WO2007008729A2

WO2007008729A2 - Local drug delivery devices

Info

Publication number: WO2007008729A2
Application number: PCT/US2006/026629
Authority: WO
Inventors: Oded Ben-Joseph; William R. Baumbach; Hariharan Shankar
Original assignee: X-Cell Medical, Inc.
Priority date: 2005-07-07
Filing date: 2006-07-07
Publication date: 2007-01-18
Also published as: WO2007008729A3

Abstract

An intravascular drug delivery device, such as a drug eluting stent, is provided for the treatment of vascular disorders including stenosis, restenosis, atherosclerosis, and vulnerable plaques. The device delivers an estrogen receptor agonist (e.g., 17β-estradiol) and a macrolide antibiotic (e.g., rapamycin, tacrolimus, and pimecrolimus).

Description

LOCAL DRUG DELIVERY DEVICES

FIELD OF THE INVENTION The present disclosure generally relates to local-delivery devices such as drug eluting stents. More particularly, such local-delivery devices are used to treat or prevent vascular diseases including restenosis, atherosclerosis, and vulnerable plaques.

BACKGROUND A variety of vascular diseases are known to affect the cardiac arteries and other large arteries in humans. A common feature of many vascular diseases is the narrowing of the blood vessel lumen. Each particular disease (e.g., stenosis/restenosis, atherosclerosis, acute myocardial infarction, coronary heart disease (CHD), etc.) is characterized by different types of occlusions and pathologies, often involving different cell types and extracellular components. For example, stenosis is a narrowing or constricting of arterial lumen usually due to atherosclerosis/coronary heart disease (CHD). Restenosis is a recurrence of stenosis after a percutaneous intervention such as angioplasty and/or stent implantation. Restenosis typically affects the large arteries of a living organism. It is believed that the underlying mechanisms of restenosis comprise a combination of effects from vessel recoil, negative vascular remodeling, thrombus formation and neointimal hyperplasia. Restenosis following balloon angioplasty is believed to be primarily a result of vessel remodeling and neointimal hyperplasia, whereas restenosis following stent implantation primarily stems from neo-intimal hyperplasia.

Treatment for stenosis and restenosis varies. Stenosis caused by CHD often forces individuals to restrict and limit their activity levels in order to avoid complications, stroke, heart attack, sudden death and loss of limb or function of a limb stemming from the stenosis. The reconstruction of blood vessels, arteries and veins may also be needed to treat individuals suffering from stenosis and restenosis. Coronary bypass procedures are utilized to revascularize the heart and restore normal blood flow. In other cases, balloon angioplasty is conducted to increase the orifice size of affected areas. Overall, these treatments address the problems associated with stenosis but, a high rate of restenosis results in the recurrence of cardiac symptoms and, in some instances, mortality. Moreover, these treatments are not preventative in nature, and therefore generally are not utilized until the patient or individual has already developed stenosis.

One type of stenosis and restenosis is atherosclerosis. Atherosclerosis affects medium and large arteries and is characterized by a patchy, intramural thickening that encroaches on the arterial lumen and, in most severe form, causes obstruction. The atherosclerotic plaque consists of an accumulation of intracellular and extracellular lipids, smooth muscle cells and connective tissue. The fatty streak is usually the earliest lesion observed in atherosclerosis and it evolves into a fibrous plaque coating the artery. Atherosclerotic vessels have reduced systolic expansion and abnormal wave propagation. Treatment of atherosclerosis is usually directed at its complications, for example, arrhythmia, heart failure, kidney failure, stroke, and peripheral arterial occlusion.

Stent implantation is frequently used to treat these various vascular diseases. An intravascular stent is a prosthesis which may be placed within a body passageway such as any vein or artery within the vascular system. Typically, the stent is inserted into a vessel and placed at a site of vascular occlusion. The stent is expanded at this site in order to contact the vessel wall, thereby widening the blood vessel and providing mechanical support for the wall. Several stent configurations' are commonly used. One example is a crimped stent. A crimped stent is transported by means of the balloon catheter into the blood vessel to the site of stent placement. The crimped stent is fixed in the vessel by increasing the internal pressure of the balloon catheter, expanding the stent and pressing it against the vessel wall. Self-expandable stents are another stent type. These stents, once placed inside the artery at the desired location in a collapsed conformation, expand as a result of the restoring forces or the ambient local conditions (flow, temperature etc.). Thus, the inserted stent expands and supports the vessel wall.

Stents and other implantable devices have been used to deliver drugs locally at the site of vascular disease or injury. Typically, the device is coated or impregnated with a therapeutic agent in a manner that results in the release of that agent over an extended period of time.

SUMMARY OF THE INVENTION The present disclosure provides a local drug-delivery device (e.g., an intravascular stent) that contains an estrogen receptor agonist and a macrolide antibiotic for the treatment of vascular disorders. In one embodiment, the estrogen receptor agonist is 17β- estradiol. In another embodiment, the macrolide antibiotic is either a rapamycin analog such as rapamycin, everolimus, ABT578, biolimus A9, and AP23573, an FK506 analog including, for example, immunosuppressive FK506 analogs such as tacrolimus and pimecrolimus, and non-immunosuppressive FK506 analogs such as JNJ460 and V-13,450.

The local drug-delivery devices are designed either for sustained therapeutic release (e.g., an intravascular stent), immediate therapeutic release (e.g., a balloon catheter or an intravascular injection system), or both (e.g., a stent having a rapid release drug- impregnated coating over a delayed/sustained release drug-impregnated coating).

The estrogen receptor agonist and the macrolide antibiotic are contained within or coated onto the device. In one embodiment in which the device is a stent, the agents are coated directly on the surface or sequestered in holes, grooves, or pores on the device. In another embodiment, the estrogen receptor agonist and the macrolide antibiotic may be present in a coating on the device (i.e., present within the same coating or different coatings). In useful embodiments, the estrogen receptor agonist and the macrolide antibiotic are present in discrete spatial locations on the stent. In this embodiment, the individual therapeutic agents may be coated directly onto the stent or present in a coating agent (e.g., polymer). For stents in which more than one therapeutic agent is applied to the stent using a coating agent, the same or different coating agents may be used.

In useful embodiments, sustained drug release devices contain between about 0.001 mg and about 1.0 mg including, for example, between about 0.005 mg and about 0.500 mg, or between about 0.010 mg and 0.300 mg of an estrogen receptor agonist and between about 0.001 mg and about 1.0 mg including, for example, between about 0.005 mg and about 0.500 mg, or between about 0.010 mg and 0.300 mg of a macrolide antibiotic.

The sustained release local drug delivery devices are designed to elute between about 0.0001 mg/day and about 1.0 mg/day of an estrogen receptor agonist and between about 0.0001 mg/day and about 1.0 mg/day of a macrolide antibiotic.

Immediate release devices of this invention contain between about 0.010 mg and about 10.0 mg (e.g., between about 0.50 mg and about 7.5 mg or between about 1.0 mg and about 5.0 mg) of an estrogen receptor agonist and between about 0.010 mg and about

10.0 mg (e.g., between about 0.50 mg and about^' 7.5 mg, or between about 1.0 mg and about 5.0 mg ) of a macrolide antibiotic.

Also provided is a method for treating or preventing a vascular disorder in a human, wherein the method comprises implanting an intravascular stent in an affected blood vessel, and the stent comprises an estrogen receptor agonist and a macrolide antibiotic which are eluted over a period of time.

It is contemplated that the estrogen receptor agonist and the macrolide antibiotic are eluted from the local delivery device over an extended period of time. The time period includes about one hour, one day, one week, one month, several months, a year, or more than a year. The estrogen receptor agonist and the macrolide antibiotic are, in particular embodiments, eluted at the same rate or at different rates and may be eluted over about the same time period or different time periods. For example, the local delivery device is designed such that the estrogen receptor antagonist is eluted over a period of two months and the macrolide antibiotic is eluted over a period of one year. By "local drug delivery device" is meant any device suitable for intravascular drug delivery that does not result in systemically therapeutic drug levels. Local drug delivery devices include, for example, drug eluting stents (DES) that are suitable for intravascular implantation, wherein one or more therapeutic agents is coated on, bonded to, impregnated within, or otherwise attached to the device is such a manner that the therapeutic agent is released in a controlled manner over a period of time. Other examples include balloon delivery systems whereby drugs are impregnated or contained within or on an intravascular balloon, and intravascular injection systems whereby drug boluses, formulated in standard excipients such as cyclodextrins or microparticles, can be locally delivered to diseased areas of the coronary vascular system. By "macrolide antibiotic" is meant any antibiotic isolated from a Streptomyces spp., or any biologically active analog thereof. Specifically included within this definition are the rapamycin analogs and the FK506 analogs. Macrolide antibiotics are also characterized by their ability to bind to human FKBP-12. Reviewed in Nghiem et al., J. Am. Acad. Dermatol, 46: 228-241, 2002. By "rapamycin analog" is meant rapamycin and any compound that is both a structural analog of rapamycin and is capable of either blocking the G₁ to S phase transition in T-cells (Siekierka, Immunol. Res., 13: 110-116, 1994) or reduce or prevent the proliferation of human smooth muscle cells in vitro (Poon et al., J Clin. Invest, 98: 2277- 2283, 1996). Rapamycin analogs include, for example, rapamycin (also known as sirolimus), everolimus, ABT578, biolimus A9, and AP23573.

By "FK506 analog" is meant tacrolimus (also known as FK506) and any compound that is both a structural analog of tacrolimus and capable of inhibiting calcineurin when bound to an FKBP (Liu et al,, Cell, 66: 807-815, 1992). Generally, FK506 analogs include both immunosuppressive analogs such as tacrolimus and pimecrolimus, and non- immunosuppressive analogs such as JNJ460 and V-13,450.

By "estrogen receptor agonist" is meant any compound that has affinity for any estrogen receptor and whose binding causes a biological effect similar to that of 17β- estradiol at the same receptor. Estrogen receptor agonists are steroidal (e.g., 17β-estradiol) or non-steroidal (e.g., diethylstilbestrol) compounds. Estrogen receptor agonists include, for example, 17β-estradiol, ethinyl estradiol, 2-rnethoxyrnethyl estradiol, tamoxifen, resveratrol, and diethylstilbestrol.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the present disclosure are set forth with particularity in the appended claims. The present disclosure, as to its organization and manner of operation, together with further objectives and advantages may be understood by reference to the following description, taken in connection with the accompanying drawings, in which:

FIGURE 1 is flowchart showing one possible set of complementary biological interactions between estradiol and pimecrolimus in the pathological process of a vascular disorder. The double arrows indicate enhancement and the crossed arrows indicate inhibition. FIGURE 2 is a line graph showing the antiproliferative effect of rapamycin (closed circles) and tacrolimus (open circles) on human smooth muscle cells in vitro.

FIGURE 3 is a line graph showing the antiproliferative effect of rapamycin alone (squares) and rapamycin in combination with 100 nM tacrolimus (circles) on human smooth muscle cells in vitro. FIGURE 4 is a line graph showing the antiproliferative effect of rapamycin on both human vascular smooth muscle cells and human vascular endothelial cells in vitro.

DETAILED DESCRIPTION

The present disclosure provides a local drug-delivery device that contains an estrogen receptor agonist and a macrolide antibiotic for the treatment of vascular disorders.

Suitable drug-eluting devices include, for example, intravascular stents and balloon catheters, useful for the treatment of restenosis, atherosclerosis, vulnerable plaque, acute myocardial infarction, coronary heart disease, and urinary tract and bile duct indications. During the treatment of vascular disorders clinicians seek to suppress a local immune response, promote the proliferation and migration of vascular endothelial cells, and inhibit the proliferation of the vascular smooth muscle cells (SMCs). A combination drug therapy using an estrogen receptor agonist (e.g., 17β-estradiol) and a macrolide antibiotic (e.g., an FK506 analog or a rapamycin analog) is capable of effecting many of these therapeutic outcomes. In particular embodiments, these compounds are locally coadministered at the site of vascular injury using a drug delivery device such as an intravascular implantable stent.

Vascular Protective Effects of Estrogen

A variety of in vitro, in vivo, and epidemiologic studies report that estrogen has beneficial effects on vascular function and health. Some studies attribute gender differences in cardiovascular disease to the protective effects of estrogen in women and premenopausal women have a lower incidence of CHD. Hormone replacement therapy (HRT) is reported to reduce the risk of coronary-artery disease in post-menopausal women. Mechanistically, estrogen is believed to have beneficial effects on lipid profile and may directly affect vascular reactivity; a component of atherosclerosis. The beneficial effects of these hormone therapies are also be applicable to males. For both genders, systemic estrogen administration is limited by side effects including the possibility of hyperplastic effects on the uterus and breast in women, and feminizing effects in men.

The mechanisms for these beneficial effects are likely multifactorial. Estrogen is believed to alter the atherogenic lipid profile and also have a direct action on blood vessel walls. Estrogen is reported to have both rapid and long-term effects on the vasculature including the local production of coagulation and fibrinolytic factors, antioxidants and the production of other vasoactive molecules, such as nitric oxide and prostaglandins, all of which are known to influence the development of vascular disease.

Estrogen, and more particularly 17β-estradiol, are useful in the treatment of vascular disorders. Estradiol has been shown to inhibit SMC proliferation and migration. By contrast, estradiol enhances endothelial cell proliferation and migration which promotes endothelialization. New et al, Catheterization and Cardiovascular Interventions, 57: 266- 271, 2002; Chandrasekar, J. Invasive Cardiology, 16: 719-722, 2004. Estradiol is also reported to have local effects on the vasculature including enhance vascular dilation, increased NO production, and reduced platelet activity. Finally, estradiol is reported to produce anti-inflammatory effects by blocking the NFKB pathway. These effects are observed for 17β-estradiol in the sub-to-low nanomolar range.

Vascular Protective Effects ofMacrolide Antibiotics Tacrolimus (FK506) and the other FK506 analogs including pimecrolimus are useful macrolide antibiotics in accordance with the principles of this disclosure. The FK506 analogs possess potent anti-inflammatory properties which are mediated through an inhibition of calcineurin activity. Without wishing to be bound by any theory, it is believed that the calcineurin inhibition results in an inhibition of IL-2, IL-3, IL-A, and TNFα production via the NFAT pathway. The FK506 analogs have also been reported as lacking the ability to significantly inhibit proliferation and migration of vascular endothelial cells. However, the lack of vascular SMC activation following vascular injury may be a result of the general anti-inflammatory effects of the FK506 analogs. Tacrolimus and many of the other potent FK506 analogs exert their biological effects in the sub-to-low nanomolar range.

Rapamycin and other rapamycin analogs are useful macrolide antibiotics in accordance with the principles of this disclosure. Rapamycin blocks IL-2-mediated T-cell proliferation and possesses anti-inflammatory activity. Rapamycin exerts in antiproliferative effects by preventing the G₁ to S phase progression of T-cells through the cell cycle through the inhibition of specific cell cyclins and cyclin-dependent protein kinases (Siekierka, Immunol. Res., 13: 110-116, 1994). Rapamycin also inhibits the proliferation and migration of other cell types including, for example, smooth muscle cells (Marx et al., Circ. Res., 16: All-All, 1995; Poon et al., J. Clin. Invest. 98: 2277-2283, 1996). Thus, it is believed that rapamycin and its functional analogs are capable of inhibiting many of the deleterious features of restenosis, including the inflammatory response, and the SMC hyperproliferative and motogenic responses. However, unlike the FK506 analogs, the rapamycin analogs are also reported to inhibit the proliferation and migration of vascular endothelial cells.

Multi-Drug Local Drug Delivery Devices

The local co-administration of an estrogen receptor agonist and a macrolide antibiotic provides therapy for vascular disorders. Both estrogen receptor agonists and macrolide antibiotics are therapeutically effective, when locally administered. Typically, the drug that elutes from such devices provides a certain dose to tissues surrounding the device, e.g., the portion of blood vessel surrounding the device and diseased tissues in close proximity downstream of the device. The amount of drug is typically insufficient to induce a significant systemic effect. For example, estradiol eluting from a stent placed in a coronary artery has a beneficial effect on early stage atherosclerotic lesions or vulnerable plaque in nearby regions of the same artery, or, in some embodiments, benefit (reduce cell death, enhance function) regions of the heart that are served by that stented artery. The estradiol eluted from the stent is, however, insufficient to affect therapeutic or toxic effects outside of the heart. Without wishing to be bound by any particular theory, the metabolic pathways through which each class of compounds exerts its beneficial effect are complementary rather than overlapping (FIGURE 1).

It is believed that estradiol promotes endothelial cell proliferation and migration which causes re-endothelialization at the site of injury. Estradiol also promotes eNOS expression which causes a local vasodilation, further relieving any constriction caused by restenosis or plaque formation. These effects together generally inhibit neointimal formation which underlies the restenotic process. Estrogen also acts directly on the SMCs to inhibit proliferation, migration, and extracellular matrix production. Finally, estrogen exerts an anti-inflammatory effect through the inhibition of the NFKB pathway. A characteristic of most macrolide antibiotics is their anti-inflammatory effects.

Many of these effects are believed to be mediated by different mechanisms than those used to produce estradiol's anti-inflammatory effect. Specifically, it is believed that the FK506 analogs block calcineurin activation which inhibits the NFAT pathway. The rapamycin analogs are believed to inhibit inflammation by blocking mTOR. Like estradiol, the rapamycin analog class of the macrolide antibiotics also inhibit SMC migration and proliferation at therapeutically relevant doses. In the case of the FK506 analogs, the beneficial effects of endothelial cell proliferation and migration, which is stimulated by estradiol, is not inhibited.

An enhanced therapeutic benefit is derived by co-administering an estrogen receptor agonist and a macrolide antibiotic to a patient having a vascular disorder. It is contemplated that these two classes of therapeutic agents are present in a single delivery device and that device is configured for either for immediate release (e.g., a balloon catheter) or extended release (e.g., a stent) of the agents at the site or region of vascular injury.

Therapeutic Dosage

The local drug delivery device contains therapeutically effective amounts of an estrogen receptor agonist and a macrolide antibiotic. It is contemplated that, because of the synergistic effect of these compounds for the treatment of vascular disorders, one or both of the estrogen receptor agonist and a macrolide antibiotic are present is amounts that would be sub-therapeutic if administered alone. Alternatively, if medically necessary, both agents are present in amounts that are therapeutic when administered individually. Generally, the local drug delivery devices of this invention that are designed for sustained drug release (e.g., stents) contain between about 0.001 mg and about 1.0 mg (e.g., between about 0.005 mg and about 0.500 mg, or between about 0.010 mg and 0.300 mg) of an estrogen receptor agonist and between about 0.001 mg and about 1.0 mg (e.g., between about 0.005 mg and about 0.500 mg, or between about 0.010 mg and 0.300 mg) of a macrolide antibiotic. Alternatively, the sustained release local drug delivery devices of this invention are designed to elute between about 0.0001 mg/day and about 1.0 mg/day of an estrogen receptor agonist and between about 0.0001 mg/day and about 1.0 mg/day of a macrolide antibiotic. In some instances, at least 10% of the total drug will be released during the first five days following implantation. Local drug delivery devices of this invention that are designed for immediate drug release (e.g., balloon catheters) are designed to administer between about 0.010 mg and about 10.0 mg (e.g., between about 0.50 mg and about 7.5 mg, or between about 1.0 mg and about 5.0 mg) of an estrogen receptor agonist and between about 0.010 mg and about 10.0 mg (e.g., between about 0.50 mg and about 7.5 mg, or between about 1.0 mg and about 5.0 mg) of a macrolide antibiotic.

Therapeutic Attachments

The therapeutic agents used in accordance with the principles of this disclosure are attached to an implantable device using any suitable means. Ih one embodiment, the therapeutic agents are attached by a physical means such as by dipping or spraying the therapeutic agent onto the device. Therapeutic attachment is enhanced for devices having grooves, holes, or pores that are capable of trapping the therapeutic-containing solution.

Generally, however, the therapeutic agents are attached using a coating. Suitable coatings include erodable and non-eroable polymers, and other types of coatings from which the therapeutics can diffuse. It is contemplated that the coating has known and controllable release characteristics, being biocompatible when implanted in animals and humans, and is non-thrombogenic when in contact with blood and the vascular system. The reactants and reaction conditions used to generate the polymer compositions disclosed herein can be modified to alter the properties of the final polymer composition. For example, properties such as the diffusion coefficients (e.g., the rate at which the therapeutic agents are able to diffuse through the polymer matrix), the rate of degradation of one or more of the polymer components, and the rate of the release of the therapeutic agents can be manipulated by altering the reaction conditions and reagents, and hence the final polymer properties, used to generate the coating polymers.

Two major classes of polymeric coatings are useful with implantable devices: biostable, or non-erodable, coatings; and bioabsorbable, or biodegradable, coatings. Examples of biostable coatings are fluorosilicone, silicone co-polymers, polyethylene glycol (PEG), poly(butyl methacrylate), poly(ethylene-co-vinyl acetate), polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, polyacrylamide, polyacrylic acid, polyhydroxyethyl methacrylate, polyethylene oxide. Examples of bioabsorbable coatings are polyglycolic acid (PGLA), polylactic acid (PLA), PGLA-PLA copolymers, polysaccharides, and phospholipids. Delivery of therapeutic agents from biostable coatings occurs via diffusion from the surface and/or interior of the coating into surrounding tissue, interstitial space, or vascular lumen. For bioabsorbable coatings, in vivo hydrolytic degradation of the polymeric coating is an additional mechanism for' release of the therapeutic agent, whereby metabolism of the polymeric coating by endogenous enzymes may also play a role (Meyers et al., J. Med. Chem. 2000, 43, 4319-4327). Important factors influencing hydrolytic degradation include water permeability, chemical structure, molecular weight, morphology, glass transition temperature, additives, and other environmental factors such as pH, ionic strength, site of implantation, etc. The duration of sustained delivery can be adjusted from few days up to one year by a person of ordinary skill in the art through proper selection of polymer and fabrication method.

In one embodiment, preparation of coated implantable devices is accomplished by dissolving the dried polymer in a suitable solvent and spin-coating, dipping, or spraying the medical device, typically using, for example, a 5 wt % in 2-propanol solution of the polymer. The selection of other suitable solvents for coating the medical devices will typically depend on the particular polymer as well as the volatility of the solvent.

One method of modulating the properties of the polymer compositions is to control the diffusion coefficient of the one or more polymer coating layers. The diffusion coefficient relates to the rate at which a compound diffuses through a coating matrix. The analyte diffusion coefficient is determined for the coating compositions of the present invention. Methods for determining diffusion coefficients are described, for example, in U.S. Patents 5,786,439 and 5,777,060.

One method for coating a local delivery device includes sequentially applying a plurality of relatively thin outer layers of a coating composition comprising a solvent mixture of polymeric silicone material, a crosslinking agent, and one or more of the therapeutic agents (see, for example, U.S. Patent No. 6,358,556). The polymeric coatings are cured in situ and the coated, cured prosthesis is sterilized in a step that includes pretreatment with argon gas plasma and exposure to gamma radiation, electron beam, ethylene oxide, and/or steam.

In another embodiment, the polymeric coating is applied as a mixture, solution or suspension of polymeric material and one or more of the therapeutic agents is dispersed in an organic vehicle or a solution or partial solution of such agents in a solvent or vehicle for the polymer and/or the therapeutic agents. Optionally the various therapeutic agents are placed within different polymer layers. The therapeutic agents are dispersed in a carrier material which is variously the polymer, a solvent, or both. In some instances, the coating is applied sequentially in one or more relatively thin layers. In some applications the coating is further characterized as an undercoat and a topcoat. The coating thickness ratio of the topcoat to the undercoat varies with the desired effect and/or the elution system. In an illustrative embodiment of a device having a plurality of coating layers, the coating on the medical device includes one or more base coatings and a top coating (see, for example, U.S. Patent No. 6,287,285).

In another embodiment, linking agents are used to encapsulate and/or link the therapeutic agent to the polymer matrix or link the various components of the polymer matrix together (e.g., the different polymers that comprise the various coating layers, the bioactive agents in the polymer matrices etc.). Such linking agents include, for example, polyester amide (PEA), polyethylene imine (PEI), avidin-biotin complexes, photolinking, functionalized liposomes, microsponges and microspheres. In another embodiment, therapeutic agents are modified by chemically linking them to a high molecular weight, water-soluble polymer carrier. This modified therapeutic agent is termed herein an agent-polymer conjugate. One special property of the agent conjugate is that the chemical linkage of the agent to the water-soluble polymer can be manipulated to hydrolytically degrade, thereby releasing biologically active agent into the environment in which they are placed.

The agent-polymer conjugate is incorporated into a controlled release matrix, formulated from a second biocompatible polymer. When implanted into a tissue such as the arterial lumen, the controlled-release matrix will release the agent-polymer conjugate which will release free agent molecules to treat the area of the tissue in the immediate vicinity of the polymer. The agent-polymer conjugates will also diffuse within the tissue, reaching a distance from the matrix because of their low rate of clearance from the tissue. As the agent conjugates diffuse, the bond between the polymer and the agent will slowly degrade in a controlled, prespecifϊed pattern, releasing the active agent into the environment in which they are placed to have its therapeutic effect. Similarly, agent- polymer conjugates are administered directly to a tissue and the elimination rate will be reduced relative to free agent.

There are several other variables, all of which can be controlled to produce a final product that is best suited for treating a certain disease with specific kinds of agents. A first variable is the size and characteristics of the water-soluble polymer carrier. Either synthetic or naturally occurring polymers may be used. While not limited to this group, some types of polymers that might be used are polysaccharides (e.g., dextran and ficoll), proteins (e.g., poly-lysine), poly(ethylene glycol), and poly(methacrylates). Different polymers produce different diffusion characteristics in the target tissue or organ as a result of their different size and shape.

The rate of hydrolytic degradation, and thus of agent release, can be also altered from minutes to months by altering the physico-chemical properties of the bonds between the agents and the polymer. While not wishing to be limited to the following types of bonds, artisans can bond therapeutic agents to water-soluble polymers using covalent bonds, such as ester, amide, amidoester, and urethane bonds. Ionic conjugates are also used. By changing the nature of the chemical association between water-soluble polymer and agent, the half-life of carrier-agent association is varied. This half-life of the agent- polymer conjugate in the environment in which it is placed determines the rate of active agent release from the polymer and, therefore, the degree of penetration that the agent- polymer conjugate can achieve in the target tissue. Other suitable hydrolytically labile bonds which can be used to link the agent to the water soluble polymer include thioester, acid anhydride, carbamide, carbonate, semicarbazone, hydrazone, oxime, iminocarbonate, phosphoester, phophazene, and anhydride bonds.

The rate of release is also affected by (a) stereochemical control (varying amounts of steric hindrance around the hydrolyzable bonds); (b) electronic control (varying electron donating/accepting groups around the reactive bond, controlling reactivity by induction/resonance); (c) varying the hydrophilicity/hydrophobicity of any optional spacer groups between the therapeutic agent and the polymer; (d) varying the length of the optional spacer groups (increasing length making the bond to be hydrolyzed more accessible to water); and (e) using bonds susceptible to cleavage by soluble blood plasma enzymes.

The properties of the controlled release matrix vary the rate of polymeric agent conjugate release into the tissue (Dang, et al, Biotechnol. Prog., 8: 527-532, 1992; Powell, et al., Brain Res., 515: 309-311, 1990; Radomsky, et al., Biol, of Repro., 47: 133-140, 1992; Saltzman, et al., Biophys. J, 55: 163-171, 1989; Chemical Engineering Science, 46: 2429-2444, 1991; J. Appl. Polymer Sd., 48: 1493-1500, 1992; Sherwood, et al., BioTechnology, 10: 1446-1449, 1992). Among the variables which affect conjugate release kinetics are: controlled release polymer composition, mass fraction of agent- polymer conjugate within the matrix (increasing mass fraction increases release rate), particle size of agent-polymer conjugate within the matrix (increasing particle size increases release rate), composition of polymeric agent conjugate particles, and polymer size (increasing surface area increases the release rate), and polymer shape of the controlled release matrix. Suitable polymer components for use as controlled-release matrices include poly(ethylene-co-vinyl acetate), poly(DL-lactide), polyglycolide), copolymers of lactide and glycolide, and polyanhydride copolymers.

As discussed in U.S. Pat. No. 6,300,458, hydroxypolycarbonates (HPC) is used as hydroxyl functional polymers that bind therapeutic agents or carbohydrate polymers chemically or via hydrogen bonding. These copolymers have properties attractive to the biomedical area as is or by conversion to the HPC product provided by hydrolysis or by in vivo enzymatic attack. A feature of these polymers is their tendency to undergo surface erosion. Heterogeneous hydrolysis theoretically preserves the mechanical strength and physical integrity of the matrix during biodegradation. To control the release process, it is useful to have a polymeric system which degrades from the surface and deters the permeation of the agent molecules. Achieving such a heterogeneous degradation usually requires the rate of hydrolytic degradation on the surface to be faster than the rate of water penetration into the bulk.

As noted above, the polymer compositions disclosed herein allow for the controlled release of therapeutic agents. This controlled release is modulated by the pH of the environment in which the polymer compositions function. In this context, one embodiment includes the controlled release of the therapeutic agents from a hydrophobic, pH-sensitive polymer matrix (see, for example, U.S. Patent No. 6,306,422). A polymer of hydrophobic and weakly acidic comonomers is used in the controlled release system. Weakly basic comonomers are used and the active agent is released as the pH drops. For example, a pH- sensitive polymer releases the therapeutic agents when exposed to a higher pH environment as the polymer gel swells. Such release can be made slow enough so that the therapeutic agent remains at significant levels for a clinically useful period of time.

Related embodiments provide additional compositions for releasing therapeutic agents using a dual phase polymeric agent-delivery composition. These dual phase polymeric compositions comprise a continuous biocompatible gel phase, a discontinuous particulate phase comprising defined microparticles, and the therapeutic agents to be delivered (see, for example, U.S. Patent No. 6,287,588). Typically in such embodiments, a microparticle containing a therapeutic agent is entrained within a biocompatible polymeric gel matrix. The therapeutic agent release is contained in the microparticle phase alone or in both the microparticles and the gel matrix. The release of the therapeutic agent is prolonged over a period of time, and the delivery is modulated and/or controlled. In addition, the second agent is loaded in the same or different microparticles and/or in the gel matrix. Alternatively, the therapeutic agent is encapsulated within nanospheres such as Trimetaspheres™, available from Luna Innovations, Blacksburg, VA.

Drug-eluting devices of this invention release a plurality of therapeutic agents. These agents are released at a constant rate or at a multi-phasic rate. For example, in one embodiment, the release comprises an initial burst or, immediate release of the therapeutic agents present at or near the surface of the coating layer, a second phase during which a release rate is slow or sometime no therapeutic agent is released, and a third phase during which most of the remainder of the therapeutic agents are released as erosion proceeds. Formulation, Therapeutic Attachment, andElution of Multiple Drugs

To effectively deliver two therapeutic drugs simultaneously from an implanted medical device such as a coronary stent, the desired dose of each is initially and individually determined based on the known activity and the desired rate of elution from the device. Subsequently, the drugs can be mixed together prior to attachment to the device or each of the drugs can be attached to the device independently.

The simplest formulation is to incorporate all therapeutic agents into a single coating. Typically, the drugs are premixed in the presence of a coating agent, such as a polymer, with defined adhesion and release characteristics such that the desired amount of each drug is attached to the device. For example, co-formulations in a single coating agent typically results in a single release rate common to both therapeutic agents. The most significant control that the practitioner has over the release profile is the relative concentration of each therapeutic in the coating. In one alternative embodiment, one or more of the therapeutics is encapsulated prior to being mixed with the coating agent. This effectively separates the therapeutic agents, preventing them from chemically or physically interacting with each other or the environment. Generally, the single coating formulations are effected as described above.

In contrast to single coatings containing multiple therapeutics, multiple coatings each containing individual therapeutics provide for different erosion rates/properties of the coating, and hence release rates of the therapeutics. Generally, each drug is independently formulated with a coating agent and applied to the device in such a way that the two formulated drugs do not mix together. This process is useful for drug combinations in which the practitioner seeks to eliminate the possibility that the drugs physically or chemically interact in a manner that alters the properties of the final product or its therapeutic efficacy. Such interactions that are desirably eliminated are those that interfere with, for example, the multi-drug formulation, coating, crimping, packaging, sterilization, storage, or deployment of the device. Individual drug formulation and device coating also allows for the practitioner to individually control the elution rates of the various drugs. This also permits the use of different coating agents that may have different erosion/release profiles. Useful embodiments include combinations of rapid release coatings with delayed or sustained release coatings.

To accomplish the application of two formulated drugs on a stent in such a way that they do not mix with each other, each drug could, for example, be applied using a drop-on- demand ink-jet printing system (e.g. similar to that disclosed in U.S. Patents 6,645,547, 6,916,379, and 7,048,962) or a piezoelectric micro-jetting device (e.g., similar to that disclosed in U.S. Patents 6,562,065 and 7,056,338) whereby each drug is delivered in discrete droplets onto the stent surface. The droplets could be delivered in such a way that the droplets from one drug are all directed to depressions, holes, or locations on the stent surface that are different from those depressions, holes, or locations to which each other drug is delivered. Thus, a stent could be coated with intermittent or alternating drug reservoirs of two or more different drugs whereby drug reservoirs containing different individual drugs do not physically touch or intermingle. Despite these drug reservoirs being separate from each other, they are nevertheless close enough together (e.g. between 1 μm and 200 μm apart) such that the treated arterial tissue receives an overall consistent and uniform dosage of each drug.

EXAMPLE 1: Effects of Tacrolimus and Rapamycin on Human Vascular SMCs The viability and proliferative capacity of human SMCs was measured by the color change of the tetrazolium salt WST-I. WST-I is modified by a mitochondrial enzyme involved in respiration and is, therefore, only active in living cells. The readout from this assay provides a linear correlation with live cell number in the conditions that are used here. Primary cells and cell culture media are obtained from Clonetics (Walkersville,

MD) and are grown at 37°C in a humidified incubator containing 5% CO₂. Primary human coronary artery smooth muscle cells (HCASMC) or other human vascular SMC are used at passage number < 10. They are grown in smooth muscle cell basal medium (modified MCDB 131), with the addition of: 5% fetal bovine serum (FBS); 0.5 μg/ml human epidermal growth factor (hEGF); 5 mg/ml insulin; 1.0 μg/ml human fibroblast growth factor; 50 mg/ml gentamycin; and 50 μg/ml amphoteracin B. In some assays, cells are induced to proliferate with other mitogens, such as thrombin or PDGF, combined with various levels of FBS. Alternatively, higher amounts of FBS, up to 10% or 20% can be used. Primary human coronary vascular endothelial cells (HCVEC) or other human endothelial cells (EC) are used at passage number < 10. They are grown in endothelial cell basal medium (modified MCDB 131), with the addition of: 5% FBS; 10 μg/ml hEGF; 1.0 mg/ml hydrocortisone; 3 mg/ml bovine brain extract; 50 mg/ml gentamycin; and 50 μg/ml amphoteracin B. In some assays, cells are induced to proliferate with other mitogens, such as thrombin or PDGF, in which case 5% FBS is replaced by 0.1% FBS.

Cells are removed from flasks by brief exposure to trypsin-EDTA (Invitrogen), followed by inactivation in complete medium, centrifugation for 5' at 2,000 rpm, and resuspension in test medium. Cells are counted using a hemocytometer and plated into 96- well tissue culture plates at 5x10³ cells/well in 50 μl test medium.

Test compounds are dissolved either in DMSO or PBS, such that the final concentration of DMSO in the assay is 0.5% or less, typically 0.2% or 0.4%. Compounds are prepared at twice the final assay concentration in test medium, and 50 μl is added to each well. The plates are then incubated for 2 - 5 days at 37°C.

At the end of the incubation period, 10 μl WST-I reagent (Roche Molecular Biochemicals, Indianapolis, IN) is added to each well, followed by incubation at 37⁰C for 90 minutes. During this time, the color change in the WST-I reagent correlates with the number of live cells in each well. At the end of the incubation period, plates containing live cells can be analyzed immediately, or 15 μl of 10% sodium dodecyl sulfate (SDS) can be added to each well, thus lysing the cells and preserving the assay for later analysis. Plates are analyzed (0.1 second/well) for absorbance at 450 nm in a Victor II plate reader (PerkinElmer, Boston, MA).

Data is expressed as arbitrary absorbance units (correlating with the number of live cells) and analyzed using Prism v 3.02 (Graphpad Software). Controls are wells that contain no cells (this absorbance reading is subtracted from all test wells) and wells in which cells do not proliferate due to presence of low serum (0.1% FBS) or added mitogens

- this number represents live cells that have not proliferated during the course of the assay, and can demonstrate the effect of compounds that are cytotoxic in addition to being antiproliferative.

This proliferation assay was conducted using the compounds rapamycin (sirolimus) and FK506 (tacrolimus). The results are shown in Figures 2 and 3.

EC₅₀ numbers refer to the calculated molar drug concentration at which the final cell number corresponds to 50% of the cell number in the untreated sample. Treatment of human coronary artery SMC with rapamycin results in a bi-phasic antiproliferation curve, whereby a portion of cellular proliferation is inhibited with a sub-nanomolar IC₅₀, (0.4 nM) and outright killing of the cells occurs in the low micromolar range (IC_5O = 5 μM) . In contrast, FK506 does not inhibit cell proliferation at sub-micromolar concentrations, while exhibiting cytotoxicity in the low micromolar range (Figure 2; IC₅₀ ⁼ 37 μM). The results of Figure 3 demonstrate that, while FK506 possesses no antiproliferative effects in the nanomolar range, it does nevertheless compete with rapamycin for similar binding sites. Thus, 100 nM FK506 causes the antiproliferation curve of rapamycin to shift more than two orders of magnitude to the right.

Figure 4 demonstrates that rapamycin is not selective in its effect vis-a-vis human vascular smooth muscle cells compared to endothelial cells. The coronary artery endothelial cells (left) were inhibited by rapamycin at an IC₅₀ = 58 pM, and the coronary artery smooth muscle cells (right) were inhibited at an IC₅₀ = 143 pM when cultured in the presence of 2.5% FBS, 20 μg/ml PDGF, and 10 μg/ml VEGF.

EXAMPLE 2: Prevention of Restenosis Using a Drug-Eluting Stent

A patient is diagnosed as having coronary artery disease characterized by atherosclerotic plaque formation. Following admission to hospital and balloon angioplasty, an intravascular stent is implanted at the site of plaque deposition. The stent contains 250 μg of 17β-estradiol and 200 μg of tacrolimus in a bioerodable polymer coating. A post-operative angiogram six months after surgery shows no significant restenosis in the area of stent implantation.

AU publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually indicated to be incorporated by reference.

Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this present disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

What is claimed is:

Claims

I . A local drug delivery device comprising an estrogen receptor agonist and a macrolide antibiotic.

2. The device of claim 1, wherein said estrogen receptor agonist is 17β- estradiol.

3. The device of claim 1 or 2, wherein said macrolide antibiotic is a rapamycin analog.

4. The device of claim 3, wherein said rapamycin analog is selected from the group consisting of rapamycin, everolimus, ABT578, biolimus A9, and AP23573.

5. The device of claim 1 or 2, wherein said macrolide antibiotic is an FK506 analog.

6. The device of claim 5, wherein said FK506 analog is selected from the group consisting of tacrolimus and pimecrolimus.

7. The device of any of claims 1-6, wherein said estrogen receptor agonist is present in an amount between about 0.001 mg and about 1.0 mg.

8. The device of any of claims 1-7, wherein said macrolide antibiotic is present in an amount between about 0.001 mg and about 1.0 mg.

9. The device of any of claims 1-8, wherein said device comprises a balloon catheter or an intravascular injection system.

10. The device of any of claim 1-8, wherein said device comprises a intravascular stent.

II. The device of claim 10, wherein said estrogen receptor agonist and said macrolide antibiotic are present in a polymeric coating on said stent.

12. The device of claim 10 or 11, wherein said estrogen receptor agonist and said macrolide antibiotic are each present at discrete spatial locations on said stent.

13. A method for treating or preventing a vascular disorder in a human, said method comprising implanting an intravascular stent in the affected blood vessel, wherein said stent comprises an estrogen receptor agonist and a macrolide antibiotic.

14. The method of claim 13, wherein said vascular disorder is selected from the group consisting of stenosis, restenosis, atherosclerosis, vulnerable plaques, acute myocardial infarction, and coronary heart disease.

15. The method of claim 13 or 14 wherein said estrogen receptor agonist is 17β- estradiol.

16. The method of any of claims 13-15, wherein said macrolide antibiotic is a rapamycin analog.

17. The method of claim 16, wherein said rapamycin analog is selected from the group consisting of rapamycin, everolimus, ABT578, biolimus A9, and AP23573.

18. The method of any of claims 13-15, wherein said macrolide antibiotic is an FK506 analog.

19. The method of claim 18, wherein said FK506 analog is selected from the group consisting of tacrolimus and pimecrolimus.

20. The method of any of claims 13-19, wherein said estrogen receptor agonist is eluted in an amount between about 0.0001 mg/day and about 1.0 mg/day for at least about 1 day.

21. The method of any of claims 13-20, wherein said macrolide antibiotic is present in an amount between about 0.0001 mg/day and about 1.0 mg/day for at least about 2 days.

22. An intravascular stent comprising an estrogen receptor agonist and a macrolide antibiotic.

23. The stent of claim 22, wherein said estrogen receptor agonist is 17β- estradiol.

24. The stent of claim 22 or 23, wherein said macrolide antibiotic is selected from the group consisting of rapamycin, everolimus, ABT578, biolimus A9, AP23573, tacrolimus and pimecrolimus.

25. The stent of any of claims 22-24, wherein said estrogen receptor agonist is present in an amount between about 0.001 mg and about 1.0 mg.

26. The stent of any of claims 22-25, wherein said macrolide antibiotic is present in an amount between about 0.001 mg and about 1.0 mg.

27. The stent of any of claims 22-26, wherein said estrogen receptor agonist and said macrolide antibiotic are each present at discrete spatial locations on said stent.