Plasma concentrations of sunitinib and its active metabolite, SU12662, were determined on days 1 and 28 of cycles 1 to 4. Plasma concentrations of both were determined predose by a liquid chromatography/mass spectrometry method at BASi (West Lafayette, IN), with a lower limit of detection of 0.1 ng/mL [23 (link)].
Predose plasma samples were collected on days 1 and 28 of each cycle for assessment of soluble proteins that may be correlates of angiogenic activity and/or pharmacodynamic inhibition of VEGF receptor-mediated signaling [24 -26 (link)]. Each of the soluble proteins was analyzed with enzyme-linked immunosorbent assay (ELISA) kits (all manufactured by R&D Systems, Minneapolis, MN). The (VEGF)-A ELISA assay measures the VEGF-A165 and VEGF-A121 isoforms. The PlGF assay primarily measures PlGF-1. sVEGFR-2 was quantified with an ELISA that measures the extracellular (soluble) domain of VEGFR-2 [27 (link)]. Similarly, an ELISA component kit that measures the extracellular (soluble) domain of VEGFR-3 was employed. Both the sVEGFR-2 and sVEGFR-3 assays are calibrated against recombinant proteins consisting of the full-length extracellular domain of the respective receptors. No cross-reactivity or interference is detected between the two receptors in the ELISA assays. Though the structural details of sVEGFR-2 and sVEGFR-3 remain to be established, plasma-derived sVEGFR-2 has been reported to be heavily glycosylated and to have a molecular weight of ~90 kDa when de-glycosylated; the size is similar to that or insect-derived recombinant sVEGFR-2, implying that endogenous sVEGFR-2 may be similar in structure to recombinant versions of the VEGFR-2 extraceullar domain [27 (link)]. All ELISA assays were run under Good Laboratory Practice conditions, and performance specifications of each ELISA were validated for their intended purpose, as per established guidelines [28 (link)].
Predose plasma samples were collected on days 1 and 28 of each cycle for assessment of soluble proteins that may be correlates of angiogenic activity and/or pharmacodynamic inhibition of VEGF receptor-mediated signaling [24 -26 (link)]. Each of the soluble proteins was analyzed with enzyme-linked immunosorbent assay (ELISA) kits (all manufactured by R&D Systems, Minneapolis, MN). The (VEGF)-A ELISA assay measures the VEGF-A165 and VEGF-A121 isoforms. The PlGF assay primarily measures PlGF-1. sVEGFR-2 was quantified with an ELISA that measures the extracellular (soluble) domain of VEGFR-2 [27 (link)]. Similarly, an ELISA component kit that measures the extracellular (soluble) domain of VEGFR-3 was employed. Both the sVEGFR-2 and sVEGFR-3 assays are calibrated against recombinant proteins consisting of the full-length extracellular domain of the respective receptors. No cross-reactivity or interference is detected between the two receptors in the ELISA assays. Though the structural details of sVEGFR-2 and sVEGFR-3 remain to be established, plasma-derived sVEGFR-2 has been reported to be heavily glycosylated and to have a molecular weight of ~90 kDa when de-glycosylated; the size is similar to that or insect-derived recombinant sVEGFR-2, implying that endogenous sVEGFR-2 may be similar in structure to recombinant versions of the VEGFR-2 extraceullar domain [27 (link)]. All ELISA assays were run under Good Laboratory Practice conditions, and performance specifications of each ELISA were validated for their intended purpose, as per established guidelines [28 (link)].
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