Src up-regulation is a common event in human cancers. In colorectal cancer, increased Src levels are an indicator of poor prognosis, and progression to metastatic disease is associated with substantial increases in Src activity. Therefore, we examined the activity of SKI-606, a potent inhibitor of Src and Abl kinases, against colon tumor lines in vitro and in s.c. tumor xenograft models. SKI-606 inhibited Src autophosphorylation with an IC 50 of f0.25 Mmol/L in HT29 cells. Phosphorylation of Tyr 925 of focal adhesion kinase, a Src substrate, was reduced by similar concentrations of inhibitor. Antiproliferative activity on plastic did not correlate with Src inhibition in either HT29 or Colo205 cells (IC 50 s, 1.5 and 2.5 Mmol/L, respectively), although submicromolar concentrations of SKI-606 inhibited HT29 cell colony formation in soft agar. SKI-606 also caused loosely aggregated Colo205 spheroids to condense into compact spheroids. On oral administration to nude mice at the lowest efficacious dose, peak plasma concentrations of f3 Mmol/L, an oral bioavailability of 18%, and a t 1/2 of 8.6 hours were observed. SKI-606 was orally active in s.c. colon tumor xenograft models and caused substantial reductions in Src autophosphorylation on Tyr 418 in HT29 and Colo205 tumors. SKI-606 inhibited HT29 tumor growth on once daily administration, whereas twice daily administration was necessary to inhibit Colo205, HCT116, and DLD1 tumor growth. These results support development of SKI-606 as a therapeutic agent for treatment of colorectal cancer. (Cancer Res 2005; 65(12): 5358-64)

Organic cation transporter (OCT) 2, multidrug and toxin extrusion protein (MATE) 1, and MATE2K mediate the renal secretion of various cationic drugs and can serve as the loci of drug-drug interactions (DDI). To support the evaluation of cynomolgus monkey as a surrogate model for studying human organic cation transporters, monkey genes were cloned and shown to have a high degree of amino acid sequence identity versus their human counterparts (93.7, 94.7, and 95.4% for OCT2, MATE1, and MATE2K, respectively). Subsequently, the three transporters were individually stably expressed in human embryonic kidney (HEK) 293 cells and their properties (substrate selectivity, time course, pH dependence, and kinetics) were found to be comparable to the corresponding human form. For example, six known human cation transporter inhibitors, including pyrimethamine (PYR), showed generally similar IC 50 values against the monkey transporters (within sixfold). Consistent with the in vitro inhibition of metformin (MFM) transport by PYR (IC 50 for cynomolgus OCT2, MATE1, and MATE2K; 1.2 6 0.38, 0.17 6 0.04, and 0.25 6 0.04 mM, respectively), intravenous pretreatment of monkeys with PYR (0.5 mg/kg) decreased the clearance (54 6 9%) and increased in the area under the plasma concentration-time curve of MFM (AUC ratio versus control = 2.23; 90% confidence interval of 1.57 to 3.17). These findings suggest that the cynomolgus monkey may have some utility in support of in vitroin vivo extrapolations (IVIVEs) involving the inhibition of renal OCT2 and MATEs. In turn, cynomolgus monkey-enabled IVIVEs may inform human DDI risk assessment. IntroductionIt is widely appreciated that renal elimination of cations, drug, toxin, or endogenous metabolite related, is achieved not only by glomerular filtration but also by active transport processes that facilitate their tubular secretion and reabsorption. Indeed, the mechanisms governing the renal elimination of organic cations have been studied in detail, and the transporters facilitating their tubular uptake and extrusion have been identified (Okuda et al., 1996;Otsuka et al., 2005;Masuda et al., 2006).Secretion of organic cations in renal proximal tubules involves at least two distinct transporters, located in the basolateral and apical membranes of proximal tubule cells (Motohashi et al., 2002Masuda et al., 2006). Specifically, organic cations, like metformin (MFM), 1-methyl-4-phenylpyridinium (MPP + ), tetraethylammonium (TEA), and cimetidine (CMD), are taken up from the circulation by organic cation transporter 2 (OCT2) expressed on the basolateral domain of renal proximal tubular cells. In turn, uptake is followed by efflux into the tubular fluid by multidrug and toxin extrusion protein (MATE) 1 and MATE2K expressed on the apical domain of the same cells. Therefore, OCT2 and MATEs function coordinately to mediate vectorial transport of certain cationic drugs, from blood to tubular fluid, which represents the tubular secretion clearance component of total renal clearance. Perturbation of c...

An LC-MS/MS assay was developed and fully validated for the simultaneous quantitation of two coadministered human monoclonal antibodies (mAbs), mAb-A and mAb-B of IgG4 subclass, in monkey serum. The total serum proteins were digested with trypsin at 50 °C for 30 min after methanol denaturation and precipitation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated with a C18 column (2.1 × 100 mm, 1.7 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. Four peptides, a unique peptide for each mAb and two confirmatory peptides from different antibody domains, were simultaneously quantified by LC-MS/MS in the multiple reaction-monitoring mode. Stable isotopically labeled peptides with flanking amino acids on C- and N-terminals were used as internal standards to minimize the variability during sample processing and detection. The LC-MS/MS assay showed lower limit of quantitation (LLOQ) at 5 μg/mL for mAb-A and 25 μg/mL for mAb-B. The intra- and interassay precision (%CV) was within 10.0% and 8.1%, respectively, and the accuracy (%Dev) was within ±5.4% for all the peptides. Other validation parameters, including sensitivity, selectivity, dilution linearity, processing recovery and matrix effect, autosampler carryover, run size, stability, and data reproducibility, were all evaluated. The confirmatory peptides played a critical role in confirming quantitation accuracy and the integrity of the drugs in the study samples. The robustness of the LC-MS/MS assay and the data agreement with the ligand binding data demonstrated that LC-MS/MS is a reliable and complementary approach for the quantitation of coadministered antibody drugs.