RESUMEN
Perturbations of the expression of transporters and drug-metabolizing enzymes (DMEs) by opioids can be the locus of deleterious drug-drug interactions (DDIs). Many transporters and DMEs are regulated by xenobiotic receptors [XRs; e.g., pregnane X receptor (PXR), constitutive androstane receptor (CAR), and Aryl hydrocarbon receptor (AhR)]; however, there is a paucity of information regarding the influence of opioids on XRs. The objective of this study was to determine the influence of oxycodone administration (15 mg/kg intraperitoneally twice daily for 8 days) on liver expression of XRs, transporters, and DMEs in rats. Microarray, quantitative real-time polymerase chain reaction and immunoblotting analyses were used to identify significantly regulated genes. Three XRs (e.g., PXR, CAR, and AhR), 27 transporters (e.g., ABCB1 and SLC22A8), and 19 DMEs (e.g., CYP2B2 and CYP3A1) were regulated (P < 0.05) with fold changes ranging from -46.3 to 17.1. Using MetaCore (computational platform), we identified a unique gene-network of transporters and DMEs assembled around PXR, CAR, and AhR. Therefore, a series of transactivation/translocation assays were conducted to determine whether the observed changes of transporters/DMEs are mediated by direct activation of PXR, CAR, or AhR by oxycodone or its major metabolites (noroxycodone and oxymorphone). Neither oxycodone nor its metabolites activated PXR, CAR, or AhR. Taken together, these findings identify a signature hepatic gene-network associated with repeated oxycodone administration in rats and demonstrate that oxycodone alters the expression of many transporters and DMEs (without direct activation of PXR, CAR, and AhR), which could lead to undesirable DDIs after coadministration of substrates of these transporters/DMEs with oxycodone.
Asunto(s)
Oxicodona/farmacología , Receptores de Droga/biosíntesis , Xenobióticos/metabolismo , Animales , Línea Celular Tumoral , Expresión Génica/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Droga/genética , Activación TranscripcionalRESUMEN
It is well established that 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) causes acute liver damage in animals and humans. The aim of this study was to identify and characterize oxidative modification and inactivation of cytosolic proteins in MDMA-exposed rats. Markedly increased levels of oxidized and nitrated cytosolic proteins were detected 12 h after the second administration of two consecutive MDMA doses (10 mg/kg each). Comparative 2-DE analysis showed markedly increased levels of biotin-N-methylimide-labeled oxidized cytosolic proteins in MDMA-exposed rats compared to vehicle-treated rats. Proteins in the 22 gel spots of strong intensities were identified using MS/MS. The oxidatively modified proteins identified include anti-oxidant defensive enzymes, a calcium-binding protein, and proteins involved in metabolism of lipids, nitrogen, and carbohydrates (glycolysis). Cytosolic superoxide dismutase was oxidized and its activity significantly inhibited following MDMA exposure. Consistent with the oxidative inactivation of peroxiredoxin, MDMA activated c-Jun N-terminal protein kinase and p38 kinase. Since these protein kinases phosphorylate anti-apoptotic Bcl-2 protein, their activation may promote apoptosis in MDMA-exposed tissues. Our results show for the first time that MDMA induces oxidative-modification of many cytosolic proteins accompanied with increased oxidative stress and apoptosis, contributing to hepatic damage.
Asunto(s)
Citosol/efectos de los fármacos , Alucinógenos/farmacología , Hígado/efectos de los fármacos , N-Metil-3,4-metilenodioxianfetamina/farmacología , Proteínas/metabolismo , Animales , Citosol/enzimología , Activación Enzimática/efectos de los fármacos , Hígado/enzimología , Masculino , Proteínas Quinasas Activadas por Mitógenos/análisis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitrógeno/metabolismo , Oxidación-Reducción , Proteínas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVES: Clinical use and illicit abuse of the potent opioid agonist oxycodone has dramatically increased over the past decade. Yet oxycodone remains one of the least studied opioids, particularly its interactions on the genomic level. The aim of this study was to examine potential alterations in gene expression of drug metabolising enzymes in the liver tissue of male Sprague-Dawley rats chronically treated with oxycodone. METHODS: Rats were administered saline or oxycodone 15 mg/kg i.p. twice a day for 8 days. Changes in RNA levels were detected using microarray analysis validated by quantitative real-time PCR; consequent changes in protein expression and functionality were further assessed by Western blotting and activity assays. KEY FINDINGS: The expression of several drug metabolising enzymes was modulated by oxycodone treatment: cytochrome P450 (CYP) 2B2, CYP2C13, CYP17A1, epoxide hydrolase 2, carboxylesterase 2, flavin-containing monooxygenase 1, glutathione S-transferase alpha 5 (rGSTA5) and CYP3A2. In particular, the mRNA level of rGSTA5 (formerly GSTYc(2)) was up-regulated by approximately 6.5 fold and CYP3A2 was down-regulated by approximately 7.0 fold. Immunoblotting assays demonstrated a corresponding significant elevation of rGSTA5 protein and repression of CYP3A2 protein. The apparent cytosolic GST activity towards 1-chloro-2,4-dinitrobenzene conjugation and reduction of cumene hydroperoxide were significantly higher in liver from oxycodone-treated rats than that of saline-treated rats. In addition, the microsomal activity of CYP3A2, measured via 6beta-hydroxylation of testosterone, was significantly decreased in oxycodone-treated rats. CONCLUSIONS: Repeated oxycodone administration is associated with a significant up-regulation of rGSTA5 and concomitant down-regulation of CYP3A2 mRNA, protein expression and functionality. These results support further in-vivo studies into the clinical impact of our findings.
Asunto(s)
Analgésicos Opioides/efectos adversos , Hidrocarburo de Aril Hidroxilasas/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutatión Transferasa/genética , Isoenzimas/genética , Hígado/enzimología , Proteínas de la Membrana/genética , Oxicodona/efectos adversos , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacocinética , Animales , Western Blotting , Citocromo P-450 CYP3A , Citosol/efectos de los fármacos , Citosol/enzimología , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Inyecciones Intraperitoneales , Hígado/efectos de los fármacos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxicodona/administración & dosificación , Oxicodona/farmacocinética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Despite numerous reports about the acute and sub-chronic toxicities caused by MDMA (3,4-methylenedioxymethamphetamine, ecstasy), the underlying mechanism of organ damage is poorly understood. The aim of this review is to present an update of the mechanistic studies on MDMA-mediated organ damage partly caused by increased oxidative/nitrosative stress. Because of the extensive reviews on MDMA-mediated oxidative stress and tissue damage, we specifically focus on the mechanisms and consequences of oxidative-modifications of mitochondrial proteins, leading to mitochondrial dysfunction. We briefly describe a method to systematically identify oxidatively-modified mitochondrial proteins in control and MDMA-exposed rats by using biotin-N-maleimide (biotin-NM) as a sensitive probe for oxidized proteins. We also describe various applications and advantages of this Cys-targeted proteomics method and alternative approaches to overcome potential limitations of this method in studying oxidized proteins from MDMA-exposed tissues. Finally we discuss the mechanism of synergistic drug-interaction between MDMA and other abused substances including alcohol (ethanol) as well as application of this redox-based proteomics method in translational studies for developing effective preventive and therapeutic agents against MDMA-induced organ damage.
Asunto(s)
Mitocondrias/efectos de los fármacos , Modelos Biológicos , Insuficiencia Multiorgánica/inducido químicamente , Insuficiencia Multiorgánica/fisiopatología , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Estrés Oxidativo/efectos de los fármacos , Vísceras/efectos de los fármacos , Animales , Alucinógenos/toxicidad , Humanos , RatasRESUMEN
Although oxycodone is the most often used opioid agonist, it remains one of the most understudied drugs. We used microarray analysis to better understand the global changes in gene expression in brain tissues of rats repeatedly treated with oxycodone. Many genes were significantly regulated by oxycodone (e.g., Fkbp5, Per2, Rt1.Dalpha, Slc16a1, and Abcg2). Validation of the microarray data by quantitative real-time-polymerase chain reaction (Q-PCR) indicated that there was a strong significant correlation (r = 0.979, p < 0.0000001) between the Q-PCR and the microarray data. Using MetaCore (a computational platform), many biological processes were identified [e.g., organic anion transport (p = 7.251 x 10(-4)) and regulation of immune response (p = 5.090 x 10(-4))]. Among the regulated genes, Abcg2 mRNA was up-regulated by 2.1-fold, which was further confirmed by immunoblotting (1.8-fold up-regulation). Testing the Abcg2 affinity status of oxycodone using an Abcg2 ATPase assay suggests that oxycodone behaves as an Abcg2 substrate only at higher concentrations (> or = 500 microM). Furthermore, brain uptake studies demonstrated that oxycodone-induced Abcg2 up-regulation resulted in a significant (p < 0.05) decrease (approximately 2-fold) in brain/plasma ratios of mitoxantrone. These results highlight markers/mediators of neuronal responses and identify regulatory pathways involved in the pharmacological action of oxycodone. These results also identify genes that potentially modulate tolerance, dependence, immune response, and drug-drug interactions. Finally, our findings suggest that oxycodone-induced up-regulation of Abcg2 enhanced the efflux of the Abcg2 substrate, mitoxantrone, limiting its brain accumulation and resulting in an undesirable drug-drug interaction. Extrapolating these results to other Abcg2 substrates (e.g., daunorubicin and doxorubicin) indicates that the brain uptake of these agents may be affected if they are administered concomitantly with oxycodone.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxicodona/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Algoritmos , Animales , Biocatálisis/efectos de los fármacos , Análisis por Conglomerados , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Interacciones Farmacológicas/fisiología , Cinética , Masculino , Proteínas de Transporte de Membrana/genética , Mitoxantrona/sangre , Mitoxantrona/metabolismo , Oxicodona/administración & dosificación , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trastornos Relacionados con Sustancias/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
The effect of progesterone and estrogen treatment on the expression and function of P-glycoprotein (P-gp) was evaluated in JAR cells and a P-gp overexpressing cell line, NCI-ADR-RES. Western blot analysis and real-time Q-PCR were used to evaluate P-gp protein and MDR1 mRNA expression respectively in the cells following incubation with progesterone (P4) and/or beta-estradiol (E2). Cellular uptake studies of the P-gp substrates, saquinavir and paclitaxel, were performed to evaluate function. Treatment with either E2 or P4 resulted in a significant increase in P-gp protein levels in the NCI-ADR-RES cells at concentrations of or greater than 100 nM or 10 nM, respectively. JAR cells also had increased levels of P-gp with 100 nM of P4 but were much more sensitive to E2 showing increased P-gp at a concentration of 1 nM. Furthermore, E2 or P4 treatment resulted in a significant decrease in cellular uptake of the P-gp substrates tested in these cells lines. Based on mRNA quantitation, a transient increase (2-fold) in MDR1 levels was observed at 8 h postincubation with either E2 or P4, while MDR1 levels remained high in the JAR cells treated with E2 for 72 h postincubation. The addition of actinomycin D, a transcription inhibitor negated the increase in P-gp by P4 and E2. P4 and E2 increase P-gp expression and function in NCI-ADR-RES and JAR cells with the ERalpha-expressing cells (JAR) much more sensitive to E2. Furthermore, transcriptional regulation by E2 and P4 likely contributes to the modulation of P-gp levels.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Estradiol/farmacología , Estrógenos/farmacología , Placenta/citología , Progesterona/farmacología , Progestinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Inhibidores de Cisteína Proteinasa/farmacología , Dactinomicina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Leupeptinas/farmacología , Embarazo , Inhibidores de la Síntesis de la Proteína/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Salvinorin A is an unregulated potent hallucinogen isolated from the leaves of Salvia divinorum. It is the only known non-nitrogenous kappa-opioid selective agonist, and rivals synthetic lysergic acid diethylamide (LSD) in potency. The objective of this study was to characterize the in vitro transport, in vitro metabolism, and pharmacokinetic properties of Salvinorin A. The transport characteristics of Salvinorin A were assessed using MDCK-MDR1 cell monolayers. The P-glycoprotein (P-gp) affinity status was assessed by the P-gp ATPase assay. In vitro metabolism studies were performed with various specific human CYP450 isoforms and UGT2B7 to assess the metabolic characteristics of Salvinorin A. Cohorts (n = 3) of male Sprague Dawley rats were used to evaluate the pharmacokinetics and brain distribution of Salvinorin A (10 mg/kg, intraperitoneal (i.p.) over a 240-min period. A validated UV-HPLC and LC/MS/MS method was used to quantify the hallucinogen concentrations obtained from the in vitro and in vivo studies, respectively. Salvinorin A displayed a high secretory transport in the MDCK-MDR1 cells (4.07 +/- 1.34 x 10(-)5 cm/s). Salvinorin A also stimulated the P-gp ATPase activity in a concentration (5 and 10 microM)-dependent manner, suggesting that it may be a substrate of (P-gp). A significant decrease in Salvinorin A concentration ranging from 14.7 +/- 0.80% to 31.1 +/- 1.20% was observed after incubation with CYP2D6, CYP1A1, CYP2C18, and CYP2E1, respectively. A significant decrease was also observed after incubation with UGT2B7. These results suggest that Salvinorin A maybe a substrate of UGT2B7, CYP2D6, CYP1A1, CYP2E1, and CYP2C18. The in vivo pharmacokinetic study showed a relatively fast elimination with a half-life (t1/2) of 75 min and a clearance (Cl/F) of 26 L/h/kg. The distribution was extensive (Vd of 47.1 L/kg); however, the brain to plasma ratio was 0.050. Accordingly, the brain half-life was relatively short, 36 min. Salvinorin A is rapidly eliminated after i.p. dosing, in accordance with its fast onset and short duration of action. Further, it appears to be a substrate for various oxidative enzymes and multi-drug resistant protein, P-gp.
Asunto(s)
Diterpenos de Tipo Clerodano/farmacocinética , Alucinógenos/farmacocinética , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Perros , Semivida , Masculino , Ratas , Ratas Sprague-Dawley , Espectrofotometría UltravioletaRESUMEN
Alcohol (ethanol) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are frequently co-abused, but recent findings indicate a harmful drug interaction between these two agents. In our previous study, we showed that MDMA exposure inhibits the activity of the acetaldehyde (ACH) metabolizing enzyme, aldehyde dehydrogenase2 (ALDH2). Based on this finding, we hypothesized that the co-administration of MDMA and ethanol would reduce the metabolism of ACH and result in increased accumulation of ACH. Rats were treated with MDMA or vehicle and then administered a single dose of ethanol. Liver ALDH2 activity decreased by 35% in the MDMA-treated rats compared to control rats. The peak concentration and the area under the concentration versus time curve of plasma ACH were 31% and 59% higher, respectively, in the MDMA-ethanol group compared to the ethanol-only group. In addition, the MDMA-ethanol group had 80% higher plasma transaminase levels than the ethanol-only group, indicating greater hepatocellular damage. Our results not only support a drug interaction between MDMA and ethanol but a novel underlying mechanism for the interaction.
Asunto(s)
Acetaldehído/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Etanol/toxicidad , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Acetaldehído/sangre , Aldehído Deshidrogenasa/antagonistas & inhibidores , Familia de Aldehído Deshidrogenasa 1 , Aldehído Deshidrogenasa Mitocondrial , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Interacciones Farmacológicas , Etanol/farmacocinética , Isoenzimas/antagonistas & inhibidores , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , N-Metil-3,4-metilenodioxianfetamina/farmacocinética , Ratas , Ratas Sprague-Dawley , Retinal-DeshidrogenasaRESUMEN
Efflux transporters such as P-glycoprotein (P-gp) play a critical role in the maternal-to-fetal and blood-to-brain transfer of many drugs. Using a mouse model, the effects of gestational age on P-gp and MRP expression in the placenta and brain were evaluated. P-gp protein levels in the placenta and brain were greater at mid-gestation (gd 13) than late-gestation (gd 18). Likewise, brain MRP1 levels were greater at mid-gestation, whereas, placental levels were greater at late-gestation. To evaluate these effects on drug disposition, concentrations of [(3)H]saquinavir, [(3)H]methadone, [(3)H]buprenorphine, and the paracellular marker, [(14)C]mannitol were measured in plasma, brain, placenta, and fetal samples after i.v. administrations to nonpregnant and pregnant mice. Following i.v. administration, [(3)H]saquinavir placenta-to-plasma and fetal-to-plasma ratios were significantly greater in late-gestation mice versus mid-gestation. Furthermore, late-gestation mice experienced significant increases in the [(3)H]saquinavir and [(3)H]methadone brain-to-plasma ratios 60 min after dosing relative to mid-gestation (p < 0.05). No significant differences were observed in these tissue-to-plasma ratios for buprenorphine or mannitol. Repeated dosing (three doses, once daily) decreased the differential uptake of [(3)H]saquinavir in brain but potentiated it in the fetus. These results suggest that differential expression of P-gp and possibly MRP1 contributes to the gestational-induced changes in brain and fetal uptake of saquinavir.
Asunto(s)
Encéfalo/metabolismo , Buprenorfina/metabolismo , Metadona/metabolismo , Placenta/metabolismo , Preñez/metabolismo , Saquinavir/metabolismo , Animales , Encéfalo/efectos de los fármacos , Buprenorfina/farmacología , Femenino , Desarrollo Fetal/efectos de los fármacos , Desarrollo Fetal/fisiología , Feto/efectos de los fármacos , Feto/metabolismo , Exposición Materna , Intercambio Materno-Fetal/efectos de los fármacos , Intercambio Materno-Fetal/fisiología , Metadona/farmacología , Ratones , Placenta/efectos de los fármacos , Embarazo , Preñez/efectos de los fármacos , Saquinavir/farmacología , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
Despite numerous reports citing the acute hepatotoxicity caused by 3,4-methylenedioxymethamphetamine (MDMA) (ecstasy), the underlying mechanism of organ damage is poorly understood. We hypothesized that key mitochondrial proteins are oxidatively modified and inactivated in MDMA-exposed tissues. The aim of this study was to identify and investigate the mechanism of inactivation of oxidatively modified mitochondrial proteins, prior to the extensive mitochondrial dysfunction and liver damage following MDMA exposure. MDMA-treated rats showed abnormal liver histology with significant elevation in plasma transaminases, nitric oxide synthase, and the level of hydrogen peroxide. Oxidatively modified mitochondrial proteins in control and MDMA-exposed rats were labeled with biotin-N-maleimide (biotin-NM) as a sensitive probe for oxidized proteins, purified with streptavidin-agarose, and resolved using 2-DE. Comparative 2-DE analysis of biotin-NM-labeled proteins revealed markedly increased levels of oxidatively modified proteins following MDMA exposure. Mass spectrometric analysis identified oxidatively modified mitochondrial proteins involved in energy supply, fat metabolism, antioxidant defense, and chaperone activities. Among these, the activities of mitochondrial aldehyde dehydrogenase, 3-ketoacyl-CoA thiolases, and ATP synthase were significantly inhibited following MDMA exposure. Our data show for the first time that MDMA causes the oxidative inactivation of key mitochondrial enzymes which most likely contributes to mitochondrial dysfunction and subsequent liver damage in MDMA-exposed animals.
Asunto(s)
Alucinógenos/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , N-Metil-3,4-metilenodioxianfetamina/farmacología , Animales , Biotina/química , Peróxido de Hidrógeno/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Maleimidas/química , Mitocondrias Hepáticas/metabolismo , Óxido Nítrico Sintasa/sangre , Oxidación-Reducción , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem/métodos , Transaminasas/sangreRESUMEN
BACKGROUND: The effects of alcohol consumption and its subsequent metabolism on drug transport, absorption and pharmacokinetics are poorly understood. This study examines the effects of the ethanol metabolite, acetaldehyde, on the clinically relevant drug transporter, PEPT1. The metabolism of ethanol and the following acetaldehyde formation is thought to modulate the uptake capacity of PEPT1 within the gastrointestinal tract for a variety of clinically important peptidomimetic drug compounds. METHODS: Glycylsarcosine ([(3)H]-GlySar), a nonhydrolysable PEPT1 specific substrate was used in our studies. In vitro uptake studies were performed in the Caco-2 and Chinese hamster ovary (CHO)-hPEPT1 cell models, measuring cellular uptake of labeled compound against increasing levels of unlabeled compound in the presence of acetaldehyde. In vivo absorption of [(3)H]-GlySar was measured in male Sprague-Dawley rats that were treated with oral dose of ethanol/disulfiram (5 g/kg / 100 mg/kg) for 6 days. These results were compared to control rats treated with saline, ethanol alone or disulfiram alone. RESULTS: In vitro uptake of [(3)H]-GlySar in CHO-hPEPT1 cells treated with 1 mM acetaldehyde was significantly decreased (p < 0.05) as compared to untreated controls. The uptake of [(3)H]-GlySar in Caco-2 cell monolayers treated with 1 mM acetaldehyde was also significantly decreased as compared to the untreated control cells. In vivo absorption of [(3)H]-GlySar in ethanol treated rats, as measured by AUC(0-12 hours) were decreased by approximately 50% versus the control rat group. CONCLUSION: The effects of acetaldehyde due to consumption of ethanol on the uptake and bioavailability of therapeutic drug compounds transported by the PEPT1 oligopeptide transporter have not been documented. In the present studies, we demonstrate that acetaldehyde significantly modulates PEPT1 function and, thereby, affects drug bioavailability. To our best knowledge, this is the first report on the effects of an ethanol metabolite on substrate absorption in the gastrointestinal tract, rather than interactions in the liver, which is an under-represented area of research in alcohol pathophysiology.
Asunto(s)
Acetaldehído/farmacología , Dipéptidos/farmacocinética , Etanol/farmacocinética , Simportadores/efectos de los fármacos , Absorción , Acetaldehído/sangre , Animales , Disponibilidad Biológica , Células CHO , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Etanol/administración & dosificación , Humanos , Hidrazinas , Concentración de Iones de Hidrógeno/efectos de los fármacos , Masculino , Transportador de Péptidos 1 , Ratas , Ratas Sprague-DawleyRESUMEN
Previous studies suggest that P-glycoprotein (P-gp) modulates the PK/PD of many compounds including opioid agonists and chemotherapeutic agents. The objective of this study was to assess the P-gp affinity status of oxycodone, the P-gp expression, and the paclitaxel's tissue distribution in oxycodone-treated rats. P-gp ATPase assay, Caco-2 transepithelial permeability studies, and mdr1a/b (-/-) mice were used to assess the P-gp affinity status of oxycodone. P-gp expression was determined by Western blot analysis while [(14)C] paclitaxel's distributions in the liver, kidney, brain, and plasma tissues were determined by liquid scintillation counter. Oxycodone stimulated the P-gp ATPase activity in a concentration-dependant manner. The Caco-2 secretory transport of oxycodone was reduced from 3.64 x 10(-5) to 1.96 x 10(-5) cm/s (p < 0.05) upon preincubation with the P-gp inhibitor, verapamil. The brain levels of oxycodone in mdr1a/b (+/+) were not detectable (<15 ng/mL) while in mdr1a/b (-/-) the average levels were 115 +/- 39 ng/mL. The P-gp protein levels were increased by 1.3-4.0 folds while paclitaxel's tissue distributions were decreased by 38-90% (p < 0.05) in oxycodone-treated rats. These findings display that oxycodone is a P-gp substrate, induces overexpression of P-gp, and affects paclitaxel's tissue distribution in a manner that may influence its chemotherapeutic activity.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Analgésicos Opioides/farmacología , Antineoplásicos Fitogénicos/farmacocinética , Oxicodona/farmacología , Paclitaxel/farmacocinética , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Adenosina Trifosfatasas/antagonistas & inhibidores , Analgésicos Opioides/metabolismo , Animales , Transporte Biológico Activo/fisiología , Células CACO-2 , Bloqueadores de los Canales de Calcio/farmacología , Cromatografía Líquida de Alta Presión , Interacciones Farmacológicas , Tolerancia a Medicamentos , Humanos , Masculino , Ratones , Ratones Noqueados , Oxicodona/metabolismo , Dimensión del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Espectrofotometría Ultravioleta , Distribución Tisular , Regulación hacia Arriba/efectos de los fármacos , Verapamilo/farmacologíaRESUMEN
MUC1 is a large transmembrane glycoprotein overexpressed by a majority of carcinomas. High expression of MUC1 is associated with aggressive tumors, and MUC1 antigen is used as a marker to monitor disease progression in breast cancer patients. Several lines of evidence strongly suggest that the overexpression of MUC1 contributes to cancer progression and metastasis. In this report, we demonstrate that the naturally occurring cancer preventative, indole-3-carbinol (I3C), inhibits the expression of MUC1 in breast cancer cells. I3C inhibited both MUC1 mRNA and protein levels in a dose- and time-dependent manner. This inhibition was seen in the estrogen responsive MCF-7 cells as well as unresponsive MDA-MB-468 cells, indicating that the inhibitory pathway is independent of estrogen receptor. Gene expression studies using the human MUC1 gene promoter connected to a luciferase reporter demonstrated that I3C inhibits the transcription of the MUC1 gene. Promoter deletion studies indicate that the region containing up to 600 bp upstream (-600) of the initiation site is sufficient for inhibition by I3C. Furthermore, I3C represses the activation of transcription mediated by the region between -600 and -450 bp. A putative xenobiotic response element was located within this region but the binding of AhR/Arnt heterodimer to this site was undetectable by electrophoretic mobility shift assays. Our results may point to the existence of a novel pathway of transcriptional inhibition by I3C in cancer cells as well as a new mechanism of MUC1 gene inhibition. Our findings might have implications in the use of I3C as a preventative as well as a therapeutic agent for breast cancer.