RESUMEN

A novel series of indazole/indole derivatives were discovered as glucagon receptor (GCGR) antagonists through scaffold hopping based on two literature leads: MK-0893 and LY-2409021. Further structure-activity relationship (SAR) exploration and optimization led to the discovery of multiple potent GCGR antagonists with excellent pharmacokinetic properties in mice and rats, including low systemic clearance, long elimination half-life, and good oral bioavailability. These potent GCGR antagonists could be used for potential treatment of type II diabetes.

Asunto(s)

Indazoles/química , Receptores de Glucagón/antagonistas & inhibidores , Humanos , Estructura Molecular , Relación Estructura-Actividad

RESUMEN

A new series of (2S,3R,4R,5S,6R)-5-fluoro-6-(hydroxymethyl)-2-aryltetrahydro-2H-pyran-3,4-diols as dual inhibitors of sodium glucose co-transporter proteins (SGLTs) were disclosed. Two methods were developed to efficiently synthesize C5-fluoro-lactones 3 and 4, which are key intermediates to the C5-fluoro-hexose based C-aryl glucosides. Compound 2b demonstrated potent hSGLT1 and hSGLT2 inhibition (IC50 = 43 nM for SGLT1 and IC50 = 9 nM for SGLT2). It showed robust inhibition of blood glucose excursion in oral glucose tolerance test (OGTT) in Sprague Dawley (SD) rats and exerted pronounced antihyperglycemic effects in db/db mice and high-fat diet-fed ZDF rats when dosed orally at 10 mg/kg.

Asunto(s)

Desoxiglucosa/uso terapéutico , Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Administración Oral , Animales , Glucemia/efectos de los fármacos , Desoxiglucosa/administración & dosificación , Desoxiglucosa/análogos & derivados , Desoxiglucosa/síntesis química , Diseño de Fármacos , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/síntesis química , Hipoglucemiantes/química , Macaca fascicularis , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Estructura Molecular , Ratas Sprague-Dawley , Ratas Zucker , Transportador 2 de Sodio-Glucosa/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/administración & dosificación , Inhibidores del Cotransportador de Sodio-Glucosa 2/síntesis química , Inhibidores del Cotransportador de Sodio-Glucosa 2/química , Relación Estructura-Actividad

RESUMEN

The discovery of a novel series of N-arylpyrroles as agonists of GPR120 (FFAR4) is discussed. One lead compound is a potent GPR120 agonist, has good selectivity for related receptor GPR40 (FFAR1), has acceptable PK properties, and is active in 2 models of Type 2 Diabetes in mice.

Asunto(s)

Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Descubrimiento de Drogas , Hipoglucemiantes/farmacología , Pirroles/farmacología , Receptores Acoplados a Proteínas G/agonistas , Animales , Relación Dosis-Respuesta a Droga , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/química , Ratones , Estructura Molecular , Pirroles/síntesis química , Pirroles/química , Relación Estructura-Actividad

RESUMEN

Although much speculation has surrounded intestinally expressed FcRn as a means for systemic uptake of orally administered immunoglobulin G (IgG), this has not been validated in translational models beyond neonates or in FcRn-expressing cells in vitro. Recently, IgG1 intestinal infusion acutely in anesthetized cynomolgus resulted in detectable serum monoclonal antibody (mAb) levels. In this study, we show that IgG2 has greater protease resistance to intestinal enzymes in vitro and mice in vivo, due to protease resistance in the hinge region. An IgG2 mAb engineered for FcRn binding, was optimally formulated, lyophilized, and loaded into enteric-coated capsules for oral dosing in cynomolgus. Small intestinal pH 7.5 was selected for enteric delivery based on gastrointestinal pH profiling of cynomolgus by operator-assisted IntelliCap System(®). Milling of the lyophilized IgG2 M428L FcRn-binding variant after formulation in 10 mmol/L histidine, pH 5.7, 8.5% sucrose, 0.04% PS80 did not alter the physicochemical properties nor the molecular integrity compared to the batch released in PBS. Size 3 hard gel capsules (23.2 mg IgG2 M428L ~3 mg/kg) were coated with hydroxypropyl methylcellulose acetate succinate for rapid dissolution at pH 7.5 in small intestine and FcRn binding of encapsulated mAb confirmed. Initial capsule dosing by endoscopic delivery into the small intestine achieved 0.2 + 0.1 ng/mL (n = 5) peak at 24 h. Weekly oral capsule dosing for 6 weeks achieved levels of 0.4 + 0.2 ng/mL and, despite increasing the dose and frequency, remained below 1 ng/mL. In conclusion, lyophilized milled mAb retains FcRn binding and molecular integrity for small intestinal delivery. The low systemic exposure has demonstrated the limitations of intestinal FcRn in non-human primates and the unfeasibility of employing this for therapeutic levels of mAb. Local mAb delivery with limited systemic exposure may be sufficient as a therapeutic for intestinal diseases.

RESUMEN

Metabolic syndrome and T2D produce significant health and economic issues. Many available animal models have monogenic leptin pathway mutations that are absent in the human population. Development of the ZDSD rat model was undertaken to produce a model that expresses polygenic obesity and diabetes with an intact leptin pathway. A lean ZDF rat with the propensity for beta-cell failure was crossed with a polygenetically obese Crl:CD (SD) rat. Offspring were selectively inbred for obesity and diabetes for >30 generations. In the current study, ZDSD rats were followed for 6 months; routine clinical metabolic endpoints were included throughout the study. In the prediabetic metabolic syndrome phase, ZDSD rats exhibited obesity with increased body fat, hyperglycemia, insulin resistance, dyslipidemia, glucose intolerance, and elevated HbA1c. As disease progressed to overt diabetes, ZDSD rats demonstrated elevated glucose levels, abnormal oral glucose tolerance, increases in HbA1c levels, reductions in body weight, increased insulin resistance with decreasing insulin levels, and dyslipidemia. The ZDSD rat develops prediabetic metabolic syndrome and T2D in a manner that mirrors the development of metabolic syndrome and T2D in humans. ZDSD rats will provide a novel, translational animal model for the study of human metabolic diseases and for the development of new therapies.

Asunto(s)

Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Resistencia a la Insulina/fisiología , Leptina/metabolismo , Síndrome Metabólico/metabolismo , Obesidad/metabolismo , Animales , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Intolerancia a la Glucosa/metabolismo , Intolerancia a la Glucosa/fisiopatología , Insulina/metabolismo , Masculino , Síndrome Metabólico/fisiopatología , Obesidad/fisiopatología , Ratas , Transducción de Señal

RESUMEN

Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-reperfusion injury (IRI), vascular leak syndrome, and acute respiratory distress syndrome (ARDS). The utility of biphenylsulfonyl-thiophene-carboxamidine small-molecule C1s inhibitors are limited by their poor in vivo pharmacokinetic properties. Pegylation of a potent analog has provided compounds with good potency and good in vivo pharmacokinetic properties.

Asunto(s)

Amidas/química , Complemento C1s/antagonistas & inhibidores , Diseño de Fármacos , Polietilenglicoles/química , Inhibidores de Proteasas/síntesis química , Tiofenos/química , Animales , Complemento C1s/metabolismo , Semivida , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Ratas

RESUMEN

Pharmacokinetic studies in mice traditionally require one animal per time point, resulting in dosing and euthanizing a large number of animals and producing suboptimal quality of pharmacokinetic data due to inter-animal variability and dosing error. These studies are time-consuming and labor-intensive. To improve the throughput and quality of pharmacokinetic evaluation in mice, we have developed a serial blood sampling methodology using the lateral saphenous vein puncture technique. Two marketed drugs, indinavir and rosuvastatin, were selected for this validation study because of their distinctly different physicochemical and pharmacokinetic properties. Each compound was dosed orally and intravenously in mice using both discrete and serial blood sampling methods. The pharmacokinetic results from serial bleeding are in excellent agreement with those from discrete sampling for both compounds. Compared to the discrete sampling, the serial sampling procedure is a more humane method, allowing for rapid and repeated sampling from the same site without the need for anesthesia. The application of this new method has led to a remarkable reduction in animal and compound usage, a significant increase in throughput and speed, and a drastic improvement in pharmacokinetic data quality. This approach is especially useful for the first-tier in vivo pharmacokinetic screening of discovery compounds.

Asunto(s)

Disponibilidad Biológica , Preparaciones Farmacéuticas/metabolismo , Farmacocinética , 2-Hidroxipropil-beta-Ciclodextrina , Administración Oral , Animales , Área Bajo la Curva , Diseño de Fármacos , Fluorobencenos/administración & dosificación , Fluorobencenos/farmacocinética , Inhibidores de la Proteasa del VIH/administración & dosificación , Inhibidores de la Proteasa del VIH/farmacocinética , Semivida , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Indinavir/administración & dosificación , Indinavir/farmacocinética , Inyecciones Intravenosas , Masculino , Ratones , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Reproducibilidad de los Resultados , Rosuvastatina Cálcica , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacocinética , beta-Ciclodextrinas

RESUMEN

In addition to matrix effects, common interferences observed in liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses can be caused by the response of drug-related metabolites to the multiple reaction monitoring (MRM) channel of a given drug, as a result of in-source reactions or decomposition of either phase I or II metabolites. However, it has been largely ignored that, for some drugs, metabolism can lead to the formation of isobaric or isomeric metabolites that exhibit the same MRM transitions as parent drugs. The present study describes two examples demonstrating that interference caused by isobaric or isomeric metabolites is a practical issue in analyzing biological samples by LC/MS/MS. In the first case, two sequential metabolic reactions, demethylation followed by oxidation of a primary alcohol moiety to a carboxylic acid, produced an isobaric metabolite that exhibits a MRM transition identical to the parent drug. Because the drug compound was rapidly metabolized in rats and completely disappeared in plasma samples, the isobaric metabolite appeared as a single peak in the total ion current (TIC) trace and could easily be quantified as the drug since it was eluted at a retention time very close to that of the drug in a 12-min LC run. In the second example, metabolism via the ring-opening of a substituted isoxazole moiety led to the formation of an isomeric product that showed an almost identical collision-induced dissociation (CID) MS spectrum as the original drug. Because two components were co-eluted, the isomeric product could be mistakenly quantified and reported by data processing software as the parent drug if the TIC trace was not carefully inspected. Nowadays, all LC/MS data are processed by computer software in a highly automated fashion, and some analysts may spend much less time to visually examine raw TIC traces than they used to do. Two examples described in this article remind us that quality data require both adequate chromatographic separations and close examination of raw data in LC/MS/MS analyses of drugs in biological matrix.

Asunto(s)

Biopolímeros/química , Biopolímeros/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray/métodos , Mezclas Complejas/química , Mezclas Complejas/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K(i)=1.2nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.

Asunto(s)

Acetamidas , Secuencias de Aminoácidos , Anticoagulantes/administración & dosificación , Diseño de Fármacos , Nitrilos , Trombina/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Acetamidas/síntesis química , Acetamidas/farmacocinética , Acetamidas/uso terapéutico , Administración Oral , Animales , Anticoagulantes/síntesis química , Anticoagulantes/farmacocinética , Sitios de Unión , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Haplorrinos , Humanos , Enlace de Hidrógeno , Modelos Químicos , Nitrilos/síntesis química , Nitrilos/farmacocinética , Nitrilos/uso terapéutico , Ratas , Relación Estructura-Actividad

RESUMEN

Complement activation has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury, acute respiratory distress syndrome, and acute transplant rejection. Even though the complement cascade provides several protein targets for potential therapeutic intervention only two complement inhibitors have been approved so far for clinical use including anti-C5 antibodies for the treatment of paroxysmal nocturnal hemoglobinuria and purified C1-esterase inhibitor replacement therapy for the control of hereditary angioedema flares. In the present study, optimization of potency and physicochemical properties of a series of thiophene amidine-based C1s inhibitors with potential utility as intravenous agents for the inhibition of the classical pathway of complement is described.

Asunto(s)

Complemento C1s/antagonistas & inhibidores , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Animales , Sitios de Unión , Semivida , Modelos Moleculares , Estructura Molecular , Ratas , Relación Estructura-Actividad

RESUMEN

The pharmacokinetics of TDP223206 was studied following single intravenous and oral administrations in rats. A mixture of TDP223206 and (14)C-TDP223206 were administered to intact and bile duct-cannulated rats. Following intravenous administration, plasma concentrations declined biphasically. The AUC(inf) increased linearly with dose but was not dose proportional. The PK parameters of TDP223206 indicated low clearance (254-386 ml/h/kg) and a moderate volume of distribution (968-1883 ml/kg). The bioavailability was 32.95% and 24.46% for 10 and 50 mg/kg oral doses, respectively. (14)C-TDP223206 was distributed widely into different tissues with small intestine, liver, kidneys and large intestine having large tissue to plasma ratios. (14)C-TDP223206 was the major circulating component in the plasma. A total of 91.2% of administered radioactivity of (14)C-TDP223206 was recovered in bile indicating that biliary excretion was the major pathway for drug elimination. (14)C-TDP223206-acyl glucuronides were the major metabolites in bile. The oxo-(14)C-TDP223206 was the major metabolite in plasma and an important metabolite in bile. Two forms of diastereomeric acyl glucuronides of (14)C-TDP223206 were detected in bile with similar LC/MS intensities suggesting a similar biotransformation capacity. Only one form of these (14)C-TDP223206-acyl glucuronides was detected in plasma suggesting that enterohepatic recirculation was related to the nature of the stereo-isomers.

Asunto(s)

Indoles/farmacocinética , Integrina alfaVbeta3/antagonistas & inhibidores , Propionatos/farmacocinética , Administración Oral , Animales , Bilis/metabolismo , Heces/química , Indoles/sangre , Indoles/orina , Inyecciones Intravenosas , Masculino , Propionatos/administración & dosificación , Propionatos/sangre , Propionatos/orina , Ratas , Ratas Sprague-Dawley , Distribución Tisular

RESUMEN

The pharmacokinetics of TDP4815 was evaluated in rats, rabbits, dogs and monkeys. After intravenous administration, TDP4815 achieved C(O) of 3255 ng/ml in rats at 5 mg/kg, 9066 ng/ml in rabbits and 7858 ng/ml in monkeys at 6 mg/kg, and 4457 ng/ml in dogs at 3 mg/kg. The clearance (C(L)) was 3105, 1692, 835 and 640 ml/h/kg in rats, rabbits, monkeys and dogs, respectively. The volume of distribution (V(Z)) was more than 3861 ml/kg in all species, except 1915 ml/kg in monkeys. The oral bioavailability was rabbit >rat> monkey compared at 100 mg/kg, but it was much higher in dogs (>64%) after oral administrations. The calculated intrinsic clearance data suggested that the clearance of dog and human was restricted by binding to the plasma protein, and the clearance of rat and monkey was dependent on both the free fraction of plasma protein binding and the liver blood flow rate. The unbound hepatic intrinsic clearance of monkey was close to its C(L) suggesting that the hepatic clearance was an important excretion in monkeys. The poor oral bioavailability in the monkey may be related to the extensive glucuronidation. The V(Z).kg and C(L).kg in test species showed good correlation with the animal body weights (R(2)=0.87 and 0.96).

Asunto(s)

Anticoagulantes/farmacocinética , Guanidina/análogos & derivados , Administración Oral , Animales , Anticoagulantes/administración & dosificación , Disponibilidad Biológica , Peso Corporal , Perros , Glucurónidos/metabolismo , Guanidina/administración & dosificación , Guanidina/farmacocinética , Humanos , Técnicas In Vitro , Inyecciones Intravenosas , Hígado/irrigación sanguínea , Hígado/metabolismo , Macaca fascicularis , Masculino , Microsomas Hepáticos/metabolismo , Unión Proteica , Conejos , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Distribución Tisular

RESUMEN

The alpha(V) integrins are key receptors involved in mediating cell migration and angiogenesis. In age-related macular degeneration (AMD) and diabetic retinopathy, angiogenesis plays a critical role in the loss of vision. These ocular vasculopathies might be treatable with a suitable alpha(V) antagonist, and an oral drug would offer a distinct advantage over current therapies. (3,S,beta,S)-1,2,3,4-Tetrahydro-beta-[[1-[1-oxo-3-(1,5,6,7-tetrahydro-1,8-naphthyridin-2-yl)propyl]-4-piperidinyl]methyl]-3-quinolinepropanoic acid (JNJ-26076713) is a potent, orally bioavailable, nonpeptide alpha(V) antagonist derived from the arginine-glycine-asparagine binding motif in the matrix protein ligands (e.g., vitronectin). This compound inhibits alpha(V)beta(3) and alpha(V)beta(5) binding to vitronectin in the low nanomolar range, it has excellent selectivity over integrins alpha(IIb)beta(3) and alpha(5)beta(1), and it prevents adhesion to human, rat, and mouse endothelial cells. JNJ-26076713 blocks cell migration induced by vascular endothelial growth factor, fibroblast growth factor (FGF), and serum, and angiogenesis induced by FGF in the chick chorioallantoic membrane model. JNJ-26076713 is the first alpha(V) antagonist reported to inhibit retinal neovascularization in an oxygen-induced model of retinopathy of prematurity after oral administration. In diabetic rats, orally administered JNJ-26076713 markedly inhibits retinal vascular permeability, a key early event in diabetic macular edema and AMD. Given this profile, JNJ-26076713 represents a potential therapeutic candidate for the treatment of age-related macular degeneration, macular edema, and proliferative diabetic retinopathy.

Asunto(s)

Permeabilidad Capilar/fisiología , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Integrina alfaV/metabolismo , Naftiridinas/administración & dosificación , Naftiridinas/farmacocinética , Quinolinas/administración & dosificación , Quinolinas/farmacocinética , Neovascularización Retiniana/metabolismo , Administración Oral , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacocinética , Animales , Disponibilidad Biológica , Permeabilidad Capilar/efectos de los fármacos , Línea Celular , Embrión de Pollo , Diabetes Mellitus Experimental/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Naftiridinas/química , Embarazo , Quinolinas/química , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Neovascularización Retiniana/tratamiento farmacológico

RESUMEN

Glutathione (GSH) has been widely used for in vitro trapping and subsequently detecting reactive metabolites using liquid chromatography-mass spectrometry. A major drawback of GSH is its low trapping efficiency for "hard" reactive metabolites such as reactive aldehydes. In the present study, a bifunctional trapping agent (gamma GSK, gamma-glutamylcysteinlysine) is investigated as an alternative of GSH for simultaneous trapping both "hard" and "soft" reactive metabolites. In microsomal incubations, soft and hard reactive metabolites are captured by conjugation to the free thiol and the amine group of gamma GSK, respectively, resulting in formation of stable peptide adducts. Similar to GSH conjugates, all gamma GSK adducts derived from both soft and hard reactive metabolites contain a gamma-glutamyl moiety and, thus, undergo a neutral loss of 129 Da under collision-induced dissociation. As a result, an NL MS/MS scan can be utilized as a generic method for rapid detecting of both hard or soft reactive metabolites. As demonstrated by a number of model compounds, this approach, in combination with the isotope trapping technique, is reliable, sensitive, and efficient and can be potentially utilized as a high-throughput method for screening and rapid identification of both soft and hard reactive metabolites. In comparison with other methods, this approach is highly efficient and suitable in drug discovery for screening a wide variety of compounds for different reactive metabolites.

Asunto(s)

Oligopéptidos/química , Cresoles/química , Cresoles/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Furanos/química , Furanos/metabolismo , Glutatión/química , Humanos , Espectrometría de Masas , Microsomas/enzimología , Estructura Molecular , Espectrometría de Masas en Tándem

RESUMEN

Metabolism and bioactivation of 3-methylindole (3MI) were investigated in human liver microsomes. The metabolism of two deuterium-labeled analogues of 3MI permitted a relatively unambiguous identification of multiple metabolites and glutathione (GSH) adducts of reactive intermediates. A total of eight oxidized metabolites were detected, five of which were assigned as previously identified 3-methyloxindole, 3-hydroxy-3-methylindolenine, 3-hydroxy-3-methyloxindole, 5-hydroxy-3-methylindole, and 6-hydroxy-3-methylindole. Among the three new metabolites, one was either 4- or 7-OH-3-methylindole, and the other two were derived from additional oxidation on the phenyl ring of 3-methyloxindole. When GSH was added to the microsomal incubations, seven conjugates that had molecular ions corresponding to the incorporation of GSH and an atom of oxygen at m/z 453 (group I) were produced, and two additional conjugates had molecular ions at m/z 437 that corresponded to the incorporation of GSH with no additional oxygen (group II). Two conjugates in group I (m/z 453) were apparently derived by GSH addition to the 5,6-epoxide metabolite of 3-methyloxindole. These two GSH adducts were tentatively identified as 5-(glutathione-S-yl)-3-methyloxindole and 6-(glutathione-S-yl)-3-methyloxindole. The most abundant conjugate in group I was identified as 3-(glutathione-S-yl)-3-methyloxindole, which substantiated the presence of the putative 2,3-epoxy-3-methylindole intermediate. The remaining four adducts in group I were likely formed by conjugation of GSH at different positions of the phenyl ring, possibly via oxidation of 5-hydroxy-3-methylindole and 6-hydroxy-3-methylindole to two very interesting new electrophilic benzoquinone imine intermediates. For the group II conjugates (m/z 437), two isomers were identified as 2-(glutathione-S-yl)-3-methylindole and 3-(glutathione-S-yl-methyl)-indole. The former adduct was primarily derived from the 2,3-epoxide intermediate by thiol conjugation followed by dehydration. The latter adduct was consistent with our previously published work on the dehydrogenation of 3MI. In those studies, we showed that the reactive intermediate, 3-methylenenindolenine, was formed by hydrogen abstraction at the methyl group and was trapped with GSH. The putative dehydrogenation bioactivation mechanism is also substantiated by the finding that CYP2E1 selectively generated 2-(glutathione-S-yl)-3-methylindole but did not produce 3-(glutathione-S-yl-methyl)-indole. In summary, the results not only confirmed the formation of 2,3-epoxide-3-methylindole in human liver microsomes but also suggested that the phenolic metabolites of 3-methylindole were dehydrogenated to previously uncharacterized reactive intermediates.

Asunto(s)

Microsomas Hepáticos/metabolismo , Escatol/metabolismo , Biotransformación , Cromatografía Líquida de Alta Presión , Glutatión/metabolismo , Humanos , Espectrometría de Masas

RESUMEN

Brush border membrane vesicles (BBMV) were prepared from the rabbit small intestine for testing drug absorption potency through the enterocyte's apical membrane, which is an important compartment for drug oral absorption. Some modifications have been made to the traditional vesicle assay for adapting it to the 96-well plate format. The accumulation of 23 reference drugs was measured, and the data showed a good correlation with human oral absorption with a correlation coefficient R=0.853 (P<0.001), with the exception of a few false positive results. As the measured drug absorption may contain a membrane/protein binding component as well as drug uptake into vesicles, these two fractions can be discriminated by changing extravesicular osmolarity using different mannitol concentrations. This model can be applied for evaluating drug absorption rate/mechanisms, and helping drug selection in early drug research and development.

Asunto(s)

Absorción Intestinal , Mucosa Intestinal/metabolismo , Preparaciones Farmacéuticas/metabolismo , Acetaminofén/administración & dosificación , Acetaminofén/farmacocinética , Administración Oral , Animales , Azlocilina/administración & dosificación , Azlocilina/farmacocinética , Transporte Biológico Activo , Cefadroxilo/administración & dosificación , Cefadroxilo/farmacocinética , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Intestino Delgado/metabolismo , Lamivudine/administración & dosificación , Lamivudine/farmacocinética , Manitol/química , Concentración Osmolar , Ouabaína/administración & dosificación , Ouabaína/farmacocinética , Preparaciones Farmacéuticas/administración & dosificación , Farmacocinética , Fenolsulfonftaleína/administración & dosificación , Fenolsulfonftaleína/farmacocinética , Conejos , Zidovudina/administración & dosificación , Zidovudina/farmacocinética

RESUMEN

The discovery, SAR, and X-ray crystal structure of novel biarylaminoacyl-(S)-2-cyano-pyrrolidines and biarylaminoacylthiazolidines as potent inhibitors of dipeptidyl peptidase IV (DPP IV) are reported.

Asunto(s)

Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Dipeptidil Peptidasa 4/química , Inhibidores Enzimáticos/farmacología , Inhibidores de Proteasas/farmacología , Animales , Antineoplásicos/farmacología , Sitios de Unión , Glucemia/metabolismo , Células CACO-2 , Dipeptidil Peptidasa 4/metabolismo , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Cinética , Ratones , Modelos Químicos , Modelos Moleculares , Ratas , Relación Estructura-Actividad

RESUMEN

Stable-isotope trapping combined with mass spectrometry (MS) neutral loss scanning has recently been developed as a high-throughput method for the in vitro screening of major reactive metabolites. In fact, detection and identification of minor reactive metabolites are equally important since the minor metabolites, even though at low levels, may be highly reactive and also play an important role in drug-induced adverse reactions. In this study, 2-acetylthiophene, clozapine, troglitazone and 7-methylindole were selected as model compounds to further validate the advantages of this method for rapid detection and structural characterization of minor glutathione (GSH) adducts derived from reactive metabolites. The utility of the current method was clearly demonstrated by successful identification of novel reactive metabolites at low levels and also minor ones either masked by non-specific responses or co-eluted with other conjugates. In comparison with existing methods, this method is sensitive, efficient, and suitable for rapid screening and more complete profiling of reactive metabolites.

Asunto(s)

Indoles/análisis , Indoles/metabolismo , Espectrometría de Masas/métodos , Naftalenos/análisis , Naftalenos/metabolismo , Preparaciones Farmacéuticas/análisis , Preparaciones Farmacéuticas/metabolismo , Cromanos/metabolismo , Clozapina/metabolismo , Glutatión/química , Humanos , Indoles/química , Marcaje Isotópico , Isótopos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Naftalenos/química , Preparaciones Farmacéuticas/química , Sensibilidad y Especificidad , Relación Estructura-Actividad , Tiazoles/metabolismo , Tiazolidinedionas/metabolismo , Factores de Tiempo , Troglitazona

RESUMEN

It has previously been proposed that 4-methylphenol (p-cresol) is metabolically activated by oxidation of the methyl group to form a reactive quinone methide. In the present study a new metabolism pathway is elucidated in human liver microsomes. Oxidation of the aromatic ring leads to formation of 4-methyl-ortho-hydroquinone, which is further oxidized to a reactive intermediate, 4-methyl-ortho-benzoquinone. This bioactivation pathway is fully supported by the following observations: 1) one major and two minor glutathione (GSH) adducts were detected in microsomal incubations of p-cresol in the presence of glutathione; 2) a major metabolite of p-cresol was identified as 4-methyl-ortho-hydroquinone in microsomal incubations; 3) the same GSH adducts were detected in microsomal incubations of 4-methyl-ortho-hydroquinone; and 4) the same GSH adducts were chemically synthesized by oxidizing 4-methyl-ortho-hydroquinone followed by the addition of GSH, and the major conjugate was identified by liquid chromatography-tandem mass spectrometry and NMR as 3-(glutathione-S-yl)-5-methyl-ortho-hydroquinone. In addition, it was found that 4-hydroxybenzylalcohol, a major metabolite derived from oxidation of the methyl group in liver microsomes, was further converted to 4-hydroxybenzaldehyde. In vitro studies also revealed that bioactivation of p-cresol was mediated by multiple cytochromes P450, but CYP2D6, 2E1, and 1A2 are the most active enzymes for formation of quinone methide, 4-methyl-ortho-benzoquinone, and 4-hydroxybenzaldehyde, respectively. Implications of the newly identified reactive metabolite in p-cresol-induced toxicity remain to be investigated in the future.

Asunto(s)

Cresoles/metabolismo , Sistema Enzimático del Citocromo P-450/fisiología , Microsomas Hepáticos/metabolismo , Glutatión/metabolismo , Humanos , Oxidación-Reducción