Deciphering the Molecular Mechanisms of Reactive Metabolite Formation in the Mechanism-Based Inactivation of Cytochrome p450 1B1 by 8-Methoxypsoralen and Assessing the Driving Effect of phe268.

This study provides a comprehensive computational exploration of the inhibitory activity and metabolic pathways of 8-methoxypsoralen (8-MP), a furocoumarin derivative used for treating various skin disorders, on cytochrome P450 (P450). Employing quantum chemical DFT calculations, molecular docking, and molecular dynamics (MD) simulations analyses, the biotransformation mechanisms and the active site binding profile of 8-MP in CYP1B1 were investigated. Three plausible inactivation mechanisms were minutely scrutinized. Further analysis explored the formation of reactive metabolites in subsequent P450 metabolic processes, including covalent adduct formation through nucleophilic addition to the epoxide, 8-MP epoxide hydrolysis, and non-CYP-catalyzed epoxide ring opening. Special attention was paid to the catalytic effect of residue Phe268 on the mechanism-based inactivation (MBI) of P450 by 8-MP. Energetic profiles and facilitating conditions revealed a slight preference for the C4'=C5' epoxidation pathway, while recognizing a potential kinetic competition with the 8-OMe demethylation pathway due to comparable energy demands. The formation of covalent adducts via nucleophilic addition, particularly by phenylalanine, and the generation of potentially harmful reactive metabolites through autocatalyzed ring cleavage are likely to contribute significantly to P450 metabolism of 8-MP. Our findings highlight the key role of Phe268 in retaining 8-MP within the active site of CYP1B1, thereby facilitating initial oxygen addition transition states. This research offers crucial molecular-level insights that may guide the early stages of drug discovery and risk assessment related to the use of 8-MP.

8-methoxypsoralen protects against acetaminophen-induced liver injury by antagonising Cyp2e1 in mice.

This study aimed to investigate the effect and mechanism of 8-methoxypsoralen (8-MOP) on acetaminophen (APAP)-induced hepatotoxicity in mice. The study found that 1 h after intraperitoneal injection of 300 mg/kg APAP, treatment with 40 mg/kg, 80 mg/kg and 120 mg/kg 8-MOP could reduce serum transaminase level and histopathological liver necrosis area. Elevated mRNA expression of liver inflammatory mediators caused by excessive APAP was also reversed. 8-MOP significantly reduced APAP-induced hepatotoxicity dose-dependently, and the highest therapeutic dose of 8-MOP (120 mg/kg) had no harmful effects on the liver. Cocktail probe assay revealed that 8-MOP can inhibit Cyp2e1 enzymatic activities of mice, thereby reducing the production of acetaminophen-cysteine (APAP-CYS), a toxic metabolite of APAP. 8-MOP had no significant effect on the protein and gene expression of Cyp2e1. The three-dimensional structures of mouse Cyp2e1 were constructed by homologous modeling. Molecular docking showed that 8-MOP had a good binding effect on the enzyme activity site of Cyp2e1. In summary, 8-MOP dose-dependently attenuated APAP-induced hepatotoxicity by binding to Cyp2e1 and occupying the active center of the enzyme, thus competitively inhibiting the oxidative metabolism of APAP, and reducing the generation of toxic product APAP-CYS.

Traumatic Brain Injury-Mediated Neuroinflammation and Neurological Deficits are Improved by 8-Methoxypsoralen Through Modulating PPARγ/NF-κB Pathway.

8-Methoxypsoralen (8-MOP) has anti-inflammatory, antioxidant and tissue-repairing abilities. Here, we probed the function and mechanism of 8-MOP in traumatic brain injury (TBI). The in-vivo TBI model was constructed in Sprague-Dawley (SD) rats using controlled cortical impact (CCI) surgery. In parallel, BV2 microglia and HT22 neurons were activated by lipopolysaccharide (LPS) to establish an in-vitro model. The modified neurological score (mNSS) and the Morris water maze experiment were employed to evaluate the rats' neurological functions. The rats' brain edema was assessed by the dry and wet method, and neuronal apoptosis in damaged brain tissues was monitored by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and Nissl's staining. Immunohistochemistry (IHC) was applied to verify Iba1-microglial activation in brain lesions of rats. The expression of inflammatory cytokines in BV2 microglia and HT22 neurons in the injured lesion of TBI rats was examined by the enzyme-linked immunosorbent assay (ELISA). The levels of iNOS, COX2, TLR4, PPARγ, STAT3, and NF-κB in brain lesions, BV2 microglia and HT22 neurons were compared by Western blot. As a result, 8-MOP administration reduced inflammation and LPS-induced neuronal damage in BV2 microglia. In vivo, 8-MOP treatment relieved neurological deficits in TBI rats, improved cognitive, learning and motor functions and mitigated brain edema and neuroinflammation induced by TBI. Furthermore, LPS or TBI activated the NF-κB and STAT3 pathways and repressed the PPARγ expression. However, 8-MOP treatment attenuated NF-κB and STAT3 phosphorylation and elevated PPARγ levels. Hence, 8-MOP exerts neuroprotective and anti-inflammatory effects in TBI rats by modulating the PPARγ/NF-κB pathway.

Anti-Inflammatory Effects of Psoralen Derivatives on RAW264.7 Cells via Regulation of the NF-κB and MAPK Signaling Pathways.

Using repositioning to find new indications for existing functional substances has become a global target of research. The objective of this study is to investigate the anti-inflammatory potential of psoralen derivatives (5-hydroxypsoralen, 5-methoxypsoralen, 8-hydroxypsoralen, and 8-methoxypsoralen) in macrophages cells. The results indicated that most psoralen derivatives exhibited significantly inhibited prostaglandin E2 (PGE2) production, particularly for 8-hydroxypsoralen (xanthotoxol) in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. In addition, xanthotoxol treatment decreased the PGE2, IL-6, and IL-1ß production caused by LPS stimulation in a concentration-dependent manner. Moreover, Western blot results showed that the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which activated with LPS treatment, were decreased by xanthotoxol treatment. Mechanistic studies revealed that xanthotoxol also suppressed LPS-stimulated phosphorylation of the inhibitor of κBα (IκBα), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) in RAW 264.7 cells. The Western blot assay results show that xanthotoxol suppresses LPS-induced p65 translocation from cytosol to the nucleus in RAW 264.7 cells. Moreover, we tested the potential application of xanthotoxol as a cosmetic material by performing human skin patch tests. In these tests, xanthotoxol did not induce any adverse reactions at a 100 µΜ concentration. These results demonstrate that xanthotoxol is a potential therapeutic agent for topical application that inhibits inflammation via the MAPK and NF-κB pathways.

Mechanistic Insights into the Ameliorating Effect of Melanogenesis of Psoralen Derivatives in B16F10 Melanoma Cells.

The objectives of this study were to investigate the melanogenetic potential of the psoralen derivatives 5-hydroxypsoralen, 5-methoxypsoralen, 8-hydroxypsoralen, 8-methoxypsoralen, and 5,8-dimethoxypsoralen in B16F10 melanoma cells. The results indicated that melanin production is significantly stimulated in B16F10 melanoma cells with 5-methoxypsoralen, 8-methoxypsoralen, and 5,8-dimethoxypsoralen, especially for 5-methoxypsoralen (bergapten), as reported previously. In addition, Western blot results showed that the protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2) increase after bergapten treatment for the first time. The results also showed that bergapten promotes the phosphorylation of Akt at Ser 473 and glycogen synthase kinase-3ß at Ser 9. Moreover, bergapten increased the content of ß-catenin in the cell cytoplasm and nucleus by reducing the phosphorylated ß-catenin (p-ß-catenin) content. The results also indicated that bergapten regulates melanogenesis by upregulating the phosphorylation of p38 and JNK-mitogen-activated protein kinase. Taken together, these findings suggest that the regulation of melanogenesis by bergapten may be mediated by the ß-catenin and MAPK signaling pathways and that bergapten might provide new insights into the pathogenesis of pigmented diseases.

Apoptosis induction by extracorporeal photopheresis is enhanced by increasing the 8-methoxypsoralen concentration and by replacing plasma with saline.

BACKGROUND: Extracorporeal photopheresis (ECP), an apheresis-based therapy for various immunological diseases, works mainly by inducing apoptosis in lymphocytes. Several factors influence the efficacy of ECP with the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet light A (UVA). This study aimed to optimize treatment by varying the 8-MOP starting concentration and the cell suspension medium. MATERIALS AND METHODS: All patients (n = 13) included in this study received photopheresis as medically indicated. Cells collected with a Spectra Optia apheresis system were suspended in plasma or physiological saline (NaCl) and incubated with 200 ng/ml versus 340 ng/ml photosensitizer before UVA irradiation (Macogenic G2 or UVA PIT system). Lymphocyte apoptosis and caspase activity were analyzed by flow cytometry and fluorimetry, and residual 8-methoxypsoralen concentrations by liquid chromatography-mass spectrometry. RESULTS: Raising the 8-MOP starting concentration significantly increased lymphocyte apoptosis, with values of 22% versus 35% (plasma) and 28%-46% (NaCl) at 24 h post-ECP and 37% versus 86% (plasma) and 74% versus 97% (NaCl) at 48 h for 200 ng/ml versus 340 ng/ml. Pre-transfusion residual 8-MOP levels were 168 ng/ml (plasma) and 162 ng/ml (NaCl) versus 290 ng/ml (plasma) and 266 ng/ml (NaCl) for the lower versus higher dose, respectively. DISCUSSION: Hence, 8-MOP concentration influences the efficacy of photopheresis as lymphocyte apoptosis rates were significantly higher with the higher starting concentration and with NaCl versus plasma. This indicates that increased 8-MOP starting doses and saline as additional suspension medium could help in improving ECP's efficacy.

Synthetically modified methoxsalen for enhanced cytotoxicity in light and dark reactions.

Linear furocoumarins, also known as psoralens, are clinically useful photo-activated pharmaceuticals employed to address hyperproliferative skin diseases. Seven diverse cytotoxic pharmacophores have been synthetically attached to 8-methoxypsoralen via a 5-amino functionality. The resulting unique set of compounds was evaluated for dark and light toxicity against PAM212 keratinocytes in culture.

8-Methoxypsoralen protects bovine mammary epithelial cells against lipopolysaccharide-induced inflammatory injury via suppressing JAK/STAT and NF-κB pathway.

Bovine mastitis is the most common disease in dairy cattle. Bacterial infections are the main cause of mastitis. Lipopolysaccharide (LPS), a major structural component of the cell wall of Escherichia coli, is a good inducer used to replicate inflammation models. 8-Methoxypsoralen (8-MOP), a formerly considered photosensitizing agent, has been used in immunotherapy. This study investigated the protective effects of 8-MOP on LPS-induced inflammatory injury in bovine mammary epithelial cells (BMECs). LPS treatment (50 µg/mL for 12 hr) caused a decrease in cell viability, morphological damage, and cell apoptosis. Pretreatment with 8-MOP at concentrations of 25 and 50 µg/ml significantly attenuated LPS-induced inflammation in BMECs. qRT-PCR analysis revealed that the messenger RNA expression of inflammatory cytokines and chemokine (interleukin-1ß [IL-1ß], IL-6, tumor necrosis factor-α, and IL-8) was suppressed by 8-MOP in LPS-stimulated BMECs. Western blot analysis showed that 8-MOP could also reduce the protein levels of cyclooxygenase-2 and promote the translocation of high-mobility group box 1 from the nucleus to cytoplasm. Furthermore, the anti-inflammatory property of 8-MOP was mediated by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells activation and STAT1 phosphorylation. Taken together, 8-MOP could protect cells from inflammatory injury induced by LPS, and may be a potential agent against bovine mastitis.

Comparison between 8-methoxypsoralen and 5-aminolevulinic acid in killing T cells of photopheresis patients ex vivo.

BACKGROUND AND OBJECTIVE: Extracorporeal photopheresis (ECP), an established modality for cutaneous T-cell lymphoma (CTCL) and graft-versus-host disease, involves ex vivo treatment of isolated leukocytes of a patient with the photosensitizing drug 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) exposure before reinfusion back to the patient. However, 8-MOP binds to both diseased and normal cells and thus kills both types of the cells after UV-A illumination with little selectivity. Clinically, this modality gives only partial response in the majority of treated patients. 5-Aminolevulinic acid (5-ALA), a precursor of the potent photosensitizer protoporphyrin IX (PpIX), has been shown to selectively induce PpIX in activated T lymphocytes (T cells) and could be an alternative for 8-MOP. The objectives of this study were to investigate ex vivo 5-ALA dark toxicity, 5-ALA-induced PpIX production, and photodynamic effect on T cells obtained from clinical ECP patients after the treatment of 5-ALA or 8-MOP plus a built-in certified UV-A source in the commercial Therakos™ Photopheresis System. MATERIALS AND METHODS: Flow cytometry was used to study dark cytotoxic effects of 5-ALA on human leukocytes, to measure the production of 5-ALA-induced PpIX in CD25+ activated T cells from both diluted mononuclear cells and undiluted buffy coat samples of ECP patients and to compare photodynamic effects on CD4+ and CD8+ T cells with 5-ALA/UV-A or 8-MOP/UV-A. RESULTS: No dark toxicity of 5-ALA on the leukocytes of ECP patients was seen at concentrations up to 10 mM for an incubation of up to 20 hours. 5-ALA-induced PpIX was produced more in CD25+ activated T cells than resting T cells in both diluted mononuclear cells and undiluted buffy coat samples, although there was a huge variation of samples from different individual patients. The CD4+ and CD8+ T cells treated with 5-ALA/UV-A were killed more than those treated with 8-MOP/UV-A. CONCLUSION: These results suggest that 5-ALA/UV-A may have the potential for improving the efficacy of ECP. Lasers Surg. Med. 50:469-475, 2018. © 2018 Wiley Periodicals, Inc.

The anti-metastatic effect of 8-MOP on hepatocellular carcinoma is potentiated by the down-regulation of bHLH transcription factor DEC1.

Despite progress in diagnostics and treatment of hepatocellular carcinoma (HCC), its prognosis remains poor. 8-Methoxypsoralen (8-MOP), a formerly considered photosensitizing agent, has been reported to induce cell apoptosis in HepG2 cells in a modest way when used alone. In this study, it was demonstrated that 8-MOP inhibited HCC HepG2 cells and SMMC-7721 cells migratory and invasive potentiality, as well as modulated the expression of various EMT-associated genes such as enhancing E-cadherin and reducing N-cadherin, vimentin, α-SMA and MMP9 in a concentration-dependent way. Differentiated embryonic chondrocyte-expressed gene 1, DEC1 (BHLHE40/Stra13/Sharp2), is a basic helix-loop-helix (bHLH) transcription factor that regulates cell growth, differentiation, apoptosis and tumorigenesis. 8-MOP suppressed the expression of DEC1 in a concentration- and time-dependent manner. Overexpression of DEC1 endorsed the HepG2 cells a higher metastatic phenotype, while totally abolished 8-MOP-repressed metastatic capability. In the meanwhile, overexpression of DEC1 promoted EMT process by suppressing expression of epithelial protein and enhancing expression of mesenchymal proteins, while potently antagonized the regulation of EMT-associated genes by 8-MOP. In vivo experiments revealed that the treatment of 8-MOP (5 or 20mg/kg) resulted in a dose-dependent decreases in the lung metastasis of hepatoma H22-transplanted mice without any obvious toxicity to the organs, as well as increased expression of E-cadherin in lung tissues. Consistently, 8-MOP down-regulated the expression of DEC1 in the lungs of tumor-bearing mice, which further confirms that DEC1 was correlated with 8-MOP-induced anti-metastatic effect. The present findings establish a function for DEC1 in HCC metastatic progression and suggest its candidacy as a novel target for the anti-metastasis effect of 8-MOP.

Assessment of 8-methosypsoralen, lomefloxacin, sparfloxacin, and Pirfenidone phototoxicity in Long-Evans rats.

Phototoxicity has a strong impact on drug development. Although several animal models have been developed to quantitatively assess human risks, none have been validated for standardized use. In this study, we validated an in vivo phototoxicity model using Long-Evans (LE) rats treated with 4 well-known phototoxic drugs, namely 8-methoxypsoralen, lomefloxacin, sparfloxacin, and pirfenidone. Daily macroscopic observations of skin and eyes, ophthalmological examinations 4 days after dosing, and blood sampling for toxicokinetics (TKs) were performed after exposure of treated animals to ultraviolet, and dose-dependent eye and/or skin reactions were noted for all compounds. Margins of safety were calculated when possible and correlated well with known relative phototoxicity of the 4 compounds. We conclude that the present in vivo phototoxicity assay using LE rats with TK analysis can be used to quantitatively predict the risk of pharmaceutical phototoxicity in humans.

Intravitreal administration of known phototoxicants in the rabbit fails to produce phototoxicity: implications for phototoxicity testing of intravitreally administered small molecule therapeutics.

CONTEXT: Intravitreal (ITV) dosing has become a clinically important route of administration for the treatment of uveitis, endophthalmitis, retinal vein occlusion, diabetic macular edema and age-related macular degeneration. Despite this, there are no validated non-clinical models of phototoxicity for ITV products. OBJECTIVE: The objective of this study was to develop an ITV rabbit model of phototoxicity for use in assessing the photosafety of small molecules therapeutics. MATERIALS AND METHODS: Dutch Belted rabbits were intravitreally injected bilaterally with four known phototoxicants: 8-methoxypsoralen, lomefloxacin, doxycycline and stannsoporfrin. Triescence(®), a non-phototoxic triamcinolone acetonide steroid formulation designed for ITV administration, was used as a negative control. One eye was then irradiated with solar-simulated ultraviolet radiation for 30 min, 1 h after dosing, while the other eye was occluded, serving as a non-irradiated control. RESULTS: Despite the direct administration of known phototoxicants into the vitreous, no evidence of ocular phototoxicity was observed in any dose group. Direct (non-phototoxic) retinal toxicity was observed in the doxycycline dose group only. CONCLUSION: These data suggest that the posterior segment of the rabbit eye is protected against phototoxicity by anatomical and/or physiological mechanisms, and is not a useful model for the assessment of phototoxicity of intravitreally administered molecules.

Phototoxicity in vitro and safety in vivo of the emulsion photosensitizer based on furanocoumarins of Heracleum sosnowskyi.

The use of plants such as giant hogweed as raw materials for the manufacture of dosage forms has been little explored. In this study, we utilized furanocoumarins from the Heracleum sosnowskyi plant to create an experimental emulsion dosage form (EmFHS). The EmFHS was finely dispersed (481.8 nm ± 71.1 nm), shelf-stable, and contained predominantly 8-methoxypsoralen at a concentration of 1 mg/ml. Phototoxicity analysis of EmFHS for THP-1 cells under UV (365 nm) irradiation showed an IC50 of 19.1 µg/ml (24 h) and 6.3 µg/ml (48 h). In relation to spheroids (L929), EmFHS exhibited a phototoxic effect in the concentration range of 31.25-125 µg/ml8-MOP. A full phototoxic effect was observed 48 h after UV irradiation. The phototoxic effect of EmFHS in vitro was dose-dependent and comparable to the effect of emulsion synthetic 8-methoxypsoralen and chlorin e6 solution. EmFHS cytotoxicity was caused solely by UV radiation, and toxicity in the dark was minimal. EmFHS, administered at a dose of 3 mg/kg8-MOP, was found to be safe after a single intravenous administration to rats. It had a photosensitizing effect in the form of local photodermatitis when exposed to UV irradiation at a dose of 44 J/cm2. The biokinetics of emulsion furanocoumarins showed that the phototoxic effect of EmFHS is due to the high penetration ability of the emulsion into cells of spheroids. At the same time, it has a low degree of cumulation when administered intravenously. The obtained data suggest that EmFHS may be a promising treatment for PUVA therapy of various dermatological diseases. Additionally, the plant Heracleum sosnowskyi shows potential as a basis for creating new dosage forms with phototherapeutic effects.

Recent insights into extracorporeal photopheresis for graft-versus-host disease.

INTRODUCTION: Extracorporeal Photopheresis (ECP) may be considered the unique large-scale cell therapy currently available. It is currently employed mainly as second-line treatment, especially in steroid-resistant or steroid-dependent Graft versus Host Disease (GvHD) with good results and very few limitations. AREAS COVERED: Many points need to be clarified regarding the ECP mechanism of action, that conditions the lack of uniqueness among the different centers, essentially cycle frequency, treatment duration, and the number of cells to be treated to obtain a response, according to the organs involved. Moreover, reliable biomarkers for prediction of response are lacking, as well as the best pharmacological combination. We will focus on the recent advances concerning ECP for GvHD treatment. We performed a systematic literature research in Pubmed and Embase as of September 2023. EXPERT OPINION: The recent studies on ECP mechanism of action along with the promising biomarkers of response, and the synergistic benefit of ECP in association with the new drugs render this therapy an important weapon for GvHD resistant to conventional treatment and can be proposed as a valid first-line therapy option with promising results. We believe that it should be used early in all categories of patients, considering its high safety profile.

New features on Pso2 protein family in DNA interstrand cross-link repair and in the maintenance of genomic integrity in Saccharomyces cerevisiae.

Pso2 protein, a member of the highly conserved metallo-ß-lactamase (MBL) super family of nucleases, plays a central role in interstrand crosslink repair (ICL) in yeast. Pso2 protein is the founder member of a distinct group within the MBL superfamily, called ß-CASP family. Three mammalian orthologs of this protein that act on DNA were identified: SNM1A, SNM1B/Apollo and SNM1C/Artemis. Yeast Pso2 and all three mammalian orthologs proteins have been shown to possess nuclease activity. Besides Pso2, ICL repair involves proteins of several DNA repair pathways. Over the last years, new homologs for human proteins have been identified in yeast. In this review, we will focus on studies clarifying the function of Pso2 protein during ICL repair in yeast, emphasizing the contribution of Brazilian research groups in this topic. New sub-pathways in the mechanisms of ICL repair, such as recently identified conserved Fanconi Anemia pathway in yeast as well as a contribution of non-homologous end joining are discussed.

8-Methoxypsoralen Induces Apoptosis by Upregulating p53 and Inhibits Metastasis by Downregulating MMP-2 and MMP-9 in Human Gastric Cancer Cells.

Furanocoumarin 8-methoxypsoralen (8-MOP) is the parent compound that naturally occurs in traditional medicinal plants used historically. 8-MOP has been employed as a photochemotherapeutic component of Psoralen + Ultraviolet A (PUVA) therapy for the treatment of vitiligo and psoriasis. Although the role of 8-MOP in PUVA therapy has been studied, little is known about the effects of 8-MOP alone on human gastric cancer cells. In this study, we observed anti-proliferative effect of 8-MOP in several human cancer cell lines. Among these, the human gastric cancer cell line SNU1 is the most sensitive to 8-MOP. 8-MOP treated SNU1 cells showed G1-arrest by upregulating p53 and apoptosis by activating caspase-3 in a dose-dependent manner, which was confirmed by loss-of-function analysis through the knockdown of p53-siRNA and inhibition of apoptosis by Z-VAD-FMK. Moreover, 8-MOPinduced apoptosis is not associated with autophagy or necrosis. The signaling pathway responsible for the effect of 8-MOP on SNU1 cells was confirmed to be related to phosphorylated PI3K, ERK2, and STAT3. In contrast, 8-MOP treatment decreased the expression of the typical metastasis-related proteins MMP-2, MMP-9, and Snail in a p53-independent manner. In accordance with the serendipitous findings, treatment with 8-MOP decreased the wound healing, migration, and invasion ability of cells in a dose-dependent manner. In addition, combination treatment with 8-MOP and gemcitabine was effective at the lowest concentrations. Overall, our findings indicate that oral 8-MOP has the potential to treat early human gastric cancer, with fewer side effects.

Photodynamic therapy fortified with topical oleyl alcohol-based transethosomal 8-methoxypsoralen for ameliorating vitiligo: Optimization and clinical study.

Vitiligo is a common autoimmune skin disorder that is characterized by patchy depigmentation of the skin due to melanocytes and melanin loss. Herein, photodynamic therapy mediated 8-methoxypsoralen (8-MOP), has been used fortified with topical oleyl alcohol-based transethosomes; to overcome the poor solubility and adverse effects associated with 8-MOP oral delivery. A 23 factorial design was used to study the formulation variables. In vitro and ex-vivo characterization besides a clinical study were conducted to assess therapeutic efficacy of the formulation. Results revealed that transethosomes were superior to transfersomes regarding drug protection from degradation. The optimized transethosomal formulation, composed of 50 mg oleyl alcohol, 10 mg Tween 80® and 20% v/v ethanol, exhibited high entrapment efficiency (83.87 ± 4.1%) and drug loading (105.0 ± 0.2%). Moreover, it showed small vesicular size (265.0 ± 2.9 nm) and PDI (0.19). The formulation depicted core and shell structure, high deformability index (12.45 ± 0.7 mL/s) and high ex-vivo skin permeation. The topical application of the developed 8-MOP transethosomal gel enhanced the effect of NB UVB radiation in the treatment of vitiligo patients and exhibited no side effects. Hence, it can be used as a future strategy for delivering 8-MOP without the need of systemic application.

8-Methoxypsoralen has Anti-inflammatory and Antioxidant Roles in Osteoarthritis Through SIRT1/NF-κB Pathway.

Osteoarthritis (OA) is mainly manifested by joint pain, stiffness and mobility disorder, which is the main cause of pain and disability in middle-aged and elderly people. In this study, we aimed to explore the role and mechanism of 8-Methoxypsoralen (8-MOP) in the OA model both in vitro and in vivo. The rat chondrocytes were treated with IL-1ß, and the proliferation, apoptosis, inflammatory reactions and oxidative stress responses were determined after treatment with different concentrations of 8-MOP. Real-time quantitative polymerase chain reaction (qRT-PCR) and/or Western blot were implemented to check the AMPK/SIRT1/NF-κB expression in chondrocytes. The NF-κB activity was determined by dual luciferase experiment. The pain threshold of OA rat model dealt with 8-MOP and/or the SIRT1 inhibitor EX527 was measured. Our results revealed that 8-MOP evidently reduced IL-1ß-mediated apoptosis and inhibition of proliferation, and mitigated the expression of inflammatory cytokines and oxidative stress factors in chondrocytes. Additionally, 8-MOP promoted phosphorylated level of AMPKα, enhanced SIRT1 expression and inhibited the phosphorylation of NF-κB. After treatment with EX527, 8-MOP-mediated protective effects on chondrocytes were mostly reversed. In vivo, 8-MOP obviously improved the pain threshold in the OA rat model and reduced the injury and apoptosis of chondrocytes in the joints. In addition, 8-MOP relieved inflammatory and oxidative stress responses in the articular cartilage via enhancing SIRT1 and repressing NF-κB activation. After the treatment with EX527, the 8-MOP-mediated protective effects were distinctly weakened. In summary, our study testified that 8-MOP alleviates pain, inflammatory and oxidative stress responses in OA rats through the SIRT1/NF-κB pathway, which is expected to become a new reagent for clinical treatment of OA.

Determination of benchmark doses for linear furanocoumarin consumption associated with inhibition of cytochrome P450 1A2 isoenzyme activity in healthy human adults.

Millions of individuals globally consume traditional herbal medicines (THMs), which contain abundant amounts of linear furanocoumarins. Linear furanocoumarins (i.e., 8-methoxypsoralen, 5-methoxypsoralen, and isopimpinellin) are inhibitors of cytochrome P450 (CYP) isoenzymes including 1A2, a major enzyme involved in drug metabolism and carcinogen bioactivation. Despite the high consumption of furanocoumarin-containing THMs, no studies have measured the furanocoumarin consumption level that triggers an inhibition to CYP1A2 activity in humans. The first objective was to verify if the potencies of the three furanocoumarins are additive towards the inhibition of CYP1A2 activity in vitro using concentration-addition and whole-mixture chemical-mixture-assessment models. A second objective was to determine the benchmark dose (BMD) with the mixtures of furanocoumarin oral doses, expressed as 8-MOP equivalents, and to assess the in vivo CYP1A2 activity, expressed as inhibition percentages. The in vitro results indicated that the three furanocoumarin inhibitory potencies were additive in the THM extracts, validating the use of the concentration-addition model in total furanocoumarin dose-equivalent calculations. Using the USEPA BMD software, the BMD was 18.9 µg 8-MOP equivalent/kg body weight. This information is crucial for furanocoumarin-related health-assessment studies and the regulation of THMs. Further studies should be performed for the remaining major metabolic enzymes to complete the safety profile of furanocoumarin-containing THMs and to provide accurate warning labelling.

Coumarins, psoralens, and quantitative 1H-NMR spectroscopy for authentication of lemon (Citrus limon [L.] Burm.f.) and Persian lime (Citrus × latifolia [Yu.Tanaka] Tanaka) juices.

Mutual adulterations of lemon and lime juices may be detected using coumarins and psoralens as markers. Poor manufacturing practices or legal but mechanically intense processing of lemons were recently suspected to lead to false accusations of deliberate adulterations with lime juices due to potentially unspecific markers. Therefore, we studied coumarin and psoralen profiles in carefully dissected flavedo, albedo, and endocarp of lime and lemon as well as in juices produced under variable mechanical stresses at laboratory and pilot plant scale. Although the marker herniarin was detectable in juices from lime and harshly extracted lemons at low levels, isopimpinellin, bergapten and the herein proposed, tentatively assigned 5-geranyloxy-8-methoxypsoralen represented unambiguously lime-specific markers. Coumarin and psoralen data also allowed differentiating juices produced at differing degrees of mechanical stress. The latter was also possible using quantitative 1H-NMR spectroscopy, which yielded best results when combined with HPLC data on coumarins and psoralens. In the future, the reported approach may be used for establishing a robust database prior to being used in industrial practice.