RESUMEN
During the key event 1 of skin sensitization defined as covalent binding or haptenization of sensitizer to either thiol or amino group of skin proteins, a sensitizer not only covalently binds with skin proteins but also interacts with nucleophilic small molecules such as glutathione (GSH). Although GSH would not be directly associated with skin sensitization, this interaction may be applied for developing an alternative test method simulating key event 1, haptenization. Thus, the aim of the present study was to examine whether N-acetyl-L-cysteine methyl ester (NACME), a thiol-containing compound, was selected as an electron donor to determine whether NACME reacted with sensitizers. Following a reaction of NACME with a sensitizer in a 96-well plate, the remaining NACME was measured spectrophotometrically using 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Following the optimization of test conditions with two different vehicles, such as acetonitrile (ACN) and dimethyl sulfoxide (DMSO), 64 test chemicals were tested to determine the predictive capacity of current NACME test method. The results obtained showed, the predictive capacity of 94.6% sensitivity, 88.9% specificity, and 92.2% accuracy utilizing DMSO as a vehicle with a cutoff NACME depletion of 5.85%. The three parameters were also over 85% in case of ACN. These values were comparable to or better than other OECD-approved test methods. Data demonstrated that a simple thiol-containing compound NACME might constitute as a reliable candidate for identifying reactive skin sensitizers, and that this method be considered as practical method as a screening tool for assessing a chemical's tendency to initiate skin sensitization.
Asunto(s)
Acetilcisteína , Acetilcisteína/análogos & derivados , Espectrofotometría , Humanos , Piel/efectos de los fármacos , Ácido Ditionitrobenzoico/química , Haptenos/toxicidad , Haptenos/química , Alternativas a las Pruebas en Animales/métodos , AnimalesRESUMEN
To initiate skin sensitization, haptens react with endogenous proteins. During this process, skin sensitizers react with small endogenous molecules containing thiol or amino groups. In this study, a simple spectrophotometric method to identify skin sensitizers in chemico was developed using the reactivity of glutathione (GSH) with test chemicals in a 96-well plate. To quantitate the remaining GSH following the reaction with a skin sensitizer, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was employed. The optimized experimental conditions included the pH- and time-dependent stability of GSH, stability of derivatized products of GSH with optimal concentration and incubation time of DTNB, incubation time of GSH with the test chemicals, and molar ratios of GSH to the test chemicals. With the optimized conditions with both acetonitrile and DMSO as vehicles and incubation of GSH with test chemicals in 1:10 and 1:15 ratios for 24 h at 4 °C, 23 skin sensitizers and 23 non-sensitizers, based on the local lymph node assay, were tested to determine the predictive capacity of individual conditions. The best result showed a predictive capacity of 95.2% sensitivity, 91.3% specificity, and 93.2% accuracy, with 93.2% consistency in three trials, when 5.8% depletion was used as a cut-off value in 1:10 of GSH:test chemicals in DMSO. It would be an economic and useful screening tool for determining the skin sensitization potential of small molecules, because the present method employs simple endogenous GSH as an electron donor for sensitizers with a spectrophotometric detection system in 96-well plates, and because the method requires neither experimental animals nor cell cultures. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00218-9.
RESUMEN
Immunomodulation is an essential consideration for cell replacement procedures. Unfortunately, lifelong exposure to nonspecific systemic immunosuppression results in immunodeficiency and has toxic effects on nonimmune cells. Here, we engineered hybrid spheroids of mesenchymal stem cells (MSCs) with rapamycin-releasing poly(lactic-co-glycolic acid) microparticles (RAP-MPs) to prevent immune rejection of islet xenografts in diabetic C57BL/6 mice. Hybrid spheroids were rapidly formed by incubating cell-particle mixture in methylcellulose solution while maintaining high cell viability. RAP-MPs were uniformly distributed in hybrid spheroids and sustainably released RAP for ~3 weeks. Locoregional transplantation of hybrid spheroids containing low doses of RAP-MPs (200- to 4000-ng RAP per recipient) significantly prolonged islet survival times and promoted the generation of regional regulatory T cells. Enhanced programmed death-ligand 1 expression by MSCs was found to be responsible for the immunomodulatory performance of hybrid spheroids. Our results suggest that these hybrid spheroids offer a promising platform for the efficient use of MSCs in the transplantation field.
Asunto(s)
Células Madre Mesenquimatosas , Esferoides Celulares , Animales , Humanos , Inmunomodulación , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Trasplante HeterólogoRESUMEN
Ethanol intake can alter pharmacokinetics by increasing the solubility or enhancing the absorption of concomitant drugs. Here, a selective, sensitive and reproducible high-performance liquid chromatography-tandem mass spectrometry method for the quantitative analysis of nicardipine in rat plasma was developed using simple protein precipitation. The calibration curve was linear over a concentration range of 1-2,000 ng/ml (r2 > 0.998). Accuracy ranged from 93.4 to 112.2% and precision was within 12.1% from three independent analytical batches. Stable conditions for the quantification of nicardipine in rat plasma were established in various conditions, including sample storage and handling. The matrix effect was negligible, and recovery was consistent at three different levels of quality control sample. The method was applied to assessment for the effect of ethanol on the pharmacokinetics of nicardipine in rats. The oral bioavailability of nicardipine was increased from 5.4 to 9.4% in Sprague-Dawley rats by concomitant oral administration of ethanol whereas the half-life was not altered. The findings indicated that concomitant ethanol intake can increase systemic drug exposure by increasing gastrointestinal absorption, especially poorly soluble drugs. This study provides an insight for further investigation of the alteration of the pharmacological effect of poorly soluble drugs owing to ethanol intake.
Asunto(s)
Nicardipino , Espectrometría de Masas en Tándem , Administración Oral , Animales , Cromatografía Líquida de Alta Presión , Etanol , Preparaciones Farmacéuticas , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosRESUMEN
A convenient fluorometrical test method to identify skin sensitizers in chemico was developed using reactivity with glutathione (GSH), a low molecular weight endogenous substance. Following incubation of test chemicals with GSH, the remaining GSH was quantitated fluorometrically by using monobromobimane (mBBr), a thiol-detecting agent, for determining % depletion of this endogenous substance by test chemicals. The experimental conditions optimized were: (1) reactivity of thiol compounds including GSH with mBBr, (2) effects of vehicles on reactivity, (3) molar ratios of GSH to test chemicals, and (4) reactivity of endogenous substance with test substances under different incubation times. When an optimized condition with DMSO as a vehicle for test chemicals and in 1:60 ratio for 24 hr at 4°C was applied to classify 48 well-known skin sensitizers and non-sensitizers, the predictive capacity was as follows: 88.2% sensitivity, 78.6% specificity, and 85.4% accuracy with 95.8% consistency of three trials when 10.3% depletion of GSH was used as a cutoff value. Because the present method employed relatively simple GSH as an acceptor for sensitizers and/or a relatively convenient fluorometric detection system in 96-well plates for a high throughput test, it would be a useful test tool for screening skin sensitization potential of test chemicals.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Compuestos Bicíclicos con Puentes/química , Fluorometría/métodos , Glutatión/análisis , Piel/efectos de los fármacos , Piel/fisiopatologíaRESUMEN
CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro-3-1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC-MS/MS using a cocktail assay. Among the four compounds, HYIpro-3-1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC50 values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro-3-1 is not dependent on preincubation time. HYIpro-3-1 showed a typical pattern of competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation, based on the Lineweaver-Burk plot, with a Ki value of 0.05 µM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro-3-1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs.
Asunto(s)
Inhibidores del Citocromo P-450 CYP1A2/farmacología , Microsomas Hepáticos/enzimología , Citocromo P-450 CYP1A1/antagonistas & inhibidores , HumanosRESUMEN
In addition to the hepatic metabolism, the role of intestinal microbiota in drug metabolism has been considered important in the biotransformation of xenobiotics. Crocin and its aglycone, crocetin, isolated from many plants, including the dried stigma of Crocus sativus and the fruit of Gardenia jasminoides, have been used in treatment of inflammation, cancer, and metabolic disorders. In this study, the effect of intestinal microbiota on the pharmacokinetics of crocin was studied following single oral treatment with 600 mg/kg crocin to male rats pre-treated with a mixture of antibiotics, such as cefadroxil, oxytetracycline, and erythromycin, for three consecutive days. Following crocin treatment, blood, urine, and feces were collected at various time points for evaluating pharmacokinetic characteristics of crocin and crocetin by using LC-MS. Results showed that intestinal absorption of crocin was relatively marginal when compared with that of crocetin, and that crocin metabolism to crocetin by intestinal microbiota would be a critical step for absorption. The present results clearly suggested that the in vivo pharmacological effects of crocin might be considered as the effects by its aglycone, crocetin, mainly, and that the metabolism of glycosidic natural products by intestinal microbiota should be considered to understand their pharmacodynamic actions.
RESUMEN
We conducted a me-too validation study to confirm the reproducibility, reliability, and predictive capacity of KeraSkin™ skin irritation test (SIT) as a me-too method of OECD TG 439. With 20 reference chemicals, within-laboratory reproducibility (WLR) of KeraSkin™ SIT in the decision of irritant or non-irritant was 100%, 100%, and 95% while between-laboratory reproducibility (BLR) was 100%, which met the criteria of performance standard (PS, WLR≥90%, BLR≥80%). WLR and BLR were further confirmed with intra-class correlation (ICC, coefficients >0.950). WLR and BLR in raw data (viability) were also shown with a scatter plot and Bland-Altman plot. Comparison with existing VRMs with Bland-Altman plot, ICC and kappa statistics confirmed the compatibility of KeraSkin™ SIT with OECD TG 439. The predictive capacity of KeraSkin™ SIT was estimated with 20 reference chemicals (the sensitivity of 98.9%, the specificity of 70%, and the accuracy of 84.4%) and additional 46 chemicals (for 66 chemicals [20 + 46 chemicals, the sensitivity, specificity and accuracy: 95.2%, 82.2% and 86.4%]). The receiver operating characteristic (ROC) analysis suggested a potential improvement of the predictive capacity, especially sensitivity, when changing cut-off (50% â 60-75%). Collectively, the me-too validation study demonstrated that KeraSkin™ SIT can be a new me-too method for OECD TG 439.
Asunto(s)
Epidermis/efectos de los fármacos , Adhesión a Directriz/normas , Irritantes/toxicidad , Modelos Biológicos , Organización para la Cooperación y el Desarrollo Económico/normas , Pruebas de Irritación de la Piel/normas , Epidermis/metabolismo , Epidermis/patología , Humanos , Irritantes/metabolismo , Pruebas de Irritación de la Piel/métodosRESUMEN
Deoxyshikonin, a natural shikonin derivative, is the major component of Lithospermum erythrorhizon and exhibits various pharmacological effects such as lymphangiogenetic, antibacterial, wound healing, and anticancer effects. To investigate the herb-drug interaction potential associated with deoxyshikonin, the inhibitory effects of deoxyshikonin on eight major cytochrome P450 (CYP) enzymes were examined using cocktail substrate-incubated human liver microsomes. Deoxyshikonin strongly inhibited CYP2B6-catalyzed bupropion hydroxylation, with a Ki value of 3.5 µM, and the inhibition was confirmed using purified human CYP2B6. The inhibition was reversible because the inhibitory effect of deoxyshikonin was not dependent on the preincubation time. The results indicated that deoxyshikonin-induced drug-drug interaction should be considered when any herb containing deoxyshikonin is used for conventional medications.
Asunto(s)
Inhibidores del Citocromo P-450 CYP2B6/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones de Hierba-Droga , Naftoquinonas/farmacología , Humanos , Microsomas Hepáticos/metabolismoRESUMEN
BACKGROUND: Voglibose, an α-glucosidase inhibitor, inhibits breakdown of complex carbohydrates into simple sugar units in intestine. Studies showed that voglibose metabolism in the liver might be negligible due to its poor intestinal absorption. Numerous microorganisms live in intestine and have several roles in metabolism and detoxification of various xenobiotics. Due to the limited information, the possible metabolism of voglibose by intestinal microbiota was investigated in vitro and in vivo. METHODS: For the in vitro study, different concentrations of voglibose were incubated with intestinal contents, prepared from both vehicle- and antibiotics-treated mice, to determine the decreased amount of voglibose over time by using liquid chromatography-mass spectrometry. Similarly, in vivo pharmacodynamic effect of voglibose was determined following the administration of voglibose and starch in vehicle- and antibiotic-pretreated non-diabetic and diabetic mice, by measuring the modulatory effects of voglibose on blood glucose levels. RESULTS: The in vitro results indicated that the remaining voglibose could be significantly decreased when incubated with the intestinal contents from normal mice compared to those from antibiotic-treated mice, which had less enzyme activities. The in vivo results showed that the antibiotic pretreatment resulted in reduced metabolism of voglibose. This significantly lowered blood glucose levels in antibiotic-pretreated mice compared to the control animals. CONCLUSION: The present results indicate that voglibose would be metabolized by the intestinal microbiota, and that this metabolism might be pharmacodynamically critical in lowering blood glucose levels in mice.
Asunto(s)
Microbioma Gastrointestinal , Animales , Diabetes Mellitus Experimental , Inhibidores de Glicósido Hidrolasas , Inositol/análogos & derivados , RatonesRESUMEN
Botulinum toxins are neurotoxic modular proteins composed of a heavy chain and a light chain connected by a disulfide bond and are produced by Clostridium botulinum. Although lethally toxic, botulinum toxin in low doses is clinically effective in numerous medical conditions, including muscle spasticity, strabismus, hyperactive urinary bladder, excessive sweating, and migraine. Globally, several companies are now producing products containing botulinum toxin for medical and cosmetic purposes, including the reduction of facial wrinkles. To test the efficacy and toxicity of botulinum toxin, animal tests have been solely and widely used, resulting in the inevitable sacrifice of hundreds of animals. Hence, alternative methods are urgently required to replace animals in botulinum toxin testing. Here, the various alternative methods developed to test the toxicity and efficacy of botulinum toxins have been briefly reviewed and future perspectives have been detailed.
RESUMEN
1. Glycyrol is a coumestan derivative that is isolated from roots of Glycyrrhiza uralensis. Glycyrol exhibits several biological effects, including anti-oxidative and anti-inflammatory effects.2. Herein, we characterized glycyrol metabolism by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM), human liver cytosol, human intestinal microsomes, or human recombinant cDNA-expressed CYPs and UGTs. The analysis was conducted using high resolution mass spectroscopy (HR-MS) on a Q ExactiveTM HF Hybride Quadrupole-Orbitrap mass spectrometer.3. NADPH-supplemented HLM generated six glycyrol metabolites (M1-M6) via hydroxylation, oxidation, and hydration; both NADPH- and UDPGA-supplemented liver microsomes generated three glucuronides (M7-M9). Reaction phenotyping revealed that CYP1A2 is the primary enzyme responsible for phase I metabolism, with minor involvement of the CYP3A4/5, CYP2D6, and CYP2E1 enzymes. Glucuronidation of glycyrol was primarily mediated by UGT1A1, UGT1A3, UGT1A9, and UGT2B7.4. In conclusion, glycyrol undergoes the efficient metabolic hydroxylation and glucuronidation reactions in human liver microsomes, which are predominantly catalyzed by CYP1A2, UGT1A1/3/9, and UGT2B7.
Asunto(s)
Flavonoides/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Humanos , Microsomas/metabolismo , Microsomas Hepáticos/metabolismo , Espectrometría de Masas en Tándem , UDP Glucuronosiltransferasa 1A9RESUMEN
Enteric-coated formulations using Eudragit® polymers have been extensively used for delivering drugs to the lower gastrointestinal tract. However, these drug-delivery systems cannot accurately deliver the therapeutic cargoes to colon because of early degradation of the polymers at alkaline pH of the small intestine. Here, we describe a precise method of delivering drugs to inflammation sites in colon using an oral drug delivery system. Tacrolimus (FK506)-loaded microspheres were prepared using a thioketal-based polymer that releases drug in response to reactive oxygen species (ROS), which are abundantly produced at the sites of inflammation in acute colitis. Orally-administered FK506-loaded thioketal microspheres (FK506-TKM) led to a substantial accumulation of FK506 in inflamed colon and effectively alleviated dextran-sulfate sodium (DSS)-induced murine colitis. At the molecular level, FK506-TKM significantly inhibited infiltration of CD4+ and CD8+ T lymphocytes in colon and differentiation of CD4+ T cells into Th1 and Th17 cells in colon-draining mesenteric lymph nodes via restricting dendritic cell migration from colon. Our findings indicate orally-administered thioketal-based drug delivery system as a promising means of treating acute inflammatory bowel diseases.
Asunto(s)
Colitis/tratamiento farmacológico , Inmunosupresores/administración & dosificación , Inflamación/tratamiento farmacológico , Tacrolimus/administración & dosificación , Animales , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Colitis/patología , Células Dendríticas/citología , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Inmunosupresores/farmacología , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Microesferas , Ácidos Polimetacrílicos/química , Especies Reactivas de Oxígeno/metabolismo , Tacrolimus/farmacología , Células TH1/citología , Células Th17/citologíaRESUMEN
Loxoprofen (LOX) is a non-selective cyclooxygenase inhibitor that is widely used for the treatment of pain and inflammation caused by chronic and transitory conditions. Its alcoholic metabolites are formed by carbonyl reductase (CR) and they consist of trans-LOX, which is active, and cis-LOX, which is inactive. In addition, LOX can also be converted into an inactive hydroxylated metabolite (OH-LOXs) by cytochrome P450 (CYP). In a previous study, we reported that CYP3A4 is primarily responsible for the formation of OH-LOX in human liver microsomes. Although metabolism by CYP3A4 does not produce active metabolites, it can affect the conversion of LOX into trans-/cis-LOX, since CYP3A4 activity modulates the substrate LOX concentration. Although the pharmacokinetics (PK) and metabolism of LOX have been well defined, its CYP-related interactions have not been fully characterized. Therefore, we investigated the metabolism of LOX after pretreatment with dexamethasone (DEX) and ketoconazole (KTC), which induce and inhibit the activities of CYP3A, respectively. We monitored their effects on the PK parameters of LOX, cis-LOX, and trans-LOX in mice, and demonstrated that their PK parameters significantly changed in the presence of DEX or KTC pretreatment. Specifically, DEX significantly decreased the concentration of the LOX active metabolite formed by CR, which corresponded to an increased concentration of OH-LOX formed by CYP3A4. The opposite result occurred with KTC (a CYP3A inhibitor) pretreatment. Thus, we conclude that concomitant use of LOX with CYP3A modulators may lead to drug-drug interactions and result in minor to severe toxicity even though there is no direct change in the metabolic pathway that forms the LOX active metabolite.
RESUMEN
It has been a challenge to develop in vitro alternative test methods for accurate prediction of metallic products which may exert skin sensitization, as several test methods adopted by OECD were relatively ineffective in assessing the capacity for metallic compounds to exert sensitizing reactions, compared with organic test substances. Based upon these findings, a system that incorporates ß-galactosidase producing E. coli cultures was tested for its predictive capacity to well-known metallic sensitizers. In this system, E. coli cells were incubated with metal salts at various concentrations and ß-galactosidase suppression by each test metal was determined. Fourteen local lymph node assay (LLNA) categorized metal salts were examined. Although color interference from metal salts was minimal, a fluorometric detection system was also employed using 4-methylumbelliferyl galactopyranoside as a substrate for ß-galactosidase to avoid the color interference, concomitantly with the original UV-spectrometric method. Data demonstrated that two detection methods were comparable and complementary. In addition, most of the metallic sensitizers were correctly identified at 0.6 and 0.8 mM concentrations. Despite the lower specificity obtained in the current study and small number of substances tested, the developed method appears to be a relatively simple and effective in vitro method for detecting metallic sensitizers. When 61 chemicals tested in the ß-galactosidase producing E. coli cultures including the present study were collectively analyzed, the prediction capacity was as high as other OECD-adopted tests: 95.6% of sensitivity, 66.7% of specificity, and 88.5% of accuracy. It is important to emphasize that animals or mammalian cell cultures were not required in the current method, which are in accordance with the EU guidelines on restricted or banned animal testing.
Asunto(s)
Dermatitis Alérgica por Contacto , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Metales/toxicidad , beta-Galactosidasa/antagonistas & inhibidores , beta-Galactosidasa/metabolismo , Alternativas a las Pruebas en Animales/métodos , Escherichia coli/enzimología , Fluorometría , Isopropil Tiogalactósido , Sensibilidad y Especificidad , Piel/efectos de los fármacos , beta-Galactosidasa/genéticaRESUMEN
With the aim of developing new effective topoisomerase IIα-targeted anticancer agents, we synthesized a series of hydroxy- and halogenated 2,4-diphenyl indeno[1,2-b]pyridinols using a microwave-assisted single step synthetic method and investigated structure-activity relationships. The majority of compounds with chlorophenyl group at 2-position and phenol group at the 4-position of indeno[1,2-b]pyridinols exhibited potent antiproliferative activity and topoisomerase IIα-selective inhibition. Of the 172 compounds tested, 89 showed highly potent and selective topoisomerase IIα inhibition and antiproliferative activity in the nanomolar range against human T47D breast (2.6 nM) cancer cell lines. In addition, mechanistic studies revealed compound 89 is a nonintercalative topoisomerase II poison, and in vitro studies showed it had promising cytotoxic effects in diverse breast cancer cell lines and was particularly effective at inducing apoptosis in T47D cells. Furthermore, in vivo administration of compound 89 had significant antitumor effects in orthotopic mouse model of breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Compuestos de Bifenilo/farmacología , Neoplasias de la Mama/tratamiento farmacológico , ADN-Topoisomerasas de Tipo II/metabolismo , Descubrimiento de Drogas , Piridinas/farmacología , Inhibidores de Topoisomerasa II/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos ICR , Microondas , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/química , Células Tumorales CultivadasRESUMEN
Host immune response remains an obstacle in cell-replacement therapy for treating type I diabetes. Long-term systemic immunosuppression results in suboptimal efficacy and adverse reactions. Thus, "cell-particle hybrids" of pancreatic islets and tissue-adhesive, polydopamine-coated, FK506-loaded biodegradable microspheres (PD-FK506-MS) were developed to locally modulate the immune response at the transplantation site. Coating of FK506-MS with PD enabled the rapid formation of stable cell-particle hybrids without significant changes in islet viability and functionality. Extremely low quantities of FK506 (approximately 600â¯ng per recipient) sustainably released from cell-particle hybrids effectively prolonged survival of xenogeneic islet graft. Interestingly, FK506 exhibited extended bioavailability in the grafts but was undetectable in systemic circulation and other tissues. Moreover, mRNA expression of inflammatory cytokines was significantly inhibited in the PD-FK506-MS-containing grafts but not in lymphoid organs. This study presents a promising platform that facilitates the translation of local immunomodulation towards an effective strategy with improved safety profiles for treating type I diabetes.
Asunto(s)
Terapia de Inmunosupresión/métodos , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/metabolismo , Microesferas , Trasplante Heterólogo/métodos , Animales , Citometría de Flujo , Prueba de Tolerancia a la Glucosa , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Polímeros/química , TacrolimusRESUMEN
The function of regulatory T (Treg) cells depends on lipid oxidation. However, the molecular mechanism by which Treg cells maintain lipid metabolism after activation remains elusive. Liver kinase B1 (LKB1) acts as a coordinator by linking cellular metabolism to substrate AMP-activated protein kinase (AMPK). We show that deletion of LKB1 in Treg cells exhibited reduced suppressive activity and developed fatal autoimmune inflammation. Mechanistically, LKB1 induced activation of the mevalonate pathway by upregulating mevalonate genes, which was essential for Treg cell functional competency and stability by inducing Treg cell proliferation and suppressing interferon-gamma and interleukin-17A expression independently of AMPK. Furthermore, LKB1 was found to regulate intracellular cholesterol homeostasis and to promote the mevalonate pathway. In agreement, mevalonate and its metabolite geranylgeranyl pyrophosphate inhibited conversion of Treg cells and enhanced survival of LKB1-deficient Treg mice. Thus, LKB1 is a key regulator of lipid metabolism in Treg cells, involved in optimal programming of suppressive activity, immune homeostasis, and tolerance.
Asunto(s)
Ácido Mevalónico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T Reguladores/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/terapia , Proliferación Celular , Colesterol/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hidroximetilglutaril-CoA Reductasas/deficiencia , Hidroximetilglutaril-CoA Reductasas/genética , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Metabolismo de los Lípidos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatos de Poliisoprenilo/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Factor de Transcripción STAT5/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/trasplanteRESUMEN
Loxoprofen is a prodrug that exerts strong analgesic and anti-inflammatory effects through its active trans-alcohol metabolite, which is produced in the liver by carbonyl reductase. Previous metabolic studies have evaluated loxoprofen, but its sulfate and taurine conjugates have not yet been studied. We characterized the metabolomic profile of loxoprofen in rat plasma, urine, and feces using high-resolution mass spectrometry. We identified 17 metabolites of loxoprofen in the three different biological matrices, 13 of which were detected in plasma and feces and 16 in urine. Amongst these metabolites, two novel taurine conjugates (M12 and M13) and two novel acyl glucuronides (M14, M15) were identified for the first time in rats. In addition, we detected three novel sulfate conjugates (M9, M10, and M11) of loxoprofen. Further study of these metabolites of loxoprofen is essential in order to assess their potency and toxicity.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacocinética , Fenilpropionatos/farmacocinética , Profármacos/farmacocinética , Administración Oral , Animales , Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/orina , Heces/química , Masculino , Metabolómica , Fenilpropionatos/sangre , Fenilpropionatos/orina , Ratas Sprague-Dawley , Sulfatos/metabolismoRESUMEN
Occupational exposure of workers to 1-bromopropane (1-BP) has raised concerns in industry for many years. Despite the known toxicity of this chemical, molecular events attributed to exposure to 1-BP have not been extensively studied. The aim of the present study was to examine the effects of 1-BP exposure on adduct formation with DNA and glutathione (GSH) in male Sprague-Dawley rats in an attempt to determine the early stages of toxicity. Following 6 h after either single or daily exposure to 1-BP for 3 days, N7-propyl guanine and S-propyl GSH were quantified in several organs by using liquid chromatography-mass spectrometry (LC-MS/MS). The results showed that N7-propyl guanine was maximally formed in liver followed by spleen, testes, and lung in both dose- and time-dependent manners. However, DNA adduct was not detected in cardiac tissue. In the case of S-propyl GSH, this compound was formed in the following order in various organs: liver > testes > spleen > kidney > lung > heart. In a subsequent in vitro study, formation of N7-propyl guanine initiated by 1-BP in calf thymus DNA was not markedly affected by addition of liver homogenates, which indicated that this chemical may be acting as a direct alkylating agent. In contrast, an in vitro study with free GSH demonstrated that 1-BP reduced GSH and elevated production of S-propyl GSH, and that the production of this adduct was significantly higher in the presence of active liver homogenates. Data indicated that formation of GSH adducts initiated by 1-BP might be associated with an enzyme-driven process. Although further characterization is necessary, it would appear that N7-propyl guanine and S-propyl GSH might serve as useful markers in cases of exposure assessment of 1-BP.