RESUMEN
A sensitive, accurate and rugged UHPLC-MS/MS method was developed and validated for the quantitation of Epothilone D (EpoD), a microtubule stabilizer in development for treatment of Alzeimer's disease, in rat plasma. The ester group in EpoD can be hydrolyzed by esterases in blood or plasma, which creates a stability concern for the bioanalysis of EpoD. Species differences in the stability of EpoD in plasma were observed. Carboxylesterases were identified as the likely esterases responsible for the hydrolysis of EpoD in plasma ex vivo, and the cause of the species different stability. Phenylmethanesulfonyl fluoride, a carboxylesterase inhibitor, was used to stabilize EpoD in rat blood during sample collection, processing, and storage. A systematic method screening and optimization strategy was used to improve the assay sensitivity and minimize potential bioanalytical risks. The stabilized plasma samples were extracted by liquid-liquid extraction. Chromatographic separation was achieved on an Acquity UPLC BEH Phenyl column with a gradient elution. EpoD and its stable isotope labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 0.100 to 100ng/mL was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV and inter-assay precision was within ±4.2% CV. The assay accuracy was within ±8.3% of the nominal values. Assay recovery of EpoD was high (â¼90%) and matrix effect was minimal (1.02-1.05). EpoD was stable in stabilized rat plasma for at least 30h at room temperature, 180 days at -20°C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in toxicology studies.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Epotilonas/sangre , Epotilonas/química , Fluoruro de Fenilmetilsulfonilo/química , Espectrometría de Masas en Tándem/métodos , Animales , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Hidrolasas de Éster Carboxílico/efectos de los fármacos , Estabilidad de Medicamentos , Epotilonas/farmacocinética , Femenino , Modelos Lineales , Fluoruro de Fenilmetilsulfonilo/farmacología , Ratas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
An UHPLC-MS/MS method was developed and validated to quantify BMS-927711, a drug candidate to treat migraine, in rat dried blood spots (DBS). The DBS samples were extracted using an improved liquid-liquid extraction (LLE) strategy involving in the sonication of DBS punches in 20% MeOH aqueous solution containing the internal standard, [(13)C2, D4]-BMS-927711, and then with a 100mM NH4OAc buffer solution, followed by an automated LLE with EtOAc-hexane (70:30, v/v). The presence of 20% MeOH as an organic modifier in the elution solution significantly improved the analyte elution efficiency and assay performance. A novel inter-well volume replacement dilution workflow was introduced for DBS sample dilution before LLE step. This was a simple two-step process, firstly a small portion of the DBS blank solution was discarded, and then the same volume of a concentrated DBS sample solution was spiked into the leftover blank solution to achieve a desired dilution. Chromatographic separation was achieved on an Acuity UPLC(®) BEH C18 column (2.1mm×50mm, 1.7µm) and the analyte was detected by selected reaction monitoring (SRM) with positive electrospray ionization on an AB Sciex Triple Quad 5500 mass spectrometer. The standard curve was linear from 5.00 to 5000ng/mL with assay precision ≤4.9% CV, and assay accuracy within ±3.1%Dev of the nominal values. Accurate sample dilution was achieved by using inter-well volume replacement with a precision of ≤4.2% CV and an accuracy of ±3.3% for dilution QC at 50,000ng/mL with 100-fold dilution (n=18). This robust UHPLC-MS/MS assay has been successfully applied to the non-clinical studies in rats. By using inter-well volume replacement workflow, accurate dilution was demonstrated using only one DBS blank sample for a typical dilution of <50-fold, and using only two blank DBS samples for a dilution of up to 625-fold. Moreover, this new workflow makes it easier to automate DBS sample dilution.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Pruebas con Sangre Seca/métodos , Piperidinas/química , Piridinas/química , Animales , Técnicas de Dilución del Indicador , Límite de Detección , Extracción Líquido-Líquido/métodos , Ratas , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos , Flujo de TrabajoRESUMEN
BMS-927711 is a calcitonin gene-related peptide (CGRP) receptor antagonist that is being developed for the treatment of migraine. A rapid, accurate and robust assay was developed and validated for the quantitation of BMS-927711 in rat, monkey, rabbit and mouse plasma using ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). A simplified method screening strategy was utilized that included a liquid-liquid extraction (LLE) methodology and eleven LC columns (ten sub-2 µm UHPLC columns and one 2.6 µm HPLC column) for screening with emphasis on the removal of phospholipids, avoidance of metabolite interference and ruggedness of LC conditions. A stable isotope labeled [(13)C2, D4]-BMS-927711 was used as the internal standard, and 50 µL of plasma samples were used for extraction by automated LLE with methyl tert-butyl ether (MTBE) in 96-well format. Chromatographic separation was achieved with an isocratic elution and a gradient column wash on a Waters Acuity UPLC(®) BEH C18 column (2.1 mm × 50 mm, 1.7 µm) with run time of 3.7 min. Positive electrospray ionization was performed using selected reaction monitoring (SRM) with transitions of m/z 535>256 for BMS-927711 and m/z 541>256 for [(13)C2, D4]-BMS-927711. The standard curve, which ranged from 3.00 to 3000 ng/mL for BMS-927711, was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within 5.2% CV, inter-assay precision was within 5.9% CV, and the assay accuracy was within ±5.2% deviation (%Dev) of the nominal values in all the species. The stability of an N-carbamoyl glucuronide metabolite was carefully investigated, and the conversion of this metabolite to BMS-927711 was minimal and manageable without a stabilization procedure. The method was successfully applied to multiple non-clinical toxicokinetic studies in different species in support of the investigative new drug (IND) filing.
Asunto(s)
Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Cromatografía Líquida de Alta Presión/métodos , Piperidinas/sangre , Piperidinas/química , Piridinas/sangre , Piridinas/química , Espectrometría de Masas en Tándem/métodos , Animales , Glucurónidos/química , Haplorrinos , Extracción Líquido-Líquido/métodos , Ratones , Conejos , Ratas , Estándares de ReferenciaRESUMEN
Automated sample extraction for regulated bioanalysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS) still presents significant challenges. A new sample preparation methodology with a simplified and completely automated workflow was developed to overcome these challenges using cap piercing for direct biofluid transfer and evaporation-free solid phase extraction (SPE). Using pierceable cap sample tubes, a robotic liquid handler was able to sample without uncapping or recapping during sample preparation. Evaporation for SPE was eliminated by using a mobile phase-compatible elution solvent followed by sample dilution prior to LC-MS/MS analysis. Presented here are three LC-MS/MS assays validated using this methodology to support three CNS drug development programs: (1) BMS-763534 and its metabolite, BMS-790318, in dog plasma; (2) BMS-694153 in monkey plasma; and (3) Pexacerfont (BMS-562086) and two metabolites, BMS-749241 and DPH-123554, in human plasma. These assays were linear from 1.00 to 1000 or 2.00 to 2000ng/mL for each analyte with excellent assay accuracy, precision and reproducibility. These assays met acceptance criteria for regulated bioanalysis and have been successfully applied to drug development study samples. The methodology described here successfully eliminated all manual intervention steps achieving fully automated sample preparation without compromising assay performance. Importantly, this methodology eliminates the potential exposure of the bioanalyst to any infectious biofluids during sample preparation.
Asunto(s)
Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Indazoles/sangre , Pirazoles/sangre , Quinazolinonas/sangre , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidores , Extracción en Fase Sólida/métodos , Manejo de Especímenes/métodos , Espectrometría de Masas en Tándem/métodos , Triazinas/sangre , Animales , Cromatografía Liquida/economía , Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Perros , Diseño de Equipo , Haplorrinos , Ensayos Analíticos de Alto Rendimiento/economía , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Indazoles/metabolismo , Pirazoles/metabolismo , Quinazolinonas/metabolismo , Sensibilidad y Especificidad , Extracción en Fase Sólida/economía , Extracción en Fase Sólida/instrumentación , Manejo de Especímenes/economía , Manejo de Especímenes/instrumentación , Espectrometría de Masas en Tándem/economía , Espectrometría de Masas en Tándem/instrumentación , Triazinas/metabolismoRESUMEN
Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) non-structural protein protease inhibitor currently in Phase III clinical trials for the treatment of HCV infection. A rugged and accurate LC-MS/MS method was developed and validated for the quantitation of asunaprevir in rat, dog, monkey, rabbit and mouse plasma. A systematic method screening and optimization strategy was applied to achieve optimized mass spectrometry, chromatography, and sample extraction conditions. The validated method utilized stable-isotope labeled D9-asunaprevir as the internal standard. The samples were extracted by liquid-liquid extraction using 10% ethyl acetate in hexane. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column. Analyte and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 5.00 to 2000ng/mL for asunaprevir, was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV, inter-assay precision was within ±4.0% CV, and the assay accuracy was within ±8.1% of the nominal values in all the species. The method was successfully applied to support multiple pre-clinical toxicokinetic studies in different species.
Asunto(s)
Antivirales/sangre , Hepacivirus/enzimología , Isoquinolinas/sangre , Inhibidores de Proteasas/sangre , Sulfonamidas/sangre , Espectrometría de Masas en Tándem/métodos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Cromatografía Liquida/métodos , Perros , Haplorrinos , Hepatitis C/tratamiento farmacológico , Ratones , Conejos , Ratas , Sensibilidad y EspecificidadRESUMEN
Quantitative determination of drug concentrations in tissue homogenates via liquid chromatography-tandem mass spectrometry (LC-MS/MS) is commonly conducted using the standards and analytical quality controls (QCs) prepared in the same matrix (tissue homogenates), to keep the matrix and its effects consistent on the analytes during sample extraction and analysis. In this manuscript, we proposed to analyze tissue homogenate samples using an LC-MS/MS assay with the standards and analytical QCs prepared in plasma after tissue homogenate samples were appropriately diluted with plasma. BMS-650032 was used as a model compound, and its validated dog plasma assay was used for dog liver sample analyses. The tissue matrix effect was evaluated by diluting liver homogenate QCs with drug-free plasma at different dilution factors to determine the minimum required dilution factor (MRDF) at which tissue matrix has insignificant impact to the plasma assay. The percentage deviation of the measured concentration from the nominal concentration was used as an indicator of the tissue matrix effect. The results suggested that the tissue matrix effect was decreased as the plasma dilution factor increased. Based on the results of the tissue matrix effect evaluation, liver homogenate samples were analyzed after appropriate dilutions with plasma at the MRDF or greater dilution factors. The results confirmed that this approach generates accurate data, and the process is very convenient and economic. This approach has been used on the analyses of different tissues (liver and brain) and biofluid (bile) to support several drug development programs.
Asunto(s)
Antivirales/sangre , Cromatografía Liquida/métodos , Hígado/química , Proyectos de Investigación , Espectrometría de Masa por Ionización de Electrospray/métodos , Extractos de Tejidos/análisis , Animales , Antivirales/farmacología , Calibración , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/virología , Perros , Hepacivirus/efectos de los fármacos , Hepacivirus/crecimiento & desarrollo , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Control de Calidad , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Extractos de Tejidos/químicaRESUMEN
We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.
Asunto(s)
Piperazinas/química , Receptor de Melanocortina Tipo 4/agonistas , Urea/análogos & derivados , Administración Oral , Animales , Disponibilidad Biológica , Modelos Animales de Enfermedad , Perros , Evaluación Preclínica de Medicamentos , Ingestión de Alimentos/efectos de los fármacos , Haplorrinos , Ratones , Obesidad/tratamiento farmacológico , Piperazinas/farmacocinética , Piperazinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptor de Melanocortina Tipo 4/genética , Receptor de Melanocortina Tipo 4/metabolismo , Relación Estructura-Actividad , Urea/química , Urea/farmacocinética , Urea/uso terapéuticoRESUMEN
We report an SAR study of MC4R analogs containing spiroindane heterocyclic privileged structures. Compound 26 with N-Me-1,2,4-triazole moiety possesses exceptional potency at MC4R and potent anti-obesity efficacy in a mouse model. However, the efficacy is not completely mediated through MC4R. Additional SAR studies led to the discovery of compound 32, which is more potent at MC4R. Compound 32 demonstrates MC4R mediated anti-obesity efficacy in rodent models.
Asunto(s)
Obesidad/tratamiento farmacológico , Receptor de Melanocortina Tipo 4/agonistas , Triazoles/farmacología , Animales , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Estructura Molecular , Ratas , Receptor de Melanocortina Tipo 4/genética , Relación Estructura-Actividad , Triazoles/química , Triazoles/uso terapéuticoRESUMEN
We report a series of potent and selective MC4R agonists based on spiroindane amide privileged structures for potential treatments of obesity. Among the synthetic methods used, Method C allows rapid synthesis of the analogs. The series of compounds can afford high potency on MC4R as well as good rodent pharmacokinetic profiles. Compound 1r (MK-0489) demonstrates MC4R mediated reduction of food intake and body weight in mouse models. Compound 1r is efficacious in 14-day diet-induced obese (DIO) rat models.
Asunto(s)
Amidas/química , Fármacos Antiobesidad/química , Obesidad/tratamiento farmacológico , Pirrolidinas/química , Receptor de Melanocortina Tipo 4/agonistas , Compuestos de Espiro/química , Amidas/farmacocinética , Amidas/uso terapéutico , Animales , Fármacos Antiobesidad/farmacocinética , Fármacos Antiobesidad/uso terapéutico , Peso Corporal/efectos de los fármacos , Humanos , Ratones , Ratones Noqueados , Pirrolidinas/farmacocinética , Pirrolidinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptor de Melanocortina Tipo 4/metabolismo , Compuestos de Espiro/farmacocinética , Compuestos de Espiro/uso terapéutico , Relación Estructura-ActividadRESUMEN
Design, synthesis, and SAR of a series of 3H-spiro[isobenzofuran-1,4'-piperidine] based compounds as potent, selective and orally bioavailable melanocortin subtype-4 receptor (MC4R) agonists are disclosed.
Asunto(s)
Piperidinas/química , Receptor de Melanocortina Tipo 4/agonistas , Administración Oral , Animales , Encéfalo/metabolismo , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Conformación Molecular , Piperidinas/síntesis química , Piperidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Melanocortina Tipo 4/metabolismo , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
SAR around non-peptidic potent bombesin receptor subtype-3 (BRS-3) agonist lead 2 is presented. Attempts to replace the carboxylic acid with heterocyclic isosteres to improve oral bioavailability and brain penetration are described.
Asunto(s)
Fármacos Antiobesidad/síntesis química , Imidazoles/química , Receptores de Bombesina/agonistas , Administración Oral , Animales , Fármacos Antiobesidad/química , Fármacos Antiobesidad/uso terapéutico , Encéfalo/metabolismo , Humanos , Imidazoles/síntesis química , Imidazoles/uso terapéutico , Ratones , Obesidad/tratamiento farmacológico , Ratas , Receptores de Bombesina/metabolismo , Relación Estructura-ActividadRESUMEN
We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective, orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent erectogenic activity in the rodent models.
Asunto(s)
Disfunción Eréctil/tratamiento farmacológico , Indanos/química , Indanos/uso terapéutico , Receptor de Melanocortina Tipo 4/agonistas , Receptor de Melanocortina Tipo 4/metabolismo , Compuestos de Espiro/química , Compuestos de Espiro/uso terapéutico , Animales , Células CHO , Cricetinae , Cricetulus , Perros , Haplorrinos , Humanos , Indanos/farmacocinética , Indanos/farmacología , Masculino , Ratones , Estructura Molecular , Unión Proteica , Ratas , Compuestos de Espiro/farmacocinética , Compuestos de Espiro/farmacología , Relación Estructura-ActividadRESUMEN
This Letter describes a series of potent and selective BRS-3 agonists containing a biarylethylimidazole pharmacophore. Extensive SAR studies were carried out with different aryl substitutions. This work led to the identification of a compound 2-{2-[4-(pyridin-2-yl)phenyl]ethyl}-5-(2,2-dimethylbutyl)-1H-imidazole 9 with excellent binding affinity (IC(50)=18 nM, hBRS-3) and functional agonist activity (EC(50)=47 nM, 99% activation). After oral administration, compound 9 had sufficient exposure in diet induced obese mice to demonstrate efficacy in lowering food intake and body weight via BRS-3 activation.
Asunto(s)
Imidazoles/química , Imidazoles/uso terapéutico , Obesidad/tratamiento farmacológico , Receptores de Bombesina/agonistas , Receptores de Bombesina/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Humanos , Imidazoles/farmacocinética , Ratones , Ratas , Relación Estructura-ActividadRESUMEN
We report SAR studies on a novel non-peptidic bombesin receptor subtype-3 (BRS-3) agonist lead series derived from high-throughput screening hit RY-337. This effort led to the discovery of compound 22e with significantly improved potency at both rodent and human BRS-3.
Asunto(s)
Descubrimiento de Drogas , Imidazoles/química , Imidazoles/farmacología , Receptores de Bombesina/agonistas , Animales , Disponibilidad Biológica , Humanos , Imidazoles/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
Discovery of a series of tert-butyl pyrrolidine derived, potent and orally bioavailable melanocortin receptor subtype-4 (MC4R) selective modulators is disclosed.
Asunto(s)
Pirrolidinas/síntesis química , Pirrolidinas/farmacocinética , Receptor de Melanocortina Tipo 4/agonistas , Administración Oral , Humanos , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , TelomerasaRESUMEN
A novel isoquinuclidine containing selective melanocortin subtype-4 receptor small molecule agonist, 3 (RY764), is reported. Its in vivo characterization revealed mechanism-based food intake reduction and erectile activity augmentation in rodents.
Asunto(s)
Compuestos Aza/farmacología , Ingestión de Alimentos/efectos de los fármacos , Erección Peniana/efectos de los fármacos , Piperazinas/farmacología , Piperidinas/farmacología , Receptor de Melanocortina Tipo 4/agonistas , Animales , Compuestos Aza/síntesis química , Humanos , Masculino , Microsomas Hepáticos/metabolismo , Piperazinas/química , Piperidinas/síntesis química , Unión Proteica , Quinuclidinas/química , Ratas , Ratas Sprague-Dawley , Roedores , Relación Estructura-Actividad , Factores de TiempoRESUMEN
We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. The 5- and 6-alkylated piperazine compounds exhibit low bioactivation potential as measured by covalent binding in microsome preparations.
Asunto(s)
Piperazinas/química , Piperazinas/farmacología , Receptor de Melanocortina Tipo 4/agonistas , Humanos , Piperazinas/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , Relación Estructura-ActividadRESUMEN
We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. Further in vivo development of lead agonist, MB243, is disclosed.
Asunto(s)
Piperazinas/farmacología , Piperidinas/farmacología , Receptor de Melanocortina Tipo 4/agonistas , Animales , Perros , Disfunción Eréctil/tratamiento farmacológico , Haplorrinos , Masculino , Ratones , Obesidad/tratamiento farmacológico , Piperazinas/química , Piperazinas/farmacocinética , Piperazinas/uso terapéutico , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/uso terapéutico , RatasRESUMEN
In the process of drug discovery, brain and plasma measurements of new chemical entities in rodents are of interest, particularly when the target receptors are in the brain. Brain-to-plasma ratios (B/P) obtained from a rodent pharmacokinetic assay are useful in helping determine which compounds are brain penetrant. The study reported here was performed to determine whether whole-body saline perfusion for complete blood removal was required to accurately measure brain tissue compound concentrations. Diazepam was used as a positive control since it is highly brain penetrant. Compound A was used as a negative control since it had known poor brain penetration. After intravenous dosing with either diazepam or compound A, rats were anesthetized and blood was collected, then the brain was removed following no perfusion or whole-body perfusion with saline. The analytes described (compound A, diazepam, and the internal standard) were recovered from plasma or brain homogenate by use of protein precipitation, and were subsequently analyzed by use of liquid chromatography/tandem mass spectrometry (LC/MS/MS). The B/P values determined by use of LC-MS were not significantly different in perfused vs. non-perfused rats (P > or = 0.05). This approach (whole brain collected from non-perfused male rats) is an attractive alternative over brain penetration studies of perfused rats, since it has markedly reduced the technical time and potential for pain and distress required for generating B/P data due to elimination of the requirement for anesthesia and surgical preparation of animals.