The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901.

A novel series of benzhydroxamate esters derived from their precursor anthranilic acids have been prepared and have been identified as potent MEK inhibitors. 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide, CI-1040, was the first MEK inhibitor to demonstrate in vivo activity in preclinical animal models and subsequently became the first MEK inhibitor to enter clinical trial. CI-1040 suffered however from poor exposure due to its poor solubility and rapid clearance, and as a result, development of the compound was terminated. Optimization of the diphenylamine core and modification of the hydroxamate side chain for cell potency, solubility, and exposure with oral delivery resulted in the discovery of the clinical candidate N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide PD 0325901.

Magnetic resonance imaging determination of tumor grade and early response to temozolomide in a genetically engineered mouse model of glioma.

PURPOSE: The median survival for patients diagnosed with glioblastoma multiforme, the most common type of brain tumor, is less than 1 year. Animal glioma models that are more predictive of therapeutic response in human patients than traditional models and that are genetically and histologically accurate are an unmet need. The nestin tv-a (Ntv-a) genetically engineered mouse spontaneously develops glioma when infected with ALV-A expressing platelet-derived growth factor, resulting in autocrine platelet-derived growth factor signaling. EXPERIMENTAL DESIGN: In the Ntv-a genetically engineered mouse model, T2-weighted and T1-weighted, contrast-enhanced magnetic resonance images were correlated with histology, glioma grade (high or low), and survival. Magnetic resonance imaging (MRI) was therefore used to enroll mice with high-grade gliomas into a second study that tested efficacy of the current standard of care for glioma, temozolomide (100 mg/kg qdx5 i.p., n=13). RESULTS: The Ntv-a model generated a heterogeneous group of gliomas, some with high-grade growth rate and histologic characteristics and others with characteristics of lower-grade gliomas. We showed that MRI could be used to predict tumor grade and survival. Temozolomide treatment of high-grade tv-a gliomas provided a 14-day growth delay compared with vehicle controls. Diffusion MRI measurement of the apparent diffusion coefficient showed an early decrease in cellularity with temozolomide, similar to that observed in humans. CONCLUSIONS: The use of MRI in the Ntv-a model allows determination of glioma grade and survival prediction, distribution of mice with specific tumor types into preclinical trials, and efficacy determination both by tumor growth and early apparent diffusion coefficient response.

Dynamic imaging of emerging resistance during cancer therapy.

One of the greatest challenges in developing therapeutic regimens is the inability to rapidly and objectively assess tumor response due to treatment. Moreover, tumor response to therapeutic intervention in many cases is transient, and progressive alterations within the tumor may mask the effectiveness of an initially successful therapy. The ability to detect these changes as they occur would allow timely initiation of alternative approaches, maximizing therapeutic outcome. We investigated the ability of diffusion magnetic resonance imaging (MRI) to provide a sensitive measure of tumor response throughout the course of treatment, possibly identifying changes in sensitivity to the therapy. Orthotopic 9L gliomas were subjected to two separate therapeutic regimens, with one group receiving a single 5-day cycle (1omega) of low-dose 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and a second group receiving two cycles at the same dose, bisected with 2 days of rest (2omega). Apparent diffusion coefficient maps were acquired before and throughout treatment to observe changes in water mobility, and these observations were correlated to standard measures of therapeutic response and outcome. Our results showed that diffusion MRI was indeed able to detect the emergence of a drug-resistant tumor subpopulation subsequent to an initially successful cycle of BCNU therapy, leading to minimal gains from a second cycle. These diffusion MRI findings were highly correlated with tumor growth delay, animal survival, and ex vivo growth inhibition assays showing emerging resistance in excised tumors. Overall, this study highlights the ability of diffusion MRI to provide sensitive dynamic assessment of therapy-induced response, allowing early opportunities for optimization of therapeutic protocols.

Plasma vascular endothelial growth factor and interleukin-8 as biomarkers of antitumor efficacy of a prototypical erbB family tyrosine kinase inhibitor.

CI-1033 (N-[4-[N-(3-chloro-4-fluorophenyl)amino-7-[3-(4-morpholynyl)propoxy]quinazolin-6-yl]acrylamide, PD 0183805-mesylate salt) was identified as a potent, selective inhibitor of erbB family tyrosine kinases, which are overexpressed in a number of solid tumors and have been shown to be involved in tumor progression. Because objective response of clinical patients to erbB-targeted therapies like CI-1033 has been observed only in a subset of cancer patients that exhibit the intended molecular targets, much emphasis has been placed on the identification of biomarkers of antitumor efficacy. Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) were considered as potential biomarkers for CI-1033 due to ease of detection in patient plasma and showed roles in angiogenesis and cancer progression and positive regulation by the erbB receptor family. In the present studies, mice bearing established xenografts (A431 epidermoid carcinoma, H125 non-small cell lung carcinoma, SF767 glioblastoma, and MDA-MB-468 mammary carcinoma) were treated with efficacious and subefficacious doses of CI-1033, and plasma levels and xenograft gene expression of VEGF and IL-8 were evaluated. Oral administration of CI-1033 to tumor-bearing mice at efficacious doses resulted in markedly decreased levels of VEGF and/or IL-8 plasma levels and tumor mRNA levels relative to vehicle-treated control mice in xenograft models that exhibited evaluable levels of these markers. In contrast, subefficacious doses of CI-1033 did not significantly affect VEGF or IL-8 levels in any of the xenograft models. These studies indicate that plasma VEGF and IL-8 may have use as biomarkers of antitumor efficacy for epidermal growth factor receptor/erbB-targeted therapies such as CI-1033 and suggest that further clinical study of these markers in cancer patients are warranted.

Radiosensitization by pan ErbB inhibitor CI-1033 in vitro and in vivo.

PURPOSE: Overexpression of the ErbB family of receptor tyrosine kinases has been associated with uncontrolled growth of many tumor types and, therefore, presents a promising molecular target for cancer therapy. CI-1033 is a small molecule tyrosine kinase inhibitor that differs from other 4-anilinoquinazolines by being a pan ErbB (instead of epidermal growth factor receptor-specific) irreversible (instead of reversible) inhibitor. Therefore, we investigated the antitumor effect of CI-1033 alone and in combination with ionizing radiation in vitro and in vivo. EXPERIMENTAL DESIGN: We selected three human colon carcinoma cell-lines (LoVo, Caco-2, which express activated epidermal growth factor receptor and ErbB-2 family members, and SW620, which does not), and analyzed the effects of CI-1033 both in vitro and in vivo. For in vivo studies LoVo and Caco-2 cells were implanted s.c. in the flank of nude mice. After the tumor reached approximately 100 mm(3), treatment was initiated with 20 mg/kg of CI-1033 (orally once daily x 5 for 3 successive weeks), radiation treatment (a total of 30 Gy given in 2 Gy once daily x 5 for 3 successive weeks), or a combination of both CI-1033 and radiation treatment. RESULTS: We found that exposure of LoVo and Caco-2, but not SW620 cells, to CI-1033 in the range of 1-3 micro M could inhibit constitutive signaling by tyrosine kinases, arrest cell growth, inhibit cells in G(1), stimulate expression of p53, and induce apoptosis. The inhibition of cell growth by CI-1033 seemed to produce only minimal radiosensitization in LoVo and Caco-2 cells. In contrast, the combination of CI-1033 and radiation produced significant (P < 0.0005 and P = 0.0002, respectively) and prolonged suppression of tumor growth in both the tumor types when compared with either treatment alone. CONCLUSIONS: These findings suggest that CI-1033 can increase the effectiveness of radiation therapy. The extent of suppression of tyrosine kinase activity by CI-1033, rather than the amount of activity in untreated cells, seemed to be more closely associated with the efficacy of combination treatment.

Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts.

PD 0332991 is a highly specific inhibitor of cyclin-dependent kinase 4 (Cdk4) (IC50, 0.011 micromol/L) and Cdk6 (IC50, 0.016 micromol/L), having no activity against a panel of 36 additional protein kinases. It is a potent antiproliferative agent against retinoblastoma (Rb)-positive tumor cells in vitro, inducing an exclusive G1 arrest, with a concomitant reduction of phospho-Ser780/Ser795 on the Rb protein. Oral administration of PD 0332991 to mice bearing the Colo-205 human colon carcinoma produces marked tumor regression. Therapeutic doses of PD 0332991 cause elimination of phospho-Rb and the proliferative marker Ki-67 in tumor tissue and down-regulation of genes under the transcriptional control of E2F. The results indicate that inhibition of Cdk4/6 alone is sufficient to cause tumor regression and a net reduction in tumor burden in some tumors.

The influence of tumor size and environment on gene expression in commonly used human tumor lines.

BACKGROUND: The expression profiles of solid tumor models in rodents have been only minimally studied despite their extensive use to develop anticancer agents. We have applied RNA expression profiling using Affymetrix U95A GeneChips to address fundamental biological questions about human tumor lines. METHODS: To determine whether gene expression changed significantly as a tumor increased in size, we analyzed samples from two human colon carcinoma lines (Colo205 and HCT-116) at three different sizes (200 mg, 500 mg and 1000 mg). To investigate whether gene expression was influenced by the strain of mouse, tumor samples isolated from C.B-17 SCID and Nu/Nu mice were also compared. Finally, the gene expression differences between tissue culture and in vivo samples were investigated by comparing profiles from lines grown in both environments. RESULTS: Multidimensional scaling and analysis of variance demonstrated that the tumor lines were dramatically different from each other and that gene expression remained constant as the tumors increased in size. Statistical analysis revealed that 63 genes were differentially expressed due to the strain of mouse the tumor was grown in but the function of the encoded proteins did not link to any distinct biological pathways. Hierarchical clustering of tissue culture and xenograft samples demonstrated that for each individual tumor line, the in vivo and in vitro profiles were more similar to each other than any other profile. We identified 36 genes with a pattern of high expression in xenograft samples that encoded proteins involved in extracellular matrix, cell surface receptors and transcription factors. An additional 17 genes were identified with a pattern of high expression in tissue culture samples and encoded proteins involved in cell division, cell cycle and RNA production. CONCLUSIONS: The environment a tumor line is grown in can have a significant effect on gene expression but tumor size has little or no effect for subcutaneously grown solid tumors. Furthermore, an individual tumor line has an RNA expression pattern that clearly defines it from other lines even when grown in different environments. This could be used as a quality control tool for preclinical oncology studies.