Wayne D. Klohs

Inhibitors of tyrosine kinase.

This review covers the literature on significant studies of small molecule inhibitors of the epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), Flk-1, and src family tyrosine kinases from 1 996 through mid-1997. During this period, there has been substantial progress in the discovery of new and highly specific tyrosine kinase inhibitors (TKIs), particularly for the EGFR family. The last 18 months saw a focused effort to discover tyrosine kinase inhibitors with increased potency, increased selectivity, better animal pharmacokinetics, and decreased toxicity. Indeed, some EGFR TKIs are now in clinical trials or are about to enter clinical trials as potential anticancer agents. Potent and selective kinase inhibitors have also been described for PDGFR, but none of these compounds have appeared to advance in the developmental process as far as kinase inhibitors for the EGFR family. Surprisingly, potent and selective inhibitors of receptors involved in neovascularization such as FGFR, Flk-1, or Flt-1 are less prevalent in the literature, and the discovery of TKIs that can inhibit angiogenesis remains a fertile area for drug discovery. Tyrosine kinases continue to remain an extremely attractive target for the design of potent and selective inhibitors that will represent an important new class of therapeutic agents for the treatment of a variety of diseases where current therapy is still insufficient.

Anti-Angiogenic Activity of Selected Receptor Tyrosine Kinase Inhibitors, PD166285 and PD173074: Implications for Combination Treatment with Photodynamic Therapy

Angiogenesis, the formation of new blood vessels from an existing vasculature, is requisite for tumor growth. It entails intercellular coordination of endothelial and tumor cells through angiogenic growth factor signaling. Interruption of these events has implications in the suppression of tumor growth. PD166285, a broad-spectrum receptor tyrosine kinase (RTK) inhibitor, and PD173074, a selective FGFR1TK inhibitor, were evaluated for their anti-angiogenic activity and anti-tumor efficacy in combination with photodynamic therapy (PDT). To evaluate the anti-angiogenic and anti-tumor activities of these compounds, RTK assays, in vitro tumor cell growth and microcapillary formation assays, in vivo murine angiogenesis and anti-tumor efficacy studies utilizing RTK inhibitors in combination with photodynamic therapy were performed. PD166285 inhibited PDGFR-β-, EGFR-, and FGFR1TKs and c-src TK by 50% (IC50) at concentrations between 7−85nM. PD173074 displayed selective inhibitory activity towards FGFR1TK at 26nM. PD173074 demonstrated (>100 fold) selective growth inhibitory action towards human umbilical vein endothelial cells compared with a panel of tumor cell lines. Both PD166285 and PD173074 (at 10nM) inhibited the formation of microcapillaries on Matrigel-coated plastic. In vivo anti-angiogenesis studies in mice revealed that oral administration (p.o.) of either PD166285 (1−25 mg/kg) or PD173074 (25−100 mg/kg) generated dose dependent inhibition of angiogenesis. Against a murine mammary 16c tumor, significantly prolonged tumor regressions were achieved with daily p.o. doses of PD166285 (5−10 mg/kg) or PD173074 (30−60 mg/kg) following PDT compared with PDT alone (p<0.001). Many long-term survivors were also noted in combination treatment groups. PD166285 and PD173074 displayed potent anti-angiogenic and anti-tumor activity and prolonged the duration of anti-tumor response to PDT. Interference in membrane signal transduction by inhibitors of specific RTKs (e.g. FGFR1TK) should result in new chemotherapeutic agents having the ability to limit tumor angiogenesis and regrowth following cytoreductive treatments such as PDT.

discovery and structure-activity studies of a novel series of pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors

Abstract The inhibition of tyrosine kinase-mediated signal transduction pathways represents a therapeutic approach to the intervention of proliferative diseases such as cancer, atherosclerosis, and restenosis. A novel series of pyrido[2,3-d]pyrimidine inhibitors of the PDGFr, bFGFr, and c-Src tyrosine kinases was developed from compound library screening and lead optimization.1 In addition, highly selective inhibitors of the FGFr tyrosine kinase were also discovered and developed from this novel series of pyrido[2,3-d]pyrimidines. The syntheses, biological evaluation, and structure-activity relationships of this series are reported.

Plasma vascular endothelial growth factor and interleukin-8 as biomarkers of antitumor efficacy of a prototypical erbB family tyrosine kinase inhibitor

CI-1033 (N-[4-[N-(3-chloro-4-fluorophenyl)amino-7-[3-(4-morpholynyl)propoxy]quinazolin-6-yl]acrylamide, PD 0183805-mesylate salt) was identified as a potent, selective inhibitor of erbB family tyrosine kinases, which are overexpressed in a number of solid tumors and have been shown to be involved in tumor progression. Because objective response of clinical patients to erbB-targeted therapies like CI-1033 has been observed only in a subset of cancer patients that exhibit the intended molecular targets, much emphasis has been placed on the identification of biomarkers of antitumor efficacy. Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) were considered as potential biomarkers for CI-1033 due to ease of detection in patient plasma and showed roles in angiogenesis and cancer progression and positive regulation by the erbB receptor family. In the present studies, mice bearing established xenografts (A431 epidermoid carcinoma, H125 non–small cell lung carcinoma, SF767 glioblastoma, and MDA-MB-468 mammary carcinoma) were treated with efficacious and subefficacious doses of CI-1033, and plasma levels and xenograft gene expression of VEGF and IL-8 were evaluated. Oral administration of CI-1033 to tumor-bearing mice at efficacious doses resulted in markedly decreased levels of VEGF and/or IL-8 plasma levels and tumor mRNA levels relative to vehicle-treated control mice in xenograft models that exhibited evaluable levels of these markers. In contrast, subefficacious doses of CI-1033 did not significantly affect VEGF or IL-8 levels in any of the xenograft models. These studies indicate that plasma VEGF and IL-8 may have use as biomarkers of antitumor efficacy for epidermal growth factor receptor/erbB–targeted therapies such as CI-1033 and suggest that further clinical study of these markers in cancer patients are warranted.

Cross resistance of pleiotropically drug resistant P338 leukemia cells to the lipophilic antifolates trimetrexate and BW 301U.

Several antifolate compounds were examined for their cytotoxic activity in a pleiotropically resistant P388 cell line (P388R). The sensitivity of P388R cells to methotrexate (MTX) and the lipophilic antifols, metoprine and methotrexate gamma-mono t-butyl ester (MTX-gamma-t-butyl ester) were comparable with that activity observed in the parental cell line (P388S). P388R cells were, however, resistant to 2 other lipophilic antifols, trimetrexate (TMQ) and BW 301U. The degree of resistance to TMQ and BW 301U was 22-fold and 15-fold, respectively and could be partially overcome by the calcium channel blocker, verapamil (VER) or the detergent Tween 80. Transport studies showed that net accumulation of trimetrexate was markedly reduced in P388R cells resulting in a steady-state level which was 25% of the sensitive line. This impaired uptake was reversed by 5 micrograms/ml VER which increased the steady-state to a level comparable to P388S. P388R also exhibited a 50% reduction in the unindirectional influx rate, however, this defect could not be reversed by VER. The resistance of P388R cells to TMQ and BW 301U and their potentiation by VER extends pleiotropic resistance to yet another class of drugs which have important clinical implications.

Evaluation of galactosyltransferase isoenzyme II in a human colon carcinoma-derived cell line, HCT-8☆

Polyacrylamide gel electrophoresis of galactosyltransferase (GT) extracted from a human colon adenocarcinoma cell line, HCT-8, demonstrated the presence of two peaks of activity: a slow-moving peak, referred to as GT-II, and a more anodally migrating peak, designated as GT-I, which was also found for normal human serum. However, if GT solubilized from HCT-8 cells was separated by isoelectric focusing, no unique isoenzymes could be detected. Total GT activity from HCT-8 cells was purified by alpha-lactalbumin-Sepharose affinity chromatography followed by ion exchange chromatography on either DEAE-cellulose or FPLC using a Mono Q anion exchange resin. Three major peaks of activity were resolved from anion exchange chromatography. Electrophoresis of each peak revealed a GT pattern identical with that originally observed for the crude (detergent) solubilized homogenate. No enrichment of either GT-I or GT-II was observed in the three enzyme fractions. The data suggest that GT-II may be an artifactual activity of cancer cells composed of GT-I associated with some contaminating protein.

Lack of effect of corticosteroids and tamoxifen on suramin protein binding and in vitro activity

Stout and colleagues [Proc Am Assoc Cancer Res 1993, 34, p. 298] previously reported that both hydrocortisone and tamoxifen increased the free fraction of suramin in human plasma. We examined several corticosteroids as well as tamoxifen for their effects on suramin protein binding and also evaluated hydrocortisone for its ability to modulate suramin activity in PC-3 and MCF-7 cells. Greater than 99% of the suramin was protein bound in undiluted human plasma. However, the free fraction of suramin was increased with the reduced plasma protein levels and increased suramin concentrations. At concentrations ranging from 1 to 30 microM, neither tamoxifen, hydrocortisone, prednisone nor dexamethasone had any effect on the binding of suramin to human plasma, regardless of protein concentrations. Similar results were observed with fetal calf serum. Hydrocortisone also had no effect on suramin activity against PC-3 and MCF-7 cell in vitro. We conclude from these studies that neither corticosteroids nor tamoxifen affect suramin protein binding or its cytotoxic activity.