem in Cannabis husbandry remains a
dearth of voucher specimens (which are prohibited by the US
Drug Enforcement Administration without Schedule I license)
and formal deposits of chemovar accessions in seed and
tissue repositories. The latter has been accomplished by GW
Pharmaceuticals, and independently by NaPro Research (Lewis
et al., 2018) in the National Collection of Industrial, Food and
Marine Bacteria (NCIMB) in Scotland. Many private companies
have eschewed sharing germplasm due to legal restrictions and
fear of loss of intellectual property.
In 1998, Professors Raphael Mechoulam and Shimon Ben-Shabat
posited that the endocannabinoid system demonstrated an
“entourage effect” in which a variety of “inactive” metabolites
and closely related molecules markedly increased the activity
of the primary endogenous cannabinoids, anandamide and
2-arachidonoylglycerol (Ben-Shabat et al., 1998). They also
postulated that this helped to explain how botanical drugs
were often more efficacious than their isolated components
(Mechoulam and Ben-Shabat, 1999). Although the single
molecule synthesis remains the dominant model for
pharmaceutical development (Bonn-Miller et al., 2018), the
concept of botanical synergy has been amply demonstrated
contemporaneously, invoking the pharmacological contributions
of “minor cannabinoids” and Cannabis terpenoids to the plant’s
overall pharmacological effect (McPartland and Pruitt, 1999;
McPartland and Mediavilla, 2001;McPartland and Russo, 2001,
2014;Russo and McPartland, 2003;Wilkinson et al., 2003;Russo,
2011). Several pertinent examples of the entourage effect in
Cannabis are illustrative:
In a randomized controlled trial of oromucosal Cannabis-
based extracts in patients with intractable pain despite optimized
opioid treatment, a THC-predominant extract failed to
demarcate favorably from placebo, whereas a whole plant extract
(nabiximols, vide infra) with both THC and cannabidiol (CBD)
proved statistically significantly better than both (Johnson et al.,
2010), the only salient difference being the presence of CBD in
the latter.
In animal studies of analgesia, pure CBD produces a
biphasic dose-response curve such that smaller doses reduce
pain responses until a peak is reached, after which further
increases in dose are ineffective. Interestingly, the application of
a full spectrum Cannabis extract with equivalent doses of CBD
eliminates the biphasic response in favor of a linear dose-response
curve such that the botanical extract is analgesic at any dose with
no observed ceiling effect (Gallily et al., 2014).
A recent study of several human breast cancer cell lines in
culture and implanted tumors demonstrated superiority of a
Cannabis extract treatment to pure THC, seemingly attributable
in the former to the presence of small concentrations of
cannabigerol (CBG) and tetrahydrocannabinolic acid (THCA)
(Blasco-Benito et al., 2018).
Anticonvulsant effects of cannabidiol were noted in animals
in the 1970s with the first human trials in 1980 (Cunha et al.,
1980). A recent experiment in mice with seizures induced by
pentylenetetrazole employed five different Cannabis extracts with
equal CBD concentrations (Berman et al., 2018). Although all
the extracts showed benefits compared to untreated controls,
salient differences were observed in biochemical profiles of
non-CBD cannabinoids, which, in turn, led to significant
differences in numbers of mice developing tonic-clonic seizures
(21.5–66.7%) and survival rates (85–100%), highlighting the
relevance of these “minor” components. This study highlights
the necessity of standardization in pharmaceutical development,
and although it could be construed to support the single
molecule therapeutic model (Bonn-Miller et al., 2018), it requires
emphasis that complex botanicals can meet American FDA
standards (Food and Drug Administration, 2015). Specifically,
two Cannabis-based drugs have attained regulatory approval,
Sativex R
(nabiximols, US Adopted Name) in 30 countries, and
Epidiolex R
in the United States.
The question then arises: Can a Cannabis preparation or single
molecule be too pure, thus reducing synergistic potential? Recent
data support this as a distinct possibility. Anecdotal information
from clinicians utilizing high-CBD Cannabis extracts to treat
severe epilepsy, such as Dravet and Lennox-Gastaut syndromes,
showed that their patients demonstrated notable improvement
in seizure frequency (Goldstein, 2016;Russo, 2017;Sulak et al.,
2017) with doses far lower than those reported in formal
clinical trials of Epidiolex, a 97% pure CBD preparation with
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Russo Conventional Breeding of Cannabis
FIGURE 2 | PhytoFactsTM depiction of cannabinoid and terpenoid content of CaryodiolTM, aka “Kashmir Blue,” a Type III, cannabidiol-, and
caryophyllene-predominant chemovar. See (Lewis et al., 2018) for details of PhytoFacts and conventional breeding methodology. Copyright© 2016 BHC Group,
LLC. All rights reserved. Any unauthorized use of this document or the images or marks above may violate copyright, trademark, and other applicable laws.
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Russo Conventional Breeding of Cannabis
THC removed (Devinsky et al., 2016, 2017, 2018;Thiele et al.,
2018). This observation was recently subjected to meta-analysis
of 11 studies with 670 patients in aggregate (Pamplona et al.,
2018). Those results showed that 71% of patients improved
with CBD-predominant Cannabis extracts vs. 36% on purified
CBD (p<0.0001). The response rate at 50% improvement
in seizure frequency was not statistically different in the two
groups and both groups achieved seizure-free status in about
10% of patients. However, the mean daily doses were markedly
divergent in the groups: 27.1 mg/kg/d for purified CBD vs.
only 6.1 mg/kg/d. for CBD-rich Cannabis extracts, a dose only
22.5% of that for CBD alone. Furthermore, the incidence of
mild and severe adverse events was demonstrably higher in
purified CBD vs. high-CBD extract patients (p<0.0001), a result
that the authors attributed to the lower dose utilized, which
was achieved in their opinion by the synergistic contributions
of other entourage compounds. Such observations support the
hypothesis of greater efficacy for Cannabis extracts combining
multiple anticonvulsant components, such as CBD, THC, THCA,
THCV, CBDV, linalool, and even caryophyllene (Lewis et al.,
These studies and others provide a firm foundation for
Cannabis synergy, and support for botanical drug development
vs. that of single components (Bonn-Miller et al., 2018),
or production via fermentation methods in yeast or other
micro-organisms. An example of the power of conventional
selective breeding is illustrated (Figure 2), in the form of
a Cannabis chemovar named CaryodiolTM for its enhanced
caryophyllene content (0.83%) as a CB2agonist, along with
highly favorable Type III THC:CBD ratio of 1:39.4. Such
a preparation portends to be applicable to treatment of
numerous clinical conditions including: pain, inflammation,
fibrotic disorders, addiction, anxiety, depression, autoimmune
diseases, dermatological conditions and cancer (Pacher and
Mechoulam, 2011;Russo, 2011;Xi et al., 2011;Russo and Marcu,
2017;Lewis et al., 2018). Producing such a combination from
microbial sources might require combinations of cannabinoids
from multiple yeast species and, as a result, it would represent
a combination product subject to a difficult regulatory path
compared to Cannabis preparations from extracts of a single
species (e.g., nabiximols) that has been accepted as a unitary
formulation in 30 countries across the globe (Food and Drug
Administration, 2015).
This article has briefly outlined recently technological
attempts to “reinvent the phytocannabinoid wheel.” Cogent
arguments would support that it can be done, but should it be
done? The data supporting the existence of Cannabis synergy
and the astounding plasticity of the Cannabis genome suggests a
reality that obviates the need for alternative hosts, or even genetic
engineering of Cannabis sativa, thus proving that, “The plant
does it better.”
The author confirms being the sole contributor of this work and
has approved it for publication.
The author appreciated the assistance of the staff of the University
of Montana Inter-Library Loan Office of Mansfield Library for
provision of research materials.
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Conflict of Interest Statement: I am Director of Research for the International
Cannabis and Cannabinoids Institute. We serve clients engaged in cannabis
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Frontiers in Plant Science | 8January 2019 | Volume 9 | Article 1969
... In addition to phytocannabinoids, the Cannabis genus also produces non-cannabinoid compounds, including terpenoids (12), which may also be medically useful (13)(14)(15). The pharmacological contributions of minor cannabinoids and non-cannabinoid compounds have been highlighted and popularized under the term "entourage effect" (16,17). Through synergistic mechanisms between different cannabis chemical components [over 500 have been identified to date (18,19)], "full-spectrum" cannabis extracts may have different and potentially superior effects to those observed with purified major cannabinoids (16,17). ...
... The pharmacological contributions of minor cannabinoids and non-cannabinoid compounds have been highlighted and popularized under the term "entourage effect" (16,17). Through synergistic mechanisms between different cannabis chemical components [over 500 have been identified to date (18,19)], "full-spectrum" cannabis extracts may have different and potentially superior effects to those observed with purified major cannabinoids (16,17). ...
... There is great genetic variability between cannabis plants (20). Each chemical variety of cannabis (chemovar or chemotype) has a specific profile of various cannabinoid and non-cannabinoid compounds depending on its genetic make-up (16,21). This profile can change depending on pre-and post-harvest environmental factors (22)(23)(24)(25). ...
... However, assuming that the effects of cannabis are solely attributable to the most abundant phytocannabinoid Δ9-tetrahydrocannabinol (THC) (Cluny et al. 2015) and/or are only mediated through CB1 may be reductive. Cannabis exposes users to a large number of phytocannabinoids, as well as to non-cannabinoid molecules, such as terpenoids (Russo 2018). The most abundant phytocannabinoids in cannabis are THC, cannabidiol (CBD), and Δ9-tetrahydrocannabivarin (THCV). ...
... Non-cannabinoid cannabis compounds such as limonene, β-caryophyllene, and other terpenes may also play a role in the corpulence lowering effect of cannabis use (Hashiesh et al. 2021;Jing et al. 2013;Scandiffio et al. 2020). Finally, a synergetic effect, resulting from the interactions between those compounds, cannot be excluded (Russo 2018). ...
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Background: Patients with chronic hepatitis C virus (HCV) infection are at greater risk of developing metabolic disorders. Obesity is a major risk factor for these disorders, and therefore, managing body weight is crucial. Cannabis use, which is common in these patients, has been associated with lower corpulence in various populations. However, this relationship has not yet been studied in persons with chronic HCV infection. Methods: Using baseline data from the French ANRS CO22 Hepather cohort, we used binary logistic and multinomial logistic regression models to test for an inverse relationship between cannabis use (former/current) and (i) central obesity (i.e., large waist circumference) and (ii) overweight and obesity (i.e., elevated body mass index (BMI)) in patients from the cohort who had chronic HCV infection. We also tested for relationships between cannabis use and both waist circumference and BMI as continuous variables, using linear regression models. Results: Among the 6348 participants in the study population, 55% had central obesity, 13.7% had obesity according to their BMI, and 12.4% were current cannabis users. After multivariable adjustment, current cannabis use was associated with lower risk of central obesity (adjusted odds ratio, aOR [95% confidence interval, CI]: 0.45 [0.37-0.55]), BMI-based obesity (adjusted relative risk ratio (aRRR) [95% CI]: 0.27 [0.19-0.39]), and overweight (aRRR [95% CI]: 0.47 [0.38-0.59]). This was also true for former use, but to a lesser extent. Former and current cannabis use were inversely associated with waist circumference and BMI. Conclusions: We found that former and, to a greater extent, current cannabis use were consistently associated with smaller waist circumference, lower BMI, and lower risks of overweight, obesity, and central obesity in patients with chronic HCV infection. Longitudinal studies are needed to confirm these relationships and to assess the effect of cannabis use on corpulence and liver outcomes after HCV cure. Trial registration: identifier: NCT01953458 .
... For example, synthetic THC alone, in manufactured products such as 'Marinol,' may produce unpleasant effects [53,54]. Whether or not distinct ratios of cannabinoids and terpenes are able to consistently yield different subjective effects or therapeutic outcomes is unknown, and a topic of debate [55]. ...
... Medical Cannabis patients report an even wider array of conditions they believe Cannabis is efficacious for, including mental health outcomes [82]. It has also been hypothesized that distinct chemotypes of Cannabis, each with different ratios of cannabinoids and terpenes, may offer distinct medical benefits and psychoactive effects [55,83]. This hypothesized "entourage effect" has been difficult to confirm experimentally due to onerous regulations that make it challenging to execute in vivo studies with controlled administration of the myriad compounds found in Cannabis. ...
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The legal status of Cannabis is changing, fueling an increasing diversity of Cannabis -derived products. Because Cannabis contains dozens of chemical compounds with potential psychoactive or medicinal effects, understanding this phytochemical diversity is crucial. The legal Cannabis industry heavily markets products to consumers based on widely used labeling systems purported to predict the effects of different “strains.” We analyzed the cannabinoid and terpene content of commercial Cannabis samples across six US states, finding distinct chemical phenotypes (chemotypes) which are reliably present. By comparing the observed phytochemical diversity to the commercial labels commonly attached to Cannabis -derived product samples, we show that commercial labels do not consistently align with the observed chemical diversity. However, certain labels do show a biased association with specific chemotypes. These results have implications for the classification of commercial Cannabis , design of animal and human research, and regulation of consumer marketing—areas which today are often divorced from the chemical reality of the Cannabis -derived material they wish to represent.
... Similarly to psychedelics, some cannabis chemovars, specially THCpredominant profiles, may induce transformative experiences [255] and may substitute more harmful drugs [299] , and although studies with isolated cannabinoids are advancing, higher quality studies using commercially-available, legally-obtained cannabis are necessary to understand this effects. As van Wel et al. [254] demonstrated in their study, experimenting with specifically selected chemovars may be an alternative to safely conduct high quality assessment and clinical studies as our understanding of the biochemical properties of cannabis advances [300] and we begin to tackle obstacles that isolated cannabinoids and the harms that uncontrolled high potency cannabis may present [ 301 , 302 ]. ...
Full-text available
Substance use disorder (SUD) is a global public health concern that affects millions of people worldwide. Considering current research, addiction has been noted as the last stage of a chronic disease that may impair brain reward circuit responses and affects personal and social life. Treatments for SUD face challenges including availability and limited pharmacological response, often resulting in low retention of patients. A growing number of studies from the 'psychedelic renaissance' have highlighted the therapeutic potential of psychedelics for several psychiatric disorders, including SUD. In this non-systematic review we discuss past and current clinical and observational studies with classic (LSD, DMT, psilocybin and mescaline) and non-classic (ibogaine, ketamine, MDMA, salvinorin A and THC) psychedelics for the treatment of SUD published until December 2021. Although results are still inconclusive for LSD, DMT, mescaline, MDMA and Salvinorin A, in general, the literature presents moderate evidence on the controlled use of psilocybin and ketamine for Alcohol Use Disorder, ketamine for management of opiate and alcohol withdrawal, and THC preparations for reducing withdrawal symptoms in Cannabis and possibly in Opioid Use Disorder. Importantly, studies suggest that psychedelics should be more effective when employed as an adjunct therapy. Extensive research is warranted to further elucidate the role of psychedelics in the treatment of SUD.
... Journal of Cannabis Research (2022) 4:30 concentrations (Turkanis et al. 1991) and linalool inhibits Na V channels at high micromolar concentrations (Leal-Cardoso et al. 2010). Mounting evidence supports synergistic interactions between the many molecular constituents of cannabis-derived products, consistent with the notion of an "entourage effect" (Russo 2019;Russo 2011). Future studies could explore whether an "entourage effect" or synergism between components within the NP contributes to its inhibition of Na V channel function. ...
Background Purified cannabidiol (CBD), a non-psychoactive phytocannabinoid, has gained regulatory approval to treat intractable childhood epilepsies. Despite this, artisanal and commercial CBD-dominant hemp-based products continue to be used by epilepsy patients. Notably, the CBD doses used in these latter products are much lower than that found to be effective in reducing seizures in clinical trials with purified CBD. This might be because these CBD-dominant hemp products contain other bioactive compounds, including phytocannabinoids and terpenes, which may exert unique effects on epilepsy-relevant drug targets. Voltage-gated sodium (Na V ) channels are vital for initiation of neuronal action potential propagation and genetic mutations in these channels result in epilepsy phenotypes. Recent studies suggest that Na V channels are inhibited by purified CBD. However, the effect of cannabis-based products on the function of Na V channels is unknown. Methods Using automated-planar patch-clamp technology, we profile a hemp-derived nutraceutical product (NP) against human Na V 1.1–Na V 1.8 expressed in mammalian cells to examine effects on the biophysical properties of channel conductance, steady-state fast inactivation and recovery from fast inactivation. Results NP modifies peak current amplitude of the Na V 1.1–Na V 1.7 subtypes and has variable effects on the biophysical properties for all channel subtypes tested. NP potently inhibits Na V channels revealing half-maximal inhibitory concentration (IC 50 ) values of between 1.6 and 4.2 μg NP/mL. Purified CBD inhibits Na V 1.1, Na V 1.2, Na V 1.6 and Na V 1.7 to reveal IC 50 values in the micromolar range. The CBD content of the product equates to IC 50 values (93–245 nM), which are at least an order of magnitude lower than purified CBD. Unlike NP, hemp seed oil vehicle alone did not inhibit Na V channels, suggesting that the inhibitory effects of NP are independent of hemp seed oil. Conclusions This CBD-dominant NP potently inhibits Na V channels. Future study of the individual elements of NP, including phytocannabinoids and terpenes, may reveal a potent individual component or that its components interact to modulate Na V channels.
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Background: Cannabis-based formulations are now widely used by patients with neurological and psychiatric problems but no studies have been published on the clinical utility of CBD/CBG enriched extracts for parkinsonism's symptoms. Objectives: To describe preliminary clinical data collection of PD and DLB patients under CBD/CBG medical prescription. Methods: Review of electronic records of 14 PD and 5 DLB patients. Four extracts were available 1) CBD broad spectrum (100 mg/ mL) 2) CBD/CBG broad spectrum (100 mg/mL 2:1) (3) CBD/CBG (2:1) + THC0.3% full spectrum (100mg/mL) 4) CBD+THC0.3% full spectrum (100 mg/mL). All the patients received authorization from ANVISA (Brazil) to import the formulations for medical use. Outcomes of each unmet need (UMN) were tabulated and graded. Results: Demographics: PD N = 14 (10 male). DLB: N = 5 (3 male). Mean age: PD: 76.2 yrs. (46-94). DLB: 82.2 yrs. (83-92). Disease duration: PD (6.57 yrs.), DLB (4.2 yrs.); PD H/Y stage (3); PD levodopa dose: 490 mg (150-900). Mean daily doses: PD CBD: 65.17 mg (8.33-125 mg), CBG: 22,50 mg (4.16-50 mg), THC: 2,32 mg (0,75-4,5 mg). DLB CBD: 52 mg (5-100 mg). CBG: 8,75 mg (2,5-15 mg), THC: 0,225 mg. Positive results were seen for RBD, insomnia, anxiety, and pain. All pain responders were on CBG and/or THC formulations. Hallucinations were also attenuated in both patient groups. Safety and tolerability were favorable in this small sample. Conclusions: Future clinical trials in Parkinson's disease and DLB with cannabinoids should focus on their potential benefit for associated anxiety, and pain. The potential anti-psychotic effects of CBD and CBD/CBG should also be further evaluated in a phase 2a clinical trial. Abstract Citation: Flávio Henrique de Rezende Costa., et al. "Parkinson's Disease and Dementia with Lewy Bodies, Patients Under Treatment with Standardized
Background Autism spectrum disorder is specifically approved for medical cannabis consumption in 20 U.S. states, the District of Columbia, and the Territory of Puerto Rico. Despite increased access, there is limited knowledge about who consumes medical cannabis, what they consume, and perceived effectiveness. We addressed these gaps by conducting a natural history study of medical cannabis consumption. Method Children and their families engaged with a large pediatric care system were recruited to complete a telephonic study regarding their medical cannabis consumption. All children had to be consuming approved medical cannabis products issued from a state that had legalized medical cannabis for the treatment of ASD or related behaviors (irritability, hyperactivity, anxiety) (N = 89). Results The sample’s ‘level of support’ and gender-ratio reflected the general autism population (~33 % requiring ‘Very Substantial Support’ and ~80 % male). The most common treatment targets were ASD behaviors (repetitive behaviors) and irritability. More children consumed compounds with high cannabidiol (CBD) and low or no tetrahydrocannabinol (THC). While dose did not affect overall perceived effectiveness, compounds with high-levels of CBD and low-levels of THC (CBD-dominant) were perceived as more effective than CBD-only. Conclusions This “real world” study revealed that medical cannabis is being used to treat a wide range of behaviors. Our study also suggests that children consume CBD-rich products, and the effectiveness of CBD-dosing may be tied to the inclusion of THC in the compound. Future research should evaluate optimal dosing with a particular focus on the CBD-to-THC ratio.
Cannabis has been well known for centuries due to its medicinal properties. In recent decades, the inclination of researchers towards its important phytoconstituents as a potential therapeutic alternative has been propounded due to the discovery of its major active constituent, i.e., Δ9-tetrahydrocannabinol (Δ9-THC). Besides this, the presence of other phytoproducts, including cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc., also contribute towards its medicinal importance. Interestingly, due to the effectiveness of cannabis against various pathological conditions, its use for medicinal purposes has been revolutionized worldwide. Despite these facts, it has become obligatory to explore synergistic interactions and mode of action of its phytoconstituents involving various biological pathways. Current advancements have allowed medical practitioners to better understand cannabis-derived products as a pharmacological choice in several conditions, including pain treatment, stress, anxiety, neurodegenerative disorders, and cancer. However, there exists a lacuna in the literature regarding its beneficial doses. Since medicinal exploration and the legalization of cannabis depend upon various factors, the present review deals with the important phytocannabinoids, their biogenesis, types of drugs obtained, mode of action, therapeutic implications, and new approaches for supporting this plant as a critical therapeutic agent for pharmaceutical drugs. Overall, this may provide an insight into the role of cannabis as a potent candidate for future drug discovery and generate efficient products for human welfare.
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Cannabis: Evolution and Ethnobotany is a comprehensive, interdisciplinary exploration of the natural origins and early evolution of this famous plant, highlighting its historic role in the development of human societies. Cannabis has long been prized for the strong and durable fiber in its stalks, its edible and oil-rich seeds, and the psychoactive and medicinal compounds produced by its female flowers. The culturally valuable and often irreplaceable goods derived from cannabis deeply influenced the commercial, medical, ritual, and religious practices of cultures throughout the ages, and human desire for these commodities directed the evolution of the plant toward its contemporary varieties. As interest in cannabis grows and public debate over its many uses rises, this book will help us understand why humanity continues to rely on this plant and adapts it to suit our needs.
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New concepts are reviewed in Cannabis systematics, including phylogenetics and nomenclature. The family Cannabaceae now includes Cannabis, Humulus, and eight genera formerly in the Celtidaceae. Grouping Cannabis, Humulus, and Celtis actually goes back 250 years. Print fossil of the extinct genus Dorofeevia (=Humularia) reveals that Cannabis lost a sibling perhaps 20 million years ago (mya). Cannabis print fossils are rare (n=3 worldwide), making it difficult to determine when and where she evolved. A molecular clock analysis with chloroplast DNA (cpDNA) suggests Cannabis and Humulus diverged 27.8 mya. Microfossil (fossil pollen) data point to a center of origin in the northeastern Tibetan Plateau. Fossil pollen indicates that Cannabis dispersed to Europe by 1.8–1.2 mya. Mapping pollen distribution over time suggests that European Cannabis went through repeated genetic bottlenecks, when the population shrank during range contractions. Genetic drift in this population likely initiated allopatric differences between European Cannabis sativa (cannabidiol [CBD]>Δ⁹-tetrahydrocannabinol [THC]) and Asian Cannabis indica (THC>CBD). DNA barcode analysis supports the separation of these taxa at a subspecies level, and recognizing the formal nomenclature of C. sativa subsp. sativa and C. sativa subsp. indica. Herbarium specimens reveal that field botanists during the 18th–20th centuries applied these names to their collections rather capriciously. This may have skewed taxonomic determinations by Vavilov and Schultes, ultimately giving rise to today's vernacular taxonomy of “Sativa” and “Indica,” which totally misaligns with formal C. sativa and C. indica. Ubiquitous interbreeding and hybridization of “Sativa” and “Indica” has rendered their distinctions almost meaningless.
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Most clinical studies of Cannabis today focus on the contents of two phytocannabinoids: (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), regardless of the fact that the plant contains over 100 additional phytocannabinoids whose therapeutic effects and interplay have not yet been fully elucidated. This narrow view of a complex Cannabis plant is insufficient to comprehend the medicinal and pharmacological effects of the whole plant. In this study we suggest a new ESI-LC/MS/MS approach to identify phytocannabinoids from 10 different subclasses, and comprehensively profile the identified compounds in diverse medical Cannabis plants. Overall, 94 phytocannabinoids were identified and used for profiling 36 of the most commonly used Cannabis plants prescribed to patients in Israel. In order to demonstrate the importance of comprehensive phytocannabinoid analysis before and throughout medical Cannabis clinical trials, treatments, or experiments, we evaluated the anticonvulsant effects of several equally high-CBD Cannabis extracts (50% w/w). We found that despite the similarity in CBD contents, not all Cannabis extracts produced the same effects. This study’s approach for phytocannabinoid profiling can enable researchers and physicians to analyze the effects of specific Cannabis compositions and is therefore critical when performing biological, medical and pharmacological-based research using Cannabis.
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This meta-analysis paper describes the analysis of observational clinical studies on the treatment of refractory epilepsy with cannabidiol (CBD)-based products. Beyond attempting to establish the safety and efficacy of such products, we also investigated if there is enough evidence to assume any difference in efficacy between CBD-rich extracts compared to purified CBD products. The systematic search took place in February/2017 and updated in December/2017 using the keywords “epilepsy” or “Dravet” or “Lennox-Gastaut” or “CDKL5” combined with “Cannabis,” “cannabinoid,” “cannabidiol,” or “CBD” resulting in 199 papers. The qualitative assessment resulted in 11 valid references, with an average impact factor of 8.1 (ranging from 1.4 to 47.8). The categorical data of a total of 670 patients were analyzed by Fischer test. The average daily dose ranged between 1 and 50 mg/kg, with treatment length from 3 to 12 months (mean 6.2 months). Two thirds of patients reported improvement in the frequency of seizures (399/622, 64%). There were more reports of improvement from patients treated with CBD-rich extracts (318/447, 71%) than patients treated with purified CBD (81/223, 36%), with statistical significance (p < 0.0001). Nevertheless, when the standard clinical threshold of a “50% reduction or more in the frequency of seizures” was applied, only 39% of the individuals were considered “responders,” and there was no difference (p = 0.56) between treatments with CBD-rich extracts (97/255, 38%) and purified CBD (94/223, 42%). Patients treated with CBD-rich extracts reported lower average dose (6.1 mg/kg/day) than those using purified CBD (27.1 mg/kg/day). The reports of mild (109/285 vs. 291/346, p < 0.0001) and severe (23/285 vs. 77/346, p < 0.0001) adverse effects were more frequent in products containing purified CBD than in CBD-rich extracts. CBD-rich extracts seem to present a better therapeutic profile than purified CBD, at least in this population of patients with refractory epilepsy. The roots of this difference is likely due to synergistic effects of CBD with other phytocompounds (aka Entourage effect), but this remains to be confirmed in controlled clinical studies.
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Conventional wisdom states Cannabis sativa originated in Asia and its dispersal to Europe depended upon human transport. Various Neolithic or Bronze age groups have been named as pioneer cultivators. These theses were tested by examining fossil pollen studies (FPSs), obtained from the European Pollen Database. Many FPSs report Cannabis or Humulus (C/H) with collective names (e.g. Cannabis/Humulus or Cannabaceae). To dissect these aggregate data, we used ecological proxies to differentiate C/H pollen, as follows: unknown C/H pollen that appeared in a pollen assemblage suggestive of steppe (Poaceae, Artemisia, Chenopodiaceae) we interpreted as wild-type Cannabis. C/H pollen in a mesophytic forest assemblage (Alnus, Salix, Populus) we interpreted as Humulus. C/H pollen curves that upsurged and appeared de novo alongside crop pollen grains we interpreted as cultivated hemp. FPSs were mapped and compared to the territories of archaeological cultures. We analysed 479 FPSs from the Holocene/Late Glacial, plus 36 FPSs from older strata. The results showed C/H pollen consistent with wild-type C. sativa in steppe and dry tundra landscapes throughout Europe during the early Holocene, Late Glacial, and previous glaciations. During the warm and wet Holocene Climactic Optimum, forests replaced steppe, and Humulus dominated. Cannabis retreated to steppe refugia. C/H pollen consistent with cultivated hemp first appeared in the Pontic-Caspian steppe refugium. GIS mapping linked cultivation with the Copper age Varna/Gumelniţa culture, and the Bronze age Yamnaya and Terramara cultures. An Iron age steppe culture, the Scythians, likely introduced hemp cultivation to Celtic and Proto-Slavic cultures.
Accumulating evidence suggests that the endocannabinoid system is a promising target for the treatment of a variety of health conditions. Two paths of cannabinoid drug development have emerged. One approach is focused on developing medications that are directly derived from the cannabis plant. The other utilizes a single molecule approach whereby individual phytocannabinoids or novel cannabinoids with therapeutic potential are identified and synthesized for pharmaceutical development. This commentary discusses the unique challenges and merits of botanical vs single molecule cannabinoid drug development strategies, highlights how both can be impacted by legalization of cannabis via legislative processes, and also addresses regulatory and public health considerations that are important to consider as cannabinoid medicine advances as a discipline.
Breast cancer is the second leading cause of death among women. Although early diagnosis and development of new treatments have improved their prognosis, many patients present innate or acquired resistance to current therapies. New therapeutic approaches are therefore warranted for the management of this disease. Extensive preclinical research has demonstrated that cannabinoids, the active ingredients of Cannabis sativa, trigger antitumor responses in different models of cancer. Most of these studies have been conducted with pure compounds, mainly Δ9-tetrahydrocannabinol (THC). The cannabis plant, however, produces hundreds of other compounds with their own therapeutic potential and the capability to induce synergic responses when combined, the so-called "entourage effect". Here, we compared the antitumor efficacy of pure THC with that of a botanical drug preparation (BDP). The BDP was more potent than pure THC in producing antitumor responses in cell culture and animal models of ER+/PR+, HER2+ and triple-negative breast cancer. This increased potency was not due to the presence of the 5 most abundant terpenes in the preparation. While pure THC acted by activating cannabinoid CB2 receptors and generating reactive oxygen species, the BDP modulated different targets and mechanisms of action. The combination of cannabinoids with estrogen receptor- or HER2-targeted therapies (tamoxifen and lapatinib, respectively) or with cisplatin, produced additive antiproliferative responses in cell cultures. Combinations of these treatments in vivo showed no interactions, either positive or negative. Together, our results suggest that standardized cannabis drug preparations, rather than pure cannabinoids, could be considered as part of the therapeutic armamentarium to manage breast cancer.
Δ9-tetrahydrocannabinolic acid (THCA) is a secondary natural product from the plant Cannabis sativa L. with therapeutic indications like analgesics for cancer pain or reducing spasticity associated with multiple sclerosis. Here, we investigated the influence of the co-expression of 12 helper protein genes from Komagataella phaffii (formerly Pichia pastoris) on the functional expression of the Δ9-tetrahydrocannabinolic acid synthase (THCAS) heterologously expressed in K. phaffii by screening 21 clones of each transformation. Our findings substantiate the necessity of a suitable screening system when interfering with the secretory network of K. phaffii. We found that co-production of the chaperones CNE1p and Kar2p, the foldase PDI1p, the UPR-activator Hac1p as well as the FAD synthetase FAD1p enhanced THCAS activity levels within the K. phaffii cells. The strongest influence showed co-expression of Hac1s - increasing the volumetric THCAS activities 4.1-fold on average. We also combined co-production of Hac1p with the other beneficial helper proteins to further enhance THCAS activity levels. An optimized strain overexpressing Hac1s, FAD1 and CNE1 was isolated that showed 20-fold increased volumetric, intracellular THCAS activity compared to the starting strain. We used this strain for a whole cell bioconversion of cannabigerolic acid (CBGA) to THCA. After 8 h of incubation at 37 °C, the cells produced 3.05 g L-1THCA corresponding to 12.5 % gTHCAgCDW-1.