RESUMEN
INTRODUCTION: Pharmacokinetic outcomes of transporter-mediated drug-drug interactions (TMDDIs) are increasingly being evaluated clinically. The goal of our study was to determine the effects of selective inhibition of multidrug and toxin extrusion protein 1 (MATE1), using famotidine, on the pharmacokinetics and pharmacodynamics of metformin in healthy volunteers. METHODS: Volunteers received metformin alone or with famotidine in a crossover design. As a positive control, the longitudinal effects of famotidine on the plasma levels of creatinine (an endogenous substrate of MATE1) were quantified in parallel. Famotidine unbound concentrations in plasma reached 1 µM, thus exceeding the in vitro concentrations that inhibit MATE1 [concentration of drug producing 50 % inhibition (IC50) 0.25 µM]. Based on current regulatory guidance, these concentrations are expected to inhibit MATE1 clinically [i.e. maximum unbound plasma drug concentration (C max,u)/IC50 >0.1]. RESULTS: Consistent with MATE1 inhibition, famotidine administration significantly altered creatinine plasma and urine levels in opposing directions (p < 0.005). Interestingly, famotidine increased the estimated bioavailability of metformin [cumulative amount of unchanged drug excreted in urine from time zero to infinity (A e∞)/dose; p < 0.005] without affecting its systemic exposure [area under the plasma concentration-time curve (AUC) or maximum concentration in plasma (C max)] as a result of a counteracting increase in metformin renal clearance. Moreover, metformin-famotidine co-therapy caused a transient effect on oral glucose tolerance tests [area under the glucose plasma concentration-time curve between time zero and 0.5 h (AUCglu,0.5); p < 0.005]. CONCLUSIONS: These results suggest that famotidine may improve the bioavailability and enhance the renal clearance of metformin.
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Antiulcerosos/farmacología , Famotidina/farmacología , Hipoglucemiantes/farmacocinética , Metformina/farmacocinética , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Adulto , Área Bajo la Curva , Glucemia , Creatinina/sangre , Creatinina/orina , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
BACKGROUND AND OBJECTIVES: In the proximal tubule, basic drugs are transported from the renal cells to the tubule lumen through the concerted action of the H(+)/organic cation antiporters, multidrug and toxin extrusion (MATE) 1 and MATE2K. Dual inhibitors of the MATE transporters have been shown to have a clinically relevant effect on the pharmacokinetics of concomitantly administered basic drugs. However, the clinical impact of selective renal organic cation transport inhibition on the pharmacokinetics and pharmacodynamics of basic drugs, such as metformin, is unknown. This study sought to identify a selective MATE2K inhibitor in vitro and to determine its clinical impact on the pharmacokinetics and pharmacodynamics of metformin in healthy subjects. METHODS: Strategic cell-based screening of 71 US Food and Drug Administration (FDA)-approved medications was conducted to identify selective inhibitors of renal organic cation transporters that are capable of inhibiting at clinically relevant concentrations. From this screen, nizatidine was identified and predicted to be a clinically potent and selective inhibitor of MATE2K-mediated transport. The effect of nizatidine on the pharmacokinetics and pharmacodynamics of metformin was evaluated in 12 healthy volunteers in an open-label, randomized, two-phase crossover drug-drug interaction (DDI) study. RESULTS: In healthy volunteers, the MATE2K-selective inhibitor nizatidine significantly increased the apparent volume of distribution, half-life, and hypoglycemic activity of metformin. However, despite achieving unbound maximum concentrations greater than the in vitro inhibition potency (concentration of drug producing 50% inhibition [IC50]) of MATE2K-mediated transport, nizatidine did not affect the renal clearance (CLR) or net secretory clearance of metformin. CONCLUSION: This study demonstrates that a selective inhibition of MATE2K by nizatidine affected the apparent volume of distribution, tissue concentrations, and peripheral effects of metformin. However, nizatidine did not alter systemic concentrations or the CLR of metformin, suggesting that specific MATE2K inhibition may not be sufficient to cause renal DDIs with metformin.
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Metformina/farmacología , Metformina/farmacocinética , Nizatidina/farmacología , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Adolescente , Adulto , Línea Celular , Estudios Cruzados , Interacciones Farmacológicas , Femenino , Células HEK293 , Semivida , Humanos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Organic anion transporter 3 (OAT3, SLC22A8), a transporter expressed on the basolateral membrane of the proximal tubule, plays a critical role in the renal excretion of organic anions including many therapeutic drugs. The goal of this study was to evaluate the in vivo effects of the OAT3-Ile305Phe variant (rs11568482), present at 3.5% allele frequency in Asians, on drug disposition with a focus on cefotaxime, a cephalosporin antibiotic. In HEK293-Flp-In cells, the OAT3-Ile305Phe variant had a lower maximum cefotaxime transport activity, Vmax , [159 ± 3 nmol*(mg protein)(-1) /min (mean ± SD)] compared with the reference OAT3 [305 ± 28 nmol*(mg protein)(-1) /min, (mean ± SD), p < 0.01], whereas the Michaelis-Menten constant values (Km ) did not differ. In healthy volunteers, we found volunteers that were heterozygous for the Ile305Phe variant and had a significantly lower cefotaxime renal clearance (CLR ; mean ± SD: 84.8 ± 32.1 mL/min, n = 5) compared with volunteers that were homozygous for the reference allele (158 ± 44.1 mL/min, n = 10; p = 0.006). Furthermore, the net secretory component of cefotaxime renal clearance (CLsec ) was reduced in volunteers heterozygous for the variant allele [33.3 ± 31.8 mL/min (mean ± SD)] compared with volunteers homozygous for the OAT3 reference allele [97.0 ± 42.2 mL/min (mean ± SD), p = 0.01]. In summary, our study suggests that a low-frequency reduced-function polymorphism of OAT3 associates with reduced cefotaxime CLR and CL(sec) .
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Antibacterianos/sangre , Antibacterianos/metabolismo , Cefotaxima/sangre , Cefotaxima/metabolismo , Riñón/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/genética , Adolescente , Adulto , Pueblo Asiatico/genética , Femenino , Frecuencia de los Genes , Células HEK293 , Humanos , Masculino , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Polimorfismo Genético , Adulto JovenRESUMEN
OBJECTIVES: Human multidrug and toxin extrusion member 1, MATE1 (SLC47A1), plays an important role in the renal and biliary excretion of endogenous and exogenous organic cations including many therapeutic drugs. In this study, we characterized the transcriptional effects of five polymorphic variants and six common haplotypes in the basal promoter region of MATE1 that were identified in 272 DNA samples from ethnically diverse US populations. METHODS: We measured luciferase activities of the six common promoter haplotypes of MATE1 using in-vitro and in-vivo reporter assays. RESULTS: Haplotypes that contain the most common variant (mean allele frequency in four ethnic groups: 0.322), g.-66T>C, showed a significant decrease in reporter activities compared to the reference. Two transcription factors, activating protein-1 (AP-1) and activating protein-2 repressor (AP-2rep), were predicted to bind to the promoter in the region of g.-66T>C. Results from electrophoretic mobility shift assays showed that the g.-66T allele, exhibited greater binding to AP-1 than the g.-66C allele. AP-2rep inhibited the binding of AP-1 to the MATE1 basal promoter region, and the effect was considerably greater for the g.-66T>C. These data suggest that the reduced transcriptional activity of g.-66T>C results from a reduction in the binding potency of the transcriptional activator, AP-1, and an enhanced binding potency of the repressor, AP-2rep to the MATE1 basal promoter region. Consistent with the reporter assays, MATE1 mRNA expression levels were significantly lower in kidney samples from individuals who were homozygous or heterozygous for g.-66T>C in comparison with samples from individuals who were homozygous for the g.-66T allele. CONCLUSION: Our study suggests that the rate of transcription of MATE1 is regulated by AP-1 and AP-2rep and that a common promoter variant, g.-66T>C may affect the expression level of MATE1 in human kidney, and ultimately result in variation in drug disposition and response.
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Proteínas de Transporte de Catión Orgánico/genética , Polimorfismo Genético , Regiones Promotoras Genéticas , Ensayo de Cambio de Movilidad Electroforética , Variación Genética , Haplotipos , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp) and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Variación Genética , Riñón/metabolismo , Hígado/metabolismo , Regiones Promotoras Genéticas , Adolescente , Adulto , Membrana Celular/metabolismo , Femenino , Frecuencia de los Genes , Humanos , Masculino , Familia de Multigenes , Polimorfismo GenéticoRESUMEN
OBJECTIVE: The goal of this study was to determine the effect of a genetic variant in the organic cation transporter 2 (OCT2), OCT2-808G/T, which results in an amino acid change, A270S, on the pharmacokinetics of the antidiabetic drug, metformin. METHODS: The uptake of metformin was performed in stably transfected HEK-293 cells expressing the empty vector (MOCK), the reference OCT2-808G, and the variant OCT2-808T. Healthy individuals with known OCT2 genotypes [14 homozygous for the OCT2 reference allele (808G/G) and nine heterozygous for the variant allele (808G/T, *3D)] were recruited to this study. Metformin concentrations in plasma and urine were measured by liquid chromatography-tandem mass spectrometry method. Creatinine levels were also measured in plasma and urine. Pharmacokinetic parameters were evaluated for both the groups. RESULTS: We observed that in HEK-293 stably transfected cells, OCT2-808T had a greater capacity to transport metformin than did the reference OCT2. Metformin pharmacokinetics was characterized in 23 healthy volunteers of Caucasian and African-American ancestries. We observed that the renal clearance (CL(R)) and the net secretion (SrCL(R)) of metformin were significantly different between the volunteers heterozygous for the variant allele (808G/T), and the volunteers homozygous for the reference allele (808G/G) (P<0.005). Multivariate analysis revealed that OCT2 genotype was a significant predictor of CL(R) and SrCL(R) of metformin (P<0.01). CONCLUSION: We conclude that genetic variation in OCT2 plays an important role in the CL(R) and SrCL(R) of metformin in healthy volunteers.
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Riñón/metabolismo , Metformina/farmacocinética , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Polimorfismo de Nucleótido Simple/genética , Transporte Biológico , Línea Celular , Ligamiento Genético , Homocigoto , Humanos , Metformina/sangre , Metformina/orina , Proteínas Mutantes/genética , Transportador 2 de Cátion Orgánico , Factores de TiempoRESUMEN
The organic cation/ergothioneine transporter OCTN1 (SLC22A4) and the high-affinity carnitine transporter OCTN2 (SLC22A5), play an important role in the disposition of xenobiotics and endogenous compounds. Here, we analyzed the sequence of the proximal promoter regions of OCTN1 and OCTN2 in four ethnic groups and determined the effects of the identified genetic variants on transcriptional activities and mRNA expression. Six variants were found in the proximal promoter of OCTN1, one of which showed high allele frequency ranging from 13 to 34% in samples from individuals with ancestries in Africa, Europe, China, and Mexico. OCTN1 haplotypes had similar activities as the reference in luciferase reporter assays. For OCTN2, three of the seven variants identified in the proximal promoter showed allele frequencies greater than 29.5% in all populations, with the exception of -207C>G (rs2631367) that was monomorphic in Asian Americans. OCTN2 haplotypes containing -207G, present in all populations, were associated with a gain of function in luciferase reporter assays. Consistent with reporter assays, OCTN2 mRNA expression levels in lymphoblastoid cell lines (LCLs) from gene expression analysis were greater in samples carrying a marker for -207G. This SNP seems to contribute to racial differences in OCTN2 mRNA expression levels in LCLs. Our study with healthy subjects (n = 16) homozygous for either -207C or -207G, showed no appreciable effect of this SNP on carnitine disposition. However, there were significant effects of gender on carnitine plasma levels (p < 0.01). Further in vivo studies of OCTN2 promoter variants on carnitine disposition and variation in drug response are warranted.
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Proteínas de Transporte de Catión Orgánico/genética , Regiones Promotoras Genéticas/genética , Carnitina/metabolismo , Línea Celular Tumoral/metabolismo , Células Cultivadas , Clonación Molecular , Etnicidad , Variación Genética , Haplotipos , Humanos , Luciferasas/genética , Polimorfismo de Nucleótido Simple , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Miembro 5 de la Familia 22 de Transportadores de Solutos , Simportadores , Distribución TisularRESUMEN
BACKGROUND: National asthma guidelines recommend that patients with persistent asthma regularly use an inhaled corticosteroid (ICS) in addition to as-needed albuterol, yet recent debates question whether this combination is equally efficacious in all ethnicities. OBJECTIVE: To examine the effect of ICS use on bronchodilator responsiveness to albuterol in 3 different ethnic populations. METHODS: A cross-sectional study of 106 Mexican Americans, 246 Puerto Ricans, and 163 African Americans with physician-diagnosed persistent asthma. Asthma severity, ethnicity, and medication use were evaluated using spirometry and questionnaires. Percentage change in forced expiratory volume in 1 second (FEV) was compared in patients who used ICSs vs those who used a short-acting beta2-agonist as their only asthma medication. RESULTS: Inhaled corticosteroid use was associated with improvements in the percentage change in FEV1 after albuterol administration in Mexican Americans (21.7%, P = .01) and Puerto Ricans (18.5%, P = .02) but not in African Americans (3.0%, P = .73). CONCLUSIONS: Inhaled corticosteroid use is associated with augmented bronchodilator responsiveness to albuterol in Mexican Americans and Puerto Ricans, but not in African Americans, with persistent asthma. This underscores the need for an improved understanding of ethnic-specific drug-drug interactions, particularly in those subgroups experiencing the highest burden of asthma morbidity and mortality in the United States.
Asunto(s)
Corticoesteroides/uso terapéutico , Albuterol/uso terapéutico , Asma/tratamiento farmacológico , Negro o Afroamericano/estadística & datos numéricos , Hispánicos o Latinos/estadística & datos numéricos , Administración por Inhalación , Adolescente , Corticoesteroides/administración & dosificación , Adulto , Factores de Edad , Albuterol/administración & dosificación , Asma/etnología , Asma/fisiopatología , Bronquios/efectos de los fármacos , Bronquios/patología , Broncodilatadores/administración & dosificación , Broncodilatadores/uso terapéutico , Niño , Estudios Transversales , Interacciones Farmacológicas , Femenino , Volumen Espiratorio Forzado/efectos de los fármacos , Volumen Espiratorio Forzado/fisiología , Humanos , Modelos Lineales , Masculino , Pruebas de Función Respiratoria , Factores Sexuales , Capacidad Vital/fisiologíaRESUMEN
Socioeconomic and environmental differences do not fully explain differences in asthma prevalence, morbidity, and mortality among Puerto Ricans, African Americans, and Mexican Americans. Differences in response to albuterol may be a factor. We compared bronchodilator responsiveness between these three populations. All groups demonstrated below expected responsiveness. Puerto Ricans of all ages and African American children with moderate-to-severe asthma demonstrated the lowest responsiveness overall. Among subjects with moderate-to-severe asthma, children were even less likely than adults to show the expected bronchodilator response. We conclude that ethnic-specific differences in bronchodilator drug responsiveness exist between Mexicans, Puerto Ricans, and African Americans with asthma. This may be of importance in asthma management.
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Albuterol/uso terapéutico , Asma/tratamiento farmacológico , Asma/etnología , Negro o Afroamericano , Broncodilatadores/uso terapéutico , Hispánicos o Latinos , Americanos Mexicanos , Adolescente , Adulto , Asma/fisiopatología , Niño , Femenino , Volumen Espiratorio Forzado/efectos de los fármacos , Humanos , Masculino , México , Ciudad de Nueva York , Puerto Rico , San Francisco , Estadísticas no Paramétricas , Capacidad Vital/efectos de los fármacosRESUMEN
BACKGROUND: OCTN1 is a multispecific transporter of organic cations and zwitterions, including several clinically important drugs as well as the antioxidant ergothioneine. OCTN1 is highly expressed in the kidney, where it is thought to aid in active secretion of organic cations, and may facilitate the active reabsorption of ergothioneine. Genetic variation in OCTN1 may help to explain interindividual variability in the pharmacokinetics of many cationic or zwitterionic drugs. METHODS: We screened for human genetic variants in the OCTN1 coding region by direct sequencing in a large sample (n=270) of ethnically diverse healthy volunteers. RESULTS: Six protein sequence-altering variants were identified, including five-amino-acid substitutions and one nonsense mutation. Two of the variants, T306I and L503F, were polymorphic, occurring at frequencies of 37 and 19%, respectively, in the total sample. Allele frequencies are varied by ethnicity. In biochemical assays, two of the variants (D165G and R282X) resulted in complete loss of transport function, and one variant (M205I) caused a reduction in activity to approximately 50% of the reference sequence protein. One variant, L503F, showed altered substrate specificity; this variant occurred at particularly high allele frequency (42%) in the European-American participants in our sample. Subcellular localization and ergothioneine inhibition kinetics were similar among the common amino-acid sequence variants of OCTN1. CONCLUSIONS: The common OCTN1-L503F variant may explain a significant amount of population variation in the pharmacokinetics of OCTN1 substrate drugs. The rare loss-of-function variants provide a rational tool for studying the importance of ergothioneine in humans in vivo.
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Proteínas de Transporte de Catión Orgánico/genética , Sustitución de Aminoácidos , Línea Celular , Codón sin Sentido , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Modelos Moleculares , Proteínas de Transporte de Catión Orgánico/química , Proteínas de Transporte de Catión Orgánico/metabolismo , Farmacogenética , Polimorfismo Genético , Estructura Secundaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Simportadores , TransfecciónRESUMEN
Metformin is among the most widely prescribed drugs for the treatment of type 2 diabetes. Organic cation transporter 1 (OCT1) plays a role in the hepatic uptake of metformin, but its role in the therapeutic effects of the drug, which involve activation of AMP-activated protein kinase (AMPK), is unknown. Recent studies have shown that human OCT1 is highly polymorphic. We investigated whether OCT1 plays a role in the action of metformin and whether individuals with OCT1 polymorphisms have reduced response to the drug. In mouse hepatocytes, deletion of Oct1 resulted in a reduction in the effects of metformin on AMPK phosphorylation and gluconeogenesis. In Oct1-deficient mice the glucose-lowering effects of metformin were completely abolished. Seven nonsynonymous polymorphisms of OCT1 that exhibited reduced uptake of metformin were identified. Notably, OCT1-420del (allele frequency of about 20% in white Americans), previously shown to have normal activity for model substrates, had reduced activity for metformin. In clinical studies, the effects of metformin in glucose tolerance tests were significantly lower in individuals carrying reduced function polymorphisms of OCT1. Collectively, the data indicate that OCT1 is important for metformin therapeutic action and that genetic variation in OCT1 may contribute to variation in response to the drug.
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Variación Genética/fisiología , Hipoglucemiantes/farmacología , Metformina/farmacología , Transportador 1 de Catión Orgánico/genética , Células 3T3-L1 , Animales , Línea Celular , Células Clonales , Femenino , Humanos , Hipoglucemiantes/antagonistas & inhibidores , Masculino , Metformina/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transportador 1 de Catión Orgánico/fisiología , Polimorfismo GenéticoRESUMEN
BACKGROUND: The prostanoid DP receptor (PTGDR) gene on chromosome 14q22.1 has been identified as an asthma susceptibility gene. A haplotype with decreased transcription factor binding and transcription efficiency was associated with decreased asthma susceptibility in African American and white subjects. The significance of PTGDR gene variants in asthma has yet to be determined in Latinos, the largest US minority population, nor has the association been replicated in other populations. OBJECTIVE: To determine the role of PTGDR gene variants in asthma susceptibility and asthma-related traits among the Mexican, Puerto Rican, and African American populations. METHODS: We determined whether single nucleotide polymorphisms (SNPs) and haplotypes in PTGDR were associated with asthma and asthma-related traits by family-based and cross-sectional cohort analyses in 336 Puerto Rican and 273 Mexican asthmatic trios and by case-control analysis among African American subjects with asthma and healthy controls (n = 352). RESULTS: We identified 13 SNPs in the PTGDR gene, and 6 were further analyzed. There was no significant association between PTGDR variants and asthma by family-based or case-control analyses. SNPs -441C and -197C and haplotype TTT showed marginal association with asthma-related traits in Mexican subjects. SNP -441 genotype TT (P = .05) and haplotype TTT (P = .02) were associated with increased IgE levels in African Americans. CONCLUSION: We conclude that the PTGDR gene is not a significant risk factor for asthma among Puerto Ricans, Mexicans, or African Americans. CLINICAL IMPLICATIONS: Asthma candidate genes provide insights to pathophysiology and potentially new therapeutic targets, although the PTGDR gene was not found to be a significant risk factor for asthma in 3 populations.
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Asma/etnología , Asma/genética , Negro o Afroamericano , Predisposición Genética a la Enfermedad , Hispánicos o Latinos , Receptores Inmunológicos/genética , Receptores de Prostaglandina/genética , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Cromosomas Humanos Par 14/genética , Estudios de Cohortes , Estudios Transversales , Femenino , Haplotipos , Humanos , Masculino , Americanos Mexicanos , Polimorfismo de Nucleótido Simple , Puerto Rico/etnologíaRESUMEN
Systemic carnitine deficiency (SCD) is a rare autosomal recessive disease resulting from defects in the OCTN2 (SLC22A5) gene, which encodes the high-affinity plasma membrane carnitine transporter. Although OCTN2 is fairly well studied in its relationship with SCD, little is known about the carrier frequency of disease-causing alleles of OCTN2, or of more common functional polymorphisms in this gene. To address these issues, we screened for genetic variants in the OCTN2 coding region by direct sequencing of the exons and flanking intronic region of OCTN2 in a large sample (n = 276) of ethnically diverse subjects. In addition, we established lymphoblastoid cell lines from subjects homozygous for either allele of the previously identified promoter region variant, -207G>C. We found eight amino acid sequence variants of OCTN2, of which three (Phe17Leu, Leu144Phe, and Pro549Ser) were polymorphic in at least one ethnic group. When assayed for functional activity by expression in human embryonic kidney 293 cells, using as probes both the endogenous substrate (l-carnitine) and the organic cation tetraethylammonium, three variants showed functional differences from the reference OCTN2 (Phe17Leu, Tyr449Asp, Val481Phe; p < 0.05). Further studies of the Phe17Leu polymorphism showed a reduced V(max) for l-carnitine transport to approximately 50% of the reference OCTN2. Confocal microscopy studies using an OCTN2-GFP fusion protein showed that Phe17Leu had distinct subcellular localization from the reference OCTN2, with diffuse cytoplasmic retention of Phe17Leu, in contrast to reference OCTN2, which localized specifically to the plasma membrane. Lymphoblasts from subjects homozygous for the -207G allele showed increased l-carnitine transport compared with the -207C/C homozygotes (p < 0.05). This study suggests that although loss-of-function mutations in OCTN2 are likely to be rare, common variants of OCTN2 found in healthy populations may contribute to variation in the disposition of carnitine and some clinically used drugs.
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Proteínas de Transporte de Catión Orgánico/genética , Polimorfismo Genético , Secuencia de Aminoácidos , Carnitina/metabolismo , Regulación de la Expresión Génica , Genética de Población , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Proteínas de Transporte de Catión Orgánico/química , Fenotipo , Regiones Promotoras Genéticas/genética , Estructura Secundaria de Proteína , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Miembro 5 de la Familia 22 de Transportadores de Solutos , Tetraetilamonio/metabolismoRESUMEN
Methotrexate (MTX) is used in patients with malignant and autoimmune diseases. This drug is primarily excreted unchanged in the urine, and its net excretion occurs via active secretory and reabsorptive processes. We characterized the interaction of MTX with human organic-anion transporting polypeptide transporter (OATP) 1A2, which is expressed in tissues important for MTX disposition and toxicity, such as the intestine, kidney, liver, and endothelial cells of the blood-brain barrier. In Xenopus laevis oocytes expressing OATP1A2, the uptake of the model substrate, estrone-3-sulfate (ES), was enhanced 30-fold compared with uninjected oocytes. MTX uptake in oocytes expressing OATP1A2 was saturable (Km = 457 +/- 118 microM; Vmax = 17.5 +/- 4.9 pmol/oocyte/60 min) and sensitive to extracellular pH. That is, acidic pHs stimulated MTX uptake by as much as 7-fold. Seven novel protein-altering variants were identified in 270 ethnically diverse DNA samples. Four protein-altering variants in OATP1A2 exhibited altered transport of ES and/or MTX. The common variant, protein reference sequence (p.) Ile13Thr, was hyperfunctional for ES and MTX and showed a 2-fold increase in the V(max) for ES. The common variant, p. Glu172Asp, exhibited reduced maximal transport capacity for ES and MTX. p. Arg168Cys was hypofunctional, and p. Asn277DEL was nonfunctional. Because of its expression on the apical membrane of the distal tubule and in tissues relevant to MTX disposition and toxicity, these findings suggest that OATP1A2 may play a role in active tubular reabsorption of MTX and in MTX-induced toxicities. Furthermore, genetic variation in OATP1A2 may contribute to variation in MTX disposition and response.
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Antagonistas del Ácido Fólico/farmacología , Metotrexato/farmacología , Transportadores de Anión Orgánico/efectos de los fármacos , Transportadores de Anión Orgánico/genética , Alelos , Secuencia de Aminoácidos , Animales , Transporte Biológico Activo/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacocinética , Variación Genética , Humanos , Concentración de Iones de Hidrógeno , Metotrexato/análogos & derivados , Metotrexato/farmacocinética , Datos de Secuencia Molecular , Oocitos/metabolismo , Xenopus laevisRESUMEN
The human organic anion transporter, OAT3 (SLC22A8), plays a critical role in renal drug elimination, by mediating the entry of a wide variety of organic anions, including a number of commonly used pharmaceuticals, into the renal proximal tubular cells. To understand the nature and extent of genetic variation in OAT3, and to determine whether such variation affects its function, we identified OAT3 variants in a large, ethnically diverse sample population and studied their transport activities in cellular assays. We identified a total of 10 distinct coding-region variants, which altered the encoded amino acid sequence, in DNA samples from 270 individuals (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). The overall prevalence of these OAT3 variants was relatively low among the screened population, with only three variants having allele frequencies of >1% in a particular ethnic group. Clones of each variant were created by site-directed mutagenesis, expressed in HEK-293 cells, and tested for function using the model substrates, estrone sulfate (ES) and cimetidine (CIM). The results revealed a high degree of functional heterogeneity among OAT3 variants, with three variants (p. Arg149Ser, p. Gln239Stop, and p. Ile260Arg) that resulted in complete loss of function, and several others with significantly reduced function. One of the more common variants (p. Ile305Phe), found in 3.5% of Asian-Americans, appeared to have altered substrate specificity. This variant exhibited a reduced ability to transport ES, but a preserved ability to transport CIM. These data suggest that genetic variation in OAT3 may contribute to variation in the disposition of drugs.
Asunto(s)
Variación Genética , Transportadores de Anión Orgánico Sodio-Independiente/genética , Transportadores de Anión Orgánico Sodio-Independiente/fisiología , Alelos , Análisis Mutacional de ADN , Etnicidad , Genética de Población , Humanos , Preparaciones Farmacéuticas/metabolismo , FarmacocinéticaRESUMEN
BACKGROUND: A recent family-based genomewide screen revealed linkage between the 5q31 region and the diagnosis of asthma, but only in those exposed to environmental tobacco smoke (ETS). Among the candidate genes in this region is CD14. METHODS: To determine whether polymorphisms in the CD14 gene are related to this gene-by-environment interaction in Latinos, we used both family-based and cross-sectional cohort analysis to test for interactions between CD14 genotypes/haplotypes, exposure to ETS, and asthma-related phenotypes in 659 Mexican and Puerto Rican families. RESULTS: We identified 21 single nucleotide polymorphisms (SNPs) in the CD14 gene by sequencing 72 Puerto Ricans, Mexicans, and African Americans with asthma. Three SNPs, -810, -159, and +1437, were further genotyped in families with asthma. Among all subjects with asthma exposed to ETS, without regard to ethnicity, CD14 +1437 genotypes were associated with asthma severity. SNP +1437 GG or GC genotypes were significantly associated with lower baseline FEV1 using both family-based (p = 0.0009) and cross-sectional cohort (p = 0.03) analyses. Subjects with asthma with the GG or GC genotypes who were exposed to ETS had mean baseline FEV1 (% predicted) values 8.6% lower than subjects not exposed to ETS (p = 0.03). As previously observed in whites, we found an interaction between plasma IgE levels, SNP -159 genotypes, and ETS exposure (p = 0.0002). The lowest IgE levels were in those subjects with the TT genotype and who were exposed to ETS regardless of ethnicity. CONCLUSIONS: Our data suggest a gene-by-environment interaction between CD14 genotypes and ETS, which affects pulmonary function and IgE levels among Latinos with asthma.
Asunto(s)
Asma/fisiopatología , Hispánicos o Latinos , Inmunoglobulina E/sangre , Receptores de Lipopolisacáridos/genética , Polimorfismo de Nucleótido Simple , Contaminación por Humo de Tabaco/efectos adversos , Adolescente , Asma/tratamiento farmacológico , Asma/etnología , Asma/genética , Broncodilatadores/uso terapéutico , Niño , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Desequilibrio de Ligamiento , Pulmón/fisiopatología , Masculino , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: In the United States, Puerto Ricans and Mexicans have the highest and lowest asthma prevalence, morbidity, and mortality, respectively. Ethnic-specific differences in the response to drug treatment may contribute to differences in disease outcomes. Genetic variants at the beta(2)-adrenergic receptor (beta(2)AR) may modify asthma severity and albuterol responsiveness. We tested the association of beta(2)AR genotypes with asthma severity and bronchodilator response to albuterol in Puerto Ricans and Mexicans with asthma. METHODS: We used both family-based and cross-sectional tests of association with 8 beta(2)AR single nucleotide polymorphisms in 684 Puerto Rican and Mexican families. Regression analyses were used to determine the interaction between genotype, asthma severity, and bronchodilator drug responsiveness. RESULTS: Among Puerto Ricans with asthma, the arginine (Arg) 16 allele was associated with greater bronchodilator response using both family-based and cross-sectional tests (p = 0.00001-0.01). We found a strong interaction of baseline FEV(1) with the Arg16Glycine (Gly) polymorphism in predicting bronchodilator response. Among Puerto Ricans with asthma with baseline FEV(1) < 80% of predicted, but not in those with FEV(1) > 80%, there was a very strong association between the Arg16 genotype and greater bronchodilator responsiveness. No association was observed between Arg16Gly genotypes and drug responsiveness among Mexicans with asthma. CONCLUSIONS: Ethnic-specific pharmacogenetic differences exist between Arg16Gly genotypes, asthma severity, and bronchodilator response in Puerto Ricans and Mexicans with asthma. These findings underscore the need for additional research on racial/ethnic differences in asthma morbidity and drug responsiveness.