AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings. Mol Cancer Ther; 9(10); 2641–51.

The large conductance, voltage-and Ca 2؉ -activated K ؉ channel (MaxiK) is expressed in several renal segments and functions in cell volume regulation and flowmediated K ؉ secretion. Previously, we cloned two MaxiK channel isoforms, named rbslo1 and rbslo2, from rabbit renal cells. rbslo1 has a 58-amino acid insertion after the S8 hydrophobic domain, whereas rbslo2 is truncated and cannot be activated. Here we use the sequence differences between the two variants to examine their plasma membrane processing. Plasma membrane localization of rbslo1 and 2 expressed in HEK293 cells was assayed by electrophysiology, immunocytochemistry, and biochemistry studies. Consistent with its functional silence, rbslo2 localized primarily within the cytoplasm, presumably in the endoplasmic reticulum and Golgi region. Coexpression with MaxiK ␤ subunits did not alter the cellular localization of either rbslo1 or rbslo2. When rbslo1 and 2 are cotransfected in non-polarized cells, they colocalized primarily within the cell with only rbslo1 detected at the plasma membrane. When transfected into polarized, medullary-thick ascending limb (mTAL) cells, rbslo1 is expressed at the apical membrane whereas the majority of rbslo2 localized throughout the cytoplasm. Given the high degree of similarity between the two isoforms, we conclude that the cytoplasmic tail of rbslo1 is important for the cell surface expression of MaxiK channels.

The caffeine content of foods and beverages varies considerably, interfering with our ability to obtain valid interpretations in many human studies with regard to the mechanism of action(s) of caffeine and/or its metabolites. The rate of metabolism of caffeine and other xanthine drugs also varies greatly from one individual to another. Therefore, it is extremely important to develop accurate, reliable analytical methods to quantify caffeine and its metabolites in simple and complex matrixes. A simple method is described for the separation and characterization of caffeine and its major metabolites employing capillary electrophoresis (CE) coupled to ultraviolet-absorption and mass spectrometry (MS) detection. After optimization of the electrophoresis separation conditions, a reliable separation of caffeine and 11 of its major metabolites was achieved in 50 mM ammonium carbonate buffer, pH 11.0. The volatile aqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by MS. The CE method achieved baseline resolution for all 12 compounds in less than 30 min. The CE-MS method is suitable for use as a routine procedure for the rapid separation and characterization of caffeine and its metabolites. The usefulness of this method was demonstrated by the extraction, separation, and identification of caffeine and its 11 metabolites from normal urine samples. The urine specimens were first acidified to obtain optimum binding efficiency to the sorbents of the off-line, solid-phase extraction procedure employed here, and an acidified eluent solvent was employed for the desorption step to maximize the recovery of the bound analytes.