The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo.

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

PI 3-kinase p110beta: a new target for antithrombotic therapy.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.

Morphine-3-glucuronide: evidence to support its putative role in the development of tolerance to the antinociceptive effects of morphine in the rat.

Antinociceptive tolerance to morphine (MOR) was induced in groups of Sprague-Dawley rats receiving continuous intravenous infusions of morphine sulphate administered by 3 different MOR dosing regimes. At appropriate intervals throughout each infusion period, antinociceptive testing was performed using the tail-flick latency test and blood samples were collected. Groups of saline (SAL)-infused control rats also underwent antinociceptive testing and blood sample collection. Complete antinociceptive tolerance developed during each MOR infusion period and was characterized by a marked decline in the degree of antinociception from values greater than 90% of the maximum possible effect (%MPE) to pre-dosing baseline values. By contrast, %MPE values in SAL-infused control animals and in sham-operated rats were not significantly different from pre-dosing values throughout the infusion period, indicating that the experimental procedures themselves did not contribute to the development of antinociceptive tolerance to MOR. In addition, the rate of MOR tolerance development was inversely proportional to the MOR infusion rate. A very significant inverse relationship was observed between the mean degree of antinociception (%MPE) and the mean plasma molar concentration ratio, [morphine-3-glucuronide]/[MOR], for each of the 3 MOR dosing regimes and for the cumulated data. This relationship showed that near-maximum antinociception was attainable at ratio values less than approximately 0.50, whilst at ratio values above approximately 1.5, little or no antinociception was observed. Although %MPE was highly inversely correlated with the mean plasma morphine-3-glucuronide (M3G) concentrations for rats receiving regimes A and B, this was not the case for rats receiving regime C where antinociceptive tolerance was partially reversed by an increase in the morphine infusion rate part-way through the infusion period. In addition, a poor relationship was observed between %MPE and the mean plasma MOR concentration, possibly due to the confounding presence of M3G in all samples. Thus, we may conclude from this study in Sprague-Dawley rats that irrespective of the rate of antinociceptive tolerance development, the level of antinociception achievable appears to be highly inversely correlated with the mean [M3G]/[MOR] plasma molar concentration ratio and poorly correlated with the plasma MOR concentration, consistent with the notion that it is perhaps the balance between the excitatory effects of M3G and the inhibitory effects of MOR at the functional level which is the important determinant. Further research is required in carefully conducted studies in cancer patients to evaluate the possible contribution of the MOR metabolites, M3G and morphine-6-glucuronide (MbG), to increasing dosing requirements of MOR.(ABSTRACT TRUNCATED AT 400 WORDS)