Tumor volume was estimated by the formula, L W W, for prolate ellipsoids, and efficacy was determined as percentage inhibition compared with vehicletreated controls.Similarly, ZD did not inhibit the activity of the serinethreonine kinases raf, MEK, and ERK. Enzyme kinetic studies to determine the Sodium salicylate characteristics of ZD inhibition of EGFR TK and the ATP concentration was varied, showed that ZD is a competitive inhibitor with respect to ATP. Samples were electrophoresed and electroblotted on to polyvinylidene difluoride membranes.For immunodetection of phosphotyrosine residues, membranes were incubated with primary antibody for hbefore incubation with secondary antibody and single conjugated antibody. Expression of cfos mRNA in mice bearing A xenografts was inhibited by ZD in a dosedependent manner after days of treatment.Either fragments of tumor tissue under anesthesia in nude mice.Tumors were allowed to establish growth after implantation before treatment commenced.Other quinazoline RTK inhibitors similarly show a high degree of selectivity for their specific targets. Importantly, ZD has no effect on the activity of the serinethreonine kinases raf, MEK, and MAPK, which transmit the EGF proliferative signal downstream of EGFR.KB cells grow well in growth factordepleted medium, and addition of EGF increases KB cell growth rate in a reproducible manner.Cytotoxicity was not observed at ZD concentrations up to M.Drug Venlafaxine hydrochloride washout studies showed that this inhibition is sustained for at least hafter a h drug treatment.Other investigators also noted sustained inhibition of autophosphorylation in vitro by ZD after drug washout and attributed this to drug sequestration in cells. Other investigators have studied the effects of ZD, alone and in combination with other drugs or radiation, on tumor cell proliferation.The effect of ZD was cytostatic, but higher doses increased apoptotic cell death, and in combination with cytotoxic drugs, ZD markedly enhanced apoptotic cell death.The IC for tumor cell growth inhibition by ZD was not strongly influenced by the level of expression of EGFR. Preliminary data indicate that combination of ZD with ionizing radiation also has additive or synergistic effects in nonsmall cell lung cancer cell lines in vitro. This model identified the structural features of quinazolines necessary for in vivo antitumor activity and indicated the importance for in vivo activity of sustaining sufficient drug concentrations in the blood to inhibit EGFRTK activity throughout each h period after onceaday dosing. ZD was chosen as a candidate for development because it achieves high and sustained blood levels in vivo over a h period. ZD was particularly effective against A xenografts, a recognized model for the testing of biological effects on EGFR signaling. Longterm treatment completely suppressed A tumor growth, and withdrawal of drug treatment allowed some tumors to resume growth in the day followup period.The majority of these tumors resumed growth in the day period after withdrawal of drug treatment, supporting the importance of continuous drug treatment to maintain antitumor activity.No evidence for the development of drug resistance emerged during these studies with A tumors because no tumor regrew during longterm ZD treatment.Dosedependent tumor growth inhibition by ZD was also demonstrated in mice bearing xenografts of human lung tumors; additionally, antitumor activity was demonstrated in a breast model, but some tumors failed to respond to drug treatment.

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