Gerald McMahon

One challenging aspect in the clinical development of molecularly targeted therapies, which represent a new and promising approach to treating cancers, has been the identification of a biologically active dose rather than a maximum tolerated dose. The goal of the present study was to identify a pharmacokinetic/pharmacodynamic relationship in preclinical models that could be used to help guide selection of a clinical dose. SU11248, a novel small molecule receptor tyrosine kinase inhibitor with direct antitumor as well as antiangiogenic activity via targeting the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), KIT, and FLT3 receptor tyrosine kinases, was used as the pharmacological agent in these studies. In mouse xenograft models, SU11248 exhibited broad and potent antitumor activity causing regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. To predict the target SU11248 exposure required to achieve antitumor activity in mouse xenograft models, we directly measured target phosphorylation in tumor xenografts before and after SU11248 treatment and correlated this with plasma inhibitor levels. In target modulation studies in vivo, SU11248 selectively inhibited Flk-1/KDR (VEGF receptor 2) and PDGF receptor beta phosphorylation (in a time- and dose-dependent manner) when plasma concentrations of inhibitor reached or exceeded 50-100 ng/ml. Similar results were obtained in a functional assay of VEGF-induced vascular permeability in vivo. Constant inhibition of VEGFR2 and PDGF receptor beta phosphorylation was not required for efficacy; at highly efficacious doses, inhibition was sustained for 12 h of a 24-h dosing interval. The pharmacokinetic/pharmacodynamic relationship established for SU11248 in these preclinical studies has aided in the design, selection, and evaluation of dosing regimens being tested in human trials.

A new class of protein tyrosine kinase inhibitors was identified that is based on an oxindole core (indolinones). Two compounds from this class inhibited the kinase activity of fibroblast growth factor receptor 1 (FGFR1) and showed differential specificity toward other receptor tyrosine kinases. Crystal structures of the tyrosine kinase domain of FGFR1 in complex with the two compounds were determined. The oxindole occupies the site in which the adenine of adenosine triphosphate binds, whereas the moieties that extend from the oxindole contact residues in the hinge region between the two kinase lobes. The more specific inhibitor of FGFR1 induces a conformational change in the nucleotide-binding loop. This structural information will facilitate the design of new inhibitors for use in the treatment of cancer and other diseases in which cell signaling by tyrosine kinases plays a crucial role in disease pathogenesis.

SU5416, a novel synthetic compound, is a potent and selective inhibitor of the Flk-1/KDR receptor tyrosine kinase that is presently under evaluation in Phase I clinical studies for the treatment of human cancers. SU5416 was shown to inhibit vascular endothelial growth factor-dependent mitogenesis of human endothelial cells without inhibiting the growth of a variety of tumor cells in vitro. In contrast, systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals, supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate gene transcription in response to peroxisome proliferators and fatty acids. PPARs also play an important role in the regulation of adipocyte differentiation. It is unclear, however, what naturally occurring compounds activate each of the PPAR subtypes. To address this issue, a screening assay was established using heterologous fusions of the bacterial tetracycline repressor to several members of the peroxisome proliferator-activated receptor (PPAR) family. This assay was employed to compare the activation of PPAR family members by known PPAR activators including peroxisome proliferators and fatty acids. Interestingly, the activation of PPARs by fatty acids was partially inhibited by the cyclooxygenase inhibitor indomethacin, which prevents prostaglandin synthesis. Indeed, prostaglandins PGA1 and 2, PGD1 and 2, and PGJ2-activated PPARs, while a number of other prostaglandins had no effect. We also screened a variety of hydroxyeicosatetraenoic acids (HETEs) for the ability to activate PPARs. 8(S)-HETE, but not other (S)-HETEs, was a strong activator of PPARα. Remarkably, PPAR activation by 8(S)-HETE was stereoselective. In addition, 8(S)-HETE was able to induce differentiation of 3T3-L1 preadipocytes. These results indicate that PPARs are differentially activated by naturally occurring eicosanoids and related molecules. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate gene transcription in response to peroxisome proliferators and fatty acids. PPARs also play an important role in the regulation of adipocyte differentiation. It is unclear, however, what naturally occurring compounds activate each of the PPAR subtypes. To address this issue, a screening assay was established using heterologous fusions of the bacterial tetracycline repressor to several members of the peroxisome proliferator-activated receptor (PPAR) family. This assay was employed to compare the activation of PPAR family members by known PPAR activators including peroxisome proliferators and fatty acids. Interestingly, the activation of PPARs by fatty acids was partially inhibited by the cyclooxygenase inhibitor indomethacin, which prevents prostaglandin synthesis. Indeed, prostaglandins PGA1 and 2, PGD1 and 2, and PGJ2-activated PPARs, while a number of other prostaglandins had no effect. We also screened a variety of hydroxyeicosatetraenoic acids (HETEs) for the ability to activate PPARs. 8(S)-HETE, but not other (S)-HETEs, was a strong activator of PPARα. Remarkably, PPAR activation by 8(S)-HETE was stereoselective. In addition, 8(S)-HETE was able to induce differentiation of 3T3-L1 preadipocytes. These results indicate that PPARs are differentially activated by naturally occurring eicosanoids and related molecules.