In Vivo Antitumour Activity

Testing of drug candidate TK inhibitors for in vivo anti-tumour activity was carried out with KB cell-derived human squamous tumour xenografts grown in nude mice. Structural chemistry necessary for in vivo anti-tumour activity, and the importance of sustaining concentrations of drug in the blood sufficient to inhibit EGFR-TK activity throughout each 24-h period following once-daily, oral dosing, were defined using this model. Iressa was chosen as a drug development candidate because it achieves high and sustained blood levels, in vivo, over a 24-h period (Barker et al. 2001). The potential spectrum of anti-tumour activity for Iressa in patients was assessed in xenografts of major solid human tumours representing a wide range of tissues of origin (Wakeling et al. 2002). The anti-tumour efficacy of Iressa varied widely between tumours. Dose-dependent growth inhibition was noted for A431 (human vulval squamous), A549 (lung), Du145 (prostate), HCT15, HT29 and LoVo (colon), and KB (oral squamous) tumours (dosing regimen 3-200 mg/kg, po, once daily). Maximum anti-tumour efficacy ranged from tumour regression of A431, through tumour stasis in A549 and Du145 tumours, to no effect on P246, (broncho-epithelial), MKN45, (gastric) and AR42 J, (pancreatic) tumours. No correlation was apparent between xenograft response to Iressa and expression levels of EGFR in the cognate tumour cell lines.

In long-term treatment studies, Iressa provided complete control of tumour growth; when drug treatment was withdrawn after 4 months, most but not all tumours resumed growth, demonstrating that Iressa has a cytostatic effect. When drug treatment was delayed until tumours were well-established, Iressa caused complete regression of all A431 tumours. Drug withdrawal again allowed tumour regrowth. No evidence for the development of drug resistance emerged during these studies with A431 tumours, since no tumour regrew during Iressa treatment (Wakeling et al. 2002). To assess the pharmacodynamic action of Iressa treatment, A431 tumours were excised 6 h after the last of four daily doses of 0, 12.5, 50 and 200 mg/kg Iressa, or at 2, 4, 6, 24, 30 and 36 h after a single 50 mg/kg dose. Total RNA was extracted from the tumours to quantify c-fos mRNA by RT-PCR. C-fos expression was inhibited in a dose-dependent manner; at the anti-tumour ED50 dose (50 mg/kg), c-fos mRNA was reduced by 94% and was completely blocked (>99%) at the 200-mg/kg dose. In mice treated with a single 50-mg/kg po dose of Iressa, c-fos expression reached a nadir (5% versus control) at 6 h and did not fully recover until 36 h later. These studies were consistent with a cytostatic effect on tumour cell growth, and suggested that continuous drug treatment is required to maintain control of tumour growth.

Xenograft studies have demonstrated Iressa's potential clinical efficacy alone (Anderson et al. 2001; Moasser et al. 2001; Fujimara et al. 2002; Sewell et al. 2002; Matsuo et al. 2003), in combination with single agent chemotherapy (Ciardiello et al. 2000; Sirotnak et al. 2000), radiotherapy (Bianco el 2002; Huang et al. 2002; Williams et al. 2002) or hormone therapy (Sirotnak et al. 2002; Schiff et al. 2004), and in combination with other targeted treatments (Moulder et al. 2001;Tortora et al. 2003). Animal model studies have also demonstrated that Iressa may have utility in early disease, for example, in the adjuvant or chemopreventative treatment of breast cancer. Iressa inhibited the proliferation of xenografts of normal breast and of ductal carcinoma in situ (Chan et al. 2001, 2002), and suppressed the development of mammary tumours in mouse mammary tumour virus (MMTV)-c-erbB2 transgenic mice (Lu et al. 2003). Since no correlation has been established between response and expression of EGFR in any of these studies, it is clear that receptor expression does not indicate the degree to which any individual tumour is dependent for growth on the EGFR signalling pathway. Biomarkers that might predict drug response or resistance have yet to be defined. Some studies have implicated persistent activation of the AKT/PKB pathway in resistance to EGFR-TK inhibition. For example, loss of PTEN (Bianco et al. 2003), high AKT activity (Janmaat et al. 2003) and increased insulin-like growth factor (IGF)-1R signalling (Chakravarti et al. 2002) all confer decreased sensitivity to anti-EGFR treatments. Comparison of gene expression profiles in Iressa-sensitive and -resistant tumour cell lines or xenografts could provide markers of activity for evaluation in biopsies from drug treated patients (Zembutsu et al. 2003).

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