Glioblastoma multiforme (GBM) is the most lethal subtype of glioma. Cannabis sativa is
used for the treatment of various medical conditions. Around 150 phytocannabinoids have been identified in C. sativa, among them Δ‐9‐tetrahydrocannabinol (THC) and cannabidiol (CBD) that trigger GBM cell death. However, the optimal combinations of cannabis molecules for anti‐GBM activity are unknown. Chemical composition was determined using high‐performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC/MS). Cytotoxic activity was determined by XTT and lactate dehydrogenase (LDH) assays and apoptosis and cell cycle by fluorescence‐
activated cell sorting (FACS). F‐actin structures were observed by confocal microscopy,
gene expression by quantitative PCR, and cell migration and invasion by scratch and transwell assays, respectively. Fractions of a high‐THC cannabis strain extract had significant cytotoxic activity against GBM cell lines and glioma stem cells derived from tumor specimens. A standard mix (SM) of the active fractions F4 and F5 induced apoptosis and expression of endoplasmic reticulum (ER)‐stress associated‐genes. F4 and F5 inhibited cell migration and invasion, altered cell cytoskeletons, and inhibited colony formation in 2 and 3‐dimensional models. Combinations of cannabis compounds
exert cytotoxic, anti‐proliferative, and anti‐migratory effects and should be examined for
efficacy on GBM in pre‐clinical studies and clinical trials.

Cannabis sativa is widely used for medical purposes and has anti-inflammatory activity. This study intended to examine the anti-inflammatory activity of cannabis on immune response markers associated with coronavirus disease 2019 (COVID-19) inflammation. An extract fraction from C. sativa Arbel strain (FCBD) substantially reduced (dose dependently) interleukin (IL)-6 and -8 levels in an alveolar epithelial (A549) cell line. FCBD contained cannabidiol (CBD), cannabigerol (CBG) and tetrahydrocannabivarin (THCV), and multiple terpenes. Treatments with FCBD and a FCBD formulation using phytocannabinoid standards (FCBD:std) reduced IL-6, IL-8, C–C Motif Chemokine Ligands (CCLs) 2 and 7, and angiotensin I converting enzyme 2 (ACE2) expression in the A549 cell line. Treatment with FCBD induced macrophage (differentiated KG1 cell line) polarization and phagocytosis in vitro, and increased CD36 and type II receptor for the Fc region of IgG (FcγRII) expression. FCBD treatment also substantially increased IL-6 and IL-8 expression in macrophages. FCBD:std, while maintaining anti-inflammatory activity in alveolar epithelial cells, led to reduced phagocytosis and pro-inflammatory IL secretion in macrophages in comparison to FCBD. The phytocannabinoid formulation may show superior activity versus the cannabis-derived fraction for reduction of lung inflammation, yet there is a need of caution proposing cannabis as treatment for COVID-19.

Cannabis sativa produces hundreds of phytocannabinoids and terpenes. Mycosis
fungoides (MF) is the most common type of cutaneous T-cell lymphoma (CTCL),
characterized by patches, plaques and tumors. Sézary is a leukemic stage of CTCL
presenting with erythroderma and the presence of neoplastic Sézary T-cells in
peripheral blood. This study aimed to identify active compounds from whole cannabis
extracts and their synergistic mixtures, and to assess respective cytotoxic activity
against CTCL cells. Ethanol extracts of C. sativa were analyzed by high-performance
liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS).
Cytotoxic activity was determined using the XTT assay on My-La and HuT-78 cell lines
as well as peripheral blood lymphocytes from Sézary patients (SPBL). Annexin V assay
and fluorescence-activated cell sorting (FACS) were used to determine apoptosis
and cell cycle. RNA sequencing and quantitative PCR were used to determine gene
expression. Active cannabis compounds presenting high cytotoxic activity on My-La
and HuT-78 cell lines were identified in crude extract fractions designated S4 and S5,
and their synergistic mixture was specified. This mixture induced cell cycle arrest and
cell apoptosis; a relatively selective apoptosis was also recorded on the malignant
CD4+CD26- SPBL cells. Significant cytotoxic activity of the corresponding mixture of
pure phytocannabinoids further verified genuine interaction between S4 and S5. The
gene expression profile was distinct in My-La and HuT-78 cells treated with the S4
and S5 synergistic mixture. We suggest that specifying formulations of synergistic
active cannabis compounds and unraveling their modes of action may lead to new
cannabis-based therapies.

Highlights

• Cannabis has been used for millennia by humanity for social, ritual, and medical purposes. Humans bred and selected for cannabis strains based on their needs.

• Today, patients are treated by cannabis ‘strains’, without a clear definition of activity or known chemical content.

• Cannabis sativa strain taxonomy is based on the content of two phytocannabinoids only, Δ9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA).

• C. sativa produces hundreds of phytomolecules, including phytocannabinoids, terpenes, and flavonoids.

• Some cannabis phytomolecules interact, causing enhanced biological activity. Defining this phenomenon, known as the ‘entourage effect’, is one of the leading challenges in the field of research.

Cannabis sativa is widely used for medical purposes. However, to date, aroma, popular strain name or the content of two phytocannabinoids—Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are mostly considered for therapeuticactivity. This is despite the hundreds of compounds in this plant and their potential synergistic interactions inmixtures. New, specific and effective cannabis-based drugs must be developed to achieve adequate medical standards for the use of cannabis. To do this, the comprehensive molecular profile of cannabis-based drugs must be defined, and mixtures of compounds should be tested for superior therapeutic activity due to synergistic effects compared to individually isolated cannabis compounds. The biological pathways targeted by these new drugs should also be characterized more accurately. For drug development and design, absorption, distribution, metabolism and elimination versus toxicity (ADME/Tox) must be characterized, and therapeutic doses identified. Promoting the qualityand therapeutic activity of herbal or synthetic cannabis products to pharma grade is a pressing need worldwide.

Mixtures of different Cannabis sativa phytocannabinoids are more active biologically than single phytocannabinoids. However, cannabis terpenoids as potential instigators of phytocannabinoid activity have not yet been explored in detail. Terpenoid groups were statistically co-related to certain cannabis strains rich in Δ9-tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), and their ability to enhance the activity of decarboxylase phytocannabinoids (i.e., THC or CBD) was determined. Analytical HPLC and GC/MS were used to identify and quantify the secondary metabolites in 17 strains of C. sativa, and correlations between cannabinoids and terpenoids in each strain were determined. Column separation was used to separate and collect the compounds, and cell viability assay was used to assess biological activity. We found that in “high THC” or “high CBD” strains, phytocannabinoids are produced alongside certain sets of terpenoids. Only co-related terpenoids enhanced the cytotoxic activity of phytocannabinoids on MDA-MB-231 and HCT-116 cell lines. This was found to be most effective in natural ratios found in extracts of cannabis inflorescence. The correlation in a particular strain between THCA or CBDA and a certain set of terpenoids, and the partial specificity in interaction may have influenced the cultivation of cannabis and may have implications for therapeutic treatments.