WO2010084384A1

WO2010084384A1 - Combinations of an antiplatelet agent and an antioxidant for the treatment of vascular disorders

Info

Publication number: WO2010084384A1
Application number: PCT/IB2009/051960
Authority: WO
Inventors: Lola Maksumova; Farkhad Muratkhodjaev
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-01-26
Filing date: 2009-05-13
Publication date: 2010-07-29
Anticipated expiration: 2011-07-26

Abstract

The present invention relates, in general, to vascular disorders, and, in particular, to a method of treating, preventing or reducing vascular disorders by administering to a mammal in need a combination comprising pharmacologically effective doses of an anti-platelet agent, such as cilostazol, and an anti-oxidant, such as N-acetyl-L-cysteine, or their pharmaceutically acceptable derivatives, salts, metabolites, or structural or functional analogues thereof.

Description

Title of Invention: COMPOSITION AND METHOD OF TREATING VASCULAR DISORDERS

Technical Field

[1] The present invention relates to vascular disorders. In particular, this invention relates to a combination composition and method for treating, preventing or reducing cardiovascular disorders such as arterial (cerebral, coronary and peripheral) occlusive diseases and vascular restenosis complications following surgical intervention of blocked arteries. Background Art

[2] Aberrant vascular remodeling is a central hallmark of the development and progression of cardiovascular disorders such as systemic atherosclerosis, coronary and peripheral arterial occlusive disease, diabetic complications, systemic scleroderma and all forms of detrimental scarring triggered by interventional therapies such as angioplasty of blocked arteries and eye surgery.

[3] After artery damage, such as during systemic atherosclerosis and angioplasty, the vascular injury is repaired by cells derived from adjacent normal tissue. To repair the injury site, vascular smooth muscle cells (VSMCs) are stimulated to proliferate and migrate by several cytokines including basic fibroblast growth factor (FGF), platelet- derived growth factor (PDGF), transforming growth factor-beta (TGF- b ), and angiotensin II. In vivo, PDGF is released by platelets at the injury site and facilitates the movement of VSMCs from the media across the elastic lamina to the intima. However, in clinically diverse conditions, excessive migration and proliferation of VSMCs plays detrimental role in neointima formation leading to the pathogenesis of vascular occlusion. Vascular restenosis, for example, remains a major long-term complication following surgical intervention of blocked arteries - angioplasty, with or without stenting. In about 35% of patients who undergo percutaneous coronary intervention, restenosis occurs within three to six months after the intervention. Agents that inhibit the proliferation and migration of VSMCs are found to be useful in the treatment of vascular disorders.

[4] The present invention provides a method and a combination composition for treating, preventing or reducing cardiovascular disorders. Disclosure of Invention Technical Problem

[5] A method of treating, preventing or reducing cardiovascular disorders and delaying disease progression associated therewith is provided. The method includes admin- istering a composition comprising cilostazol in combination with N-acetyl-L-cysteine. In some embodiments, the composition comprises pharmacologically acceptable derivatives, salts, metabolites or structural or functional analogues of either or both of cilostazol and N-acetyl-L-cysteine. These agents are present in the amounts that, when administered to a mammal in need, are sufficient and effective to reduce cardiovascular disorder .

[6] A composition comprising a pharmacologically effective dose of cilostazol and a pharmacologically effective dose of N-acetyl-L-cysteine is also provided . In some embodiments of the invention, the composition comprises pharmacologically acceptable derivatives, salts, metabolites or structural or functional analogues of either or both of cilostazol and N-acetyl-cysteine.

[7] The composition may include cilostazol and N-acetyl-L-cysteine in a dosage unit form. In some embodiments, the dosage unit form may include in the range of 50 mg to 200 mg cilostazol, and in the range of 200 mg to 1800 mg of N- acetyl-L-cysteine. Brief Description of Drawings

[8] In drawings which show non-limiting embodiments of the invention:

[9] Fig. 1 illustrates the effect of cilostazol on PDGF mediated migration of human coronary artery smooth muscle cells. Left panel (A) represents absolute mean and standard deviation of the migrated cells from 3 randomly selected microscopic fields. Right panel (B) depicts relative % inhibition of PDGF- mediated migration by various concentrations of cilostazol.

[10] Fig. 2 illustrates the effect of N-acetyl-L-cysteine on PDGF mediated migration of human coronary artery smooth muscle cells. Left panel (A) represents absolute mean and standard deviation of the migrated cells from 3 randomly selected microscopic fields. Right panel (B) depicts relative % inhibition of PDGF mediated migration by various concentration of N-acetyl-L-cysteine.

[11] Fig. 3 illustrates % inhibition of PDGF-mediated migration of human coronary artery smooth muscle cells by the pharmaceutical composition of a combination of cilostazol with N-acetyl-L-cysteine. A range of therapeutic concentrations of cilostazol (0.1-50 micro M) was mixed with a range of N-acetyl-cysteine concentrations (5- 5000 micro M) in the screen plate.

[12] Fig. 4 illustrates % inhibition of PDGF mediated migration of human coronary artery smooth muscle cells by the pharmaceutical composition of a combination of cilostazol with N-acetyl-L-cysteine. Results are from the second run (confirmation plate) with mean + SD (n=3). Description

[13] Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[14] The inventors have shown cilostazol in combination with N-acetyl-L-cysteine exhibit substantial synergistic and super- additive anti-migration effect in PDGF-mediated migration of human coronary smooth muscle cells.

[15] PDGF induced migration of vascular smooth muscle cells is a major mediator of cardiovascular disorders. Therefore, suppression of PDGF- mediated migration of vascular smooth muscle cells using a combination of cilostazol and NAC can be successfully used to treat cardiovascular disorders. The inventors have found that cilostazol plus N-acetyl-cysteine combinations of the invention result in the enhancement of the anti-migration activity of the cilostazol by several folds when the said compound is combined with a sub-therapeutic dose of N-acetyl-L-cysteine, even when N-acetyl-L-cysteine is administered at a dose lower than that known to be effective. For example, cilostazol is often administered at 200mg/day orally, while N- acetyl-cysteine is generally taken in amounts between 1200-1800 mg/day. The inventors have shown a several fold increase in the potency and safety of the cilostazol by combining it, at 50 mg/day, with 600 mg/day NAC.

[16] Accordingly, the present invention relates to a method of treating vascular disorders in mammals. In one embodiment, the method comprises administering to a mammal in need of such treatment an effective amount of a composition comprising effective dose of cilostazol and N-acetyl-L-cysteine . Structural and functional analogs, metabolites and derivatives of each of these compounds are known, and any of these analogs can be used in the anti-migration combination.

[17] The terms 'treat' and 'treatment' are used broadly to denote therapeutic and prophylactic interventions that favourably alter a pathological state. Treatments include procedures that moderate or reverse the progression of, reduce the severity of, prevent, or cure a disease. As used herein, the term 'vascular disorders' includes all forms of systemic atherosclerosis and diabetic complications; coronary, cerebral and peripheral arterial occlusive disease (and its symptoms such as intermittent claudication); all forms of interventional therapy triggered vascular remodelling such as restenosis of blood vessels after balloon angioplasties, atherectomies and stenting; systemic and local scleroderma, macular degeneration and retinopathy.

[18] Preferred active agents for use in the present invention include cilostazol or any pharmaceutically acceptable derivatives, salts or metabolites, as well as any structural or functional analogs thereof . While the use of N-acetyl-L-cysteine is also preferred, pharmaceutically acceptable derivatives, salts and metabolites or structural and functional analogs of N-acetyl-L-cysteine may also be used.

[19] The amount of active agents (e.g., cilostazol and N-acetyl-L-cysteine) administered can vary with the patient, the route of administration and the result sought. Optimum dosing regimens for particular patients can be readily determined by one skilled in the art. For example, the daily dose of cilostazol can be from 50 mg to 200 mg combined with a daily dose of N-acetyl-L-cysteine from 200 mg to 1800 mg. The ratio of agents used can also range. Administration of each compound of the combination may be by any dose ratio that results in a concentration of the compound that, combined with the other compound, is a pharmacologically effective dose.

[20] The individual components of the composition can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.

[21] The active agents of the invention (which may be, for example, cilostazol in combination with N-acetyl-cysteine) can be administered in any convenient manner, such as orally, by inhalation, sub lingually, rectally, parentally (including subcutaneously, intrathecally, intramuscularly or intravenously), or transdermally.

[22] The active ingredients may be administered in the form of a pharmaceutical composition or compositions that contain one or both in an admixture with a pharmaceutical carrier. Each compound is admixed with a suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The pharmaceutical composition can be in dosage unit form such as tablet, capsule, sprinkle capsule, pill, granule, powder, syrup, suspension, emulsion, solution, gel, paste, ointment, cream, lotion, plaster, drench, suppository, enema, injectable, implant, spray or aerosol. The composition can also be present in a transdermal delivery system, which may be, by way of example, a skin patch.

[23] A large variety of delivery vehicles for administering the composition are contemplated as within the scope of the present invention when containing therapeutic amounts of cilostazol and N-acetyl-L-cysteine. Suitable delivery vehicles include, but are not limited to, microcapsules or microspheres; liposomes and other lipid-based release systems; absorbable and/or biodegradable mechanical barriers, polymeric or gel-like materials, or drug eluting stents.

[24] The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice. Sustained or controlled release formulations can also be used. Each compound of the combination may be formulated in a variety of ways that are known in the art. For example, the first agent (cilostazol) and the second agent (N-acetyl-L-cysteine) may be formulated together or separately. Desirably, the two components are formulated together for simultaneous administration. Such co- formulated compositions can include the two agents formulated together in the same pills, capsule, liquid, etc. The individually or separately formulated agents can be packaged together as a co-packaged product. Non-limiting examples include two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc.

[25] In accordance with the invention, a composition of cilostazol and N- acetyl-L-cysteine is an effective treatment for vascular disorders and provides an effective means of delaying disease progression associated therewith. The composition can be more effective than, for example, cilostazol or N-acetyl-L-cysteine treatment alone and with fewer side effects. Lower doses of both types of medication can be used in the compound treatment, thereby further reducing the overall side effect burden. It is a particular advantage that, because of synergistic and super-additive effect on administration, the amounts of cilostazol and N-acetyl-L-cysteine which are to be administered can be reduced to those amounts which, on administration alone, show only a minimal pharmacological effects so that, at the same time, side effects which are elicited by high doses of these medicaments can be diminished. This is of great importance because it is known that N-acetyl- L-cysteine can, in the customary doses, elicit undesired side effects such as nausea, vomiting, headache, dry mouth, dizziness, or abdominal pain (Whyte, Francis et al. 2007). Cilostazol may show undesired side effects in the forms of headache, diarrhea and dizziness (Ahn, Jeong et al. 2008).

[26] When used in combination, it is now possible to reduce drastically the dose of cilostazol necessary for humans, as well as the amount of N-acetyl-L-cysteine below the dose of each compound that would be pharmacologically effective when compound is used in isolation, so that there is an even greater improvement in the general toxi- cological tolerability with therapeutic efficacy.

[27] As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Example

[28] The composition comprising cilostazol and N-acetyl-L-cysteine was investigated by employing a modified Boyden chamber method.

[29] Method

[30] Primary human coronary artery smooth muscle cells were grown in 231 medium supplemented with smooth muscle growth supplement (Invitrogen) and antibiotics. Media was changed every 3-4 days. Cells from the third or fourth passages were used in the experiments.

[31] Briefly, serum starved cells were detached, resuspended in migration medium (231 medium containing 0.5% BSA) and counted. 1 x 10⁵ cells in 0.3ml were added to the upper chamber of the transwell on a collagen-coated polycarbonate membrane containing 8 microm pores (Costar). Smooth muscle cells were allowed 30 minutes to attach to the membrane before addition of the inhibitors. Then inhibitors (cilostazol, N- acetyl-L-cysteine or combination thereof) were added. Cells were then exposed to recombinant human PDGF in the lower chamber for 4 hours, after which nonmigrated cells were removed from the upper chamber using a cotton swab. The cells remaining on the inserts were fixed with 3.7% formaldehyde and stained with crystal violet solution. The migrated cells were counted in three randomly selected fields (xlO objective).

[32] Results

[33] The inventors first examined the effect of the two therapeutic compounds: cilostazol and N-acetyl-L-cysteine in the above described experimental methodology. In the negative control (in the absence of exogenous PDGF) number of spontaneous migrated cells was limited. In contrast, addition of recombinant human PDGF induced migration by 10-15 folds (21.3+3.6 vs. 302.7 + 4.7; P<0.0001).

[34] Cilostazol, an anti-platelet and vasodilator drug widely used for the treatment of peripheral arterial disease, has reportedly inhibited the proliferation and migration of rat aorta SMCs (Takahashi, Oida et al. 1992). After oral administration of the usual therapeutic dose of 200mg/day cilostazol, the plasma concentration has been reported to reach 1-10 microM (Bramer, Forbes et al. 1999). We have tested a range of concentrations of cilostazol (0.1-50 microM) on human coronary artery smooth muscle cells stimulated by human PDGF. Cilostazol demonstrated an inhibitory effect in dose- dependent manner, whereas cilostazol at lower concentrations, i.e. 0.1, 1, and 5 microM showed no significant inhibition; 10 microM cilostazol demonstrated 49.7+4.7% (P<0.001) inhibition; and at 50 microM inhibition has reached 93+7% (P<0.0001) (Fig.l).

[35] N-acetyl-L-cysteine is the acetylated form of the aminoacid cysteine. It contains a free sulphydryl group capable of rupturing disulphide bonds and reducing other compounds. In this study, the inventors have documented that N-acetyl-L-cysteine is capable of inhibiting PDGF mediated migration of vascular smooth muscle cells. 5, 50 and 100 microM were not effective but 500 microM of NAC caused 33+6.8 suppression (P<0.001), ImM - 57.33+7.2% (P<0.001), and 5mM - 93.47+1.9% (P<0.001) (Fig. 2).

[36] Next, the inventors tested the effect of the combination compositions of cilostazol with N-acetyl-L-cysteine. The inventors found that the combination of cilostazol with N-acetyl-L-cysteine has substantial antimigration activity against PDGF- stimulated primary human coronary artery smooth muscle cells. Thus, combination compositions of cilostazol with N-acetyl-L-cysteine could be very useful for the treatment of vascular disorders. Together, cilostazol and N-acetyl-L-cysteine were able to suppress PDGF mediated migration to a greater extent than either compound alone. Specifically, as seen in Fig. 3, cilostazol at maximal therapeutic dose (50 m M) can suppress the PDGF-induced migration by almost 100%. The same level of suppression can be achieved by only 5 micro M of cilostazol in combination with ImM N- acetyl-L-cysteine. Further, the addition of ImM of N-acetyl-L-cysteine to 1 m M of cilostazol resulted in super- additive and synergistic effect (81.8%), compared to cilostazol alone (4.2%) and N-acetyl-L-cysteine alone (55.9%). This represents a significant shift in the potency of both cilostazol and N-acetyl-L-cysteine. Strong synergistic enhancement of cilostazol by N-acetyl-L-cysteine was observed in the second run with 3 replicates to confirm the observation (Fig.4). Reference List

[37] Ahn, Y., M. H. Jeong, et al. (2008). 'Randomized comparison of cilostazol vs clopidogrel after drug-eluting stenting in diabetic patients— clilostazol for diabetic patients in drug-eluting stent (CIDES) trial.' Circ J72(l): 35-9.

[38] Bramer, S. L., W. P. Forbes, et al. (1999). 'Cilostazol pharmacokinetics after single and multiple oral doses in healthy males and patients with intermittent claudication resulting from peripheral arterial disease.' Clin Pharmacokinet37 Suppl 2: 1-11.

[39] Takahashi, S., K. Oida, et al. (1992). 'Effect of cilostazol, a cyclic AMP phosphodiesterase inhibitor, on the proliferation of rat aortic smooth muscle cells in culture.' J Cardiovasc PharmacoPOfo): 900-6.

[40] Whyte, I. M., B. Francis, et al. (2007). 'Safety and efficacy of intravenous N- acetylcysteine for acetaminophen overdose: analysis of the Hunter Area Toxicology Service (HATS) database.' Curr Med Res Opw23(10): 2359-68.

Claims

[Claim 1] A method of treating, preventing or reducing a vascular disorder in a mammal comprising administering a combination of a pharmacologically effective dose of an anti-platelet agent and a pharmacologically effective dose of an anti-oxidant.

[Claim 2] A method according to aspect 1 wherein the anti-platelet agent of the combination is cilostazol, or a pharmaceutically acceptable derivative, salt, metabolite, or structural or functional analogue thereof.

[Claim 3] A method according to aspect 1 wherein the anti-oxidant is N- acetyl-L-cysteine, or a pharmaceutically acceptable derivative, salt, metabolite, or structural or functional analogue thereof.

[Claim 4] A method according to aspect 1 wherein the anti-platelet agent and the anti-oxidant are administered separately.

[Claim 5] A method according to aspect 1 wherein theanti-platelet agent and the anti-oxidant are administered concurrently.

[Claim 6] A method according to aspect 1 wherein the anti-platelet agent is cilostazol, or a pharmaceutically acceptable metabolite, salt, or derivative thereof; and wherein the anti-oxidant is N-acetyl-L-cysteine, or a pharmaceutically acceptable derivative, salt, metabolite, or structural or functional analogue thereof.

[Claim 7] A method according to aspect 6 wherein the daily dose of cilostazol is in the range of 50 mg to 200 mg; and wherein the daily dose of N- acetyl-L-cysteine is in the range of 200 mg to 1800 mg.

[Claim 8] A method according to aspect 1 wherein both the anti-platelet agent and the anti-oxidant are administered by any suitable means for oral, parenteral, rectal, cutaneous, nasal, vaginal, inhalant use.

[Claim 9] A method according to aspect 1 wherein either or both of the antiplatelet agent and the anti-oxidant are admixed with a pharmaceutical carrier before administration.

[Claim 10] A composition comprising a pharmacologically effective dose of an anti-platelet agent and a pharmacologically effective dose of an antioxidant.

[Claim 11] A composition comprising a pharmacologically effective dose of cilostazol, or a pharmaceutically acceptable metabolite, salt, or derivative thereof; and N-acetyl-L-cysteine, or a pharmaceutically acceptable derivative, salt, metabolite, or structural or functional analogue thereof.

[Claim 12] A composition according to aspect 10 wherein the anti-platelet agent and the anti-oxidant are in dosage unit form.

[Claim 13] A composition according to aspect 12 wherein the composition is in the form of a tablet, capsule, granule, powder, syrup, suspension, emulsion, solution, gel, paste, ointment, cream, lotion, plaster, skin patch, drench, suppository, enema, injectable, implant, spray or aerosol.

[Claim 14] A composition according to aspect 12 further comprising a pharmaceutically acceptable carrier.

[Claim 15] A composition according to aspect 12 wherein the composition is delivered by a suitable vehicle such as microcapsules or microspheres, liposomes and other lipid-based release systems, absorbable and/or biodegradable mechanical barriers, polymeric or gel-like materials or drug eluting stents.

[Claim 16] A composition according to aspect 11 wherein the pharmacologically effective dose of cilostazol and the pharmacologically effective dose of N-acetyl-L-cysteine are effective in combination to treat a vascular disorder.

[Claim 17] A composition according to aspect 15 wherein the pharmacologically effective dose of cilostazol is below a dose of cilostazol that would be pharmacologically effective if the cilostazol were administered in isolation, and wherein the pharmacologically effective dose of N- acetyl-L-cysteine is below a dose of N-acetyl-L-cysteine that would be pharmacologically effective if the N-acetyl-L-cysteine were administered in isolation.

[Claim 18] A method according to aspect 1 wherein the vascular disorder is one of systemic atherosclerosis and diabetic complications, cerebral, coronary and peripheral occlusive disease, intermittent claudication, liver vascular fibrosis, kidney vascular fibrosis, interventional therapy triggered vascular remodelling such as restenosis of blood vessels after angioplasties, atherectomies and stenting; systemic and local scleroderma, macular degeneration and retinopathy.

[Claim 19] A method according to aspect 13 wherein the pharmacologically effective dose of the anti-platelet agent and the pharmacologically effective dose of the anti-oxidant are effective in combination to treat the vascular disorder.

[Claim 20] A method, composition or use comprising any new, inventive and useful feature, combination of features, or subcombination of features, described or clearly inferred herein.