Medicinal Cannabis: Extended Stability of Cannabis Extracts Produced Using a New Ethanol-based Extraction Method.

Cannabis as a therapeutic agent is increasing in popularity all around the globe, particularly in Western countries, and its potential is now well assessed. On the other hand, each country has its own regulation for the preparation of cannabis macerated oils; in Italy, there are only a few preparation methods allowed. With this work, we aim to perform a stability study of cannabis oils produced with a novel method for the extraction of cannabinoids from cannabis inflorescence. Three different varieties of cannabis were used, with and without the adding of tocopherol acetate as an antioxidant. Cannabinoids were extracted using ethanol at room temperature; then, the solvent was evaporated under reduced pressure and the preparations reconstituted with olive oil. In this work, we assessed the stability of both cannabinoids and terpenes in these formulas over 8 months. Cannabinoid stability was assessed by monitoring the concentrations of THC and CBD, while terpene stability was assessed by monitoring ß-Caryophyllene and α-Humulene concentrations. Stability of the extracts was not influenced by the presence of tocopherol acetate, though refrigeration seems to be detrimental for a long storage of products, especially regarding THC concentrations. The improvements offered by this method reside in the flexibility in controlling the concentration of the extract and the ability to produce highly concentrated oils, alongside the possibility to produce standardized oils despite the variability of the starting plant material.

Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

A sensitive method for determining UDP-glucose: ceramide glucosyltransferase (UGCG) activity in biological samples using deuterated glucosylceramide as acceptor substrate.

Glucosylceramide synthase (UGCG) is a key enzyme in the biosynthesis of glycosphingolipids and its activity is related to the resistance to anticancer drugs and is involved in the derangement of metabolism in various diseases. Moreover, UGCG acts as a major controller of the balanced levels of individual brain sphingolipids that may trigger neurodegeneration in Gaucher disease and in Parkinson disease associated to pathogenic variants in the glucocerebrosidase-encoding gene GBA. We have developed an effective method for determining UGCG activity in vitro using deuterated ceramide as an acceptor, and quantitation of the formed deuterated glucosylceramide by liquid chromatography coupled with tandem mass spectrometry. The method enabled us to determine the kinetic parameters of UGGC and the effect of the inhibitor GZ667161 on the enzyme activity expressed in model cells, as well as to measure UGCG specific activity in human fibroblasts using a simple crude cell homogenate. This novel approach may be useful in determining the actual UGCG activity levels in patient cells and tissues of animal models of diseases, and to study novel drugs targeting glycosphingolipid metabolism.

Recent advances of drugs monitoring in oral fluid and comparison with blood.

The use of alternative matrices in toxicological analyses has been on the rise in clinical and forensic settings. Oral fluid (OF), as non-invasive fluid, has attracted attention in the field of drug screening, both for therapeutic and forensic purposes, as well as for medical diagnosis, clinical management, on-site (real time) doping and for monitoring environmental exposure to toxic substances. A good correlation between OF and blood is now established for drug concentrations. Therefore, OF might be a potential substitute of blood, especially for long-term surveillance (e.g., therapeutic drugs) or to screen a large number of patients, as well as for the development of salivary point-of-care technologies. In this review, we aimed to summarize and critically evaluate the current literature that focused on the comparison of drugs detection in OF and blood specimens.

Development of AMBER Parameters for Molecular Simulations of Selected Boron-Based Covalent Ligands.

Boron containing compounds (BCCs) aroused increasing interest in the scientific community due to their wide application as drugs in various fields. In order to design new compounds hopefully endowed with pharmacological activity and also investigate their conformational behavior, the support of computational studies is crucial. Nevertheless, the suitable molecular mechanics parameterization and the force fields needed to perform these simulations are not completely available for this class of molecules. In this paper, Amber force field parameters for phenyl-, benzyl-, benzylamino-, and methylamino-boronates, a group of boron-containing compounds involved in different branches of the medicinal chemistry, were created. The robustness of the obtained data was confirmed through molecular dynamics simulations on ligand/ß-lactamases covalent complexes. The ligand torsional angles, populated over the trajectory frames, were confirmed by values found in the ligand geometries, located through optimizations at the DFT/B3LYP/6-31g(d) level, using water as a solvent. In summary, this study successfully provided a library of parameters, opening the possibility to perform molecular dynamics simulations of this class of boron-containing compounds.

Fractioning and Compared 1H NMR and GC-MS Analyses of Lanolin Acid Components.

The management of food and food-related wastes represents a growing global issue, as they are hard to recycle and dispose of. Foremost, waste can serve as an important source of biomasses. Particularly, fat-enriched biomasses are receiving more and more attention for their role in the manufacturing of biofuels. Nonetheless, many biomasses have been set aside over the years. Wool wax, also known as lanolin, has a huge potential for becoming a source of typical and atypical fatty acids. The main aim of this work was to evaluate and assess a protocol for the fractioning of fatty acids from lanolin, a natural by-product of the shearing of sheep, alongside the design of a new and rapid quantitative GC-MS method for the derivatization of free fatty acids in fat mixtures, using MethElute™. As the acid portion of lanolin is characterized by the presence of both aliphatic and hydroxylated fatty acids, we also evaluated a procedure for the parting of these two species, by using NMR spectroscopy, benefitting of the different solubilities of the components in organic solvents. At last, we evaluated and quantified the fatty acids and the α-hydroxy fatty acids present in each attained portion, employing both analytical and synthetic standards. The performed analyses, both qualitative and quantitative, showed a good performance in the parting of the different acid components, and GC-MS allowed to speculate that the majority of α-hydroxylated fatty acids is formed of linear saturated carbon chains, while the totality of properly said fatty acids has a much more complex profile.

Computational Design, Synthesis, and Biophysical Evaluation of ß-Amido Boronic Acids as SARS-CoV-2 Mpro Inhibitors.

The COVID-19 pandemic has given a strong impetus to the search for antivirals active on SARS-associated coronaviruses. Over these years, numerous vaccines have been developed and many of these are effective and clinically available. Similarly, small molecules and monoclonal antibodies have also been approved by the FDA and EMA for the treatment of SARS-CoV-2 infection in patients who could develop the severe form of COVID-19. Among the available therapeutic tools, the small molecule nirmatrelvir was approved in 2021. It is a drug capable of binding to the Mpro protease, an enzyme encoded by the viral genome and essential for viral intracellular replication. In this work, by virtual screening of a focused library of ß-amido boronic acids, we have designed and synthesized a focused library of compounds. All of them were biophysically tested by microscale thermophoresis, attaining encouraging results. Moreover, they also displayed Mpro protease inhibitory activity, as demonstrated by performing enzymatic assays. We are confident that this study will pave the way for the design of new drugs potentially useful for the treatment of SARS-CoV-2 viral infection.

Focused Design of Novel Cyclic Peptides Endowed with GABARAP-Inhibiting Activity.

(1) Background: Disfunctions in autophagy machinery have been identified in various conditions, including neurodegenerative diseases, cancer, and inflammation. Among mammalian autophagy proteins, the Atg8 family member GABARAP has been shown to be greatly involved in the autophagy process of prostate cancer cells, supporting the idea that GABARAP inhibitors could be valuable tools to fight the progression of tumors. (2) Methods: In this paper, starting from the X-ray crystal structure of GABARAP in a complex with an AnkirinB-LIR domain, we identify two new peptides by applying in silico drug design techniques. The two ligands are synthesized, biophysically assayed, and biologically evaluated to ascertain their potential anticancer profile. (3) Results: Two cyclic peptides (WC8 and WC10) displayed promising biological activity, high conformational stability (due to the presence of disulfide bridges), and Kd values in the low micromolar range. The anticancer assays, performed on PC-3 cells, proved that both peptides exhibit antiproliferative effects comparable to those of peptide K1, a known GABARAP inhibitor. (4) Conclusions: WC8 and WC10 can be considered new GABARAP inhibitors to be employed as pharmacological tools or even templates for the rational design of new small molecules.

Functional Lipids in Autoimmune Inflammatory Diseases.

Lipids are apolar small molecules known not only as components of cell membranes but also, in recent literature, as modulators of different biological functions. Herein, we focused on the bioactive lipids that can influence the immune responses and inflammatory processes regulating vascular hyperreactivity, pain, leukocyte trafficking, and clearance. In the case of excessive pro-inflammatory lipid activity, these lipids also contribute to the transition from acute to chronic inflammation. Based on their biochemical function, these lipids can be divided into different families, including eicosanoids, specialized pro-resolving mediators, lysoglycerophospholipids, sphingolipids, and endocannabinoids. These bioactive lipids are involved in all phases of the inflammatory process and the pathophysiology of different chronic autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, type-1 diabetes, and systemic lupus erythematosus.

Impact of Lipid Sources on Quality Traits of Medical Cannabis-Based Oil Preparations.

The feasibility of the use of two lipid sources and their impact on the cannabinoid profile, terpene fingerprint, and degradation products in medical cannabis oil preparations during 3 months of refrigerated storage time were investigated. LCHRMS-Orbitrap® and HS-SPME coupled to GC-MS for the investigation of targeted and untargeted cannabinoids, terpenes, and lipid degradation products in Bedrocan® and Bediol® macerated oils were used as analytical approaches. As regards the cannabinoid trend during 90 days of storage, there were no differences between PhEur-grade olive oil (OOPH) and medium-chain triglycerides oil (MCT oil) coupled to a good stability of preparations for the first 60 days both in Bedrocan® and Bediol® oils. MCT lipid source extracted a significant concentration of terpenes compared to olive oil. Terpenes showed a different scenario since MCT oil displayed the strongest extraction capacity and conservation trend of all compounds during the shelf life. Terpenes remained stable throughout the entire storage period in MCT formulations while a significant decrease after 15 and 30 days in Bediol® and Bedrocan® was observed in olive oil. Therefore, MCT oil could be considered a more suitable lipid source compared to olive oil involved in the extraction of medical cannabis for magistral preparations.