RESUMO
A platform that incorporates computational library design, parallel solution-phase synthesis, continuous flow hydrogenation, and automated high throughput purification and reformatting technologies was applied to the production of a 120-member library of 1-aryl-4-aminopiperidine analogues for drug discovery screening. The application described herein demonstrates the advantages of computational library design coupled with a flexible, modular approach to library synthesis. The enabling technologies described can be readily adopted by the traditional medicinal chemist without extensive training and lengthy process development times.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Simulação de Dinâmica Molecular , Piperidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Algoritmos , Animais , Linhagem Celular , Permeabilidade da Membrana Celular/fisiologia , Humanos , Microssomos/química , Microssomos/metabolismo , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Ratos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Solubilidade , SuínosRESUMO
The goal of this work was to obtain full pharmacokinetic profiles from individual mice with the use of an automated blood sampling system and dried blood spot (DBS) technique. AMG 517, a potent and selective vanilloid receptor (VR1) antagonist, was dosed to mice (n=3) intravenously and blood samples were collected using the automated blood sampling system with the "no blood waste" method. The collected blood samples were a mixture of 25 µL blood and 50 µL of heparinized saline solution. Two 15 µL aliquots were manually spotted onto a DBS card and dried at room temperature for at least 2h before being stored in zip bags with desiccant. The remaining samples (45 µL) were stored at -70°C until analysis. Both the DBS and the whole blood samples (diluted with saline (1:2, v/v)) were extracted and analyzed by liquid chromatography-tandem mass spectrometry. The overall extraction recovery of the analyte from the dried blood spots was determined to be about 90%. The pharmacokinetic parameters calculated using the whole blood or the DBS concentration data were comparable, and were obtained from only 3 mice, whereas conventional sampling and analysis would have required up to 27 mice to achieve the same result. The analyte was shown to be stable in the diluted whole blood (blood:saline 1:2) at room temperature for at least 4h and in the DBS for at least 34 days when stored at room temperature. These results indicated that the automated blood sampling system and DBS collection are promising techniques to obtain full pharmacokinetic profiles from individual mice and reduce the use of animals.
Assuntos
Benzotiazóis/sangue , Coleta de Amostras Sanguíneas/métodos , Cromatografia Líquida/métodos , Teste em Amostras de Sangue Seco/métodos , Pirimidinas/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Benzotiazóis/administração & dosagem , Benzotiazóis/farmacocinética , Calibragem , Técnicas de Laboratório Clínico/métodos , Monitoramento de Medicamentos/métodos , Masculino , Camundongos , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A LC-MS/MS method was developed for the determination of a p38 MAP kinase inhibitor (Compound I) and its amide hydrolyzed metabolite (M7) in Cynomolgus monkey plasma over the concentration range of 1.00-1000ng/mL. Stable isotope labeled compounds (d(3)-Compound I and d(3)-M7) were used as internal standards (IS). Samples were prepared using protein precipitation in the 96-well format with a 30µL plasma sample volume. Chromatographic separation was performed with reversed-phase liquid chromatography on a Varian Monochrom C(18) (100mm×2.00mm, 5µm) analytical column. The mobile phases were 5mM ammonium formate in acetonitrile/water (95/5, v/v) pH 7.0 and 5mM ammonium formate in acetonitrile/water (5/95, v/v) pH 7.0. Gradient elution, at a flow rate of 550µL/min, was used to separate Compound I and M7. Positive atmospheric pressure chemical ionization was utilized with detection by multiple reaction monitoring (MRM). Total run time was about 3.2min. This method was validated following the current Food and Drug Administration (FDA) guidance for bioanalytical method validation. The intra- and inter-day precision (% CV) and accuracy (% bias) at all concentrations tested were below 15% for both analytes. The mean recoveries for Compound I, M7, d(3)-Compound I, and d(3)-M7 were 106%, 107%, 108% and 105%, respectively. The method was successfully applied to support a GLP toxicokinetic study in Cynomolgus monkeys after oral administration of Compound I. A total of 48 samples (â¼12.5% of the total number of samples) were selected for incurred sample reanalysis (ISR). The % difference between the reassay concentrations and the original concentrations were all less than 20% of their mean values and met the acceptance criteria for ISR.
Assuntos
Amidas/química , Inibidores Enzimáticos/análise , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Acetonitrilas/química , Administração Oral , Animais , Área Sob a Curva , Calibragem , Técnicas de Química Analítica , Cromatografia Líquida/métodos , Inibidores Enzimáticos/sangue , Feminino , Formiatos/química , Íons , Macaca fascicularis , Masculino , Controle de Qualidade , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Água/químicaRESUMO
INTRODUCTION: Regulatory guidelines recommend the use of conscious, unrestrained animals for comprehensive cardiovascular safety assessment of a new therapeutic agent. Cardiovascular safety pharmacology studies normally use internal telemetry (surgical implants) in free-moving animals to monitor key ECG endpoints, like the QTc interval, but this technical approach is highly resource intensive. In toxicology studies, ECG recording is also typically performed under chemical or physical restraint, which has a number of disadvantages, e.g., anesthesia confounds, handling stress and limited data collection. External telemetry for ECG recording has the potential to overcome many of these restraint limitations, with the benefit of being a surgically non-invasive method. To evaluate this method, we used two jacket systems: Data Sciences International (DSI) JET and Integrated Telemetry Systems (ITS) PhysioJacket in implanted beagle dogs. METHODS: Heart rate and cardiac intervals were monitored continuously for 22-24 h following oral administration of vehicle (water) or 1 mg/kg E-4031. Data obtained from each jacket system was compared with implant-derived data in the same animal. RESULTS: Significant increases in QT/QTcV (25-30 ms) were noted following treatment with 1 mg/kg E-4031 in both external jacket systems and with implanted telemetry. Throughout the recording periods, the normal variations in heart rate and ECG intervals observed in conscious dogs as detected with the jacket systems, mirrored the changes observed via implant telemetry. DISCUSSION: The overall findings from this study support the use of external telemetry technology as a viable alternative to implants. The data demonstrated that jackets were sufficiently sensitive to detect QT/QTcV changes following E-4031 administration, that were comparable to those derived from implants. As such, this method is an invaluable tool for obtaining high quality ECG data from repeat-dose toxicology studies.