Correlation of antiangiogenic and antitumor efficacy of N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA), an orally-active, selective matrix metalloproteinase inhibitor

The antiangiogenic activity and antitumor efficacy of a newly developed matrix metalloproteinase (MMP) inhibitor were examined. N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA) potently inhibits MMP-2, -9, and -14, but not MMP-1, -3, or -7. In contrast, (-)BPHA, an enantiomer of BPHA, was inactive against all MMPs tested. Daily oral administration of 200 mg/kg BPHA, but not (-)BPHA in mice resulted in potent inhibition of tumor-induced angiogenesis, primary tumor growth, and liver metastasis. The growth inhibition activity of BPHA was 48% and 45% in a B16-BL6 melanoma and F2 hemangio-endothelioma model, respectively. BPHA also showed 42% inhibition of the liver metastasis of C-1H human colon carcinoma cells. These results indicate that selective MMP inhibition is correlated with antiangiogenic and antitumor efficacy and that the selective MMP inhibitor BPHA has therapeutic potential.     

Inhibition of gelatinolytic activity in tumor tissues by synthetic matrix metalloproteinase inhibitor: application of film in situ zymography.

Inhibition of gelatinolytic activity in implanted tumor tissues by oral administration of N-biphenyl sulfonyl-phenylalanine hydroxamic acid (BPHA), a selective matrix metalloproteinase (MMP) inhibitor, was demonstrated by means of film in situ zymography (FIZ). Active-MMP-2 but not pro-MMP-2 showed gelatinolytic activity in FIZ, whereas both forms of MMP-2 were found to be active in conventional zymography. A mixture of either tissue inhibitors of metalloproteinase-2 or BPHA with active-MMP-2 resulted in inhibition of gelatinolytic activity in FIZ but not in zymography. Thus, FIZ, but not zymography, could detect net MMP activity in tumor tissues. When a specimen from Ma44 human lung cancer xenograft was subjected to FIZ, gelatinolytic activity was markedly detected with precise localization in the tumor tissues. The gelatinolytic activity detected in Ma44 tumor tissues was found to be mainly derived from MMPs because the gelatin-degrading activity was inhibited by pretreatment of the tumor specimen with MMP inhibitors. Oral administration of BPHA but not (-)BPHA, an enantiomer of BPHA lacking MMP inhibitory activity, successfully inhibited the MMP activity localized in Ma44 tumor tissues in both a dose-dependent and time-dependent manner. The data presented in this report showed for the first time that oral administration of synthetic MMP inhibitor could inhibit the net activity of MMP activity in tumor tissues, suggesting the usefulness of the FIZ technique for determining the effective dose of MMP inhibitor in clinical studies.     

An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been implicated in the progression of several human cancers and are attractive therapeutic targets. PF-2341066 was identified as a potent, orally bioavailable, ATP-competitive small-molecule inhibitor of the catalytic activity of c-Met kinase. PF-2341066 was selective for c-Met (and anaplastic lymphoma kinase) compared with a panel of >120 diverse tyrosine and serine-threonine kinases. PF-2341066 potently inhibited c-Met phosphorylation and c-Met-dependent proliferation, migration, or invasion of human tumor cells in vitro (IC(50) values, 5-20 nmol/L). In addition, PF-2341066 potently inhibited HGF-stimulated endothelial cell survival or invasion and serum-stimulated tubulogenesis in vitro, suggesting that this agent also exhibits antiangiogenic properties. PF-2341066 showed efficacy at well-tolerated doses, including marked cytoreductive antitumor activity, in several tumor models that expressed activated c-Met. The antitumor efficacy of PF-2341066 was dose dependent and showed a strong correlation to inhibition of c-Met phosphorylation in vivo. Near-maximal inhibition of c-Met activity for the full dosing interval was necessary to maximize the efficacy of PF-2341066. Additional mechanism-of-action studies showed dose-dependent inhibition of c-Met-dependent signal transduction, tumor cell proliferation (Ki67), induction of apoptosis (caspase-3), and reduction of microvessel density (CD31). These results indicated that the antitumor activity of PF-2341066 may be mediated by direct effects on tumor cell growth or survival as well as antiangiogenic mechanisms. Collectively, these results show the therapeutic potential of targeting c-Met with selective small-molecule inhibitors for the treatment of human cancers.     

Antitumor effect of a new selective matrix metalloproteinase inhibitor, MMI-166, on experimental pancreatic cancer

The antitumor effect of a new matrix metalloproteinase inhibitor, MMI-166, which is a selective inhibitor of MMP-2 and -9, was examined in the hamster pancreatic cancer cell line PGHAM-1. In vitro, MMI-166 inhibited the gelatinase activity of MMP-2 and -9 derived from PGHAM-1 cells, and dose-dependently inhibited invasion of PGHAM-1 through a basement membrane-like barrier. MMI-166 showed no apparent cytotoxicity to PGHAM-1 cells in culture at 100 microgram/ml. MMI-166 (200 mg/kg) or vehicle were administered orally, once daily, from day 1 until day 21 after implantation in the orthotopic implantation model of PGHAM-1. MMI-166 significantly reduced the incidence of liver surface metastasis from 66.7% to 20.0%, and it reduced the number of liver surface metastases per animal from 6.17 to 2.00, but this reduction was not significant. MMI-166 significantly reduced the volume of pancreatic tumors from 718.3 +/- 220.0 mm(3) to 222.8 +/- 85.4 mm(3). Treatment of pancreatic tumors with MMI-166 caused a significant reduction in the microvessel density from 37.90 +/- 10.18/mm(2) to 16.16 +/- 3.15/mm(2) and a significant increase in apoptotic index from 1.75 +/- 0.41% to 3.96 +/- 0.38%, but there was no significant difference between tumor cell proliferation in the MMI-166-group and the control group. These results showed that selective MMP inhibition could limit both cancer spread and angiogenesis in pancreatic cancer. The selective MMP-2 and -9 inhibitor MMI-166 may be of therapeutic use in the treatment of pancreatic cancer because of its inhibitory effect on invasion and angiogenesis.     

The matrix metalloproteinase inhibitor prinomastat enhances photodynamic therapy responsiveness in a mouse tumor model

Photodynamic therapy (PDT) clinical results are promising; however, tumor recurrences can occur and, therefore, methods for improving treatment efficacy are needed. PDT elicits direct tumor cell death and microvascular injury as well as expression of angiogenic, inflammatory, and prosurvival molecules. Preclinical studies combining antiangiogenic drugs or cyclooxygenase-2 inhibitors with PDT show improved treatment responsiveness (A. Ferrario et al., Cancer Res 2000;60:4066-9; A. Ferrario et al., Cancer Res 2002;62:3956-61). In the present study, we evaluated the role of Photofrin-mediated PDT in eliciting expression of matrix metalloproteinases (MMPs) and modulators of MMP activity. We also examined the efficacy of a synthetic MMP inhibitor, Prinomastat, to enhance tumoricidal activity after PDT, using a mouse mammary tumor model. Immunoblot analysis of extracts from PDT-treated tumors demonstrated strong expression of MMPs and extracellular MMP inducer along with a concomitant decrease in expression of tissue inhibitor of metalloproteinase-1. Gelatin zymography and enzyme activity assays performed on protein extracts from treated tumors confirmed the induction of both latent and enzymatically active forms of MMP-9. Immunohistochemical analysis indicated that infiltrating inflammatory cells and endothelial cells were primary sources of MMP-9 expression after PDT, whereas negligible expression was observed in tumor cells. Administration of Prinomastat significantly improved PDT-mediated tumor response (P = 0.02) without affecting normal skin photosensitization. Our results indicate that PDT induces MMPs and that the adjunctive use of an MMP inhibitor can improve PDT tumor responsiveness.     

Prevention of liver metastasis of human colon cancer by selective matrix metalloproteinase inhibitor MMI-166.

MMI-166 is a selective inhibitor of matrix metalloproteinase (MMP)-2 and MMP-9. Mice implanted a human colon cancer orthotopically received 200 mg/kg of MMI-166 orally for 5 weeks. Gelatin zymography demonstrated that the administration of MMI-166 remarkably decreased the active MMP-2 expression. Histological examination revealed that MMI-166 showed prominent effect on reduction of the invasive feature of the cancer cells and showed inhibitory effect on tumor vasculature, resulting in the significant decrease of microvessel density of the implanted tumor and liver metastasis compared with the control group. Conclusively, MMI-166 is a potent antiangiogenic oral agent for a human colon cancer.     

Antimetastatic activity of a novel mechanism-based gelatinase inhibitor

Matrix metalloproteinases (MMPs), and in particular gelatinases (MMP-2 and MMP-9), play a key role in cancer progression. However, clinical trials in which MMP inhibitors were tested in cancer patients have been disappointing. Whereas many reasons have been postulated to explain the failure of the clinical trials, lack of inhibitor selectivity was a major limitation. Thus, despite the consensus opinion that MMP-mediated proteolysis is essential for cancer progression and that certain MMPs represent important targets for intervention, effective and selective inhibition of those MMPs remains a major challenge in drug development. We previously reported the first mechanism-based MMP inhibitor, designated SB-3CT, which is a selective gelatinase inhibitor. Here we report that SB-3CT (5-50 mg/kg/d) is a potent inhibitor of liver metastasis and increases survival in an aggressive mouse model of T-cell lymphoma. This study shows that mechanism-based inhibition of gelatinases represents a novel approach to inhibitor design that promises to be a successful anticancer therapy.     

Endostatin inhibits endothelial and tumor cellular invasion by blocking the activation and catalytic activity of matrix metalloproteinase

Here we report the inhibition of cellular invasion by a recombinant mouse endostatin and the possible mechanism of the inhibition. Endostatin significantly reduced endothelial as well as tumor cellular invasion into the reconstituted basement membrane in vitro. Gelatin zymographic analysis revealed that the activation of promatrix metalloproteinase-2 (proMMP-2) that was secreted from endothelial cells was blocked upon endostatin treatment. Studies with recombinant MMPs confirmed that endostatin inhibited proMMP-2 activation, mediated by both membrane-type 1 MMP and 4-aminophenylmercuric acetate. Furthermore, enzymatic assays using a peptide substrate demonstrated that endostatin inhibited the catalytic activities of both MMP-2 and membrane-type 1 MMP. Finally, coimmunoprecipitation experiments revealed that endostatin formed a stable complex with proMMP-2. These novel findings would, at least in part, explain the mechanism of the potent antiangiogenic and antitumor activities of endostatin.     

A novel matrix metalloproteinase inhibitor,FYK-1388 suppresses tumor growth,metastasis and angiogenesis by human fibrosarcoma cell line

The matrix metalloproteinases (MMPs) are likely to contribute to tumor cell invasion, metastasis and angiogenesis. Several MMP inhibitors have been developed, recently and their anti-tumor efficacy is being evaluated in clinical trials. FYK-1388 is a novel broad MMP inhibitor which blocks the activity of MMP-1, -2, -3, -7, -9, -13 and -14 (MT-MMP-1). It is especially effective against MMP-2 and -9 more so than other MMP inhibitors such as Marimastat, Ro 32-3555 and D-2163. Here, we investigated the anti-tumor efficacy of FYK-1388 using the human fibrosarcoma cell line HT-1080. These cells produced MMP-2 and -9, which FYK-1388 inhibited at a dose of 10(-8) M. FYK-1388 at 0.2 mg/mouse/day significantly suppressed tumor growth when given by s.c. injection for 22 days, experimental lung metastasis after 5 days s.c. injection and also suppressed tumor-induced angiogenesis in the dorsal air sac assay after 7 days s.c. injection. In the MTT assay, FYK-1388 had no effect on the in vitro growth of HT-1080 cells. These results suggest that FYK-1388 possesses anti-tumor efficacy as a result of inhibiting angiogenesis through the suppression of MMP-2 and -9 activity.     

A phase I pharmacokinetic and pharmacodynamic study of s-3304, a novel matrix metalloproteinase inhibitor, in patients with advanced and refractory solid tumors.

PURPOSE: Matrix metalloproteinases (MMP) play a fundamental role in cancer development and progression. S-3304 is a potent, orally active, noncytotoxic inhibitor of MMPs, primarily MMP-2 and MMP-9, that prolongs survival in mice xenografts and is well tolerated in healthy volunteers. EXPERIMENTAL DESIGN: The aims of this phase I clinical trial were to determine the maximum tolerated dose, dose-limiting toxicities, pharmacokinetic profile, and intratumoral MMP inhibitory activity of single-agent S-3304 in advanced and refractory solid tumors. MMP activity was determined by film in situ zymography (FIZ). Patients had tumor biopsies before and after S-3304 administration and were also evaluated for response and survival. RESULTS: Four dose levels were explored [DL1-DL4 or 800, 1,600, 2,400, and 3200 mg twice daily (BID), respectively], and 32 patients were enrolled. Toxicities were mostly gastrointestinal. The maximum tolerated dose was not reached, but dose escalations beyond DL4 were impractical (number of capsules needed). S-3304 steady-state concentrations were reached by day 8, and day 1 mean C(max) and AUC(0-8) increases were less than dose proportional. After S-3304 administration, 17 of 18 patients experienced inhibition of MMP activity by FIZ. Strong mean inhibition of MMP activity was observed in DL1 to DL3. The negative mean inhibitory activity calculated for DL4 was due to one patient with a 397% MMP activity increase. CONCLUSION: S-3304 is safe, well tolerated, and achieves plasma concentrations above those required to inhibit MMP-2 and MMP-9. Its intratumoral MMP inhibitory activity has been shown using FIZ, which is useful as a biomarker with this and other MMP inhibitors.     

Neovastat, a naturally occurring multifunctional antiangiogenic drug, in phase III clinical trials.

Recent studies have indicated that bone marrow angiogenesis is increased in multiple myeloma, suggesting that treatment with an antiangiogenic agent might be useful. Among the new antiangiogenic drugs in development, Neovastat (AE-941; Aeterna Laboratories, Quebec City, Canada) can be classified as a naturally occurring multifunctional antiangiogenic agent. It has a marked inhibitory effect on the formation of blood vessels in the chicken embryo vascularization assay (EVT) and endothelial cell proliferation. Furthermore, in vivo experiments showed that oral administration of Neovastat blocks the formation of blood vessels in Matrigel implants containing basic fibroblast growth factor (bFGF). The antiangiogenic activity of Neovastat was found to be associated with two mechanisms of action. In addition to the inhibition of the matrix metalloproteinase activities (MMP-2, MMP-9, and MMP-12), Neovastat inhibits vascular endothelial growth factor (VEGF) binding to endothelial cells, VEGF-dependent tyrosine phosphorylation, and VEGF-induced vascular permeability in mice. Neovastat was also found to have a significant antitumor activity. Oral administration of Neovastat in mice with subcutaneous grafted breast cancer (DA3) cells showed a significant reduction in tumor volume. Neovastat also decreased the number of lung metastases in the Lewis lung carcinoma model. Interestingly, the effect of Neovastat was additive to cisplatin in this model. Furthermore, no treatment-related mortality or loss of body weight was observed. Also, toxicology studies in rats and monkeys demonstrate no dose-limiting toxicity or target organ damage after 1 year of chronic exposure, thus suggesting that Neovastat could be safely administered in humans. Four clinical studies have been conducted to establish the dosing, safety, and early efficacy of Neovastat administered orally. In the oncology field, 482 patients have received Neovastat, of which 146 with solid tumors were exposed to the drug for more than 6 months. Two phase III clinical trials are currently underway. A phase III double-blind placebo-controlled study is being conducted to evaluate the efficacy of Neovastat in addition to induction chemotherapy/radiotherapy combined modality treatment in patients with unresectable non-small cell lung cancer stage IIIA and IIIB. A second phase III randomized, double-blind placebo-controlled study evaluates the efficacy of Neovastat as a monotherapy in metastatic renal cell carcinoma patients who have progressed following a first-line immunotherapy. Neovastat efficacy is also being evaluated in a registration phase II trial in patients with early relapse or refractory multiple myeloma.     

Quercetin inhibition of tumor invasion via suppressing PKC delta/ERK/AP-1-dependent matrix metalloproteinase-9 activation in breast carcinoma cells

Quercetin (QUE; 3,5,7,3',4'-tetrahydroxyflavone) has been shown to possess several beneficial biological activities including antitumor, anti-inflammation and antioxidant properties; however, the effects of QUE in preventing invasion by breast carcinoma cells are still undefined. Increases in the protein, messenger RNA and enzyme activity levels of matrix metalloproteinase (MMP)-9 were observed in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells, and these were blocked by QUE, but not by quercitrin or rutin. A translocation of protein kinase C (PKC)delta from the cytosol to the membrane followed by activation of extracellular signal-regulated kinase (ERK) and c-Jun/activator protein-1 (AP-1) by TPA was demonstrated, and TPA-induced MMP-9 activation and migration were inhibited by the pan PKC inhibitor, GF109203X, the specific PKCdelta inhibitor, rottlerin, an ERK inhibitor (PD98059) and an AP-1 inhibitor (curcumin). Application of QUE significantly suppressed TPA-induced activation of the PKCdelta/ERK/AP-1-signaling cascade. To elucidate the importance of hydroxyl (OH) substitutions to QUE's inhibition of tumor migration, several structurally related flavones of QUE including 3',4'-diOH, 3',4'-diOCH(3), 3,5,7-triOH, 3,4',4'-triOH, 3,3',4'-triOCH(3), luteolin and fisetin were used. Results suggested that OH groups at both C3' and C4' play central roles in QUE's inhibition of TPA-induced MMP-9 activation and migration, and an additional OH at C3, C5 or C7 may increase the inhibitory potency of the 3',4'-diOH flavone against TPA-induced MMP-9 activity and migration. The antitumor invasion and migration effects of breast carcinoma cells induced by QUE with the structure-activity relationship analysis were identified.     

Inhibition of angiogenesis and metastasis in two murine models by the matrix metalloproteinase inhibitor, BMS-275291.

BMS-275291 is an p.o. bioavailable, sulfhydryl-based matrix metalloproteinase (MMP) inhibitor currently in clinical development for the treatment of cancer. This inhibitor was designed to potently inhibit MMP activities while minimally affecting those of other metalloproteases (e.g., sheddases) involved in the release of cell-associated molecules such as tumor necrosis factor-alpha, tumor necrosis factor-alpha receptor, interleukin-6 receptor, or L-selectin. In vitro, BMS-275291 is a potent inhibitor (nM) of the activities of MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14. BMS-275291 inhibits tumor growth in a B16BL6 model of experimental metastasis, and in this model, BMS-275291 treatment results in a dose-dependent reduction in the number of lung metastases compared with vehicle controls. BMS-275291 also inhibits angiogenesis in a murine angiogenesis model, where once daily treatment with BMS-275291 results in a dose-dependent inhibition of endothelial cell migration into s.c. implanted Matrigel plugs. Pharmacokinetic studies demonstrated that the plasma concentrations of parent BMS-275291 in mice exceeds the in vitro IC(50) values for MMP-1, MMP-2, MMP-7, MMP-9, and MMP-14 for at least 4 h after the administration of a therapeutic dose of BMS-275291. Taken together, these data demonstrate that BMS-275291 inhibits MMP activities that contribute to tumor metastasis and angiogenesis.     

Quantification of matrix metalloproteinase activity in plasma of patients enrolled in a BAY 12-9566 phase I study

The expression of matrix metalloproteinases (MMPs) is often associated with invasiveness or grade of tumours. Increased blood levels of MMP proteins, including MMP-1, MMP-2, MMP-3 and MMP-9 have been detected in various types of cancers. With the exception of one study, MMPs in serum and plasma have been determined using ELISA. In the present study we measured the activity of the MMPs found in human plasma samples using gelatin enzymography and fluorimetric degradation assays. We used plasma samples from healthy control subjects and cancer patients enrolled in a dose-finding study for the MMP inhibitor, BAY 12-9566, to assess the activity of MMPs found in plasma and screen for efficacy of the MMP inhibitor. BAY 12-9566 has inhibitory activity toward MMP-2, MMP-3 and MMP-9. Patients with advanced solid tumours were enrolled in our study and plasma was collected on day 1 before dosing and at steady-state of the drug on day 15. Our results show that BAY 12-9566 was effective in lowering the plasma gelatinolytic activity in the group of 29 patients when considering the data obtained from a fluorimetric gelatinase assay. The data obtained from gelatin enzymography, however, did not reach significance. The fluorimetric degradation assay could be a useful tool to screen plasma from cancer patients in other clinical trials assessing MMP inhibitors.     

Assay of matrix metalloproteases types 8 and 9 by ELISA in human breast cancer.

Results from model tumour systems suggest that either increased levels of certain metalloproteases (MMPs) or decreased levels of their inhibitors correlate with metastatic potential. In this study, levels of two MMPs, i.e. MMP-8 and -9, and their inhibitor tissue inhibitor of metalloprotease type 1 (TIMP-1) were measured by enzyme-linked immunosorbent assay in human breast tumours. Levels of MMP-8 and -9 correlated significantly with each other, but neither MMP correlated with urokinase plasminogen activator. Levels of both MMP-8 and -9 were also significantly related to levels of TIMP-1. In contrast, neither MMP correlated with plasminogen activator inhibitor. No relationship was found between MMP-8, MMP-9 or TIMP-1 and either tumour size or metastasis to axillary nodes. MMP-8 and -9 levels were inversely related to levels of oestrogen receptors. MMP-8 but not MMP-9 levels were also inversely correlated with progesterone receptor levels. It is concluded that the assay for MMP-8 and -9 described here will permit the evaluation of these proteases as prognostic markers in cancer.     

Therapy of head and neck squamous cell carcinoma with replicative adenovirus expressing tissue inhibitor of metalloproteinase-2 and chemoradiation

Recent studies have demonstrated the efficacy of targeted therapy combined with radiotherapy in head and neck squamous cell carcinoma (HNSCC). We hypothesized that a combination treatment including a replicating adenovirus armed with tissue inhibitor of metalloproteinase-2 (TIMP-2), radiation and Cisplatin will augment treatment response and reduce tumor growth in vivo of HNSCC xenografts. Both single-agent (TIMP-2 virus, radiation and Cisplatin) and the combination therapies were evaluated in vitro and in vivo. The efficacy of both single-agent and combination therapies in vivo was determined by monitoring tumor growth and immunohistochemistry. Treatment with replicative Ad-TIMP-2 virus and radiation decreased cell viability in vitro and resulted in an additional antiangiogenic response in vivo. Tumor response rates to treatment with replicative Ad-TIMP-2, radiation, Cisplatin or combination therapies ranged from limited inhibition of tumor growth of the single-agent therapy to a statistically significant additive antitumor response with the combination therapies. Replicative Ad-TIMP-2+radiation+Cisplatin in the SCC1 nude mice demonstrated the greatest response rates in tumor growth and angiogenesis. Combination of Ad-TIMP-2 gene therapy with radiation and the triple treatment group resulted in an augmented therapeutic response. This is the first report of the potential benefits of combining radiation and MMP inhibitor treatment.     

Antitumor efficacy of a novel polymer-peptide-drug conjugate in human tumor xenograft models

We have designed a new dextran-peptide-methotrexate conjugate to achieve tumor-targeted delivery of chemotherapeutics. The dextran carrier was selected to allow passive targeting and enhanced permeation and retention (EPR). The peptide linker has also been optimized to allow drug release in the presence of matrix-metalloproteinase-2 (MMP-2) and matrix-metalloproteinase-9 (MMP-9), 2 important tumor-associated enzymes. The new conjugate was assessed for its in vivo antitumor efficacy and systemic side effects. It was compared with free methotrexate (MTX) and a similar conjugate, differing by an MMP-insensitive linker, at equivalent intraperitoneal dosages. The MMP-sensitive conjugate demonstrated tolerable in vivo side effects and effective inhibition of in vivo tumor growth by 83% in each of the 2 separate tumor models that overexpress MMP (HT-1080 and U-87). The antiproliferative effect of the drug contributed to the inhibition of tumor growth. In contrast, free MTX resulted in no significant tumor reduction in the same models. Neither free MTX nor the conjugate caused any tumor inhibition in the mice bearing RT-112, a slower growing model that does not overexpress MMP. MMP-insensitive conjugates, though able to inhibit tumor growth, caused toxicity in the small intestine and bone marrow.     

In vivo target modulation and biological activity of CHIR-258, a multitargeted growth factor receptor kinase inhibitor, in colon cancer models.

PURPOSE: To evaluate the therapeutic and biological effects of CHIR-258, an orally bioavailable, potent inhibitor of class III-V receptor tyrosine kinases, in colon cancer models. EXPERIMENTAL DESIGN: The pharmacologic activity of CHIR-258 was characterized by monitoring target modulation as well as by evaluating the antitumor and antiangiogenic effects in human colon xenograft models. RESULTS: CHIR-258 inhibits vascular endothelial growth factor receptor 1/2, fibroblast growth factor receptor 1/3, and platelet-derived growth factor receptor beta (PDGFRbeta) and shows both antitumor and antiangiogenic activities in vivo. Treatment of KM12L4a human colon cancer cells with CHIR-258 resulted in a dose-dependent inhibition of vascular endothelial growth factor receptor 1 and PDGFRbeta phosphorylation and reduction of phosphorylated extracellular signal-regulated kinase (ERK) levels, indicating modulation of target receptors and downstream signaling. In vivo administration of CHIR-258 resulted in significant tumor growth inhibition and tumor regressions, including large, established tumors (500-1,000 mm(3)). Immunohistochemical analysis showed a reduction of phosphorylated PDGFRbeta and phosphorylated ERK in tumor cells after oral dosing with CHIR-258 compared with control tumors. These changes were accompanied by decreased tumor cell proliferation rate and reduced intratumoral microvessel density. CHIR-258 inhibited the phosphorylation of PDGFRbeta and ERK phosphorylation in tumors within 2 hours following dosing and the inhibitory activity was sustained for >24 hours. Significant antitumor activity was observed with intermittent dosing schedules, indicating a sustained biological activity. CONCLUSION: These studies provide evidence that biological activity of CHIR-258 in tumors correlates with efficacy and aids in the identification of potential biomarkers of this multitargeted receptor tyrosine kinase inhibitor. CHIR-258 exhibits properties that make it a promising candidate for clinical development in a variety of solid and hematologic malignancies.