RESUMEN

Uricosuric agents lower serum uric acid levels by increasing urinary excretion via inhibition of urate transporter 1 (URAT1), urate reabsorption transporter in the renal proximal tubules. Probenecid and benzbromarone have been used as uricosurics, but these drugs inhibit organic anion transporters (OATs) in addition to URAT1. In this study, we investigated whether uricosuric agents interacted with adefovir, known as a substrate for OAT1, using Sprague-Dawley (SD) rats. Furthermore, involvement of other transporters, multi-drug resistance protein 2 (MRP2) in this interaction was examined using Mrp2-deficient rats. Probenecid and lesinurad increased plasma adefovir concentrations and decreased kidney-to-plasma partition coefficient (Kp) in these rats, presumably by inhibiting Oat1. Although benzbromarone had no effect on plasma adefovir concentration, it increased the Kp to 141% in SD rats. Since this effect was abolished in Mrp2-deficient rats, together with the MRP2 inhibition study, it is suggested that benzbromarone inhibits Mrp2-mediated adefovir excretion from the kidney. In contrast, dotinurad, a novel uricosuric agent that selectively inhibits URAT1, had no effect on the plasma and kidney concentrations of adefovir. Therefore, due to the lack of interaction with adefovir, dotinurad is expected to have low drug-drug interaction risk mediated by OAT1, and also by MRP2.

Asunto(s)

Transportadores de Anión Orgánico , Uricosúricos , Ratas , Animales , Uricosúricos/farmacología , Benzbromarona , Probenecid/farmacología , Probenecid/metabolismo , Ácido Úrico , Ratas Sprague-Dawley , Riñón/metabolismo , Transportadores de Anión Orgánico/metabolismo

RESUMEN

Dotinurad, a novel selective urate reabsorption inhibitor, is used to treat hyperuricemia. In humans, orally administered dotinurad is excreted mainly as glucuronide and sulfate conjugates in urine. To identify the isoforms of UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) involved in dotinurad glucuronidation and sulfation, microsome and cytosol fractions of liver, intestine, kidney, and lung tissues (cytosol only) were analyzed along with recombinant human UGT and SULT isoforms. Dotinurad was mainly metabolized to its glucuronide conjugate by human liver microsomes (HLMs), and the glucuronidation followed the two-enzyme Michaelis-Menten equation. Among the recombinant human UGT isoforms expressed in the liver, UGT1A1, UGT1A3, UGT1A9, and UGT2B7 catalyzed dotinurad glucuronidation. Based on inhibition analysis using HLMs, bilirubin, imipramine, and diflunisal decreased glucuronosyltransferase activities by 45.5%, 22.3%, and 22.2%, respectively. Diflunisal and 3'-azido-3'-deoxythymidine, in the presence of 1% bovine serum albumin, decreased glucuronosyltransferase activities by 21.1% and 13.4%, respectively. Dotinurad was metabolized to its sulfate conjugate by human liver cytosol (HLC) and human intestinal cytosol (HIC) samples, with the sulfation reaction in HLC samples following the two-enzyme Michaelis-Menten equation and that in HIC samples following the Michaelis-Menten equation. All eight recombinant human SULT isoforms used herein catalyzed dotinurad sulfation. Gavestinel decreased sulfotransferase activity by 15.3% in HLC samples, and salbutamol decreased sulfotransferase activity by 68.4% in HIC samples. These results suggest that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7, whereas its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. SIGNIFICANCE STATEMENT: The identification of enzymes involved in drug metabolism is important to predicting drug-drug interactions (DDIs) and interindividual variability for safe drug use. The present study revealed that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7 and that its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. Therefore, dotinurad, a selective urate reabsorption inhibitor, is considered safe for use with a small risk of DDIs and low interindividual variability.

Asunto(s)

Benzotiazoles/metabolismo , Glucuronosiltransferasa/metabolismo , Sulfotransferasas/metabolismo , Ácido Úrico/metabolismo , Algoritmos , Benzotiazoles/farmacocinética , Citosol/metabolismo , Glucurónidos/metabolismo , Humanos , Hiperuricemia/tratamiento farmacológico , Técnicas In Vitro , Intestinos/metabolismo , Isoenzimas/metabolismo , Hígado/metabolismo , Tasa de Depuración Metabólica , Microsomas Hepáticos/metabolismo , Sulfatos/metabolismo

RESUMEN

The effect of concomitant drugs having a cytochrome P450 (CYP) 3A inhibitory potency on tacrolimus exposure was predicted from in vitro metabolism results. In this study, the IC50 values of concomitant drugs on the formation of M-I, the major metabolite of tacrolimus, were determined, and the effect on oral exposure (AUCp.o.) of tacrolimus was assessed from static models. When an absorbed fraction (Fa ) of 0.97, intestinal wall availability (Fg) of 0.27 and fraction metabolized by CYP3A (fm(CYP3A)) of 0.8 were used, the least bias was observed for the prediction of the AUCp.o. of tacrolimus. The relationship of the IC50 values of 11 inhibitors between tacrolimus and typical CYP3A substrates (midazolam and testosterone) was also analysed. A strong correlation was found between the IC50 values of tacrolimus and typical CYP3A substrates (r(2) ≥ 0.85). The predictability of the effect of inhibitors on tacrolimus AUCp.o. was investigated based on the same static models with the use of published IC50 values for midazolam and testosterone. The bias for the prediction of tacrolimus AUCp.o. was minimal with the use of IC50 values determined using tacrolimus itself as a substrate. These results suggest that tacrolimus itself is still the best choice for predicting the AUCp.o. of tacrolimus, although our findings suggest that midazolam or testosterone may be used instead of tacrolimus to estimate roughly (predicted AUCp.o. within an approximately 2-fold range of observed values) the effect of CYP3A inhibitors on the tacrolimus AUCp.o.

Asunto(s)

Inhibidores del Citocromo P-450 CYP3A/farmacología , Inmunosupresores/farmacología , Tacrolimus/farmacología , Área Bajo la Curva , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas , Humanos , Microsomas Hepáticos/metabolismo , Midazolam/farmacología , Modelos Biológicos , Testosterona/farmacología

RESUMEN

Telaprevir, a chronic hepatitis C virus (HCV) protease inhibitor, is known to be a cytochrome P450 (CYP) 3A4/5 substrate and inhibitor. In the present study, the in vitro inhibitory effect of telaprevir on the metabolism of tacrolimus in human liver microsomes was investigated using 13-O-demethyltacrolimus (M-I) as a monitor metabolite. Telaprevir inhibited M-I formation in a time-dependent fashion with rate of enzyme inactivation (kinact ) and concentration to reach 50% of kinact (KI ) values of 0.113 min(-1) and 0.511 µm, respectively. Using the inhibition parameters generated, in vitro-in vivo extrapolations were performed to evaluate the clinical relevance of the effect of telaprevir on the area under the curve versus time (AUC) of tacrolimus. When 750 mg of telaprevir was administered orally, the intestinal wall availability (Fg ) of tacrolimus was estimated to be increased 3.7- to 7.0-fold. The hepatic intrinsic clearance (CLint ) of tacrolimus was also estimated to be decreased 4.4- to 19-fold. These results suggest that the increased AUC of tacrolimus in the presence of telaprevir was caused by intestinal and hepatic metabolism inhibition. In addition, the inhibitory effect of telaprevir on the hepatic uptake of tacrolimus was also examined using human cryopreserved hepatocytes. However, no significant inhibitory effect was noted, suggesting that the effect of telaprevir on hepatic transporters did not contribute to the increase in tacrolimus exposure.

Asunto(s)

Hígado/metabolismo , Modelos Biológicos , Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacología , Tacrolimus/metabolismo , Administración Oral , Área Bajo la Curva , Biotransformación , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Hepacivirus/enzimología , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Valor Predictivo de las Pruebas , Tacrolimus/farmacocinética , Factores de Tiempo , Distribución Tisular

RESUMEN

1. Glucuronidation via UDP-glucuronosyltransferase (UGT) in the intestine has been reported to influence the pharmacokinetics (PK) of drugs; however, information concerning the differences in activity between species is limited. Here, we investigated the in vitro and in vivo activities of intestinal glucuronidation for 17 UGT substrates in humans, rats, dogs and monkeys. 2. Although in vitro intrinsic clearance (CLint,u,UGT) in intestinal microsomes showed a good correlation between humans and laboratory animals, values tended to be lower in humans than in laboratory animals. The ratio of CLint,u,UGT in the absence and presence of bovine serum albumin differed between species. In vivo, the fraction of drug absorbed (FaFg) in humans correlated with that in dogs and monkeys, but not in rats. 3. While an inverse correlation between CLint,u,UGT and FaFg was observed in each species, the CLint,u,UGT values in the intestinal microsomes corresponding to FaFg values in dogs were three to four times higher than in other animals. 4. These results indicate the need for a degree of caution when extrapolating PK data from laboratory animals to humans.

Asunto(s)

Glucuronatos/metabolismo , Glucuronosiltransferasa/metabolismo , Mucosa Intestinal/metabolismo , Tasa de Depuración Metabólica/fisiología , Animales , Ácido Benzoico/química , Ácido Benzoico/farmacocinética , Cromatografía Liquida , Perros , Humanos , Macaca fascicularis , Microsomas/metabolismo , Modelos Biológicos , Ratas , Albúmina Sérica Bovina , Especificidad de la Especie , Espectrometría de Masas en Tándem

RESUMEN

We investigated whether the effects of intestinal glucuronidation on the first-pass effect can be predicted from in vitro data for UDP-glucuronosyltransferase (UGT) substrates. Human in vitro intrinsic glucuronidation clearance (CL(int, UGT)) for 11 UGT substrates was evaluated using pooled intestinal microsomes (4.00-4620 µl · min⁻¹ · mg⁻¹) and corrected by the free fraction in the microsomal mixture (CLu(int), (UGT) = 5.2-5133 µl · min⁻¹ · mg⁻¹). Eleven UGT substrates were stable against intestinal cytochrome P450, indicating intestinal glucuronidation has a main effect on human intestinal availability. Oral absorbability intestinal availability (F(a)F(g)) values were calculated from in vivo pharmacokinetic parameters in the literature (F(a)F(g) = 0.01-1.0). It was found that CLu(int, UGT) and human F(a)F(g) have an inverse relationship that can be fitted to a simplified intestinal availability model. Experiments using Supersomes from insect cells expressing UGT isoforms showed that the substrates used were conjugated by various UGT isoforms. These results suggest that combining the simplified intestinal availability model and in vitro conjugation assay make it possible to predict human F(a)F(g) regardless of UGT isoform.

Asunto(s)

Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Intestinos/enzimología , Modelos Biológicos , Preparaciones Farmacéuticas/metabolismo , Administración Oral , Biotransformación , Permeabilidad de la Membrana Celular , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Absorción Intestinal , Isoenzimas , Cinética , Tasa de Depuración Metabólica , Microsomas/enzimología , Preparaciones Farmacéuticas/administración & dosificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato

RESUMEN

1. As intestinal glucuronidation has been suggested to generate the low oral bioavailability (F) of drugs, estimating its effects would be valuable for selecting drug candidates. Here, we investigated the absorption and intestinal availability (F(a)F(g)) in animals, and intrinsic clearance via UDP-glucuronosyltransferase (UGT) in intestinal microsomes (CL(int,UGT)) for three drug candidates possessing a carboxylic acid group, in an attempt to estimate the impact of intestinal glucuronidation on F and select potential drug candidates with high F in humans. 2. The F(a)F(g) values of the three test compounds were low in rats and monkeys (0.16-0.51), and high in dogs (≥0.81). Correspondingly, the CL(int,UGT) values were high in rats and monkeys (101-731 µL/min/mg), and low in dogs (≤ 59.6 µL/min/mg). A good inverse correlation was observed between F(a)F(g) and CL(int,UGT), suggesting that intestinal glucuronidation was a major factor influencing F(a)F(g) of these compounds. 3. By applying this correlation to F(a)F(g) in humans using human CL(int,UGT) values (26.9-114 µL/min/mg), compounds 1-3 were predicted to have relatively high F(a)F(g). 4. Our approach is expected to be useful for estimating the impact of intestinal glucuronidation on F in animals and semiquantitatively predicting human F for drug candidates.

Asunto(s)

Bioquímica/métodos , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Mucosa Intestinal/metabolismo , Administración Intravenosa , Administración Oral , Animales , Perros , Glucurónidos/administración & dosificación , Glucurónidos/química , Glucurónidos/farmacocinética , Haplorrinos , Humanos , Isoenzimas/metabolismo , Masculino , Membranas Artificiales , Metaboloma , Microsomas/metabolismo , Permeabilidad , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Estándares de Referencia , Especificidad por Sustrato

RESUMEN

UDP-glucuronosyltransferase (UGT) is highly expressed in the small intestine and catalyzes the glucuronidation of small molecules, which may affect the oral bioavailability of drugs. However, no method of predicting the in vivo observed fraction of absorbed drug (F(a)F(g)) affected by UGT has yet been established. Here, we investigated the relationship between F(a)F(g) and in vitro clearance of nine UGT substrates (ketoprofen, tolcapone, telmisartan, raloxifene, entacapone, resveratrol, buprenorphine, quercetin, and ezetimibe) via UGT in intestinal microsomes (CL(int, UGT)) in rats. F(a)F(g) was calculated from pharmacokinetic parameters after intravenous and oral administration or using the portal-systemic concentration difference method, with values ranging from 0.027 (ezetimibe) to 1 (tolcapone). Glucuronides of model compounds were observed in the portal plasma after oral administration, with CL(int, UGT) values ranging from 57.8 (tolcapone) to 19,200 µL/min/mg (resveratrol). An inverse correlation between F(a)F(g) and CL(int, UGT) was observed for most compounds and was described using a simplified intestinal availability model reported previously. This model gave accurate predictions of F(a)F(g) values for three in-house compounds. Our results show that F(a)F(g) in rats is affected by UGT and can be predicted using CL(int, UGT). This work should hasten the development of a method to predict F(a)F(g) in humans.

Asunto(s)

Glucuronosiltransferasa/metabolismo , Mucosa Intestinal/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Evaluación Preclínica de Medicamentos/métodos , Predicción , Glucurónidos/metabolismo , Masculino , Tasa de Depuración Metabólica/fisiología , Microsomas/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

To estimate the influence of repeated administration of drug metabolism inhibitors on the extent of drug interaction, we investigated the effects of single intravenous or repeated oral administration of itraconazole on the pharmacokinetics of midazolam in rats. In the single administration study, the plasma concentration of itraconazole was maintained by intravenous infusion, and midazolam was administered into the portal vein to investigate its kinetics. In the repeated administration study, the kinetics of midazolam was investigated after seven-day oral treatment with itraconazole. The in vitro metabolism of midazolam and the contents of cytochrome P450 were investigated using liver microsomes from the itraconazole-treated rats. The area under the curve (AUC) of midazolam was increased by 1.45- or 1.44-fold after single or repeated itraconazole treatment, respectively. Meanwhile, the liver concentrations of itraconazole after single administration and repeated administration were 38.2 and 20.3 (nmol/g), respectively. In vitro maximum metabolic reaction velocity (V(max)) and Michaelis-Menten constant (K(m)) of midazolam were increased from 2.26 to 3.84 (nmol/min/mg protein) and from 8.28 to 13.0 (microM) by single itraconazole treatment, respectively, and decreased from 2.23 to 1.17 (nmol/min/mg protein) and from 7.86 to 4.47 (microM) by repeated treatment, respectively. Correspondingly, the content of CYP3A2 was significantly altered by single or repeated itraconazole administration. The increases in AUC could be predicted only when the changes in V(max) and K(m) were taken into consideration, in addition to the hepatic unbound concentration of itraconazole. In conclusion, changes in enzyme kinetics should be taken into account to predict the extent of drug interaction after repeated treatment with inhibitors.