RESUMEN
The mammalian target of rapamycin (mTOR) is a protein kinase that shows key involvement in age-related disease and promises to be a target for treatment of cancer. In the present study, the elimination of potent ATP-competitive mTOR inhibitor 3-(6-amino-2-methylpyrimidin-4-yl)-N-(1H-pyrazol-3-yl)imidazo[1,2-b]pyridazin-2-amine (compound 1) is studied in bile duct-cannulated rats, and the metabolism of compound 1 in liver microsomes is compared across species. Compound 1 was shown to undergo extensive N-glucuronidation in bile duct-catheterized rats. N-glucuronides were detected on positions N1 (M2) and N2 (M1) of the pyrazole moiety as well as on the primary amine (M3). All three N-glucuronide metabolites were detected in liver microsomes of the rat, dog, and human, while primary amine glucuronidation was not detected in cynomolgus monkey. In addition, N1- and N2-glucuronidation showed strong species selectivity in vitro, with rat, dog, and human favoring N2-glucuronidation and monkey favoring N1-glucuronide formation. Formation of M1 in monkey liver microsomes also followed sigmoidal kinetics, singling out monkey as unique among the species with regard to compound 1 N-glucuronidation. In this respect, monkeys might not always be the best animal model for N-glucuronidation of uridine diphosphate glucuronosyltransferase (UGT) 1A9 or UGT1A1 substrates in humans. The impact of N-glucuronidation of compound 1 could be more pronounced in higher species such as monkey and human, leading to high clearance in these species. While compound 1 shows promise as a candidate for investigating the impact of pan-mTOR inhibition in vivo, opportunities may exist through medicinal chemistry efforts to reduce metabolic liability with the goal of improving systemic exposure.
Asunto(s)
Glucurónidos/metabolismo , Compuestos Heterocíclicos con 2 Anillos/metabolismo , Microsomas Hepáticos/enzimología , Pirimidinas/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Perros , Femenino , Compuestos Heterocíclicos con 2 Anillos/farmacología , Humanos , Cinética , Macaca fascicularis , Espectroscopía de Resonancia Magnética , Masculino , Microsomas Hepáticos/efectos de los fármacos , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Espectrometría de Masas en TándemRESUMEN
The impact of inhibitor depletion on the determination of shifted IC50 (IC50 determined after 30 minutes of preincubation with inhibitor) is examined. In addition, IC50-shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as reversible and time-dependent inhibition. Anomalies such as a smaller-than-expected shift in IC50 and even increases in IC50 with preincubation were explained by the depletion of inhibitor during the preincubation. The IC50-shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methods, it is recommended that IC50-shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC50-shift methods, shifted IC50 should be calculated using observed inhibitor concentrations at the end of the incubation rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes, such as competitive inhibition, enzyme inactivation, and inhibitor depletion, can be used to describe accurately the observed IC50 and shifted IC50 curves. For compounds showing an IC50 fold shift >1.5 based on the observed inhibitor concentrations, reanalyzing the IC50-shift data using the mechanistic model appeared to allow for reasonable estimation of Ki, KI, and kinact directly from the IC50 shift experiments.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Citocromo P-450 CYP3A , Inhibidores del Citocromo P-450 CYP3A , Humanos , Concentración 50 Inhibidora , Modelos Biológicos , Nicardipino/farmacología , Saquinavir/farmacología , Factores de TiempoRESUMEN
Compound 1 [(E)-4-fluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1S,4S)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], a new, potent, selective anaplastic lymphoma kinase (ALK) inhibitor with potential application for the treatment of cancer, was selected as candidate to advance into efficacy studies in mice. However, the compound underwent mouse-specific enzymatic hydrolysis in plasma to a primary amine product (M1). Subsequent i.v. pharmacokinetics studies in mice showed that compound 1 had high clearance (CL) and a short half-life. Oral dose escalation studies in mice indicated that elimination of compound 1 was saturable, with higher doses achieving sufficient exposures above in vitro IC(50). Chemistry efforts to minimize hydrolysis resulted in the discovery of several analogs that were stable in mouse plasma. Three were taken in vivo into mice and showed decreased CL corresponding to increased in vitro stability in plasma. However, the more stable compounds also showed reduced potency against ALK. Kinetic studies in NADPH-fortified and unfortified microsomes and plasma produced submicromolar K(m) values and could help explain the saturation of elimination observed in vivo. Predictions of CL based on kinetics from hydrolysis and NADPH-dependent pathways produced predicted hepatic CL values of 3.8, 3.0, 1.6, and 1.2 l/hâ kg for compound 1, compound 2 [(E)-3,5-difluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], compound 3 [(E)-3-chloro-5-fluoro-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)benzamide], and compound 4 [(E)-N-(6-((4-(2-hydroxypropan-2-yl)piperidin-1-yl)methyl)-1-((1s,4s)-4-(isopropylcarbamoyl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-ylidene)-3-(trifluoromethyl)benzamide], respectively. The in vivo observed CLs for compounds 1, 2, 3, and 4 were 5.52, 3.51, 2.14, and 2.66 l/hâ kg, respectively. These results indicate that in vitro metabolism kinetic data, incorporating contributions from both hydrolysis and NADPH-dependent metabolism, could be used to predict the systemic CL of compounds cleared via hydrolytic pathways provided that the in vitro assays thoroughly investigate the processes, including the contribution of other metabolic pathways and the possibility of saturation kinetics.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico , Animales , Área Bajo la Curva , Cromatografía Liquida , Hidrólisis , Concentración 50 Inhibidora , Masculino , Ratones , Inhibidores de Proteínas Quinasas/sangre , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Nor-BNI, GNTI and JDTic induce κ opioid antagonism that is delayed by hours and can persist for months. Other effects are transient. It has been proposed that these drugs may be slowly absorbed or distributed, and may dissolve in cell membranes, thus slowing elimination and prolonging their effects. Recent evidence suggests, instead, that they induce prolonged desensitization of the κ opioid receptor. METHODS: To evaluate these hypotheses, we measured relevant physicochemical properties of nor-BNI, GNTI and JDTic, and the timecourse of brain and plasma concentrations in mice after intraperitoneal administration (using LC-MS-MS). RESULTS: In each case, plasma levels were maximal within 30 min and declined by >80% within four hours, correlating well with previously reported transient effects. A strong negative correlation was observed between plasma levels and the delayed, prolonged timecourse of κ antagonism. Brain levels of nor-BNI and JDTic peaked within 30 min, but while nor-BNI was largely eliminated within hours, JDTic declined gradually over a week. Brain uptake of GNTI was too low to measure accurately, and higher doses proved lethal. None of the drugs were highly lipophilic, showing high water solubility (> 45 mM) and low distribution into octanol (log D7.4 < 2). Brain homogenate binding was within the range of many shorter-acting drugs (>7% unbound). JDTic showed P-gp-mediated efflux; nor- BNI and GNTI did not, but their low unbound brain uptake suggests efflux by another mechanism. CONCLUSIONS: The negative plasma concentration-effect relationship we observed is difficult to reconcile with simple competitive antagonism, but is consistent with desensitization. The very slow elimination of JDTic from brain is surprising given that it undergoes active efflux, has modest affinity for homogenate, and has a shorter duration of action than nor-BNI under these conditions. We propose that this persistence may result from entrapment in cellular compartments such as lysosomes.
Asunto(s)
Guanidinas/farmacocinética , Morfinanos/farmacocinética , Naltrexona/análogos & derivados , Antagonistas de Narcóticos/farmacocinética , Piperidinas/farmacocinética , Receptores Opioides kappa/metabolismo , Tetrahidroisoquinolinas/farmacocinética , Animales , Transporte Biológico , Encéfalo/metabolismo , Guanidinas/sangre , Interacciones Hidrofóbicas e Hidrofílicas , Inyecciones Intraperitoneales , Células LLC-PK1 , Masculino , Ratones , Morfinanos/sangre , Naltrexona/sangre , Naltrexona/farmacocinética , Antagonistas de Narcóticos/sangre , Permeabilidad , Piperidinas/sangre , Porcinos , Tetrahidroisoquinolinas/sangreRESUMEN
Prediction of human volume of distribution at steady state (V(ss)) before first administration of a new drug candidate to humans has become an important part of the drug development process. This study examines the assumptions behind interspecies scaling techniques used to predict human V(ss) from preclinical data, namely the equivalency of V(ss,u) and/or f(ut) across species. In addition, several interspecies scaling techniques are evaluated side by side using a set of 67 reference compounds where observed V(ss) from rats, dogs, monkeys, and humans were compiled from the literature and where plasma protein binding was determined across species using an ultracentrifugation technique. Species similarity in V(ss,u) or f(ut) does not appear to be the norm among rats, dogs, monkeys, or humans. Despite this, interspecies scaling from rats, dogs, and monkeys is useful and can provide reasonably accurate predictions of human V(ss), although some interspecies scaling approaches were better than others. For example, the performance of the common V(ss,u) or f(ut) equivalency approaches using average V(ss,u) or f(ut) across three preclinical species was superior to allometric scaling techniques. In addition, considering data from several preclinical species, using the equivalency approach, was superior to scaling from any single species. Although the mechanistic tissue composition equations available in the Simcyp population-based pharmacokinetic simulator did not necessarily provide the most accurate predictions, and the equations used likely need refinement, they still provide the best opportunity for a mechanistic understanding and prediction of human V(ss).
Asunto(s)
Preparaciones Farmacéuticas/metabolismo , Farmacocinética , Animales , Perros , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Haplorrinos , Humanos , Unión Proteica , Ratas , Especificidad de la Especie , Distribución TisularRESUMEN
To predict volume of distribution at steady-state (V(ss)), empirical (e.g., allometry) and mechanistic (using physicochemical property data and plasma protein binding) methods have been used. None of these approaches has been able to predict V(ss) accurately for the total compliment of a wide range of drugs. Therefore, alternative approaches would be of value. This study evaluates the utility of in vitro nonspecific tissue-binding measurements in predicting V(ss) for a wide range of drugs in rats. Literature as well as proprietary compounds were studied. It was found that in vitro tissue-binding measurements combined with calculated effects of the pH partition hypothesis often predict V(ss) more accurately than other available mechanistic methods and that this approach can compliment existing methods. The V(ss) values for some compounds were not accurately predicted using either nonspecific tissue-binding experiments or other available mechanistic methods. The V(ss) for these drugs may not be describable by nonspecific tissue binding alone; there may be significant specific components to the mechanism of distribution for these drugs, such as pH-dependent uptake into lysosomes (primarily strongly basic drugs), active transport, and/or enterohepatic recirculation. A lack of prediction for certain drugs warrants further investigation into these mechanisms and their application to more accurate prediction of V(ss) by mechanistic means.
Asunto(s)
Modelos Biológicos , Preparaciones Farmacéuticas/metabolismo , Farmacocinética , Extractos de Tejidos/metabolismo , Algoritmos , Animales , Fenómenos Químicos , Concentración de Iones de Hidrógeno , Masculino , Preparaciones Farmacéuticas/sangre , Preparaciones Farmacéuticas/química , Plasma/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Glycine receptors (GlyRs) are pentameric glycine-gated chloride ion channels that are enriched in the brainstem and spinal cord where they have been demonstrated to play a role in central nervous system (CNS) inhibition. Herein we describe two novel classes of glycine receptor potentiators that have been developed using similarity- and property-guided scaffold hopping enabled by parallel synthesis and pharmacophore-based virtual screening strategies. This effort resulted in the identification of novel, efficient and modular leads having favorable in vitro ADME profiles and high CNS multi-parameter optimization (MPO) scores, exemplified by azetidine sulfonamide 19 and aminothiazole sulfone (ent2)-20.
Asunto(s)
Descubrimiento de Drogas , Receptores de Glicina/antagonistas & inhibidores , Sulfonamidas/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Because of its strong genetic validation, NaV1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [ Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation . WO 2014201206, 2014 ] of NaV1.7, which demonstrate nanomolar inhibition of NaV1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a NaV1.7-dependent model of histamine-induced pruritus (itch) and additionally in a capsaicin-induced nociception model of pain without any confounding effect in open-field activity.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Quinolonas/química , Sulfonamidas/química , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacología , Animales , Capsaicina , Línea Celular , Perros , Histamina , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Dolor/inducido químicamente , Dolor/prevención & control , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Prurito/inducido químicamente , Prurito/prevención & control , Quinolonas/administración & dosificación , Quinolonas/síntesis química , Quinolonas/farmacocinética , Quinolonas/farmacología , Ratas , Relación Estructura-Actividad , Sulfonamidas/administración & dosificación , Sulfonamidas/síntesis química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacocinética , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
The majority of potent and selective hNaV1.7 inhibitors possess common pharmacophoric features that include a heteroaryl sulfonamide headgroup and a lipophilic aromatic tail group. Recently, reports of similar aromatic tail groups in combination with an acyl sulfonamide headgroup have emerged, with the acyl sulfonamide bestowing levels of selectivity over hNaV1.5 comparable to the heteroaryl sulfonamide. Beginning with commercially available carboxylic acids that met selected pharmacophoric requirements in the lipophilic tail, a parallel synthetic approach was applied to rapidly generate the derived acyl sulfonamides. A biaryl acyl sulfonamide hit from this library was elaborated, optimizing for potency and selectivity with attention to physicochemical properties. The resulting novel leads are potent, ligand and lipophilic efficient, and selective over hNaV1.5. Representative lead 36 demonstrates selectivity over other human NaV isoforms and good pharmacokinetics in rodents. The biaryl acyl sulfonamides reported herein may also offer ADME advantages over known heteroaryl sulfonamide inhibitors.
Asunto(s)
Benzamidas/química , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Sulfonamidas/química , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Animales , Benzamidas/síntesis química , Benzamidas/farmacocinética , Benzamidas/farmacología , Línea Celular , Femenino , Histamina , Humanos , Masculino , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Prurito/inducido químicamente , Prurito/tratamiento farmacológico , Ensayo de Unión Radioligante , Ratas , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/síntesis química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacocinética , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
Species and tissue differences in the activity of three major classes of esterases, carboxylesterase (CE), butyrylcholinesterase (BChE) and paraoxonase (PON), were studied. Substantial species differences in activity of these esterases were observed between the mouse, rat, dog monkey and human. Such species differences must be considered when using these preclinical species to optimize the pharmacokinetic properties of ester compounds intended for human use.
Asunto(s)
Arildialquilfosfatasa/metabolismo , Butirilcolinesterasa/metabolismo , Carboxilesterasa/metabolismo , Animales , Perros , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Macaca fascicularis , Ratones , Modelos Animales , Ratas , Especificidad de la EspecieRESUMEN
The relationship between time-dependent inactivation (TDI) and IC50 is examined using a consolidated method for evaluating CYP450 inhibition during drug discovery. An IC50 fold-shift of >1.5 indicated significant TDI potency. Further, the "shifted IC50" could be used to estimate, the K(I) and TDI potency ratio k(inact)/K(I) to within 2-fold in most cases.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Inhibidores del Citocromo P-450 CYP2D6 , Inhibidores del Citocromo P-450 CYP3A , Inhibidores Enzimáticos/farmacología , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A , Inhibidores Enzimáticos/administración & dosificación , Humanos , Concentración 50 Inhibidora , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Modelos Biológicos , Factores de TiempoRESUMEN
In vitro studies were conducted to identify the major metabolites of eplerenone (EP) and the cytochrome p450 (p450) isozymes involved in its primary oxidative metabolism in humans and dogs. The major in vitro metabolites were identified as 6 beta-hydroxy EP and 21-hydroxy EP in both humans and dogs. EP was metabolized by cDNA-expressed human CYP3A4 and dog CYP3A12 but only minimally by human CYP3A5. In human microsomes, inhibition of total metabolism by the CYP3A-selective inhibitors ketoconazole, troleandomycin, and 6',7'-dihydroxybergamottin, each at 10 micro M concentration, was 83 to 95%, whereas inhibition with inhibitors selective for other p450 isozymes was minimal. In dog liver microsomes, the percentages of inhibition were 53 to 76% with the CYP3A-selective inhibitors. A monoclonal anti-CYP3A4 antibody inhibited EP metabolism by 84%, whereas other monoclonal antibodies had minimal effects. The formation of 6 beta-hydroxy and 21-hydroxy metabolites in human liver microsomes was best correlated with CYP3A-selective dextromethorphan N-demethylation and testosterone 6 beta-hydroxylation activities. EP moderately inhibited only CYP3A (testosterone 6 beta-hydroxylase) activity in human liver microsomes by 23, 34 and 45% at concentrations of 30, 100, and 300 micro M, respectively. With human microsomes, the V(max) and K(m) for 6 beta-hydroxylation and 21-hydroxylation were 0.973 nmol/min/mg and 217 micro M, and 0.143 nmol/min/mg and 211 micro M, respectively. The human hepatic clearance calculated from total in vitro EP metabolism was 2.30 ml/min/kg, which agrees with in vivo data. In conclusion, 6 beta- and 21-hydroxylation of EP is primarily catalyzed by CYP3A4 in humans and CYP3A12 in dogs. Also, it is unlikely that EP would substantially inhibit the metabolism of other drugs that are metabolized by CYP3A4 or other p450 isoforms.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Espironolactona/análogos & derivados , Espironolactona/metabolismo , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP3A , Perros , Eplerenona , Humanos , Microsomas Hepáticos/enzimología , Oxidorreductasas N-Desmetilantes/metabolismo , Especificidad de la Especie , Espironolactona/químicaRESUMEN
A pharmacokinetics and metabolism study was conducted in eight healthy human volunteers. After oral administration of [14C]eplerenone (EP) at a dose of 100 mg per person as an aqueous solution, blood, saliva, breath, urine, and fecal samples were collected at various time points. All matrices were analyzed for total radioactivity and/or for EP and its open-lactone-ring form (EPA). EP was well absorbed, and a mean EP Cmax of 1.72 mug/ml was achieved 1.2 h postdose. After the Cmax, plasma concentrations of EP declined with a half-life of 3.0 h. Plasma concentrations of EPA were much lower than EP concentrations, and the area under the plasma-concentration time curve (AUC) for EPA was only 4% of the EP AUC. Plasma protein binding was moderate (33-60%) but concentration-dependent over the therapeutic concentration range. EP and its metabolites did not preferentially partition into the red blood cells and blood concentrations of total radioactivity were lower than plasma concentrations. Approximately 66.6% and 32.0% of the radioactive dose were excreted in urine and feces, respectively. The majority of urinary and fecal radioactivity was due to metabolites, indicating extensive metabolism of EP. The major metabolic pathways were 6beta- and/or 21-hydroxylation and 3-keto reduction. There was no evidence for any alteration of the 9,11-epoxide ring or the methyl ester. As a percentage of dose, the primary metabolic products excreted in urine and feces included 6beta-hydroxy-EP (6beta-OHEP) (32.0%), 6beta,21-OHEP (20.5%), 21-OHEP (7.89%), and 2alpha,3beta,21-OHEP (5.96%). The amounts of the other metabolites excreted were less than 5% each.