Tapinarof (3,5-dihydroxy-4-isopropylstilbene) is a therapeutic agent used in the treatment of psoriasis (VTAMA®). In this study, we examined the redox behaviour, (photo)stability, (photo)toxicity and (bio)transformation of tapinarof in the context of a structure-activity relationship study. Selected derivatives of the structurally related tapinarof were investigated, namely resveratrol, pterostilbene, pinosylvin and its methyl ether. Tapinarof undergoes electrochemical oxidation in a neutral aqueous medium at a potential of around +0.5 V (vs. Ag|AgCl|3M KCl). The anodic reaction of this substance is a proton-dependent irreversible and adsorption-driven process. The pKa value of tapinarof corresponds to 9.19 or 9.93, based on empirical and QM calculation approach, respectively. The oxidation potentials of tapinarof and its analogues correlate well with their HOMO (highest occupied molecular orbital) energy level. The ability to scavenge the DPPH radical decreased in the order trolox ≥ resveratrol > pterostilbene > tapinarof > pinosylvin ≫ pinosylvin methyl ether. It was also confirmed that tapinarof, being a moderate electron donor, is able to scavenge the ABTS radical and inhibit lipid peroxidation. The 4'-OH group plays a pivotal role in antioxidant action of stilbenols. During the stability studies, it was shown that tapinarof is subject to spontaneous degradation under aqueous conditions, and its degradation is accelerated at elevated temperatures and after exposure to UVA (315-399 nm) radiation. In aqueous media at pH 7.4, we observed an ∼50 % degradation of tapinarof after 48 h at laboratory temperature. The main UVA photodegradation processes include dihydroxylation and hydration. In conclusion, the phototoxic effect of tapinarof on a human keratinocytes cell line (HaCaT) was evaluated. Tapinarof exhibited a clear phototoxic effect, similar to phototoxic standard chlorpromazine. The IC50 values of the cytotoxicity and phototoxic effects of tapinarof correspond to 27.6 and 3.7 μM, respectively. The main HaCaT biotransformation products of tapinarof are sulfates and glucuronides.
- MeSH
- antioxidancia farmakologie chemie MeSH
- buněčné linie keratinocytů HaCaT MeSH
- fototoxická dermatitida MeSH
- keratinocyty * účinky léků metabolismus účinky záření MeSH
- kůže účinky léků metabolismus účinky záření patologie MeSH
- lidé MeSH
- oxidace-redukce * MeSH
- resveratrol farmakologie analogy a deriváty chemie MeSH
- stilbeny * farmakologie chemie MeSH
- ultrafialové záření MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: The aim of this study was to: (1) evaluate the anti-inflammatory effects of cannabidiol (CBD) on primary cultures of human gingival fibroblasts (HGFs) and (2) to clinically monitor the effect of CBD in subjects with periodontitis. BACKGROUND: The use of phytocannabinoids is a new approach in the treatment of widely prevalent periodontal disease. MATERIALS AND METHODS: Cannabinoid receptors were analyzed by western blot and interleukin production detected using enzyme immunoassay. Activation of the Nrf2 pathway was studied via monitoring the mRNA level of heme oxygenase-1. Antimicrobial effects were determined by standard microdilution and 16S rRNA screening. In the clinical part, a placebo-control double-blind randomized study was conducted (56 days) in three groups (n = 90) using dental gel without CBD (group A) and with 1% (w/w) CBD (group B) and corresponding toothpaste (group A - no CBD, group B - with CBD) for home use to maintain oral health. Group C used dental gel containing 1% chlorhexidine digluconate (active comparator) and toothpaste without CBD. RESULTS: Human gingival fibroblasts were confirmed to express the cannabinoid receptor CB2. Lipopolysaccharide-induced cells exhibited increased production of pro-inflammatory IL-6 and IL-8, with deceasing levels upon exposure to CBD. CBD also exhibited antimicrobial activities against Porphyromonas gingivalis, with an MIC of 1.5 μg/mL. Activation of the Nrf2 pathway was also demonstrated. In the clinical part, statistically significant improvement was found for the gingival, gingival bleeding, and modified gingival indices between placebo group A and CBD group B after 56 days. CONCLUSIONS: Cannabidiol reduced inflammation and the growth of selected periodontal pathogenic bacteria. The clinical trial demonstrated a statistically significant improvement after CBD application. No adverse effects of CBD were reported by patients or observed upon clinical examination during the study. The results are a promising basis for a more comprehensive investigation of the application of non-psychotropic cannabinoids in dentistry.
- MeSH
- antiflogistika terapeutické užití farmakologie MeSH
- chlorhexidin terapeutické užití farmakologie analogy a deriváty MeSH
- dospělí MeSH
- dvojitá slepá metoda MeSH
- faktor 2 související s NF-E2 MeSH
- fibroblasty * účinky léků MeSH
- gingiva * účinky léků MeSH
- gingivitida * farmakoterapie MeSH
- hemoxygenasa-1 MeSH
- interleukin-6 analýza MeSH
- interleukin-8 účinky léků MeSH
- kanabidiol * farmakologie terapeutické užití MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- parodontitida farmakoterapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- randomizované kontrolované studie MeSH
This study examined the biotransformation of phytocannabinoids in human hepatocytes. The susceptibility of the tested compounds to transformations in hepatocytes exhibited the following hierarchy: cannabinol (CBN) > cannabigerol (CBG) > cannabichromene (CBC) > cannabidiol (CBD). Biotransformation included hydroxylation, oxidation to a carboxylic acid, dehydrogenation, hydrogenation, dehydration, loss/shortening of alkyl, glucuronidation and sulfation. CBN was primarily metabolized by oxidation of a methyl to a carboxylic acid group, while CBD, CBG and CBC were preferentially metabolized by direct glucuronidation. The study also screened for the activity of recombinant human cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), which could catalyze the hydroxylation and glucuronidation of the tested compounds, respectively. We found that CBD was hydroxylated mainly by CYPs 2C8, 2C19, 2D6; CBN by 1A2, 2C9, 2C19 and 2D6; and CBG by 2B6, 2C9, 2C19 and 2D6. CBC exhibited higher susceptibility to CYP-mediated transformation than the other tested compounds, mainly with CYPs 1A2, 2B6, 2C8, 2C19, 2D6 and 3A4 being involved. Further, CBD was primarily glucuronidated by UGTs 1A3, 1A7, 1A8, 1A9 and 2B7; CBN by 1A7, 1A8, 1A9 and 2B7; CBG by 1A3, 1A7, 1A8, 1A9, 2B4, 2B7 and 2B17; and the glucuronidation of CBC was catalyzed by UGTs 1A1, 1A8, 1A9 and 2B7.
- MeSH
- biotransformace MeSH
- glukuronosyltransferasa metabolismus MeSH
- jaterní mikrozomy * metabolismus MeSH
- kyseliny karboxylové MeSH
- lidé MeSH
- systém (enzymů) cytochromů P-450 * metabolismus MeSH
- uridindifosfát metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cannabidiol (CBD) and cannabigerol (CBG) are the two main non-psychotropic phytocannabinoids with high application potential in drug development. Both substances are redox-active and are intensively investigated for their cytoprotective and antioxidant action in vitro. In this study, we focused on an in vivo safety evaluation and the effect of CBD and CBG on the redox status in rats in a 90-d experiment. The substances were administered orogastrically in a dose of 0.66 mg synthetic CBD or 0.66 mg/1.33 mg CBG/kg/day. CBD produced no changes in the red or white blood count or biochemical blood parameters in comparison to the control. No deviations in the morphology or histology of the gastrointestinal tract and liver were observed. After 90 d of CBD exposure, a significant improvement in redox status was found in the blood plasma and liver. The concentration of malondialdehyde and carbonylated proteins was reduced compared to the control. In contrast to CBD, total oxidative stress was significantly increased and this was accompanied by an elevated level of malondialdehyde and carbonylated proteins in CBG-treated animals. Hepatotoxic (regressive changes) manifestations, disruption in white cell count, and alterations in the ALT activity, level of creatinine and ionized calcium were also found in CBG-treated animals. Based on liquid chromatography-mass spectrometry analysis, CBD/CBG accumulated in rat tissues (in the liver, brain, muscle, heart, kidney and skin) at a low ng level per gram. Both CBD and CBG molecular structures include a resorcinol moiety. In CBG, there is an extra dimethyloctadienyl structural pattern, which is most likely responsible for the disruption to the redox status and hepatic environment. The results are valuable to further investigation of the effects of CBD on redox status and should contribute towards opening up critical discussion on the applicability of other non-psychotropic cannabinoids.
- MeSH
- kanabidiol * toxicita MeSH
- kanabinoidy * toxicita MeSH
- krysa rodu rattus MeSH
- oxidace-redukce MeSH
- vápník MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lipid nitroalkenes - nitro-fatty acids (NO2-FAs) are formed in vivo via the interaction of reactive nitrogen species with unsaturated fatty acids. The resulting electrophilic NO2-FAs play an important role in redox homeostasis and cellular stress response. This study investigated the physicochemical properties and reactivity of two NO2-FAs: 9/10-nitrooleic acid (1) and its newly prepared 1-monoacyl ester, (E)-2,3-hydroxypropyl 9/10-nitrooctadec-9-enoate (2), both synthesized by a direct radical nitration approach. Compounds 1 and 2 were investigated in an aqueous medium and after incorporation into lipid nanoparticles prepared from 1-monoolein, cubosomes 1@CUB and 2@CUB. Using an electrochemical analysis and LC-MS, free 1 and 2 were found to be unstable under acidic conditions, and their degradation occurred in an aqueous environment within a few minutes or hours. This degradation was associated with the production of the NO radical, as confirmed by fluorescence assay. In contrast, preparations 1@CUB and 2@CUB exhibited a significant increase in the stability of the loaded 1 and 2 up to several days to weeks. In addition to experimental data, density functional theory-based calculation results on the electronic structure and structural variability (open and closed configuration) of 1 and 2 were obtained. Finally, experiments with a human HaCaT keratinocyte cell line demonstrated the ability of 1@CUB and 2@CUB to penetrate through the cytoplasmic membrane and modulate cellular pathways, which was exemplified by the Keap1 protein level monitoring. Free 1 and 2 and the cubosomes prepared from them showed cytotoxic effect on HaCaT cells with IC50 values ranging from 1 to 8 μM after 24 h. The further development of cubosomal preparations with embedded electrophilic NO2-FAs may not only contribute to the field of fundamental research, but also to their application using an optimized lipid delivery vehicle.
- MeSH
- dusíkaté sloučeniny MeSH
- faktor 2 související s NF-E2 MeSH
- KEAP-1 MeSH
- lidé MeSH
- mastné kyseliny * MeSH
- oxid dusnatý * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
2,3-Dehydrosilybin A and 2,3-dehydrosilybin B are a pair of enantiomers formed by the oxidation of the natural flavonolignans silybin A and silybin B, respectively. However, the antioxidant activity of 2,3-dehydrosilybin molecules is much stronger than that of their precursors. Here, we investigated the biotransformation of pure 2,3-dehydrosilybin A and 2,3-dehydrosilybin B in isolated human hepatocytes, and we also aimed to identify human UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) with activity toward their respective enantiomers. After incubation with hepatocytes, both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B were converted to hydroxyl derivatives, methylated hydroxyl derivatives, methyl derivatives, sulfates, and glucuronides. The products of direct conjugations predominated over those of oxidative metabolism, and glucuronides were the most abundant metabolites. Furthermore, we found that recombinant human UGTs 1A1, 1A3, 1A7, 1A8, 1A9, and 1A10 were capable of catalyzing the glucuronidation of both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B. UGTs 1A1 and 1A7 showed the highest activity toward 2,3-dehydrosilybin A, and UGT1A9 showed the highest activity toward 2,3-dehydrosilybin B. The sulfation of 2,3-dehydrosilybin A and B was catalyzed by SULTs 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, and 1E1, of which SULT1A3 exhibited the highest activity toward both enantiomers. We conclude that 2,3-dehydrosilybin A and B are preferentially metabolized by conjugation reactions, and that several human UGT and SULT enzymes may play a role in these conjugations.
- Publikační typ
- časopisecké články MeSH
- MeSH
- kanabidiol * farmakologie MeSH
- krysa rodu rattus MeSH
- tetrahydrokanabinol * farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- dopisy MeSH
In this contribution, a comprehensive study of the redox transformation, electronic structure, stability and photoprotective properties of phytocannabinoids is presented. The non-psychotropic cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), cannabichromene (CBC), and psychotropic tetrahydrocannabinol (THC) isomers and iso-THC were included in the study. The results show that under aqueous ambient conditions at pH 7.4, non-psychotropic cannabinoids are slight or moderate electron-donors and they are relatively stable, in the following order: CBD > CBG ≥ CBN > CBC. In contrast, psychotropic Δ9-THC degrades approximately one order of magnitude faster than CBD. The degradation (oxidation) is associated with the transformation of OH groups and changes in the double-bond system of the investigated molecules. The satisfactory stability of cannabinoids is associated with the fact that their OH groups are fully protonated at pH 7.4 (pKa is ≥ 9). The instability of CBN and CBC was accelerated after exposure to UVA radiation, with CBD (or CBG) being stable for up to 24 h. To support their topical applications, an in vitro dermatological comparative study of cytotoxic, phototoxic and UVA or UVB photoprotective effects using normal human dermal fibroblasts (NHDF) and keratinocytes (HaCaT) was done. NHDF are approx. twice as sensitive to the cannabinoids' toxicity as HaCaT. Specifically, toxicity IC50 values for CBD after 24 h of incubation are 7.1 and 12.8 μM for NHDF and HaCaT, respectively. None of the studied cannabinoids were phototoxic. Extensive testing has shown that CBD is the most effective protectant against UVA radiation of the studied cannabinoids. For UVB radiation, CBN was the most effective. The results acquired could be used for further redox biology studies on phytocannabinoids and evaluations of their mechanism of action at the molecular level. Furthermore, the UVA and UVB photoprotectivity of phytocannabinoids could also be utilized in the development of new cannabinoid-based topical preparations.
- MeSH
- antioxidancia * farmakologie MeSH
- kanabidiol * MeSH
- lidé MeSH
- tetrahydrokanabinol MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Silybum marianum (milk thistle) is a medicinal plant used for producing the hepatoprotective remedy silymarin. Its main bioactive constituents, including silybin and related flavonolignans, can be metabolized directly by phase II conjugation reactions. This study was designed to identify UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of six silymarin flavonolignans, namely silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin. UHPLC-MS analyses showed that all of the tested compounds, both individually and in silymarin, were glucuronidated by human liver microsomes, and that glucuronidation was the main metabolic transformation in human hepatocytes. Further, each compound was glucuronidated by multiple recombinant human UGT enzymes. UGTs 1A1, 1A3, 1A8 and 1A9 were able to conjugate all of the tested flavonolignans, and some of them were also metabolized by UGTs 1A6, 1A7, 1A10, 2B7 and 2B15. In contrast, no glucuronides were produced by UGTs 1A4, 2B4, 2B10 and 2B17. With silymarin, we found that UGT1A1 and, to a lesser extent UGT1A9, were primarily responsible for the glucuronidation of the flavonolignan constituents. It is concluded that the metabolism of silymarin flavonolignans may involve multiple UGT enzymes, of which UGT1A1 appears to play the major role in the glucuronidation. These results may be relevant for future research on the metabolism of flavonolignans in humans.
- MeSH
- dospělí MeSH
- flavonolignany metabolismus MeSH
- glukuronidy metabolismus MeSH
- glukuronosyltransferasa metabolismus MeSH
- hepatocyty metabolismus MeSH
- jaterní mikrozomy metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- ostropestřec mariánský metabolismus MeSH
- silibinin metabolismus MeSH
- silymarin analogy a deriváty metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
