WO2007065016A2

WO2007065016A2 - Methods and compositions to improve activity and reduce toxicity of stents

Info

Publication number: WO2007065016A2
Application number: PCT/US2006/046296
Authority: WO
Inventors: Jessie L.S. Au; Guillaume M. Wientjes
Original assignee: Au Jessie L S; Wientjes Guillaume M
Priority date: 2005-12-02
Filing date: 2006-12-04
Publication date: 2007-06-07
Also published as: WO2007065016A3

Abstract

Disclosed is a method of improving the efficacy of endovascular drug- releasing stents implanted in a subject, wherein one or more modulators of drug transport are administered at such a time as is beneficial to maintain nearby tissue concentrations of the drug released by the stent within their therapeutically effective range, wherein one or more modulators of drug activity are administered at such a time as is beneficial to prevent restenosis, and wherein one or more modulators of drug activity are administered at such a time as is beneficial to promote the early healing or reendothelialization process.

Description

METHODS AND COMPOSITIONS TO IMPROVE ACTIVITY AND REDUCE TOXICITY OF STENTS

Related Information

This application claims priority to provisional application serial number 60/74185, filed December 2. 2005, entitled "Methods and Compositions to Improve Efficacy of Drug-Coated Stents". This application is cross-referenced to U.S. Patent No. 6,599,912, entitled "Methods and Compositions for Modulating Cell Proliferation and Cell Death", filed on June 5, 2000, the disclosure of which is expressly incorporated herein by reference.

Field

This disclosure relates generally to biomedical, intraluminal, or intraductal stents. More specifically, the disclosure relates to methods of improving the efficacy of prevention of restenosis occurring in radiation or drug-coated endovascular stents. In another aspect, this disclosure provides methods to reduce the smooth muscle cell toxicity of suramin, a drug that generally enhances the cytotoxicity of anticancer drugs.

Background

Stents. A stent is a hollow, tubular, endovascular implant inserted into a blood vessel to provide mechanical support to the vessel, to prevent a vessel from collapsing, and to maintain and/or re-establish a flow channel during or following angioplasty. A stent can also be an intraluminal implant inside a duct.

Response of arteries to stentinq. Angioplasty and stenting cause damage to vessel walls. The endothelium is a single layer of endothelial cells lining the vascular wall. Re-endothelialization re-establishes the luminal endothelial monolayer that blocks circulating cells from interaction with underlying extracellular matrix and smooth muscle cells. Accelerating the re- endothelialization of the damaged arterial segment following stenting has the benefit of reducing the amount of neointimal hyperplasia and stent thrombosis. The ability of endothelium to repair itself depends on both the migration of surrounding mature endothelial cells and on the adhesion of circulating endothelial progenitor cells that differentiate into endothelial-like cells, to the injured area (Ong, Arch MaI Coeυr, 98, 123, 2005). Inflammation due to stenting often leads to neointima, or growth inside the stent. This process is restenosis, which involves the release of growth factors that stimulate smooth muscle cells to migrate and proliferate, as well as activating endothelial cells at the injury sites. Restenosis occurs secondary to the accumulation of smooth muscle cells and the extracellular matrix molecules (e.g., proteoglycans, hyaluronan and collagen) synthesized by these cells. The latter accounts for over 50% of the volume of neointima (Farb, Circulation, 99, 44, 1999; Schwartz, J Am Coll Cardiol, 20, 1284, 1992; Garratt, J Am Coll Cardiol, 17, 442, 1991 ; Carter, J Am Coll Cardiol, 24, 1398, 1994). After stenting, blood clots may form and thrombosis is present at least on a microscopic level on stent surfaces. Thrombosis plays a significant role in the early stages of restenosis by establishing a biocompatible matrix on the surfaces of the stent whereupon smooth muscle cells may subsequently attach and multiply. In animals, restenosis consists of a series of intravascular changes

(Edelman, Am J Cardiol, 81, 4E, 1998). During the first 3 days, a fibrin-rich thrombus containing entrapped erythrocytes and platelets is formed. From day 3 to day 8, the thrombus is covered by a thin layer of endothelial-like cells and is infiltrated by inflammatory cells. Starting from day 5 and lasting for about 20 days, smooth muscle cells proliferate and replace the residual thrombus, resulting in the neointimal expansion. In about 28 days after stenting, the neointima reaches the maximal thickness and the proliferation rate of smooth muscle cells returns to a resting level. In about 3 to 6 months, the neointima shrinks and remodeling occurs. The complete healing of the arterial wall after coronary stenting requires at least 18 months.

Methods to prevent neointimal growth include the use of radiation and drug-coated stents.

Radiation stents. The art describes radiation stents using gamma- and beta-emitters or intra-luminal brachytherapy (Bhargava, Lancet, 359, 543, 2002; Teirstein, N Engl J Med, 336, 1697, 1997; Leon, N Engl J Med, 344, 250, 2001; Waksman, Circulation, 102, e9046, 2000; Popma, Circulation, 106, a 1090, 2002). The effects of radiation are dose- and distance-dependent. Beneficial effects are observed when the dose was higher than 15 or 20 Gy given at 1.5 mm from the center of the source, but not when the dose is lower than 10 Gy (Wiedermann, J Am Coll Cardiol 23, 1491, 1994; Waksman, Circulation, 91, 1539, 1995; Waksman, Circulation, 92, 1383, 1995). Restenosis is observed at the proximal and/or distal end of an irradiated segment, presumably due to the lower radiation dose at the ends (Sabate, Circulation, 101, 2467, 2000; Albiero, Circulation, 101 , 2454, 2000). Drug-coated stents. The art describes the coating of stents with agents that inhibit one or more processes in neointimal growth. Such agents may be dispersed or dissolved in either a bio-durable or bio-erodable polymer matrix attached to the surface of the stent wires. After implantation, the entrapped agent is released from the stent to the surrounding tissues through diffusion and/or upon erosion of the polymer.

Coating of stent surface with heparin and other anti-platelet or anti- thrombolytic agents interferes with the blood-clot formation, or prevents the attachment of platelets (a precursor to thrombin) on the stent surface (e.g., U.S. Patents No. 6,231 ,600 and 5,288,711). The art also describes the use of agents that inhibit smooth muscle cell proliferation. Examples include a quinazolinone derivative (U.S. Patent No. 6,159,488), a cytotoxic agent (paclitaxel, e.g., U.S. Patents No. 6,171 ,609), a metal (silver, U.S. Patent No. 5,873,904), a membrane-stabilizing agent with antiinflammatory properties (tranilast, U.S. Patent No. 5,733,327), an immunosuppressant (rapamycin, e.g. U.S. Patents Nos. 5,288,711 and 6,153,252), and, inter alia, rapamycin derivatives, rapamycin analogues, camptothecin, dexamethasone, and 5-fluorouracil (U.S. Patent Appl. 20050070997 dated March 31 , 2005).

The art further describes using systemic administration of a monocyclic triene immunosuppressant everolimus and related compounds to treat restenosis (PCT Publication No. WO 97/35575).

Unresolved problems for radiation and drug-coated stents. For bare metal stents without drug coating, in-stent restenosis occurs in up to 40% of patients at 6 months after implantation. The use of drug-encoated stents lowers the rate of restenosis to about 10% after 6 months (Radke, Eur Heart J, 24, 266, 2003; Colombo, Circulation, 108, 257, 2003). Restenosis was present in 60% of patients at 12 months for paclitaxel-eluting stents, and 26% at 12 months for rapamycin-eluting stents (Liistro, Circulation, 105, 1883, 2002; Colombo, Circulation, 109, 1244, 2004; Presbitero, ltal Heart J, 6, 514, 2005). A 10% rate translates to about 100,000 patients per year. The inability of drug-encoated stents to prevent restenosis at later times, e.g., after 6 or 12 months, is likely due to the depletion of the drug being released from the stent.

A second problem with drug-encoated stents is the drug toxicity to vessels. For instance, vessels treated with paclitaxel-eluting stents exhibit chronic low-grade inflammation, intra-intimal hemorrhage, and poor healing of the endothelium (Farb, Circulation, 104, 473, 2001). Similarly, higher radiation doses of >15 Gy resulted in inadequate endothelial recovery at 1 and 3 months in human patients. A discontinuous endothelium renders the luminal surface prothrombotic and increases the risk of thrombotic occlusions (Salame, Circulation, 101 , 1087, 2000). The unwanted toxicity of drug-coated stents is likely caused by the high drug concentration due to the initial burst drug release from stents (Hwang, Circulation, 104, 600, 2001 ; Creel, Circ Res, 86, 879, 2000). For example, the in vitro release of paclitaxel from stents shows a burst release of 36% of the dose in the first day followed by a slower release over 2 months (Drachman, J Am Coll Cardiol 36, 2325, 2000). The release of cytochalasin D from polymer-coated stents showed similar burst followed by a gradual elution over time (Verhoeven, J Control Release, 96, 113, 2004). Furthermore, the toxicity of drug-encoated stents limits the amount of drug to be loaded on a stent, which in turn limits the duration for successful prevention of restenosis. Improvement of the efficacy of radiation and drug-coated stents would require extending the desired restinosis-preventing effect and reducing the unwanted toxicity of radiation and drugs, and promoting the re-endothelialization process. Another approach is to use drugs that can provide the desired restinosis-preventing effects without causing the unwanted toxicity. This application describes methods to attain these goals.

Art on drug efflux mdr1 gene and its product p-glvcoprotein. The protein product of mdr1 gene is p-glycoprotein (Pgp). Pgp is a member of a superfamily of drug transport proteins known as ATP-binding cassette (ABC) transporters and is most highly expressed in the epithelial cells of the intestine, kidney, liver, pancreas, and capillary endothelia of the brain and testes. The transporters help form the blood-brain and blood-testes barriers (Johnstone, Trends Biochem Sci., 25, 1 , 2000). Pgp is primarily present on the apical surface of the cell and the transport is outwards, suggesting a physiological role of cell protection (Schinkel, PNAS, 94, 4028, 1997). A wide range of structurally- unrelated hydrophobic compounds, Including, inter alia, agents that inhibit cell proliferation including paclitaxel, rapamycin, doxorubicin, and camptothecin, are substrates for the Pgp transporter (Johnstone, Trends Biochem Sci., 25, 1 , 2000).

The art describes the detection of Pgp in heart muscle fibers (Thiebaut, J.

Histochern Cytochem, 37, 159, 1989), normal cardiac muscle cells (Garberoglio, Arch Path Lab Med, 116, 1055, 1992), diseased cardiac muscle cells

(Lazarowski, J Histochem Cytochem, 53, 845, 2005), and in endothelium of both arterioles and capillaries in human hearts (Meissner, J Histochem Cytochem 50,

1351, 2002). The art also recognizes the use' of Pgp inhibitors to reduce the efflux and to enhance the activity of anticancer drugs that are Pgp substrates, in cancer cells (Sikic, Cancer Chemother Pharmacol, 40, S 13, 1997). However, the art does not recognize the use of Pgp inhibitors to inhibit the efflux or to enhance the activity of drugs used to coat stents in smooth muscle cells. The art also does not recognize the use of Pgp inhibitor to improve the effectiveness of drug- eluting stents. The art recognizes the expression of mdr1 gene in human atherosclerotic lesions and proliferating human smooth muscle cells (Batetta, Cell MoI Life Sci, 58, 1113, 2001). The art also recognizes that mdr1 gene is involved in the transport of free cholesterol from the plasma membrane to the endoreticulum (Batetta, 2001), a step required for cholesterol esterification, which is associated with accelerating the cell cycle progression and the proliferation of smooth muscle cells from atherosclerotic lesions. Inhibition of the cholesterol esterification pathway causes arrest in the G1 phase of the cell cycle and inhibits the proliferation of smooth muscle cells (Batetta, FASEB J, online 10.1096/fj.02- 0396fje, 2003). However, the art does not recognize the use of Pgp inhibitors to inhibit the proliferation of smooth muscle cells or the use of Pgp inhibitors to coat stents or to improve the effectiveness of stents coated with other drugs that are Pgp substrates or non-substrates.

Art on mdr1 and paclitaxel-induced apoptosis. The inventors previously disclosed that mdr1 expression positively correlates with the extent of paclitaxel-induced apoptosis in human tumor samples and cultured cancer cells (Gan, Clin. Cancer Res., 4, 2949, 1998; Li, Pharmaceut Res 18, 907, 2001). This relationship is unexpected in view of the drug efflux function of Pgp and in view of the generally accepted pharmacological principle that more drug would result in greater effect rather than less effect. The inventors have further disclosed that the higher paclitaxel-induced apoptosis in mdr7-transfected cancer cells is not related to the drug efflux property of mdr1 or Pgp (Li, Pharmaceut Res, 18, 907, 2001). The art does not recognize the use of Pgp inhibitors to reduce the paclitaxel-induced apoptosis or cytotoxicity in cancer cells, or in smooth muscle cells. The art further does not recognize the dual use of Pgp inhibitors to improve the efficacy of paclitaxel-encoated stents: (a) protecting endovascular cells against the cytotoxicity of paclitaxel when paclitaxel is present at high concentrations, e.g., during the initial burst release or when a significant amount of drug is being released from the stent, and (b) enhancing the cytotoxicity of paclitaxel when paclitaxel is present at low concentrations, e.g., when most of the drug load has been released, removed or depleted.

Art on Pgp expression and efflux of paclitaxel from cells. The inventors previously disclosed that the relative importance of Pgp-mediated efflux of paclitaxel is dependent on the extracellular paclitaxel concentrations. In human breast cancer cells transfected with the mdr1 gene and showing 9-times higher Pgp protein levels compared to the untransfected parent cells, the Pgp efflux was the major mode of drug efflux accounting for 86% of the total efflux at low paclitaxel concentrations of 1 nM or less, whereas the Pgp efflux was a minor mode of efflux accounting for only 34% of the total efflux at higher paclitaxel concentrations (Jang, J. Pharmacol. Exp. Therap., 298, 1236, 2001). The art does not provide information on the relative importance of Pgp efflux in smooth muscle cells that have natural mdr1 gene expression (i.e., no genetic manipulation). The art further does not disclose the use of Pgp inhibitors to reduce the paclitaxel efflux from smooth muscle cells.

Art on protection against toxicity of radiation or druα-encoated stents. The inventors have disclosed using a combination of aFGF and bFGF to protect against the toxicity of cancer chemotherapeutic agents in normal, noncancerous cells that are rapidly proliferating, such as cells in hair follicles and gastrointestinal crypts (US Patent No. 6,599,912). The present invention discloses using a combination of aFGF and bFGF to protect endovascular cells against the unwanted toxicity of drug-encoated stents and radiation stents.

Art on suramin as a chemosensitizer and radiosensitizer. The art recognizes that suramin has activity against cancer when used alone or in combination with chemotherapy. However, the development of suramin as an anticancer drug has shown that suramin produces significant hematologic toxicity but limited efficacy in human patients with prostate cancer, such that the application to use suramin as an anticancer drug was disapproved by the US Food and Drug Administration. The inventors have disclosed a new use of suramin, given at lower doses that do not cause significant hematologic toxicity, as a chemosensitizer or radiosensitizer to enhance the efficacy of chemotherapy or radiation in the treatment of cancer (US Patent No. 6,599,912).

In the same patent, the inventors further disclosed that the chemosensitization effect of suramin is abolished when the suramin dosage is higher than 100 mcM, The art describes that suramin at concentrations of 500 mcM, or about twice the dosage used for systemic administration, is sufficient to completely neutralize the proliferation stimulation by 10% human whole blood serum (Engisch, J Vascular Interventional Radiology 11 , 639, 2000). The same disclosure recognizes the use of suramin for prevention of restenosis by local delivery, and recommends against using systemic delivery of suramin for prevention of restenosis. However, the art does not recognize the use of suramin at concentrations or dosages below 500 mcM or, more preferably, below 100 mcM for coating stents. The art also does not recognize the use of suramin at concentrations below 100 mcM to enhance the efficacy of chemotherapy that are used to coat stents or radiation stents. The art further does not recognize the use of systemic administration of suramin, at any dose, to enhance the efficacy of radiation or drug-eluting stents.

Art on suramin toxicity to smooth muscle cells. The inventors have disclosed that suramin, at concentrations below 100 mcg/ml (equal to about 75 mcM), has either no or insignificant cytotoxicity to tumor cells (U.S. Patent No. 6,599,912). The present invention discloses the unexpected discovery that suramin, at concentrations below 75 mcM, exhibits significant cytotoxicity in smooth muscle cells. Another surprising element of this discovery is the much greater activity or potency of suramin in smooth muscle cells compared to the earlier report (Engisch, 2000). For example, the present discovery shows that suramin at concentrations below 75 mcM reduced the total cell number by more than 75% compared to cells that were not treated with suramin, whereas the earlier report shows that 300 mcM suramin was required to produce a 20% reduction of the proliferation stimulation by 10% human whole blood serum. The inventors discovered that cancer patients who were treated with a combination of chemotherapy with suramin used as a chemosensitizer often experienced muscle weakness or fatigue. The art does not recognize that suramin, when given at the concentrations for producing chemosensitization, produces cytotoxicity in smooth muscle cells, which is much greater compared to the suramin cytotoxicity in cancer cells.

The inventors disclose the use of suramin-encapulated nanoparticles to reduce the muscle weakness or fatigue associated with the use of suramin. The property of tumor targeting by the "enhanced penetration and retention" effect that is observed for nano-sized particles has been described in the literature (Ogihara, Eur. J. Nucl. Med., 11 , 405, 1986; Li, Cancer Chemother. Pharmacol., 46, 416, 2000; Bourdon, Photochem. Photobiol. Sci., 1, 709, 2002; Alexiou, J. Drug Target, 11 , 139, 2003), but has not been applied to reduce fatigue.

Summary

The inventors discovered several agents and classes of agents that have the following desired properties for improving the efficacy of stents: (a) Pgp inhibitors (e.g., Valspodar, cyclosporin A, verapamil) or FGF inhibitors (e.g., suramin), which produce cytotoxicity in smooth muscle cells;

(b) Pgp inhibitors, which improve the retention of drugs used to encoat stents;

(c) Pgp inhibitors or FGF inhibitors, which promote the activity of drugs or radiation used to encoat stents. This effect of Pgp inhibitors is most pronounced when cells are treated with low paclitaxel concentrations (equal to or less than 100 nM for 96 hours).

(d) Pgp inhibitors or a combination of aFGF and bFGF, which protect smooth muscle cells against the unwanted toxicity of drugs or radiation used to encoat stents. This protective effect of Pgp inhibitors is observed when cells are treated with high paclitaxel concentrations (above 100 nM for 9.6 hours).

(e) Pgp inhibitors, which have the dual functions of protecting against the toxicity of high paclitaxel concentrations and promoting the paclitaxel activity at lower paclitaxel concentrations.

This disclosure describes methods and compositions to use these discoveries to improve the efficacy of stents.

A reduced cytotoxicity of radiation or drugs to endovascular cells, e.g., endothelial or smooth muscle cells, especially shortly after stenting when active wound healing is ongoing, would improve the healing of the damaged tissues. A promotion of the wound healing process would improve the healing of the damaged tissues. Enhancing the cytotoxicity of radiation or drugs at later times when most of the radiation or drug dose has been depleted would extend the duration of the effectiveness of stents against restenosis. The present disclosure uses, inter alia, inhibition of the mdr1 gene product Pgp, to increase the concentration and retention of drugs that are Pgp substrates, in smooth muscle cells. Enhancement of the concentration of cytotoxic agents in smooth muscle cells is expected to yield several advantages. One such advantage is that a lower release rate of cytotoxic agent from the drug-releasing stent is needed to maintain the desired cell growth inhibitory action. As the maximal amount of drug that can be contained in a stent is limited, the required release rate of cytotoxic agent is one of the limiting factors restricting the duration of time that growth inhibitory concentrations can be maintained and the process of restenosis can be retarded or prevented. Another advantage is to promote the activity of agents used to coat stents. This application discloses the unexpected, higher sensitivity of smooth muscle cells to cytotoxicity of suramin relative to tumor cells, and the occurrence of muscle weakness and fatigue in patients receiving suramin in the treatment of a cancer. The overall cytotoxicity of suramin in porcine artery smooth muscle cells shows that 40 mcM suramin was sufficient to produce 50% cytotoxicity (referred to as IC50). This value is substantially lower than the IC50 concentrations determined in cancer cell lines (Song, Cancer Res., 61, 6145, 2001 ; Zhang, J. Pharmacol. Exp. Ther., 299, 426, 2001), indicating the greater susceptibility of muscle cells to the suramin activity. The inventors previously disclosed using suramin at concentrations or at doses that deliver plasma concentrations of less than 100 mcM, more preferably less than 70 mcM, to improve the therapeutic outcome of chemotherapy in the treatment of patients with cancer (U.S. Patent No. 6,599,912). This application further discloses that suramin at concentrations below 100 mcM also can enhance the muscle cell toxicity of chemotherapy used to treat cancer patients. Based on these unexpected findings, the inventors disclose two new uses of suramin. The first aspect is the use of suramin at concentrations below 100 mcM, preferably below 70 mcM, or at doses that deliver less than 100 mcM, preferably below 70 mcM, to the diseased vessels by either systemic administration (e.g., inter alia, by an intravenous injection) or locally (e.g., inter alia, by coating stents with suramin) to reduce restenosis. The second aspect is the use of suramin at concentrations below 100 mcM or at doses that deliver less than 100 mcM to the diseased vessels by either systemic administration {e.g., inter alia, by an intravenous injection) or locally (e.g., inter alia, by coating stents with suramin) to improve the activity of agents (e.g., inter alia, paclitaxel, rapamycin) used to coat stents for the purpose of reducing restenosis.

In a related aspect, the inventors disclose the use of suramin encapsulated in drug delivery systems that provide lower delivery to muscles, relatively to other tissues, inter alia tumor tissue, to reduce the exposure of muscle tissues and, hence, the undesired muscle-related toxicity of suramin.

Brief Description of the Drawings

For a fuller understanding of the nature and advantages of the present invention, reference should be had to the following detailed description taken in connection with the accompanying drawings, in which: Figure 1 depicts the presence of Pgp protein in smooth muscle cells.

Microscopic cross sections of dog and human arteries and cultured smooth muscle cells were immunohistochemically stained for Pgp. Positive staining from the chromogen DAB (brown color) indicates the presence of Pgp in the smooth muscle cells of the arterial wall and in cultured cells. Figure 2 depicts that Pgp inhibitors (Valspodar, cyclosporine A, verapamil) improved the retention of cytotoxic agents in smooth muscle cells. Cultured smooth muscle cells were loaded with doxorubicin or rhodamine and then placed in fresh medium (without doxorubicin or rhodamine). Both agents show fluorescence. The efflux of doxorubicin and rhodamine, both emitting fluorescence, from cells over time was monitored using fluorescence microscopy. Control cells were treated similarly with the exception that no Pgp inhibitors were applied. The results show that treatment with Pgp inhibitors reduces the efflux from, and increases the retention of doxorubicin or rhodamine in cells.

Figure 3 depicts the dual effects of Pgp inhibitors (Valspodar, cyclosporine A, verapamil) on the overall cytotoxicity of paclitaxel in porcine artery smooth muscle cells. The drug effects were measured using the MTT assay. Pgp inhibitors promoted the activity of paclitaxel at pacfitaxel concentrations of 100 nM or lower, but protected against the paclitaxel activity at paclitaxel concentrations of 1000 nM or higher. Detailed Description

Before further describing the invention, certain terms employed in the specification, examples and appended claims are, for convenience, collected here. /. Definitions

All definitions of U.S. Patent No. 6,599,912 are included herein by reference.

The terms "drug-coated stent" or "drug-eluting stent" or "drug-releasing stent" or "drug-encoated stent", are used interchangeably. These terms refe^r to an endovascular, intraluminal, or intraductal stent that has been prepared to release a pharmacologically active agent over time. The terms "drug", "cytotoxic drug", "cytotoxic agent", "anticancer agent", and "antitumor agent" are used interchangeably herein and refer to agents that have the property of inhibiting the growth or proliferation (e.g., a cytostatic agent), or inducing the killing, of hyperproliferative cells. Preferably, the cytotoxic agent inhibits or reverses the development or progression of a benign hyperplastic growth, or neointimal growth.

The term "released cytotoxic agent" refers to cytotoxic agents that are used to coat drug-encoated stents and are released from stents. A released cytotoxic agent is one or more of paclitaxel, paclitaxel derivatives and analogues, cytochalasin D, rapamycin, rapamycin derivatives and analogues, camptothecin, dexamethasone, and 5-fluorouracil, a quinazolinone derivative, metallic silver, tranilast, everolimus and related compounds, or other agents that inhibit smooth muscle cell proliferation and or migration and/or inflammatory processes. As used herein, a "therapeutically effective amount" of an agent refers to an amount of such agents which in combination is effective, upon single- or multiple-dose administration to the subject, e.g., a patient, at inhibiting the growth or proliferation, or inducing the killing, of hyperproliferative cells.

The term "protective agents" refers to agents that protect endovascular tissues or cells from the unwanted toxicity of radiation or drug-eluting stents.

The term "modulating agent" or "modulator" refers to agents that change the pharmacological effect of a cytotoxic agent that is formulated in a drug- encoated stent. A modulating agent can be a transport inhibitor, for example a Pgp inhibitor. A modulating agent can also be a protective agent, for example a combination of aFGF and bFGF. A modulating agent can also be a sensitizing agent, for example suramin.

The terms "enhancing agent" or "enhancing agents" refer to agents that enhance the activity of radiation or cytotoxic drugs used to coat stents.

The term "modulating agent" or "modulator" refers to agents that change the pharmacological effect of the inhibitor of cell migration or cell proliferation that is formulated in a drug-encoated stent. A modulating agent can be a transport inhibitor, for example a Pgp inhibitor. A modulating agent can also be a protective agent, for example a combination of aFGF and bFGF. A modulating agent can also be a sensitizing agent, for example suramin. The terms "enhancing agent" or "enhancing agents" refer to agents that enhance the activity of radiation or cytotoxic drugs used to coat stents.

The terms "systemic administration", "administered by systemic means", or "administered by a systemic route" refer to administration of an agent by a systemic route. For example, the agent is administered parenterally (e.g., subcutaneously, intravenously, intramuscularly, intraperitoneally, intradermally, intrathecal^, etc.), orally, nasally, intrapulmonary by inhalation, rectally, and/or transdermally.

The term "administered locally or regionally" refer to administration of an agent, e.g., endovascularly, intraluminally, intra-arterially, intraductal, or in the tissue surrounding the stented vessel.

As used herein, the term "fibroblast growth factor" or "FGF" refers to a member of a family of polypeptides that are potent regulators of a variety of cellular processes including proliferation, differentiation, migration, morphogenesis, tissue maintenance and in wound healing and repair (Clarke, J Cell Sci, 106, 121, 1993; Cuevas, Biochem Biophys Res Commun 156, 611 , 1988; Burgess, Ann Rev Biochem 58, 575, 1989; Rifkin, J Cell Biol, 109, 1 , 1989). The FGF family currently includes at least 19 structurally and functionally related proteins, including acidic and basic FGF₁ FGF-1 and FGF-2 respectively; inn (FGF-3); hst (FGF-4); FGF-5; hsfZ (FGF-6); keratinocyte growth factor (FGF- 7); androgen-induced growth factor (FGF-8); glia-activating factor (FGF-9); FGF- 10-19 (Galzie, Z., Biochem Cell Biol, 75, 669, 1997; Yamasaki, J Biol Chem, 271 , 15918, 1996; Smallwood, Proc Natl Acad Sci USA, 93, 9850, 1996; McWhirter, J. R., Development, 124, 3221, 1997; Hoshikawa, Biochem. Biophys. Res. Comm, 244, 187, 1998; Hu, MoI Cell Biol, 18, 6063, 1998; Nishimura, T., Biochim Biophys Acta, 1444, 148, 1999). Preferably, the term FGF refers to acidic and basic FGF, FGF-1 and FGF-2, respectively (reviewed in (Galzie, Biochem Cell Biol, 75, 669, 1997; Burgess, W.H. and Maciag, Ann Rev. Biochem, 58, 575, 1989).

The term "FGF antagonists" refers to molecules that antagonize FGF actions. An FGF antagonist refers to an agent that inhibits (completely or partially) the activity, production, stability, of an FGF molecule. Preferably, the FGF antagonist is an inhibitor of bFGF, aFGF, or an inhibitor of both.

The term "FGF agonist" refers to an agent that has FGF-like activity, potentiates the activity, production, stability, of an FGF molecule, or activates FGF receptors.

Examples of chemicals that may antagonize FGF action include, inter alia, suramin, structural analogs of suramin, pentosan polysulfate, scopolamine, angiostatin, sprouty, estradiol, carboxymethylbenzylamine dextran (CMDB7), suradista, insulin-like growth factor binding protein-3, ethanol, heparin (e.g., 6-O- desulfated heparin), low molecular weight heparin, protamine sulfate, cyclosporin A, or RNA ligands for bFGF. The FGF antagonist may also be a small molecule, e.g., a member of a combinatorial library. The FGF antagonist also may be an interferon.

As used herein, the language "subject" or "patient" is intended to include human and non-human animals. Preferred human animals include a human patient having a disorder characterized by the aberrant activity of a hyperproliferative cell. The term "non-human animals" of the invention includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, etc. Preferably, the subject is a human patient, e.g., a patient receiving implementation of an endovascular stent, or a cancer patient, e.g., a patient with a malignant or benign hyperplastic disorder.

The terms "inhibiting restenosis", "inhibiting neointimal growth", "preventing restenosis", or "protecting against restenosis", refers to inhibiting, slowing, interrupting, arresting or stopping the processes involved in the neointimal growth and restenosis, and does not necessarily indicate a total elimination of the neoplastic growth.

As used herein, "apoptosis" refers to a form of cell death that can occur naturally or upon treatment with a cytotoxic agent or a drug. The terms "induce", "enhance", "inhibit", "potentiate", "elevate", "increase", "decrease" or the like, denote quantitative differences between two states, refer to at least statistically significant differences between the two states. For example, "an amount effective to inhibit growth of hyperproliferative cells" means that the rate of growth of the cells will at least statistically significantly differ from the untreated cells. Such terms are applied herein to, for example, rates of cell proliferation.

The terms "drug loading", "loading of drug" or "drug load" are used interchangeably and refer to the amount of a drug loaded on a stent.

The term "Pgp inhibitors" refers to agents that inhibit the mdr1 product Pgp- Pgp inhibitor is one or more of the following compounds: 17-ally!amino-17- demethoxygeldanamycin (17-AAG), amiodarone, amitriptyline, amprenavir, arsenic trioxide, atazanavir, atorvastatin, azelastine, azidopine, bepridil, biricodar (VX 710), bromocriptine, 5-bromotetrandrine, capsaicin, carvedilol, celecoxib, cephalosporines, cepharanthine, chlorpromazine, clarithromycin, clofazimine, clozapine, coumarin, CP 100356, Cremophor EL, curcumin, cyclosporin A, daidsein, desipramine, dexverapamil, diclofenac, dihydro-beta-agarofuran sesquiterpenes, diltiazem, dipyridamole, disulfiram, diterpenic lactones, DM27, doxepin, elacridar (GF120918), ellipticines, emitine, endotoxin, erythromycin, evodiamine, felodipin, FG020326, flupentixol, fluphenazine, gallopamil, gefitinib, genistein, GG918, glibenclamide, grepafloxacin, haloperidol, imipramine, indinavir, isradipine, itraconazole, ivermectin, kaempferol, ketoconazole, laniquidar (R101933), lansoprazole, levofloxacin, lipopolysaccharide, loperamide, medroxyprogesterone, mefloquine, meloxicam, methoxypolyethylene glycol- block-Polycaprolactone diblock copolymer, miconazole, MS209, nefazodone, nelfinavir, nifedipine, ningalins, nobiletin, norverapamil, NS-398, OC 144-093 (ONT-093), ofloxacin, omeprazole, orataxel (BAY59-8862), pantoprazole, paroxetine, PCB-126, phenethyl isothiocyanate, phenothiazines, phenytoiπ, pimozide, pluronic P85, polysorbate 80, progesterone, promethazine, propafenone, propranolol, PSC 833 (valspodar), quercetin (QU), quinidine, quinine, reserpine, rifampin, ritonavir, RNAi₁ saquinavir, schisandrin B, selamectin, sertraline, simvastatin, sparfloxacin, spironolactone, tacrolimus, tamoxifen, tariquidar (XR9576), terfenadine, testosterone, tetranadrfne, thioridazine, TRA96023, trifluoperazine, triflupromazine, Triton X-100, Tween 80, valinomycin, vanadate, verapamil, yohimbine, or zosuquidar (LY335979). A modulator or chemosensitizer is one or more of the following compounds: AG17 (3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile), Andrographolid (AG), Bcl2 antisense oligonucleotides, Benzinidazole, Bortezomib (Velcade), Budesonide, Buthionine sulfoximine (BSO), CHK1 inhibitors, CHK2 inhibitors, Citalopram, Clomipramine, COX-2 inhibitors, Dexamethasone (DEX), Dipyridamole (DP), EGFR inhibitors, Fluoxetine (prozac), GEM231 (antisense oligonucleotide to Rlalpha-subunit of cAMP-dependent protein kinase (PKA), GPI 15427 (PARP-1 inhibitor), Inostamycin, Malagashanine, Metoclopramide, Misonidazole, MDM2 antisense oligonucleotides, NLCQ-1 (4-[3-(2- nitroimidazolyl)-propylamino]-7-chloroquinoline hydrochloride), NLCPQ-1 (9-[3-(2- Nitro-i-imidazolyOpropylaminol-cyclopentenoIblquinoline hydrochloride), Perillyl alcohol (POH), Phenoxodiol, PK11195 (carboxamide derivative of isoquinolone), PG490-88 (a derivative of triptolide), Reserpine, Rituximab (chimeric anti-human CD20 monoclonal antibody), Ro 03-8799 (pimonidazole), SR 2508 (etanidazole), STI571 , Suramin, Survivin antisense oligonucleotides, THNLA-1, tirapazamine (3-amino-1 ,2,4-benzotriazine 1 ,4-di-N-oxide), Wortmannin.

A modulator or radiosensitizer is one or more of the following compounds: i-^'.S'-dideoxy-alpha-D-erythro-hex^'-enopyranosyO^-nitroimidazole (RA-263), 1-(2-hydroxy-3-methoxypropyl)-2-chloro-4-nitroimidazole [P40], 2-nitroimidazole- 1-acetohydroxamate (KIH-802), 3-aminobenzamide (3-ABA), 4-[3-(2-Nitro-1- imidazolyl)propylamino]-7-chloroquinoline hydrochloride (NLCQ-1), 5-[3-(2-nitro- 1-imidazoyl)-propyl]-phenanthridinium bromide (2-NLP-3 or NLP-1), 5'- bromodeoxyuridine (BrdU), 5-Chlorodeoxycytidine (CIdC)₁ 5-fluorodeoxycytidine (FdC), 5-lodo-2-pyrimidinone-2'-deoxyribose (IPdR), 5-iodo-2'-deoxyuridine (IdUrd), 5-Nitro-4-(N,N-dirnethylaminopropylamino)quinoline (5-nitraquine), 5- Phenoxysulphonyl-1-methyl-4-nitroimidazole (NSC 38087), 8-nitrocaffeine, 9-[3- (2-Nitro-1-imidazolyl)propylamino]-cyclopentenotb]quinoline hydrochloride

(NLCPQ-1), acridine-linked 2-nitroimidazole (NLA-1), doxorubicin, AK-2123, AT1727 (a derivative of ICRF 154), Azomycin riboside, Benzotriazine SR 4233, Bromomisonidazole, carboplatin, CI-1010((R)-alpha-[[(2-bromoethyl)- amino]methyl]-2-nitro-1 H-imidazole-1-ethanol monohydrobromide) (a prodrug), or its drug MR (PD 146923), Cisplatin, doranidazole (PR-350), etanidazole, (E)-2'- Deoxy-(fluoromethylene)cytidine (FMdC), Gadolinium (111) texaphyrin (Gd-Tex) (NSC 695238), Gemcitabine (a'^'-difluorodeoxycytidine), H4U, Hydrogen peroxide, Hydroxyurea, Haloacetylcarbamoyl-2-nitroimidazples, including chloro (KIN-1800, TX-1835, and TX-1836) and bromo derivatives (TX-1844, TX-1845, and TX-1846), Lonidamine, JM216 (a platinum analogue), KIN804, KlN-806, KU- 2285 (a fluorinated 2-nitroimidazole), Metronidazole, Misonidazole (Ro 07-0582), Navelbine, Nicotinamide, Nimorazole, Nitroimidazole, N-methylformamide (NMF), N-(Phosphonacetyl)-L-aspartate (PALA), Paclitaxel, Paranitroacetophenone, Photofrin, Pimonidazole, Potassium 2-nitroimidazole-1-acetohydroxamate (KIH- 802), Pt(Rh-123)2, RAF antisense oligonucleotide, RK-28 (a 2-nitroimidazole nucleoside analogue), Ro 03-8799 (pimonidazole), RP170, RSU-1069 (a nitroimidazole-aziridine), RuCI2(DMSO)2(4-nitroimidazole)2, Sanazole (AK- 2123), SR 2508 (etanidazole), suramin, tirapazamine (3-amino-1 ,2,4- benzotriazine 1 ,4-di-N-oxide), Topotecan, TX-1877, Vinorelbine, Withaferin A, Wortmannin.

A "modulator" or "growth factor inhibitor" is one or more of the following compounds: suramin, structural analogs of suramin, anti-FGF antibodies, anti- FGF receptor antibodies, pentosan polysulfate, scopolamine, aπgiostatin, sprouty, estradiol, carboxymethylbenzylamine dextran (CMDB7), suradista, insulin-like growth factor binding protein-3, ethanol, heparin (e.g., 6-O-desulfated heparin), low molecular weight heparin, heparan sulfate, protamine sulfate, transforming growth factor beta, cyclosporin A, or RNA ligands for bFGF, Thalidomide, Linomide, Lenalidomide, TKI-258, CHIR258, PD173074, SU5402. As used herein, the term "gelatin" refers to a denatured form of the connective tissue protein collagen. Gelatin aggregates, formed in solution, are stabilized by cross-linking the protein chains. Gelatin is available in different protein chain lengths, indicated by different Bloom numbers. Larger Bloom numbers indicate longer chain lengths.

Detailed Description

The present disclsoure is based, at least in part, on the following discoveries:

1. Pgp inhibitors, at concentrations that have been safely used in humans, are able to produce cytotoxicity in smooth muscle cells.

2. Use of Pgp inhibitors reduces the efflux of cytotoxic agents from smooth muscle cells. Use of Pgp inhibitors also promotes the effectiveness of a cytotoxic agent released from a drug-releasing stent. Combined use of these Pgp inhibitors and a cytotoxic agent encoated on a drug-eluting stent improves the effectiveness of drug-encoated stents. 3. Pgp inhibitors have dual effects on the cytotoxicity of paclitaxel. Pgp inhibitors enhanced the activity of paclitaxel at low paclitaxel concentrations (equal to or below 100 nM, 96 hour treatment), but protected against the toxicity of paclitaxel at high paclitaxel concentrations (over 1000 nM, 96 hour treatment).

4. Combined use of the two fibroblast growth factors, aFGF and bFGF, protects cells from cytotoxic injuries from cytotoxic agents. Applying these growth factors in stents protects the cells from the undesired cytotoxicity of a cytotoxic agent that is released from a drug-encoated stent.

5. FGF inhibitors such as suramin enhance the activity of cytotoxic agents in proliferating cells, e.g., smooth muscle cells. Combined use of these FGF inhibitors and a cytotoxic agent encoated on a drug-eluting stent improves the effectiveness of drug-encoated stents. 6. Suramin, at concentrations that do not have cytotoxicity in cancer cells but are sufficient to promote the cytotoxicity of other anticancer drugs, produces cytotoxicity in smooth muscle cells.

In one aspect, cytotoxic drugs that can produce cytotoxicity to smooth muscle cells are used to encoat stents, in order to reduce restenosis.

In an embodiment, these cytotoxic drugs are Pgp inhibitors. In a preferred embodiment, the Pgp inhibitor is valspodar, cyclosporine A, or verapamil.

In another embodiment, these cytotoxic drugs are FGF antagonists. In a preferred embodiment, the FGF antagonist is suramin. In another embodiment, suramin is given at dosages that deliver less than 100 mcM, preferably below 70 mcM, to smooth muscle cells.

In another aspect, the invention provides for a composition being a drug- coated stent wfth one or more enhancing agent formulated in the stent coating.

In another aspect, an enhancing agent is used to increase the effectiveness of the released cytotoxic agent.

In another aspect, the invention provides for a method of improving the functionality of drug-coated stents by systemic administration of one or more enhancing agents.

In an embodiment, an enhancing agent is used to increase the effectiveness of the released cytotoxic agent when the amount of the cytotoxic agent released from the stents are by themselves insufficient for the desired level of control of smooth muscle cell migration or proliferation.

In a preferred embodiment, the enhancing agent is a Pgp inhibitor. In a preferred embodiment, the Pgp inhibitor is valspodar, cyclosporine A, or verapamil.

In a preferred embodiment, the enhancing agent is an FGF antagonist. In a preferred embodiment, the enhancing agent is suramin. In another embodiment, suramin is given at dosages that deliver less than 100 mcM, preferably below 70 mcM, to smooth muscle cells. In one aspect, inhibition of cellular transporters is used to reduce the drug efflux from smooth muscle cells, and thereby increase the intracellular drug concentration and consequently the duration of effective prevention of restenosis.

In a preferred embodiment, the cellular transporter is Pgp and inhibition is accomplished by a Pgp inhibitor. In a preferred embodiment, the Pgp inhibitor is valspodar, cyclosporine A, or verapamil.

In another aspect, the invention provides for a composition consisting of a drug-encoated stent loaded with a cytotoxic agent and a protective agent that protects cells from injuries, where the loading of the cytotoxic agent on the stent is increased relative to the loading that can be safely allowed on a stent that does not contain a protective agent, and where the protective agent prevent endothelial cell injury during the initial period of a high drug release, thus providing for a stent with an increased functional lifespan.

In another aspect, protective agents that protect cells from the unwanted toxicity of cytotoxic drugs protect cells from excessive death or damage during the early burst release of the cytotoxic drug from the drug-coated stent. in a preferred embodiment, these protective agents are fibroblast growth factors.

In a preferred embodiment, the fibroblast growth factors are aFGF and bFGF. In another embodiment, these protective agents are Pgp inhibitors.

In a further aspect, an enhancing agent is used to increase the duration of effective control of restenosis. One limitation of drug-eluting stents is that the drug release rate declines over time, with as a consequence that the local concentrations of the cytotoxic agent are reduced below the levels needed to inhibit the proliferation and migration of the smooth muscle cells. The inventors disclose that an enhancing agent enhances the cytotoxicity of paclitaxel, rapamycin, or doxorubicin.

In an embodiment, the enhancing agent enhances the activity of the released cytotoxic agent, preferably when the concentrations of the cytotoxic agent are below the threshold levels required to produce therapeutically significant anti-migration or anti-proliferation effects, at least about 10% inhibition of migration or proliferation of smooth muscle cells. Concentrations of the cytotoxic agent will fall below effective levels when the rate of drug release from the stent declines to a rate substantially below the desired rate, measured after the initial burst release has subsided, usually one to several days after initiation of exposure of the stent to an aqueous environment. A rate substantially below the desired rate will be approximately 10% of the desired rate, or more preferably 20% of the desired rate, or even more preferably 30% of the desired rate, or even more preferably 40% of the desired rate, or most preferably 50% of the desired rate. The time at which drug release from the stent has declined substantially from the desired rate can be determined by in vitro experimentation, using stents of the same design as the stent implanted in the patient.

In one embodiment, the enhancing agent is a Pgp inhibitor.

In another embodiment, the Pgp inhibitor is formulated in the drug-coated stent.

In a preferred embodiment, the Pgp inhibitor is formulated in one or more of multiple drug-releasing layers, and the start of release of the Pgp inhibitor is delayed until the time that the efficacy of the cytotoxic agent is insufficient to inhibit smooth muscle cell proliferation. In another embodiment of the invention, the Pgp inhibitor is administered by a systemic route. In another embodiment, the Pgp inhibitor is administered locally or regionally, e.g., intraluminally, intraductally, intra-arterially, or in the tissue surrounding the stented vessel. In a preferred embodiment, the Pgp inhibitor is administered at a regimen known to result in effective Pgp inhibition in smooth muscle tissue.

In a preferred embodiment, the Pgp inhibitor is one or more of cyclosporin A, verapamil, or Valspordar.

In a preferred embodiment, the released cytotoxic agent is one or more of paclitaxel, paclitaxel derivatives and analogues, rapamycin, rapamycin derivatives and analogues, everolimus and related compounds. In another embodiment, Pgp inhibition is used in a patient who is implanted with a stent. In a preferred embodiment, the patient is a mammal. In an even more preferred embodiment, the patient is a human.

In another aspect, Pgp inhibition is used to overcome the negative consequences of the initial burst release of the cytotoxic agent. Ip- this aspect, the stent is loaded with a reduced amount of drug to prevent excessive initial drug release, while chemotherapeutic efficacy is maintained by inhibition of Pgp- mediated efflux, starting at a time that the initial burst release has subsided.

In another aspect, a combination of aFGF and bFGF is used to encoat the stents. In a preferred embodiment, aFGF and bFGF are formulated in the drug eluting polymer layer on the stent, and are released over a time period of about five days. In an even more preferred embodiment, aFGF and bFGF are released from a stent that also releases a cytotoxic agent. In this embodiment, the combined aFGF and bFGF will have two beneficial effects. Most drug-releasing stents show a burst release where initial local drug concentrations exceed the concentrations required for impeding smooth muscle cell proliferation. The ensuing cytotoxicity to endothelial and smooth muscle cells gives rise to inflammatory processes, and an undesirable reduction in the re-endothelialization and wound healing process. The inventors have disclosed in U.S. Patent No. 6,559,912, the combined use of aFGF and bFGF protects cells from the cytotoxic action of paclitaxel and other cytotoxic agents and, therefore, overcomes the problem associated with the initial burst release of paclitaxel from paclitaxel- releasing stents. In addition, the combined fibroblast growth factors will stimulate the wound healing process. In a related aspect, a later part of the wound healing process will be improved. After initial re-endothelialization has taken place, the presence of growth factors that stimulate smooth muscle cell proliferation is undesirable as it contributes to restenosis. The initial re-endothelialization process requires approximately 8 days. Application of growth factor inhibitors, or a general inhibitor of various growth factors, after this initial vascular repair period is therefore desirable and is disclosed.

In a preferred embodiment, the general inhibitor of various growth factors is suramin. As described in our U.S. Patent No. 6,559,912, suramin provides general inhibition of a number of growth factors, including aFGF, bFGF, insulin like growth factor, and other growth factors. In a preferred embodiment, suramin is administered at a dosing regimen that results in plasma concentrations of less than about 100 mcM, starting eight days after implantation of the vascular stent, and continuing for as long as the risk of restenosis exists, or about up to 18 months. In an even more preferred embodiment, plasma concentrations are maintained between about 10 and about 50 mcM.

In another preferred embodiment, suramin is formulated in the drug- releasing layer of the stent, and will provide local concentrations in the tissue of less than about 100 mcM, or more preferably less than about 70 mcM, with the initiation of suramin release delayed by several days and most preferably at least eight days.

In another aspect, the invention provides for a composition consisting of a drug-encoated stent loaded with a cytotoxic agent and an agent that protects endothelial cells from injuries, where the loading of the cytotoxic agent on the stent is increased relative to the loading that can be safely allowed on a stent that does not contain protective agents, and where the protective agents prevent endothelial cell injury during the initial period of a high drug release, thus providing for a stent with an increased functional lifespan.

In another aspect, the stent is an ionizing-radiation releasing stent. In another aspect, the invention provides compositions for the local release of the modulating agents.

In one embodiment, the modulating- agent is formulated in a drug releasing polymer layer coated on a stent.

In a preferred embodiment, the modulating agent is formulated in one or more of multiple drug-releasing layers. ,

In a related embodiment, the modulating agent is formulated in one or more of multiple drug-releasing layers, and the start of release of the modulator is delayed until the time that the presence of the modulator is most effective.

In one embodiment, the modulating agent is a Pgp inhibitor. In another embodiment, the modulator is a chemosensitizer or a protective agent.

In a preferred embodiment, the Pgp inhibitor is one or more of cyclosporin A, Valspodar, or verapamil.

In a preferred embodiment, the chemosensitizer is suramin.

In an even more preferred form of this embodiment, the chemosensitizer is suramin, and is formulated to provide interstitial concentrations between 10 and 50 mcM.

In another further embodiment, the protective agent is a growth factor. In a preferred form of this embodiment, the growth factors are a combination of aFGF and bFGF. In an even more preferred form of this embodiment, the growth factors are a combination of aFGF and bFGF and are present at the site of endothelial cell growth in a therapeutically effective amount.

In another embodiment, the composition is a stent coated with a drug- releasing layer, where the drug-releasing layer contains more than one modulator.

In a preferred embodiment, the cytotoxic agent and the modulator or modulators are present in separate layers of the drug-releasing coating.

In another aspect, the stent is an ionizing radiation releasing stent, and the modulator is released from a drug-releasing layer coated on the stent. In one embodiment, the modulator is a radiosensitizer or a protective agent.

In a preferred form of this embodiment, the radiosensitizer is suramin.

In an even more preferred form of this embodiment, the radiosensitizer is suramin, and is formulated to provide interstitial concentrations between 10 and 50 mcM.

In another embodiment, the protective agent is a growth factor. In a preferred form of this embodiment, the growth factors are a combination of aFGF and bFGF.

In another aspect, this invention discloses the use of suramin at concentrations below 100 mcM or at doses that deliver less than 100 mcM, pfreferably below 70 mcM, to the diseased vessels by either systemic means

(inter alia, by an intravenous injection) or locally (inter alia, by coating stents with suramin) to reduce restenosis.

In another aspect, this invention discloses the use of suramin at concentrations below 100 mcM or at doses that deliver less than 100 mcM, preferably below 70 mcM, to the diseased vessels by either systemic means

(inter alia, by an intravenous injection) or locally (inter alia, by coating stents with suramin) to improve the activity of agents (inter alia, paclitaxel, rapamycin) used to coat stents for the purpose of reducing restenosis. In a related aspect, the inventors disclose the use of suramin encapsulated in drug delivery systems that are not localized in muscles, to reduce the exposure of muscle tissues and hence the undesired muscle-related toxicity of suramin. In one embodiment, this formulation is not deposited preferentially in muscle tissues. In one embodiment, the suramin dose is formulated in a form that selectively targets tumors. In one embodiment, this formulation is a nanoparticulate formulation that selectively deposits in tumor tissue through the "enhanced penetration and retention" effect. In a preferred embodiment, the nanoparticulate formulation is a liposomal formulation. In a further preferred embodiment, the liposomes are surface-modified with polyethylene glycol to further enhance the selective uptake by tumors. In another preferred embodiment, this formulation is an albumin nanoparticle formulation that is loaded with suramin through the tight binding of suramin to albumin. In yet another embodiment, suramin is delivered in a complex containing tumor- recognizing antibodies on its surface, where the tumor-recognizing antibodies cause the dosage form to accumulate selectively in tumor tissue.

The following examples show how the present invention has been practiced, but should not be construed as limiting the present invention.

EXAMPLES The examples are provided merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

The reagents and experimental protocols used in the appended examples are briefly described below. Materials & Methods. Chemicals and reagents were obtained from commercial sources, or the National Cancer Institute (Bethesda, MD). All reagents or methods described in U.S. Patent No. 6,599,912, are included by reference.

Tissues and cultures. Porcine and human coronary artery smooth muscle cells were cultured in smooth muscle cell growth media (Cell Applications, Inc.). Coronary artery and aorta tissue were surgically removed from dogs or mice. Human coronary artery paraffin tissue sections were obtained from BioChain-Diagnosis. Human aorta specimens were obtained from Cooperative Human Tissue Network. Inhibition of colony formation. This was measured using the clonogenic assay. Porcine vascular smooth muscle cells were seeded in 24-well plates at a concentration of 4x10⁴ cells per well or 60 mm tissue culture dishes at a density of 350 cells per dish. After culturing for 24 hours, cells were treated with drugs. Suramin treatments were initiated 2 hours prior to adding other drugs. Pgp inhibitors were given concomitantly with paclitaxel or rapamycin. For paclitaxel, the treatments were for 36 hours, and cells were then resuspended in fresh culture media and allowed to grow for 10 days. For rapamycin, treatments were continuous over 7 days (Kawakami, Clin Cancer Res, 8, 3503, 2002). Cells were stained with crystal violet (Delaney, Clin Cancer Res, 6 2860, 2000). The colonies, defined as clusters containing more than 50 cells, were visualized and counted.

Inhibition of DNA synthesis. This was studied by monitoring the incorporation of the DNA precursor bromodeoxyuridine (BrdU) into cells, as previously described. Porcine vascular smooth muscle cells were seeded at a concentration of 2*10³ cells/well into 96 well flat-bottom plates and allowed to attach overnight. The culture medium was replaced with drug containing medium. Cells were treated with drugs for 96 hours, and then processed using the BrdU ELISA assay (Zhang, J Pharmacol Exp Ther, 299, 426, 2001). Overall cytotoxicity. This was measured using the microtetrazolium assay (MTT) as previously described (Denizot, J Immunol Methods, 89, 271 , 1986). This assay measures the ability of cells to reduce the MTT dye, and is generally used as a measurement of the total cell number. Cells were seeded and treated with drugs as described above for inhibition of DNA synthesis. The treatments with doxorubicin, either alone or in combination with other drugs, were for 36 hours. The treatments with paclitaxel, alone or in combination with other drugs, were for 96 hours. Afterward, the samples were processed using the MTT assay.

Analysis of drug effects. The drug effects were measured by comparing the numbers of colonies or cells or the extent of BrdU incorporation of the drug- treated samples to the values of control samples without drug treatment. The effects were plotted against drug concentrations. The plots were analyzed to obtain the drug concentrations required to produce 10%, 25%, and 50% of cytotoxicity (referred to as IC10, IC25, and IC50, respectively). The methodologies are as described in the inventors' previous publications (e.g., Song, Cancer Res, 61 , 6145, 2001 ; Zhang, J Pharmacol Exp Ther, 299, 426, 2001).

Pgp expression in coronary artery and aorta tissue. The immunohistochemical staining for Pgp was performed as described previously (Gan, Clin Cancer Res, 4, 2949, 1998). The primary Pgp antibody was JSB-1 and C-494 (Zymed Laboratories, San Francisco, CA). The secondary antibody was biotinylated goat anti-rabbit immunoglobulin. The chromogen was diaminobenzidine (DAB, brown color).

Pgp inhibitors retarded the efflux of cytotoxic drugs from smooth muscle cells. Cells were seeded in 24-well plate and cultured for 1-2 days. The study of efflux of doxorubicin and rhodamine was conducted by monitoring the disappearance of the fluorescence signal from cells over time, as described in Example 4. The efflux of paclitaxel was monitored by following the decline of the cell-associated drug concentrations over time, using procedures previously described by the inventors (Jang, J. Pharmacol. Exp. Then, 298, 1236, 2001). Cells were treated with drug-containing medium, then washed two or three times with 1 ml of ice-cold phosphate-buffered saline, followed by incubation in either drug-free medium or medium containing Valspodar, cyclosporin A or verapamil, respectively. At predetermined time points, cells were washed with phosphate- buffered saline and analyzed.

EXAMPLE 1

Cytotoxicity of Pgp inhibitors in smooth muscle cells. The cytotoxicity of Pgp inhibitors was measured in three different assays: (a) clonogenicity, (b) DNA synthesis, and (c) overall cytotoxicity as measured by the MTT assay which measures the total cell number. The results were expressed as % of control, where a number lower than 100% indicates inhibition. Three Pgp inhibitors, Valspodar, cyclosporin A and verapamil, were used at concentrations known to effectively inhibit Pgp action, and have been safely given to humans (Krishna, Eur J Pharm Set, 11 , 265, 2000; Ozols, J Clin One, 5, 641 , 1987; Masip, Bioorg Med Chem, 13, 1923, 2005; Krishna, Eur J Pharm Sci, 11 , 265, 2000; Rishi, Nucleic Acids Res, 26, 4529, 1998; Tavares, J Cardiovasc Pharmacol, 31 , 46, 1998).

The results outlined below indicate that the three Pgp inhibitors produce toxicity in smooth muscle cells. However, the three inhibitors produced different effects in the three types of cytotoxicity assays. In the clonogenic assay, Valspodar was least active whereas cyclosporine A and verapamil have activities similar to the two drugs, i.e., rapamyciπ and paclitaxel, that are used to coat the drug-encoated stents approved by the US Food and Drug Administration and have shown to be effective in humans. For the inhibition of DNA synthesis, all three Pgp inhibitors have activities comparable to or greater than that of rapamycin. For the reduction of total cell number, cyclosporine A was most active and produced overall cytotoxicity that is comparable to that of paclitaxel.

TABLE 1

EXAMPLE 2

Arterial smooth muscle cells and arteries express Pgp. As shown in Figure 1 , arterial tissues of canine and human origin and the cultured porcine and human artery smooth muscle cells, which are easily recognized by their elongated shape, show strong Pgp expression (brown color staining). For the cultured smooth muscle cells, the porcine cells showed more intense staining comparing to human cells.

EXAMPLE 3 Pgp inhibitors retard the efflux of doxorubicin and rhodamine from smooth muscle cells. Porcine or human arterial smooth muscle cells were seeded in 24- well plates. Twenty-four hours later, doxorubicin or rhodamine, both at 50 mcM, were added. Doxorubicin and rhodamine are Pgp substrates and show flouresence. One hour later, the culture medium was replaced and refreshed every hour with medium containing a Pgp inhibitor. At predetermined times, the culture medium was removed and the wells were washed. The fluorescence remaining inside the cells was observed under the fluorescent microscope. The exposure time for collecting fluorescence signal was 3 second for doxorubicin and 0.6 second for rhodamine. Microphotographs are shown in Figure 2. The results show that in the absence of Pgp inhibitors, no fluorescence signals were observed in human cells after 1 hour for doxorubicin and after 16 hours for rhodamine, and no signals were observed in porcine cells after 6 hours for doxorubicin and after 3 hours for rhodamine. In the presence of Pgp inhibitors, fluorescence signals were retained at these time points. This data indicates that Pgp inhibitors retarded the efflux of the Pgp substrates doxorubicin and rhodamine from smooth muscle cells.

EXAMPLE 4

Pgp inhibitors retard the efflux of paclitaxel from smooth muscle cells. Cells were loaded with paclitaxel by incubation with 10 nM paclitaxel for 24 hours, then washed, and incubated with or without one of the Pgp inhibitors. The amount of cell-associated paclitaxel was studied over time. The results outlined below indicate that addition of Pgp inhibitors enhanced the amount retained in cells,- indicating that Pgp inhibition increases the retention of paclitaxel in smooth muscle cells. TABLE 2

EXAMPLE 5

Pgp inhibitors enhance the cytotoxicity of cytotoxic agents in porcine arterial smooth muscle cells. The effects of Pgp inhibitors on the overall cytotoxicity of paclitaxel and doxorubicin were studied using the MTT assay. The effects of Pgp inhibitors on the ability of rapamycin to inhibit DNA synthesis were measured using the BrdU assay. The effects of Pgp inhibitors on the ability of rapamycin to inhibit clonogenicity were measured using the clonogenic assay. Treatments containing paclitaxel or rapamycin, alone or in combination with Pgp inhibitors, were for 96 hours, and treatments containing doxorubicin were for 36 hours. The effects of Pgp inhibitors, when used alone without paclitaxel or rapamycin, are as described in the aforementioned examples. The results outline below show that Pgp inhibitors cause a leftward shift in the dos e-response curves, and reduced the concentrations of paclitaxel and doxorubicin required to produce 10%, 25%, and 50% and 75% of cytotoxicity (referred to as IC10, IC25, IC50, and IC75, respectively). The Pgp inhibitors further increased the maximum cytotoxicity (referred to as Emax).

TABLE 3

EXAMPLE 6

Dual effects of Pgp inhibitors on paclitaxel-induced cytotoxicity. This study demonstrates the dual effects of Pgp inhibitors in smooth muscle cells. The overall cytotoxicity was studied using the MTT assay. The results, shown in Figure 3, show that Pgp inhibitors enhanced the activity of paclitaxel at low paclitaxel concentrations (equal to or below 100 nM), but protected against the toxicity of paclitaxel at high paclitaxel concentrations (over 1000 nM). This data 0 indicates that adding Pgp inhibitors preferentially improve the paclitaxel activity when the drug release from stent is slow, e.g., when most of the drug has been released or when the drug is no longer present at sufficient quantity to prevent restenosis. This data further indicates that Pgp inhibitors can protect smooth muscle cells against the toxicity of paclitaxel at the time of rapid drug release or 5 when the paclitaxel is present at high concentrations, e.g., during the initial burst release or when the endothelium is most susceptible to paclitaxel toxicity. EXAMPLE 7

Overall cytotoxicity of suramin in smooth muscle cells. The overall cytotoxicity of suramin to porcine coronary artery smooth muscle cells was established using the MTT assay. In this assay, cells were treated with suramin for 96 hours. This treatment time was selected because the inventors have shown that in human cancer patients, the elimination of an intravenous dose of suramin, to improve the antitumor effects of chemotherapy, is very slow with an elimination half-life of more than 7 day (Villalona-Calero, CHn. Cancer Res., 9, 3303, 2003). The results show significant overall cytotoxicity for suramin. The respective IC10, IC25, IC50, IC75 and IC90 values were 15, 25, 40, 65 and 120 mcM.

EXAMPLE 8 Suramin enhances the ability of paclitaxel and rapamycin to inhibit the clonogenicity of smooth muscle cells. Example 7 shows the overall cytotoxicity of suramin after treatment for 96 hours. To investigate whether suramin, given to improve the antitumor effect of chemotherapy in patients with cancer, also enhanced the toxicity of chemotherapy to smooth muscle cells, the inventors selected different treatment conditions where suramin by itself did not produce toxicity, namely a shorter suramin treatment time and the clonogenic assay. In this way, the ability of suramin to enhance the toxicity of chemotherapy can be readily concluded. Cells were plated and treated with suramin, paclitaxel or rapamycin, singly or in combination, for 36 hours, and then processed for the measurement of clonogenicity. The results show that under these conditions, suramin alone at 10 and 30 mcM did not affect the clonogenicity of smooth muscle cells whereas 20 mcM suramin enhanced the ability of rapamycin and paclitaxel to reduce the clonogenicity. TABLE 4

EXAMPLE 9

Suramin enhances the toxicity of radiation. The inventors have previously disclosed that suramin, a nonspecific inhibitor of various growth factors, enhances the antitumor activity of chemotherapeutic agents with diverse chemical structures and action mechanisms. The present study evaluated whether suramin enhances the tumor response to radiotherapy in three human xenograft tumors (pharynx FaDu, pancreatic Hs766T, and prostatic PC-3). WeII- established subcutaneous tumors implanted on the right hind limb of immunodeficient mice received intraperitoneal injections of physiologic saline or suramin (10 mg/kg for PC-3 and FaDu, 30 mg/kg for Hs766T tumors) one day prior to radiation (10.8 Gy, single dose). Animals bearing FaDu and Hs766T tumors received a single suramin dose, whereas animals bearing PC-3 tumors received a total of 6 suramin treatments on a twice-weekly schedule. The results showed that in all three tumors, single agent suramin had no activity whereas irradiation alone either reduced tumor growth (FaDu and Hs766T) or caused tumor regression (PC-3). Addition of suramin to radiation further reduced the tumor size by ~50-85% compared to the radiation groups, on day 21. A parallel study in Hs766T tumors showed that the antitumor effect of radiation (10.8 Gy) plus suramin was similar to the effect attained at a 50% higher radiation dose (16.2 Gy). In FaDu tumors, the apoptotic index increased from ~1% for control and single agent suramin groups to 6% after irradiation, and 11 % after irradiation plus suramin. This example shows that nontoxic doses of suramin significantly enhanced the radiation response.

EXAMPLE 10

Treatment of a cancer patient with suramin nanoparticles to reduce the toxicity of suramin on muscle tissue, while maintaining its chemosensitizing efficacy. The inventors have disclosed that suramin, when maintained at plasma concentrations below 70 mcM, enhances the efficacy of cancer chemotherapy. While suramin at these low doses did not produce life threatening toxicity or serious hematologic toxicity in patients, it produced an unexpected, increased incidence of fatigue and muscle weakness. The present invention discloses the higher toxicity of suramin in smooth muscle cells compared to cancer cells. For example, 50% inhibition of proliferation of porcine smooth muscle cells occurs at a concentration of 40 mcM, while up to 10-fold higher concentrations are required to produce toxicity in tumor cells (Song, Cancer Res., 61 , 6145-6150, 2001; Zhang, J. Pharmacol. Exp. Ther., 299, 426- 433, 2001). This discovery has led to the present invention of using suramin- encapulated nanoparticles to reduce the exposure of muscle tissues to suramin. Nanoparticles, due to leakiness of vasculatures in tumors, are preferentially delivered to and retained in tumors (Jang, Pharmaceut. Res., 20, 1337, 2003). For example, the cancer patient is a patient with stage IHB or stage IV non-small- cell lung cancer, who is treated in cycles of three weeks with paclitaxel (200 mg/m²) and carboplatin (AUC of 6). To enhance the efficacy of the cancer chemotherapy, while reducing suramin's undesired side effects in the muscle tissue, suramin is administered in nanoparticulate form at a dose of that will deliver tumor tissue concentrations of less than 100 mcM and preferably less than 70 mcM, and more preferably less than 50 mcM, during the presence of effective chemotherapy concentrations. For example, the patient is a patient with chemotherapy-pre-treated non-small-cell lung cancer, who receives docetaxel (56 or 75 mg/m² every three weeks), combined with suramin formulated in a nanoparticulate dosage form. For example, the patient is a patient with metastatic breast cancer, who is treated with weekly paclitaxel at a dose of 80 mg/m², combined with suramin formulated in a nanoparticulate dosage form.

The nanoparticulate dosage form consists of pegylated long-circulating liposomes loaded with suramin. The lipid components will be hydrogenated soy phosphatidylcholine, methoxypolyethyleneglycol-distearoyl phosphatidyl- ethanolamine and cholesterol at an approximate 51 :5:44 molar ratio. Preparation of the liposomes begins by dissolving the lipid components in ethanol followed by addition to an aqueous suramin solution. The resulting liposomes are then sized by extrusion through polycarbonate membranes and diafiltered to removed unencapsulated suramin. The process produces liposomes with an average particle size of about 85 nm, without unencapsulated drug in the stored preparation.

Alternatively, the nanoparticulate dosage form consists of proteinaceous nanoparticles, for example albumin nanoparticles. This type of nanoparticles is a member of a family of promising colloidal drug delivery system with many advantages, including their biodegradability, minimal toxicity, controlled drug- release properties, and easy conjugation with tumor cell surface-recognizing molecules. Albumin nanoparticles are formed by solvent desolvation (e.g., by ethanol) of an aqueous solution containing both albumin and suramin, followed by cross-linking with glutaraldehyde. Nanoparticles with a mean size of less than about 100 nm are formed. The suramin-loaded nanoparticles are separated from free suramin by passing through a gel permeation chromatography column (e.g., Sephadex).

Conclusion

While the invention has been described with reference to preferred embodiments, those skilled in the art will understand that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. In this application all units are in the metric system and all amounts and percentages are by weight, unless otherwise expressly indicated. Also, all citations referred herein are expressly incorporated herein by reference.

Claims

We claim:

1. A method of improving the efficacy of endovascular drug-releasing stents implanted in a subject, comprising: administering one or more of:

(a) modulators of the transport of the drug released by the stent at such a time as is beneficial to maintain nearby tissue concentrations of the drug within their therapeutically effective range,

(b) modulators of drug sensitivity at such a time as is beneficial to maintain control over the proliferation of vascular smooth muscle cells;

(c) growth factors at such a time as is beneficial to maintain control over the healing process at the implantation site; or

(d) inhibitors of growth factors at such a time as is beneficial to maintain control over the healing process at the implantation site.

2. The method of claim 1, wherein the modulator of drug transport comprises an inhibitor of ABC transporters.

3. The method of claim 2, wherein the ABC transporter comprises Pgp.

4. The method of claim 1 , wherein the modulator of drug transport is one or more of the following agents: 17-allylamino-17-demethoxygeldanamycin (17-AAG), amiodarone, amitriptyline, amprenavir, arsenic trioxide, atazanavir, atorvastatin, azelastine, azidopine, bepridil, biricodar (VX 710), bromocriptine, 5-bromotetrandrine, capsaicin, carvedilol, celecoxib, cephalosporines, cepharanthine, chlorpromazine, clarithromycin, clofazimine, clozapine, coumarin, CP 100356, Cremophor EL, curcumin, cyclosporin A, daidsein, desipramine, dexverapamil, diclofenac, dihydro- beta-agarofuran sesquiterpenes, diltiazem, dipyridamole, disulfiram, diterpenic lactones, DM27, doxepin, elacridar (GF120918), ellipticines, emitine, endotoxin, erythromycin, evodiamine, felodipin, FG020326, flupentixol, fluphenazine, gallopamil, gefitinib, genistein, GG918, glibenclamide, grepafloxacin, haloperidol, imipramine, indinavir, isradipine, itraconazole, ivermectin, kaempferol, ketoconazole, laniquidar (R101933), lansoprazole, levofloxacin, lipopolysaccharide, loperamide, medroxyprogesterone, mefloquine, meloxicam, methoxypolyethylene glycol-block-Polycaprolactone diblock copolymer, miconazole, MS209, nefazodone, nelfinavir, nifedipine, ningalins, nobiletin, norverapamil, NS-

398, OC 144-093 (ONT-093), ofloxacin, omeprazole, orataxel (BAY59- 8862), pantoprazole, paroxetine, PCB-126, phenethyl isothiocyanate, phenothiazines, phenytoin, pimozide, pluronic P85, polysorbate 80, progesterone, promethazine, propafenone, propranolol, PSC 833 (valspodar), quercetin (QU), quinidine, quinine, reserpine, rifampin, ritonavir, RNAi, saquinavir, schisandrin B, selamectin, sertraline, simvastatin, sparfloxacin, spironolactone, tacrolimus, tamoxifen, tariquidar (XR9576), terfenadine, testosterone, tetranadrine, thioridazine, TRA96023, trifluoperazine, triflupromazine, Triton X-100, Tween 80, valinomycin, vanadate, verapamil, yohimbine, or zosuquidar (LY335979).

5. The method of claim 1 , wherein the modulator of drug transport is one or more of Valspodar, cyclosporin A, or verapamil.

6. The method of claim 1 , wherein administration is by oral or parenteral route.

7. The method of claim 1 , wherein the modulator of drug transport is released from the endovascular stent.

8. The method of claim 1 , wherein administration of the modulator starts at the time that the rate of drug release from the stent declines to below about 50% of the desired rate.

9. The method of claim 1 , wherein said modulator of drug transport is encapsulated in nanoparticles.

10. The method of claim 9, wherein said nanoparticles are one or more of albumin nanoparticles, gelatine nanoparticles, or liposomes.

11. The method of claim 1 , where the modulator of drug sensitivity comprises a chemosensitizer.

12. The method of claim 11 , where the modulator of drug sensitivity is one or more of the following agents: AG17 (3,5-di-tert-butyl-4- hydroxybenzylidene-malononitrile), Andrographolid (AG), Bcl2 antisense oligonucleotides, Benzinidazole, Bortezomib (Velcade), Budesonide, Buthionine sulfoximine (BSO), CHK1 inhibitors, CHK2 inhibitors, Citalopram, Clomipramine, COX-2 inhibitors, Dexamethasone (DEX), Dipyridamole (DP), EGFR inhibitors, Fluoxetine (prozac), GEM231

(antisense oligonucleotide to Rlalpha-subunit of cAMP-dependent protein kinase (PKA), GPI 15427 (PARP-1 inhibitor), Inostamycin, Malagashanine, Metoclopramide, Misonidazole, MDM2 antisense oligonucleotides, NLCQ-1 (4-[3-(2-nitroimidazolyl)-propylamino]-7- chloroquinoline hydrochloride), NLCPQ-1 (9-[3-(2-Nitro-1- imidazolyl)propylamino]-cyclopenteno[b]quinoline hydrochloride), Perillyl alcohol (POH), Phenoxodiol, PK11195 (carboxamide derivative of isoquinolone), PG490-88 (a derivative of triptolide), Reserpine, Rituximab (chimeric anti-human CD20 monoclonal antibody), Ro 03-8799 (pimonidazole), SR 2508 (etanidazole), STI571 , Suramin, Survivin antisense oligonucleotides, THNLA-1, tirapazamine (3-amino-1 ,2,4- benzotriazine 1 ,4-di-N-oxide), Wortmannin.

13. The method of claim 12, where the modulator of drug sensitivity comprises suramin.

14. The method of claim 13, where suramin concentrations are maintained below about 100 mcM.

15. The method of claim 14, where suramin concentrations are maintained between about 10 and about 50 mcM.

16. The method of claim 1, where the growth factor is one or more of the following: acidic and basic FGF, FGF-1 and FGF-2 respectively; int2 (FGF-3); hst (FGF-4); FGF-5; hsi2 (FGF-6); keratinocyte growth factor (FGF-7); androgen-induced growth factor (FGF-8); glia-activating factor (FGF-9); or FGF-10 - FGF-24.

17. The method of claim 16, where the growth factor is one or more of aFGF and bFGF.

18. The method of claim 16, where the growth factors are administered during the early phases of the healing process, while endothelialization occurs.

19. The method of claim 18, where the growth factors are administered during the first eight days after stent placement.

20. The method of claim 25, where the growth factor inhibitor is one or more of the following: suramin, structural analogs of suramin, anti-FGF antibodies, anti-FGF receptor antibodies, pentosan polysulfate, scopolamine, angiostatin, sprouty, estradiol, carboxymethylbenzylamine dextran (CMDB7), suradista, insulin-like growth factor binding protein-3, ethanol, heparin (e.g., 6-O-desulfated heparin), low molecular weight heparin, heparan sulfate, protamine sulfate, transforming growth factor beta, cyclosporin A, or RNA ligands for bFGF, Thalidomide, Linomide,

Lenalidomide, TKI-258, CHIR258, PD173074, or SU5402.

21. The method of claim 20, where the growth factor inhibitor comprises suramin.

22. The method of claim 21, where suramin concentrations are maintained below about 100 mcM.

23. The method of claim 22, where suramin concentrations are maintained between about 10 and about 50 mcM.

24. The method of claim 20, wherein the growth factor inhibitor is administered during the later phases of the healing process, while endothelialization is completed.

25. The method of claim 20, wherein the growth factor inhibitors are administered starting about 8 days after stent placement.

26. The method of claim 20, wherein said GF inhibitors are administered after the expected useful life of said stents.

27. The method of claim 20, wherein said GF inhibitors are administered systemically or by oral administration.

28. The method of claim 20, where the growth factor inhibitors are released from the endovascular stent.

29. The method of claim 11, where the stent is not a drug-releasing stent, but is instead an ionizing radiation releasing stent and the modulator of drug sensitivity is instead a radiosensitizer.

30. The method of claim 29, where the radiosensitizer is one or more of the following: 1-(2\3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2- nitroimidazote (RA-263), 1-(2-hydroxy-3-methoxypropyl)-2-chloro-4- nitroimidazole [P40], 2-nitroimidazole-1-acetohydroxamate (KIH-802), 3- aminobenzamide (3-ABA), 4-[3-(2-Nitro-1-imidazolyl)propylamino]-7- chloroquinoline hydrochloride (NLCQ-1), 5-[3-(2-nitro-1-imidazoyl)-propyl]- phenanthridinium bromide (2-NLP-3 or NLP-1), 5'-bromodeoxyuridine (BrdU), 5-Chlorodeoxycytidine (CIdC), 5-fluorodeoxycytidine (FdC), 5- lodo-2-pyrimidinone-2'-deoxyribose (IPdR), 5-iodo-2'-deoxyuridine

(IdUrd), 5-Nitro-4-(N,N-dimethylaminopropylamino)quinoline (5- nitraquine), 5-Phenoxysul phony 1-1 -methyl-4-nitroimidazole (NSC 38087), 8-nitrocaffeine, 9-[3-(2-Nitro-1 -imidazolyl)propylamino]- cyclopenteno[b]quinoline hydrochloride (NLCPQ-1), acridine-linked 2- nitroimidazole (NLA-1), doxorubicin, AK-2123, AT1727 (a derivative of

ICRF 154), Azomycin riboside, Benzotriazine SR 4233, Bromomisonidazole, carboplatin, CI-1010((R)-alpha-[[(2-bromoethyl)- amino]methyl]-2-nitro-1 H-imidazole-1-ethanol monohydrobromide) (a prodrug), or its drug HR (PD 146923), Cisplatin, doranidazole (PR-350), etanidazole, (E)-2'-Deoxy-(fluoromethylene)cytidine (FMdC), Gadolinium (III) texaphyrin (Gd-Tex) (NSC 695238), Gemcitabine (2',2'- diflυorodeoxycytidine), H4U, Hydrogen peroxide, Hydroxyurea, Haloacetylcarbamoyl-^-nitroimidazoles, including chloro (KIN-1800, TX- 1835, and TX-1836) and bromo derivatives (TX-1844, TX-1845, and TX- 1846), Lonidamine, JM216 (a platinum analogue), KIN804, KIN-806, KU-

2285 (a fluorinated 2-nitroimidazole), Metronidazole, Misonidazole (Ro 07-0582), Navelbine, Nicotinamide, Nimorazole, Nitroimidazole, N- methylformamide (NMF), N-(Phosphonacetyl)-L-aspartate (PALA), Paclitaxel, Paranitroacetophenone, Photofrin, Pimonidazole, Potassium 2- nitroimidazole-1-acetohydroxamate (KIH-802), Pt(Rh-123)2, RAF antisense oligonucleotide, RK-28 (a 2-nitroimidazole nucleoside analogue), Ro 03-8799 (pimonidazole), RP170, RSU-1069 (a nitroimidazole-aziridine), RuCI2(DMSO)2(4-nitroimidazole)2, Sanazole (AK-2123), SR 2508 (etanidazole), suramin, tirapazamine (3-amino-1,2,4- benzotriazine 1 ,4-di-N-oxide), Topotecan, TX-1877, Vinorelbine,

Withaferin A, or Wortmannin.

31. The method of claim 29, where the radiation sensitizing agent is suramin.

32. The method of claim 29, where suramin concentrations are maintained below about 100 mcM.

33. The method of claim 32, where suramin concentrations are maintained between about 10 and about 50 mcM.

34. An apparatus comprising a drug-releasing stent which further releases one or more of modulators of drug transport, modulators of drug sensitivity, growth factors, or inhibitors of growth factors.

35. A method of reducing or preventing the toxicity associated with the use of suramin as a chemosensitizer or radiosensitizer in a patient treated for a cancer, comprising of the administration of suramin in a nanoparticulate dosage form that selectively targets tumor tissue.

36. The method of claim 35, where suramin is formulated in liposomes.

37. The method of claim 36, where the liposomes are pegylated.

38. The method of claim 35, where suramin is formulated in albumin nanoparticles.

39. The method of claim 35, where suramin is formulated in gelatin nanoparticles.