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SUGEN, Inc., Preclinical Research and Translational Medicine, South San Francisco, California, USA
1Correspondence: SUGEN, Inc., 230 E. Grand Ave., South San Francisco, CA 94080, USA. E-mail: Douglas-Laird{at}sugen.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: angiogenesis • kinase inhibitor • VEGF • platelet-derived growth factor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The exact nature of this switch is unknown, but many of its key mediators have been characterized. These include transcription factors (such as hypoxia-inducible factors 1 and 2), metalloproteinases, and receptor tyrosine kinases (RTKs) (2
3
4
5)
. RTKs participate in the transmission of proliferation, migration, differentiation, and survival signals from tumor cells and neighboring host-derived stromal cells to endothelial cells that comprise the tumor vasculature (4
, 5)
. These RTKs include the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR; angiopoietin and ephrin receptors, which are largely dedicated to angiogenesis; and fibroblast and platelet-derived growth factor receptors (FGFR and PDGFR), which play a role in angiogenesis in addition to other diverse normal and aberrant signaling processes (4
5
6
7
8)
.
RTKs have proved to be attractive targets for therapeutic intervention, and much progress has been made in recent years in developing selective inhibitors of their activity (5
, 9)
. We recently reported the development of a novel small molecule oxindole compound, SU6668, which is a selective inhibitor of Flk-1/KDR, PDGFRß, and FGFR (10)
. In biochemical assays, SU6668 was a competitive (with respect to ATP) inhibitor of Flk-1/KDR, PDGFRß, and FGFR1, with IC50 values of 2.1, 0.008, and 1.2 µM, respectively. In cellular assays, it inhibited VEGF- and PDGF-dependent signaling with IC50 values of 0.5 and 1.0 µM, respectively (see ref 10
and G. Li, unpublished observations).
The ability of SU6668 to inhibit signaling via Flk-1/KDR and PDGFR makes SU6668 well adapted to inhibit multiple tumor signal transduction pathways critical to tumor growth and survival. Consistent with these properties, SU6668 potently inhibits angiogenic signaling in cellular assays and exhibits strong in vivo antiangiogenic activity in intravital tumor microscopy studies (10)
. In contrast, SU6668 is not a potent inhibitor of human cancer cells grown in culture (10)
. Once-daily oral administration of SU6668 inhibited the subcutaneous (s.c.) growth of a panel of human tumor xenografts of diverse origin implanted in athymic mice (10)
. Strikingly, SU6668 treatment was shown to stably regress large (400–2000 mm3) established A431 tumor xenografts (10)
. After withdrawal of treatment with SU6668, the majority of tumors remained stably regressed for the remainder of the lives of the mice (see ref 10
and J. Carver, unpublished observations). When regrowth did occur after cessation of treatment, tumors could be induced to regress again by a second round of SU6668 treatment (10)
.
The ability of SU6668 to rapidly regress A431 tumors was somewhat unexpected, given that antiangiogenic compounds administered as single agents might be predicted to lead to tumor growth arrest rather than tumor regression. To understand the activity of SU6668 in mouse xenograft models and to develop potential biomarker candidates for SU6668 clinical development, we undertook additional studies to examine the responses of a diverse set of large established tumor xenografts to SU6668 treatment. In these studies, we found SU6668 treatment to be highly efficacious, resulting in tumor regression, growth arrest, or growth delay in all models examined. We then set out to characterize the processes underlying SU6668-driven tumor responses at the histological, molecular, and biochemical levels. We also explored the pharmacokinetic/pharmacodynamic relationship for SU6668-induced target modulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A431 (human epidermoid), Colo205 (human colon), H460 (human non-small cell lung carcinoma), SF767T (human glioma), C6 (rat glioma), and A375 (human melanoma) tumor cells were obtained and cultured as described (11)
. HT29 and PC3 cells were obtained from the National Cancer Institutes and cultured respectively in McCoy’s 5a or RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies, Rockville, MD). Tumor cells (3x106 [SF767T, C6], 1x107 [PC3], or 5x106 cells [all other cell lines]) were implanted s.c. into the hind flank of mice on day 0 as described previously (12)
. To facilitate their growth, we suspended PC3 cells in Matrigel (Becton Dickinson Labware, Franklin Lakes, NJ) diluted 1:1 in phosphate-buffered saline (PBS) before implantation with a 25 gauge needle. Daily treatment with SU6668 or vehicle commenced when tumors had reached the indicated average sizes. SU6668 was delivered orally (PO) by gavage. In all cases, a Cremophor-based vehicle was used (10)
except for in vivo Flk-1/KDR phosphorylation studies, where a carboxy methylcellulose suspension was used. We selected 200 mg/kg once daily as the standard dosage of SU6668 for these studies on the basis of earlier work showing this was the maximally efficacious dosage against newly implanted A431 tumors (10)
. Tumor growth was measured twice a week using vernier calipers for the duration of treatment. Tumor volumes were calculated as a product of length x width x height.
Histology and immunohistochemistry
Tumor specimens were harvested; samples were fixed in 10% buffered formalin for 24 h, then transferred to 70% EtOH. The specimens were subsequently paraffin-embedded and sectioned. General tissue morphology and mitotic figures were visualized by hematoxylin and eosin staining. Apoptotic cells, tumor microvessels, and proliferating cells were visualized using immunohistochemical detection of active caspase-3, CD31, and Ki-67, respectively. Active caspase-3 was detected using a 1:400 dilution of a polyclonal anticleaved caspase-3 antibody (New England BioLabs, Beverly, MA, cat#9661) and visualized using a biotinylated anti-rabbit antibody (Vector Laboratories, Burlingame, CA). CD31 was detected using a 1:100 dilution of the rat anti-mouse monoclonal antibody MEC 13.3 (BD PharMingen, San Diego, CA, cat#01951) and visualized using the labeled streptavidin biotin plus kit (DAKO, Carpinteria, CA). Ki-67 was detected using a 1:250 dilution of monoclonal MIB-1 (Immunotech, Westbrook, ME; cat#0505) and visualized using a biotinylated polyclonal mouse anti-rat antibody (Zymed, South San Francisco, CA). All immunostained sections were counterstained using hematoxylin.
Three or four fields per tumor were scored (in blinded fashion) for mitotic figures at 400x magnification using a Leitz Laborlux S microscope (Leica, Deerfield, IL) and results were averaged. Tumor microvessel elements were scored independently (three or four fields per tumor) at 100x magnification by two blinded observers and tumor microvessel densities (i.e., number of microvessel elements/field) were calculated. The extensive tumor destruction induced by SU6668 at 24 h in A431 tumors precluded scoring of three entire fields in some of the tumors.
Transcript imaging using DNA arrays
RNA was prepared from tumors by using the Nucleospin RNA II kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. For screening for changes in transcript levels for genes implicated in angiogenesis, 32P-labeled transcript-specific cDNA probes were generated from 6 µg of total cellular RNA and used to probe Human Angiogenesis-1 Superarrays (SuperArray, Bethesda, MD, cat#hGEA9908030) according to the manufacturer’s instructions. Data were collected on a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and analyzed using ArrayVision version 5.1 software (Imaging Research, Ontario, Canada).
In vivo target modulation studies
For Flk-1/KDR modulation studies, mice bearing A375 xenografts (
300 mm3) were treated with a single oral dose of vehicle or SU6668 (50, 100, or 200 mg/kg). Subsequently, they received an intravenous (i.v.) injection of VEGF (1 µg) via their tail vein 30 min before death at the indicated times post-treatment with SU6668. Tumors were resected, snap-frozen on dry ice, pulverized, and lysed in HNTG buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1.5 mM MgCl2, 10% v/v glycerol, 1% Triton X-100, 1% sodium orthovanadate, 2 mM NaF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A). Phosphotyrosine-containing proteins were immunoprecipitated from 2 mg of total protein from individual tumors by using an antiphosphotyrosine agarose-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA; sc-508AC), resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and immunoblotted using an antibody recognizing total Flk-1/KDR (Santa Cruz Biotechnology; sc-315). Comparable Flk-1/KDR levels in lysates were verified by parallel Flk-1/KDR immunoprecipitation and immunoblotting.
For PDGFRß modulation studies, mice bearing SF767T xenografts (300–400 mm3) were treated with a single oral dose of vehicle or SU6668. Tumors were resected, snap-frozen on dry ice, pulverized, and lysed in HNTG buffer (also containing 0.1 mM phenylmethylsulfonylfluoride). Total PDGFRß was immunoprecipitated from 1 mg of total protein from individual tumors by using an anti-PDGFRß polyclonal antibody (Upstate Biotechnology, Charlottesville, VA), resolved by SDS-PAGE, transferred to nitrocellulose, and sequentially immunoblotted using antibodies recognizing phosphotyrosine and total PDGFRß (loading control).
Miles assay for vascular permeability
The Miles assay for vascular permeability (13)
was adapted to athymic mice as follows. Mice were given a single oral dose of SU6668 or its vehicle alone. Five hours later, 100 µL of a 0.5% solution of Evan’s blue dye (Sigma, St. Louis, MO) was administered i.v. via the tail vein. One hour later, mice were injected intradermally (on their back) with 100 ng of VEGF (in 20 µL of PBS) or PBS alone. After 1 h, the extent of VEGF-dependent dye leakage from the vasculature into skin was assessed visually, photographed, and qualitatively scored.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To determine whether this potent activity against large established tumors was unique to the A431 xenograft model, we examined the response to SU6668 treatment of different tumor xenografts established in athymic mice.
Oral SU6668 treatment at 200 mg/kg once and twice daily induced growth arrest and regression, respectively, of large ( 500 mm3) established Colo205 (colon-derived) tumors (Fig. 1
). Histological examination of tumors removed at the end of the study revealed that in contrast to tumors from vehicle-treated animals, tumors from animals treated with SU6668 twice daily were completely acellular, with the exception of a thin (several cells deep) front of apparently intact tumor cells (Fig. 1)
. Surprisingly, the extent of tumor tissue damage observed at the histological level was almost as great after once-daily administration, even though these tumors did not decrease in size (Fig. 1)
. The results of similar studies carried out using large tumor xenografts established from multiple cancer cell lines are summarized in Table 1
. In all cases, a dramatic response to SU6668 treatment was seen. This took the form of tumor regression, tumor growth arrest (stasis), or tumor growth delay. End-of-study histological examination of resected tumors showed that SU6668 induced a loss of cellularity in all models except the C6 rat glioma xenograft, which exhibited steady, albeit delayed, tumor growth despite protracted treatment with SU6668 for 13 days (Table 1)
. The ability of SU6668 to induce loss of cellularity was dose dependent (Table 1)
.
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Mechanisms underlying the effect of SU6668 on established tumors
Having characterized the response of numerous tumor xenografts to SU6668 treatment, we examined the bases for tumor regression at histological and molecular levels in the most responsive tumor model identified to date, the A431 human epidermoid carcinoma xenograft. A431 tumors were established to average volumes of 900 mm3, and once daily treatment at 200 mg/kg was initiated. Tumors were harvested at multiple time points (1, 6, 12, and 24 h and 37 days) from three animals per time point after the initiation of SU6668 treatment. Resected tumors were then subdivided, with half fixed for histological examination and the remaining half frozen for molecular analyses.
To determine whether apoptosis might be implicated in SU6668-driven A431 tumor regression, we immunostained tumor histological sections using an antibody specific for active caspase-3 as a marker for apoptosis (Fig. 2
A). In the growing front of untreated tumors, occasional apoptotic tumor cells were evident, but apoptotic endothelial cells (readily identifiable based on cell and vessel morphology) were seldom seen. In contrast, numerous apoptotic endothelial cells (often contiguous) were evident by 6 h after the initiation of treatment (Fig. 2A
). By 24 h, the former growing tumor front was greatly eroded and flanked by active caspase-3-positive tumor cells and cellular debris (Fig. 2A
). These observations were reproduced in a second study, in which starting A431 tumor size was 500 mm3 (J. Carver and D. Laird, unpublished observations). By 24 h, tumor microvessel density (MVD), as detected by anti-CD31 immunostaining, was reduced by 50% relative to pretreatment values in surviving islands of tumor tissue (Fig. 2B
, Table 2
).
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Ki-67 immunostaining of the resected tumors was used to visualize proliferating cells. By 24 h after the initiation of treatment, a dramatic reduction in vital tumor tissue was evident. Moreover, the remaining cells exhibited reduced levels of Ki-67 positivity, indicating reduced proliferation and/or reduced viability (Fig. 3
). Consistent with this finding, the mitotic index of the surviving cells was reduced by 70% at 24 h (Table 2)
compared with the tumors harvested from untreated animals. Tumor tissue continued to deteriorate over time. By 37 days of treatment, the residual tumor mass (regressed 70% from its starting volume; J. Carver, unpublished observations) was almost completely acellular (Fig. 3)
.
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SU6668 treatment exerted early antivascular effects in three other tumor xenografts: Colo205, SF767T, and C6 (Table 2)
. The reduction of MVD after 3 days of treatment at 200 mg/kg in SF767T and C6 tumors was comparable to that seen for A431 tumors after a single treatment (50% reduction). Colo205 tumors were less sensitive, with a significant reduction in tumor MVD evident at 3 days only after administration at 200 mg/kg twice daily (Table 2)
. In Colo205 and C6 tumors, reductions in tumor cell proliferation were evident (Table 2)
, which correlated with the extent of reduction in tumor MVD, supporting the hypothesis that reduced proliferation is secondary to reduced tumor vascularization. SF767T tumor cell proliferation was not significantly reduced at 3 days (Table 2)
; in a separate study, treatment for 11 days at 200 mg/kg/day induced a reduction of 72% (P=0.001) in the mitotic index in this model. SU6668 treatment induced tumor cell apoptosis in Colo205 and (to a lesser extent) in SF767T tumors, with protracted kinetics relative to A431 tumors (J. Carver and D. Laird, unpublished observations).
To further explore these histological changes as potential biomarkers for SU6668 action in vivo and subsequently in the clinic, we examined their dose and time dependence. We found that SU6668-induced reductions in tumor MVD and proliferation were dose dependent in A431 tumors, with a maximal response evident at 200 mg/kg (Fig. 4
). Similarly, SU6668-induced changes in tumor MVD were dose dependent in C6 tumors (Fig. 5
). Reductions in tumor MVD were found to be progressive in SF767T tumors, where MVD was reduced by 46% at 3 days (P=0.05; see Table 2
) and 80% (P=0.01) at 13 days relative to predose tumors. Colo205 tumors were selected for a study of the time and dose dependence of apoptosis because they are highly responsive to SU6668 but have somewhat delayed kinetics (Fig. 1
, Table 1
). SU6668 administered at 200 mg/kg once daily induced tumor cell apoptosis that was evident by 1 day after treatment and progressively increased until most of the tumor mass was destroyed (Fig. 6
). This process was accelerated and enhanced after administration of SU6668 at 200 mg/kg twice daily (Fig. 6)
. Hence, SU6668-induced histological changes in tumors were shown to be both dose and time dependent.
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The induction of VEGF expression, mediated by hypoxia-inducible factor 1 
, is a sensitive indicator of hypoxic status (2)
. Consistent with the rapid killing of tumor vessels evident 6 h after SU6668 treatment (Fig. 2A
and J. Carver and D. Laird, unpublished observations), VEGF transcript levels were found to be elevated two- to threefold in RNA isolated from A431, Colo205, and SF767T tumors 6 h after treatment (C6 tumors were not evaluated), returning to near-normal levels by 24 h (Table 3
).
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SU6668 inhibits Flk-1/KDR and PDGFRß in vivo
Because SU6668 is an inhibitor of Flk-1/KDR and PDGFRß in biochemical and cellular assays (10)
, we anticipated its ability to induce tumor microvessel apoptosis and reduce tumor MVD stemmed from inhibition of these targets in tumors. To determine directly whether SU6668 inhibits Flk-1/KDR signaling in vivo, the ability of SU6668 to inhibit total receptor phosphotyrosine levels in tumors was determined. The human melanoma xenograft A375 was selected because the tumor cells themselves express KDR (14)
, resulting in an enhanced overall level of Flk-1/KDR in tumor xenografts beyond that contributed by the vasculature alone (SU6668 is highly efficacious in suppressing the growth of newly implanted A375 tumors but has not been evaluated in efficacy studies using large established A375 tumors [J. Carver and D. Laird, unpublished observations]). Total phosphotyrosine-containing proteins were immunoprecipitated from tumor lysates and immunoblotted for Flk-1/KDR. Thus, the quantity of Flk-1/KDR immunoprecipitated reflects Flk-1/KDR phosphorylation status, a readout for Flk-1/KDR activity. Flk-1/KDR phosphorylation levels were unchanged in vehicle-treated animals but suppressed by SU6668 in a dose- and time-dependent manner (Fig. 7
). Treatment at 200 mg/kg was associated with sustained inhibition of Flk-1/KDR phosphorylation starting at or before 1.5 h after treatment and persisting until at least 12 h after treatment. Partial recovery was seen at 24 h after treatment. SU6668 administered at 100 mg/kg was highly efficacious, with inhibition evident from 1.5 h through at least 9 h and partial recovery at 16 h. In contrast, SU6668 administered at 50 mg/kg was less efficacious, with strong inhibition evident only 3–6 h after treatment and partial inhibition sustained through 12 h. Total Flk-1/KDR levels in lysates were determined by parallel Flk-1/KDR immunoprecipitation and immunoblotting and generally found to be comparable between samples (Fig. 7)
. Pharmacokinetic analyses of plasma samples taken from the animals used in this and similar target modulation studies indicate that inhibition of Flk-1/KDR phosphorylation in tumors is associated with sustained SU6668 plasma concentrations of 
1 µg/ml (J. Sukbuntherng, J. Haznedar, and D. Mendel, unpublished observations).
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To confirm that SU6668 inhibits Flk-1/KDR-dependent signaling in the vasculature, we assessed the ability of SU6668 to inhibit VEGF-induced vascular permeability in mouse skin using the Miles assay (13)
. In vehicle-pretreated mice, an intradermal VEGF injection induced substantial leakage of Evan’s blue dye from the circulation. In contrast, pretreatment with SU6668 for 6 h inhibited dye leakage into the skin in a dose-dependent manner (Fig. 8
). Consistent with the effects on tumor Flk-1/KDR phosphorylation described previously, administration at 200 and 75 mg/kg almost completely abolished VEGF-induced dye leakage whereas administration at 25 mg/kg was ineffective. These data demonstrate, using independent biochemical and biological readouts, that SU6668 inhibits Flk-1/KDR in vivo in a dose-dependent manner.
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In biochemical and cellular assays, SU6668 is also a potent inhibitor of PDGFRß, which is implicated in the proliferation and survival of host cells supporting tumor growth, survival, and angiogenesis (15
, 16)
. To assess whether SU6668 inhibits PDGFRß signaling in vivo, the ability of SU6668 to inhibit PDGFRß phosphorylation in tumors was determined. For these studies, we used the SF767T human glioma xenograft, which expresses PDGFRß. We found that SU6668 could inhibit PDGFRß phosphorylation in SF767T tumors (Fig. 9
). Hence, we have confirmed that SU6668 potently inhibits its putative targets Flk-1/KDR and PDGFRß in tumors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. VEGF is the critical factor promoting survival of immature endothelial cells in normal and oncogenic contexts, and its withdrawal induces apoptotic death in cultured human umbilical vein endothelial cells (18)
and in tumor endothelia (19)
. PDGF and FGF are proliferative factors for endothelial cells and supporting host cells such as fibroblasts and pericytes, which provide survival factors and mechanical support to the tumor vasculature (5
, 15
, 16
, 20)
. SU6668 was selected for development for its ability to inhibit VEGF, PDGF, and FGF receptors, suggesting it should function as a potent antiangiogenic and antitumor agent (10)
.
In athymic mice, SU6668 was efficacious against all newly implanted s.c. tumor xenografts tested (10)
. SU6668 treatment of murine colon tumor cells introduced into murine livers via intrasplenic injection was recently shown to result in elevated levels of apoptosis in tumor microvessels and tumor cells (21)
. This broad efficacy is consistent with SU6668 targeting primarily tumor vasculature rather than the tumor cells themselves. We have shown here that it is also efficacious against large established epidermoid (A431), colon (Colo205 and HT29), prostate (PC3), lung (H460), and glioma (SF767T and C6) tumor xenografts in athymic mice (Table 1)
. Of the seven tumors evaluated, SU6668 treatment resulted in regression of four (A431, Colo205, HT29, and PC3), growth arrest of two (H460 and SF767T), and growth delay of one (C6). Histological examination of A431, Colo205, H460, SF767T, and C6 tumors after protracted treatment with SU6668 revealed a dramatic loss in tumor cellularity in all cases except C6 (Table 1)
.
Because of these effects after chronic administration of SU6668, we examined the acute effects of SU6668 on tumors at the histological, molecular, and biochemical levels. SU6668 treatment induced extensive A431 tumor vessel apoptosis at 6 h, followed by apoptosis of tumor cells evident at 24 h. This sequence of events is mirrored in observations of cultured human endothelial cells in which SU6668 was found to counteract VEGF-induced survival after serum withdrawal, using cleavage of the caspase-3 substrate poly-ADP-ribosyl polymerase as a readout for apoptosis (G. Li, unpublished observations). Such a mechanism of action is consistent with the antiangiogenic activity of SU6668 in newly implanted tumors and its comparative lack of potency against cultured tumor cells (10)
. Most critical, the rapid induction of apoptosis in tumor vessels by SU6668 is consistent with its ability to inhibit Flk-1/KDR in vasculature (Miles assay) and to inhibit Flk-1/KDR and PDGFRß in tumors. The data presented here can be synthesized into a model for SU6668-induced A431 tumor killing whereby SU6668 treatment blocks survival signals received by vessels via Flk-1/KDR and PDGFRß, resulting in vessel death by apoptosis, which in turn leads to tumor cell stress and subsequent apoptosis.
SU6668 treatment rapidly induced apoptosis of tumor microvessels (evident 6 h after treatment) in Colo205 and SF767T tumors (J. Carver and D. Laird, unpublished observations), resulting in decreased tumor MVD, evident by 3 days after treatment (Table 2)
. A similar rapid reduction in MVD was evident in C6 tumors from treated animals (Table 2
, Fig. 5
; acute caspase-3 activation was not examined in this model). SU6668 treatment rapidly (detected 6 h after treatment) induced expression of VEGF transcripts in multiple tumor models, suggesting that vessel destruction resulted in tumors suffering hypoxic stress. As in A431 tumors, these responses in tumor vasculature were associated with extensive tumor cell apoptosis, most dramatically in Colo205 tumors (Fig. 6)
. In all models examined, histological changes induced by SU6668 (in MVD, proliferation, and apoptosis) occurred in a dose- and time-dependent manner. Hence, these markers show potential utility as biomarkers for monitoring clinical responses to SU6668 and are being evaluated for that purpose in ongoing clinical trials.
In A431, Colo205, and H460 tumors, SU6668 ultimately induced complete or near-complete tumor destruction. However, in SF767T tumors extended SU6668 treatment resulted in only partial loss of cellularity (Table 1)
and decreased tumor vascularization and proliferation. Moreover, C6 tumors exhibited reduced MVD and proliferation but continued to grow slowly without evident loss of cellularity (Tables 1
and 2)
. Because all tumor models examined so far are fast growing in vivo, differences in tumor growth rates are unlikely to be the key determinants of differential tumor responses to SU6668 treatment. The bases for these differential responses to treatment are unclear, but might include either or both of two possibilities. First, there may be qualitative or quantitative differences in the growth/survival factors supplied by the tumor cells, in basal functional vascularization or in the extent of microvessel maturation (i.e., the extent to which microvessels are mechanically supported by pericytes (22
, 23)
. Indeed, preliminary analyses suggest there are substantial qualitative and quantitative differences in the angiogenic ligands expressed by these tumor xenografts (G. Li, unpublished observations). Second, there may be intrinsic differences in the resistance of tumor cell lines to hypoxic stress. This is consistent with the continued survival/delayed growth of SF767T and C6 tumors in treated animals despite the profound antiangiogenic effect of acute and chronic SU6668 administration (Tables 1
and 2
; J. Carver and D. Laird, unpublished observations). Ongoing preclinical studies with SU6668 aim to correlate pretreatment tumor properties with differing responses of tumors to SU6668 treatment and to identify which early tumor responses are predictive for ultimate therapeutic outcome. The ability to predict outcome on the basis of either or both underlying basal tumor characteristics and early tumor responses, and to stratify patients accordingly, could have great clinical significance.
We have also explored the pharmacokinetic/pharmacodynamic relationship for inhibition of Flk-1/KDR phosphorylation by SU6668 in vivo (Fig. 7)
. To our knowledge, this is the first time that dose- and time-dependent target modulation has been demonstrated for a small molecule RTK inhibitor in vivo. These studies demonstrate that SU6668 inhibits Flk-1/KDR phosphorylation when its plasma levels are maintained 1 µg/ml. High levels of efficacy do not require complete target inhibition for the full 24 h interval between treatments, but rather a protracted period of target inhibition (Fig. 7)
. This is consistent with the relatively rapid induction of tumor microvessel apoptosis after SU6668 administration (detectable 6 h after treatment; Fig. 2A
). Moreover, SU6668 exhibits comparable activity in vivo when assessed by three different readouts: inhibition of RTK phosphorylation, inhibition of VEGF-induced vascular permeability, and inhibition of tumor growth. For example, SU6668 administered at 75 mg·kg-1·day-1 significantly inhibits the growth of A431 tumors (10)
and inhibits VEGF-induced vascular permeability (Fig. 8)
, whereas lower dosages are not significantly inhibitory in both assays. These data are, in turn, consistent with the dramatically superior performance of SU6668 administered at 100 mg/kg compared with 50 mg/kg in reducing Flk-1/KDR phosphotyrosine levels in tumors (Fig. 7)
. In addition to Flk-1/KDR and PDGFRß (whose inhibition in vivo is shown in Fig. 9
), SU6668 also inhibits FGFR1 in biochemical and cellular assays (10)
. Its cellular activity vs. FGFR1 is somewhat weaker than against Flk-1/KDR and PDGFRß; therefore, in vivo target modulation efforts focused on Flk-1/KDR and PDGFRß. Hence, the contribution, if any, of FGFR1 inhibition to the antitumor effects documented here is unknown.
In summary, these studies have demonstrated that the unanticipated activity of SU6668 against large, established tumors is not unique to A431 tumors. Rather, it appears to be a more general property of the compound. Tumor regression is due to killing of tumor vessels, with consequent severe damage to or death of the tumor itself. The ability of SU6668 to kill tumor vessels presumably stems from its ability to block receptor tyrosine kinase activity, as evidenced by its ability to persistently inhibit Flk-1/KDR and PDGFRß phosphorylation in tumors at efficacious doses. Thus, we have elucidated for the first time mechanisms underlying the antitumor activity of SU6668 and have explored the PK/PD relationship underlying its effects. These insights are now incorporated into the ongoing clinical development of SU6668, in which changes in tumor histology and target kinase activity after treatment are currently being explored.
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ACKNOWLEDGMENTS |
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; Tinya Abrams, Robert Wild, Alyssa Morimoto, and Gerald McMahon for helpful discussions; Beverly Smolich, Laura Shawver, and Sara Courtneidge for insightful comments on the manuscript; and Sean Paxton and Barbara Remley for expert assistance during preparation of the manuscript.
Received for publication November 12, 2001.
Revision received February 6, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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