Tricyclic Indazoles-A Novel Class of Selective Estrogen Receptor Degrader Antagonists.

Herein, we report the identification and synthesis of a series of tricyclic indazoles as a novel class of selective estrogen receptor degrader antagonists. Replacement of a phenol, present in our previously reported tetrahydroisoquinoline scaffold, with an indazole group led to the removal of a reactive metabolite signal in an in vitro glutathione trapping assay. Further optimization, guided by X-ray crystal structures and NMR conformational work, varied the alkyl side chain and pendant aryl group and resulted in compounds with low turnover in human hepatocytes and enhanced chemical stability. Compound 9 was profiled as a representative of the series in terms of pharmacology and demonstrated the desired estrogen receptor α degrader-antagonist profile and demonstrated activity in a xenograft model of breast cancer.

Combined Inhibition of mTOR and CDK4/6 Is Required for Optimal Blockade of E2F Function and Long-term Growth Inhibition in Estrogen Receptor-positive Breast Cancer.

The cyclin dependent kinase (CDK)-retinoblastoma (RB)-E2F pathway plays a critical role in the control of cell cycle in estrogen receptor-positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor-resistant cell lines reactivate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR.

Discovery of a Potent, Selective, Orally Bioavailable, and Efficacious Novel 2-(Pyrazol-4-ylamino)-pyrimidine Inhibitor of the Insulin-like Growth Factor-1 Receptor (IGF-1R).

Optimization of cellular lipophilic ligand efficiency (LLE) in a series of 2-anilino-pyrimidine IGF-1R kinase inhibitors led to the identification of novel 2-(pyrazol-4-ylamino)-pyrimidines with improved physicochemical properties. Replacement of the imidazo[1,2-a]pyridine group of the previously reported inhibitor 3 with the related pyrazolo[1,5-a]pyridine improved IGF-1R cellular potency. Substitution of the amino-pyrazole group was key to obtaining excellent kinase selectivity and pharmacokinetic parameters suitable for oral dosing, which led to the discovery of (2R)-1-[4-(4-{[5-chloro-4-(pyrazolo[1,5-a]pyridin-3-yl)-2-pyrimidinyl]amino}-3,5-dimethyl-1H-pyrazol-1-yl)-1-piperidinyl]-2-hydroxy-1-propanone (AZD9362, 28), a novel, efficacious inhibitor of IGF-1R.

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models.

Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307-18. ©2016 AACR.

Prevention of fostamatinib-induced blood pressure elevation by antihypertensive agents.

Fostamatinib is a tyrosine kinase inhibitor with activity against spleen tyrosine kinase which has completed clinical trials for patients with rheumatoid arthritis. In clinical studies fostamatinib treatment was associated with a small elevation of systemic arterial blood pressure (BP), a similar finding to that seen with other kinase inhibitors, especially those that inhibit VEGFR2 signaling. We have investigated the link between fostamatinib-induced blood pressure elevation and plasma levels of the fostamatinib-active metabolite R940406 in conscious rats and found the time course of the BP effect correlated closely with changes in R940406 plasma concentration, indicating a direct pharmacological relationship. Free plasma levels of R940406 produced in these studies (up to 346 nmol/L) span the clinically observed mean peak free plasma concentration of 49 nmol/L. We have demonstrated that the blood pressure elevation induced by fostamatinib dosing can be successfully controlled by a variety of methods, notably simple drug withdrawal or codosing with a range of standard antihypertensive agents such as atenolol, captopril, and nifedipine. These findings support potential methods of maintaining patient safety while on fostamatinib therapy. Furthermore, we have demonstrated, using nifedipine as an example agent, that this blood pressure control was not achieved by reduction in plasma exposure of R940406, suggesting that potential benefits from the pharmacology of the investigational drug can be maintained while blood pressure control is managed by use of standard comedications.

In vitro pharmacological profiling of R406 identifies molecular targets underlying the clinical effects of fostamatinib.

Off-target pharmacology may contribute to both adverse and beneficial effects of a new drug. In vitro pharmacological profiling is often applied early in drug discovery; there are fewer reports addressing the relevance of broad profiles to clinical adverse effects. Here, we have characterized the pharmacological profile of the active metabolite of fostamatinib, R406, linking an understanding of drug selectivity to the increase in blood pressure observed in clinical studies. R406 was profiled in a broad range of in vitro assays to generate a comprehensive pharmacological profile and key targets were further investigated using functional and cellular assay systems. A combination of traditional literature searches and text-mining approaches established potential mechanistic links between the profile of R406 and clinical side effects. R406 was selective outside the kinase domain, with only antagonist activity at the adenosine A3 receptor in the range relevant to clinical effects. R406 was less selective in the kinase domain, having activity at many protein kinases at therapeutically relevant concentrations when tested in multiple in vitro systems. Systematic literature analyses identified KDR as the probable target underlying the blood pressure increase observed in patients. While the in vitro pharmacological profile of R406 suggests a lack of selectivity among kinases, a combination of classical searching and text-mining approaches rationalized the complex profile establishing linkage between off-target pharmacology and clinically observed effects. These results demonstrate the utility of in vitro pharmacological profiling for a compound in late-stage clinical development.

AZD2014, an Inhibitor of mTORC1 and mTORC2, Is Highly Effective in ER+ Breast Cancer When Administered Using Intermittent or Continuous Schedules.

mTOR is an atypical serine threonine kinase involved in regulating major cellular functions, such as nutrients sensing, growth, and proliferation. mTOR is part of the multiprotein complexes mTORC1 and mTORC2, which have been shown to play critical yet functionally distinct roles in the regulation of cellular processes. Current clinical mTOR inhibitors only inhibit the mTORC1 complex and are derivatives of the macrolide rapamycin (rapalogs). Encouraging effects have been observed with rapalogs in estrogen receptor-positive (ER(+)) breast cancer patients in combination with endocrine therapy, such as aromatase inhibitors. AZD2014 is a small-molecule ATP competitive inhibitor of mTOR that inhibits both mTORC1 and mTORC2 complexes and has a greater inhibitory function against mTORC1 than the clinically approved rapalogs. Here, we demonstrate that AZD2014 has broad antiproliferative effects across multiple cell lines, including ER(+) breast models with acquired resistance to hormonal therapy and cell lines with acquired resistance to rapalogs. In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials.

Optimization of a Novel Binding Motif to (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic Acid (AZD9496), a Potent and Orally Bioavailable Selective Estrogen Receptor Downregulator and Antagonist.

The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.

Targeting non-small cell lung cancer cells by dual inhibition of the insulin receptor and the insulin-like growth factor-1 receptor.

Phase III trials of the anti-insulin-like growth factor-1 receptor (IGF1R) antibody figitumumab in non-small cell lung cancer (NSCLC) patients have been discontinued owing to lack of survival benefit. We investigated whether inhibition of the highly homologous insulin receptor (IR) in addition to the IGF1R would be more effective than inhibition of the IGF1R alone at preventing the proliferation of NSCLC cells. Signalling through IGF1R and IR in the NSCLC cell lines A549 and Hcc193 was stimulated by a combination of IGF1, IGF2 and insulin. It was inhibited by antibodies that block ligand binding, αIR3 (IGF1R) and IR47-9 (IR), and by the ATP-competitive small molecule tyrosine kinase inhibitors AZ12253801 and NVPAWD742 which inhibit both IGF1R and IR tyrosine kinases. The effect of inhibitors was determined by an anchorage-independent proliferation assay and by analysis of Akt phosphorylation. In Hcc193 cells the reduction in cell proliferation and Akt phosphorylation due to anti-IGF1R antibody was enhanced by antibody-mediated inhibition of the IR whereas in A549 cells, with a relatively low IR:IGF1R expression ratio, it was not. In each cell line proliferation and Akt phosphorylation were more effectively inhibited by AZ12253801 and NVPAWD742 than by combined αIR3 and IR47-9. When the IGF1R alone is inhibited, unencumbered signalling through the IR can contribute to continued NSCLC cell proliferation. We conclude that small molecule inhibitors targeting both the IR and IGF1R more effectively reduce NSCLC cell proliferation in a manner independent of the IR:IGF1R expression ratio, providing a therapeutic rationale for the treatment of this disease.

Novel imidazo[1,2-a]pyridine based inhibitors of the IGF-1 receptor tyrosine kinase: optimization of the aniline.

Following the discovery of imidazopyridine 1 as a potent IGF-1R tyrosine kinase inhibitor, the aniline part has been modified with the aim to optimize the properties of this series. The structure-activity relationships against IGF-1R kinase activity as well as inhibition of the hERG ion channel are discussed.

Novel thienopyrimidine and thiazolopyrimidine kinase inhibitors with activity against Tie-2 in vitro and in vivo.

The SAR and improvement in potency against Tie2 of novel thienopyrimidine and thiazolopyrimidine kinase inhibitors are reported. The crystal structure of one of these compounds bound to the Tie-2 kinase domain is consistent with the SAR. These compounds have moderate potency in cellular assays of Tie-2 inhibition, good physical properties, DMPK, and show evidence of in vivo inhibition of Tie-2.

The use and refinement of rodent models in anti-cancer drug discovery: a review.

This review describes the changing use of tumour models in rodents (predominantly mice) as employed over the last four decades in anti-cancer drug discovery, and the refinements in the experimental methods used. Such models are required to examine the complexities of cancer biology (e.g. tumour angiogenesis, invasion and metastasis, host immunity factors) and the impact of potential therapies (e.g. drug pharmacokinetics, pharmacodynamics and therapeutic index), and they have produced efficacious human therapeutics. Animal welfare considerations have driven refinements to animal models of cancer over time, with the most dramatic refinements being facilitated by the move away from inherently cytotoxic therapeutic approaches toward targeted inhibitors of disease-related processes. Whereas, four decades ago, the impact of disease burden was used as an endpoint in the absence of defined mechanistic parameters, acute pharmacodynamic measures are now increasingly used to minimise the adverse effects of disease and experimental procedures in a given animal. The changes in the UK guidelines on the use of rodents in preclinical cancer testing are also used as an illustration of the progressive refinement in tumour models and drug testing.

Preclinical anticancer activity of the potent, oral Src inhibitor AZD0530.

AZD0530, an orally available Src inhibitor, demonstrated potent antimigratory and anti-invasive effects in vitro, and inhibited metastasis in a murine model of bladder cancer. Antiproliferative activity of AZD0530 in vitro varied between cell lines (IC(50) 0.2 ->10µM). AZD0530 inhibited tumor growth in 4/10 xenograft models tested and dynamically inhibited in vivo phosphorylation of Src substrates paxillin and FAK in both growth-inhibition-resistant and -sensitive xenografts. The activity of AZD0530 in NBT-II bladder cancer cells in vitro was consistent with inhibition of cell migration and stabilization of cell-cell adhesion. These data suggest a dominant anti-invasive pharmacology for AZD0530 that may limit tumor progression in a range of cancers. AZD0530 is currently in Phase II clinical trials.

Inhibition of vascular endothelial growth factor-a signaling induces hypertension: examining the effect of cediranib (recentin; AZD2171) treatment on blood pressure in rat and the use of concomitant antihypertensive therapy.

PURPOSE: Inhibition of vascular endothelial growth factor-A (VEGF) signaling is a key therapeutic approach in oncology given the role of VEGF in angiogenesis and vascular permeability in solid tumors. Clinical trials examining VEGF signaling inhibitors commonly report hypertension. We examined the effect of cediranib, a highly potent VEGF signaling inhibitor, on the blood pressure of rats and the ability of standard antihypertensive agents to modulate the consequences of VEGF signaling inhibition. EXPERIMENTAL DESIGN: The ability of cediranib to induce hypertensive changes and the effect of giving antihypertensive therapy were investigated in conscious, unrestrained telemetered rats. Two antihypertensive agents were studied: captopril, an angiotensin-converting enzyme inhibitor, and nifedipine, a dihydropyridine calcium channel blocker. The antitumor activity of cediranib, alone and in combination with nifedipine, was also evaluated in a LoVo human colorectal tumor xenograft model in nude rats. All treatments were given orally. RESULTS: Administration of 0.1 to 1.5 mg/kg/d of cediranib for 4 consecutive days induced a relatively mild hypertensive effect, elevating diastolic blood pressure by 10 to 14 mmHg. Dosing 3 mg/kg/d cediranib for 4 days induced a marked hypertension of 35 to 50 mmHg. Captopril (30 mg/kg, qd) was effective at lowering a 10 mmHg increase in blood pressure but not a 35 to 50 mmHg increase. However, the latter was rapidly reversed by administration of nifedipine (10 mg/kg, bd). Coadministration of nifedipine did not negatively affect the antitumor activity of cediranib (1.5 mg/kg/d). CONCLUSIONS: Hypertension is a direct consequence of inhibiting VEGF signaling but can be controlled with appropriately selected, standard antihypertensive medication.

Effect of pretreatment with atenolol and nifedipine on ZD6126-induced cardiac toxicity in rats.

Antivascular agents that act by destabilizing microtubules, such as ZD6126 (N-acetylcolchinol-O-phosphate), are associated with adverse cardiovascular effects, including transient hypertension, cardiac ischemia, myocardial infarction, and increases in circulating levels of markers of cardiac damage (e.g., troponins). We investigated mechanisms underlying these effects of ZD6126 in rats by continuously monitoring their heart rate and blood pressure and by assessing heart histopathology and plasma troponin T levels. ZD6126 induced acute transient hemodynamic changes (hypertension and delayed tachycardia), which were associated with statistically significant increases in circulating troponin T levels (median level 3 hours after treatment with vehicle or 12.5 mg/kg ZD6126: <9 pg/mL and 563 pg/mL, respectively; P <.001 [two-sided Wilcoxon rank sum test]) and in the incidence of left ventricular myocardial fiber necrosis (incidence 24 hours after treatment with vehicle or 12.5 mg/kg ZD6126: 0/10 rats and 9/10 rats, respectively; P <.001 [two-sided Wilcoxon rank sum test]). Pretreatment of rats with atenolol and nifedipine ameliorated the acute hemodynamic changes and prevented ZD6126-induced increases in both troponin T and myocardial necrosis but did not prevent ZD6126-induced tumor necrosis in an Hras5 tumor xenograft model in nude rats. Our findings suggest that ZD6126-induced acute hemodynamic changes are a prerequisite for cardiac damage in rats.

N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5- (tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor.

Src family kinases (SFKs) are nonreceptor tyrosine kinases that are reported to be critical for cancer progression. We report here a novel subseries of C-5-substituted anilinoquinazolines that display high affinity and specificity for the tyrosine kinase domain of the c-Src and Abl enzymes. These compounds exhibit high selectivity for SFKs over a panel of recombinant protein kinases, excellent pharmacokinetics, and in vivo activity following oral dosing. N-(5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530) inhibits c-Src and Abl enzymes at low nanomolar concentrations and is highly selective over a range of kinases. AZD0530 displays excellent pharmacokinetic parameters in animal preclinically and in man (t(1/2) = 40 h). AZD0530 is a potent inhibitor of tumor growth in a c-Src-transfected 3T3-fibroblast xenograft model in vivo and led to a significant increase in survival in a highly aggressive, orthotopic model of human pancreatic cancer when dosed orally once daily. AZD0530 is currently undergoing clinical evaluation in man.

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.

Discovery of a new class of anilinoquinazoline inhibitors with high affinity and specificity for the tyrosine kinase domain of c-Src.

Deregulated activity of the nonreceptor tyrosine kinase c-Src is believed to result in signal transduction, cytoskeletal and adhesion changes, ultimately promoting a tumor-invasive phenotype. We report here the discovery of a new class of anilinoquinazoline inhibitors with high affinity and specificity for the tyrosine kinase domain of the c-Src enzyme. Special attention was directed toward finding inhibitors selective against KDR tyrosine kinase in order to ensure that the in vivo profile of a specific Src inhibitor could be determined. The 4-aminobenzodioxole quinazoline series gave compounds with excellent potency and selectivity. The most interesting compounds were evaluated in vivo and displayed good pharmacokinetics following oral dosing. Compounds such as the aminobenzodioxoles were shown to be potent inhibitors of tumor growth in a c-Src-transformed 3T3 xenograft model in vivo, resulting in more than 90% growth inhibition at doses as low as 6 mg/kg po once daily. Src tyrosine kinase inhibitors such as these may provide a novel therapeutic modality for targeting cancer invasion and metastasis.

ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration.

ZD6474 [N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine]is a potent, p.o. active, low molecular weight inhibitor of kinase insert domain-containing receptor [KDR/vascular endothelial growth factor receptor (VEGFR) 2] tyrosine kinase activity (IC(50) = 40 nM). This compound has some additional activity versus the tyrosine kinase activity of fms-like tyrosine kinase 4 (VEGFR3;IC(50) = 110 nM) and epidermal growth factor receptor (EGFR/HER1; IC(50) = 500 nM) and yet demonstrates selectivity against a range of other tyrosine and serine-threonine kinases. The activity of ZD6474 versus KDR tyrosine kinase translates into potent inhibition of vascular endothelial growth factor-A (VEGF)-stimulated endothelial cell (human umbilical vein endothelial cell) proliferation in vitro (IC(50) = 60 nM). Selective inhibition of VEGF signaling has been demonstrated in vivo in a growth factor-induced hypotension model in anesthetized rat: administration of ZD6474 (2.5 mg/kg, i.v.) reversed a hypotensive change induced by VEGF (by 63%) but did not significantly affect that induced by basic fibroblast growth factor. Once-daily oral administration of ZD6474 to growing rats for 14 days produced a dose-dependent increase in the femoro-tibial epiphyseal growth plate zone of hypertrophy, which is consistent with inhibition of VEGF signaling and angiogenesis in vivo. Administration of 50 mg/kg/day ZD6474 (once-daily, p.o.) to athymic mice with intradermally implanted A549 tumor cells also inhibited tumor-induced neovascularization significantly (63% inhibition after 5 days; P < 0.001). Oral administration of ZD6474 to athymic mice bearing established (0.15-0.47 cm(3)), histologically distinct (lung, prostate, breast, ovarian, colon, or vulval) human tumor xenografts or after implantation of aggressive syngeneic rodent tumors (lung, melanoma) in immunocompetent mice, produced a dose-dependent inhibition of tumor growth in all cases. Statistically significant antitumor activity was evident in each model with at least 25 mg/kg ZD6474 once daily (P < 0.05, one-tailed t test). Histological analysis of Calu-6 tumors treated with 50 mg/kg/day ZD6474 for 24 days showed a significant reduction (>70%) in CD31 (endothelial cell) staining in nonnecrotic regions. ZD6474 also restrained growth of much larger (0.9 cm(3) volume) Calu-6 lung tumor xenografts and induced profound regression in established PC-3 prostate tumors of 1.4 cm(3) volume. ZD6474 is currently in Phase I clinical development as a once-daily oral therapy in patients with advanced cancer.