Purpose: Axitinib (AG-013736) is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases 1to 3 that is in clinical development for the treatment of solid tumors. We provide a comprehensive description of its in vitro characteristics and activities, in vivo antiangiogenesis, and antitumor efficacy and translational pharmacology data. Experimental Design: The potency, kinase selectivity, pharmacologic activity, and antitumor efficacy of axitinib were assessed in various nonclinical models. Results: Axitinib inhibits cellular autophosphorylation of VEGF receptors (VEGFR) with picomolar IC 50 values. Counterscreening across multiple kinase and protein panels shows it is selective for VEGFRs. Axitinib blocks VEGF-mediated endothelial cell survival, tube formation, and downstream signaling through endothelial nitric oxide synthase, Akt and extracellular signal-regulated kinase. Following twice daily oral administration, axitinib produces consistent and dose-dependent antitumor efficacy that is associated with blocking VEGFR-2 phosphorylation, vascular permeability, angiogenesis, and concomitant induction of tumor cell apoptosis. Axitinib in combination with chemotherapeutic or targeted agents enhances antitumor efficacy in many tumor models compared with single agent alone. Dose scheduling studies in a human pancreatic tumor xenograft model show that simultaneous administration of axitinib and gemcitabine without prolonged dose interruption or truncation of axitinib produces the greatest antitumor efficacy. The efficacious drug concentrations predicted in nonclinical studies are consistent with the range achieved in the clinic. Although axitinib inhibits platelet-derived growth factor receptors and KIT with nanomolar in vitro potencies, based on pharmacokinetic/pharmacodynamic analysis, axitinib acts primarily as a VEGFR tyrosine kinase inhibitor at the current clinical exposure. Conclusions: The selectivity, potency for VEGFRs, and robust nonclinical activity may afford broad opportunities for axitinib to improve cancer therapy.

PURPOSE. Endothelial precursor cells (EPCs) derived from hematopoietic stem cells (HSCs) have been shown to contribute to choroidal neovascularization by signaling through the SDF-1/CXCR4 axis. In a prevention and treatment/intervention modality of the laser choroidal neovascularization (CNV) model, the efficacy of CXCR4 inhibition on reducing choroidal leakage and angiogenesis was evaluated. METHODS. CNV in rats was generated by focal rupture of Bruch's membrane with an 810-nm diode laser. In the prevention mode, a CXCR4 antagonist (AMD3100) was delivered via an osmotic pump 1 day after laser induction. In the intervention mode, AMD3100 delivery commenced 14 days after laser induction. Inhibition of CXCR4 was determined through leukocyte and SDF-1 actin polymerization blood biomarker assays. Leakage was assessed by fluorescein angiography, and CNV lesion size was quantified after isolectin B4 endothelial cell staining. SU14813, an anti-VEGFR, PDGFR-beta, KIT, and FLT3 inhibitor, was also assessed in an intervention study protocol. RESULTS. Inhibition of CXCR4 was demonstrated by an increase in the number of blood leukocytes, and diminished SDF-1 induced actin polymerization in whole blood. CNV leakage and neovascularization were inhibited when the dose regimen was initiated 1 day after laser-induced CNV induction. AMD3100 did not show efficacy when administered 14 days after lasering. Treatment with SU14813 significantly decreased CNV leakage and lesion size in an intervention modality. CONCLUSIONS. Inhibition of CXCR4 may be useful in preventing neovascularization but does not appear to have an effect on already established angiogenesis. A multiple receptor tyrosine kinase (RTK) inhibitor approach shows promise for the treatment of wet age-related macular degeneration.

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Abstract-Prostaglandin E 2 (PGE 2 ) is an endogenous hormone of adrenal zona glomerulosa cells and is released in response to stimulation by agonists such as angiotensin II (Ang II). It stimulates the release of aldosterone from cultured bovine adrenal zona glomerulosa cells. These studies were designed to determine whether this steroidogenic effect of PGE 2 was mediated by an EP 1 , EP 2 , or EP 3 receptor. Prostaglandin E 2 and 11-deoxy PGE 1 , an EP 2 -selective agonist, stimulated aldosterone release in a concentration-related manner with an ED 50 of 300 nmol/L for PGE 2 and 2 mol/L for 11-deoxy PGE 1 . The maximal effect of PGE 2 was less than that of angiotensin II. 17-Phenyl trinor PGE 2 , an EP 1 -selective agonist, required concentrations of 100 mol/L to stimulate aldosterone release and sulprostone, an EP 3 /EP 1 -selective agonist, failed to alter aldosterone release. The EP 1 -selective antagonist SC19220 failed to alter basal or PGE 2 -stimulated aldosterone release over a range of concentrations. PGE 2 and 11-deoxy PGE 1 also stimulated an increase in both intracellular and extracellular cAMP. This increase was time-and concentration-related. The ED 50 for PGE 2 was 9.8 mol/L. 17-Phenyl trinor PGE 2 and sulprostone were without effect. Using fura-2 loaded cells, PGE 2 (2 mol/L), dibutyryl cAMP (2 mmol/L), and Ang II (2 mol/L) increased intracellular calcium over basal concentrations by 5.5-fold, 3-fold, and 6.2-fold, respectively. Like PGE 2 , dibutyryl cAMP also stimulated aldosterone release. PGE 2 -and dibutyryl cAMP-induced aldosterone release were blocked by the calcium channel inhibitor diltiazem. These studies indicate that PGE 2 is a potent stimulus for aldosterone release and that the effect is mediated by EP 2 receptors. Both cAMP and calcium appear to mediate the steroidogenic effect of PGE 2 and calcium seems to be distal to cAMP. (Hypertension. 1998;31:575-581.)Key Words: zona glomerulosa Ⅲ cyclic AMP Ⅲ calcium Ⅲ receptors, prostanoid Ⅲ angiotensin II T here are five classes of prostanoid (P) receptors designated as EP, FP, DP, IP, and TP, corresponding to their naturally occurring agonists, prostaglandin E 2 , prostaglandin F 2␣ , prostaglandin D 2 , prostaglandin I 2 , and thromboxane A 2 , respectively.1 Synthetic and natural analogues of these prostaglandins also exist that possess selectivity at these five classes of receptors. 2,3The EP receptor has been subclassified into three subtypes: EP 1 , EP 2 , and EP 3 . [4][5][6][7][8] Based on the analysis of Coleman et al, 1,7 Eglin and Whiting, 3 and Muallem et al, 9 we know that prostanoid receptors differ in the second messengers that mediate their biological effects. Agonists acting on the EP 1 , FP, and TP receptors stimulate the IP 3 /DAG pathway and exert their effects through an increase in intracellular calcium. The EP 2 , DP, or IP receptor agonists stimulate adenylyl cyclase and the accumulation of cAMP. Finally, EP 3 receptor activation may increase IP 3 /DAG formation or inhibit adenylyl cyclase.There is considerable evid...

Age-related macular degeneration (AMD) is the major cause of blindness for people over 60. In the "wet" form of AMD compounds targeting growth factor signaling pathways such as VEGF have been a major focus for therapeutic interventions. In a previously developed rat model of CNV, we utilized two receptor tyrosine kinase inhibitors (RTKi) to block VEGFR-1, VEGFR-2 and PDGFR signaling following the establishment of CNV. AAV-VEGF 165 was injected into the subretinal space of rats at postnatal days 15-17. Six weeks later, a suspension of RTK inhibitors, AG013764 or AG013711, was injected intraperitoneally (IP, twice daily) or intravitreally (every five days) over a two week period. FITC-dextran whole-mounts of RPE-choroid-sclera were prepared after the animals were sacrificed. CNV area was quantified using Neurolucida to measure the hyperfluorescence on FITC-dextran whole-mounts. Histology and immunohistochemistry were performed as described previously. VEGF expression in control and treated eyes was confirmed by immunohistochemistry and histological sections indicated recovery of retinal morphology and CNV reduction in treated eyes. In the animals IP injected with AG013764 or AG013711 the mean CNV level was reduced by 25 to 33% compared to control, but this effect did not achieve statistical significance. Intravitreal injections of AG013764 or AG013711 reduced the level of CNV by approximately 60% compared to control (p< 0.005 or p< 0.05, respectively). These data show that two RTK inhibitors, AG013764 or AG013711, delivered intravitreally, significantly reduce blood vessel proliferation in this AAV-VEGF 165 model of CNV.