RESUMEN

This study proposes a new alternative for template removal from molecularly imprinted polymers by heat activated persulfate. It is known that trace amounts of template molecule remains in the polymer network after extraction by current methodologies leading to bleeding and incomplete removal of template which could compromise final determination of target analytes especially in trace analysis. A previously developed molecularly imprinted polymer specially designed for Coenzyme Q10 (CoQ10) extraction was employed as a model to test this template elimination approach. This polymer is based on methacrylic acid and ethylene glycol dimethylacrylate as monomers and Coenzyme Q0 as template. This coenzyme has the same quinone group as the CoQ10. Selectivity was analyzed comparing the recovery of CoQ10 and ubichromenol, a CoQ10 related substance. Chemical degradation using heat-activated persulfate allows the elimination of the template molecule with a high level of efficiency, being a simple and ecological methodology, yielding a polymer that exhibits comparable selectivity and imprinting effect with respect to traditional extraction methods.

Asunto(s)

Impresión Molecular , Polímeros Impresos Molecularmente , Ubiquinona , Calor , Polímeros/química , Impresión Molecular/métodos

RESUMEN

BACKGROUND: Ursodeoxycholic acid (UDCA) is a therapeutic agent used for the treatment of cholestatic hepatobiliary diseases in pediatric patients. It is a bile acid that presents high lipophilicity, and it belongs to Class II of the Biopharmaceutical Classification System (BCS), which exhibits low water solubility and high intestinal permeability, which leads to poor oral absorption. The objective of this work was to design and optimize UDCA nanosuspensions by means of the precipitation-ultrasonication method to improve the solubility, dissolution, and oral bioavailability of UDCA. METHODS: A three-level, three-factor Box-Behnken design was used to optimize formulation variables and obtain uniform, small-particle-size UDCA nanosuspensions. The independent variables were: stabilizer percentage (X1), amplitude (X2), and sonication time (X3), and the dependent variable was the particle size (Y1). In the precipitation-ultrasonication method, UDCA was dissolved in acetone:PEG 400 (1:1 v/v) and quickly incorporated into the antisolvent (pre-cooled aqueous dispersion of HPMC E-15 0.3%), by means of intense sonication at 50 W for 5 min, controlling temperature through an ice water bath. The lyophilization efficacy was evaluated by means of a cryoprotective efficacy test, working with 10% maltose at -80 °C. The nanosuspensions were characterized by dynamic light scattering (DLS), X-ray diffraction, and scanning electron microscopy (SEM). The physicochemical stability was determined at 25 °C and 4 °C at 7, 14, 30, and 60 days, and the UDCA content was analyzed via HPLC-UV. An in vitro dissolution assay and an oral bioavailability study were performed in male Wistar rats. RESULTS: A significant impact was achieved in the optimized nanosuspension with 0.3% (stabilizer), 50 W (amplitude), and 5 min (sonication time), with a particle size of 352.4 nm, PDI of 0.11, and zeta potential of -4.30 mV. It presented adequate physicochemical stability throughout the study and the UDCA content was between 90% and 110%. In total, 86% of UDCA was dissolved in the in vitro dissolution test. The relative oral bioavailability was similar without significant statistical differences when comparing the lyophilized nanosuspension and the commercial tablet, the latter presenting a more erratic behavior. The pharmacokinetic parameters of the nanosuspension and the commercial tablet were Tmax (1.0 ± 0.9 h vs. 2.0 ± 0.8 h, respectively), Cmax (0.558 ± 0.118 vs. 0.366 ± 0.113 µM, respectively), ΔCmax (0.309 ± 0.099 vs. 0.232 ± 0.056, respectively), AUC (4.326 ± 0.471 vs. 2.188 ± 0.353 µg/mL.h, respectively, p < 0.02), and IAUC0-24h (2.261 ± 0.187 µg/mL.h vs. 1.924 ± 0.440 µg/mL.h, respectively). CONCLUSIONS: The developed nanosuspension presents an appropriate dosage and administration for pediatric patients. On the other hand, it exhibits an adequate absorption and UDCA oral bioavailability.

RESUMEN

BACKGROUND: Increasing evidence suggests that glaucoma affects the ocular surface. We aimed to investigate the cellular mechanisms underlying the glaucoma-associated corneal alterations in an animal model. METHODS: Wistar rats underwent the cauterization of two episcleral veins of the left eye to elevate the intraocular pressure (ipsilateral, G-IL). Control animals received a sham procedure (C-IL). Contralateral eyes did not receive any procedure (G-CL or C-CL). Enzymes related to the redox status, oxidative damage to macromolecules, and inflammatory markers were assessed in corneal lysates. RESULTS: Compared to C-IL, NOX4, NOX2, and iNOS expression was increased in G-IL (68%, p < 0.01; 247%, p < 0.01; and 200%, p < 0.001, respectively). We found an increase in SOD activity in G-IL (60%, p < 0.05). The GSH/GSSG ratio decreased in G-IL (80%, p < 0.05), with a decrease in GR activity (40%, p < 0.05). G-IL displayed oxidative (90%, p < 0.01) and nitrosative (40%, p < 0.05) protein damage, and enhanced lipid peroxidation (100%, p < 0.01). G-IL group showed an increased in CD45, CD68 and F4/80 expression (50%, p < 0.05; 190%, p < 0.001 and 110%, p < 0.05, respectively). G-CL displayed a higher expression of Nrf2 (60%, p < 0.001) and increased activity of SOD, CAT, and GPx (60%, p < 0.05; 90%, p < 0.01; and 50%, p < 0.05, respectively). CONCLUSIONS: Glaucoma induces a redox imbalance in the ipsilateral cornea with an adaptive response of the contralateral one. GENERAL SIGNIFICANCE: Our study provides a possible mechanism involving oxidative stress and inflammation that explains the corneal alterations observed in glaucoma. We demonstrate that these changes extend not only to the ipsilateral but also to the contralateral cornea.

Asunto(s)

Glaucoma , Ratas , Animales , Ratas Wistar , Estrés Oxidativo/fisiología , Oxidación-Reducción , Córnea/metabolismo , Superóxido Dismutasa/metabolismo

RESUMEN

Supplementation with Coenzyme Q10 (CoQ10), in patients with its deficiency, has greater odds of success if the treatment is carried out early with an appropriate formulation. For neonatal CoQ10 deficiency, infant formula supplementation could be an attractive option. However, solid CoQ10 cannot be solubilized or dispersed in milk matrix leading to an inefficient CoQ10 dosage and poor intestinal absorption. We developed and characterized a high-dose CoQ10 oil-in-water (O/W) nanoemulsion suitable to supplement infant formula without modifying its organoleptic characteristics. CoQ10 powder and soy lecithin were solubilized in an oil phase consisted of Labrasol® and LabrafacTM. The aqueous phase was Tween 80, TPGS, methylparaben and propylparaben. O/W nanoemulsion was prepared by adding dropwise the oil phase to the aqueous phase under stirring to a final concentration of CoQ10 9.5 % w/w followed by ultrasonic homogenization. Pharmacotechnical parameters were determined. This formulation resulted to be easily to be dispersed in milk matrix, stable for at least 90 days, with no cytotoxicity in in vitro assays, and higher bioavailability than CoQ10 powder. CoQ10 nanoemulsion supplementation in the infant formula facilitates the individualized administration for the child with accurate dosage, overcome swallowing difficulties and in turn could increase the treatment adherence and efficacy.

Asunto(s)

Fórmulas Infantiles , Ubiquinona , Humanos , Recién Nacido , Disponibilidad Biológica , Suplementos Dietéticos , Polvos , Lactante

RESUMEN

Coenzyme Q10 (CoQ10) supplementation has demonstrated to be safe and effective in primary and secondary CoQ10 deficiencies. Previously, we have designed a high-dose CoQ10 oleogel (1 g/disk) with excipients used in quantities that do not represent any toxic risk. However, it was necessary to demonstrate their safety in the final formulation. Following this purpose, an acute toxicity study of the oleogel in rats was performed. Furthermore, the genotoxic risk was evaluated in human volunteers after CoQ10 supplementation with oleogel and compared to the solid form (1 g/three 00-size-capsules). In addition, the general health status and possible biochemical changes of the participants were determined using serum parameters. Results suggested the absence of adverse effects caused by the interaction of the components in the oleogel formulation. Therefore, we conclude that the designed novel high-dose CoQ10 oleogel was safe for oral consumption.

RESUMEN

RESUMEN Introducción: Resultados de nuestro laboratorio sugieren que la disfunción mitocondrial en el corazón precede a la falla miocárdica asociada a la hiperglucemia sostenida. Objetivo: Estudiar los eventos tempranos que ocurren en las mitocondrias de corazón en un modelo de diabetes mellitus tipo 1. Materiales y métodos: Ratas Wistar macho fueron inyectadas con estreptozotocina (STZ; 60 mg/kg, ip) y sacrificadas 10 o 14 días posinyección. Se obtuvo la fracción mitocondrial de corazón. Resultados: El consumo de O2 en estado 3 en presencia de malato-glutamato (21%) o succinato (16%) y las actividades de los complejos I-III (27%), II-III (24%) y IV (22%) fueron menores en los animales diabéticos a los 14 días posinyección. Cuando los animales se sacrificaron al día 10, solo el consumo de O2 en estado 3 en presencia de sustratos del complejo I (23%) y su control respiratorio (30%) fueron menores en las ratas inyectadas con STZ, de acuerdo con una reducción en la actividad del complejo I-III (17%). Estos cambios se acompañaron de un aumento en las velocidades de producción de H2O2 (117%), NO (30%) y ONOO- (∼225%), en la expresión de mtNOS (29%) y en la [O2 -]ss (∼150%) y [NO]ss (∼30%), junto con una disminución de la actividad de la Mn-SOD (15%) y la [GSSG+GSH]mitocondrial (28%), sin cambios en la expresión de PGC-1α. Conclusión: La disfunción del complejo I y el aumento en la generación de H2O2, NO y ONOO- pueden considerarse señales subcelulares prodrómicas del deterioro de la función mitocondrial que precede a la disfunción cardíaca en la diabetes.

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ABSTRACT Background: Previous results from our laboratory suggest that heart mitochondrial dysfunction precedes myocardial failure associated with sustained hyperglycemia. Purpose: The aim of this study was to analyze the early events that take place in heart mitochondria in a type 1 diabetes mellitus (DM) model. Methods: Male Wistar rats were injected with streptozotocin (STZ; 60 mg/kg, ip.) to induce DM. They were euthanized 10 or 14 days later and the heart mitochondrial fraction was obtained. Results: State 3 O2 consumption in the presence of malate-glutamate (21%) or succinate (16%), and complex I-III (27%), II-III (24%) and IV (22%) activities were lower in diabetic animals 14 days after STZ injection. When animals were euthanized at day 10, only state 3 O2 consumption sustained by complex I substrates (23%) and its corresponding respiratory control (30%) were lower in rats injected with STZ, in agreement with reduced complex I-III activity (17%). These changes were accompanied by increased H2O2 (117%), NO (30%) and ONOO- (~225%) production rates, mtNOS expression (29%) and O2 - (~150%) and NO (~30%) steady-state concentrations, together with a decrease in Mn-SOD activity (15%) and mitochondrial [GSSG+GSH] (28%), without changes in PGC-1α expression. Conclusion: Complex I dysfunction and increased H2O2, NO and ONOO- production rates can be considered subcellular prodromal signals of the mitochondrial damage that precedes myocardial dysfunction in diabetes.

RESUMEN

Ursodeoxycholic acid (UDCA) is used in the oral therapy of hepatobiliary cholestatic diseases. Due to UDCA low aqueous solubility, two pediatric oral suspensions (25 mg/mL) were formulated with a few excipients, suspension A (SA) and suspension B (SB) with a vehicle, including two suspending agents. Physical, chemical and microbiological stability and a rheological study were performed at three different conditions (5 °C ± 3 °C, 25 °C ± 2 °C/60% RH ± 5% RH and 40 °C ± 2 °C/75% RH ± 5% RH) for 120 days. Moreover, dissolution study, content uniformity, related substances, and a study of relative oral bioavailability were also carried out. Both suspensions were physically, chemically and microbiologically stable throughout the study. SA and SB can be stored at 25 °C and 5 °C for at least 120 days whereas SA can be kept at 40 °C for at least 90 days and SB for 120 days. They both met USP specifications for dissolution, content uniformity, and related substances. SA and SB showed an improved relative oral bioavailability compared to the solid dosage form and they both displayed similar relative oral bioavailability with no significant differences between them. The developed suspensions proved to be safe and adequate and they are ideal for pediatric use for their acceptability, accurate dose administration and treatment adherence.

Asunto(s)

Colagogos y Coleréticos/administración & dosificación , Excipientes/química , Ácido Ursodesoxicólico/administración & dosificación , Administración Oral , Animales , Disponibilidad Biológica , Química Farmacéutica , Colagogos y Coleréticos/química , Colagogos y Coleréticos/farmacocinética , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Humedad , Masculino , Ratas , Ratas Sprague-Dawley , Reología , Solubilidad , Suspensiones , Temperatura , Ácido Ursodesoxicólico/química , Ácido Ursodesoxicólico/farmacocinética

RESUMEN

AIM: Hereditary hemochromatosis (HH) is a group of inherited disorders that causes a slow and progressive iron deposition in diverse organs, particularly in the liver. Iron overload induces oxidative stress and tissue damage. Coenzyme Q10 (CoQ10) is a cofactor in the electron-transport chain of the mitochondria, but it is also a potent endogenous antioxidant. CoQ10 interest has recently grown since various studies show that CoQ10 supplementation may provide protective and safe benefits in mitochondrial diseases and oxidative stress disorders. In the present study we sought to determine CoQ10 plasma level in patients recently diagnosed with HH and to correlate it with biochemical, genetic, and histological features of the disease. METHODS: Plasma levels of CoQ10, iron, ferritin, transferrin and vitamins (A, C and E), liver tests (transaminases, alkaline phosphatase and bilirubin), and histology, as well as three HFE gene mutations (H63D, S654C and C282Y), were assessed in thirty-eight patients (32 males, 6 females) newly diagnosed with HH without treatment and in twenty-five age-matched normolipidemic healthy subjects with no HFE gene mutations (22 males, 3 females) and without clinical or biochemical signs of iron overload or liver diseases. RESULTS: Patients with HH showed a significant decrease in CoQ10 levels respect to control subjects (0.31 ±â€¯0.03 µM vs 0.70 ±â€¯0.06 µM, p < 0.001, respectively) independently of the genetic mutation, cirrhosis, transferrin saturation, ferritin level or markers of hepatic dysfunction. Although a decreasing trend in CoQ10 levels was observed in patients with elevated iron levels, no correlation was found between both parameters in patients with HH. Vitamins C and A levels showed no changes in HH patients. Vitamin E was significantly decreased in HH patients (21.1 ±â€¯1.3 µM vs 29.9 ±â€¯2.5 µM, p < 0.001, respectively), but no correlation was observed with CoQ10 levels. CONCLUSION: The decrease in CoQ10 levels found in HH patients suggests that CoQ10 supplementation could be a safe intervention strategy complementary to the traditional therapy to ameliorate oxidative stress and further tissue damage induced by iron overload.

Asunto(s)

Ataxia , Hemocromatosis , Enfermedades Mitocondriales , Debilidad Muscular , Ubiquinona/deficiencia , Ataxia/epidemiología , Estudios de Casos y Controles , Femenino , Hemocromatosis/sangre , Hemocromatosis/epidemiología , Hemocromatosis/genética , Humanos , Masculino , Enfermedades Mitocondriales/epidemiología , Debilidad Muscular/epidemiología , Ubiquinona/análogos & derivados , Ubiquinona/sangre

RESUMEN

Exposure to ambient air particulate matter (PM) is associated with increased cardiorespiratory morbidity and mortality. In this context, alveolar macrophages exhibit proinflammatory and oxidative responses as a result of the clearance of particles, thus contributing to lung injury. However, the mechanisms linking these pathways are not completely clarified. Therefore, the oxinflammation phenomenon was studied in RAW 264.7 macrophages exposed to Residual Oil Fly Ash (ROFA), a PM surrogate rich in transition metals. While cell viability was not compromised under the experimental conditions, a proinflammatory phenotype was observed in cells incubated with ROFA 100 µg/mL, characterized by increased levels of TNF-α and NO production, together with PM uptake. This inflammatory response seems to precede alterations in redox metabolism, characterized by augmented levels of H2O2, diminished GSH/GSSG ratio, and increased SOD activity. This scenario resulted in increased oxidative damage to phospholipids. Moreover, alterations in mitochondrial respiration were observed following ROFA incubation, such as diminished coupling efficiency and spare respiratory capacity, together with augmented proton leak. These findings were accompanied by a decrease in mitochondrial membrane potential. Finally, NADPH oxidase (NOX) and mitochondria were identified as the main sources of superoxide anion () in our model. These results indicate that PM exposure induces direct activation of macrophages, leading to inflammation and increased reactive oxygen species production through NOX and mitochondria, which impairs antioxidant defense and may cause mitochondrial dysfunction.

Asunto(s)

Macrófagos Alveolares/efectos de los fármacos , Mitocondrias/efectos de los fármacos , NADPH Oxidasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Material Particulado/toxicidad , Superóxidos/metabolismo , Contaminantes Atmosféricos/toxicidad , Animales , Antioxidantes/metabolismo , Ceniza del Carbón/toxicidad , Peróxido de Hidrógeno/metabolismo , Inflamación , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Ratones , Mitocondrias/inmunología , Mitocondrias/metabolismo , Oxidación-Reducción , Estrés Oxidativo/inmunología , Células RAW 264.7 , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy specific liver disease characterized by pruritus, elevated serum bile acids and abnormal liver function that may be associated with severe adverse pregnancy outcomes. We previously reported that plasma coenzyme Q10 (CoQ10) is decreased in women with ICP as it is its analogue coenzyme Q9 (CoQ9) in rats with ethinyl estradiol (EE)-induced cholestasis. The aim of the present study was to evaluate the possible therapeutic role of CoQ10 in experimental hepatocellular cholestasis and to compare it with ursodeoxycholic acid (UDCA) supplementation. Bile acids, CoQ9, CoQ10, transaminases, alkaline phosphatase, retinol, α-tocopherol, ascorbic acid, thiobarbituric acid reactive substances, carbonyls, glutathione, superoxide dismutase and catalase were assessed in plasma, liver and/or hepatic mitochondria in control and cholestatic rats supplemented with CoQ10 (250 mg/kg) administered alone or combined with UDCA (25 mg/kg). CoQ10 supplementation prevented bile flow decline (P < 0.05) and the increase in serum alkaline phosphatase and bile acids, particularly lithocholic acid (P < 0.05) in cholestatic rats. Furthermore, it also improved oxidative stress parameters in the liver, increased both CoQ10 and CoQ9 plasma levels and partially prevented the fall in α-tocopherol (P < 0.05). UDCA also prevented cholestasis, but it was less efficient than CoQ10 to improve the liver redox environment. Combined administration of CoQ10 and UDCA resulted in additive effects. In conclusion, present findings show that CoQ10 supplementation attenuated EE-induced cholestasis by promoting a favorable redox environment in the liver, and further suggest that it may represent an alternative therapeutic option for ICP.

Asunto(s)

Colestasis Intrahepática/tratamiento farmacológico , Suplementos Dietéticos , Complicaciones del Embarazo/tratamiento farmacológico , Ubiquinona/análogos & derivados , Animales , Catalasa/metabolismo , Colestasis Intrahepática/metabolismo , Femenino , Glutatión/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Embarazo , Complicaciones del Embarazo/metabolismo , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Ubiquinona/farmacología , Ubiquinona/uso terapéutico , Ácido Ursodesoxicólico/uso terapéutico

RESUMEN

Coenzyme Q10 (CoQ10) is essential in mitochondrial bioenergetics and is a potent endogenous antioxidant. Low CoQ10 levels are associated with neurodegenerative, metabolic, muscular and cardiovascular disorders. Early treatment with high doses (5-50 mg/kg/day) demonstrated to limit the onset and progression of neuropathology. Recently, we developed an oleogel matrix able to support a high dose of oil-dissolved CoQ10, easy to swallow by CoQ10-deficient patients who suffer from secondary dysphagia. In the present study, we evaluated the bioavailability of oleogel-dissolved CoQ10 and plasma antioxidant status in healthy adults in single-dose and repeated-dose studies. The single-dose study demonstrated that, in terms of CoQ10 bioavailability, 1 g CoQ10/5g oleogel-disk was equivalent to the solid form (1 g CoQ10/three 00-size-capsules), whereas the repeated-dose study (14-days-administration) demonstrated a significantly higher increase in plasma CoQ10 when administered through the oleogel, which could be compatible with the levels necessary to achieve an adequate therapeutic response. Also, a trend to a higher plasma apparent half-life (greater than24 h) was observed for the oleogel-loaded-CoQ10. In conclusion, the oleogel matrix does not compromise the oil-dissolved CoQ10 bioavailability and can prevent the non-adherence to this vital supplementation in patients with high CoQ10 requirements. No significant variation in the plasma antioxidant status (vitamins A, E and C, glutathione and TBARs) was observed.

Asunto(s)

Antioxidantes/administración & dosificación , Portadores de Fármacos , Ubiquinona/análogos & derivados , Administración Oral , Adulto , Antioxidantes/química , Antioxidantes/farmacocinética , Disponibilidad Biológica , Biomarcadores/sangre , Cápsulas , Estudios Cruzados , Composición de Medicamentos , Femenino , Semivida , Humanos , Masculino , Persona de Mediana Edad , Compuestos Orgánicos/química , Ubiquinona/administración & dosificación , Ubiquinona/química , Ubiquinona/farmacocinética

RESUMEN

AIM: Intrahepatic cholestasis of pregnancy (ICP) is considered a high-risk condition because it may have serious consequences for the fetus health. ICP is characterized by the accumulation of bile acids in maternal serum which contribute to an imbalance between the production of reactive oxygen species and the antioxidant defenses increasing the oxidative stress experienced by the fetus. Previously, it was reported a significant decrease in plasma coenzyme Q10 (CoQ10) in women with ICP. CoQ10 is a redox substance integrated in the mitochondrial respiratory chain and is recognized as a potent antioxidant playing an intrinsic role against oxidative damage. The objective of the present study was to investigate the levels of CoQ10 in umbilical cord blood during normal pregnancy and in those complicated with ICP, all of them compared to the maternal ones. METHODS: CoQ10 levels and bile acid levels in maternal and umbilical cord blood levels during normal pregnancies (n=23) and in those complicated with ICP (n=13), were investigated. RESULTS: A significant decrease in neonate CoQ10 levels corrected by cholesterol (0.105±0.010 vs. 0.069±0.011, P<0.05, normal pregnancy vs. ICP, respectively), together with an increase of total serum bile acids (2.10±0.02 vs. 7.60±2.30, P<0.05, normal pregnancy vs. ICP, respectively) was observed. CONCLUSIONS: A fetus from an ICP mother is exposed to a greater risk derived from oxidative damage. The recognition of CoQ10 deficiency is important since it could be the starting point for a new and safe intervention strategy which can establish CoQ10 as a promising candidate to prevent the risk of oxidative stress.

Asunto(s)

Ataxia/sangre , Ácidos y Sales Biliares/sangre , Colestasis Intrahepática/sangre , Sangre Fetal/química , Enfermedades Mitocondriales/sangre , Debilidad Muscular/sangre , Complicaciones del Embarazo/sangre , Ubiquinona/análogos & derivados , Ubiquinona/deficiencia , Adulto , Ataxia/diagnóstico , Biomarcadores/sangre , Peso al Nacer , Colesterol/sangre , Ácido Cólico/sangre , Estudios Transversales , Femenino , Feto/metabolismo , Edad Gestacional , Humanos , Recién Nacido , Enfermedades Mitocondriales/diagnóstico , Debilidad Muscular/diagnóstico , Oxidación-Reducción , Estrés Oxidativo , Embarazo , Estudios Prospectivos , Especies Reactivas de Oxígeno/metabolismo , Ubiquinona/sangre , Adulto Joven

RESUMEN

The aim of the study was to evaluate the time course of the effects of urban air pollutants on the ocular surface, focusing on the morphological changes, the redox balance, and the inflammatory response of the cornea. 8-week-old mice were exposed to urban or filtered air (UA-group and FA-group, respectively) in exposure chambers for 1, 2, 4, and 12 weeks. After each time, the eyes were enucleated and the corneas were isolated for biochemical analysis. UA-group corneas exhibited a continuous increase in NADPH oxidase-4 levels throughout the exposure time, suggesting an increased production of reactive oxygen species (ROS). After 1 week, an early adaptive response to ROS was observed as an increase in antioxidant enzymes. After 4 weeks, the enzymatic antioxidants were decreased, meanwhile an increase of the glutathione was shown, as a later compensatory antioxidant response. However, redox imbalance took place, evidenced by the increased oxidized proteins, which persisted up to 12 weeks. At this time point, corneal epithelium hyperplasia was also observed. The inflammatory response was modulated by the increase in IL-10 levels after 1 week, which early regulates the release of TNF-α and IL-6. These results suggest that air pollution alters the ocular surface, supported by the observed cellular hyperplasia. The redox imbalance and the inflammatory response modulated by IL-10 play a key role in the response triggered by air pollutants on the cornea. Taking into account this time course study, the ocular surface should also be considered as a relevant target of urban air pollutants.

Asunto(s)

Contaminantes Atmosféricos/toxicidad , Contaminación del Aire/efectos adversos , Epitelio Corneal/patología , Animales , Brasil , Ciudades , Epitelio Corneal/efectos de los fármacos , Hiperplasia/inducido químicamente , Hiperplasia/patología , Interleucina-10/metabolismo , Masculino , Ratones , NADPH Oxidasa 4/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Pruebas de Toxicidad Subaguda , Pruebas de Toxicidad Subcrónica

RESUMEN

Sildenafil is a phosphodiesterase type 5 inhibitor which confers cardioprotection against myocardial ischaemia/reperfusion (I/R) injury. The aim of this study was to determine if Trx1 participates in cardioprotection exerted by sildenafil in an acute model of I/R, and to evaluate mitochondrial bioenergetics and cellular redox status. Langendorff-perfused hearts from wild type (WT) mice and a dominant negative (DN-Trx1) mutant of Trx1 were assigned to placebo or sildenafil (0.7 mg/kg i.p.) and subjected to 30 min of ischaemia followed by 120 min of reperfusion. WT + S showed a significant reduction of infarct size (51.2 ± 3.0% vs. 30 ± 3.0%, p < .001), an effect not observed in DN-Trx. After I/R, sildenafil preserved state 3 oxygen consumption from WT, but had a milder effect in DN-Trx1 only partially protecting state 3 values. Treatment restored respiratory control (RC) after I/R, which resulted 8% (WT) and 24% (DN-Trx1) lower than in basal conditions. After I/R, a significant increase in H2O2 production was observed both for WT and DN-Trx (WT: 1.17 ± 0.13 nmol/mg protein and DN-Trx: 1.38 ± 0.12 nmol/min mg protein). With sildenafil, values were 21% lower only in WT I/R. Treatment decreased GSSG levels both in WT and DN-Trx1. In addition, GSSG/GSH2 ratio was partially restored by sildenafil. Also, an increase in p-eNOS/eNOS even before the myocardial ischaemia was observed with sildenafil, both in WT (14%, p > .05) and in DN-Trx (35%, p < .05). Active Trx1 is required for the onset of the cardioprotective effects of sildenafil on I/R injury, together with the preservation of cellular redox balance and mitochondrial function.

Asunto(s)

Mitocondrias/efectos de los fármacos , Isquemia Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Citrato de Sildenafil/uso terapéutico , Animales , Masculino , Ratones , Ratones Transgénicos , Inhibidores de Fosfodiesterasa 5/farmacología , Citrato de Sildenafil/farmacología

RESUMEN

Coenzyme Q10 (CoQ10) is an important cofactor in the mitochondrial respiratory chain and a potent endogenous antioxidant. CoQ10 deficiency is currently associated with numerous diseases like mitochondrial and neurodegenerative pathologies, in which the earliest diagnosis and treatment with CoQ10 supplementation becomes paramount for patient's treatment. Consequently, the determination of CoQ10 levels in different biological matrices positions as a fundamental tool. Urine is an attractive and non-invasive alternative source to tissue, blood or other biofluids for CoQ10 analysis. However, it poses an analytical challenge, as it generally requires a complex sample preparation, with multiple steps. In this work we developed and validated a molecularly imprinted polymer solid phase extraction (MIP-SPE) followed by a HPLC-MS/MS method for the analysis of CoQ10 in urine. The MIP-SPE method developed is simple and fast compared to previously traditional reported methods, with reduced processing time, improved sample cleaning and excellent recovery values, along with its inherent high selectivity. The developed chromatographic method was validated according to FDA guidelines, and demonstrated to be suitable for the analysis of CoQ10 in urine samples with LOQ and LOD values of 0.6 ng/mL and 0.2 ng/mL of CoQ10 in urine respectively. Recovery values at three concentration levels were higher than 90.0%.The proposed method is amenable to be applied in pediatric patients due to the low sample requirement and useful for diagnosis and post-treatment control.

Asunto(s)

Impresión Molecular/métodos , Extracción en Fase Sólida/métodos , Ubiquinona/análogos & derivados , Adolescente , Adulto , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Límite de Detección , Modelos Lineales , Masculino , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Ubiquinona/aislamiento & purificación , Ubiquinona/orina , Adulto Joven

RESUMEN

A simple and highly sensitive CE-UV method was applied in the determination of l-ctrulline, which was developed from an oral formulation for pediatric use. The novel method was based on the analysis of l-citrulline for direct ultraviolet detection at 198 nm. The BGE consisted of 10 mM sodium tetraborate and 50 mM SDS at pH 9, and the electrophoretic parameters were optimized. The method was validated in terms of specificity, linearity, LOD, LOQ, precision, accuracy, and robustness. The LOD and LOQ obtained were 1.36 and 4.54 µg/mL, respectively. In addition, the method offers higher sensitivity and specificity compared with the results obtained from HPLC method using UV-detectors, in which l-citrulline needs to be derivatizated. Furthermore, low cost and simplicity of the system allowed the rapid and simple quantitation of l-citrulline in the oral formulation for quality control and stability indicated method.

Asunto(s)

Citrulina/análisis , Electroforesis Capilar/métodos , Soluciones Farmacéuticas/análisis , Espectrofotometría Ultravioleta/métodos , Cromatografía Capilar Electrocinética Micelar , Citrulina/química , Límite de Detección , Modelos Lineales , Soluciones Farmacéuticas/química , Reproducibilidad de los Resultados

RESUMEN

Ischemic postconditioning (PostC) reduces infarct size in healthy experimental models. However, if protective effects of PostC are abolished during early stages of atherosclerotic and if this is related with a disbalance in mitochondrial energetics and alterations in thioredoxin-1 (Trx1) is still unknown. The objectives were to generate a murine high-fat diet (HFD)-fed model that developed in a phenotype consistent with early stages of atherosclerosis to then evaluate whether HFD exposure increased oxidative stress and consequently abolished the cardioprotection conferred by PostC. We used C57/BL6 mice fed with control diet (CD) or HFD for 12 weeks. Isolated mice hearts were subjected to 30 min of ischemia and 120 min of reperfusion (I/R group). For PostC group, after ischemia, six cycles of reperfusion/ischemia were performed (10 s per cycle) at the onset of reperfusion. In CD group, the PostC reduced infarct size (CD-I/R: 52.14 ± 2.8 vs. CD-PostC: 36.58 ± 1.8, P < 0.05) and increased phosphorylation of GSK3ß (CD-PostC: 2.341 ± 1.03 vs. CD-Baseline: 0.923 ± 0.41 AUOD, P < 0.05), and this cardioprotection was abolished in HFD-exposed mice. HFD increased hydrogen peroxide levels, produced a shift towards an oxidized intracellular environment (GSSG/GSH2), and increased Trx1 expression with higher fractions of oxidized protein. State 3 mitochondrial oxygen consumption in basal conditions decreased 24% in HFD-exposed mice and PostC improved state 3 values only in CD mice. Cellular redox state and mitochondrial bioenergetics were altered in HFD-exposed mice. We demonstrated that alterations in redox state at early stages of atherosclerosis abolished cardioprotective mechanisms, such as those induced by PostC, even with increased Trx1 levels.

Asunto(s)

Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Poscondicionamiento Isquémico , Daño por Reperfusión Miocárdica/etiología , Tiorredoxinas/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Oxidación-Reducción

RESUMEN

Coenzyme Q10 (CoQ10) is a mitochondrial respiratory cofactor and potent endogenous antioxidant. In CoQ10-deficient patients, early treatment with high-oral doses (5-50 mg/kg/day) can limit the progression of renal disease and the onset of neurological manifestations. Crystalline CoQ10 is lipophilic, water-insoluble, and poorly absorbed in the gut. Here, CoQ10 showed low bulk density, another important disadvantage in solid oral formulations. Thus, we propose the use of oleogels to maintain dissolved a high-dose of CoQ10 in medium-chain triglyceride (MCT) oil, using ethylcellulose (EC) for gelling, and a surfactant (sorbitan monostearate -SMS- or lecithin). "True gels" were only obtained with the surfactant presence. Thermoreversible oleogels with 1 g of dissolved CoQ10 per 5 g-disk were successfully developed with proved stability and solubility for 12 months (25.0 °C). SMS was better than lecithin as a surfactant because it allowed lower syneresis, higher CoQ10 retention for 12 months, and notably higher oxidative-stability of the MCT-oil, best immobilized by its true gel network. Plastic deformation without fracture was determined under compression, emulating the soft deformation behavior inside the mouth. SMS-oleogels allowed loading a maximal solubilized CoQ10 dose with maximal stability, and may be easier to swallow by CoQ10-deficient patients who suffer from secondary dysphagia.

Asunto(s)

Antioxidantes/administración & dosificación , Celulosa/análogos & derivados , Tensoactivos/química , Ubiquinona/análogos & derivados , Administración Oral , Antioxidantes/química , Celulosa/química , Química Farmacéutica/métodos , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Hexosas/química , Lecitinas/química , Compuestos Orgánicos , Solubilidad , Triglicéridos/química , Ubiquinona/administración & dosificación , Ubiquinona/química

RESUMEN

OBJECTIVES: Conduct a preliminary comparison of the bioavailability between two formulations: commercial grade coenzyme Q10 (CoQ10) powder (solid formulation) and a new oil-in-water liquid emulsion and their effect on other antioxidants. METHODS: Six healthy individuals participated in a randomized, crossover, open, consecutive design, with a 2-week washout period. Pharmacokinetic parameters were assessed after a single and multiple intakes of 250 mg CoQ10 given daily for 1 week. KEY FINDINGS: The differences in the pharmacokinetic parameters of maximum plasma concentration, area under the curve between 0-360 and 0-4 h, elimination half-life were statistically significant with a relative bioavailability of 489% increase over solid CoQ10 formulation. A multiple dose supplementation increased plasma CoQ10 levels in both formulations, liquid emulsion performing better (2.4- vs 3.9-fold for solid and liquid formulation, respectively) without modifications on other antioxidants. Furthermore, the plasma CoQ10 at 7th day was statistically different between formulations (P < 0.05). CONCLUSIONS: The results obtained showed that liquid emulsion improves the bioavailability of CoQ10 respect to solid form which not only facilitates the individualized administration for the child but in turn could increase the therapeutic efficacy, which should be confirmed by further studies.

Asunto(s)

Ubiquinona/análogos & derivados , Adolescente , Adulto , Antioxidantes/farmacocinética , Disponibilidad Biológica , Química Farmacéutica/métodos , Estudios Cruzados , Suplementos Dietéticos , Emulsiones/farmacocinética , Femenino , Semivida , Humanos , Masculino , Medicina de Precisión/métodos , Ubiquinona/metabolismo , Ubiquinona/farmacocinética , Adulto Joven

RESUMEN

A stereoselective CD-MEKC system has been developed for the quality control of Montelukast (MK), commercialized as a pure enantiomer. The proposed method is the first one that allows the simultaneous determination of MK, its enantiomeric form, diasteroisomers and its main degradation compound (MK sulphoxide). CD-MEKC system is composed of 10 mM SDS, 10 mM sulfobutylether-ß-CD, 10 mM TM-ß-CD, and 20 mM borate buffer at pH 9.0. Combination of these two CDs allows high baseline enantioresolution between MK and its enantiomeric impurity, but also, between the diasteroisomeric forms. Moreover, a multivariate design was applied to optimize operational parameters. The method was designed to meet with requirements of the official pharmacopoeias and fully validated according to international guidelines. Linearity of MK was demonstrated in the range from 10.0 to 100.0 µg/mL (r(2) = 0.9908) with a LOD and LOQ of 0.30 and 0.90 µg/mL, respectively. Intra and interday precision were evaluated and RSD values were below 2%, and also, accuracy expressed as percentage of recovery was in a range from 99.0 to 101.9 for the three assayed levels. The method allows determining 0.02% w/w of the enantiomeric and diasteroisomeric impurities, and 0.01% w/w of MK sulphoxide. Robustness was evaluated by a Plackett and Burman design. Finally, the CD-MEKC system was successfully applied to the determination of related substances in MK bulk drug and its quantification in two pediatric pharmaceutical dosage forms.

Asunto(s)

Acetatos/análisis , Electroforesis Capilar/métodos , Antagonistas de Leucotrieno/análisis , Quinolinas/análisis , Ciclopropanos , Límite de Detección , Estándares de Referencia , Reproducibilidad de los Resultados , Estereoisomerismo , Sulfuros