WO2007080598A2

WO2007080598A2 - A pharmaceutical composition for inhibiting/reducing neointimal proliferation and restenosis.

Info

Publication number: WO2007080598A2
Application number: PCT/IN2006/000008
Authority: WO
Inventors: Baskaran Chandrasekar
Original assignee: Baskaran Chandrasekar
Priority date: 2006-01-10
Filing date: 2006-01-10
Publication date: 2007-07-19
Also published as: WO2007080598A3

Abstract

This invention relates to a pharmaceutical composition for inhibiting / reducing neointimal proliferation. This pharmaceutical composition consists of diphenyleneiodonium and/or its chemical equivalents either alone or in combination with any pharmaceutically effective compound capable of preventing restenosis. This composition is administered locally into the wall of the coronary artery during the percutaneous coronary intervention procedure.

Description

TITLE OF THE INVENTION

A pharmaceutical composition for inhibiting / reducing neointimal proliferation and restenosis.

FIELD OF THE INVENTION

The present invention relates generally to a composition for the prevention of restenosis after percutaneous coronary intervention, and specifically to the inhibition/reduction of neointimal formation following percutaneous coronary intervention by inhibition / reduction of oxidative stress and cell cycle inhibition.

BACKGROUND OFTHE INVENTION

Proliferation of vascular smooth muscle cells and endothelial damage are important underlying causes of restenosis following angioplasty. These have led to therapies with the anti-proliferative agents rapamycin and paclitaxel, and therapies aimed to facilitate endothelial regeneration in the management of restenosis that have been well described in the literature. The role of another important mechanism, the redox process, in neointimal formation after arterial injury is becoming increasingly evident (Leite PF, et al. Redox processes underlying the vascular repair reaction. World J Surg. 2004;28:331-6; Azevedo LC, et al. Oxidative stress as a signaling mechanism of the vascular response to injury: the redox hypothesis of restenosis. Cardiovasc Res. 2000;47:436-45.). Increased oxidative stress has been demonstrated after angioplasty, and has been implicated in the pathophysiology of restenosis (Kanellakis P, et al. Angioplasty-induced superoxide anions and neointimal hyperplasia in the rabbit carotid artery: suppression by the isoflavone trans- tetrahydrodaidzein. Atherosclerosis 2004;176:63-72; Cipollone F, et al. High preprocedural non-HDL cholesterol is associated with enhanced oxidative stress and monocyte activation after percutaneous coronary intervention : possible implications in restenosis. Heart 2003;89:773-9; Imai K, et al. Lipid peroxidation may predict restenosis after coronary balloon angioplasty. Jpn Circ J 2001;65:495-9). Oxidative stress after arterial injury is an early event, and is likely to act as an amplifier of the late cellular response (Azevedo LC, et al. Oxidative stress as a signaling mechanism of the vascular response to injury: the redox hypothesis of restenosis. Cardiovasc Res. 2000;47:436~45; Janiszewski M, et al. Oxidized thiols markedly amplify the vascular response to balloon injury in rabbits through a redox active metal-dependent pathway. Cardiovasc Res. 1998;39:327-38). There is evidence supporting a role for early redox processes in apoptotic cell loss and NF-kappa B activation (Azevedo LC, et al. Oxidative stress as a signaling mechanism of the vascular response to injury: the redox hypothesis of restenosis. Cardiovasc Res. 2000;47:436-45), reduced nitric oxide bioavailability (Leite PF, et al. Redox processes underlying the vascular repair reaction. World J Surg. 2004;28:331-6), and induction of vascular smooth muscle cell apoptosis (Pollman MJ, et al. Determinants of vascular smooth muscle cell apoptosis after balloon angioplasty injury. Influence of redox state and cell phenotype. Circ Res. 1999;84:113-21) - the mechanisms by which redox process is postulated to influence neointimal formation after injury. After vascular injury, activation of enzymes such as NADPH oxidase lead to a marked increase in superoxide (reactive oxygen metabolites) generation in the vascular wall, proportional to the degree of injury (Leite PF, et al. Redox processes underlying the vascular repair reaction. World J Surg. 2004;28:331-6; Souza HP, et al. Vascular oxidant stress early after balloon injury: evidence for increased NAD(P)H oxidoreductase activity. Free Radic Biol Med. 2000; 28: 1232-1242; Szδcs K, et al. Upregulation of nox-based NAD(P)H oxidases in restenosis after carotid injury. Arterioscler Thromb Vase Biol. 2002; 22: 21-27).

Inhibition of redox process after arterial injury has been associated with reduced intimal hyperplasia (Kanellakis P, et al. Angioplasty-induced superoxide anions and neointimal hyperplasia in the rabbit carotid artery: suppression by the isoflavone trans-tetrahydrodaidzein. Atherosclerosis 2004;176:63-72; Muscoli C, et al. The protective effect of superoxide dismutase mimetic M40401 on balloon injury- related neointimal formation: role of the lectin-like oxidized low-density lipoprotein receptor- 1. J Pharmacol Exp Ther. 2004;311 :44-50; Kuo MD, et al. Local resistance to oxidative stress by overexpression of copper-zinc superoxide dismutase limits neointimal formation after angioplasty. J Endovasc Ther. 2004; 11:585-94). Systemic administration of a NADPH oxidase inhibitor, the chimeric peptide gp9 Ids-tat, has been demonstrated to reduce neointimal formation by >60% following vascular injury (Jacobson GM, et al. Novel NAD(P)H oxidase inhibitor suppresses angioplasty- induced superoxide and neointimal hyperplasia of rat carotid artery. Circ Res 2003;92:637-43). This is greater than the reduction in neointimal area reported in experimental studies with rapamycin (Suzuki T, et al. Stent-based delivery of sirolimus reduces neointimal formation in a porcine coronary model. Circulation 2001;104:l 188-93; Klugherz BD, et al. Twenty-eight-day efficacy and phamacokinetics of the sirolimus-eluting stent. Coron Artery Dis 2002;13:183-8) and paclitaxel (Heldman AW, et al. Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosis. Circulation 2001; 103:2289-95; Herdeg C, et al. Local paclitaxel delivery for the prevention of restenosis: biological effects and efficacy in vivo. J Am Coll Cardiol 2000;35:1969-76). Recently, perivascular delivery of adenovirus-mediated NADPH oxidase inhibitor (Ad- PDGFbetaR-gp91ds/eGFP) has been demonstrated to reduce neointimal formation after injury in a rat carotid model (Dourron HM₅ et al. Perivascular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced neointimal proliferation of rat carotid artery. Am J Physiol Heart Circ Physiol. 2005;288:H946-53).

In U.S. Patent Nos. 6242473, 6407133, 6462067, and 6730692 to Hellstrand et al., the use of diphenyleneiodonium for inhibiting and reducing reactive oxygen metabolites has been described. The use of diphenyleneiodonium was for inhibiting the oxidative stress associated with several medical conditions where reactive oxygen metabolites were thought to contribute to the disease state. Diphenyleneiodonium was administered by oral, subcutaneous, intravenous, intraperitoneal, or intramuscular injection, by inhalation, or by infusion devices such as syringe pumps, auto injector systems and minipumps, by implantable or injectable polymer matrices, and transdermal formulations. The administration of diphenyleneiodonium directly into the arterial wall (intra-mural) has not been described in the art. Also, none of the above patents describe the application of diphenyleneiodonium for the prevention of restenosis following percutaneous coronary intervention. Restenosis is not a disease, but is a complication of the percutaneous coronary intervention procedure. In addition to inhibition of oxidative stress, the antiproliferative effect of diphenyleneiodonium could also result from arrest of cell cycle division by inhibition of G(I) and G(2) phases of cell division (Scaife RM. Selective and irreversible cell cycle inhibition by diphenyleneiodonium. MoI Cancer Ther 2005;4:876-84; Scaife RM. G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium. MoI Cancer Ther 2004;3: 1229-37) in the inhibition/reduction of neointimal proliferation. This effect assumes importance because cellular proliferation is the most important underlying pathologic process in the development of restenosis.

This clearly indicates that the local direct injection (intra-mural) of diphenyleneiodonium into the arterial wall, its application for the inhibition/reduction of neointimal proliferation in the prevention of restenosis, and its application as an inhibitor of G(I) and G(2) phases of cell division in the inhibition/reduction of neointimal proliferation is non-obvious over the art of record, and constitutes as novel in the present invention.

SUMMARY OF THE INVENTION

Diphenyleneiodonium is a powerful inhibitor of the enzyme NADPH oxidase. In ex-vivo animal experiments, arterial injury resulted in increase of oxidative stress that was inhibited by the presence of diphenyleneiodonium, due to a decrease in NF- kappaB activation (Souza HP, et al. Vascular oxidant stress early after balloon injury: evidence for increased NAD(P)H oxidoreductase activity. Free Radic Biol Med. 2000; 28: 1232-1242). Diphenyleneiodonium inhibits migration of vascular smooth muscle cells by inhibiting activation of p38 MAPK by reactive oxygen species (Wang Z, et al. Reactive oxygen species-sensitive p38 MAPK controls thrombin-induced migration of vascular smooth muscle cells. J MoI Cell Cardiol. 2004;36:49-56.). Diphenyleneiodonium also leads to arrest of cell proliferation by the inhibition of G(I) and G(2) phases of cell division cycle (Scaife RM. Selective and irreversible cell cycle inhibition by diphenyleneiodonium. MoI Cancer Ther 2005;4:876-84; Scaife RM. G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium. MoI Cancer Ther 2004;3: 1229-37). Diabetic patients characteristically exhibit elevated vascular NADPH levels and may particularly benefit from the administration of diphenyleneiodonium during percutaneous coronary intervention.

Tetraploidization has been reported with diphenyleneiodonium, a finding that has also been documented with paclitaxel, an inhibitor of cell division that is currently being used in the prevention of restenosis (Chen JG, et al. Gene expression and mitotic exit induced by microtubule-stabilizing drugs. Cancer Res 2003;63:7891-9; Andreassen PR, et al. Chemical induction of mitotic checkpoint override in mammalian cells results in aneuploidy following a transient tetraploid state. Mutat Res 1996;372: 181-94). However, with diphenyleneiodonium, tetraploidization has been observed only in cells that have previously been mitotically arrested before exposure to diphenyleneiodonium (Scaife RM. Selective and irreversible cell cycle inhibition by diphenyleneiodonium. MoI Cancer Ther 2005;4:876-84; Scaife RM. G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium. MoI Cancer Ther 2004;3: 1229-37).

Important differences exist between diphenyleneiodonium and paclitaxel (Scaife RM. G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium. MoI Cancer Ther 2004;3: 1229-37). Unlike paclitaxel which is a microtubule stabilizer that mediates block of mitosis, diphenyleneiodonium treated cells are arrested in the cell cycle prior to mitosis. Also, diphenyleneiodonium impairs cyclin Bl accumulation, which has not been reported with paclitaxel. Furthermore, importantly, paclitaxel unlike diphenyleneiodonium, has not been demonstrated as an inhibitor of oxidative stress.

In animal studies, no evidence of toxicity has been demonstrated after long- term systemic administration of diphenyleneiodonium in doses of 1 mg/kg everyday (Kono H, et al. Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcohol-induced liver injury in the rat. Am J Physiol Gastrointest Liver Physiol 2001; 280: G1005-G1012).

The present invention relates to a pharmaceutical composition for inhibiting / reducing neointimal proliferation and preventing restenosis following percutaneous coronary intervention comprising diphenyleneiodonium and / or its functional equivalents either alone or in combination with one or more pharmaceutically effective compound capable of preventing restenosis said composition capable of being administered locally into the wall of the coronary artery.

This invention also provides a method of treating neointimal proliferation and preventing restenosis comprising the step of administering locally into the wall of the coronary artery a pharmaceutical composition containing diphenyleneiodonium and / or its functional equivalents either alone or in combination with one or more pharmaceutically effective compounds capable of preventing restenosis.

The present invention further provides a method of administering a pharmaceutically effective dosage of diphenyleneiodonium and / or its functional equivalents either alone or in combination with one or more pharmaceutically effective compounds capable of preventing restenosis locally for inhibiting / reducing neointimal proliferation and restenosis after percutaneous coronary intervention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Systemic administration of diphenyleneiodonium may not achieve a local concentration of the agent sufficient to produce a significant effect. Administration of higher systemic doses to produce the desired effect may result in intolerance or adverse effects. Locally delivering diphenyleneiodonium directly into the wall of the coronary artery would eliminate any adverse effects associated with systemic administration. As the diphenyleneiodonium is delivered directly to the desired site of action, the dose required to produce the desired effect is also markedly reduced, further decreasing the incidence of any possible adverse effect.

In an embodiment of the invention, the local delivery of diphenyleneiodonium can be performed at the end of balloon angioplasty procedure with the help of a variety of drug delivery catheters. The use of various drug delivery catheters for injection of drugs directly into the wall of a coronary artery is well described and is known to those skilled in the art (Chandrasekar B, et al. Local delivery in coronary artery disease: an overview for the interventional cardiologist. Indian Heart Journal 1999; 51: 21-6.). These include, but are not limited to a double-balloon catheter, porous balloon catheter, infusion sleeve catheter, coil balloon catheter (Dispatch catheter), iontophoretic balloon catheter, the infiltrator, and the hydrogel-coated balloon catheter. In one embodiment, an aqueous solution of diphenyleneiodonium (1 ml to 10 ml) may be injected into the wall of the coronary artery using designated drug delivery catheters. Alternatively, the diphenyleneiodonium may be administered in the form of a suspension of bio-degradable, polymer-derived, encapsulated microspheres or nano-spheres carrying the diphenyleneiodonium. In another embodiment, the diphenyleneiodonium can be delivered in the form of a viscous material or gel using a hydrogel-coated balloon catheter. In another embodiment, the diphenyleneiodonium can be delivered using iontophoresis.

In yet another embodiment of the invention, diphenyleneiodonium can be delivered locally into the wall of the coronary artery by means of a drug-eluting stent. The drug-eluting stent may be metallic, made of metal alloy, non-metallic, or biodegradable. The diphenyleneiodonium may be released from the drug-eluting stent either with or without the use of polymers or other bio-degradable materials as drug carriers. Alternatively, the diphenyleneiodonium may be released directly from a bare drug-eluting stent. The use of drug-eluting stents for the local delivery of drugs or agents into the wall of a coronary artery is well known to those skilled in the art (de Scheerder I, et al. The Elutes clinical study: 12 -month clinical follow-up. Circulation 2002;106;II-394; Park SJ, et al. A paclitaxel-eluting stent for the prevention of coronary restenosis. N Engl J Med 2003;348:1537-45; Moses JW, et al. Sirolimus- eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med 2003;349:1315-23; Colombo A, et al. Randomized study to assess the effectiveness of slow- and moderate-release polymer-based paclitaxel- eluting stents for coronary artery lesions. Circulation 2003;108:788-94)

In another embodiment, diphenyleneiodonium may be administered by means of any intravascular device (implantable or non-implantable) capable of achieving a sustained local concentration of diphenyleneiodonium in the wall of the coronary arteries.

The dose of diphenyleneiodonium intended to be delivered in the present invention varies from 1.0 microgram/kg to 100 microgram/kg. This is well within the toxicity limit of diphenyleneiodonium. In animal studies, systemic doses of diphenyleneiodonium as high as 1000 microgram/kg administered every day for as long as 4 weeks have demonstrated to be safe with no evidence of toxicity (Kono H, et al. Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcohol-induced liver injury in the rat. Am J Physiol Gastrointest Liver Physiol 2001; 280: G1005-G1012). The dose of diphenyleneiodonium intended to be delivered in the present invention is only a fraction of the dose used for systemic administration in animal studies, and, more importantly, will be administered to the patient only at the time of the percutaneous coronary intervention, and not everyday.

Drugs, chemicals, pharmaceutical agents, genetic material and peptides that are functional equivalents of diphenyleneiodonium are included within the scope of this invention. The description of functional equivalence to diphenyleneiodonium will be easily obvious to those skilled in the art.

There is some evidence from in-vitro studies that diphenyleneiodonium may inhibit proliferation of human umbilical vein endothelial cells (Balcerczyk A, et al. Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium. Biochem Pharmacol. 2005 ;69: 1263-73). An adverse effect of diphenyleneiodonium on endothelium has not been demonstrated thus far.

In another embodiment of the invention, diphenyleneiodonium may be administered locally into the wall of the coronary artery in combination with another drug, hormone, chemical, pharmaceutical agent, genetic material, or peptide. Such combinations may include but are not limited to combination of diphenyleneiodonium with one or more of antiproliferative agents, or immunosuppressive agents, or agents that enhance endothelial proliferation and function, hormones such as 17beta- estradiol, anti-inflammatory agents such as dexamethasone, inhibitors of extracellular matrix synthesis, and sulfated polysaccharides (fucoidan, fucoidin, sulfated fucans, etc.), and heparin. The above said combination may either be administered as a single injection, or, as separate injections sequentially. Alternatively, one of the two agents in tihe combination (either diphenyleneiodonium or the second agent) may be administered as an injection and this is followed sequentially by delivery of the other agent in the form of a drug-eluting stent. In yet another embodiment, both the agents (diphenyleneiodonium and the second agent) may be administered loaded onto the same drug-eluting stent. The local delivery of diphenyleneiodonium may also be combined with intravascular brachytherapy.

In another embodiment of the invention, diphenyleneiodonium, either alone or in combination as discussed above, may be administered locally into the wall of peripheral arteries such as the arteries supplying the limbs, renal arteries or carotid arteries for the inhibition/reduction of neointimal proliferation following percutaneous intervention of these arteries. The following example is given to help to further understand the scope of the present invention. The example is for illustrative purposes only and should not be construed as in anyway to limit the scope of the invention.

EXAMPLE

Local delivery of diphenyleneiodonium by a drug-eluting stent for the prevention of restenosis following angioplasty in a porcine coronary model

Animal preparation

Juvenile farm pigs (weighing 20 - 25 kg) are used for the experiment. A total of 16 animals (normal = 10, diabetic = 6) is intended to be studied. In 6 animals diabetes will be induced by intravenous injection of alloxan monohydrate as described in literature (Kjems LL, et al. Decrease in β-cell mass leads to impaired pulsatile insulin secretion, reduced postprandial hepatic insulin clearance, and relative hyperglucagonemia in the minipig. Diabetes 2001;50:2001-12.).

Before the procedure, animals are premedicated with 350 mg of acetylsalicylic acid and 75 mg clopidogrel for 2 days prior to procedure and until euthanasia. AU invasive procedures are performed under general anesthesia. Activated coagulation time will be maintained at > 300 seconds throughout the procedure.

A baseline coronary angiography is performed. Using standard percutaneous coronary intervention equipment, the left anterior descending artery and the right coronary artery of each animal are subjected to balloon angioplasty, and randomized either to drug group or control group. A 3x18 mm stent is deployed in the left anterior descending artery and the right coronary artery of each animal so that one artery receives the drug-eluting stent and the other receives the control stent. Stents are deployed at a 14 atmosphere pressure inflation for 30 seconds, to achieve a stent/artery ratio of 1.2-1.3:1 at full expansion.

Coronary angiography After 28 days, the animals are scheduled to undergo coronary angiography. Quantitative coronary , analysis is performed using a computerized edge-detection algorithm. The following coronary artery diameter measurements are made using diastolic frames: (1) basal diameter before injury, (2) diameter at full stent expansion, (3) stent/artery ratio [(2)/(l)], (4) minimal lumen diameter (MLD) of the stented segment at 4 weeks, (5) %-diameter stenosis, and (6) late lumen loss [(2) - (4)].

Histology and immunohistochemistry

Following coronary angiography, the animals are euthanized. The stented artery segments are harvested and embedded in acrylic plastic and cut into 3 blocks containing the proximal, middle, and distal portions of the stent. Three cross sections are cut from each of these blocks and stained with elastic van Gieson or Movat pentachrome (Heldman AW, et al. Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosis. Circulation 2001;103:2289-95). Histomorphometric analysis of the tissue sections is performed by computerized video imaging. The external elastic lamina area (EEL), internal elastic lamina area (IEL) and lumen area are measured, and the %-morphologic stenosis 100 (1 - lumen/IEL area) is calculated. The neointimal thickness (in millimeters) is measured halfway between each pair of strut openings (in-between distance) and averaged over all tissue cross sections. Neointimal thickness is also measured at each strut site (strut-lumen distance) (Heldman AW, et al. Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosis. Circulation 2001; 103:2289-95).

The degree of re-endothelialization is determined by immunohistochemistry using goat polyclonal anti-mouse platelet/endothelial cell adhesion molecule- 1 (PECAM-I; CD-31) IgG. The lumen circumference and the sum-total of the luminal border staining positively for PECAM-I expression are measured for each section. The degree of reendothelialization is evaluated by the percentage of vascular lumen covered by endothelial cells staining positively for PECAM-I. Inflammation score for each section is obtained by dividing the aggregate of inflammation score around each strut by the total number of struts (Kornowski R, et al. In-stent restenosis: contributions of inflammatory responses and arterial injury to neointimal hyperplasia. J Am Coll Cardiol 1998;31:224-30). Injury score is determined as previously defined (Schwartz RS, et al. Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model. J Am Coll Cardiol 1992;19:267-74).

Although the invention has been described in detail with particular reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents.

Claims

CLAIMS :

1. A pharmaceutical composition for inhibiting /reducing neointimal proliferation and preventing restenosis following percutaneous coronary intervention comprising diphenyleneiodonium and / or its functional equivalents either alone or in combination with one or more pharmaceutically effective compounds capable of preventing restenosis said composition capable of being administered locally into the wall of the coronary artery.

2. A method of treating neointimal proliferation and preventing restenosis comprising the step of administering locally into the wall of the coronary artery

^• a pharmaceutical composition containing diphenyleneiodonium and / or its functional equivalents either alone or in combination with one or more pharmaceutically effective compounds capable of preventing restenosis.

3. The method according to claim 2, wherein said pharmaceutical composition contains diphenyleneiodonium and / or its functional equivalents in combination with one or more of antiproliferative agents or immunosuppressive agents, or agents that enhance endothelial proliferation and function, or hormones such as 17beta-estradiol, or anti-inflammatory agents such as dexamethasone, or inhibitors of extracellular matrix synthesis, or sulfated polysaccharides (fucoidan, fucoidin, sulfated fucans, etc.), or heparin.

4. The method according to claim 2, wherein said pharmaceutical composition is in the form of an aqueous solution, a suspension, a viscous gel, or a drug- eluting stent.

5. A method of administering locally a pharmaceutically effective dosage of diphenyleneiodonium and / or its functional equivalents either alone or in combination with one or more pharmaceutically effective compounds capable of preventing restenosis for inhibiting / reducing neointimal proliferation and restenosis after percutaneous coronary intervention.

6. The method according to claim 5, wherein said dosage is administered locally into the wall of the artery by injecting diphenyleneiodonium and / or its functional equivalents in combination with one or more pharmaceutically effective compounds either as a composition or sequentially.

7. The method according to claim 5, wherein said administration is effected by means of a drug eluting stent, or, by means of any intravascular device (implantable or non-implantable) capable of achieving a sustained local concentration of diphenyleneiodonium in the wall of the coronary arteries.

8. Use of diphenyleneiodonium for inhibiting / reducing neointimal proliferation and restenosis.