Development and Validation of a GC-FID Method for the Quantitation of Δ 8-Tetrahydrocannabinol and Impurities Found in Synthetic Δ 8-Tetrahydrocannabinol and Vaping Products.

Concerns about health hazards associated with the consumption of trans-delta-8-tetrahydrocannabinol products were highlighted in public health advisories from the U. S. Food and Drug Administration and U. S. Centers for Disease Control and Prevention. Simple and rapid quantitative methods to determine trans-delta-8-tetrahydrocannabinol impurities are vital to analyze such products. In this study, a gas chromatography-flame ionization detection method was developed and validated for the determination of delta-8-tetrahydrocannabinol and some of its impurities (recently published) found in synthesized trans-delta-8-tetrahydrocannabinol raw material and included olivetol, cannabicitran, Δ 8-cis-iso-tetrahydrocannabinol, Δ 4-iso-tetrahydrocannabinol, iso-tetrahydrocannabifuran, cannabidiol, Δ 4,8-iso-tetrahydrocannabinol, Δ 8-iso-tetrahydrocannabinol, 4,8-epoxy-iso-tetrahydrocannabinol, trans-Δ 9-tetrahydrocannabinol, 8-hydroxy-iso-THC, 9α-hydroxyhexahydrocannabinol, and 9ß-hydroxyhexahydrocannabinol. Validation of the method was assessed according to the International Council for Harmonization guidelines and confirmed linearity with R2 ≥ 0.99 for all the target analytes. The limit of detection and limit of quantitation were 1.5 and 5 µg/mL, respectively, except for olivetol, which had a limit of detection of 3 µg/mL and a limit of quantitation of 10 µg/mL. Method precision was calculated as % relative standard deviation and the values were less than 8.4 and 9.9% for the intraday precision and inter-day precision, respectively. The accuracy ranged from 85 to 118%. The method was then applied to the analysis of 21 commercially marketed vaping products claiming to contain delta-8-tetrahydrocannabinol. The products analyzed by this method have various levels of these impurities, with all products far exceeding the 0.3% of trans-Δ 9-tetrahydrocannabinol limit for hemp under the Agriculture Improvement Act of 2018. The developed gas chromatography-flame ionization detection method can be an important tool for monitoring delta-8-tetrahydrocannabinol impurities in commercial products.

Human quad liver-on-chip system as a tool toward bridging the gap between animals and humans regarding toxicology and pharmacology of a cannabidiol-rich cannabis extract.

Cannabidiol (CBD) is a major phytocannabinoid from Cannabis sativa. It is currently widely available and widely used in the USA, but despite its rapid progress to market, the pharmacology and toxicology of both CBD and cannabidiol-rich cannabis extracts (CRCE) remain largely unknown. The goals of this study were to investigate the potential of a novel human microphysiological system to emulate CRCE-induced hepatotoxicity and pharmacological properties demonstrated in animal models. For this purpose, C57BL6/J male mice were subjected to dosing with either 0, 61.5, 184.5, or 615 mg/kg of CRCE for 10 days. The liver-on-chip system, incorporating human primary hepatocytes, sinusoidal endothelial cells, as well as Kupffer and stellate cells was subjected to 0, 300, 1,200, or 4,400 ng/mL of CRCE (8 h exposure followed by 16 h washout) for 5 days. Administration of CRCE in mice resulted in nearly 4-fold elevations of plasma ALT at 615 mg/kg (p < 0.01) and a dose-dependent decrease in intrahepatic miR-122. Elevated levels of ALT, paralleled by decreased intrahepatic and increased effluent levels of miR-122, were also observed in the liver-on-chip, although these results were not statistically significant. Exposure to CRCE resulted in a robust and dose-dependent induction of key cytochrome P450 enzymes, namely Cyp1a2, Cyp2b6 (CYP2B10), Cyp2e1, and Cyp2c9 (CYP2C19) in both mouse livers and liver-on-chip. The results of this study demonstrate the congruence between the responses observed in mouse and human liver-on-chip experimental systems and provide evidence of the potential microphysiological systems hold for translating animal data into clinical practice.

Chemistry and Pharmacology of Delta-8-Tetrahydrocannabinol.

Cannabis sativa is one of the oldest plants utilized by humans for both economic and medical purposes. Although the use of cannabis started millennia ago in the Eastern hemisphere, its use has moved and flourished in the Western nations in more recent centuries. C. sativa is the source of psychoactive cannabinoids that are consumed as recreational drugs worldwide. The C21 aromatic hydrocarbons are restricted in their natural occurrence to cannabis (with a few exceptions). Delta-9-tetrahydrocannabinol (Δ9-THC) is the main psychoactive component in cannabis, with many pharmacological effects and various approved medical applications. However, a wide range of side effects are associated with the use of Δ9-THC, limiting its medical use. In 1966, another psychoactive cannabinoid, Delta-8-tetrahydrocannabinol (Δ8-THC) was isolated from marijuana grown in Maryland but in very low yield. Δ8-THC is gaining increased popularity due to its better stability and easier synthetic manufacturing procedures compared to Δ9-THC. The passing of the U.S. Farm Bill in 2018 led to an increase in the sale of Δ8-THC in the United States. The marketed products contain Δ8-THC from synthetic sources. In this review, methods of extraction, purification, and structure elucidation of Δ8-THC will be presented. The issue of whether Δ8-THC is a natural compound or an artifact will be discussed, and the different strategies for its chemical synthesis will be presented. Δ8-THC of synthetic origin is expected to contain some impurities due to residual amounts of starting materials and reagents, as well as side products of the reactions. The various methods of analysis and detection of impurities present in the marketed products will be discussed. The pharmacological effects of Δ8-THC, including its interaction with CB1 and CB2 cannabinoid receptors in comparison with Δ9-THC, will be reviewed.

Isolation and Characterization of Impurities in Commercially Marketed Δ8-THC Products.

Qualitative analysis of several commercial products containing Δ8-tetrahydrocannabinol (Δ8-THC) as a major component using GC-MS resulted in the identification of several impurities along with Δ8-THC. In an attempt to isolate and identify these impurities, a commercial Δ8-THC distillate was selected for the isolation work. Eleven impurities were isolated using a variety of chromatographic techniques, and their chemical structures were determined. These include Δ4,8-iso-tetrahydrocannabinol (1), Δ4-iso-tetrahydrocannabinol (2), Δ8-cis-iso-tetrahydrocannabinol (3), 4,8-epoxy-iso-tetrahydrocannabinol (4), 8-hydroxy-iso-tetrahydrocannabinol (5), 9ß-hydroxyhexahydrocannabinol (6), 9α-hydroxyhexa-hydrocannabinol (7), iso-tetrahydrocannabifuran (8), cannabicitran (CBT, 9), olivetol (10), and Δ9-THC (11). The chemical structures of the purified compounds were determined using several spectroscopic methods, including 1D (1H, 13C, and DEPT-135) and 2D (COSY, HMQC, HMBC, and NOESY) NMR, LC-MS, and GC-MS. Other naturally occurring cannabinoids and impurities were also identified in GC-MS chromatograms but were not isolated. These were cannabidiol (CBD, 12), cannabinol (CBN, 13), hexahydrocannabinol (HHC, 14), and Δ8-tetrahydrocannabivarin (Δ8-THCV, 15). The chemical structure of Δ8-THCV (15), for which a standard was not available, was confirmed by partial synthesis and NMR analysis. This is the first report for many of the above compounds as well as Δ8-THCV as impurities in Δ8-THC products.

Development and Validation of a GC-FID Method for the Quantitation of 20 Different Acidic and Neutral Cannabinoids.

For decades, Cannabis sativa had been illegal to sell or consume around the world, including in the United States. However, in light of the recent 2018 Farm Bill and the legalization of hemp across the US, various cannabis preparations have flooded the market, making it essential to be able to quantitate the levels of the different acidic and neutral cannabinoids in C. sativa and to have a complete cannabinoid profile of the different chemovars of the cannabis plant. A GC-FID method was developed and validated for the analysis of 20 acidic and neutral cannabinoids as trimethylsilyl (TMS) derivatives. The analyzed cannabinoids include cannabidivarinic acid (CBDVA), cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabielsoic acid (CBEA), cannabicyclolic acid (CBLA), cannabichromenic acid (CBCA), trans-Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA), trans-Δ9-tetrahydrocannabinolic acid A (Δ9-THCAA), cannabigerolic acid (CBGA), cannabidiol (CBD), cannabicyclol (CBL), cannabidivarin (CBDV), trans-Δ9-tetrahydrocannabivarin (THCV), cannabichromene (CBC), trans-Δ8-tetrahydrocannabinol (Δ8-THC), trans-Δ9-tetrahydrocannabinol (Δ9-THC), cannabigerol (CBG), cannabinol (CBN), cannabicitran (CBT), and cannabielsoin (CBE). The method limit of detection (LOD) was as low as 0.1 µg/mL, while the limit of quantitation ranged from 0.25 µg/mL to 0.5 µg/mL. The precision (%RSD) was < 10%, while trueness ranged from 90 - 107%. The developed method is simple, accurate, and sensitive for the quantitation of all 20 acidic and neutral cannabinoids. Finally, the proposed method was successfully applied to the quantitation of the cannabinoids in different cannabis chemovars grown at the University of Mississippi.

Effect of Gamma Irradiation on Cannabinoid, Terpene, and Moisture Content of Cannabis Biomass.

In recent years, cannabis has been proposed and promoted not only as a medicine for the treatment of a variety of illnesses, but also as an industrial crop for different purposes. Being an agricultural product, cannabis inflorescences may be contaminated by environmental pathogens at high concentrations, which might cause health problems if not controlled. Therefore, limits have to be placed on the levels of aerobic bacteria as well as yeast and mold. To ensure the safety of cannabis plant material and related products, a remediation process has to be put in place. Gamma irradiation is a sterilization process mainly used for pharmaceuticals, foods, cosmetics, agricultural, and herbal products including cannabis plant material. This study was designed to determine the effect of irradiation on the microbial count as well as on the chemical and physical profiles of the cannabis biomass, particularly cannabinoids, terpenes, and moisture content. The full cannabinoid profile was measured by GC/FID and HPLC analysis, while terpene profile and moisture content were determined using GC/MS and Loss on Drying (LoD) methods, respectively. Analyses were conducted on the samples before and after gamma irradiation. The results showed that the minimum and maximum doses were 15 and 20.8 KiloGray (KGY), respectively. Total Aerobic Microbial Count (TAMC) and Total Yeast and Mold Count (TYMC) were determined. The study showed that irradiation has no effect on the cannabinoids and little effect on terpenes and moisture content, but it did result in the virtual sterilization of the plant material, as evidenced by the low levels of bacterial and fungal colony-forming units (CFUs) < 10 after gamma irradiation.

Microbial Biotransformation of Cannabidiol (CBD) from Cannabis sativa.

Microbial biotransformation of cannabidiol was assessed using 31 different microorganisms. Only Mucor ramannianus (ATCC 9628), Beauveria bassiana (ATCC 7195), and Absidia glauca (ATCC 22 752) were able to metabolize cannabidiol. M. ramannianus (ATCC 9628) yielded five metabolites, namely, 7,4″ß-dihydroxycannabidiol (1: ), 6ß,4″ß-dihydroxycannabidiol (2: ), 6ß,2″ß-dihydroxycannabidiol (3: ), 6ß,3″α-dihydroxycannabidiol (4: ), and 6ß,7,4″ß-trihydroxycannabidiol (5: ). B. bassiana (ATCC 7195) metabolized cannabidiol to afford six metabolites identified as 7,3″-dihydroxycannabidivarin (6: ), 7-hydroxycannabidivarin-3″-carboxylic acid (7: ), 3″-hydroxycannabidivarin (8: ), 4″ß-hydroxycannabidiol (9: ), and cannabidivarin-3″-carboxylic acid (10: ) along with compound 1: . Incubation of cannabidiol with A. glauca (ATCC 22 752) yielded three metabolites, 6α,3″-dihyroxycannabidivarin (11: ), 6ß,3″-dihyroxycannabidivarin (12: ), and compound 6: . All compounds were evaluated for their antimicrobial and antiprotozoal activity.

Design, synthesis, and anti-proliferative evaluation of new quinazolin-4(3H)-ones as potential VEGFR-2 inhibitors.

Inhibiting VEGFR-2 has been set up as a therapeutic strategy for treatment of cancer. Thus, nineteen new quinazoline-4(3H)-one derivatives were designed and synthesized. Preliminary cytotoxicity studies of the synthesized compounds were evaluated against three human cancer cell lines (HepG-2, MCF-7 and HCT-116) using MTT assay method. Doxorubicin and sorafenib were used as positive controls. Five compounds were found to have promising cytotoxic activities against all cell lines. Compound 16f, containing a 2-chloro-5-nitrophenyl group, has emerged as the most active member. It was approximately 4.39-, 5.73- and 1.96-fold more active than doxorubicin and 3.88-, 5.59- and 1.84-fold more active than sorafenib against HepG2, HCT-116 and MCF-7 cells, respectively. The most active cytotoxic agents were further evaluated in vitro for their VEGFR-2 inhibitory activities. The results of in vitro VEGFR-2 inhibition were consistent with that of the cytotoxicity data. Molecular docking of these compounds into the kinase domain, moreover, supported the results.

Discovery of new quinoxaline-2(1H)-one-based anticancer agents targeting VEGFR-2 as inhibitors: Design, synthesis, and anti-proliferative evaluation.

VEGF/VEGFR2 pathway is the crucial therapeutic target in the treatment of cancer. So that, a new series of quinoxaline-2(1H)-one derivatives were designed and synthesized. The synthesized compounds were tested against three human cancer cell lines (HepG-2, MCF-7 and HCT-116) aiming to evaluate its anti-proliferative activities. Doxorubicin as a universal anticancer drug and sorafenib as a potent VEGFR-2 inhibitor were used as positive controls. The data obtained from biological activity were found highly correlated with that obtained from molecular modeling studies. The most sensitive cell line to the influence of our new derivatives was HCT-116. Compounds 13b, 15, 16e and 17b exert the highest cytotoxic activities against the tested cell lines. Overall, compound 15 was the most active member with IC50 values of 5.30, 2.20, 5.50 µM against HepG-2, MCF-7 and HCT-116, respectively. Compounds 15 and 17b showed better anti-proliferative activities than doxorubicin and sorafenib against the three cancer cell lines. Additionally, compound 16e showed better anti-proliferative activities than doxorubicin and sorafenib against HepG-2 and HCT-116 but exhibited lower activity against MCF-7 cell line. In addition, the most promising members were further evaluated for their inhibitory activities against VEGFR-2. Compounds 15 and 17b potently inhibited VEGFR-2 at lower IC50 values of 1.09 and 1.19 µM, respectively, compared to sorafenib (IC50 = 1.27 µM). Moreover, docking studies were conducted to investigate the binding pattern of the synthesized compounds against the prospective molecular target VEGFR-2.

Design, molecular docking, in vitro, and in vivo studies of new quinazolin-4(3H)-ones as VEGFR-2 inhibitors with potential activity against hepatocellular carcinoma.

A series of new VEGFR-2 inhibitors were designed, synthesized and evaluated for their anti-proliferative activities against hepatocellular carcinoma (HepG-2 cell line). Compound 29b (IC50 = 4.33 ± 0.2 µg/ml) was found to be the most potent derivative as it has showed to be more active than doxorubicin (IC50 = 4.50 ± 0.2 µg/ml) and 78% of sorafenib activity (IC50 = 3.40 ± 0.25 µg/ml). The inhibitory profiles against VEGFR-2 were also assessed for the most promising candidates (16b, 20c, 22b, 24a, 24b, 28c, 28e, 29a, 29b and 29c). Compounds 29b, 29c and 29a exhibited potent inhibitory activities towards VEGFR-2 at IC50 values of 3.1 ± 0.04, 3.4 ± 0.05 and 3.7 ± 0.06 µM, respectively, comparing sorafenib (IC50 = 2.4 ± 0.05 µM). Furthermorer, compound 29b induced apoptosis and arrested the cell cycle growth at G2/M phase. Additionally, in vivo antitumor experiments revealed that compounds 29b and 29c have significant tumor growth inhibition. The test of immuno-histochemical expression of activated caspase-3 revealed that there is a time-dependent increase in cleaved caspase-3 protein expression upon exposure of HepG-2 cells to compound 29b. Moreover, the fibroblastic proliferative index test revealed that compound 29b could attenuate liver fibrosis. Docking studies also supported the results concluded from the biological screening via prediction of the possible binding interactions of the target compounds with VEGFR-2 active sites using the crystal structure of VEGFR-2 downloaded from the Protein Data Bank, (PDB ID: 2OH4) using Discovery Studio 2.5 software. Further structural optimization of the most active candidates may serve as a useful strategy for getting new lead compounds in search for powerful and selective antineoplastic agents.

Cannabinoids, Phenolics, Terpenes and Alkaloids of Cannabis.

Cannabis sativa is one of the oldest medicinal plants in the world. It was introduced into western medicine during the early 19th century. It contains a complex mixture of secondary metabolites, including cannabinoids and non-cannabinoid-type constituents. More than 500 compounds have been reported from C. sativa, of which 125 cannabinoids have been isolated and/or identified as cannabinoids. Cannabinoids are C21 terpeno-phenolic compounds specific to Cannabis. The non-cannabinoid constituents include: non-cannabinoid phenols, flavonoids, terpenes, alkaloids and others. This review discusses the chemistry of the cannabinoids and major non-cannabinoid constituents (terpenes, non-cannabinoid phenolics, and alkaloids) with special emphasis on their chemical structures, methods of isolation, and identification.

Validating a predictive model of cannabinoid inheritance with feral, clinical, and industrial Cannabis sativa.

PREMISE: How genetic variation within a species affects phytochemical composition is a fundamental question in botany. The ratio of two specialized metabolites in Cannabis sativa, tetrahydrocannabinol (THC) and cannabidiol (CBD), can be grouped into three main classes (THC-type, CBD-type, and intermediate type). We tested a genetic model associating these three groups with functional and nonfunctional alleles of the cannabidiolic acid synthase gene (CBDAS). METHODS: We characterized cannabinoid content and assayed CBDAS genotypes of >300 feral C. sativa plants in Minnesota, United States. We performed a test cross to assess CBDAS inheritance. Twenty clinical cultivars obtained blindly from the National Institute on Drug Abuse and 12 Canadian-certified grain cultivars were also examined. RESULTS: Frequencies of CBD-type, intermediate-type, and THC-type feral plants were 0.88, 0.11, and 0.01, respectively. Although total cannabinoid content varied substantially, the three groupings were perfectly correlated with CBDAS genotypes. Genotype frequencies observed in the test cross were consistent with codominant Mendelian inheritance of the THC:CBD ratio. Despite significant mean differences in total cannabinoid content, CBDAS genotypes blindly predicted the THC:CBD ratio among clinical cultivars, and the same was true for industrial grain cultivars when plants exhibited >0.5% total cannabinoid content. CONCLUSIONS: Our results extend the generality of the inheritance model for THC:CBD to diverse C. sativa accessions and demonstrate that CBDAS genotyping can predict the ratio in a variety of practical applications. Cannabinoid profiles and associated CBDAS segregation patterns suggest that feral C. sativa populations are potentially valuable experimental systems and sources of germplasm.

Cannabis Inflorescence for Medical Purposes: USP Considerations for Quality Attributes.

There is an active and growing interest in cannabis female inflorescence (Cannabis sativa) for medical purposes. Therefore, a definition of its quality attributes can help mitigate public health risks associated with contaminated, substandard, or adulterated products and support sound and reproducible basic and clinical research. As cannabis is a heterogeneous matrix that can contain a complex secondary metabolome with an uneven distribution of constituents, ensuring its quality requires appropriate sampling procedures and a suite of tests, analytical procedures, and acceptance criteria to define the identity, content of constituents (e.g., cannabinoids), and limits on contaminants. As an independent science-based public health organization, United States Pharmacopeia (USP) has formed a Cannabis Expert Panel, which has evaluated specifications necessary to define key cannabis quality attributes. The consensus within the expert panel was that these specifications should differentiate between cannabis chemotypes. Based on the secondary metabolite profiles, the expert panel has suggested adoption of three broad categories of cannabis. These three main chemotypes have been identified as useful for labeling based on the following cannabinoid constituents: (1) tetrahydrocannabinol (THC)-dominant chemotype; (2) intermediate chemotype with both THC and cannabidiol (CBD); and (3) CBD-dominant chemotype. Cannabis plants in each of these chemotypes may be further subcategorized based on the content of other cannabinoids and/or mono- and sesquiterpene profiles. Morphological and chromatographic tests are presented for the identification and quantitative determination of critical constituents. Limits for contaminants including pesticide residues, microbial levels, mycotoxins, and elemental contaminants are presented based on toxicological considerations and aligned with the existing USP procedures for general tests and assays. The principles outlined in this review should be able to be used as the basis of public quality specifications for cannabis inflorescence, which are needed for public health protection and to facilitate scientific research on cannabis safety and therapeutic potential.

Design, synthesis, molecular modeling, in vivo studies and anticancer evaluation of quinazolin-4(3H)-one derivatives as potential VEGFR-2 inhibitors and apoptosis inducers.

Inhibiting VEGFR-2 has been set up as a therapeutic strategy for treatment of cancer. Accordingly, new quinazoline-based derivatives having the structural features of VEGFR-2 inhibitors were designed and synthesized. Anti-proliferative activities were evaluated against three human cancer cell lines (HepG-2, MCF-7 and HCT-116) using MTT assay method. Doxorubicin and sorafenib were used as positive controls. Compounds 26b, 29a, 29b and 30 showed excellent anti-cancer activities against all cell lines. Moreover, compound 31 was the most active with IC 50 values of 3.97 ±â€¯0.2, 4.83 ±â€¯0.2 and 4.58 ±â€¯0.3 µM, respectively. The most active cytotoxic agents were further evaluated in vitro for their VEGFR-2 inhibitory activities, compound 31 showed a high activity against VEGFR-2 with an IC50 value of 2.5 ±â€¯0.04 µM, almost equal to that of sorafenib (IC50 = 2.4 ±â€¯0.05 µM). Further studies revealed the ability of this promising quinazoline derivative 31 to induce apoptosis and arrest cell cycle growth at G2/M phase. In vivo antitumor activities of the synthesized compounds revealed that compounds 30 and 31 possessed significant tumor growth inhibition effect. Molecular docking studies were also performed and finally we can say that VEGFR-2 inhibition confers the reported cytotoxic activities.

Discovery of new quinazolin-4(3H)-ones as VEGFR-2 inhibitors: Design, synthesis, and anti-proliferative evaluation.

Sixteen novel quinazoline-based derivatives were designed and synthesized via modification of the VEGFR-2 reported inhibitor 7 in order to increase the binding affinity of the designed compounds to the receptor active site. The designed compounds were evaluated for their VEGFR-2 inhibitory effects. Inhibiting VEGFR-2 has been set up as a therapeutic strategy for treatment of cancer. The bioactivity of the new compounds was performed against HepG-2, MCF-7 and HCT-116 cell lines. Doxorubicin and sorafenib were used as positive controls. Compound 18d was observed to have promising cytotoxic activity (IC50 = 3.74 ± 0.14, 5.00 ± 0.20 and 6.77 ± 0.27 µM) in comparison to the reference drug doxorubicin (IC50 = 8.28, 9.63 and 7.67 µM) and sorafenib (IC50 = 7.31, 9.40 and 7.21 µM). The most active compounds were tested for their in vitro VEGFR-2 inhibitory activities. Results of VEGFR-2 inhibition were consistent with that of the cytotoxicity data. Thus, compound 18d showed VEGFR-2 inhibitory activity (IC50 = 0.340 ± 0.04 µM) superior to that of the reference drug, sorafenib (IC50 = 0.588 ± 0.06 µM). Furthermore, docking study was performed in order to understand the binding pattern of the new compounds into VEGFR-2 active site. Docking results attributed the potent VEGFR-2 inhibitory effect of the new compounds as they bound to the key amino acids in the active site, Glu883 and Asp1044, as well as their hydrophobic interaction with the receptor hydrophobic pocket. Results of cytotoxic activities, in vitro VEGFR-2 inhibition together with docking study argument the advantages of the synthesized analogues as promising anti-angiogenic agents.

Design, synthesis, molecular modeling, in vivo studies and anticancer activity evaluation of new phthalazine derivatives as potential DNA intercalators and topoisomerase II inhibitors.

Herein we report the design and synthesis of a new series of phthalazine derivatives as Topo II inhibitors and DNA intercalators. The synthesized compounds were in vitro evaluated for their cytotoxic activities against HepG-2, MCF-7 and HCT-116 cell lines. Additionally, Topo II inhibitory activity and DNA intercalating affinity were investigated for the most active compounds as a potential mechanism for the anticancer activity. Compounds 15h, 23c, 32a, 32b, and 33 exhibited the highest activities against Topo II with IC50 ranging from 5.44 to 8.90 µM, while compounds 27 and 32a were found to be the most potent DNA binders at IC50 values of 36.02 and 48.30 µM, respectively. Moreover, compound 32a induced apoptosis in HepG-2 cells and arrested the cell cycle at the G2/M phase. Besides, compound 32a showed Topo II poisoning effect at concentrations of 2.5 and 5 µM, and Topo II catalytic inhibitory effect at a concentration of10 µM. In addition, compound 32b showed in vivo a significant tumor growth inhibition effect. Furthermore, molecular docking studies were carried out against DNA-Topo II complex and DNA to investigate the binding patterns of the designed compounds.

Safety and Molecular-Toxicological Implications of Cannabidiol-Rich Cannabis Extract and Methylsulfonylmethane Co-Administration.

Cannabidiol (CBD) is a biologically active, non-psychotropic component of Cannabis sativa whose popularity has grown exponentially in recent years. Besides a wealth of potential health benefits, ingestion of CBD poses risks for a number of side effects, of which hepatotoxicity and CBD/herb-drug interactions are of particular concern. Here, we investigated the interaction potential between the cannabidiol-rich cannabis extract (CRCE) and methylsulfonylmethane (MSM), a popular dietary supplement, in the mouse model. For this purpose, 8-week-old male C57BL6/J mice received MSM-containing water (80 mg/100 mL) ad libitum for 17 days. During the last three days of treatment, mice received three doses of CRCE administered in sesame oil via oral gavage (123 mg/kg/day). Administration of MSM alone did not result in any evidence of liver toxicity and did not induce expression of mouse cytochrome P450 (CYP) enzymes. Administration of CRCE did produce significant (p < 0.05) increases in Cyp1a2, Cyp2b10, Cyp2c29, Cyp3a4, Cyp3a11, Cyp2c65, and Cyp2c66 messenger RNA, however, this effect was not amplified by MSM/CRCE co-treatment. Similarly, no evidence of liver toxicity was observed in MSM/CRCE dosed mice. In conclusion, short-term MSM/CRCE co-administration did not demonstrate any evidence of hepatotoxicity in the mouse model.

New trends in cannabis potency in USA and Europe during the last decade (2008-2017).

Through the potency monitoring program at the University of Mississippi supported by National Institute on Drug Abuse (NIDA), a total of 18108 samples of cannabis preparations have been analyzed over the last decade, using a validated GC/FID method. The samples are classified as sinsemilla, marijuana, ditchweed, hashish, and hash oil (now referred to as cannabis concentrate). The number of samples received over the last 5 years has decreased dramatically due to the legalization of marijuana either for medical or for recreational purposes in many US states. The results showed that the mean Δ9-THC concentration has increased dramatically over the last 10 years, from 8.9% in 2008 to 17.1% in 2017. The mean Δ9-THC:CBD ratio also rose substantially from 23 in 2008 to 104 in 2017. There was also marked increase in the proportion of hash oil samples (concentrates) seized (0.5-4.7%) and their mean Δ9-THC concentration (6.7-55.7%) from 2008 to 2017. Other potency monitoring programs are also present in several European countries such as The Netherlands, United Kingdom, France, and Italy. These programs have also documented increases in Δ9-THC concentrations and Δ9-THC:CBD ratios in cannabis. These trends in the last decade suggest that cannabis is becoming an increasingly harmful product in the USA and Europe.

Correction to: New trends in cannabis potency in USA and Europe during the last decade (2008-2017).

In the original publication, table 2 contained data that should not have been included in table 2 and also was not discussed in the body of the manuscript.

Analysis of Terpenes in Cannabis sativa L. Using GC/MS: Method Development, Validation, and Application.

Terpenes are the major components of the essential oils present in various Cannabis sativa L. varieties. These compounds are responsible for the distinctive aromas and flavors. Besides the quantification of the cannabinoids, determination of the terpenes in C. sativa strains could be of importance for the plant selection process. At the University of Mississippi, a GC-MS method has been developed and validated for the quantification of terpenes in cannabis plant material, viz., α-pinene, ß-pinene, ß-myrcene, limonene, terpinolene, linalool, α-terpineol, ß-caryophyllene, α-humulene, and caryophyllene oxide. The method was optimized and fully validated according to AOAC (Association of Official Analytical Chemists) guidelines against reference standards of selected terpenes. Samples were prepared by extraction of the plant material with ethyl acetate containing n-tridecane solution (100 µg/mL) as the internal standard. The concentration-response relationship for all analyzed terpenes using the developed method was linear with r2 values > 0.99. The average recoveries for all terpenes in spiked indoor cultivated samples were between 95.0 - 105.7%, with the exception of terpinolene (67 - 70%). The measured repeatability and intermediate precisions (% relative standard deviation) in all varieties ranged from 0.32 to 8.47%. The limit of detection and limit of quantitation for all targeted terpenes were determined to be 0.25 and 0.75 µg/mL, respectively. The proposed method is highly selective, reliable, and accurate and has been applied to the simultaneous determination of these major terpenes in the C. sativa biomass produced by our facility at the University of Mississippi as well as in confiscated marijuana samples.