RESUMEN
We optimized the structure of an active metabolite (1) of WAY-123783, which was obtained from mouse urine after oral administration, to improve selectivity for SGLT2 and oral bioavailability. O-glucoside derivative 24 (remogliflozin etabonate) was subsequently identified as a potent, highly selective, and orally available SGLT2 inhibitor.
Asunto(s)
Glucósidos/química , Glucósidos/farmacología , Pirazoles/química , Pirazoles/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/química , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Administración Oral , Animales , Disponibilidad Biológica , Células COS , Chlorocebus aethiops , Descubrimiento de Drogas , Glucosuria , Ratones , Estructura Molecular , Pirazoles/metabolismo , Transportador 2 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/metabolismoRESUMEN
Transient receptor potential melastatin 8 (TRPM8), a temperature-sensitive ion channel responsible for detecting cold, is an attractive molecular target for the treatment of pain and other disorders. We have previously discovered a selective TRPM8 antagonist, KPR-2579, which inhibited bladder afferent hyperactivity induced by acetic acid instillation into the bladder. However, additional studies have revealed potential adverse effects with KPR-2579, such as the formation of a reactive metabolite, CYP3A4 induction, and convulsions. In this report, we describe the optimization of α-phenylglycinamide derivatives to mitigate the risk of these adverse effects. The optimal compound 13x exhibited potent inhibition against icilin-induced wet-dog shakes and cold-induced frequent voiding in rats, with a wide safety margin against the potential side effects.
Asunto(s)
Canales Catiónicos TRPM/antagonistas & inhibidores , Administración Oral , Animales , Conducta Animal/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Estructura Molecular , Pirimidinonas , Ratas , Ratas Sprague-Dawley , Factores de Riesgo , Relación Estructura-Actividad , Canales Catiónicos TRPM/metabolismoRESUMEN
AIM: Doxapram, a respiratory stimulant, is used to treat apnea. A reliable method of determining doxapram in blood is required for monitoring purposes. RESULTS: Doxapram, keto-doxapram (active metabolite) and propranolol (internal standard) were extracted from human serum by protein precipitation and plate filtration. Molecular ions were generated by electrospray ionization in positive ion mode, and the ions were analyzed using a triple-quadrupole mass spectrometer. The calibration curves were linear from 20 to 5000 ng/ml. The method was validated and the selectivity, reproducibility and stability met the acceptance criteria. CONCLUSION: An LC-MS/MS method was successfully developed for determining doxapram and keto-doxapram in human serum. The method can be used to monitor doxapram and keto-doxapram concentrations in blood.
Asunto(s)
Cromatografía Liquida/métodos , Doxapram/análogos & derivados , Doxapram/sangre , Espectrometría de Masas en Tándem/métodos , Calibración , Monitoreo de Drogas , Estabilidad de Medicamentos , Humanos , Límite de Detección , Propranolol/sangre , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cold and chemical substances, and antagonists of this channel have been considered to be effective for pain and urinary diseases. N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride (AMTB), a TRPM8 antagonist, was proposed to be effective for overactive bladder and painful bladder syndrome; however, there is a potential risk of low blood pressure. We report herein the synthesis and structure-activity relationships of novel phenylglycine derivatives that led to the identification of KPR-2579 (20l), a TRPM8 selective antagonist. KPR-2579 reduced the number of icilin-induced wet-dog shakes and rhythmic bladder contraction in rats, with no negative cardiovascular effects at the effective dose.
Asunto(s)
Glicina/análogos & derivados , Canales Catiónicos TRPM/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Glicina/síntesis química , Glicina/química , Glicina/farmacología , Humanos , Estructura Molecular , Relación Estructura-Actividad , Canales Catiónicos TRPM/metabolismoRESUMEN
UNLABELLED: This study aimed to determine the population pharmacokinetics of doxapram in low-birth-weight (LBW) infants. A total of 92 serum concentration measurements that were obtained from 34 Japanese neonates were analyzed using nonlinear mixed-effect modeling (NONMEM). Estimates generated by NONMEM indicated that clearance of doxapram (CL; L/kg/h) was affected by postmenstrual age (PMA; weeks), body weight (BW; g), and aspartate aminotransferase (AST; IU/L). In addition, the volume of distribution (Vd; L/kg) was affected by gestational age (GA; weeks). The final pharmacokinetic model was as follows: CL = BW / PMA × 0.0453 × serum AST(-0.373); Vd = 2.54 (if GA >28 weeks) and Vd = 2.54 × 2.11 (if GA ≤28 weeks). The interindividual variabilities in CL and Vd were 39.9 and 83.0 %, respectively, and the residual variability was 20.9 %. To clarify the reasons for large interindividual variations, the enzymes involved in the metabolic pathway of doxapram were also determined. We found that doxapram was metabolized by CYP3A4/5. CONCLUSION: We report the population pharmacokinetics of doxapram in neonates and the involvement of CYP3A4/5 in its metabolism. The final model of population pharmacokinetics may be useful for formulating a safe and effective dosage regimen and for predicting serum doxapram concentrations in neonates.
Asunto(s)
Apnea/metabolismo , Estimulantes del Sistema Nervioso Central/farmacocinética , Doxapram/farmacocinética , Recién Nacido de Bajo Peso , Apnea/tratamiento farmacológico , Pueblo Asiatico , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450 , Método Doble Ciego , Femenino , Humanos , Recién Nacido , Recien Nacido Prematuro , Japón , Masculino , Espectrometría de Masas , Modelos BiológicosRESUMEN
The use of human induced pluripotent stem (iPS) cells would be of great value for a variety of applications involving drug development studies. Several reports have been published on the differentiation of human iPS cells into hepatocyte-like cells; however, the cells were insufficient for application in drug metabolism studies. In this study, we aimed to establish effective methods for differentiation of human iPS cells into hepatocytes. Two human iPS cell lines were differentiated by addition of activin A, dimethyl sulfoxide, hepatocyte growth factor, oncostatin M, and dexamethasone. The differentiated cells expressed hepatocyte markers and drug-metabolizing enzymes, revealing that the human iPS cells were differentiated into hepatocyte-like cells. Expression of CYP3A4 and UGT1A1 mRNAs increased with treatment with typical inducers of the enzymes, and the response of the cells against the inducers was similar to that of human hepatocytes. Furthermore, the drug-metabolizing activity of CYP3A4, as monitored by testosterone 6ß-hydroxylase activity, was elevated by these inducers. In conclusion, we established methods for differentiation of hepatocyte-like cells expressing drug metabolizing activity from human iPS cells. The hepatocyte-like cells derived from human iPS cells will be useful for drug metabolism studies.
Asunto(s)
Diferenciación Celular/fisiología , Hepatocitos/metabolismo , Hepatocitos/fisiología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Activinas/metabolismo , Diferenciación Celular/genética , Línea Celular , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Dexametasona/metabolismo , Dimetilsulfóxido/metabolismo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Inactivación Metabólica/genética , Inactivación Metabólica/fisiología , Oncostatina M/metabolismo , ARN Mensajero/genética , Esteroide Hidroxilasas/metabolismoRESUMEN
Sodium glucose co-transporter 1 (SGLT1) plays a dominant role in the absorption of glucose in the gut and is considered a promising target in the development of therapeutic options for postprandial hyperglycemia. Previously, we reported potent and selective SGLT1 inhibitors 1 and 2 showing efficacy in oral carbohydrate tolerance tests in diabetic rat models. In a pharmacokinetic (PK) study of 2, excessive systemic exposure to metabolites of 2 was observed, presumably due to the high permeability of its aglycone (2a). To further improve SGLT1 inhibitory activity and reduce aglycone permeability, a series of 4-benzyl-5-isopropyl-1H-pyrazol-3-yl ß-D-glycopyranoside derivatives bearing novel hydrophilic substitution groups on the phenyl ring were synthesized and their inhibitory activity toward SGLTs was evaluated. Optimized compound 14c showed an improved profile satisfying both higher activity and lower permeability of its aglycone (22f) compared with initial leads 1 and 2. Moreover, the superior efficacy of 14c in various carbohydrate tolerance tests in diabetic rat models was confirmed compared with acarbose, an α-glucosidase inhibitor (α-GI) widely used in the clinic.
Asunto(s)
Diseño de Fármacos , Glicósidos/farmacología , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Glicósidos/síntesis química , Glicósidos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Relación Estructura-ActividadRESUMEN
We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6ß-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Células Madre Embrionarias/citología , Hepatocitos/citología , Albúminas/genética , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Células Cultivadas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Haplorrinos , Hepatocitos/metabolismo , Receptor X de Pregnano , Receptores de Hidrocarburo de Aril/genética , Receptores de Esteroides/genética , Rifampin/farmacología , Esteroide Hidroxilasas/metabolismo , alfa-Fetoproteínas/genéticaRESUMEN
Sodium glucose co-transporter 1 (SGLT1) plays a dominant role in the absorption of glucose in the gut and is considered a promising target in the development of treatments for postprandial hyperglycemia. A series of 4-benzyl-1H-pyrazol-3-yl ß-d-glucopyranoside derivatives have been synthesized, and its inhibitory activity toward SGLTs has been evaluated. By altering the substitution groups at the 5-position of the pyrazole ring, and every position of the phenyl ring, we studied the structure-activity relationship (SAR) profiles and identified a series of potent and selective SGLT1 inhibitors. Representative derivatives showed a dose-dependent suppressing effect on the escalation of blood glucose levels in oral mixed carbohydrate tolerance tests (OCTT) in streptozotocin-nicotinamide-induced diabetic rats (NA-STZ rats).
Asunto(s)
Glucósidos/química , Hipoglucemiantes/síntesis química , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Animales , Glucemia/análisis , Cristalografía por Rayos X , Diabetes Mellitus Experimental/tratamiento farmacológico , Glucósidos/síntesis química , Glucósidos/uso terapéutico , Humanos , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/química , Hipoglucemiantes/uso terapéutico , Conformación Molecular , Ratas , Transportador 1 de Sodio-Glucosa/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Relación Estructura-ActividadRESUMEN
There is a great deal of interest in differentiation of human embryonic stem cells (hESCs) into hepatocyte-like cells for application in pharmaceutical screening. Cytochrome P450 (CYP) 1A is involved in the metabolic activation of procarcinogenic compounds as well as in detoxification of drugs. We differentiated hESCs into hepatocyte-like cells (hESC-derived hepatocyte-like cells) and examined whether CYP1A was induced in these cells by typical inducers of CYP1A. hESC-derived hepatocyte-like cells expressed albumin, α-fetoprotein, CYP3A4, CYP3A7, CYP1A1, CYP1A2, and UDP-glucuronyl transferase (UGT) 1A1 mRNA. The levels of CYP1A1, CYP1A2, and UGT1A1 mRNA expression were increased by omeprazole and 3-methylcholanthrene. Furthermore, the enzyme activity of CYP1A was also increased by these compounds. In conclusion, hESC-derived hepatocyte-like cells are available for the detection of CYP1A inducers.
Asunto(s)
Citocromo P-450 CYP1A1/biosíntesis , Células Madre Embrionarias/química , Células Madre Embrionarias/citología , Hepatocitos/química , Hepatocitos/citología , Adulto , Albúminas/metabolismo , Animales , Biotransformación/efectos de los fármacos , Biotransformación/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Descubrimiento de Drogas , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/enzimología , Inducción Enzimática/efectos de los fármacos , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Inactivación Metabólica , Masculino , Metilcolantreno/farmacología , Ratones , Persona de Mediana Edad , Omeprazol/farmacología , ARN Mensajero/genética , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismoRESUMEN
We have developed concentrative nucleoside transporter 2 (CNT2) inhibitors as a novel pharmacological approach for improving hyperuricemia by inhibiting intestinal absorption of purines. Dietary purine nucleosides are absorbed in the small intestines by CNTs expressed in the apical membrane. In humans, the absorbed purine nucleosides are rapidly degraded to their final end product, uric acid, by xanthine oxidase. Based on the expression profile of human CNTs in digestive tract tissues, we established a working hypothesis that mainly CNT2 contributes to the intestinal absorption of purine nucleosides. In order to confirm this possibility, we developed CNT2 inhibitors and found that (2R,3R,4S,5R)-2-(6-amino-8-{[3'-(3-aminopropoxy)-biphenyl-4-ylmethyl]-amino}-9H-purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (KGO-2142) and 1-[3-(5-{[1-((2R,3R,4S,5R)-3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-benzimidazol-2-ylamino]-methyl}-2-ethoxyphenoxy)-propyl]-piperidine-4-carboxylic acid amide (KGO-2173) were inhibitory. These CNT2 inhibitors had potent inhibitory activity against inosine uptake via human CNT2, but they did not potently interfere with nucleoside uptake via human CNT1, CNT3 or equilibrative nucleoside transporters (ENTs) in vitro. After oral administration of KGO-2173 along with [(14)C]-inosine, KGO-2173 significantly decreased the urinary excretion of radioactivity at 6 and 24h in rats. Since dietary purine nucleosides are not utilized in the body and are excreted into the urine rapidly, this decrease in radioactivity in the urine represented the inhibitory activity of KGO-2173 toward the absorption of [(14)C]-inosine in the small intestines. KGO-2142 almost completely inhibited dietary RNA-induced hyperuricemia and the increase in urinary excretion of uric acid in cebus monkeys. These novel CNT2 inhibitors, KGO-2142 and KGO-2173, could be useful therapeutic options for the treatment of hyperuricemia.
Asunto(s)
Furanos/farmacología , Absorción Intestinal/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Nucleósidos de Purina/metabolismo , Defectos Congénitos del Transporte Tubular Renal/tratamiento farmacológico , Defectos Congénitos del Transporte Tubular Renal/metabolismo , Cálculos Urinarios/tratamiento farmacológico , Cálculos Urinarios/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células COS , Cebus , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Furanos/química , Furanos/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Inosina/metabolismo , Masculino , ARN de Hongos/administración & dosificación , ARN de Hongos/farmacología , Ratas , Ratas Sprague-Dawley , Defectos Congénitos del Transporte Tubular Renal/sangre , Ácido Úrico/sangre , Cálculos Urinarios/sangreRESUMEN
To improve the in vitro potency of the c-Src inhibitor 1a and to address its hERG liability, a structure-activity study was carried out, focusing on two regions of the lead compound. The blockade of the delayed cardiac current rectifier K(+) (I(Kr)) channel was overcome by replacing the ethylenediamino group with an amino alcohol group at the 7-position. In addition, modifying the substituents at the 5-position and the side chain groups on the amino alcohols at the 7-position enhanced the intracellular c-Src inhibitory activity and increased central nervous system (CNS) penetration. In the present study, 6l exhibited significant in vivo efficacy in a middle cerebral artery (MCA) occlusion model in rats.
Asunto(s)
Bloqueadores de los Canales de Potasio/farmacología , Pirazoles , Pirimidinas , Familia-src Quinasas/antagonistas & inhibidores , Animales , Técnicas Químicas Combinatorias , Arteria Cerebral Media/efectos de los fármacos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacología , Pirimidinas/síntesis química , Pirimidinas/química , Pirimidinas/farmacología , RatasRESUMEN
We synthesized a series of novel 2-anilinopyrazolo[1,5-a]pyrimidine derivatives and evaluated their ability to inhibit c-Src kinase; 7-(2-amino-2-methylpropylamino)-5-cyclopropyl-2-(3,5-dimethoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide 7o and 7-(2-amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide 7f showed potent inhibitory activity. Compound 7f inhibited c-Src selectively and exhibited satisfactory central nervous system (CNS) penetration. Furthermore, 7f.HCl reduced the infarct volume in vivo in a rat middle cerebral artery (MCA) occlusion model when administrated intraperitoneally.
Asunto(s)
Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/química , Accidente Cerebrovascular/tratamiento farmacológico , Familia-src Quinasas/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Masculino , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Espectrofotometría InfrarrojaRESUMEN
We synthesized and evaluated a series of C-5 substituted imidazo[1,5-a]pyrazine derivatives to identify potent c-Src inhibitors as potential therapeutic agents for acute ischemic stroke. Among these compounds, compound 14c.HCl demonstrated remarkable central nervous system (CNS) penetration and significant neuroprotective efficacy in vivo in rat models.
Asunto(s)
Isquemia Encefálica/complicaciones , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Imidazoles/síntesis química , Imidazoles/farmacología , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Pirazinas/síntesis química , Pirazinas/farmacología , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/etiología , Familia-src Quinasas/antagonistas & inhibidores , Enfermedad Aguda , Animales , Cristalografía por Rayos X , Inhibidores Enzimáticos/sangre , Ensayo de Inmunoadsorción Enzimática , Humanos , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Conformación Molecular , Fármacos Neuroprotectores/sangre , Ratas , Espectrofotometría Infrarroja , Relación Estructura-ActividadRESUMEN
By adding high concentrations of test drugs to an Ussing chamber with rat jejunum, we established a system that yields very high correlations between the rat absorption percentage and the membrane permeability, and that can accurately predict the absorption percentage for rats. An advantage of this technique is that, unlike the results obtained using Caco-2, the slope of the absorption/membrane-permeability curve is gentle, which facilitates a more exact prediction of the absorption percentage. In addition, the results obtained with this technique demonstrated that it could be used to evaluate the absorption percentage of drugs with an affinity for P-glycoprotein (P-gp), which cannot be assessed using Caco-2. This method also allows for cassette screening, which would facilitate evaluation of the contribution of P-gp to absorption in the small intestine. Cassette screening showed that absorption of fexofenadine was unaffected by combination with the P-gp substrate ketoconazole. Consistent with this finding, in vivo studies showed that ketoconazole did not affect the Fa Fg for fexofenadine, a pharmacokinetic parameter that reflects absorption and bioavailability in the small intestine. This confirms the usefulness of the Ussing chamber for cassette screening and also suggests that intestinal P-gp has a minimal contribution to drug absorption.