RESUMEN
Mcl-1, a pro-survival member of the Bcl-2 protein family, is an attractive target for cancer therapy. We have recently identified the natural product marinopyrrole A (maritoclax) as a novel small molecule Mcl-1 inhibitor. Here, we describe the structure-activity relationship study of pyoluteorin derivatives based on maritoclax. To date, we synthesized over 30 derivatives of maritoclax and evaluated their inhibitory actions and cytotoxicity toward Mcl-1-dependent cell lines. As a result, several functional groups were identified in the pyoluteorin motif that significantly potentiate biological activity. A number of such derivatives, KS04 and KS18, interacted with Mcl-1 in a conserved fashion according to NMR spectroscopy and molecular modeling. KS04 and KS18 induced apoptosis selectively in Mcl-1-dependent but not Bcl-2-dependent K562 cells through selective Mcl-1 down-regulation, and synergistically enhanced apoptosis in combination with ABT-737. Moreover, the intraperitoneal administration of KS18 (10 mg/kg/d) and ABT-737 (20 mg/kg/d) significantly suppressed the growth of ABT-737-resistant HL-60 xenografts in nude mice without apparent toxicity. Overall, we identified the pharmacophore of pyoluteorin derivatives that act as potent and promising Mcl-1 antagonists against Mcl-1-dependent hematological cancers.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Hematológicas/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fenoles/farmacología , Pirroles/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Femenino , Neoplasias Hematológicas/patología , Humanos , Modelos Moleculares , Conformación Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Fenoles/química , Fenoles/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis/efectos de los fármacos , Pirroles/química , Pirroles/metabolismo , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Lipid matrix particles (LMP) may be used as better carriers for poorly water-soluble drugs than liquid lipid carriers because of reduced drug mobilization in the formulations. However, the digestion process of solid lipid particles and their effect on the absorption of poorly water-soluble drugs are not fully understood. This study aimed at investigating the effect of particle size of LMP on drug release in vitro as well as absorption in vivo in order to get a better understanding on the effect of degradation of lipid particles on drug solubilisation and absorption. Fenofibrate, a model poorly water-soluble drug, was incorporated into LMP in this study using probe ultrasound sonication. The resultant LMP were characterised in terms of particle size, size distribution, zeta potential, entrapment efficiency, in vitro lipolysis and in vivo absorption in rat model. LMP of three different particle sizes i.e. approximately 100 nm, 400 nm, and 10 µm (microparticles) were produced with high entrapment efficiencies. The in vitro lipolysis study showed that the recovery of fenofibrate in the aqueous phase for 100 nm and 400 nm LMP was significantly higher (p<0.05) than that of microparticles after 30 min of lipolysis, suggesting that nano-sized LMP were digested to a larger extent due to greater specific surface area. The 100 nm LMP showed faster initial digestion followed by 400 nm LMP and microparticles. The area under the plasma concentration-time curve (AUC) following oral administration of 100 nm LMP was significantly higher (p<0.01) than that of microparticles and fenofibrate crystalline suspension (control). However, no significant difference was observed between the AUCs of 100 nm and 400 nm LMP. The same rank order on the in vivo absorption and the in vitro response was observed. The recovery (%) of fenofibrate partitioning into the aqueous phase during in vitro lipolysis and the AUC of plasma concentration-time curve of fenofibric acid was in the order of 100 nm LMP>microparticles>control. In summary, the present study demonstrated the particle size dependence of bioavailability of fenofibrate loaded LMP in rat model which correlates well with the in vitro drug release performed in the biorelevant medium.
Asunto(s)
Fenofibrato/análogos & derivados , Lípidos/química , Absorción , Animales , Área Bajo la Curva , Fenofibrato/química , Fenofibrato/farmacocinética , Lipólisis/fisiología , Masculino , Tamaño de la Partícula , Ratas , Ratas Sprague-Dawley , Solubilidad , Agua/químicaRESUMEN
A simple, reproducible and fast (4 min chromatogram) method of liquid chromatography in tandem with mass spectrometry (LC/MS-MS) was developed to determine simultaneously the plasma levels of albendazole (ABZ) and its metabolite albendazole sulfoxide (ABZOX) for pharmacokinetic and clinical analysis. Each plasma sample was extracted by solid phase extraction (SPE) using phenacetin as internal standard (IS). The extracted sample was eluted with a Zorbax XDB-CN column using an isocratic method. The mobile phase consisting of water with 1% acetic acid (40%, A) and MeOH (60%, B), was used at a flow rate of 1 mL/min. ABZ and ABZOX were detected and identified by mass spectrometry with electrospray ionization (ESI) in the positive ion and multiple-reaction monitoring (MRM) mode. The method was linear in the range of 5-1000 ng/mL for ABZ and 10-1500 ng/mL (full validation) or 10-5000 ng/mL (partial validation) for ABZOX, with 5 and 10 ng/mL lower limit of quantification (LLOQ) for ABZ and ABZOX, respectively. The tests of accuracy and precision, matrix effect, extraction recovery and stability of the samples for both ABZ and ABZOX did not deviate more than 20% for the LLOQ and no more than 15% for other quality controls (QCs), according to regulatory agencies.
Asunto(s)
Albendazol/análogos & derivados , Albendazol/sangre , Extracción en Fase Sólida , Albendazol/química , Cromatografía Líquida de Alta Presión , Humanos , Espectrometría de Masas en TándemRESUMEN
PURPOSE: The quality of bioanalytical data is dependent upon selective, sensitive, and reproducible analytical methods. With evolving technologies available, bioanalytical scientists must assess which is most appropriate for their molecule through proper method validation. For an early stage PEGylated insulin program, the characteristics of four platforms, ELISA, ECL, Gyrolab, and LC-MS/MS, were evaluated using fit-for-purpose method development and validation, while also evaluating costs. METHOD: Methods selected for validation required acceptable performance based on satisfaction of a priori criteria prior to proceeding to subsequent stages of validation. LBA pre-validation included reagent selection, evaluation of matrix interference, and range determination. LC-MS/MS pre-validation included selection of a signature peptide; optimization of sample preparation, HPLC, and LC-MS/MS conditions; and calibration range determination. Pre-study validation tested accuracy and precision (mean bias criteria±30%; precision≤30%). Pharmacokinetic (PK) parameters were estimated for an in vivo study with WinNonlin noncompartmental analysis. Statistics were performed with JMP using ANOVA and Tukey-Kramer post hoc analysis. A cost analysis was performed for a 200-sample PK study using the methods from this study. RESULTS: All platforms, except Gyrolab, were taken through validation. However, a typical Gyrolab method was included for the cost analysis. Ranges for the ELISA, ECLA, and LC-MS/MS were 8.52-75, 2.09-125, and 100-1000 ng/mL, respectively, and accuracy and precision fell within a priori criteria. PK samples were analyzed in the 3 validated methods. PK profiles and parameters are similar for all methods, except LC-MS/MS, which differed at t=24h and with AUC0-24. Further investigation into this difference is warranted. The cost analysis identified the Gyrolab platform as the most expensive and ELISA as the least expensive, with method specific consumables attributing significantly to costs. CONCLUSIONS: ECLA had a larger dynamic range and sensitivity, allowing accurate assessment of PK parameters. Although this method was more expensive than the ELISA, it was the most appropriate for the early stage PEGylated insulin program. While this case study is specific to PEGylated human insulin, it highlights the importance of evaluating and selecting the most appropriate platform for bioanalysis during drug development.