RESUMEN
As part of the formulation of a cell-based pharmaceutical product, cells were harvested from mice and incubated in a cocktail containing cell culture media and high levels of trifluoroacetic acid (TFA). The cells were washed with a phosphate-buffered saline solution to remove residual cell culture media and other reagents before the cells were infused back into the mice from which they originated. Because of the potentially toxic nature of the TFA, the cells were washed multiple times and the final wash was monitored for residual TFA in order to demonstrate the efficient removal of the reagent before the cell product could be reintroduced into the test animal. This report describes the method that was developed incorporating anion-exchange chromatography with suppressed conductivity detection for the analysis of residual TFA (down to 50 ng/ml) in the presence of high concentrations of phosphate and chloride interferences. The ultimate sensitivity of the method was improved by selectively removing halide anions using a silver cartridge before sample analysis. The method proved to be rugged and reproducible enough to be validated and used to monitor residual TFA levels in cell washes in support of an acute toxicological study. Results demonstrating the method's sensitivity, selectivity, precision and linearity were reported.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Fosfatos/química , Cloruro de Sodio/química , Ácido Trifluoroacético/análisis , Tampones (Química) , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
As a highly lipophilic drug, propofol may interact with lipophilic domains in addition to its likely primary site of action on the gamma-aminobutyrateA) (GABA(A)) receptor. Likely candidates for such interaction are the G protein-coupled membrane receptors for lipid intercellular mediators. The phospholipid lysophosphatidate (LP) has attracted attention as such a signaling molecule. It has a variety of biological actions, including vasoconstriction. We therefore studied the interaction between propofol and the LP receptor. Intracellular Ca2+ release in response to LP was assessed by measuring C1- flux through Ca(2+)-activated C1- channels in Xenopus oocytes. The average charge movement in response to LP 10(-7)M was 2.0 +/- 0.2 microCoulombs. Propofol in Intralipid (0.01%) dose-dependently inhibited LP signaling (50% inhibitory concentration [IC50] 5.38 microM). Propofol 28 microM inhibited LP signaling by 81%. Intralipid (0.01%) was without effect. To ascertain that intracellular signaling pathways and the Ca(2+)-activated C1- channel were not affected by propofol, we tested the effects of propofol (5.6 microM) on currents induced by methylcholine (10(-7)M) in oocytes expressing the m1 muscarinic acetylcholine receptor. No inhibition was observed. As both receptors share the same intracellular signaling pathway, we conclude that clinically relevant concentrations of propofol most likely inhibit the LP receptor or its G protein. Inhibition of LP signaling may explain some of propofol's vasodilating actions.
Asunto(s)
Anestésicos Intravenosos/farmacología , Lisofosfolípidos/antagonistas & inhibidores , Propofol/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Anestésicos Intravenosos/administración & dosificación , Animales , Calcio/farmacocinética , Canales de Cloruro/efectos de los fármacos , Colina/administración & dosificación , Colina/análogos & derivados , Colina/farmacología , Relación Dosis-Respuesta a Droga , Electroquímica , Emulsiones Grasas Intravenosas/administración & dosificación , Emulsiones Grasas Intravenosas/farmacología , Femenino , Antagonistas de Receptores de GABA-A , Proteínas de Unión al GTP/antagonistas & inhibidores , Lisofosfolípidos/farmacología , Antagonistas Muscarínicos/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Propofol/administración & dosificación , Receptores Muscarínicos/efectos de los fármacos , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , Xenopus laevisRESUMEN
Fourteen cats with advanced mammary adenocarcinoma were treated with doxorubicin HCl and cyclophosphamide. All cats had inoperable or recurrent disease, and 9 cats had metastasis to the thorax. Eleven of the cats were available for long-term evaluation; 3 had complete response to chemotherapy, with survival times of 180, 283, and 344 days; 2 had partial response, with survival times of 45 and 149 days; 2 had stabilization of disease, with survival times of 170 and 182 days; and 4 had no response, with survival times of 4, 47, 67, and 106 days.