Vascular endothelial growth factor receptors (VEGFRs) are a set of three homologous transmembrane receptor tyrosine kinases that bind vascular endothelial growth factors (VEGFs). VEGFRs are expressed throughout the vascular and lymphatic systems and by some cells of the immune and nervous systems. Dimeric VEGF binding to the VEGFR extracellular domains promotes receptor dimerization and mutual receptor subunit tyrosine transphosphorylation of the intracellular domains. Specific proteins bind these receptor phosphotyrosine residues initiating a signaling cascade that culminates in cellular migration, mitosis, and survival promoting the growth of blood and lymphatic vessels, respectively, termed 'angiogenesis' and 'lymphangiogenesis'. Alternative splicing generates soluble VEGFRs that can sequester VEGF ligands and inhibit VEGFR activation.

Antiangiogenic agents such as vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors may prove most efficacious in the setting of early disease and in the prevention of dissemination and growth of micrometastases. This hypothesis was tested in a metastatic orthotopic rat model of breast cancer with the use of a novel orally bioavailable VEGFR2 kinase inhibitor, MK-0888. Mat B III rat mammary cancer cells were implanted into the mammary fat pads of syngeneic female F344 rats. Primary tumor growth was very aggressive, with micrometastases detected 8 days after cell implantation in ipsilateral axillary and inguinal lymph nodes. Lung metastases were detected 15 days after cell implantation by histological analysis. MK-0888 suppressed primary and metastatic tumor growth and reduced the incidence of metastasis in a dose- and schedule-dependent manner. Inhibitions of primary and metastatic tumor growth, as well as intratumoral antiangiogenesis effects, were detected in situ by immunohistochemical analysis of tumor cells, endothelial cell proliferation, microvascular density, and blood vessel maturity. In the Mat B HI rat mammary cancer metastasis model, our results provide further evidence supporting the ongoing clinical development of VEGFR2 kinase inhibitors, as well as clinically applicable in situ detection and verification of the inhibitor effect in tumor and metastasis biopsies. (The J Histotechnol 33(1):15-24, 2010)

Background: Although therapy for CAD has advanced with current therapies, a large number of pts have refractory angina not amenable to revascularization resulting in significant morbidity and morta...

Modifications to the basic side-chain of early lead structures of the indolyl quinolinone class of KDR kinase inhibitors resulted in improved pharmacokinetic and ancillary profiles. Specifically, compounds bearing 5-amido- and 5-sulphonamido-indolyl substituents exhibited lower plasma clearance and weaker binding affinity for the I(Kr) potassium channel hERG.

PURPOSE To test the hypothesis that dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) provides a useful in vivo measure of passive blood retinal barrier permeability surface area product (BRB PS) in experimental diabetic retinopathy. METHODS BRB PS (cm(3)/min) was measured using DCE-MRI and Gd-DTPA (MW 590 Da) in urethane-anesthetized control rats, sodium iodate-treated rats, rats receiving intravitreally injected human serum albumin (HSA) or vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), or in rats that were diabetic for 2, 4, 6, or 8 months. RESULTS Sodium iodate-treated rats exhibited an eightfold increase (P < 0.05) in BRB PS compared to that in control animals. Furthermore, in iodate-treated rats, the average vitreous signal enhancement was linearly dependent on Gd-DTPA dose (r = 0.91, P < 0.0001). Six hours postinjection, VEGF/VPF-treated rats exhibited a threefold increase in BRB PS (P < 0.05) compared to eyes injected with HSA. In 2-, 4-, and 6-month diabetic rats, BRB PS was not significantly different (P > 0.05) from control BRB PS values. After 8 months of diabetes, a twofold increase (P < 0.05) in PS over control PS values was found. DCE-MRI demonstrated that the BRB becomes leaky immediately before death, possibly causing an artificial increase in retinal permeability in methods that require enucleation or retinal isolation to assess permeability. CONCLUSIONS DCE-MRI provides a sensitive, noninvasive, and linear assay that accurately measures, without potential artifacts associated with death and enucleation, passive BRB PS in experimental diabetes. DCE-MRI BRB PS measurements are expected to provide a useful surrogate marker of drug treatment efficacy.

A strategy for antagonizing vascular endothelial growth factor (VEGF) -induced angiogenesis is to inhibit the kinase activity of its receptor, kinase insert domain-containing receptor (KDR), the first committed and perhaps the last unique step in the VEGF signaling cascade. We synthesized a novel ATP-competitive KDR tyrosine kinase inhibitor that potently suppresses human and mouse KDR activity in enzyme (IC(50) = 7.8-19.5 nM) and cell-based assays (IC(50) = 8 nM). The compound was bioavailable in vivo, leading to a dose-dependent decrease in basal- and VEGF-stimulated KDR tyrosine phosphorylation in lungs from naïve and tumor-bearing mice (IC(50) = 23 nM). Pharmacokinetics and pharmacodynamics guided drug dose selection for antitumor efficacy studies. HT1080 nude mice xenografts were treated orally twice daily with vehicle, or 33 or 133 mg/kg of compound. These doses afforded trough plasma concentrations approximately equal to the IC(50) for inhibition of KDR autophosphorylation in vivo for the 33 mg/kg group, and higher than the IC(99) for the 133 mg/kg group. Chronic treatment at these doses was well-tolerated and resulted in dose-dependent inhibition of tumor growth, decreased tumor vascularization, decreased proliferation, and enhanced cell death. Antitumor efficacy correlated with inhibition of KDR tyrosine phosphorylation in the tumor, as well as in a surrogate tissue (lung). Pharmacokinetics and pharmacodynamics assessment indicated that the degree of tumor growth inhibition correlated directly with the extent of inhibition of KDR tyrosine phosphorylation in tumor or lung at trough. These observations highlight the need to design antiangiogenic drug regimens to ensure constant target suppression and to take advantage of PD end points to guide dose selection.

5629 Flt-3 is a hematopoietic progenitor cell receptor that promotes stem cell proliferation, development and survival. Flt-3 also plays a role in leukemogenesis, being both highly expressed and frequently mutated in adult and pediatric acute leukemia. Mutations in Flt-3 result in a constitutive activation of the receptor tyrosine kinase, conferring ligand independence for growth and for activation of Flt-3 signaling pathways. The most frequently identified mutation in Flt-3 is an internal tandem duplication (ITD), an in-frame insertion of 2-60 amino acids which duplicates a sequence within the juxtamembrane domain of Flt-3. Additionally, point mutations or a single-residue deletion in the activation loop of the tyrosine kinase domain have been identified. The presence of these mutations correlates with an increased risk for relapse and is an indicator of poor prognosis for response to currently available therapy. The transforming potential of Flt-3 was shown to be dependent on its kinase activity, a finding that has led several companies to develop small molecule kinase inhibitors for treating Flt-3-driven leukemias. To provide proof-of-concept support for a leukemic indication for Flt-3 inhibitors, we cloned the most prevalent Flt-3 mutations and established cell-based assays to evaluate their sensitivity to inhibitors. Compounds in the indolyl quinolinone series were identified as potent inhibitors of wild-type as well as of mutant Flt-3 proteins in biochemical (IC50 range = 9-24 nM) and cellular phosphorylation assays (IC50 = 1-76 nM). Inhibition of receptor phosphorylation resulted in dose-dependent anti-proliferative effects, including G1 arrest and apoptosis in cells dependent on Flt-3 for growth. In vivo studies in nude mice bearing MV4;11 (ITD-Flt-3) human xenografts demonstrated that chronic treatment with the inhibitor led to inhibition of tumor Flt-3 activity and caused tumor regressions. These observations provided support for a clinical trial of a novel orally bioavailable Flt-3 kinase inhibitor in AML patients. Pharmacodynamic responses were observed in bone marrow and peripheral blood from leukemic patients with an estimated potency of 61 nM against constitutively activated Flt-3.

This study was designed to test the ability of adenovirus-delivered vascular endothelial growth factor (Ad-VEGF) to stimulate angiogenesis and arteriogenesis in the rabbit hindlimb following the induction of ischemia and to evaluate the functional changes in the collateral circulation. Ten days after the surgical induction of hindlimb ischemia, either a control virus (1 x 10(9) pfu) or an adenovirus containing the gene for VEGF(165) (1 x 10(6), 1 x 10(7), 1 x 10(8), or 1 x 10(9) pfu) was administered intramuscularly into the ischemic limb. Thirty days after administration of the adenoviral vectors, skeletal muscle capillary density was assessed and angiography was performed as markers of angiogenesis and arteriogenesis, respectively. Hindlimb blood flow was directly measured and hyperemic tests were performed to evaluate the functional improvements in collateral blood flow. Animals treated with Ad-VEGF at 1 x 10(8) and 1 x 10(9) pfu showed elevated levels of circulating VEGF and dose-dependent hindlimb edema. These doses also led to a robust angiogenic response (i.e., increase in capillary density), but failed to improve collateral blood flow. Consistent with the lack of a functional response, there was no angiographic evidence of enhanced arteriogenesis with any dose of Ad-VEGF. Following the induction of hindlimb ischemia, administration of Ad-VEGF stimulated capillary sprouting (i.e., angiogenesis), but did not increase the growth and development of larger conduit vessels (i.e., arteriogenesis) or improve collateral blood flow. These results support the concept that VEGF may not be expected to have therapeutic utility for the treatment of peripheral or myocardial ischemia.

There is ample therapeutic opportunity for the use of antiangiogenic inhibitors in the clinic, as there are several human diseases that are dependent upon angiogenesis [1]. However, no disease has attracted as much attention as a target for antiangiogenic therapy as malignant disorders. There is a vast amount of literature acting as proof-of-principle for the use of angiogenic inhibitors as effective agents for blocking tumour-induced angiogenesis and subverting tumour growth and disease dissemination. One of the unique attractions of targeting tumour angiogenesis is that vascular endothelial cells are a genetically stable population in which acquisition of therapeutic resistance might be less efficient than in genetically unstable tumour cells [2,3]. This review covers inhibitors that target the tumour angiogenic agent vascular endothelial growth factor and its receptors as one such antiangiogenic approach. Many agents in this class are in clinical trials with limited reports of toxicity and some early evidence of clinical benefit.

La presente invention se rapporte a des procedes in vivo permettant de mesurer la capacite d'un compose a inhiber l'activite de type recepteur de kinases. Les exemples presentes montrent une correlation directe entre l'inhibition in vivo des kinases KDR et la concentration de l'inhibiteur dans le plasma et le sang circulant. Ces donnees sont utilisees pour predire et valider des mesures in vitro non quantifiables, telles que des valeurs de la concentration inhibitrice IC50 de cellules endotheliales murines. La puissance in vivo d'un compose, determinee par une analyse conforme a la presente invention, peut permettre la selection de quantites dosees et de frequences destinees a des etudes de modeles animaux precliniques et a des etudes cliniques humaines concues pour generer des profils d'innocuite, de puissance et d'efficacite pour l'inhibiteur respectif.