Pharmacokinetic and pharmacodynamic assessments of the dipeptidyl peptidase-4 inhibitor PHX1149: double-blind, placebo-controlled, single- and multiple-dose studies in healthy subjects.

BACKGROUND: PHX1149 is a dipeptidyl peptidase-4 (DPP4) inhibitor that is currently in clinical development for the treatment of type 2 diabetes mellitus. PHX1149 is a small (molecular weight = 241.16 Da), highly water-soluble (>2 g/mL), orally active molecule with a selectivity index of 15- to 319-fold relative to those of other members of the DPP family. The biochemical median inhibitory concentration of DPP4 is 2.5 nmol/L. OBJECTIVE: The aim of these 2 double-blind, randomized, placebo-controlled studies was to examine the pharmacokinetic (PK) parameters and pharmacodynamic (PD) properties and tolerability of single and multiple ascending doses of PHX1149 in healthy human subjects. METHODS: Healthy men and women aged 18 to 60 years were recruited to participate in a single- or a multiple-dose study in which sequential dose escalation paradigm was used. In the single-dose study, subjects were given a single oral dose of PHX1149 50 to 500 mg or placebo; in the multiple-dose study, subjects were given PHX1149 at doses from 50 to 400 mg or placebo QD for 13 days. There was no intrasubject dose escalation. Blood samples were collected from each subject at a series of time points ranging from 1 hour before to 24 hours after dosing on day 1 in the single- dose study and on days 1, 7, and 13 in the multiple-dose study. PK and PD analyses were performed in plasma samples to determine Cmax, Tmax, AUC0-t, AUC0-infinity, and DPP4 enzymatic activity. The drug accumulation index was also calculated for each dose of PHX1149 in the multiple-dose study. Adverse events (AEs) were monitored in both studies through physical examinations, including measurement of vital signs, and clinical laboratory testing. In both studies, electrocardiography was performed. RESULTS: The single- and multiple-dose studies enrolled 30 and 28 subjects, respectively, for a total enrollment in the 2 studies of 58 healthy adult subjects. The distribution of male and female subjects was 14 (47%) and 16 (53%), respectively, in the single- dose study and 16 (57%) and 12 (43%) in the multiple- dose study. In the single-dose study, 28 (93%) subjects were white; in the multidose study, all subjects were white. The mean (SD) ages in the 2 studies were 51 (10) and 51 (12) years, respectively; and mean (SD) body weights were 89.0 (10.8) and 81.1 (10.9) kg, respectively. PHX1149 exhibited dose-proportional increases in mean Cmax AUC0-t, and AUC0-infinity across the evaluated dose ranges. Tmax ranged from 2 to 4 hours, and t1/2 ranged from approximately 10 to 13 hours. No drug accumulation was observed. Plasma DPP4 inhibition at 24 hours was >or=50% in the multiple-dose study for doses of >or=100 mg. PHX1149 400 mg achieved approximately 90% 24-hour plasma DPP4 inhibition in the multiple-dose study. All AEs were characterized as mild, with the exception of 1 case of moderate edema, which occurred 17 days after the end of dosing in the multiple-dose study (50-mg dose group) and was considered unrelated to the study drug. Adverse events were experienced by 47% of all subjects studied in the single-dose study and 93% of subjects in the multiple-dose study. The rates of AEs were comparable between the study and placebo groups. CONCLUSIONS: The PK parameters and PD properties of PHX1149 were suitable (eg, tl/2, DPP4 inhibition) for once-daily dosing in this group of 58 healthy subjects. All doses were well tolerated.

Contribution of individual targets to the antitumor efficacy of the multitargeted receptor tyrosine kinase inhibitor SU11248.

Recent achievements in the development of multitargeted molecular inhibitors necessitate a better understanding of the contribution of activity against individual targets to their efficacy. SU11248, a small-molecule inhibitor targeting class III/V receptor tyrosine kinases, including the platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptors, KIT and FLT3, exhibits direct effects on cancer cells as well as antiangiogenic activity. Here, we investigated the contributions of inhibiting individual SU11248 target receptors to its overall antitumor efficacy in tumor models representing diverse signaling paradigms. Consistent with previous results, SU11248 was highly efficacious (frequently cytoreductive) in all models tested. To elucidate the specific contributions of inhibition of PDGF and VEGF receptors to the in vivo efficacy of SU11248, we employed two selective inhibitors, SU10944 (VEGF receptor inhibitor) and Gleevec (PDGF receptor inhibitor). SU10944 alone induced a tumor growth delay in all models evaluated, consistent with a primarily antiangiogenic mode of action. In contrast, Gleevec resulted in modest growth inhibition in tumor models in which the cancer cells expressed its targets (PDGFRbeta and KIT), but was not efficacious against tumors not driven by these target receptor tyrosine kinases. Strikingly, in all but one tumor model evaluated, the antitumor efficacy of SU10944 combined with Gleevec was similar to that of single-agent SU11248, and was greatly superior to that of each compound alone, indicating that the antitumor potency of SU11248 in these models stems from combined inhibition of both PDGF and VEGF receptors. The one exception was a model driven by an activated mutant of FLT3, in which the activity of SU11248, which targets FLT3, was greater than that of SU10944 plus Gleevec. Moreover, SU10944 combined with Gleevec inhibited tumor neoangiogenesis to an extent comparable to that of SU11248. Thus, the potent efficacy of SU11248 in models representing diverse signaling paradigms results from simultaneous inhibition of individual target receptors expressed both in cancer cells and in the tumor neovasculature, supporting the hypothesis that multitargeted inhibitors have the cumulative antitumor efficacy of combined single-target inhibitors.

SU14813: a novel multiple receptor tyrosine kinase inhibitor with potent antiangiogenic and antitumor activity.

Receptor tyrosine kinases (RTK), such as vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor receptor (KIT), and fms-like tyrosine kinase 3 (FLT3), are expressed in malignant tissues and act in concert, playing diverse and major roles in angiogenesis, tumor growth, and metastasis. With the exception of a few malignancies, seemingly driven by a single genetic mutation in a signaling protein, most tumors are the product of multiple mutations in multiple aberrant signaling pathways. Consequently, simultaneous targeted inhibition of multiple signaling pathways could be more effective than inhibiting a single pathway in cancer therapies. Such a multitargeted strategy has recently been validated in a number of preclinical and clinical studies using RTK inhibitors with broad target selectivity. SU14813, a small molecule identified from the same chemical library used to isolate sunitinib, has broad-spectrum RTK inhibitory activity through binding to and inhibition of VEGFR, PDGFR, KIT, and FLT3. In cellular assays, SU14813 inhibited ligand-dependent and ligand-independent proliferation, migration, and survival of endothelial cells and/or tumor cells expressing these targets. SU14813 inhibited VEGFR-2, PDGFR-beta, and FLT3 phosphorylation in xenograft tumors in a dose- and time-dependent fashion. The plasma concentration required for in vivo target inhibition was estimated to be 100 to 200 ng/mL. Used as monotherapy, SU14813 exhibited broad and potent antitumor activity resulting in regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. Treatment in combination with docetaxel significantly enhanced both the inhibition of primary tumor growth and the survival of the tumor-bearing mice compared with administration of either agent alone. In summary, SU14813 inhibited target RTK activity in vivo in association with reduction in angiogenesis, target RTK-mediated proliferation, and survival of tumor cells, leading to broad and potent antitumor efficacy. These data support the ongoing phase I clinical evaluation of SU14813 in advanced malignancies.

Simultaneous inhibition of the receptor kinase activity of vascular endothelial, fibroblast, and platelet-derived growth factors suppresses tumor growth and enhances tumor radiation response.

We have investigated the effect of simultaneous inhibition of multiple angiogenic growth factor signaling pathways on tumor growth, tumor blood perfusion, and radiation-induced tumor-growth delay using SU6668, an inhibitor of the receptor-tyrosine kinase activity of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). The SCK mammary carcinoma, FSaII fibrosarcoma, and CFPAC human pancreatic carcinoma were grown s.c. in the hind leg of A/J mice, C3H mice, and Balb/cAnNCrl-nuBr nude mice, respectively. Daily i.p. injection of 100 mg/kg of SU6668 markedly suppressed the growth of these three tumor types. SU6668 also markedly prolonged the survival time of host mice bearing SCK tumors, which appeared to be caused by a reduction of metastatic tumor growth in the lung. There was little or no change in normal tissue blood perfusion, whereas in SCK tumors the perfusion decreased by 50% at 1 h after a single i.p. injection of SU6668, slightly recovered at 4 h, and completely recovered by 8 h. Interestingly, the tumor blood flow was significantly increased above the baseline level 24 h after SU6668 injection. After extended daily i.p. injections of SU6668, the tumor blood flow in all of the three tumor types studied was markedly decreased compared with control. The observed effects of this drug on tumor blood perfusion may partially explain the effectiveness of the compound in suppressing tumor growth and extending survival of tumor-bearing mice. We also observed that daily SU6668 administration and a single dose of 15 Gy of X-irradiation was significantly more effective than either treatment alone in suppressing tumor growth. Our results suggest that SU6668 increased the radiosensitivity of tumor blood vessels. We conclude that SU6668 is a potent therapeutic agent potentially useful to suppress tumor growth and enhance the response of tumors to radiotherapy.

A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been implicated in the development and progression of several human cancers and are attractive targets for cancer therapy. PHA-665752 was identified as a small molecule, ATP-competitive, active-site inhibitor of the catalytic activity of c-Met kinase (K(i) 4 nM). PHA-665752 also exhibited >50-fold selectivity for c-Met compared with a panel of diverse tyrosine and serine-threonine kinases. In cellular studies, PHA-665752 potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes such as cell growth (proliferation and survival), cell motility, invasion, and/or morphology of a variety of tumor cells. In addition, PHA-665752 inhibited HGF-stimulated or constitutive phosphorylation of mediators of downstream signal transduction of c-Met, including Gab-1, extracellular regulated kinase, Akt, signal transducer and activator of transcription 3, phospholipase C gamma, and focal adhesion kinase, in multiple tumor cell lines in a pattern correlating to the phenotypic response of a given tumor cell. In in vivo studies, a single dose of PHA-665752 inhibited c-Met phosphorylation in tumor xenografts for up to 12 h. Inhibition of c-Met phosphorylation was associated with dose-dependent tumor growth inhibition/growth delay over a repeated administration schedule at well-tolerated doses. Interestingly, potent cytoreductive activity was demonstrated in a gastric carcinoma xenograft model. Collectively, these results demonstrate the feasibility of selectively targeting c-Met with ATP-competitive small-molecules and suggest the therapeutic potential of targeting c-Met in human cancers.

Gene expression profiling of human colon xenograft tumors following treatment with SU11248, a multitargeted tyrosine kinase inhibitor.

Biomarkers that indicate biological activity and/or efficacy are a potentially useful tool in the development of molecularly targeted therapeutics. It is useful, though challenging, to identify biomarkers during preclinical development in order to impact decision-making during early clinical development. SU11248 is an oral, selective multitargeted tyrosine kinase inhibitor currently in Phase II oncology clinical trials. It exhibits direct antitumor and antiangiogenic activity via inhibition of the receptor tyrosine kinases PDGFR, VEGFR, KIT and FLT3. To identify clinically translatable biomarkers of SU11248 activity, expression profiling was performed on Colo205 human xenograft tumors following treatment with SU11248. Over 100 transcripts changed in abundance in SU11248 as compared to vehicle-treated tumors. Nine candidate transcripts, chosen based on putative function, were also analysed and validated by TaqMan. One such potential biomarker, cadherin-11, was further evaluated at the protein level and was found to have increased expression in xenograft tumors after SU11248 treatment. Interestingly, cadherin-11 expression was also detected via immunohistochemical analysis of archived solid tumors, indicating the technical feasibility of translating this putative biomarker to clinical studies. Importantly, SU11248 treatment also resulted in increased expression of cadherin-11 protein in human tumor biopsies in three out of seven patients examined and confirms the feasibility of using transcriptional profiling of preclinical models to identify clinically translatable biomarkers.

SU6668 inhibits Flk-1/KDR and PDGFRbeta in vivo, resulting in rapid apoptosis of tumor vasculature and tumor regression in mice.

SU6668 is a small molecule inhibitor of the angiogenic receptor tyrosine kinases Flk-1/KDR, PDGFRbeta, and FGFR1. In mice, SU6668 treatment resulted in regression or growth arrest of all large established human tumor xenografts examined associated with loss of tumor cellularity. The events underlying loss of tumor cellularity were elucidated in detail in several tumor models. SU6668 treatment induced apoptosis in tumor microvessels within 6 h of the initiation of treatment. Dose-dependent decreases in tumor microvessel density were observed within 3 days of the first treatment. These changes were accompanied by decreased tumor cell proliferation and increased tumor cell apoptosis. Rapid increases in VEGF transcript levels were seen, consistent with the induction of tumor hypoxia. Using Western blot analyses, we determined that these in vivo antiangiogenic and proapoptotic effects of SU6668 occur at doses comparable to those required to inhibit Flk-1/KDR and PDGFRbeta phosphorylation in tumors. Potent, dose-dependent inhibition of Flk-1/KDR activity in vivo was independently demonstrated using vascular permeability as a readout. These data demonstrate that SU6668-induced inhibition of angiogenic receptor tyrosine kinase activity in vivo is associated with rapid vessel killing in tumors, leading to broad and potent antitumor effects.

The combination of the tyrosine kinase receptor inhibitor SU6668 with paclitaxel affects ascites formation and tumor spread in ovarian carcinoma xenografts growing orthotopically.

PURPOSE: The purpose of this study was to investigate the antitumor activity of SU6668, tyrosine kinase inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2), fibroblast growth factor receptor 1 (FGFR1), and platelet-derived growth factor receptor beta (PDGFRbeta), as single-agent therapy and in combination with paclitaxel on ovarian carcinoma xenograft models transplanted in the peritoneal cavity of nude mice. EXPERIMENTAL DESIGN: HOC22 and HOC79 ascites-producing human ovarian carcinoma xenografts were transplanted i.p. into nude mice. SU6668 was given p.o. (200 mg/kg, daily) as a single agent or in combination with paclitaxel i.v. (6 mg/kg/dose every other day or 20 mg/kg/dose weekly). Tumor burden was evaluated at the end of the treatment period as ascites volume and tumor cells, VEGF, FGF-2, and PDGF levels in ascites, and involvement of the organ of the peritoneal cavity. Response was evaluated as percentage increment of life span (%ILS). RESULTS: SU6668 affected ascites formation and tumor burden in the peritoneal cavity of nude mice bearing HOC22 and HOC79 xenografts. Decreased levels of VEGF and PDGF in ascites paralleled this effect. The overall survival of the mice bearing HOC xenograft (HOC79 less response than HOC22) was significantly increased by the treatment with SU6668. The magnitude of the effects depended on the length of treatment and tumor burden at the beginning of treatment. The combination of SU6668 with paclitaxel significantly prolonged the survival of mice bearing HOC79, compared with single therapies. SU6668-based combination therapy was more effective with paclitaxel given at the optimal dose and schedule (20 mg/kg every 7 days for 3 doses) than at the same total dose but split (6 mg/kg every 2 days for 10 doses). However, a similar outcome was observed when giving high-dose paclitaxel (20 mg/kg every 7 days for 3 doses) in monotherapy or split low-dose paclitaxel (6 mg/kg every 2 days for 10 doses) but in combination with SU6668. The addition of paclitaxel, by either schedule, to SU6668 treatment inhibited tumor spread in the peritoneal organs (omentum, pancreas, and diaphragm) even at low doses of paclitaxel. A greater effect was observed with prolonged treatments. CONCLUSIONS: This study shows that SU6668 in combination with paclitaxel inhibits ovarian carcinoma progression in the peritoneal cavity, by blocking ascites formation and tumor spread. Because an adequate schedule and dose of the combination might be as effective as conventional chemotherapy, this should be considered as a therapeutic alternative. These findings provide a rationale for the clinical evaluation of combination therapies affecting multiple biological targets in this tumor type.

Proof of target for SU11654: inhibition of KIT phosphorylation in canine mast cell tumors.

PURPOSE: The purpose of this study was to evaluate the effect of the receptor tyrosine kinase inhibitor SU11654 on the activity of its molecular target KIT in canine mast cell tumors (MCT) and correlate target inhibition with mutational status of the c-kit juxtamembrane domain and SU11654 plasma concentration. EXPERIMENTAL DESIGN: Tumor biopsies were obtained from dogs with advanced MCTs before and 8 h after administration of a single oral dose of SU11654, previously shown to be active in dogs with MCTs. Blood samples were taken to determine the plasma concentration of SU11654. Levels of phosphorylated KIT and ERK1/2 were assessed in tumor biopsies by Western blot. Tumors were analyzed by PCR for the presence or absence of an internal tandem duplication (ITD) in the juxtamembrane domain of c-kit. RESULTS: Fourteen dogs with advanced MCTs were enrolled in the study; 11 of these were evaluable for KIT target modulation (the remaining tumor specimens had inevaluable amounts of total KIT protein). Of these, eight MCTs showed reduced levels of phosphorylated KIT relative to total KIT after treatment with SU11654, compared with pretreatment biopsies. All four evaluable MCTs expressing ITD mutant c-kitshowed modulation of KIT phosphorylation, as did four of seven tumors expressing non-ITD c-kit. Phosphorylated ERK1/2 was modulated in seven tumors; this did not correlate with inhibition of KIT phosphorylation CONCLUSION: SU11654 treatment at the efficacious dose results in inhibition of KIT phosphorylation in canine MCTs.

Phase I dose-escalating study of SU11654, a small molecule receptor tyrosine kinase inhibitor, in dogs with spontaneous malignancies.

PURPOSE: The purpose of the following study was to investigate the safety and efficacy of the novel multitargeted indolinone receptor tyrosine kinase (RTK) inhibitor, SU11654, using a canine model of spontaneous tumors. This p.o. bioavailable compound exhibits potent inhibitory activity against members of the split kinase family of RTKs, including vascular endothelial growth factor receptor, platelet-derived growth factor receptor, Kit, and Flt-3, resulting in both direct antitumor and antiangiogenic activity. EXPERIMENTAL DESIGN: This was a Phase I trial in which successive cohorts of dogs with spontaneous tumors that had failed standard treatment regimens received escalating doses of SU11654 as oral therapy. Pharmacokinetics, toxicity, and tumor response were assessed. RESULTS: Fifty-seven dogs with a variety of cancers were enrolled; of these, 10 experienced progressive disease within the first 3 weeks. Measurable objective responses were observed in 16 dogs (including 6 complete responses), primarily in mast cell tumors (n = 11), mixed mammary carcinomas (n = 2), soft tissue sarcomas (n = 2), and multiple myeloma (n = 1), for an overall response rate of 28% (16 of 57). Stable disease of sufficient duration to be considered clinically meaningful (>10 weeks) was seen in an additional 15 dogs, for a resultant overall biological activity of 54% (31 of 57). CONCLUSIONS: This study provides the first evidence that p.o. administered kinase inhibitors can exhibit activity against a variety of spontaneous malignancies. Given the similarities of canine and human cancers with regard to tumor biology and the presence of analogous RTK dysregulation, it is likely that such agents will demonstrate comparable antineoplastic activity in people.

An innovative phase I clinical study demonstrates inhibition of FLT3 phosphorylation by SU11248 in acute myeloid leukemia patients.

PURPOSE: Obtaining direct and rapid proof of molecular activity in early clinical trials is critical for optimal clinical development of novel targeted therapies. SU11248 is an oral multitargeted kinase inhibitor with selectivity for fms-related tyrosine kinase 3/Flk2 (FLT3), platelet-derived growth factor receptor alpha/beta, vascular endothelial growth factor receptor 1/2, and KIT receptor tyrosine kinases. FLT3 is a promising candidate for targeted therapy in acute myeloid leukemia (AML), because activating mutations occur in up to 30% of patients. We conducted an innovative single-dose clinical study with a primary objective to demonstrate inhibition of FLT3 phosphorylation by SU11248 in AML. EXPERIMENTAL DESIGN: Twenty-nine AML patients each received a single dose of SU11248, escalated from 50 to 350 mg, in increments of 50 mg and cohorts of three to six patients. FLT3 phosphorylation and plasma pharmacokinetics were evaluated at seven time points over 48 h after SU11248 administration, and FLT3 genotype was determined. Study drug-related adverse events occurred in 31% of patients, mainly grade 1 or 2 diarrhea and nausea, at higher dose levels. RESULTS: Inhibition of FLT3 phosphorylation was apparent in 50% of FLT3-wild-type (WT) patients and in 100% of FLT3-mutant patients. FLT3 internal tandem duplication (ITD) mutants showed increased sensitivity relative to FLT3-WT, consistent with preclinical predictions. The primary end point, strong inhibition of FLT3 phosphorylation in >50% patients, was reached in 200 mg and higher dose cohorts. Downstream signaling pathways were also inhibited; signal transducer and activator of transcription 5 (STAT5) was reduced primarily in internal tandem duplication patients and at late time points in FLT3-WT patients, whereas extracellular signal-regulated kinase (ERK) activity was reduced in the majority of patients, independent of FLT3 inhibition. CONCLUSIONS: This novel translational study bridges preclinical models to the patient setting and provides the first evidence of anti-FLT3 activity in patients. Proof of target inhibition accomplishes a crucial milestone in the development of novel oncology therapeutics.

In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship.

One challenging aspect in the clinical development of molecularly targeted therapies, which represent a new and promising approach to treating cancers, has been the identification of a biologically active dose rather than a maximum tolerated dose. The goal of the present study was to identify a pharmacokinetic/pharmacodynamic relationship in preclinical models that could be used to help guide selection of a clinical dose. SU11248, a novel small molecule receptor tyrosine kinase inhibitor with direct antitumor as well as antiangiogenic activity via targeting the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), KIT, and FLT3 receptor tyrosine kinases, was used as the pharmacological agent in these studies. In mouse xenograft models, SU11248 exhibited broad and potent antitumor activity causing regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. To predict the target SU11248 exposure required to achieve antitumor activity in mouse xenograft models, we directly measured target phosphorylation in tumor xenografts before and after SU11248 treatment and correlated this with plasma inhibitor levels. In target modulation studies in vivo, SU11248 selectively inhibited Flk-1/KDR (VEGF receptor 2) and PDGF receptor beta phosphorylation (in a time- and dose-dependent manner) when plasma concentrations of inhibitor reached or exceeded 50-100 ng/ml. Similar results were obtained in a functional assay of VEGF-induced vascular permeability in vivo. Constant inhibition of VEGFR2 and PDGF receptor beta phosphorylation was not required for efficacy; at highly efficacious doses, inhibition was sustained for 12 h of a 24-h dosing interval. The pharmacokinetic/pharmacodynamic relationship established for SU11248 in these preclinical studies has aided in the design, selection, and evaluation of dosing regimens being tested in human trials.

SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer.

The purpose of this study was to evaluate the activity of the indolinone kinase inhibitor SU11248 against the receptor tyrosine kinase KIT in vitro and in vivo, examine the role of KIT in small cell lung cancer (SCLC), and anticipate clinical utility of SU11248 in SCLC. SU11248 is an oral, multitargeted tyrosine kinase inhibitor with direct antitumor and antiangiogenic activity through targeting platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor, KIT, and FLT3 receptors. Treatment of the KIT-expressing SCLC-derived NCI-H526 cell line in vitro with SU11248 resulted in dose-dependent inhibition of stem cell factor-stimulated KIT phosphotyrosine levels and proliferation. The biological significance of KIT inhibition was evaluated in vivo by treating mice bearing s.c. NCI-H526 tumors with SU11248 or another structurally unrelated KIT inhibitor, STI571 (Gleevec), which is also known to inhibit Bcr-Abl and PDGFRbeta. SU11248 treatment resulted in significant tumor growth inhibition, whereas inhibition from STI571 treatment was less dramatic. Both compounds reduced phospho-KIT levels in NCI-H526 tumors, with a greater reduction by SU11248, correlating with efficacy. Likewise, phospho-PDGFRbeta levels contributed by tumor stroma and with known involvement in angiogenesis were strongly inhibited by SU11248 and less so by STI571. Because platinum-based chemotherapy is part of the standard of care for SCLC, SU11248 was combined with cisplatin, and significant tumor growth delay was measured compared with either agent alone. These results expand the profile of SU11248 as a KIT signaling inhibitor and suggest that SU11248 may have clinical potential in the treatment of SCLC via direct antitumor activity mediated via KIT as well as tumor angiogenesis via vascular endothelial growth factor receptor FLK1/KDR and PDGFRbeta.

Preclinical evaluation of the tyrosine kinase inhibitor SU11248 as a single agent and in combination with "standard of care" therapeutic agents for the treatment of breast cancer.

SU11248 is an oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities through targeting platelet-derived growth factor receptor, vascular endothelial growth factor receptor, KIT, and FLT3, the first three of which are expressed in human breast cancer and/or its supporting tissues. The purpose of the present studies was to demonstrate the potent anticancer activity of SU11248 alone or in combination with conventional cytotoxic agents against several distinct preclinical models of breast cancer. SU11248 was administered as a monotherapy to (1) mouse mammary tumor virus-v-Ha-ras mice and 7,12-dimethylbenz(a)anthracene-treated rats bearing mammary tumors and (2) mice bearing human breast cancer xenografts of s.c. MX-1 tumors and osseous metastasis of a MDA-MB-435-derived cell line (435/HAL-Luc). SU11248 was also administered in combination with docetaxel both in xenograft models and in combination with 5-fluorouracil and doxorubicin in the MX-1 model. SU11248 treatment potently regressed growth of mammary cancers in mouse mammary tumor virus-v-Ha-ras transgenic mice (82% regression) and 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats (99% regression at the highest dose; P < 0.05 for both). This agent also inhibited MX-1 tumor growth by 52%, with markedly enhanced anticancer effects when administered in combination with docetaxel, 5-fluorouracil, or doxorubicin compared with either agent alone (P < 0.05). SU11248 treatment in combination with docetaxel effectively prolonged survival of mice, with 435/HAL-Luc cancer xenografts established in bone compared with either agent alone (P < 0.05). These results demonstrate that SU11248 is effective in preclinical breast cancer models and suggest that it may be useful in the treatment of breast cancer in the clinic.

Src family kinase activity is required for signal tranducer and activator of transcription 3 and focal adhesion kinase phosphorylation and vascular endothelial growth factor signaling in vivo and for anchorage-dependent and -independent growth of human tumor cells.

The Src family kinases (SFKs) Src and Yes are believed to play critical roles in tumor growth, angiogenesis, invasion, and dissemination. Using a panel of highly selective and structurally diverse Src inhibitors, we found that phosphorylation of signal transducer and activator of transcription 3 [STAT3 (Y705)] and focal adhesion kinase [FAK (Y861)] was SFK dependent in cultured human colon, breast, lung, and ovarian tumor cells. These findings were reproduced in vivo in target modulation studies using tumors derived from fibroblasts overexpressing activated Src. Additionally, treatment of mice with multiple Src inhibitors resulted in inhibition of phosphorylation of FAK (Y861) and of a putative Src autophosphorylation epitope (Y419) in HT-29 human colon tumor xenografts. Next we pharmacologically examined the requirement for SFKs in asynchronous proliferation of human tumor cells. At concentrations sufficient to selectively inhibit Src, structurally diverse Src inhibitors inhibited growth of cultured human colon, breast, and lung cells on plastic under low serum conditions. In addition, these compounds inhibited anchorage-independent growth of HT-29 human colon tumor cells in soft agar. The role of SFK activity in vascular endothelial growth factor signaling was also evaluated. Inhibition of SFK signaling using structurally distinct Src inhibitors resulted in complete inhibition of vascular endothelial growth factor-dependent vascular permeability in vivo. These data demonstrate that STAT3 (Y705) and FAK (Y861) phosphoepitopes are SFK-dependent in tumor cells and reveal a requirement for SFK function in tumor cell proliferation and vascular permeability.

CCR 20th Anniversary Commentary: Determining a Pharmacokinetic/Pharmacodynamic Relationship for Sunitinib­A Look Back.

The article by Mendel and colleagues, published in the January 1, 2003, issue of Clinical Cancer Research, described their novel preclinical approach to developing a thorough understanding of the exposure-activity relationship for sunitinib, a multitargeted receptor tyrosine kinase inhibitor being developed for oncology therapy. This work successfully set exposure guidelines to identify a biologically active dose in early clinical trials.

SU11248 inhibits tumor growth and CSF-1R-dependent osteolysis in an experimental breast cancer bone metastasis model.

The aim of the study was to investigate inhibitory effects of the receptor tyrosine kinase (RTK) inhibitor SU11248 against CSF-1R and osteoclast (OC) formation. We developed an in vivo model of breast cancer metastasis to evaluate efficacy of SU11248 against tumor growth and tumor-induced osteolysis in bone. The in vitro effects of SU11248 on CSF-1R phosphorylation, OC formation and function were evaluated. Effects on 435/HAL-Luc tumor growth in bone were monitored by in vivo bioluminescence imaging (BLI), and inhibition of osteolysis was evaluated by measurement of serum pyridinoline (PYD) concentration and histology. Phosphorylation of the receptor for M-CSF (CSF-1R) expressed by NIH3T3 cells was inhibited by SU11248 with an IC50 of 50-100 nM, consistent with CSF-1R belonging to the class III split kinase domain RTK family. The early M-CSF-dependent phase of in vitro murine OC development and function were inhibited by SU11248 at 10-100 nM. In vivo inhibition of osteolysis was confirmed by significant lowering of serum PYD levels following SU11248 treatment of tumor-bearing mice (P = 0.047). Using BLI, SU11248 treatment at 40 mg/kg/day for 21 days showed 64% inhibition of tumor growth in bone (P = 0.006), and at 80 mg/kg/day showed 89% inhibition (P = 0.001). Collectively, these data suggest that SU11248 may be an effective and tolerated therapy to inhibit growth of breast cancer bone metastases, with the additional advantage of inhibiting tumor-associated osteolysis.

Small molecule tyrosine kinase inhibitors: clinical development of anticancer agents.

Numerous small molecule synthetic tyrosine kinase inhibitors are in clinical development for the treatment of human cancers. These fall into three broad categories: inhibitors of the epidermal growth factor receptor tyrosine kinase family (e.g., Iressa trade mark and Tarceva trade mark ), inhibitors of the split kinase domain receptor tyrosine kinase subgroup (e.g., PTK787/ZK 222584 and SU11248) and inhibitors of tyrosine kinases from multiple subgroups (e.g., Gleevec trade mark ). In addition, agents targeting other tyrosine kinases implicated in cancer, such as Met, Tie-2 and Src, are in preclinical development. As experience is gained in the clinic, it has become clear that unleashing the full therapeutic potential of tyrosine kinase inhibitors will require patient preselection, better assays to guide dose selection, knowledge of mechanism-based side effects and ways to predict and overcome drug resistance.

Effects of SU5416, a small molecule tyrosine kinase receptor inhibitor, on FLT3 expression and phosphorylation in patients with refractory acute myeloid leukemia.

Acute myeloid leukemia (AML) is associated with dysregulated hematopoietic cell proliferation and increased bone marrow angiogenesis, each regulated by signaling through receptor tyrosine kinases (RTKs). SU5416 is a small molecule inhibitor of VEGF receptors, c-kit and FLT3 and therefore provides a novel opportunity to target both angiogenesis and proliferation in AML. SU5416 was assessed in a phase II hematological malignancy trial in the US, where partial responses were observed in two of 33 patients. Since AML provides a unique platform to evaluate mechanism of action of small molecule inhibitors, investigation of the effect of SU5416 on FLT3 expression and phosphorylation in blood and bone marrow was an additional focus of this trial. Phosphorylated FLT3 was detected by immunoprecipitation/Western analysis in peripheral blood samples from 17 of 22 patients, and seven exhibited strong inhibition of phosphorylation immediately following a 1h SU5416 infusion, demonstrating that SU5416 can modulate RTK phosphorylation in humans. Although no clear correlation with clinical response was observed, analysis of patient plasma drug levels suggested that a threshold SU5416 concentration of 15 microM was associated with FLT3 inhibition. This observation was supported by data from an ex vivo model where AML cells were spiked into human blood, established to mimic the clinical setting and enable more rigorous analysis of effect of SU5416. In addition, FLT3 protein levels were downregulated in patient bone marrow samples, analyzed by an RIA assay. To identify putative predictors of response, patient plasma was analyzed for levels of secreted ligands of SU5416 targets; SCF and FLT3 ligand. Baseline levels of SCF in patients with stable or progressive disease were significantly higher than those in normal donors, whereas FLT3 ligand levels in patients who exhibited progressive disease were significantly lower than those in normal donors. The translational and clinical analyses described in this report provide some insights into the mechanism and duration of action of SU5416.

Expression profiling of blood samples from an SU5416 Phase III metastatic colorectal cancer clinical trial: a novel strategy for biomarker identification.

BACKGROUND: Microarray-based gene expression profiling is a powerful approach for the identification of molecular biomarkers of disease, particularly in human cancers. Utility of this approach to measure responses to therapy is less well established, in part due to challenges in obtaining serial biopsies. Identification of suitable surrogate tissues will help minimize limitations imposed by those challenges. This study describes an approach used to identify gene expression changes that might serve as surrogate biomarkers of drug activity. METHODS: Expression profiling using microarrays was applied to peripheral blood mononuclear cell (PBMC) samples obtained from patients with advanced colorectal cancer participating in a Phase III clinical trial. The PBMC samples were harvested pre-treatment and at the end of the first 6-week cycle from patients receiving standard of care chemotherapy or standard of care plus SU5416, a vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitor. Results from matched pairs of PBMC samples from 23 patients were queried for expression changes that consistently correlated with SU5416 administration. RESULTS: Thirteen transcripts met this selection criterion; six were further tested by quantitative RT-PCR analysis of 62 additional samples from this trial and a second SU5416 Phase III trial of similar design. This method confirmed four of these transcripts (CD24, lactoferrin, lipocalin 2, and MMP-9) as potential biomarkers of drug treatment. Discriminant analysis showed that expression profiles of these 4 transcripts could be used to classify patients by treatment arm in a predictive fashion. CONCLUSIONS: These results establish a foundation for the further exploration of peripheral blood cells as a surrogate system for biomarker analyses in clinical oncology studies.