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1.
Pharmaceutics ; 16(6)2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38931889

RESUMO

Drug absorption via chylomicrons holds significant implications for both pharmacokinetics and pharmacodynamics. However, a mechanistic understanding of predicting in vivo intestinal lymphatic uptake remains largely unexplored. This study aimed to delve into the intestinal lymphatic uptake of drugs, investigating both enhancement and inhibition using various excipients through our previously established in vitro model. It also examined the applicability of the model by assessing the lymphatic uptake enhancement of a lymphotropic formulation with linoleoyl polyoxyl-6 glycerides using the same model. The model successfully differentiated among olive, sesame, and peanut oils in terms of lymphatic uptake. However, it did not distinguish between oils containing long-chain fatty acids and coconut oil. Coconut oil, known for its abundance of medium-chain fatty acids, outperformed other oils. This heightened uptake was attributed to the superior emulsification of this oil in artificial chylomicron media due to its high content of medium-chain fatty acids. Additionally, the enhanced uptake of the tested formulation with linoleoyl polyoxyl-6 glycerides underscored the practical applicability of this model in formulation optimization. Moreover, data suggested that increasing the zeta potential of Intralipid® using sodium lauryl sulfate (SLS) and decreasing it using (+/-) chloroquine led to enhanced and reduced uptake in the in vitro model, respectively. These findings indicate the potential influence of the zeta potential on intestinal lymphatic uptake in this model, though further research is needed to explore the possible translation of this mechanism in vivo.

2.
J Pharm Biomed Anal ; 235: 115631, 2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37586306

RESUMO

A selective and sensitive liquid chromatography mass spectrometry assay was developed for the detection of cannflavin A, B, and C in hemp extract specimens. A deuterated analog cannabidiol-D3 was used as the internal standard and the isocratic method used a mobile phase consisting of acetonitrile and water with 0.1 % formic acid [83:17]. Detection was carried out by electrospray positive ionization in single-ion monitoring mode through a C-18 analytical column. The assay (total run time <20 min) had excellent linearity and a lower limit of quantification of 0.5 µg/mL and a limit of detection of 0.25 µg/mL with a 10 µL injection. The method possessed suitable measures of stability, sensitivity, and selectivity for detecting cannflavins in several specimen types. The method was successfully applied to the analysis of samples of cannflavin release from prototype topical formulations.


Assuntos
Cannabis , Cannabis/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida/métodos , Extratos Vegetais , Cromatografia Líquida de Alta Pressão/métodos , Reprodutibilidade dos Testes
3.
Antibiotics (Basel) ; 12(2)2023 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-36830231

RESUMO

BACKGROUND: Piperacillin (Pip)/tazobactam (Taz) is a broad-spectrum antimicrobial agent that has been commonly used in the intensive care unit for severe and life-threatening infections. Recent evidence suggests that therapeutic drug monitoring (TDM) for Pip could be beneficial in clinical practice to facilitate dose optimization and increase the odds of treatment success. The aim was to develop and validate a sensitive and simple high-performance liquid chromatography (HPLC) method for the simultaneous quantification of Pip and Taz in human plasma. METHODS: Samples (0.3 mL) were deproteinized with acetonitrile. The supernatant was evaporated and then reconstituted and injected into the HPLC. The chromatographic analysis was carried out by using the C18 column and gradient elution with the acetonitrile:water mobile phase mixture with 0.1% trifluoracetic acid at a flow rate of 0.8 mL/min using a UV detector at 218 nm. RESULTS: The method had acceptable linearity (r2 > 0.99) over the concentration ranges of 0.5-400 µg/mL and 1-100 µg/mL for Pip and Taz, respectively. The method demonstrated acceptable inter- and intra-day precision and accuracy within ±20% with adequate stability results. CONCLUSION: The developed method is sensitive and simple and utilizes simple sample preparation and elution steps, making it suitable and practical for Pip/Taz TDM.

4.
Res Pharm Sci ; 18(1): 1-15, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36846734

RESUMO

Background and purpose: Lysosomal-targeted drug delivery can open a new strategy for drug therapy. However, there is currently no universally accepted simulated or artificial lysosomal fluid utilized in the pharmaceutical industry or recognized by the United States Pharmacopeia (USP). Experimental procedure: We prepared a simulated lysosomal fluid (SLYF) and compared its composition to a commercial artificial counterpart. The developed fluid was used to test the dissolution of a commercial product (Robitussin®) of a lysosomotropic drug (dextromethorphan) and to investigate in-vitro lysosomal trapping of two model drugs (dextromethorphan and (+/-) chloroquine). Findings/Results: The laboratory-prepared fluid or SLYF contained the essential components for the lysosomal function in concentrations reflective of the physiological values, unlike the commercial product. Robitussin® passed the acceptance criteria for the dissolution of dextromethorphan in 0.1 N HCl medium (97.7% in less than 45 min) but not in the SLYF or the phosphate buffer media (72.6% and 32.2% within 45 min, respectively). Racemic chloroquine showed higher lysosomal trapping (51.9%) in the in-vitro model than dextromethorphan (28.3%) in a behavior supporting in-vivo findings and based on the molecular descriptors and the lysosomal sequestration potential of both. Conclusion and implication: A standardized lysosomal fluid was reported and developed for in-vitro investigations of lysosomotropic drugs and formulations.

5.
Free Radic Biol Med ; 192: 77-83, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36113706

RESUMO

Commercial cannabis oil products are widely available in Canada even though there is a significant gap in scientific information regarding them. Oils, such as vegetable oils, are known to undergo oxidative changes through free radical mechanisms when they are heated or aged, but the cannabis oils used in this study did not have expiry dates or best-before usage dates. This led to the question of how these products would be affected with time. We hypothesized that cannabis oils would produce increased concentrations of free radicals in aging-simulated conditions, which would be related to a decrease in cannabidiol (CBD) or Δ9-tetrahydrocannabinol (THC) content. Cannabis oils and their respective vehicles (oils) were heated using two protocols: One (moderate aging method) used a 2-day heating protocol at 50 °C, and the other (enhanced aging method) used a 14-day heating protocol at 70 °C. We used electron paramagnetic resonance (EPR) spectroscopy for free radical analysis using the spin trapping technique using 200 mM PBN and 0.02 mM CuCl2 (for peroxide breakdown to free radicals). For active ingredient analysis (CBD, THC), we used LC/MS. Cannabis oils that contained unsaturated oils as their vehicles, such as olive or sunflower oil, all showed varying degrees of free radical formation. In both aged and unaged oils containing CBD or THC, less free radical formation was detected compared to the vehicle controls. Cannabis oils using medium-chain triglycerides (MCT) showed little or no free radical formation. The most significant decrease in CBD or THC was observed in the products using sunflower oil, to a lesser extent in MCT oil, and THC also decreased in olive oil. These findings are important for consumers and policymakers considering using such products in hot beverages or cooking and highlighting the importance of appropriate storage conditions.


Assuntos
Canabidiol , Cannabis , Cannabis/química , Dronabinol/análise , Radicais Livres , Calefação , Azeite de Oliva/química , Peróxidos , Óleos de Plantas/química , Óleo de Girassol , Triglicerídeos
6.
Artigo em Inglês | MEDLINE | ID: mdl-35032893

RESUMO

The development of a selective and sensitive high-performance liquid chromatographic tandem mass spectrometric method for the determination of cycloheximide (CHX) in rat blood and plasma is described. The extraction of CHX and colchicine as internal standard from blood fluid (0.1 mL) was achieved using n-hexane: dichloromethane: isopropanol (20:10:1 v/v/v). The mobile phase, a combination of methanol:10 mM ammonium acetate (85:15, v/v), was pumped at 0.2 mL/min through a C18 analytical column with a run time of 3.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay exhibited excellent linearity (r2 > 0.999) in peak area response over the concentration ranges of 2-1000 ng CHX /mL blood fluid. The mean absolute recoveries for 20, 100 and 500 ng/mL CHX in blood fluid using the present extraction procedure were > 97%. The intra- and inter-day coefficients of variation in the plasma and blood and mean error were < 13% at different concentrations. Samples had limited stability at room temperature, and speedy processing is needed. After intravenous administration, rats had measurable concentrations of CHX for up to 24 h after dosing with 1 mg/kg of cycloheximide. The method displayed a high caliber of sensitivity and selectivity for detecting very low concentrations of CHX in rats.


Assuntos
Antibacterianos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Cicloeximida/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Antibacterianos/farmacocinética , Cicloeximida/farmacocinética , Masculino , Plasma/química , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade
7.
Biomed Chromatogr ; 35(2): e4971, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32840898

RESUMO

Nimodipine is a dihydropyridine calcium channel blocker that exhibits higher selectivity toward cerebral blood vessels compared with other members of the same class. It has been shown to improve outcomes and prevent delayed cerebral ischemia in the setting of aneurysmal subarachnoid hemorrhage, a life-threatening brain bleed. Nimodipine is a chiral compound and it is marketed as a racemic mixture of (+)-R and (-)-S enantiomers. (-)-S-Nimodipine is approximately twice as potent a vasorelaxant as the racemic mixture and is more rapidly eliminated than the (+)-R counterpart following oral dosing. Few analytical procedures have been reported to determine nimodipine enantiomers in biological samples; however, the reported methods were time-consuming, involved multistep extraction procedures and required large sample volumes. Herein, we present an LC-MS/MS method for quantifying nimodipine enantiomers in human plasma using a small sample volume (0.3 ml) and a single liquid-liquid extraction step. The peak area ratios were linear over the tested concentration ranges (1.5-75 ng/ml) with r2 > 0.99. The intraday CV and percentage error were within ±14% while the interday values were within ±13%, making this analytical method feasible for research purposes and pharmacokinetic studies.


Assuntos
Cromatografia Líquida/métodos , Nimodipina/sangue , Nimodipina/química , Espectrometria de Massas em Tandem/métodos , Humanos , Modelos Lineares , Nimodipina/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estereoisomerismo , Hemorragia Subaracnóidea/tratamento farmacológico , Vasodilatadores/sangue , Vasodilatadores/química , Vasodilatadores/farmacocinética
8.
Front Immunol ; 12: 779119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35095853

RESUMO

Disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) is a ubiquitously expressed membrane-bound enzyme that mediates shedding of a wide variety of important regulators in inflammation including cytokines and adhesion molecules. Hepatic expression of numerous cytokines and adhesion molecules are increased in cholestatic liver diseases including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), however, the pathophysiological role of ADAM17 in regulating these conditions remains unknown. Therefore, we evaluated the role of ADAM17 in a mouse model of cholestatic liver injury due to bile duct ligation (BDL). We found that BDL enhanced hepatic ADAM17 protein expression, paralleled by increased ADAM17 bioactivity. Moreover, inhibition of ADAM17 bioactivity with the specific inhibitor DPC 333 significantly improved both biochemical and histological evidence of liver damage in BDL mice. Patients with cholestatic liver disease commonly experience adverse behavioral symptoms, termed sickness behaviors. Similarly, BDL in mice induces reproducible sickness behavior development, driven by the upregulated expression of cytokines and adhesion molecules that are in turn regulated by ADAM17 activity. Indeed, inhibition of ADAM17 activity significantly ameliorated BDL-associated sickness behavior development. In translational studies, we evaluated changes in ADAM17 protein expression in liver biopsies obtained from patients with PBC and PSC, compared to normal control livers. PSC and PBC patients demonstrated increased hepatic ADAM17 expression in hepatocytes, cholangiocytes and in association with liver-infiltrating immune cells compared to normal controls. In summary, cholestatic liver injury in mice and humans is associated with increased hepatic ADAM17 expression. Furthermore, inhibition of ADAM17 activity improves both cholestatic liver injury and associated sickness behavior development, suggesting that ADAM17 inhibition may represent a novel therapeutic approach for treating patients with PBC/PSC.


Assuntos
Proteína ADAM17/metabolismo , Colestase/metabolismo , Comportamento de Doença/fisiologia , Hepatopatias/metabolismo , Fígado/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Ductos Biliares/metabolismo , Colangite Esclerosante/metabolismo , Modelos Animais de Doenças , Hepatócitos/metabolismo , Inflamação/metabolismo , Ligadura/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
9.
CMAJ Open ; 8(1): E75-E82, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32046972

RESUMO

BACKGROUND: Guidelines recommend that patients with coronary artery disease (CAD) carry and immediately use acetylsalicylic acid (ASA) and sublingually administered nitroglycerin at the onset of chest pain; however, compliance with these recommendations is poor. We designed and tested a compact on-person storage device for these medications. METHODS: We designed an airtight, light-proof and chemically inert holder to carry four 81-mg ASA tablets and three 0.3-mg Nitrostat (nitroglycerin, Pfizer) tablets. After establishing the temperatures ranges in wallets and pockets, we tested nitroglycerin dissolution and release of the stored Nitrostat tablets across a range of relevant temperatures and a variety of tablet enclosure systems. RESULTS: Microcalorimeter thermal conduction studies as well as dissolution and release testing showed that nitroglycerin tablets were stable at temperatures ranging from -20°C to 60°C for 1 week. In testing up to 24 weeks, 0.3-mg Nitrostat tablets enclosed completely in polytetrafluoroethylene (PTFE) performed similarly to those stored in the manufacturer's borosilicate glass packaging across a wide range of temperatures relevant to on-person carriage. Real-world on-person testing for 24 weeks confirmed these results. Non-PTFE enclosures performed poorly. INTERPRETATION: The PTFE enclosure with a PTFE-coated cap liner maintained long-term performance of 0.3-mg Nitrostat tablets under laboratory and real-world conditions. This storage device is now commercially available as the SMHeartCard to improve compliance and provide immediate access to emergency cardiac medications.


Assuntos
Aspirina , Embalagem de Medicamentos , Serviços Médicos de Emergência , Nitroglicerina , Aspirina/administração & dosagem , Doença da Artéria Coronariana , Estabilidade de Medicamentos , Serviços Médicos de Emergência/métodos , Humanos , Nitroglicerina/administração & dosagem , Comprimidos , Temperatura
10.
Biochem Biophys Res Commun ; 516(2): 373-380, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31213295

RESUMO

There is a growing surge of investigative research involving the beneficial use of cannabinoids as novel interventional alternatives for multiple sclerosis (MS) and associated neuropathic pain (NPP). Using an experimental autoimmune encephalomyelitis (EAE) animal model of MS, we demonstrate the therapeutic effectiveness of two cannabinoid oil extract formulations (10:10 & 1:20 - tetrahydrocannabinol/cannabidiol) treatment. Our research findings confirm that cannabinoid treatment produces significant improvements in neurological disability scoring and behavioral assessments of NPP that directly result from their ability to reduce tumor necrosis factor alpha (TNF-α) production and enhance brain derived neurotrophic factor (BDNF) production. Henceforth, this research represents a critical step in advancing the literature by scientifically validating the merit for medical cannabinoid use and sets the foundation for future clinical trials.


Assuntos
Canabinoides/administração & dosagem , Canabinoides/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Óleos de Plantas/uso terapêutico , Animais , Comportamento Animal , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Composição de Medicamentos , Encefalomielite Autoimune Experimental/genética , Feminino , Esclerose Múltipla/genética , Neuralgia/tratamento farmacológico , Ratos Endogâmicos Lew , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
11.
J Pharm Pharm Sci ; 21(1s): 309s-324s, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30407907

RESUMO

PURPOSE: Liver fatty acid binding protein (FABP1) is a cytoplasmic polypeptide that transports substrates throughout the cytosol and functions as an antioxidant. A common polymorphic variant, FABP1 T94A has a minor allele frequency of 26-38%, 8.3±1.9% homozygous in the human population. The purpose of this study was to mutate and isolate recombinant rat FABP1 to the T94A variant to evaluate the mutant's antioxidant activity using in vitro studies. METHODS: Site-directed mutagenesis was used to generate a mutation in rat cDNA within a pGEX-6p-2 vector. This plasmid was transformed into competent cells and cultured for expression of FABP1 T94A mutant. The mutated protein was purified using GSTrap Fastflow columns within an ÄKTA FPLC system. A 2,7-dichlorofluorescein (DCF) assay was used to screen the T94A variant antioxidant activity. Additionally, Thiobarbituric Acid Reactive Substances (TBARS) assay was used in determining T94A mutant antioxidant activity in hydrophilic and lipophilic environments through the use of the azo compounds AAPH and MeO-AMVN, respectively and in the presence and absence of the long-chain fatty acid palmitate and α-bromo palmitate. RESULTS: Although the FABP1 T94A (20 µM) mutant significantly reduced DCF fluorescence compared to control (no protein; P< 0.001), there were no significant difference when compared to the wild-type (WT) FABP1. T94A was able to diminish the formation of malondialdehyde (MDA) in both lipophilic and hydrophilic systems. There were significant differences between T94A mutant and WT FABP1 at concentrations 1 and 10 µM (P< 0.05) in the hydrophilic milieu, however, this was not seen at 20 µM and also not seen in the lipophilic milieu at all concentrations. When T94A was pre-incubated with the long-chain fatty acids palmitate or α -bromo palmitate, MDA formation was decreased in both lipid peroxidation systems. There were no statistical differences between the WT FABP1 and T94A bound with fatty acids in both lipid peroxidation systems, however, there was a slight statistical difference when the T94A and WT FABP1 bound α-Br-PA in the AAPH lipid peroxidation system only. CONCLUSIONS: The T94A has antioxidant activity in both hydrophilic and lipophilic environments. The T94A variant of FABP1 does not have a loss of function in regard to acting as an antioxidant but the extent of function may be influenced by ligand binding. We conclude that populations having the minor T94A allele frequency would have similar ROS scavenging potential as those with nascent FABP1.


Assuntos
Antioxidantes/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Animais , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Fluoresceínas/química , Corantes Fluorescentes/química , Mutagênese Sítio-Dirigida , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
12.
J Pharm Pharm Sci ; 21(1s): 29683, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29702047

RESUMO

PURPOSE: MyoNovin is a novel skeletal muscle-regenerating compound developed through synthesis of two nitro groups onto a guaifenesin backbone to deliver nitric oxide to skeletal muscle with a potential to treat muscle atrophy. The purpose of this study was to utilize in silico, in vitro, and in vivo approaches to characterize MyoNovin and examine its safety, biodistribution, and feasibility for drug delivery. METHODS: In silico software packages were used to predict the physicochemical and biopharmaceutical properties of MyoNovin. In vitro cardiotoxicity was assessed using human cardiomyocytes (RL-14) while effects on CYP3A4 metabolic enzyme and antioxidant activity were examined using commercial kits. A novel HPLC assay was developed to measure MyoNovin concentration in serum, and delineate initial pharmacokinetic and acute toxicity after intravenous administration (20 mg/kg) to male Sprague-Dawley rats. RESULTS: MyoNovin showed relatively high lipophilicity with a LogP value of 3.49, a 20-fold higher skin permeability (19.89 cm/s*107) compared to guaifenesin (0.66 cm/s*107), and ~10-fold higher effective jejunal permeability (2.24 cm/s*104) compared to guaifenesin (0.26 cm/s*104). In vitro, MyoNovinwas not cytotoxic to cardiomyocytes at concentrations below 8 µM and did not inhibit CYP3A4 or show antioxidant activity. In vivo, MyoNovin had a short half-life (t1/2) of 0.16 h, and a volume of distribution Vss of 0.62 L/kg. Biomarkers of MyoNovincardiac and renal toxicity did not differ significantly from baseline control levels. CONCLUSIONS: The predicted high lipophilicity and skin permeability of MyoNovin render it a potential candidate for transdermal administration while its favourable intestinal permeation suggests it may be suitable for oral administration. Pharmacokinetics following IV administration of MyoNovin were delineated for the first time in a rat model. Preliminary single 20 mg/kg dose assessment of MyoNovin suggest no influenceon cardiac troponin or ß-N-Acetylglucosaminidase. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.


Assuntos
Guaifenesina/análogos & derivados , Músculo Esquelético/efeitos dos fármacos , Nitratos/farmacologia , Troponina I/sangue , Animais , Guaifenesina/administração & dosagem , Guaifenesina/farmacologia , Humanos , Injeções Intravenosas , Masculino , Músculo Esquelético/metabolismo , Nitratos/administração & dosagem , Nitratos/sangue , Ratos , Ratos Sprague-Dawley
13.
J Innate Immun ; 8(6): 617-629, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27560480

RESUMO

NK cells play a central role in innate immunity, acting directly through cell-mediated cytotoxicity and by secreting cytokines. TNFα activation of TNFR2 enhances NK cell cytotoxicity, but its effects on the other essential function of NK cells - cytokine production, for which IFNγ is paramount - are poorly defined. We identify the expression of both TNFα receptors on human peripheral blood NK cells (TNFR2 > TNFR1) and show that TNFα significantly augments IFNγ production from IL-2-/IL-12-treated NK cells in vitro, an effect mimicked by a TNFR2 agonistic antibody. TNFα also enhanced murine NK cell IFNγ production via TNFR2 in vitro. In a mouse model characterized by the hepatic recruitment and activation of NK cells, TNFR2 also regulated NK cell IFNγ production in vivo. Specifically, in this model, after activation of an innate immune response, hepatic numbers of TNFR2-expressing and IFNγ-producing NK cells were both significantly increased; however, the frequency of IFNγ-producing hepatic NK cells was significantly reduced in TNFR2-deficient mice. We delineate an important role for TNFα, acting through TNFR2, in augmenting cytokine-induced NK cell IFNγ production in vivo and in vitro, an effect with significant potential implications for the regulation of innate and adaptive immune responses.


Assuntos
Células Matadoras Naturais/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Imunidade Inata , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-12/imunologia , Interleucina-2/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/imunologia
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