USE OF TYRPHOSTINS IN TREATING RESTENOSIS FIELD OF THE INVENTION The present invention relates generally to the field of pharmaceutical compositions and medical treatments. More specifically, the present invention relates to pharmaceutical compositions for treating restenosis and methods of using same. BACKGROUND OF THE INVENTION Angiotensin II (Angll) is a peptide hormone that promotes the growth and proliferation of vascular smooth muscle cells (SMCs). Although Angll associates with high affinity to two distinct G protein-coupled receptors, ATi and AT2, the mitogenic actions of Angll are primarily exerted through the AT-i receptor (Touyz and Schiffrin, 2000, Pharmacol Rev 52: 639-672). Coupling of the ATi receptor to both Gi and Gq indicates why decreased cAMP levels and hydrolysis of phosphoinositides by phospholipase A2 are important routes by which stimulation of cell proliferation is accomplished, as indicated by the need for Ca2+ mobilization and protein kinase C activation to induce c-fos gene expression (Taubman et al., 1989, JBC 264: 526-530). Nevertheless, evidence of increased protein tyrosine phosphorylation following Angll stimulation (Molloy et al., 1993, JBC 268: 7338-77345; Marrero et al., 1995, Cardiovasc Res 30: 530-536) suggests tyrosine kinase activation is also essential for SMC proliferation (Leduc et al., 1995, Mol Pharmacol 48: 582-592). Activation of Src kinase by Angll (Paxton et al., 1994, Biochem Biophys Res Commun 200: 260-267; Ishida et al., 1995, Circ Res 77: 1053-1059) has been implicated in the tyrosine phosphorylation of FAK and Pyk2, key players in the assembly of focal adhesions. While concurrent modification of paxillin, tensin and p130Cas (Leduc and Meloche, 1995, JBC 270: 4401-4404; Li and Earp, 1997, JBC 272: 14341-14348; Zhu et al., 1998, JBC 273: 33864-33875) produces changes in cytoskeletal organization typically associated with cell shape, modification of Pyk2 has also been reported to activate Erk1/2, JNK, and p70S6K (Li and Earp, 1997; Lev et al., 1995, Nature 376: 737-745; Graves et al, 1997, JBC 272: 1920-1928), critical mediators of cell proliferation. Likewise, activation of the JAK family of tyrosine kinases in response to Angll treatment, and subsequent phosphorylation of STAT transcription factors, is required for induction of c-fos and c-jun gene expression. More recently, several studies have suggested that tyrosine kinase receptors function as intermediates for G protein-coupled receptor signalling. For example, Voisin et al (Voisin et al, 2002, Am J Physiol Cell Physiol 283: C446-C455) have reported activation of the EGF receptor by Angll is necessary for increasing the rate of protein synthesis. While similar roles have also been suggested for both PDGF and IGF-1
receptors (Kim et al, 2000, Arterioscler Thromb Vase Biol 20: 2539-2545; Mondorf et al, 2000, FEBS Lett 472: 129-132; Du et al, 1996, Biochem Biophys Res Commun 218: 934- 939; Ali et al, 1997, JBC 272: 12373-12379), detailed functional and mechanistic studies examining the role of these receptors in Angll-dependent processes have yet to be reported. Activation of phosphatidylinositol 3-kinase (PI3-kinase) occurs in SMCs treated with Angll and PI3-kinase is required for cell proliferation (Saward and Zahradka, 1997, Circ Res 81: 249-257). In that study, we demonstrated that formation of PI-3,4,5-P3 (PIP3) was correlated with tyrosine phosphorylation of the p85 subunit and its translocation to the nuclear periphery. Furthermore, immunoprecipitation conclusively showed that the p110 isoform typically associated with tyrosine kinase receptors was responsible for catalyzing formation of PIP3. Although these observations implicated tyrosine kinase receptors in the activation of PI3-kinase by Angll, a mechanism for linking these distinct events was not apparent. PI3-kinase has been shown to interact with phosphorylated SH2-domain proteins such as Grb2 and IRS-1 (Vanhaesebroeck and Waterfield, 1999, Exp Cell Res 253: 239- 254). Since treatment with Angll results in tyrosine phosphorylation of both IRS-1 and She (Ali et al, 1997; Yoshizumi et al, 2001 , Mol Pharmacol 60: 656-665; Velloso et al, 1996, PNAS USA 93: 12490-12495), the insulin or the IGF-1 receptor may be implicated in downstream signalling from the ATi receptor. Furthermore, these observations suggest a mechanism by which PI3-kinase activation can be achieved by G protein-coupled receptors. We therefore examined the possibility that the IGF-1 receptor has a role in Angll-dependent signal transduction. A direct link between the IGF-1 receptor and Angll- dependent PI3-kinase activation was identified. Furthermore, our data show that IGF-1 receptor transactivation is essential for the activation of specific signal transduction pathways by Angll. Restenosis is caused by vascular stress or injury and leads to vessel wall thickening and loss of blood flow. These stresses may be, for example, mechanical, hypoxia, injury, shear-stress, pharmacological, infectious, inflammatory, oxidative, immunogenic, diabetic or pressure. The normal arterial vessel wall consists of a regular arrangement of endothelial, smooth muscle and fibroblast cells, present in three distinct layers of endothelium, media and adventitia. A single layer of endothelial cells forms the luminal barrier to blood-borne signals that modulate vascular function. The adventitia, which forms the outer layer around the artery, consists primarily of extracellular matrix as well as some fibroblasts, nerve fibres and microvessels. The media consists of numerous
layers of smooth muscle cells (SMCs) intermixed with extracellular matrix that is bound by the internal and external elastic lamina. The response to injury or other stress stimuli varies between the different cellular components of the vessel. Endothelial cells are capable of proliferation and migration, properties that permit re-endothelialization of the vessel after denudation or injury (Reidy, 1985, Lab Invest 53: 513-520). Medial SMCs are also able to reversibly modulate their phenotype which allows for their proliferation and/or migration into the intima at the site of injury (Schwartz et al, 1995, Circ Res 77: 445-465). It is these characteristics that lead to the adaptive and pathogenic growth of SMCs which is key to vascular remodelling and lesion formation. This is of particular concern for the treatment of coronary disease, wherein a common treatment for constricted, clogged or narrowed coronary arteries is balloon angioplasty. Angioplasty involves the use of a balloon-tipped catheter which is inserted into the heart's vessels to open partially blocked, or stenotic, coronary arteries. While balloon angioplasty does widen the restricted artery, a significant number of patients have renewed narrowing of the widened segment soon after the procedure. This subsequent narrowing of the artery is called restenosis and can necessitate the repetition of the angioplasty procedure or require alternative treatment such as coronary bypass graft surgery. Furthermore, restenosis occurs as a result of trauma to the vessel regardless of the method by which the injury is inflicted. Therefore, restenosis is not exclusive to angioplasty and is a common result of other (cardiac or peripheral) revascularization procedures (eg. stenting) or procedures involving vascular grafting (eg. bypass surgery, organ transplantation). It is also a problem associated with hemoaccess and other procedures involving long term intravenous delivery. Restenosis appears to be a response to injury of arterial wall, and appears to consist of the following events: platelet adhesion and aggregation on the damaged endothelium; release of platelet-derived growth factors; inflammation of the injured zone (Kornowski et al, 1998, J Am Coll Cardiaol 31: 224-230); secretion of specification chemotactic proteins from the damaged cells leading to recruitment of monocytes to the site of injury (Furukawa et al, 1999, Circ Res 84: 306-314); differentiation of monocytes into macrophages that produce matrix metalloproteinases required for cell migration; dedifferentiation of the smooth muscle cells after their activation by the growth factors; migration and proliferation of transformed smooth muscle cells, with secretion of extracellular matrix material; and re-growth of endothelium over the injured area. PCT Application WO 93/23067 teaches the use of fragments of IGF-1 as
antagonists for treating cell proliferative disorders such as cancer, restenosis and asthma. US Patent 6,368,826 describes an IGF-1 receptor binding protein (IIP-10) and its use in treating cancers, diabetes, neurodegenerative disorders and bone diseases. US Patent 6,518,238 teaches the use of IGF or IGF complexed with IGFBP-3 for treating psychological and/or metabolic disorders. PCT WO01/72771 teaches the use of short peptides having IGF-1 binding domains as IGF-1 receptor agonists and antagonists. PCT WO02/102805 teaches the use of cyclolignans as IGF-1 auto-phosphorylation decoy substrates. The cyclolignans are not structurally related to AG1024 or AG538. US Patent 5,789,427, US Patent 5,763,441 , US Patent 5,773,476, US Patent 5,849,742, US Patent 5,712,395, US Patent 6,358,954, US Patent 5,914,343 and PCT Application WO 99/67636 identify AG 1024 as "M14" and describe compounds structurally similar to AG538 (see Fig 2c) but containing an additional amide bond. Specifically, these patents teach the use of these compounds for treating cell proliferative disorders, primarily cancers (US Patent 5,773,476) as well as vasculogenesis and angiogenesis of tumors (US Patent 5,763,441) which are distinctly different diseases from restenosis. SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided a method of treating or preventing restenosis comprising: administering an effective amount of a pharmaceutical composition having an active ingredient selected from the group consisting of AG538, AG1024 or a mixture thereof to an individual in need of such treatment. According to a second aspect of the invention, there is provided a pharmaceutical composition for treating restenosis, said pharmaceutical composition having as an active ingredient AG538, AG1024 or a combination thereof. According to a third aspect of the invention, there is provided a medical device coated with an effective amount of a pharmaceutical composition having as an active ingredient AG538, AG1024 or a combination thereof. According to a fourth aspect of the invention there is provided a method of manufacturing a pharmaceutical composition comprising: combining an effective amount of AG538, AG1024 or a mixture thereof with a suitable carrier. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Angiotensin II stimulates tyrosine phosphorylation of the IGF-1 receptor. Panel A: Quiescent SMCs were stimulated with Angll (1 μM) and the cells lysed at the
indicated time points. IGF-1 receptor phosphorylation was monitored by Western blotting with phosphorylation-specific antibody (dilution 1 :1000). Panel B: Intensities of , phosphorylated IGF-1 receptor bands from three independent experiments were quantified by scanning densitometry and normalized to control bands for each experiment. Values were subsequently averaged and plotted as means ± SEM. Panel C: SMCs treated with and without 1 μM Angll for 10 min were lysed with RIPA buffer and phosphorylated IGF-1 and insulin receptors immunoprecipitated with phospho-specific antibody. Two immunoprecipitations were performed for each sample and the recovered protein was blotted onto the same membrane. The membrane was subsequently divided, with one portion probed with antibody to the unphosphorylated IGF-1 receptor (1 :1000 dilution) and the other section probed with antibody to the unphosphorylated insulin receptor (1 :1000 dilution). The blots were subsequently stripped and probed with the other antibody. The results from one experiment are shown. Two independent experiments were conducted to verify the results. Figure 2: Characteristics of IGF-1 receptor phosphorylation following Angll stimulation of quiescent SMCs. Panel A: SMCs were stimulated for 10 min with varying concentrations of Angll. IGF-1 receptor phosphorylation in the lysates was assessed by Western blot analysis with both PY100 and phospho-specific antibody. The data for PY100 is shown in the inset. The band intensities were quantified by scanning densitometry and means ± SEM (derived from three independent experiments as described for Figure 1) were plotted. Students t-test was used to determine statistically significant differences from unstimulated control (* p<0.05). Panel B: Quiescent SMCs were pretreated for 15 min with inhibitors prior to stimulation with 1 μM Angll for 10 min. IGF-1 receptor phosphorylation was monitored by Western blotting as previously described for Figure 1. All inhibitors were used at a concentration of 10 μM. Panels C,D: Neutralizing antibody (30 μg/mL Sm1.2, 1 μg/mL Ab-1) was added to the culture media 15 min prior to stimulants (10 μM Angll, 0.1 μM IGF-1). Cells were lysed 10 min after addition of stimulants and IGF-1 receptor phosphorylation was monitored by Western blotting. Sm1.2 (C) and Ab-1 (D) were employed in separate experiments. Figure 3: AG1024 and AG538 are specific inhibitors of the IGF-1 receptor kinase and block activation by Angll. Quiescent SMCs were pretreated for 15 min with various receptor tyrosine kinase inhibitors as indicated, and each inhibitor was used at a concentration of 5 μM. Phosphorylation of the IGF-1 receptor was monitored in samples stimulated with 0.1 μM IGF-1 (panel A) while phosphorylation of p70S6K was used to assess activation with 1 μg/mL EGF (panel B). Cells were lysed for Western blot analysis
at 10 min post-treatment. Phosphorylation-specific antibodies were diluted 1 :1000 prior to use. Panel C: Quiescent SMCs were stimulated with 1 μM Angll for 10 min following 15 min pretreatment with varying concentrations of AG1024. IGF-1 receptor phosphorylation was monitored by Western blot analysis. A representative blot is shown in the inset. Band intensity was quantified by scanning densitometry and means ± SEM (derived from three independent experiments as described for Figure 1) were plotted. Students t-test was used to determine statistically significant differences from unstimulated control (* p<0.05). Figure 4: Angll-dependent activation of PI3-kinase and its downstream effectors is blocked by inhibitors of the IGF-1 receptor kinase. Panel A: Quiescent SMCs were stimulated with 1 μM Angll for 10 min, then lysed. Inhibitors (5 μM) were added 15 min prior to Angll. Tyrosine phosphorylation of the PI3-kinase p85 subunit was determined by Western blot analysis after immunoprecipitation. Panel B: Activation of PDK1 and p70S6K was measured by Western blot analysis with phosphorylation-specific antibodies (diluted 1:1000) after treatment with 1 μM Angll or 0.1 μM IGF-1 in the presence of 5 μM inhibitors. Figure 5: AG1024 does not inhibit MAP kinase activation by Angll. Phospho- specific antibodies were employed to monitored MAP kinase activation in response to 10 min incubation with 1 μM Angll or 0.1 μM IGF-1. Inhibitors added 15 min prior to stimulation were used at a concentration of 5 μM. Panel A: Samples were analyzed by Western blotting, with antibodies were diluted 1 :1000. Panel B: Immunostaining for phosphorylated MAP kinase (antibody diluted 1 :100) is shown for control quiescent cells (A), as well as cells stimulated with Angll (B), Angll plus AG1024 (C) or IGF-1 (D). Treatment conditions were identical to those described for panel A. Figure 6: Signalling pathways activated by Angll and mediated by IGF-1 receptor transactivation. Figure 7: Graph showing the concentration effect of AG1024 on neointimal formation. DESCRIPTION OF THE PREFERRED EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. DEFINITIONS As used herein, "effective amount" refers to the administration of an amount of a
given compound that achieves the desired effect, that is, to treat or prevent restenosis, as discussed below. As used herein and as discussed above, "vascular stenosis" refers to vessel wall thickening, clogging or constriction and loss of blood flow. The stresses leading to stenosis may be, for example, mechanical, hypoxia, injury, shear-stress, pharmacological, infectious, inflammatory, oxidative, immunogenic, diabetic or pressure. As used herein and as discussed above, "angioplasty" refers to procedures and methods involved in the opening or unclogging of blocked arteries. In some instances, angioplasty involves the use of a balloon-tipped catheter which is inserted into the heart's vessels to open partially blocked, or stenotic, coronary arteries. While balloon angioplasty does widen the restricted artery, a significant number of patients have renewed narrowing of the widened segment soon after the procedure. This subsequent narrowing of the artery is called restenosis and can necessitate the repetition of the angioplasty procedure or require alternative treatment such as coronary bypass graft surgery. "Tyrphostins" refers to a family of tyrosine kinase inhibitors. See for example US Patent 6,426,366 which is incorporated herein by reference. AG1024 refers to 3-bromo-5-t-butvl-4-hvdroxv-benzylidenemalonjMI-e. AG538 refers to -alpha;c ang,-(3,4-dihydroxy) cinnamoyl-(3',4'-dihydroxyphenyJl_ ketone. As used herein, the term "treating" in its various grammatical forms refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causitive agent other abnormal condition. Described herein is a method of treating or preventing restenosis comprising administering to an individual in need of such treatment an effective amount of AG1024 and/or AG538. Also described is a pharmaceutical composition comprising as the active ingredient AG1024 and/or AG538 as well as medical devices coated or impregnated with said pharmaceutical composition. Angiotensin II (Angll) activates phosphatidylinositol 3-kinase (PI3-kinase), a known effector of receptor tyrosine kinases. Treatment of smooth muscle cells (SMCs) with Angll has also been shown to promote phosphorylation of various tyrosine kinase receptors. We therefore investigated the relationship between Angll and IGF-1 receptor activation in SMCs with a phosphorylation-specific antibody. Our experiments showed that IGF-1 receptor phosphorylation was maximally stimulated within 10 minutes by Angll. Inclusion of an IGF-1 neutralizing antibody in the culture media did not prevent IGF-1 receptor
phosphorylation following Angll treatment, which argues that a paracrine/autocrine loop is not required. Furthermore, this process was blocked by losartan and PP-1, indicating stimulation of IGF-1 receptor phosphorylation occurs via AT! receptor-dependent activation of Src kinase. The functional significance of IGF-1 receptor transactivation was examined with selective inhibitors of the IGF-1 receptor kinase (AG1024, AG538). When Angll-treated cells were incubated with AG1024 and AG538, phosphorylation of the regulatory p85 subunit of PI3-kinase was blocked. Furthermore, phosphorylation of the downstream factors PDK-1 and p70S6K did not occur. In contrast, AG1024 did not prevent MAP kinase activation by Angll. Transactivation of the IGF-1 receptor is therefore a critical mediator. of PI3-kinase activation by Angll, but is not required for stimulation of the MAP kinase cascade. Angiotensin II (Angll) is a critical element in the vascular response to injury, operating primarily to promote conversion of smooth muscle cells (SMCs) to a phenotypic state which permits migration and proliferation (Hayashy et al, 1998, JBC 273: 28860- 28867). As part of this process, Angll stimulates tyrosine phosphorylation of key protein mediators of intracellular signalling pathways associated with migration and proliferation (Yin et al, 2003, Int J Biochem Cell β/o/ 35: 780-783). However, mechanistic information detailing how G-protein-coupled Angll receptors stimulate tyrosine phosphorylation is limited. In this investigation, we examined the role of the IGF-1 receptor kinase as an intermediate for the transduction of signals originating from Angll receptors. Our rationale for examining the IGF-1 receptor was based on published evidence that showed both the IGF-1 receptor and IRS-1 are tyrosine phosphorylated in response to Angll (Du et al, 1996; Ali et al, 1997). In this study, we employed a phosphorylation-specific antibody to establish that IGF-1 receptor activation was time- and Angll concentration-dependent. Furthermore, phosphorylation of the IGF-1 receptor was shown to be ATi receptor- dependent and mediated by Src kinase. However, binding of IGF-1 to the receptor was not required for transactivation, since neutralizing antibodies did not prevent IGF-1 receptor phosphorylation by Angll. The functional significance of IGF-1 receptor participation was demonstrated with selective inhibitors of the IGF-1 receptor tyrosine kinase, which revealed transactivation was required for the stimulation of PI3-kinase and its downstream effectors, PDK1 and p70S6K, but not MAP kinase. These results indicate IGF-1 receptor transactivation has a critical role in the cellular actions of Angll. Receptor transactivation is a relatively recent concept, originating from evidence that phosphorylation of receptor tyrosine kinases occurs in response to agonists of G protein-coupled receptors (Luttrell, 2002, Can J Physiol Pharmacol 80: 375-382; Daub et
al, 1996, Nature 379: 557-560; Marinissen and Gutkind, 2001 , Trends Pharmacol Sci 22: 368-376). In SMCs treated with Angll, tyrosine phosphorylation of the EGF, PDGF and IGF-1 receptors has been observed [reviewed in Saito and Berk, 2001 , J Mol Cell Cardiol 33: 3-7; Eguchi and Inagami, 2000, Regul Pept 91: 13-20]. However, the initial reports examining this phenomenon suggested that receptor tyrosine kinase activation was triggered by the release of growth factors in response to G protein-coupled receptor stimulation (Crowley et al, 1995, Am J Physiol 269: H1641-H1647). This mechanism does not appear to operate for Angll-dependent activation of the PDGFβ-receptor, since neither conditioned media nor neutralizing antibody blocked PDGF receptor phosphorylation in SMCs (Linseman et al, 1995, JBC 270: 12563-12568). However, there are also indications that the PDGF receptor is not activated in response to Angll treatment of SMCs (Eguchi et al, 1998, JBC 273: 8890-8896), and this disparity remains unresolved. In contrast, EGF receptor phosphorylation has been independently confirmed and shown to mediate, for example, ERK1/2 phosphorylation, c-fos expression and protein synthesis (Voisin et al, 2002; Eguchi et al, 1998; Eguchi et al, 1999, JBC 274: 36843- 36851). The mechanism which mediates EGF receptor activation, however, has yet to be clarified. The failure of neutralizing antibodies to prevent EGF receptor phosphorylation (Saito and Berk, 2001) suggests that release of EGF is not a prerequisite for this process. On the other hand, metalloproteinase cleavage of proheparin-binding EGF has been reported to precede receptor dimerization and autophosphorylation (Eguchi et al, 2001 , JBC 276: 7957-7962). Delafontaine and colleagues were the first to identify IGF-1 receptor activation in response to stimulation with Angll (Du et al, 1996). Since neutralizing IGF-1 antibody was found to prevent the mitogenic effects of Angll, it was concluded that secretion of IGF-1 was essential for receptor activation (Delafontaine et al, 1995, JBC 270: 14383-14388). Although this observation does not agree with our data, it may be argued that two distinct events were being compared. It has been previously established that the IGF-1 receptor has a role in mitogenesis, since IGF-1 receptor activation is necessary for advancing past the G1 restriction point (Baserga, 1992, Ann NY Acad Sc/663: 154-157). Furthermore, the temporal separation between transactivation (minutes post-stimulus) and cell cycle progression (hours post-stimulus) unequivocally demonstrates that these are independent processes. Thus, the ability to inhibit cell proliferation with a neutralizing antibody cannot be equated with the lack of an effect on receptor transactivation, which we assayed directly in this study. Rather, transactivation of the IGF-1 receptor by the AT! receptor occurs in the absence of IGF-1 secretion, while progression through G1 phase requires
IGF-1 synthesis and secretion prior to IGF-1 receptor activation. The latter view is supported by evidence showing Angll also stimulates both IGF-1 and IGF-1 receptor gene expression (Brink et al, 1999, Hypertension 34: 1053-1059). The ATi receptor is a member of the G protein-coupled receptor family which operates through specific heterotrimeric G proteins. Although the ATi receptor involves Gq-mediated activation of various phospholipases [reviewed in Touyz and Schiffrin, 2000], participation of the non-receptor tyrosine kinases Src and Jak has also been reported (Yin et al, 2003). Src kinase has been identified as the leading candidate for mediating receptor tyrosine kinase transactivation in response to Angll (Eguchi et al, 1998; Bokemeyer et al, 2000, Kidney Int 58: 549-558; Luttrell et al, 1997, JBC 272: 4637-4644), and we have similarly shown Src kinase is required for IGF-1 receptor transactivation. It has been speculated that Src kinase may phosphorylate RTKs directly (Biscardi et al, 1999, JBC 274: 8335-8343; Wu et al, 2002, JBC 277: 24252-24257). Recently, however, Seta and Sadoshima (Seta and Sadoshima, 2003, JBC 278: 9019-9026) reported that tyrosine phosphorylation of the ATi receptor (Y-319) is required for EGF receptor transactivation. Mutation of this amino acid, while preventing EGF receptor phosphorylation, did not decrease Src kinase activation in response to Angll. These data therefore imply that Src kinase functions indirectly, while also confirming that activation of Src kinase must precede RTK transactivation. However, the link between Src kinase and G proteins (either Gα or Gβγ subunits) still remains to be resolved, and there is speculation that this step may involve a G protein-independent process (Heuss and Gerber, 2000, Trends Neurosci 23: 469-475). Nevertheless, our data implicate Src kinase in the activation of MAP kinase, and similar conclusions have been reached by other investigators studying both SMCs and other cell types (Weng and Shukla, 2002, Biochim Biophys Ada 1589: 285-297; Touyz et al, 2001 , Hypertension 38: 56-64; Ishida et al, 1998, Circ Res 82: 7-12). Interestingly, it has been recently shown that MAP kinase activation in hepatocytes requires transactivation of the PDGF receptor (Weng and Shukla, 2002). There has been limited study of IGF-1 receptor function in relation to Angll. Our data implies IGF-1 receptor phosphorylation is required for the activation of PI3-kinase by Angll. Velloso et al (Velloso et al, 1996) previously showed that PI3-kinase associates with phosphorylated IRS-1 and IRS-2 following Angll stimulation of heart tissue, and Angll also triggers phosphorylation of IRS-1 in SMCs (Ali et al, 1997). Although we did not examine IRS-1 , inhibition with AG1024 established that PI3-kinase was downstream of the IGF-1 receptor. Furthermore, experiments with AG1024 and LY294002 indicated that
p70S6K and PDK-1 also followed PI3-kinase. Based on these results, Src kinase can be viewed as controlling two distinct pathways leading from the ATi receptor (Figure 6). First, Src kinase controls MAP kinase activity and nuclear translocation. This process is necessary for immediate early gene expression. However, tyrosine kinase-independent activation of MAP kinase by the IGF-1 receptor could also explain why this event is both Src kinase-dependent and AG1024-insensitive (Yau et al, 1999, Eur J Biochem 266: 1147-1157). Second, Src kinase mediates IGF-1 receptor transactivation, which is required for PI3-kinase activation. In this pathway, the IGF-1 receptor likely functions as a scaffold for p85 binding, and synthesis of PI3-P results in PDK-,1 activation, p70S6K phosphorylation and increased protein synthesis. How does this scheme reconcile our evidence that MAP kinase phosphorylation is blocked by inhibition of PI3-kinase (Yau et al, 2003, Eur J Biochem 270: 101-110)? It is becoming obvious that each PI3-kinase isoform has a distinct function in the transduction of signals within cells. While we specifically examined the classical tyrosine kinase receptor-activated p85/p110 PI3-kinase (Class lA) during the course of this study, it is recognized that other PI3-kinase isoforms are equally sensitive to LY294002 (Vanhaesebroeck and Waterfield, 1999). Consequently, inhibition of MAP kinase by LY294002 could implicate another class of PI3-kinases in the modulation of MAP kinase cascades. The p110 isoform of PI3-kinase (Class lB) may be particularly relevant to Angll stimulation, since its activation is mediated by GPγ subunits (Vanhaesebroeck and Waterfield, 1999). A role for both PI3-kinase isoforms in the transduction of signals from the AT! receptor may therefore be projected. Appreciating these distinctions may also help alleviate the confusion surrounding the roles of individual PI3-kinase isoforms in signalling by G protein-coupled receptors. As can be seen in Figure 7, AG1024 is effective at dosages of 10"6M to 10"9M. Thus, in one embodiment of the invention, AG1024 and/or AG538 is arranged to be delivered at a local concentration of between 10"6M to 10"9M. In yet other embodiments, AG1024 and/or AG538 may be delivered at a local concentration of between 10"6M to 10" 9M. As discussed below, in some embodiments, AG1024 and/or AG538 is combined with an adhesive agent in a pharmaceutical composition that can be applied locally. In some embodiments, AG1024, AG538 or combinations thereof in a therapeutically effective amount may be combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non- biodegradable. Exemplary examples of carriers include, but are by no means limited to, for example, poly(ethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic
acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, mathacrylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like. Standard excipients include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,. hydroxypropylmethycellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars and starches. See, for example. Remington: The Science and Practice of Pharmacy, 1995, Gennaro ed. As will be apparent to one knowledgeable in the art, specific carriers and carrier combinations known in the art may be selected based on their properties and release characteristics in view of the intended use. Specifically, the carrier may be pH-sensitive, thermo-sensitive, thermo-gelling, arranged for sustained release or a quick burst. In some embodiments, carriers of different classes may be used in combination for multiple effects, for example, a quick burst followed by sustained release. As discussed herein, in one embodiment, the pharmaceutical composition comprising AG1024, AG538 or a combination thereof is combined with an adhesive agent. As a result of this arrangement, the pharmaceutical composition can be localized to the intended area, for example, a damaged vessel, thereby limiting side effects. Furthermore, in preferred embodiments, the combination has suitable release kinetics so that delivery of the active agent occurs over an extended period of time which may be necessary to ensure efficacy. As will be appreciated by one knowledgeable in the art, the adhesive agent is non-toxic. In one embodiment, AG1024 and/or AG538 is suspended in a non- toxic, biodegradable fibrin glue (Grecto et al, 1991 , J Biomed Mater Res 25:39-51 ; Zilch and Lambiris, 1986, Arch Orthop Trauma Surg 106:36-41), which consists of separate fibrinogen and thrombin components purified from human or bovine plasma (Senderoff et. al, 1991 , J Parenteral Sci Technol 45:2-6; Katz and Spera, 1998, Medical Device and Diagnostic Industry Magazine, April). One example of a fibrin glue is Tisseel™ (Immuno AG, Vienna, Austria). In addition to Tisseel, adhesive biomaterials are being manufactured by Thermogenesis (Rancho Cordova, CA), Fusion Medical Technologies, Inc. (Mountain View, CA), and CryoLife, Inc. (Kennesaw, GA), which offers photoactivated fibrin sealants. V.I. Technologies (New York City) is developing a fibrin sealant similar to Tisseel, as are
Haemacure Corp. (Sarasota, FL), Convatec/Bristol-Myers Squibb (Skillman, NJ), and BioSurgical Corp. (Pleasanton, CA). Also applicable are conventional hemostatic agents that work on various stages of the coagulation cascade; for example, agents such as Surgicel®, Gelfoam®, and Avitene® activate the first stage of the coagulation pathway. Hydrogels can also be used for this application (Dagani, 1997 Chemical & Engineering News, June 9, 1997). Finally, the glue produced by mussels has received interest for bonding materials (Morgan, 1990, The Scientist 4 (April 30):1). It has been found equal or better to fibrin glue (Pitman et al, 1989, Bull Hosp Jt Dis Orthop Inst 49:213-20), and is capable of attaching molecules as small as proteins to a surface (Burzio et al, 1996, Anal Biochem. 241:190-4). This mixture can be applied externally onto the vessel. It is of note that fibrin glues have been successfully used to deliver growth factors to promote angiogenesis (Fasol et al, 1994, Thorac Cardiovasc Surg 107: 1432-1439) or inhibit intimal hyperplasia (Zarge et al, 1997, J Vase Surg 25: 840-848), and to deliver antibiotics in vitro (Greco et al, 1991 , J Biomed Mater Res 25: 39-51). This approach allows for the delivery of pharmacologically potent concentrations locally, without any significant release of the drug systemically. It is of note that other suitable biocompatible or biodegradable adhesives known in the art may also be used. Furthermore, AG1024 and/or AG538 are arranged to be delivered at a local concentration when used in this combination. It is important to note that a therapeutic dosage of AG1024 and/or AG538 will of course depend on at least the age, weight and condition of the patient as well as the location of treatment, where appropriate. In yet other embodiments, the pharmaceutical composition comprising AG1024, AG538 or a combination thereof may be contained within or adapted to be released by a surgical or medical device, for example, stents, catheters, prostheses, sutures and the like. In these embodiments, pharmaceutical composition comprising AG1024, AG538 or a combination thereof at concentrations or dosages described above may be incorporated into nylon microcapsules and applied to the surface of the stent or device. Alternatively, the device may be coated with a film composed of, for example, cellulose, hyaluronic acid, chitosan, ethylene vinyl acetate, or poly lactic acid, impregnated with the pharmaceutical composition comprising as an active ingredient a therapeutic amount of AG1024, AG538 or a combination thereof. Yet further, the device may be coated with a thermo-sensitive gel such that the pharmaceutical composition comprising AG1024, AG538 or a combination thereof is released when the device is implanted. Typically, stents are used to expand the lumen of a body passageway. This involves inserting the stent into the passageway such that the passageway is expanded.
In general, a preinsertion examination, for example, either a diagnostic imaging procedure or direct visualization at the time of surgery is performed to determine the appropriate location for stent insertion. First, a guide wire is advanced through the proposed site of insertion. A delivery catheter is then passed over the guide wire, allowing insertion of the catheter into the desired position. The stent is then expanded by means known in the art. The stent may be coated for example by spraying or dipping the stent with or in the pharmaceutical composition comprising AG1024, AG538 or a combination thereof described above, or the stent may be coated with an absorption-promoting substance, such as hydrogel, first. Alternatively, the stent may be surrounded in a sleeve, mesh or other structure impregnated with the pharmaceutical composition comprising AG1024, AG538 or a combination thereof and arranged to release the pharmaceutical composition comprising a therapeutic amount of AG1024, AG538 or a combination thereof over time. In other embodiments, a pharmaceutical composition comprising AG1024, AG538 or a combination thereof at concentrations or dosages described above may be encapsulated for delivery. Specifically, AG1024 and/or AG538 may be encapsulated in biodegradable microspheres, microcapsules, microparticles, or nanospheres. The delivery vehicles may be composed of, for example, hyaluronic acid, polyethylene glycol, poly(lactic acid), gelatin, poly(E-caprolactone), or a poly(lactic-glycolic) acid polymer. Combinations may also be used, as, for example, gelatin nanospheres may be coated with a polymer of poly(lactic-glycolic) acid. As will be apparent to one knowledgeable in the art, these and other suitable delivery vehicles may be prepared according to protocols known in the art and utilized for delivery of the pharmaceutical composition comprising AG1024, AG538 or a combination thereof. In some embodiments, the delivery vehicle may be coated with an adhesive for localizing the pharmaceutical composition comprising AG1024, AG538 or a combination thereof to the area of interest. Alternatively, the delivery vehicle may be suspended in saline and used as a nanospray for aerosol dispersion onto an area of interest. Furthermore, the delivery vehicle may be dispersed in a gel or paste, thereby forming a nanopaste for coating a tissue or tissue portion. It is of note that the pharmaceutical composition comprising AG1024, AG538 or a combination thereof as described above may be combined with permeation enhancers known in the art for improving delivery. Examples of permeation enhancers include, but are by no means limited to those compounds described in U.S. Pat. Nos. 3,472,931 3,527,864; 3,896,238; 3,903,256; 3,952,099; 4,046,886; 4,130,643; 4,130,667; 4,299,826 4,335,115; 4,343,798; 4,379,454; 4,405,616; 4,746,515; 4,788,062; 4,820,720; 4,863,738 4,863,970; and 5,378,730; British Pat. No. 1 ,011 ,949; and Idson, "1975, J. Pharm. Sci.
64:901-924. In some embodiments, the pharmaceutical composition comprising AG1024, AG538 or a combination thereof in any suitable form as described above, may be combined with biological or synthetic targetting molecules, for example, site-specific binding proteins, antibodies, lectins or ligands, for targetting the pharmaceutical composition comprising AG1024, AG538 or a combination thereof to a specific region or location. Furthermore, it is known that there are inflammatory and proliferative components that contribute to the development of an arteriosclerotic lesion. Specifically, Angll is a critical element in the vascular response to injury, as Angll promotes conversion of SMCs to a phenotypic state which permits migration and proliferation by phosphorylating a number of tyrosine kinase receptors, including PI3-kinase. Because AG1024 and AG538 prevent at least phosphorylation of PI3-kinase, these compounds constitute an effective treatment for restenosis, as described herein. For this reason, pharmaceutical composition comprising a therapeutically effective amount of AG1024, AG538 or a combination thereof should be able to restrict progression of this condition, that is, reduce the incidence and/or severity of the lesions. Furthermore, the incidence or severity of symptoms associated with all vascular procedures involving grafting, puncturing or producing intimal damage can be reduced by the above-described compounds, as could inflammation and/or irritation accompanying valve replacements, catheters, prosthesis, implanted devices, pacemakers, nerve stimulators, patches, organ transplants, small vessel vaeulopathy, wound repair, or psoriasis. Thus, the symptoms associated with any inflammation or inflammatory disease that is localized to a defined region can be ameliorated using the pharmaceutical composition' comprising a therapeutically effective amount of AG1024, AG538 or a combination thereof described above. In these embodiments, the pharmaceutical composition comprising AG1024, AG538 or a combination thereof may be localized through the use of an adhesive, impregnated mesh or targetting molecule as described herein, or the device or organ may be coated or infused with the pharmaceutical composition comprising AG1024, AG538 or a combination thereof as described herein. The pharmaceutical composition comprising AG1024, AG538 or a combination thereof could also be sprayed or applied to tissue grafts or organs prior to transplantation. Specifically, graft rejection is characterized by lesion formation, inflammation and necrosis. The pharmaceutical composition comprising a therapeutically effective amount of AG1024, AG538 or a combination thereof will accomplish at least one of the following: prolong the life of the graft; decrease the side effects associated with immunosuppressive therapy and
decrease accelerated atherosclerosis associated with transplants. In other embodiments, a mesh coated or arranged to release the pharmaceutical composition comprising AG1024, AG538 or a combination thereof may be used in lieu of spray application. Alternatively, the sprays or meshes could also be used to treat, for example, venous leg ulcers, skin grafts, post-operative hypertrophy, hyperplasia, hypertrophic burn scars, hypertrophic gastropathy, cardiac hypertrophy associated with congestive heart failure and hypertrophic cardiopathy, or hypertension. For example, hypertension is an increase in smooth muscle cell volume within a blood vessel due to excessive pressure, lack of oxygen/nutrients or enhanced production of hypertrophy-inducing factors released as a result of trauma distinct from the site of action (for example, kidney disease). Also, hypertrophic cardiac disease (for example, congestive heart failure, hypertrophic cardiomyopathy, valve replacement surgery) results from an increase in cardiomyocyte volume as a result of hypoxia, surgical intervention or genetic defect. Cellular hypertrophy and inflammation occur in the region affected by the causative factor. Thus, these disorders also require cell migration and differentiation, meaning that the pharmaceutical composition comprising AG1024, AG538 or a combination thereof may alleviate some of the associated symptoms. The following Examples are provided to illustrate, but not limit, the invention. MATERIALS and METHODS Cell Culture: Primary cultures of porcine coronary artery smooth muscle cells (SMCs) were generated from the left anterior descending coronary artery (LAD) by an explant organ culture method (Saward and Zahradka, 1997, Mol Cell Biochem 176: 53- 59). To obtain a quiescent cell population, SMCs were grown to 70% confluence in D- MEM containing 20% FBS, 2 mM glutamine, 50 μg/mL streptomycin, 50 μg/mL penicillin, and subsequently incubated in serum-free D-MEM supplemented with 11 μg/mL pyruvate, 5 μg/mL transferrin, 10"9 M selenium, 2X10"4 M ascorbate and 10"8 M insulin for 5 days. Cells were used only after the second passage to maintain consistency between cultures. Western Blot Analysis: Western blotting of cellular proteins (10 μg) separated by SDS/polyacrylamide gel electrophoresis in a 7.5% gel and transferred to PVDF membrane (Roche) was conducted as previously described (Yau et al, 1999). Horseradish peroxidase-.(HRP)-conjugated secondary antibody (1 :10,000 diluted) was detected using the ECL chemiluminescent system (Amersham). Band intensity was quantified by scanning densitometry. Antibodies employed during the course of this investigation were obtained from either Cell Signaling (phospho-p44/42 MAP kinase (thr202/tyr204), phospho-p70S6K (thr389), phospho-PDK1 (ser241), phospho-insulin (tyr1146)/IGF-1
(tyr1131) receptor, PY100), Upstate Biotechnology (PI3-kinase p85 subunit) or Santa Cruz Biotechnology (IGF-1 receptor β-subunit, insulin receptor β-subunit). Immunoprecipitation: Cell lysates were prepared from 100-mm culture dishes by addition of either 1.0 mL lysis buffer (1% NP-40, 20 mM Tris-HCI (pH 7.5), 10% glycerol, 137 mM NaCI, 1 mM MgCI2, 1 mM PMSF, 0.4 mM orthovanadate, 1 mM NaF) or RIPA buffer (150 mM NaCI, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCI (pH 8.0), 1 mM EGTA, 1 mM EDTA). The plates were scraped and the lysates cleared by centrifugation (10 min, 12,000χg at 4°C). Protein concentrations were measured and aliquots of 100 μg protein were mixed for 2 h at 4°C with Protein G Sepharose (Amersham Pharmacia). Protein G Sepharose was subsequently removed by centrifugation at 12,000*g for 5 min at 4DC. Each aliquot was then mixed over 2.5 - 4 h at 4πC with 4 μg of antibody. Protein G Sepharose was added for an additional 0.5 - 2 h. The Protein G Sepharose beads were subsequently collected by centrifugation (12,000*g for 5 min at 4°C) and washed 4 times with 1.0 mL lysis buffer. The beads were then suspended in 2* SDS sample buffer, heated 5 min at 95°C and centrifuged for 5 min at 12,000χg. Western blot analysis was used to examine the protein content of the supernatant. Immunocytochemical Analysis: SMCs grown on Superfrost Plus glass slides (Fisher Scientific) were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 prior to antibody treatment. The slides were blocked for 60 min at room temperature, rinsed with TBS and incubated with primary antibody to phosphorylated MAP kinase (Cell Signaling, diluted 1 :100 in 1% BSA/TBS-T) for 60 min at room temperature (Yau et al, 1999). Primary antibodies were detected with Cy3-coupled secondary antibody (Jackson Laboratories, diluted 1 :400 with 1% BSA/TBS-T). Nuclei were visualized with Hoescht No. 33342 (0.5 mg/mL diluted 1 :4000 in TBS). Images were captured with a DAGE-MTI CCD camera and associated software. Data Analysis: Quantification of data obtained on film or autoradiographs was accomplished with a BioRad Model GS800 Imaging Densitometer under non-saturating conditions. Background subtraction was achieved by reading the absorbance of an equal sized region directly adjacent (above, below or beside) to the band. Although multiple exposures were acquired to ensure the absence of film saturation, the experimental figures typically show longer exposures selected specifically for visual presentation and not used for data analysis. Data were quantified and presented as means ± SEM of individual experiments conducted in triplicate. Student's t-test was used to compare treatment means versus controls. Statistical significance was set at p<0.05. RESULTS
Angiotensin II stimulates IGF-1 receptor phosphorylation: Quiescent SMCs were treated with 1 μM Angll and harvested at various time points over 60 min. Cell extracts were subsequently analyzed by Western blot analysis for IGF-1 receptor modification. The phospho-specific antibody employed recognizes phosphorylation of the β-subunit at Tyr1131. Although a band at 95 kDa was detected in unstimulated cells, the intensity of this band increased considerably following stimulation (Figure 1A). Maximum band intensity (2.2-fold above control) was reached within 10 min, and was sustained over a period of 120 min. Phosphorylation of this 95 kDa band was also observed following treatment with 0.1 μM IGF-1 , indicating that this band likely represents the IGF-1 receptor. Since the antibody used to monitor IGF-1 receptor phosphorylation cross-reacts with the insulin receptor, an immunoprecipitation approach was employed to verify the identity of the 95 kDa phosphorylated band. Quiescent cells were treated with 1 μM Angll for 10 min, lysed in RIPA buffer, and the samples were subsequently immunoprecipitated with antibody recognizing the phosphorylated IGF-1 receptor. The protein present in the immunoprecipitate was visualized by Western blotting with antibody to the IGF-1 receptor β-subunit (Figure 1C). Furthermore, there was an increase in immunoprecipitated receptor protein from the Angll sample relative to untreated control. In contrast, a much less intense band was detected with antibody specific for the insulin receptor β-subunit (Figure 1C), which may be attributable to a low level of expression in SMCs (Bornfeldt et al, 1991, Diabetologia 34: 307-313; Lee et al, 1988, In Vitro Cell Dev Biol 24: 921-926). As well, there was no obvious difference in band intensity between the treated and untreated samples. These data therefore indicate that Angll activates the IGF-1 receptor rather than the insulin receptor. IGF-1 receptor phosphorylation is ATt receptor and Src kinase-dependent: Receptor-driven processes demonstrate both concentration-dependence and saturation kinetics. To establish that Angll-dependent phosphorylation of the IGF-1 receptor is receptor mediated, the effect of Angll concentration was examined. Quiescent SMCs were treated with varying amounts of Angll (10"9 - 10"5 M) for 10 min and cell extracts were subsequently examined by Western blotting. Increased phosphorylation of the 95-kDa β- subunit was observed with increasing Angll (inset Figure 2A). Quantitative analysis of the data by scanning densitometry indicated maximal phosphorylation was reached with 1 μM Angll (Figure 2A). These results support a role for the Angll receptor in IGF-1 receptor transactivation. Angll operates via two distinct receptors which can be distinguished by selective receptor antagonists. Quiescent SMCs were pre-treated with either losartan (ATi receptor
antagonist) or PD123319 (AT2 receptor antagonist) for 15 min before addition of Angll (1 .μM). Stimulation with Angll increased IGF-1 receptor phosphorylation after 10 min relative to untreated control (Figure 2B). The intensity of this band was reduced in the presence of losartan, but not PD123319 (Figure 2B). These results indicate transactivation of the IGF- 1 receptor occurs via the AT! receptor. Additionally, the signalling intermediates coupling these receptors were examined with inhibitors selective for PI3-kinase, protein kinase C and Src kinase (Davies et al, 2000, Biochem J 351: 95-105; Herbert et al, 1990, Biochem Biophys Res Commun 172: 993-999; Hanke et al, 1996, J Biol Chem 271: 695-701). It was observed that only PP-1 , a Src kinase inhibitor, prevented IGF-1 receptor phosphorylation in the presence of Angll (Figure 2B). These data agree with other studies that have indicated Src kinase has a pivotal role in the transactivation of receptor tyrosine kinases (eg. EGF receptor) by GPCR (eg. Angll receptor) (Eguchi et al, 1998; Bokemeyer, 2000). Neutralizing antibody treatment does not prevent IGF-1 receptor phosphorylation: Paracrine/autocrine stimulation by IGF-1 and IGF-1 -independent transactivation represent the two mechanisms by which Angll treatment could induce phosphorylation of the IGF-1 receptor (Luttrell, 2002). To distinguish between these possibilities, we employed a neutralizing antibody to IGF-1 derived from clone Sm1.2 (Upstate) that has previously been shown to inhibit IGF-1. receptor activation (Russell et al, 1984, PNAS USA 81: 2389- 2392). It was assumed that the antibody would interfere with the actions of IGF-1 if it is secreted in response to Angll treatment. As seen previously, Angll stimulated phosphorylation of a 95 kDa band (Figure 2C). Pretreatment of the cells with Sm1.2 antibody, however, did not reduce the intensity of this band after Angll treatment (Figure 2C). The effectiveness of the antibody was confirmed by adding it to cells prior to treatment with IGF-1 , and under these conditions phosphorylation of the 95 kDa band was reduced to near basal levels (Figure 2C). Similar results (Figure 2D) were obtained with a neutralizing IGF-1 receptor antibody (Ab-1 , clone IR-3, Oncogene), which blocks binding of IGF-1 to the receptor (Rohlik et al, 1987, Biochem Biophys Res Commun 149: 276- 281). The IGF-1 receptor tyrosine kinase can be selectively inhibited: Although inhibitors of tyrosine kinases are commercially available, few of these compounds have shown selectivity for either the insulin or IGF-1 receptors. We therefore tested several compounds that have been reported to inhibit the IGF-1 receptor kinase, and compared their actions with those of other receptor tyrosine kinase inhibitors. The panel of inhibitors included a PDGF receptor inhibitor (AG1295), an EGF receptor inhibitor (AG1478) and
two putative IGF-1 receptor inhibitors (AG538, AG1024) (Kovalenko et al, 1994, Cancer Res 54: 6106-6114; Levitzki and Gazit, 1995, Science 267: 1782-1788; Blum et al, 2000, Biochemistry 39: 15705-15712; Parrizas et al, 1997, Endocrinology 138: 1427-1433). Quiescent SMCs were stimulated with IGF-1 (0.1 μM), which resulted in an increase in IGF-1 receptor phosphorylation relative to control (Figure 3A). Addition of EGF and PDGF receptor kinase inhibitors prior to IGF-1 receptor treatment had no effect on IGF-1 receptor phosphorylation. In contrast, both IGF-1 receptor kinase inhibitors decreased phosphorylation to near control levels. To verify that the IGF-1 receptor kinase inhibitors did not affect other receptor tyrosine kinases, SMCs were treated with EGF (1 μg/mL) and p70S6K phosphorylation was monitored in the presence of these inhibitors. Under these conditions, only the selective EGF receptor kinase inhibitor, AG1478, prevented p70S6 phosphorylation (Figure 3B). Neither AG1024 nor AG538 influenced this process, which suggests these compounds are specific for the IGF-1 receptor kinase. The putative insulin/IGF-1 receptor inhibitor HNMPA-(AM)3 (hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester, (Saperstein et al, 1989, Biochemistry 28: 5694-5701) was also tested in these experiments. This compound consistently killed the cells when concentrations capable of preventing receptor phosphorylation (>100 μM) were employed, even with only a 15 minute pre-incubation (data not shown). HNMPA-(AM)3 was therefore not used. Although AG1024 and AG538 effectively inhibited IGF-1 receptor activation, we also tested their ability to block receptor phosphorylation following stimulation with Angll. Quiescent cells were pretreated for 15 min with varying concentrations of AG1024 or AG538 (1 nM - 10 μM), then exposed to 1 μM Angll. Western blot analysis of samples prepared 10 min after Angll addition confirmed that the IGF-1 receptor was phosphorylated (Figure 3C). AG1024 inhibited IGF-1 receptor phosphorylation with an EC50 of 0.23 ± .07 μM, and exhibiting the greatest potency at 1-10 μM. Similar results were obtained with AG538 (data not shown). Since 10 μM was slightly toxic to the cells over the maximum 30 min exposure period, a concentration of 5 μM was used for all experiments. IGF-1 receptor kinase inhibitors prevent PI3-kinase activation: ATi receptor stimulation by Angll increases PI3-kinase activity in SMCs (Saward and Zahradka, 1997). Concurrent with this change in activity, the p85 regulatory subunit undergoes tyrosine phosphorylation (Saward and Zahradka, 1997). To determine whether p85 phosphorylation was IGF-1 receptor-dependent, quiescent SMCs were stimulated with Angll in the presence and absence of AG1024. Cells were lysed after 10 min, and p85 was immunoprecipitated. Western blotting with PY100, an antibody selective for phosphorylated tyrosine residues, was then used to compare levels of p85 modification.
This experiment revealed that p85 was phosphorylated in response to 1 μM Angll treatment (Figure 4A). In the presence of either AG1024 or AG538, however, p85 phosphorylation was reduced to basal levels (Figure 4A). These data suggest that activation of PI3-kinase requires transactivation of the IGF-1 receptor. PI3-kinase mediates a variety of distinct intracellular signalling pathways, including those leading to protein synthesis. PI3-kinase controls this process through the sequential activation of PDK-1 and p70S6K (Vanhaesebroeck et al, 2001 , Annu Rev Biochem 70: 535-602). We therefore monitored PDK-1 and p70S6K to determine whether their phosphorylation was inhibited with AG1024. As expected, the phosphorylation of PDK-1 and p70S6K was increased in response to Angll and inhibited by LY294002, a selective inhibitor of PI3-kinase (Figure 4B). Like LY294002, AG1024 also prevented PDK-1 and p70S6K phosphorylation (Figure 4B). These data indicate that IGF-1 receptor transactivation regulates specific signalling pathways downstream of PI3-kinase. ATi receptor-dependent stimulation of MAP kinase is IGF-1 receptor independent: Our studies have indicated that PI3-kinase activation mediates stimulation of the MAP kinase cascade in response to Angll (Yau et al, 2003). Activation of Erk1/2 MAP kinase by PI3-kinase, however, apparently involves a cascade that is distinct from the PDK-1 and p70S6K-dependent pathway that leads to protein synthesis (Vanhaesebroeck et al, 2001). Therefore, to determine whether IGF-1 receptor transactivation participates in all PI3-kinase-mediated processes, we examined the effect of IGF-1 receptor inhibition with AG1024 on MAP kinase activation by Angll. Quiescent SMCs were treated with 1 μM Angll, and harvested after 5 min for assessment of MAP kinase phosphorylation by Western blotting. The degree of phosphorylation is directly linked to activity (Yau and Zahradka, 1997, Mol Cell Biochem 172: 59-66). As we have seen previously (Yau et al, 2003), Angll stimulated a rapid increase in MAP kinase phosphorylation, and this response was also obtained with IGF-1 (Figure 5A). However, the degree of MAP kinase phosphorylation was not reduced in the presence of AG1024. Interestingly, inhibition of Src kinase with PP-1 prevented MAP kinase phosphorylation, suggesting the existence of a Src kinase-dependent pathway that branches prior to reaching the IGF-1 receptor. To verify the observation that MAP kinase activation occurs independent of IGF-1 receptor phosphorylation, SMCs were immunologically stained for phosphorylated MAP kinase (Figure 5B). In this experiment, strong staining was observed in the nuclei of Angll and IGF-1 treated cells (panels B and D, respectively) relative to untreated control (panel A). As was seen by Western blotting, AG1024 did not affect the stimulation produced with Angll treatment (panel C). These data indicate that IGF-1 receptor transactivation . is
essential for PI3-kinase activation in response to Angll, but is apparently not required for MAP kinase phosphorylation. AG1024 is capable of preventing neointimal formation in an organ culture model of restenosis. Porcine hearts were obtained from the local abattoir, transported to the laboratory on ice, and the left descending coronary artery carefully exposed. The coronary arteries were injured in situ by inflation of an angioplasty catheter (3.5 mm x 20 mm), then carefully dissected free of the underlying heart tissue. Each injured or non-injured control vessel was divided into 4 segments of 5 mm and individual segments were placed into 24- well dishes with 1.0 ml growth medium (Dulbecco's modified Eagle medium containing 20% fetal bovine serum) plus various concentrations of AG1024. The vessel segments were incubated in a standard C02 incubator for 14 days, with medium plus treatments refreshed every second day. Neointimal formation was quantified by morphometric analysis of each vessel segment. Briefly, the segments were frozen in O.C.T. medium after removal of 1.5 mm from each end. Sections of 7 μm obtained with a cryostat were stained with Lee's methylene blue. Digital photographs were prepared of each section and StainPoint software employed to quantify the neointimal index, a ratio of the intimal and medial areas. This method is described in detail in several publications from this laboratory (Wilson et al 1999 Cardiovasc Res 42:761-772, Yau et al 2001 Am J Physiol 281 :H1648-H1656, Zahradka et al 2002 J Mol Cell Cardiol 34:1609-1621). Figure 7 demonstrates the results of one experiment conducted to determine the concentration effect of AG1024 on neointimal formation. Non-injured (NI) and balloon- injured (Bl) segments incubated in the absence of AG1024 served as negative and positive controls, respectively. The AG1024 concentrations are as indicated. A total of 10 segments were used for each experimental condition. Statistically significant differences (P<0.05) from the Bl injured control were identified by 1-way ANOVA. While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.