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Policosanols (PCs) are bioactive compounds extracted from different natural waxes. In this work, the purification, characterization and assessment of the antioxidant and anti-inflammatory activity was carried out on PCs from an innovative source, i.e. a waxy material from supercritical-fluid extraction (SFE) of non-psychoactive Cannabis sativa L. (hemp) inflorescences. Starting from this material, PCs were obtained by microwave-assisted trans -esterification and hydrolysis, followed by preparative liquid chromatography under normal phase conditions. The purified product was characterized using high-performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). In vitro cell-free and cell-based antioxidant and anti-inflammatory assays were then performed to assess their bioactivity. HPLC-ELSED analysis of the purified mixture from hemp wax revealed C 26 OH and C 28 OH as the main compounds. In vitro assays indicated an inhibition of intracellular reactive oxygen species (ROS) production, a reduction of nuclear factor kappa B (NF-κB) activation and of the activity of the neutrophil elastase. Immunoblotting assays allowed us to hypothesize the mechanism of action of the compounds of interest, given the higher levels of MAPK-activated protein kinase 2 (MK2) and heme oxygenase-1 (HO-1) protein expression in the PC pretreated HaCaT cells. In conclusion, even if more research is needed to unveil other molecular mechanisms involved in hemp PC activity, the results of this work suggest that these compounds may have potential for use in oxinflammation processes., Competing Interests: Federica Pellati reports financial support was provided by University of Modena and Reggio Emilia, Department of Life Sciences (FAR2023). The other authors have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

This study reports on the synthesis and evaluation of novel compounds replacing the nitrogen-containing heterocyclic ring on the chemical backbone structure of cytochrome P450 17α-hydroxylase/12,20-lyase (CYP17A1) inhibitors with a phenyl bearing a sulfur-based substituent. Initial screening revealed compounds with marked inhibition of CYP17A1 activity. The selectivity of compounds was thereafter determined against cytochrome P450 21-hydroxylase, cytochrome P450 3A4, and cytochrome P450 oxidoreductase. Additionally, the compounds showed weak inhibitory activity against aldo-keto reductase 1C3 (AKR1C3). The compounds' impact on steroid hormone levels was also assessed, with some notable modulatory effects observed. This work paves the way for developing more potent dual inhibitors specifically targeting CYP17A1 and AKR1C3.

Bevacizumab (BVZ) is a monoclonal antibody that binds to human vascular endothelial growth factor A (VEGF-A) and inhibits the interaction between VEGF-A and VEGF receptors, thus blocking the angiogenesis. Repeated intravitreal injections of BVZ for the treatment of ocular pathologies that present an excessive proliferation results in a low patience compliance. BVZ is specially indicated for the treatment of diabetic and degenerative retinopathy. In the present study, we designed lipid nanoparticles (NPs) as a BVZ sustained drug delivery system for reducing the frequency of administration. We used a simple and highly efficient procedure, "Cold dilution of microemulsions", to obtain spherical NPs with mean diameters of 280-430 nm, Zeta potentials between -17 and -31 mV, and drug entrapment efficiencies between 50 to 90%. This study focused on the biochemical and biophysical stabilities of BVZ after entrapment in NPs. SDS-PAGE electrophoretic analysis and circular dichroism, dynamic light scattering, and scanning electron microscopy were used to characterize BVZ-loaded NPs. The biocompatibility was assessed by in vitro cell compatibility studies using the ARPE-19 cell line. Thus, in this work, a stable BVZ-loaded system was obtained. In addition, several studies have shown that BVZ is released slowly from the lipid matrix and that this system is biocompatible. The results are promising and the developed NPs could be exploited to create a new, potentially effective and minimally invasive treatment of intraocular diseases.

Purpose: Oropharynx squamous cell carcinoma (OPSCC) is a subtype of head and neck squamous cell carcinoma (HNSCC) arising from the base of the tongue, lingual tonsils, tonsils, oropharynx or pharynx. The majority of HPV-positive OPSCCs has a good prognosis, but a fraction of them has a poor prognosis, similar to HPV-negative OPSCCs. An in-depth understanding of the molecular mechanisms underlying OPSCC is mandatory for the identification of novel prognostic biomarkers and/or novel therapeutic targets., Methods: 14 HPV-positive and 15 HPV-negative OPSCCs with 5-year follow-up information were subjected to gene expression profiling and, subsequently, compared to three extensive published OPSCC cohorts to define robust biomarkers for HPV-negative lesions. Validation of Aldo-keto-reductases 1C3 (AKR1C3) by qRT-PCR was carried out on an independent cohort (n = 111) of OPSCC cases. In addition, OPSCC cell lines Fadu and Cal-27 were treated with Cisplatin and/or specific AKR1C3 inhibitors to assess their (combined) therapeutic effects., Results: Gene set enrichment analysis (GSEA) on the four datasets revealed that the genes down-regulated in HPV-negative samples were mainly involved in immune system, whereas those up-regulated mainly in glutathione derivative biosynthetic and xenobiotic metabolic processes. A panel of 30 robust HPV-associated transcripts was identified, with AKR1C3 as top-overexpressed transcript in HPV-negative samples. AKR1C3 expression in 111 independent OPSCC cases positively correlated with a worse survival, both in the entire cohort and in HPV-positive samples. Pretreatment with a selective AKR1C3 inhibitor potentiated the effect of Cisplatin in OPSCC cells exhibiting higher basal AKR1C3 expression levels., Conclusions: We identified AKR1C3 as a potential prognostic biomarker in OPSCC and as a potential drug target whose inhibition can potentiate the effect of Cisplatin.

Bevacizumab (BVZ), a recombinant humanized monoclonal antibody, has recently been proposed as a topical application in the treatment of anterior segment neovascularization; however, as there are some disadvantages in the administration of common eye-drops, ophthalmic topical drug delivery systems are under study to improve the precorneal residence time, reducing the frequency of administration. In this work, oil-in-water and water-in-oil BVZ-loaded microemulsions are developed, able to increase their viscosity, either by the formation of a liquid-crystalline structure upon aqueous dilution, thanks to the presence of Epikuron ® 200 and polysorbate 80, or by body-temperature-induced jellification for the presence of Pluronic ® F127 aqueous solution as an external phase. In oil-in-water microemulsion, hydrophobic ion pairs of BVZ were also prepared, and their incorporation was determined by release studies. Microemulsions were characterized for rheological behavior, corneal opacity, in vitro corneal permeation, and adhesion properties. The studied microemulsions were able to incorporate BVZ (from 1.25 to 1.6 mg/mL), which maintained dose-dependent activity on retinal pigment epithelial ARPE-19 cell lines. BVZ loaded in microemulsions permeated the excised cornea easier (0.76-1.56% BVZ diffused, 4-20% BVZ accumulated) than BVZ commercial solution (0.4% BVZ diffused, 5% accumulated) and only a mild irritation effect on the excised cornea was observed. The good adhesion properties as well the increased viscosity after application, under conditions that mimic the corneal environment (from 1 × 10 3 to more than 100 × 10 3 mPa·s), might prolong precorneal residence time, proving these systems could be excellent topical BVZ release systems.

Endophthalmitis is a rare, but serious, intravitreal inflammatory disorder that can arise after cataract surgery. The intracameral injection of 1 mg cefuroxime (CEF) followed by three-times daily antibiotic topical administration for a week is generally recognized as the routine method of prophylaxis after cataract surgery. This procedure is controversial because of both the low efficacy and the low adherence to therapy by elderly patients. A unique slow release antibiotic intravitreal injection could solve these problems. The objective of the present study was to design ophthalmic nanocomposite delivery systems based on in situ gelling formulations that undergo sol-to-gel transition upon change in temperature to prolong the effect of CEF. Oil in water (O/W) microemulsion (µE) and solid lipid nanoparticles (SLN), obtained with an innovative formulation technology called cold microemulsion dilution , were evaluated as ocular drug delivery systems for CEF. Drug entrapment efficiency up to 80% was possible by esterifying CEF with 1-dodecanol to obtain dodecyl-CEF (dCEF). Both dCEF-loaded SLN and µE were then added with Pluronic®F127 (20% w / v ) to obtain a nanocomposite hydrogel-based long acting system. The prepared thermosensitive formulations were evaluated for their physical appearance, drug content, gelation temperature, injectability and rheological properties, in vitro release studies and stability studies. Moreover, cell proliferation assays on human retinal pigment epithelial ARPE-19 cells were performed to evaluate the influence of this innovative system on the cellular viability. In addition, minimal inhibitory concentration (MIC) was assessed for both CEF and dCEF, revealing the need of dCEF hydrolysis for the antimicrobial activity. Although further experimental investigations are required, the physico-chemical characterization of the nanocomposite hydrogels and the preliminary in vitro release studies highlighted the potential of these systems for the sustained release of CEF.

Tetrahydroisoquinoline (THIQ) is a key structural component in many biologically active molecules including natural products and synthetic pharmaceuticals. Here, we report on the use of transition-metal mediated [2 + 2 + 2] cyclotrimerisation of alkynes to generate tricyclic THIQs with potential to selectively inhibit AKR1C3., (This journal is © The Royal Society of Chemistry 2019.)

Aldo-keto reductase 1C3 (AKR1C3) is an attractive target in drug design for its role in resistance to anticancer therapy. Several nonsteroidal anti-inflammatory drugs such as indomethacin are known to inhibit AKR1C3 in a nonselective manner because of COX-off target effects. Here we designed two indomethacin analogues by proposing a bioisosteric connection between the indomethacin carboxylic acid function and either hydroxyfurazan or hydroxy triazole rings. Both compounds were found to target AKR1C3 in a selective manner. In particular, hydroxyfurazan derivative is highly selective for AKR1C3 over the 1C2 isoform (up to 90-times more) and inactive on COX enzymes. High-resolution crystal structure of its complex with AKR1C3 shed light onto the binding mode of the new inhibitors. In cell-based assays (on colorectal and prostate cancer cells), the two indomethacin analogues showed higher potency than indomethacin. Therefore, these two AKR1C3 inhibitors can be used to provide further insight into the role of AKR1C3 in cancer., Competing Interests: The authors declare no competing financial interest.

There is a growing body of evidence to suggest that cannabinoids are beneficial for a range of clinical conditions, including pain, inflammation, epilepsy, sleep disorders, the symptoms of multiple sclerosis, anorexia, schizophrenia and other conditions. The transformation of cannabinoids from herbal preparations into highly regulated prescription drugs is therefore progressing rapidly. The development of such drugs requires well-controlled clinical trials to be carried out in order to objectively establish therapeutic efficacy, dose ranges and safety. The low oral bioavailability of cannabinoids has led to feasible methods of administration, such as the transdermal route, intranasal administration and transmucosal adsorption, being proposed. The highly lipophilic nature of cannabinoids means that they are seen as suitable candidates for advanced nanosized drug delivery systems, which can be applied via a range of routes. Nanotechnology-based drug delivery strategies have flourished in several therapeutic fields in recent years and numerous drugs have reached the market. This review explores the most recent developments, from preclinical to advanced clinical trials, in the cannabinoid delivery field, and focuses particularly on pain and inflammation treatment. Likely future directions are also considered and reported.

A comparative study on post-squalene sterol synthesis in intact yeast cells and spheroplasts was carried out with strains from three genera (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris) as well as with engineered S. cerevisiae cells altered in regard to the late ergosterol synthesis pathway. A common outcome of incubation experiments with radioactive acetate was that in intact cells the metabolic pathway flows till its specific end product (ergosterol and its precursor, depending on the enzyme deficiency), whereas in spheroplasts the pathway was stalled some step upstream. For example, in spheroplasts from wt strains, non-cyclic triterpenes squalene and oxidosqualene accumulated as though the metabolic path was kept from producing steroid-shaped molecules different from the end product. Accumulation of non-cyclic triterpenes was observed also in spheroplasts from S. cerevisiae cells lacking 3-ketosteroid reductase activity, an enzyme belonging to the C4-demethylase complex. When production of cyclic triterpenes was compromised by loss or poor functionality of oxidosqualene cyclase (EC 5.4.99.7), the difference between intact cells and spheroplasts was still remarkable, yet limited to the different oxido/dioxidosqualene ratio. The characteristics of spheroplasts as non-proliferating cells may partially explain the observed differences in post-squalene pathway from intact cells. We cannot say if the difference in metabolic pathways in spheroplasts and intact cells is a rule. We think, however, that it is worthwhile to search for an answer, as a wider picture of the points where the metabolic pathways are stalled in spheroplasts could provide original ideas about the metabolic network in yeast.

Aberrant cholesterol homeostasis and biosynthesis has been observed in different tumour types. This paper investigates the role of the post-squalenic enzyme of cholesterol biosynthesis, oxidosqualene cyclase (OSC), in regulating tumour angiogenesis and metastasis dissemination in mouse models of cancer. We showed that Ro 48-8071, a selective inhibitor of OSC, reduced vascular density and increased pericyte coverage, with a consequent inhibition of tumour growth in a spontaneous mouse model of pancreatic tumour (RIP-Tag2) and two metastatic mouse models of human colon carcinoma (HCT116) and pancreatic adenocarcinoma (HPAF-II). Remarkably, the inhibition of OSC hampered metastasis formation in HCT116 and HPAF-II models. Ro 48-8071 induced tumour vessel normalization and enhanced the anti-tumoral and anti-metastatic effects of 5-fluorouracil (5-FU) in HCT116 mice. Ro 48-8071 exerted a strong anti-angiogenic activity by impairing endothelial cell adhesion and migration, and by blocking vessel formation in angiogenesis assays. OSC inhibition specifically interfered with the PI3K pathway. According to in vitro results, Ro 48-8071 specifically inhibited Akt phosphorylation in both cancer cells and tumour vasculature in all treated models. Thus, our results unveil a crucial role of OSC in the regulation of cancer progression and tumour angiogenesis, and indicate Ro 48-8071 as a potential novel anti-angiogenic and anti-metastatic drug.

In oxidosqualene cyclases (OSCs), an enzyme which has been extensively studied as a target for hypocholesterolemic or antifungal drugs, a lipophilic channel connects the surface of the protein with the active site cavity. Active site and channel are separated by a narrow constriction operating as a mobile gate for the substrate passage. In Saccharomyces cerevisiae OSC, two aminoacidic residues of the channel/constriction apparatus, Ala525 and Glu526, were previously showed as critical for maintaining the enzyme functionality. In this work sixteen novel mutants, each bearing a substitution at or around the channel constrictions, were tested for their enzymatic activity. Modelling studies showed that the most functionality-lowering substitutions deeply alter the H-bond network involving the channel/constriction apparatus. A rotation of Tyr239 is proposed as part of the mechanism permitting the access of the substrate to the active site. The inhibition of OSC by squalene was used as a tool for understanding whether the residues under study are involved in a pre-catalytic selection and docking of the substrate oxidosqualene.

erg26-1ts cells harbor defects in the 4alpha-carboxysterol-C3 dehydrogenase activity necessary for conversion of 4,4-dimethylzymosterol to zymosterol. Mutant cells accumulate toxic 4-carboxysterols and are inviable at high temperature. A genetic screen aimed at cloning recessive mutations remediating the temperature sensitive growth defect has resulted in the isolation of four complementation groups, ets1-4 (erg26-1ts temperature sensitive suppressor). We describe the characterization of ets1-1 and ets2-1. Gas chromatography/mass spectrometry analyses demonstrate that erg26-1ts ets1-1 and erg26-1ts ets2-1 cells do not accumulate 4-carboxysterols, rather these cells have increased levels of squalene and squalene epoxide, respectively. ets1-1 and ets2-1 cells accumulate these same sterol intermediates. Chromosomal integration of ERG1 ERG7 at their loci in erg26-1ts ets1-1 and erg26-1ts and ets2-1 mutants, respectively, results in the loss of accumulation of squalene and squalene epoxide, re-accumulation of 4-carboxysterols and cell inviability at high temperature. Enzymatic assays demonstrate that mutants harboring the ets1-1 allele have decreased squalene epoxidase activity, while those containing the ets2-1 allele show weakened oxidosqualene cyclase activity. Thus, ETS1 and ETS2 are allelic to ERG1 and ERG7, respectively. We have mapped mutations within the erg1-1/ets1-1 (G247D) and erg7-1/ets2-1 (D530N, V615E) alleles that suppress the inviability of erg26-1ts at high temperature, and cause accumulation of sterol intermediates and decreased enzymatic activities. Finally using erg1-1 and erg7-1 mutant strains, we demonstrate that the expression of the ERG25/26/27 genes required for zymosterol biosynthesis are coordinately transcriptionally regulated, along with ERG1 and ERG7, in response to blocks in sterol biosynthesis. Transcriptional regulation requires the transcription factors, Upc2p and Ecm22p.

Squalene-hopene cyclase (SHC) is a useful model enzyme for predicting molecular interactions with oxidosqualene cyclase (OSC). Structure--activity relationships were investigated for numerous coumarin-derived inhibitors of SHC, and structural simplifications are suggested. Both umbelliferone and 2,4-dihydroxyacetophenone provide convenient starting nuclei for the design of SHC inhibitors. Derivatives bearing an omega-epoxyfarnesyl moiety or just a plain alkyl chain showed an inhibitory effect on a recombinant SHC from Alicyclobacillus acidocaldarius expressed in Escherichia coli.

Oxidosqualene cyclase of the yeast encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study (Athenstaedt, K., Zweytick, D., Jandrositz, A, Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448), Erg7p was identified as a component of yeast lipid particles. Here, we present evidence that Erg7p is almost exclusively associated with this compartment as shown by analysis of enzymatic activity, Western blot analysis, and in vivo localization of Erg7p-GFP. Occurrence of oxidosqualene cyclase in other organelles including the endoplasmic reticulum is negligible. In an erg7 deletion strain or in wild-type cells treated with an inhibitor of oxidosqualene cyclase, the substrate of Erg7p, oxidosqualene, accumulated mostly in lipid particles. Storage in lipid particles of this intermediate produced in excess may provide a possibility to exclude this membrane-perturbing component from other organelles. Thus, our data provide evidence that lipid particles are not only a depot for neutral lipids, but also participate in coordinate sterol metabolism and trafficking and serve as a storage site for compounds that may negatively affect membrane integrity.