Stephen R. Wedge

Guidelines for the welfare and use of animals in cancer research

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

AZD2171: A Highly Potent, Orally Bioavailable, Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor for the Treatment of Cancer

Inhibition of vascular endothelial growth factor-A (VEGF) signaling is a promising therapeutic approach that aims to stabilize the progression of solid malignancies by abrogating tumor-induced angiogenesis. This may be accomplished by inhibiting the kinase activity of VEGF receptor-2 (KDR), which has a key role in mediating VEGF-induced responses. The novel indole-ether quinazoline AZD2171 is a highly potent (IC50 < 1 nmol/L) ATP-competitive inhibitor of recombinant KDR tyrosine kinase in vitro. Concordant with this activity, in human umbilical vein endothelial cells, AZD2171 inhibited VEGF-stimulated proliferation and KDR phosphorylation with IC50 values of 0.4 and 0.5 nmol/L, respectively. In a fibroblast/endothelial cell coculture model of vessel sprouting, AZD2171 also reduced vessel area, length, and branching at subnanomolar concentrations. Once-daily oral administration of AZD2171 ablated experimental (VEGF-induced) angiogenesis in vivo and inhibited endochondral ossification in bone or corpora luteal development in ovary; physiologic processes that are highly dependent upon neovascularization. The growth of established human tumor xenografts (colon, lung, prostate, breast, and ovary) in athymic mice was inhibited dose-dependently by AZD2171, with chronic administration of 1.5 mg per kg per day producing statistically significant inhibition in all models. A histologic analysis of Calu-6 lung tumors treated with AZD2171 revealed a reduction in microvessel density within 52 hours that became progressively greater with the duration of treatment. These changes are indicative of vascular regression within tumors. Collectively, the data obtained with AZD2171 are consistent with potent inhibition of VEGF signaling, angiogenesis, neovascular survival, and tumor growth. AZD2171 is being developed clinically as a once-daily oral therapy for the treatment of cancer.

Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials

*Department of Medical Oncology, Chafing Cross Hospital, Fulham Palace Road, London W6 8RF, U.K. t Cancer Research Campaign Experimental Cancer Chemotherapy Research Group, Cancer Research Laboratories, Department of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 ZRD, U.K.

AZD1152, a Selective Inhibitor of Aurora B Kinase, Inhibits Human Tumor Xenograft Growth by Inducing Apoptosis

Drugs Dynamics Institute, College of Pharmacy, University of Texas at Austin, Austin, TX 78712-7074, U.S.A.

ZD1839 ('Iressa'), a specific oral epidermal growth factor receptor-tyrosine kinase inhibitor, potentiates radiotherapy in a human colorectal cancer xenograft model.

Purpose: In the current study, we examined the in vivo effects of AZD1152, a novel and specific inhibitor of Aurora kinase activity (with selectivity for Aurora B). Experimental Design: The pharmacodynamic effects and efficacy of AZD1152 were determined in a panel of human tumor xenograft models. AZD1152 was dosed via several parenteral (s.c. osmotic mini-pump, i.p., and i.v.) routes. Results: AZD1152 potently inhibited the growth of human colon, lung, and hematologic tumor xenografts (mean tumor growth inhibition range, 55% to ≥100%; P < 0.05) in immunodeficient mice. Detailed pharmacodynamic analysis in colorectal SW620 tumor-bearing athymic rats treated i.v. with AZD1152 revealed a temporal sequence of phenotypic events in tumors: transient suppression of histone H3 phosphorylation followed by accumulation of 4N DNA in cells (2.4-fold higher compared with controls) and then an increased proportion of polyploid cells (>4N DNA, 2.3-fold higher compared with controls). Histologic analysis showed aberrant cell division that was concurrent with an increase in apoptosis in AZD1152-treated tumors. Bone marrow analyses revealed transient myelosuppression with the drug that was fully reversible following cessation of AZD1152 treatment. Conclusions: These data suggest that selective targeting of Aurora B kinase may be a promising therapeutic approach for the treatment of a range of malignancies. In addition to the suppression of histone H3 phosphorylation, determination of tumor cell polyploidy and apoptosis may be useful biomarkers for this class of therapeutic agent. AZD1152 is currently in phase I trials.

Preclinical evaluation of polymer-bound doxorubicin

The effect of ZD1839 (‘Iressa’), a specific inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor, on the radiation response of human tumour cells (LoVo colorectal carcinoma) was evaluated in vitro and in vivo. ZD1839 (0.5 μM, incubated days 1–5) significantly increased the anti-proliferative effect of fractionated radiation treatment (2 Gy day−1, days 1–3) on LoVo cells grown in vitro (P=0.002). ZD1839 combined with either single or fractionated radiotherapy in mice bearing LoVo tumour xenografts, also produced a highly significant increase in tumour growth inhibition (P⩽0.001) when compared to treatment with either modality alone. The radio-potentiating effect of ZD1839 was more apparent when radiation was administered in a fractionated protocol. This phenomenon may be attributed to an anti proliferative effect of ZD1839 on tumour cell re-population between radiotherapy fractions. These data suggest radiotherapy with adjuvant ZD1839 could enhance treatment response. Clinical investigation of ZD1839 in combination with radiotherapy is therefore warranted.

ZD6474, a Potent Inhibitor of Vascular Endothelial Growth Factor Signaling, Combined With Radiotherapy: Schedule-Dependent Enhancement of Antitumor Activity

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers (Mw approximately 20 000) were synthesised to contain doxorubicin (DOX) ( approximately 7 wt% ) attached to the polymer backbone via biodegradable Gly-Phe-Leu-Gly peptidyl spacers (polymer 1) and optionally in addition (polymer 2) the carbohydrate moiety galactosamine (approximately 4 mol%) to facilitate liver targeting. Both polymeric DOX conjugates were degraded by isolated rat lysosomal enzymes in vitro and within rat liver following intravenous (i.v.) administration, to liberate free DOX in a time-dependent fashion. Total release of DOX occurred over 24–48 h. When administered either intraperitoneally (i.p.) at doses 2.5–50 mg DOX/kg (triplicate doses) or i.v. (single dose, 13–100 mg DOX/kg) to mice bearing L1210 leukaemia inoculated i.p., both polymers 1 and 2 showed good antitumour activity. The highest T/C seen for conjugates after i.p. treatment was > 762 compared with a T/C of 214 seen for free Dox. At 30 mg/kg given i.p. polymer 2 produced a large number of long-term survivors 720. A variety of dosing schedules were used to test the activity of polymer-DOX against a number of solid tumour models; M5076, P388, B16 melanoma, Walker sarcoma and the xenograft LS174T. In each case treatment with polymer-bound DOX produced an increase in survival time greater than seen with free DOX and experiments with P388 and Walker sarcoma showed remarkable tumour regression (in some cases tumours completely disappeared). B16 melanoma and M5076 were used as a liver metastatic model in particular to study the antitumour activity of the liver-targeted polymer 2. Although polymer 2 did decrease tumour burden attributable to liver metastases it was not significantly better than polymer 1 or free DOX. In these studies HPMA copolymer-DOX conjugates expressed a wide range of antitumour activities, but their overall mechanism of action is still unclear. Certainly these macromolecular produgs are subject to intracellular lysosomal activation, they have the ability to concentrate drug in solid tumours, and with incorporation of targeting residues can promote organ-specific or tumour-specific uptake. In addition covalent conjugation markedly reduces all aspects of DOX-associated toxicity.

IN VITRO EVALUATION OF TEMOZOLOMIDE COMBINED WITH X-IRRADIATION

Purpose: Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis and acts as a radiation survival factor for endothelial cells. ZD6474 (N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine) is a potent VEGF receptor 2 (KDR) tyrosine kinase inhibitor (TKI) that has additional activity versus the epidermal growth factor receptor. This study was designed to determine the efficacy of combining ZD6474 and radiotherapy in vivo. Experimental Design: The Calu-6 (non–small-cell lung cancer) tumor model was selected because it was found to be unresponsive to treatment with a selective epidermal growth factor receptor TKI but responds significantly to treatment with selective VEGF receptor TKIs. Tumor-bearing mice received either vehicle or ZD6474 (50 mg/kg, by mouth, once daily) for the duration of the experiment, with or without radiotherapy (3 × 2 Gy, days 1–3). Two combination schedules were examined: (a) ZD6474 given before each dose of radiation (concurrent schedule); and (b) ZD6474 given 30 minutes after the last dose of radiotherapy (sequential schedule). Results: The growth delay induced using the concurrent schedule was greater than that induced by ZD6474 or radiation treatment alone (22 ± 1 versus 9 ± 1 and 17 ± 2 days, respectively; P = 0.03 versus radiation alone). When administered sequentially, the growth delay was markedly enhanced (36 ± 1 days; P < 0.001 versus radiation alone or the concurrent schedule). Intravenous administration of Hoechst 33342 showed a trend toward reduced tumor perfusion after ZD6474 treatment, and a pairwise comparison (versus control) was significant after three doses of ZD6474 (P = 0.05 by one-tailed t test). Thus, impaired reoxygenation between fractions in the concurrent protocol may be the causal basis for the schedule dependency of the radiopotentiation observed. Conclusions: ZD6474 may be a successful adjuvant to clinical radiotherapy, and scheduling of the treatments could be important to ensure optimal efficacy.

ZD6474 - a novel inhibitor of VEGFR and EGFR tyrosine kinase activity

The In vitro cytotoxicity of 8-carbamoyl-3-methylimldazo[ 5,1-d]-1,2,3,5-tetrazine-4(3 H)-one (temozolomide) with concurrent X-lrradiation was examined in a human glioblastoma cell line (U373MG) as a potential radio-chemotherapeutlc treatment for malignant glioma. The combination was also examined in a human colorectal adenocarcinoma (Mawi) which had 100-fold greater O6-alkylguanine-DNA alkyltransferase (AGT) activity, a DNA-repair protein which confers resistance to temozolomide. A comparison of IC50 values indicated U373MG to be over 32-fold more sensitive to temozolomide than Mawi, but slightly more resistant to X-irradlation (p < 0.035; unpaired two-tailed Mest). Temozolomide and X-irradiatlon proved largely additive in U373MG by Isobologram analysis (50% iso-effect) and the addition of 10 µ M temozolomide to 1-2 Gy of X-irradiation increased cell kill by 2.5- to 3.0-fold. However, the combination was antagonistic in Mawi: an effect attributed to AGT induction by X-irradiation as the antagonism was removed by coincubation with the AGT inhibitor O6-benzylguanine (O6-BG 1 µ M; 24 h). O6-BG did not affect the radiation doseresponse curve, but significantly increased temozolomide cytotoxicity (p < 0.015). In conclusion, the combination of temozolomide with radiation is at best additive, but could nonetheless be of considerable therapeutic benefit in glioma, particularly if administered for prolonged periods. If AGT induction compromises the efficacy of this therapy, it may be circumvented with an appropriate inhibitor such as O6-BG.

Use of dynamic contrast-enhanced MRI to evaluate acute treatment with ZD6474, a VEGF signalling inhibitor, in PC-3 prostate tumours

Angiogenesis is crucial for maintaining the supply of oxygen and nutrients required to support solid tumour growth. Inhibitors of tumour blood vessel formation are therefore being sought, in particular, inhibitors of vascular endothelial growth factor-A (VEGF)-signalling, which has a pivotal role in stimulating neovascular growth and survival. ZD6474 is an orally bioavailable inhibitor of VEGF receptor-2 tyrosine kinase activity that in preclinical studies has been shown to inhibit both VEGF-induced signalling in endothelial cells and tumour-induced angiogenesis. Consistent with inhibition of angiogenesis, once-daily oral dosing of ZD6474 produced significant broad-spectrum antitumour activity in a panel of histologically diverse human tumour xenografts. In addition to its antiangiogenic properties, ZD6474 also has activity against the epidermal growth factor receptor (EGFR) tyrosine kinase, which could impart a direct inhibitory effect on tumour cell growth and survival. This may be particularly relevant in tumours with a dependency upon EGFR signalling, for example in certain tumours harbouring activating mutations in EGFR. RET kinase has also been identified as a third target for ZD6474. This review summarises preclinical studies with this unique agent and considers its future direction in cancer treatment.