Evaluating Sex Differences in Efficacy, Safety and Pharmacokinetics in Patients Treated with Cannabis by a Metered-Dose Inhaler.

BACKGROUND: Clinical studies on medical cannabis (MC) treatment have shown sex-related differences, including higher susceptibility to adverse events among women and greater analgesia among men. Here, we used the Syqe metered-dose inhaler (MDI) and a single chemovar to analyze sex differences. METHODS: A total of 1249 Israeli chronic pain patients were assessed for pain intensity, sleep and adverse events (AEs) over 240 days. RESULTS: Following the first two weeks, no significant sex differences were found in the effectiveness or safety of MC treatment (p > 0.05). Inhaled Δ9-THC doses did not vary significantly between sexes (p > 0.05) except in the first month of treatment. Pain reduction and sleep improvement were similar for both sexes (p > 0.05). The overall rate of AEs was equal and relatively low at 10% (n = 65, 10% of women and n = 60, 10% of men; χ2 (1) = 0.05, p = 0.820). A secondary analysis of pharmacokinetic data showed no significant differences between sexes in Δ9-THC and its metabolite pharmacokinetics, cardiovascular measures, or AE severity (p > 0.05). CONCLUSIONS: Uniform MC treatment via the Syqe MDI showed no sex differences in short-term effectiveness, safety and pharmacokinetics, nor in long-term effects, under "real-life" conditions. These findings provide insights into MC treatment which may inform clinical practice and policy-making in the field.

Decreased melanoma CSF-1 secretion by Cannabigerol treatment reprograms regulatory myeloid cells and reduces tumor progression.

During solid tumor progression, the tumor microenvironment (TME) evolves into a highly immunosuppressive milieu. Key players in the immunosuppressive environment are regulatory myeloid cells, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), which are recruited and activated via tumor-secreted cytokines such as colony-stimulating factor 1 (CSF-1). Therefore, the depletion of tumor-secreted cytokines is a leading anticancer strategy. Here, we found that CSF-1 secretion by melanoma cells is decreased following treatment with Cannabis extracts. Cannabigerol (CBG) was identified as the bioactive cannabinoid responsible for the effects. Conditioned media from cells treated with pure CBG or the high-CBG extract reduced the expansion and macrophage transition of the monocytic-MDSC subpopulation. Treated MO-MDSCs also expressed lower levels of iNOS, leading to restored CD8+ T-cell activation. Tumor-bearing mice treated with CBG presented reduced tumor progression, lower TAM frequencies and reduced TAM/M1 ratio. A combination of CBG and αPD-L1 was more effective in reducing tumor progression, enhancing survival and increasing the infiltration of activated cytotoxic T-cells than each treatment separately. We show a novel mechanism for CBG in modulating the TME and enhancing immune checkpoint blockade therapy, underlining its promising therapeutic potential for the treatment of a variety of tumors with elevated CSF-1 expression.

Poly(ethylene glycol)-b-poly(epsilon-caprolactone) nanoparticles as a platform for the improved oral delivery of cannabidiol.

Cannabidiol (CBD), a non-psychoactive constituent of Cannabis, has proven neuroprotective, anti-inflammatory and antioxidant properties though his therapeutic use, especially by the oral route, is still challenged by the poor aqueous solubility that results in low oral bioavailability. In this work, we investigate the encapsulation of CBD within nanoparticles of a highly hydrophobic poly(ethylene glycol)-b-poly(epsilon-caprolactone) block copolymer produced by a simple and reproducible nanoprecipitation method. The encapsulation efficiency is ~ 100% and the CBD loading 11% w/w (high performance liquid chromatography). CBD-loaded nanoparticles show a monomodal size distribution with sizes of up to 100 nm (dynamic light scattering), a spherical morphology, and the absence of CBD crystals (high resolution-scanning electron microscopy and cryogenic-transmission electron microscopy) which is in line with a very efficient nanoencapsulation. Then, the CBD release profile from the nanoparticles is assessed under gastric- and intestine-like conditions. At pH 1.2, only 10% of the payload is released after 1 h. Conversely, at pH 6.8, a release of 80% is recorded after 2 h. Finally, the oral pharmacokinetics is investigated in rats and compared to a free CBD suspension. CBD-loaded nanoparticles lead to a statistically significant ~ 20-fold increase of the maximum drug concentration in plasma (Cmax) and a shortening of the time to the Cmax (tmax) from 4 to 0.3 h, indicating a more complete and faster absorption than in free form. Moreover, the area-under-the-curve (AUC), a measure of oral bioavailability, increased by 14 times. Overall results highlight the promise of this simple, reproducible, and scalable nanotechnology strategy to improve the oral performance of CBD with respect to common oily formulations and/or lipid-based drug delivery systems associated with systemic adverse effects.

Correction: Blal et al. The Effect of Cannabis Plant Extracts on Head and Neck Squamous Cell Carcinoma and the Quest for Cannabis-Based Personalized Therapy. Cancers 2023, 15, 497.

In the original publication [...].

The Effect of Cannabis Plant Extracts on Head and Neck Squamous Cell Carcinoma and the Quest for Cannabis-Based Personalized Therapy.

Cannabis sativa plants have a wide diversity in their metabolite composition among their different chemovars, facilitating diverse anti-tumoral effects on cancer cells. This research examined the anti-tumoral effects of 24 cannabis extracts representative of three primary types of chemovars on head and neck squamous cell carcinoma (HNSCC). The chemical composition of the extracts was determined using High-Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS). The most potent anti-tumoral extracts were type III decarboxylated extracts, with high levels of Cannabidiol (CBD). We identified extract 296 (CAN296) as the most potent in inducing HNSCC cell death via proapoptotic and anti-proliferative effects. Using chemical fractionation of CAN296, we identified the CBD fraction as the primary inducer of the anti-tumoral activity. We succeeded in defining the combination of CBD with cannabichromene (CBC) or tetrahydrocannabinol (THC) present in minute concentrations in the extract, yielding a synergic impact that mimics the extract's full effect. The cytotoxic effect could be maximized by combining CBD with either CBC or THC in a ratio of 2:1. This research suggests using decarboxylated CBD-type extracts enriched with CBC for future preclinical trials aimed at HNSCC treatment.

Solid-State Microwave Drying for Medical Cannabis Inflorescences: A Rapid and Controlled Alternative to Traditional Drying.

Introduction: As the medical use of Cannabis is evolving there is a greater demand for high-quality products for patients. One of the main steps in the manufacturing process of medical Cannabis is drying. Most current drying methods in the Cannabis industry are relatively slow and inefficient processes. Materials and Methods: This article presents a drying method based on solid-state microwave (MW) that provides fast and uniform drying, and examines its efficiency for drying Cannabis inflorescences compared with the traditional drying method. We assessed 67 cannabinoids and 36 terpenoids in the plant in a range of drying temperatures (40°C, 50°C, 60°C, and 80°C). The identification and quantification of these secondary metabolites were done by chromatography methods. Results: This method resulted in a considerable reduction of drying time, from several days to a few hours. The multiple frequency-phase combination states of the system allowed control and prediction of moisture levels during drying, thus preventing overdrying. A drying temperature of 50°C provided the most effective results in terms of both short drying time and preservation of the composition of the secondary metabolites compared with traditional drying. At 50°C, the chemical profile of phytocannabinoids and terpenoids was best kept to that of the original plant before drying, suggesting less degradation by chemical reactions such as decarboxylation. The fast-drying time also reduced the susceptibility of the plant to microbial contamination. Conclusion: Our results support solid-state MW drying as an effective postharvest step to quickly dry the plant material for improved downstream processing with a minimal negative impact on product quality.

The Effectiveness and Safety of Medical Cannabis for Treating Cancer Related Symptoms in Oncology Patients.

The use of medical cannabis (MC) to treat cancer-related symptoms is rising. However, there is a lack of long-term trials to assess the benefits and safety of MC treatment in this population. In this work, we followed up prospectively and longitudinally on the effectiveness and safety of MC treatment. Oncology patients reported on multiple symptoms before and after MC treatment initiation at one-, three-, and 6-month follow-ups. Oncologists reported on the patients' disease characteristics. Intention-to-treat models were used to assess changes in outcomes from baseline. MC treatment was initiated by 324 patients and 212, 158 and 126 reported at follow-ups. Most outcome measures improved significantly during MC treatment for most patients (p < 0.005). Specifically, at 6 months, total cancer symptoms burden declined from baseline by a median of 18%, from 122 (82-157) at baseline to 89 (45-138) at endpoint (-18.98; 95%CI= -26.95 to -11.00; p < 0.001). Reported adverse effects were common but mostly non-serious and remained stable during MC treatment. The results of this study suggest that MC treatment is generally safe for oncology patients and can potentially reduce the burden of associated symptoms with no serious MC-related adverse effects.

Identification of novel proteins in the Dictyostelium discoideum chemorepulsion pathway using REMI.

Chemorepulsion, the biased migration of a cell away from a signal, is essential for many biological processes and the ability to manipulate chemorepulsion could lead to new therapeutics for a variety of diseases. However, little is known about eukaryotic cell chemorepulsion. Utilizing the model organism Dictyostelium discoideum, we previously identified an endogenous chemorepellent protein secreted by D. discoideum cells called AprA, and proteins involved in the AprA-induced chemorepulsion pathway including the G protein-coupled receptor GrlH, G beta and G protein alpha 8 protein subunits, protein kinase A, components of the mammalian target of rapamycin complex 2 (mTORC2), phospholipase A, PTEN and a PTEN-like phosphatase (CnrN), a retinoblastoma orthologue (RblA), extracellular signal-regulated kinase 1 (Erk1), p-21 activated protein kinase D (PakD), and the Ras proteins RasC and RasG. In this report, we used a genetic screen to identify 17 additional proteins involved in the AprA-induced chemorepulsion pathway .

Cannabis for Medical Use: Versatile Plant Rather Than a Single Drug.

Medical Cannabis and its major cannabinoids (-)-trans-Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are gaining momentum for various medical purposes as their therapeutic qualities are becoming better established. However, studies regarding their efficacy are oftentimes inconclusive. This is chiefly because Cannabis is a versatile plant rather than a single drug and its effects do not depend only on the amount of THC and CBD. Hundreds of Cannabis cultivars and hybrids exist worldwide, each with a unique and distinct chemical profile. Most studies focus on THC and CBD, but these are just two of over 140 phytocannabinoids found in the plant in addition to a milieu of terpenoids, flavonoids and other compounds with potential therapeutic activities. Different plants contain a very different array of these metabolites in varying relative ratios, and it is the interplay between these molecules from the plant and the endocannabinoid system in the body that determines the ultimate therapeutic response and associated adverse effects. Here, we discuss how phytocannabinoid profiles differ between plants depending on the chemovar types, review the major factors that affect secondary metabolite accumulation in the plant including the genotype, growth conditions, processing, storage and the delivery route; and highlight how these factors make Cannabis treatment highly complex.

Sex differences in medical cannabis-related adverse effects.

ABSTRACT: Studies have shown that women are more susceptible to adverse effects (AEs) from conventional drugs. This study aimed to investigate the differences of medical cannabis (MC)-related AEs between women and men in patients with chronic noncancer pain (CNCP). This is a cross-sectional study of adult patients licensed for MC treatment who were also diagnosed as patients with CNCP by a physician. Data included self-reported questionnaires and comprehensive MC treatment information. Simultaneously, identification and quantification of phytocannabinoids and terpenoids from the MC cultivars were performed. Comparative statistics were used to evaluate differences between men and women. Four hundred twenty-nine patients with CNCP (64% males) reported fully on their MC treatment. Subgrouping by sex demonstrated that the weight-adjusted doses were similar between men and women (0.48 [0.33-0.6] gr for men and 0.47 [0.34-0.66] gr for women). Nonetheless, women reported more than men on MC-related AEs. Further analysis revealed that women consumed different MC cultivar combinations than men, with significantly higher monthly doses of the phytocannabinoids CBD and CBC and significantly lower monthly doses of the phytocannabinoid 373-15c and the terpenoid linalool. Our findings demonstrate sex differences in MC-related AEs among patients with CNCP. Women are more susceptible to MC-related AEs, presumably because of both the inherent sex effect and the consumption of specific phytocannabinoid compositions in the MC cultivar(s). The understanding of these differences may be crucial for planning MC treatments with safer phytocannabinoid and terpenoid compositions and to better inform patients of expected AEs.

Fertilization Following Pollination Predominantly Decreases Phytocannabinoids Accumulation and Alters the Accumulation of Terpenoids in Cannabis Inflorescences.

In the last decades, growing evidence showed the therapeutic capabilities of Cannabis plants. These capabilities were attributed to the specialized secondary metabolites stored in the glandular trichomes of female inflorescences, mainly phytocannabinoids and terpenoids. The accumulation of the metabolites in the flower is versatile and influenced by a largely unknown regulation system, attributed to genetic, developmental and environmental factors. As Cannabis is a dioecious plant, one main factor is fertilization after successful pollination. Fertilized flowers are considerably less potent, likely due to changes in the contents of phytocannabinoids and terpenoids; therefore, this study examined the effect of fertilization on metabolite composition by crossbreeding (-)-Δ9-trans-tetrahydrocannabinol (THC)- or cannabidiol (CBD)-rich female plants with different male plants: THC-rich, CBD-rich, or the original female plant induced to develop male pollen sacs. We used advanced analytical methods to assess the phytocannabinoids and terpenoids content, including a newly developed semi-quantitative analysis for terpenoids without analytical standards. We found that fertilization significantly decreased phytocannabinoids content. For terpenoids, the subgroup of monoterpenoids had similar trends to the phytocannabinoids, proposing both are commonly regulated in the plant. The sesquiterpenoids remained unchanged in the THC-rich female and had a trend of decrease in the CBD-rich female. Additionally, specific phytocannabinoids and terpenoids showed an uncommon increase in concentration followed by fertilization with particular male plants. Our results demonstrate that although the profile of phytocannabinoids and their relative ratios were kept, fertilization substantially decreased the concentration of nearly all phytocannabinoids in the plant regardless of the type of fertilizing male. Our findings may point to the functional roles of secondary metabolites in Cannabis.

Specific phytocannabinoid compositions are associated with analgesic response and adverse effects in chronic pain patients treated with medical cannabis.

Medical cannabis (MC) treatment for chronic pain is increasing, but evidence regarding short- and long-term efficacy and associated adverse effects (AEs) of the different cannabis plant components is limited. Most reports focus on two phytocannabinoids, (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). This study, aimed to identify patterns of phytocannabinoid compositions associated with MC treatment response and with related AEs. Participants in this multicenter prospective cohort were patients with chronic non-cancer pain that were prescribed MC by physicians. Data was collected before MC treatment, at one month (short-term) and at 12 months (long-term). Simultaneously, liquid chromatography mass spectrometry identification and quantification of phytocannabinoids from the cultivars were performed. The monthly dose of each phytocannabinoid for each patient was z-scaled and clustered into ten groups to assess the difference in analgesic treatment response (≥30%/50% pain intensity reduction) and AEs rates. We identified ten clusters that had similar analgesic treatment response rates. However, there were significant differences in AEs rates both at short- and long-term. We identified specific phytocannabinoid compositions that were associated with overall AEs rates (5% compared to 53% at short-term and 44% at long-term) and with specific AEs rates such as MC related central nervous system, gastrointestinal and psychological AEs. To conclude, Evaluating only Δ9-THC or CBD is insufficient to find associations with MC related AEs. Therefore, comprehensive profiling of phytocannabinoids is needed to discover associations to related AEs and help physicians prescribe safer cannabis with less AEs while still relieving pain.

Short-Term Medical Cannabis Treatment Regimens Produced Beneficial Effects among Palliative Cancer Patients.

In the last decade the use of medical cannabis (MC) for palliative cancer treatment has risen. However, the choice between products is arbitrary and most patients are using Tetrahydrocannabinol (THC)-dominant cannabis products. In this study, we aimed to assess the short-term outcomes of MC treatment prescribed by oncologists in relation to the type of cannabis they receive. A comparative analysis was used to assess the differences in treatment effectiveness and safety between THC-dominant (n = 56, 52%), cannabidiol (CBD)-dominant (n = 19, 18%), and mixed (n = 33, 30%) MC treatments. Oncology patients (n = 108) reported on multiple symptoms in baseline questionnaires, initiated MC treatment, and completed a one-month follow-up. Most parameters improved significantly from baseline, including pain intensity, affective and sensory pain, sleep quality and duration, cancer distress, and both physical and psychological symptom burden. There was no significant difference between the three MC treatments in the MC-related safety profile. Generally, there were no differences between the three MC treatments in pain intensity and in most secondary outcomes. Unexpectedly, CBD-dominant oil treatments were similar to THC-dominant treatments in their beneficial effects for most secondary outcomes. THC-dominant treatments showed significant superiority in their beneficial effect only in sleep duration compared to CBD-dominant treatments. This work provides evidence that, though patients usually consume THC-dominant products, caregivers should also consider CBD-dominant products as a useful treatment for cancer-related symptoms.

Direct binding of MEK1 and MEK2 to AKT induces Foxo1 phosphorylation, cellular migration and metastasis.

Crosstalk between the ERK cascade and other signaling pathways is one of the means by which it acquires its signaling specificity. Here we identified a direct interaction of both MEK1 and MEK2 with AKT. The interaction is mediated by the proline rich domain of MEK1/2 and regulated by phosphorylation of Ser298 in MEK1, or Ser306 in MEK2, which we identified here as a novel regulatory site. We further developed a blocking peptide, which inhibits the interaction between MEK and AKT, and when applied to cells, affects migration and adhesion, but not proliferation. The specific mechanism of action of the MEK-AKT complex involves phosphorylation of the migration-related transcription factor FoxO1. Importantly, prevention of the interaction results in a decreased metastasis formation in a breast cancer mouse model. Thus, the identified interaction both sheds light on how signaling specificity is determined, and represents a possible new therapeutic target for metastatic cancer.

Palmoplantar Keratoderma in Slurp2-Deficient Mice.

SLURP1, a member of the lymphocyte antigen 6 protein family, is secreted by suprabasal keratinocytes. Mutations in SLURP1 cause a palmoplantar keratoderma (PPK) known as mal de Meleda. SLURP2, another secreted lymphocyte antigen 6 protein, is encoded by a gene located ?20 kb downstream from SLURP1. SLURP2 is produced by suprabasal keratinocytes. To investigate the importance of SLURP2, we first examined Slurp2 knockout mice in which exon 2-3 sequences had been replaced with lacZ and neo cassettes. Slurp2(-/-) mice exhibited hyperkeratosis on the volar surface of the paws (i.e., palmoplantar keratoderma), increased keratinocyte proliferation, and an accumulation of lipid droplets in the stratum corneum. They also exhibited reduced body weight and hind limb clasping. These phenotypes are similar to those of Slurp1(-/-) mice. To solidify a link between Slurp2 deficiency and palmoplantar keratoderma and to be confident that the disease phenotypes in Slurp2(-/-) mice were not secondary to the effects of the lacZ and neo cassettes on Slurp1 expression, we created a new line of Slurp2 knockout mice (Slurp2X(-/-)) in which Slurp2 was inactivated with a simple nonsense mutation. Slurp2X(-/-) mice exhibited the same disease phenotypes. Thus, Slurp2 deficiency and Slurp1 deficiencies cause the same disease phenotypes.

Mice that express farnesylated versions of prelamin A in neurons develop achalasia.

Neurons in the brain produce lamin C but almost no lamin A, a consequence of the removal of prelamin A transcripts by miR-9, a brain-specific microRNA. We have proposed that miR-9-mediated regulation of prelamin A in the brain could explain the absence of primary neurological disease in Hutchinson-Gilford progeria syndrome, a genetic disease caused by the synthesis of an internally truncated form of farnesyl-prelamin A (progerin). This explanation makes sense, but it is not entirely satisfying because it is unclear whether progerin-even if were expressed in neurons-would be capable of eliciting neuropathology. To address that issue, we created a new Lmna knock-in allele, Lmna(HG-C), which produces progerin transcripts lacking an miR-9 binding site. Mice harboring the Lmna(HG-C) allele produced progerin in neurons, but they had no pathology in the central nervous system. However, these mice invariably developed esophageal achalasia, and the enteric neurons and nerve fibers in gastrointestinal tract were markedly abnormal. The same disorder, achalasia, was observed in genetically modified mice that express full-length farnesyl-prelamin A in neurons (Zmpste24-deficient mice carrying two copies of a Lmna knock-in allele yielding full-length prelamin A transcripts lacking a miR-9 binding site). Our findings indicate that progerin and full-length farnesyl-prelamin A are toxic to neurons of the enteric nervous system.

The MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation.

The MAPK cascades are central signaling pathways that regulate a wide variety of stimulated cellular processes, including proliferation, differentiation, apoptosis and stress response. Therefore, dysregulation, or improper functioning of these cascades, is involved in the induction and progression of diseases such as cancer, diabetes, autoimmune diseases, and developmental abnormalities. Many of these physiological, and pathological functions are mediated by MAPK-dependent transcription of various regulatory genes. In order to induce transcription and the consequent functions, the signals transmitted via the cascades need to enter the nucleus, where they may modulate the activity of transcription factors and chromatin remodeling enzymes. In this review, we briefly cover the composition of the MAPK cascades, as well as their physiological and pathological functions. We describe, in more detail, many of the important nuclear activities of the MAPK cascades, and we elaborate on the mechanisms of ERK1/2 translocation into the nucleus, including the identification of their nuclear translocation sequence (NTS) binding to the shuttling protein importin7. Overall, the nuclear translocation of signaling components may emerge as an important regulatory layer in the induction of cellular processes, and therefore, may serve as targets for therapeutic intervention in signaling-related diseases such as cancer and diabetes. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.

Determination of ERK activity: anti-phospho-ERK antibodies and in vitro phosphorylation.

The ERK signaling cascade is composed of several protein kinases that sequentially activate each other by phosphorylation. This pathway is a central component of a complex signaling network that regulates important cellular processes including proliferation, differentiation, and survival. In most of these cases, the ERK cascade is activated downstream of the small GTPase Ras that, upon activation, recruits and activates the first tier in the cascade, which contains the Raf kinases. Afterward the signal is further transmitted by MEKs, ERKs, and often RSKs in the MAPKK, MAPK, and MAPKAPKs tiers of the cascade, respectively. ERKs and RSKs can further disseminate the signal by phosphorylating and modulating the activity of a large number of regulatory proteins including transcription factors and chromatin modifying enzymes. Understanding the mechanisms of activation and the regulation of the various components of this cascade will enhance our insight into the regulation of the ERK-dependent cellular processes in normal cells or of their malfunctioning in various diseases, including cancer. In this chapter, we describe methods used to determine the activity of ERKs, which upon slight modifications can also be used for the study of other signaling kinases, either within the cascade or in other pathways. These methods have been successfully applied to study the ERK signaling cascades in a variety of tissue-cultured cell lines, homo-genized animal organs, and lower organisms. As such, the use of these methods should expand our knowledge on the regulation of many distinct systems and upon induction of various stimulations.