Medical devices containing rapamycin analogs

The invention relates to a medical device comprising a support structure and a therapeutic substance. New chemical compounds with immunomodulatory activity, synthetic intermediates useful in the preparation of new compounds, and in particular macrolide immunomodulators, have been disclosed.

Also discussed are semi-synthetic rapamycin analogs, preparations for their preparation, pharmaceutical compositions containing such compounds, and their use.

Since its introduction, the compound cyclosporin (cyclosporin A) has found wide application in the field of organ transplantation and immunomodulation and has led to a significant increase in the proportion of successful transplant operations. Recently, several classes of macrocyclic compounds with potent immunomodulatory effects have been discovered. Okuhara et al. in EP 184162, published June 11, 1986, claims a number of macrocyclic compounds isolated from the genus Streptomyces, including the immunosuppressant FK-506, a 23-membered macrocyclic lactone which has been isolated from a strain of S. tsukubaensis.

Other related natural products, such as FR-900520 and FR-900523, differing from FK-506 with an alkyl substituent at C-21, were isolated from S. hygroscopicus yakushimnaensis.

Another analog, FR-900525, produced by S. tsukubaensis, differs from FK-506 in the replacement of a pipecolinic acid residue with a proline group. Adverse side effects associated with cyclosporin and FK-506, such as nephrotoxicity, have led to the continuation of the search for immunosuppressant compounds with increased efficacy and safer, including immunosuppressive agents that are effective when applied topically but are ineffective when applied systemically (US 5,457,111).

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces hygroscopicus that has shown antifungal activity, especially against Candida albicans, both in vitro and in vivo (C. Vezina et al., J. Antibiot. 1975, 28, 721, SNSehgal et al., J Antibiot. 1975, 28, 727, HA Baker et al., J. Antibiot. 1978, 31, 539, US 3,929,992 and US 3,993,749).

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Rapamycin, alone (US 4,885,171) or in combination with picibanil (US 4,401,653) has been shown to have anti-tumor activity. In 1977, rapamycin also proved effective as an immunosuppressant in an experimental model of allergic encephalitis, a model for multiple sclerosis, in assisted arthritis, a model for rheumatoid arthritis, and was found to be effective in inhibiting the formation of Ig-E-like antibodies ( R. Martel et al., Can. J. Physiol. Pharmacol., 1977, 55, 48).

The immunosuppressive effects of rapamycin were also reported in FASEB, 1989, 3, 3411 due to its ability to prolong the survival time of organ transplants in incompatible

In rodent tissue (R. Morris, Med. Sci. Ras., 1989, 17, 877). The ability of rapamycin to inhibit T cell activation was discovered by M. Strauch (FASEB, 1989, 3, 3411). For a review of these and other biological effects of rapamycin, see Transplantation Reviews, 1992, 6, 39-87.

Rapamycin has been shown to reduce neointimal hyperplasia in animal models and reduce restenosis in humans. Evidence has been published to suggest that rapamycin also has anti-inflammatory effects, a property that further justifies its choice as a treatment for rheumatoid arthritis. Since both cellular proliferation and inflammation are believed to cause stenosis after balloon angioplasty and stent insertion, it has been proposed to use rapamycin and its analogs to prevent restenosis.

Monoester and diester rapamycin derivatives (esterification at positions 31 and 42) have proved to be useful as antifungal agents (US 4,316,885) and as water-soluble rapamycin precursors (US 4,650,803).

Fermentation and purification of rapamycin and 30-demethoxyrapamycin are described in the literature (C. Vezina et al. J. Antibiot. (Tokyo), 1975, 28 (10), 721, SN Sehgal et al., J. Antibiot. (Tokyo), 1975. , 28 (10), 727, 1983, 36 (4), 351, NL Pavia et al., J. Natural Products, 1991, 54 (1), 167-177).

Numerous chemical modifications of rapamycin have been obtained. These include mono- and diester preparations of rapamycin derivatives (WO 92/05179), rapamycin 27-oximes (EP0 467606), rapamycin 42-oxo analog (US 5,023,262), bicyclic rapamycins (US 5,120,725), rapamycin dimers (US 5,120,727), silyl rapamycins (US 5,120,842) and arylsulfonates and sulfamates (US 5,177,203). Rapamycin has recently been synthesized in its naturally occurring enantiomeric form (KC Nicolaou et al., J. Am. Chem. Soc, 1993, 115, 4419-4420, SL Schreiber, J. Am. Chem. Soc, 1993, 115, 7906-7907, SJ Danishefsky, J. Am. Chem. Soc, 1993, 115, 9345-9346.

It is known that rapamycin, like FK-506, binds to FKBP-12 (Siekierka, JJ, Hung,

SHY, Poe, M., Lin CS, Sigal NH Nature, 1989, 341, 755-757, Harding, MW, Galat, A., Uehling, DE, Schreiber, SL Nature, 1989, 341, 758-760, Dumont, FJ, Melino, MR, Staruch, MJ, Koprak,

SL, Fischer, PA, Sigal, NHJ Immunol. 1990, 144, 1418-1424, Bierer, BE, Schreiber, SL, Burakoff, SJ Eur. J. Immunol. 1991, 21, 439-445, Fretz, H., Albers, MW, Galat, A., Standaert, RF, Lane, WS, Burakoff, SJ, Bierer, BE, Schreiber, SLJ Am. Chem. Soc. 1991, 113, 1409-1411). Recently, it was discovered that the rapamycin / FKBP-12 complex binds to yet another protein, other than calcineurin, a protein that inhibits the FK-506 / FKBP-12 complex (Brown, EJ, Albers, MW, Shin, TB, Ichikawa, K. , Keith, CT, Lane, WS, Schreiber, SL Nature 1994, 369, 756-758, Sabatini, DM, Erdjument-Bromage, H., Lui, M., Tempest, P., Snyder, SH Cell, 1994, 78 , 35-43).

Percutaneous transluminal coronary angioplasty (PTCA) was developed by Andreas Gruntzig in the 1970s. The first canine coronary vasodilation was performed on September 24, 1975, and results demonstrating the use of PTCA were presented at the American Heart Associacion's annual meeting the following year. Shortly thereafter in Zurich, Switzerland, the first human patient was tested, followed by the first American patients in San Francisco and New York. While this procedure changed the treatment of interventional cardiology in the treatment of patients with obstructive coronary artery disease, it did not provide a long-term solution. Patients only achieved a temporary reduction in chest pain associated with occlusion of the vessels.

It was often necessary to repeat the procedure. It was found that the presence of changes associated with restenosis severely limits the usefulness of the new procedure. In the late 1980s, stents were used to maintain the patency of vessels after angioplasty. The use of stents is associated with 90% of angioplasty procedures performed today. Before stenting, the proportion of restenosis ranged from 30% to 50% of patients who underwent balloon angioplasty. The rate of relapse after dilatation of restenosis in the stent may be as high as 70% in selected patient subsets, while the rate of angiographic restenosis after stent relocation is approximately 20%. Stent placement reduces restenosis by 15% to 20%. The percentages shown are probably the best results that can be achieved with purely mechanical stenting. The lesion resulting from restenosis is primarily caused by the growth of new internal tissue, which differs significantly from atherosclerotic disease in terms of both its time course and its histopathological appearance. Restenosis is the process of healing the damaged walls of the coronary artery, with new internal tissue significantly influencing the lumen of the vessel. Against change

Brachytherapy has been shown to be effective when it comes to restenosis in the stent. However, radiation is limited by feasibility and cost, and by ongoing safety and durability problems.

Accordingly, it is desirable to reduce the number of restenosis by at least 50% of the current level. It is for this reason that the intervention device environment is making great efforts to manufacture and evaluate drug-eluting stents. If successful, such devices would have many advantages, largely because they would not require complementary therapies, either in the form of postoperative techniques or through chronic oral pharmacotherapy.

Brief description of the drawings

Figure 1 shows the blood concentrations ± SEM (n = 3) of tetrazole-containing rapamycin analogues administered to monkeys.

Figure 2 is a side elevation view of a stent suitable for use in the present invention.

Figure 3A is a cross-sectional view of a section of vessel in which a polymer-only coated stent has been placed.

Figure 3B is a cross-sectional view of a section of vessel in which a polymer and drug coated stent has been placed.

The present invention relates to a medical device comprising a support structure and a therapeutic substance that is deposited on the support structure. The therapeutic substance has the formula:

<img file="PL209153B1_D0002.tif" />

or a pharmaceutically acceptable salt or prodrug thereof. The prodrug is an ester of an acetyl, ethanoyl, pivaloyl, pivaloyloxymethyl, acetoxymethyl, phthalidyl, methoxymethyl or indanyl group and a C-31 hydroxyl group or the prodrug is an ester of a natural amino acid and a C-31 hydroxyl group. In contrast, the support structure is selected from the group of coronary stents, peripheral stents, catheters, arteriovenous grafts, vascular bypass grafts, and drug delivery balloons used in the vascular system. The medical device according to the invention shows a controlled release of therapeutic substances over a period of several hours to several weeks. The support structure of the medical device includes a coating that contains a therapeutic substance. Preferably the coating is polymeric, and preferably it is biostable or biodegradable.

The medical device according to the invention has a therapeutic substance of the formula:

PL 209 153 B1

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The invention relates to a medical device comprising a support structure with a coating on its surface which contains a drug substance, such as a drug. Support structures for medical devices suitable for use in the present invention include coronary stents, peripheral stents, catheters, arteriovenous grafts, bypass grafts, and drug delivery balloons used in the vasculature. Suitable medications include,

<img file="PL209153B1_D0005.tif" />

Or a pharmaceutically acceptable salt or precursor thereof, including

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or a pharmaceutically acceptable salt or precursor thereof (optionally hereinafter referred to as

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or a pharmaceutically acceptable salt or precursor thereof,

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Or a pharmaceutically acceptable salt or precursor thereof (hereinafter optionally referred to as SDZ RAD or 40-O- (2-hydroxyethyl) rapamycin),

<img file="PL209153B1_D0009.tif" />

or a pharmaceutically acceptable salt or precursor thereof (hereinafter optionally referred to as A-94507).

Coatings suitable for use in the present invention include polymeric coatings, which may include any polymeric material in which the therapeutic agent, i.e., drug, is substantially soluble. The coating can be hydrophilic, hydrophobic, biodegradable or non-biodegradable. The medical device reduces restenosis in the vascular system. Direct intracoronary administration of a drug such as A-179578 is expected to reduce the rate of restenosis to about 0% to 25%.

Definitions of terms used

The term drug precursor refers to compounds which are rapidly converted in vivo to the parent compound of the above formula, for example by hydrolysis in blood. For a detailed discussion, see T. Higuchi and V. Stella, Pro-drugs as Novel Delivery systems, vol. 14 ACS Symposium Series and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Associacion and Pergamon Press, 1987 .

Pharmaceutically acceptable drug precursors refer to precursors to compounds which, within the normal judgment of the practitioner, are suitable for use in contact with human and lower mammalian tissues without undue toxicity, irritation and allergic response, and correspond to a reasonable benefit / risk ratio and are effective for the intended purpose. use and where possible as ionic zwitterionic forms of the described compounds. Particularly preferred pharmaceutically acceptable drug precursors are the C-31 hydroxyl group precursor esters of these compounds.

The term precursor esters refers to any of several ester-forming groups that are hydrolyzed under physiological conditions. Examples of precursor ester groups include acetyl, ethanoyl, pivaloyl, pivaloyloxymethyl, acetoxymethyl, phthalidyl, methoxymethyl, indanyl groups and the like, as well as ester groups derived from the coupling of amino acids of natural or synthetic origin to the C-31 hydroxyl group of the compounds.

The term carrier structure means a structure that may contain or carry a pharmaceutically acceptable carrier or vehicle, which, carrier or vehicle, may contain one or more agents or therapeutic substances, e.g. one or more drugs and / or other compounds. The support structure is usually made of metal or a polymeric material.

PL 209 153 B1

Solutions

In one embodiment of the invention there is a compound having the formula:

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In another embodiment of the invention there is a compound having the formula:

<img file="PL209153B1_D0011.tif" />

Preparation of compounds

The compounds can be obtained by various synthetic routes.

A typical procedure is shown in Scheme 1.

PL 209 153 B1

Scheme 1

<img file="PL209153B1_D0012.tif" />

epimeric mixture (B / C)

As shown in Scheme 1, conversion of the C-42 hydroxyl rapamycin to a leaving trifluoromethanesulfonate or fluorosulfonate group gave A. Replacement of the leaving group with tetrazole in the presence of a blocked, non-nucleophilic base such as 2,6-lutidine or, preferably, diisopropylethylamine, gave the epimers B and C. which were separated and purified by flash column chromatography.

PL 209 153 B1

Synthetic methods

Illustrative of the preparation methods of the compounds are provided.

Example 1

42-Epi- (tetrazolyl) rapamycin (less polar isomer)

Example 1A

A solution of rapamycin (100 mg, 0.11 mmol) in dichloromethane (0.6 mL) at 78 ° C under nitrogen was treated sequentially with 2,6-lutidine (53 µl, 0.46 mmol, 4.3 eq.) and trifluoromethanesulfonic anhydride (37 µL, 0.22 mmol) then stirred for 15 minutes, warmed to room temperature and eluted through a pad of silica gel (6 mL) with diethyl ether. The fractions containing the triflate were collected and concentrated to give the expected compound in the form of an amber colored foam.

Example 1B

42-Epi- (tetrazolyl) rapamycin (less polar isomer)

A solution of Example 1A in isopropyl acetate (0.3 ml) was treated with 1H-tetrazole (35 mg, 0.5 mmol) and then stirred for 18 hours. This mixture was partitioned between water (10 ml) and ether (10 ml). The organic layer was washed with brine (10 ml) and dried (Na2SO4). Concentration of the organic layer gave a sticky yellow solid which was purified by silica gel chromatography (3.5 g, 70-230 mesh) eluting with hexane (10 mL), hexane: ether (4: 1 (10 mL), 3: 1 (10 mL), 1: 1 (10 mL)), ether (30 mL), hexane: acetone (1: 1 (30 mL)). One of the isomers was collected in the ether fractions. MS (ESI) m / e 966 (M) "

Example 2

42-Epi- (tetrazolyl) rapamycin (more polar isomer)

Example 2A

42-Epi- (tetrazolyl) rapamycin (more polar isomer)

Collecting the slower moving band from the chromatography column using hexane: acetone (1: 1) mobile phase in Example 1B gave the expected compound. MS (ESI) m / e 966 (M).

In vitro biological activity test

The immunosuppressive activity of the compounds in question was compared with that of rapamycin and two rapamycin analogs: 40-epi-N- [2'-pyridone] rapamycin and 40-epi-N- [4'-pyridone] -rapamycin, as claimed in US 5,527,907. The activity was determined using a test mixed human lymphocyte (MLR) reaction described by Kino, T. et al in Transplantation Proceedings, XIX (5): 36-39, Suppl. 6 (1987). The results of the test show that these compounds are effective immunomodulators at nanomolar concentrations, as shown in Table 1.

Table 1

<td>Example</td><td>Human MLR IC50 ± S. E. M. (nM)</td>

<td>Rapamycin</td><td> 0,91 ± 0,36</td>

<td>2-pyridone</td><td> 12,39 ± 5,3</td>

<td>4-pyridone</td><td> 0,43 ± 0,20</td>

<td>Example 1</td><td> 1,70 ± 0,48</td>

<td>Example 2</td><td> 0,66 ± 0,19</td>

The pharmacokinetic properties for Example 1 and Example 2 were determined after a single intravenous dose of 2.5 mg / kg administered to a cynomolgus monkey (n = 3 per group). Each compound was made up as a solution of 2.5 mg / ml in 20% ethanol: 30% propylene glycol: 2% cremophor EL: 48% 5% dextrose in water as vehicle. An intravenous dose of 1 ml / kg was administered as a single single dose (-1-2 minutes) into the monkey's saphenous vein. Before dosing and 0.1 (IV only), 0.25, 0.5, 1, 1.5, 2, 4, 6, 9, 12, 24, and 30 hours after dosing, each animal's femoral vein or artery was collected blood samples. EDTA preserved samples were thoroughly mixed and extracted before being analyzed.

A blood sample (1.0 ml) was haemolysed with 20% methanol in water (0.5 ml) containing the internal standard. Hemolyzed samples were extracted with a mixture of ethyl acetate and hexane (1: 1 (v / v), 6.0 ml). The organic layer was evaporated to dryness at room temperature under a stream of nitrogen. The samples were redissolved in methanol: water (1: 1, 150 µΙ). The title compounds (50 µL injections) were separated from impurities using reverse phase HPLC with UV detection. The samples were held

PL 209 153 B1 during the low temperature (4 ° C) test. All samples from each study were analyzed separately by HPLC.

The areas under the curve (AUC) of the measurements for Example 1, Example 2 and the internal standard were determined using the Sciex MacQuan ™ software. Calibration curves were derived from the peak area ratio (parent drug / internal standard) of the blood standards using least squares linear regression over the theoretical concentration ratio. The methods were linear for both compounds within the standard curve (correlation&gt; 0.99) with a defined limit of quantitation of 0.1 ng / ml. Maximum blood concentration (CMAX) and time to reach maximum blood concentration (TMAX) were read directly from the blood concentration / time data. Blood concentration data was fitted to an exponential curve using CSTRIP to obtain pharmacokinetic parameters. The parameters found were then determined using NONLIN84. The area under the blood concentration-time curve between hours 0 to t (time of last measurable blood concentration) after administration (AUC0-t) was calculated using the linear trapezoidal method for the blood concentration-time runs. The residual area was extrapolated to infinity, defined as the final measured blood concentration (Ct) divided by the last elimination rate constant (β) and added to the AUC0-t to give the total area under the curve (AUC0-t).

As shown in Figure 1 and Table 1, Example 1 and Example 2 have a surprisingly much lower half-time terminal elimination compared to rapamycin. Only both of these compounds provide sufficient efficacy and a shorter terminal half-life (Table 2).

Table 2

<td>Relationship</td><td>AUC ng-h / ml</td><td>^vol1/2 (hours)</td>

<td>Rapamycin</td><td> 6,87</td><td> 16,7</td>

<td>2-pyridone</td><td> 2,55</td><td> 2,8</td>

<td>4-pyridone</td><td> 5,59</td><td> 13,3</td>

<td>Example 1</td><td> 2,35</td><td> 5,0</td>

<td>Example 2</td><td> 2,38</td><td> 6,9</td>

The described compounds show immunomodulatory activity in mammals (especially in humans). As immunosuppressants, they are useful in the treatment and prevention of diseases mediated by the immune system such as resistance in organ or tissue transplants such as heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, intestine, limb, muscle, nerves, duodenum, small intestine, Langerhans cell and the like and transplant / host diseases induced by bone marrow transplantation or autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's goiter, multiple sclerosis, severe myasthenia gravis, type I diabetes mellitus, uveitis, allergic encephalitis , glomerulonephritis and the like. Further applications include the treatment and prevention of inflammatory and hyperplasia related skin diseases and skin symptoms associated with immune mediated diseases such as psoriasis, atopic dermatitis, contact dermatitis and other eczema dermatitis, seborrheic dermatitis, lichen planus, pemphigus, bullous eruption, epidermal blistering, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophils, lupus erythematosus, acne and alopecia areata, various eye diseases (autoimmunity and others) such as keratoconjunctivitis, vernal conjunctivitis, Behcet-related uveitis, keratitis, herpetic keratitis, keratoconus, dystrophy corneal epithelium, corneal endosperm, and ophthalmic pemphigus. The subjects of action of the compounds in question are additionally reversible diseases of the respiratory tract, including conditions such as asthma (e.g. bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), especially chronic or chronic asthma (e.g. latent asthma and airway hyperresponsiveness), bronchitis, allergic rhinitis and the like. Inflammation of the mucosa and blood vessels such as gastric ulcer, vascular damage caused by ischemic diseases, and thrombosis. Diseases related to excessive vascular growth such as smooth muscle cell proliferation, relapse

Vasoconstriction and obstruction, especially as a result of biological or mechanical damage to the vessels, can be treated with these compounds.

The compounds or drugs described herein can be applied to stents that are to be coated with polymeric compounds. Incorporation of the compound or drug into the polymer coating of the stent may be accomplished by immersing the polymer coated stent in a solution containing the compound or drug for a sufficient period of time (such as, for example, five minutes) and then drying the coated stent, preferably with dry air, for a sufficient period of time ( such as 30 minutes). The polymer coated stent containing the compound or drug can then be introduced into the coronary vessel by insertion from a balloon catheter. Other devices besides stents that can be used to deliver drugs into the vasculature include grafts, catheters, and balloons. Compounds or drugs that can be used in place of these drugs include A-94507 and SDZ RAD. The coating may contain any polymeric material in which the therapeutic agent, i.e. drug, is substantially soluble. The coating is intended to serve as a controlled release vehicle for a medicament or as a reservoir of a medicament to be delivered to a lesion. The coating may be polymeric, and may be hydrophilic, hydrophobic, biodegradable, or non-biodegradable. The material for the polymer coating can be selected from the group consisting of polycarboxylic acids, cellulose polymers, gelatin, polyvinylpyrrolidone, maleic anhydride polymers, polyamides, polyvinyl alcohols, polyethylene oxides, glocosaminoglycans, polysaccharides, polyesters, polybortho-esters, polybortic esters, polyurethanes, polycarbonates, polypropylenes, polylactic acids, polycaprolactones, polyhydroxybutyrate valerates, polyacrylamides, polyethers and their copolymers. Coatings made of polymeric dispersions such as polyurethane dispersions (BAYHYDROL etc.) and acrylic acid latex dispersions may also be used with the medicaments of the invention.

Biodegradable polymers that can be used in the invention include polymers such as poly (L-lactic acid), poly (DL-lactic acid), polycaprolactone, poly (hydroxybutyrate), polyglycolide, poly (diaxanone), poly (hydroxyvalerate, polyorthoester, copolymers such as such as poly (lactide-co-glycolide), polyhydroxy (butyrate-co-valerate), polyglycolide-co-trimethylene carbonate), polyanhydrides, polyphosphoesters, polyphosphoester-urethane, polyamino acids, polycyanoacrylates, biomolecules such as fibrin, fibrin, cellulose, starch, collagen and hyaluronic acid, and mixtures thereof. Biologically stable materials suitable for use include polymers such as polyurethane, silicones, polyesters, polyolefins, polyamides, polycaprolactam, polyimide, polyvinyl chloride, polyvinyl methyl ether, polyvinyl alcohol, acrylic polymers and copolymers, polyacrylonitrile, copolymers with vinyl and styrene olefin monomers. such as styrene-acrylonitrile copolymers, ethylene-methyl methacrylate copolymers, ethylene-vinyl acetate), polyethers, rayon, cellulose derivatives (such as cellulose acetate, cellulose nitrate, cellulose propionate, etc.), Parylene and its derivatives, and mixtures and copolymers thereof.

Another polymer that can be used in the invention is poly (MPCW: LAMX: HPMAy TSMA2) where w, x, y, and z are the mole fractions of the monomers used in the preparation of the polymer and MPC is the 2-methacryloyloxyethylphosphorylcholine unit, LMA is the lauryl methacrylate unit , HPMA is 2-hydroxypropyl methacrylate unit and TSMA is trimethoxysilylpropyl methacrylate unit. A drug impregnated stent can be used to maintain patency of a coronary artery previously obstructed by a thrombus and / or atherosclerotic plaque. The incorporation of an anti-proliferative agent reduces the rate of intra-stent obstruction recurrence.

Other conditions treated include, but are not limited to, bowel disease, inflammatory bowel disease, necrotizing enterocolitis, bowel inflammation / allergies such as celiac disease, proctitis, eosinophilic granuloma, mastocytosis, Crohn's disease, and ulcerative inflammation. colon, nervous diseases such as dermatomyositis, Guillain-Barre syndrome, Menier's disease, neuritis, polyneuritis, mononeritis and root and nerve disease, endocrine diseases such as hyperthyroidism and Basedow's disease, blood diseases such as red blood cell aplasia, aplastic anemia, hypoplastic anemia, Werlhof's disease, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, and megaloblastic anemia red blood cells, bone diseases such as osteoporosis, respiratory diseases such as sarcoidosis, pulmonary fibrosis and idiopathic interstitial pneumonia, skin diseases such as dermatomyositis, vitiligo, ichthyosis, photoallergic sensitivity and cutaneous T-cell lymphoma, cardiovascular diseases such as hardening of the arteries, atherosclerosis, aortic syndrome, nodular inflammation arteries and myocardial fibrosis, collagen diseases such as

Scleroderma, Wegener's granulomatosis and Sjogren's syndrome, obesity, eosinophilic fasciitis, periodontal disease such as gingivitis, periodontitis, alveolar bone and dentin, nephrotic syndrome such as glomerulonephritis, male pattern baldness, and senile alopecia hair loss or causing knots and / or promoting hair formation and hair growth, muscle wasting, pyoderma and Sezary's syndrome, Addison's disease, Diseases mediated by active oxygen such as organ damage such as ischemia reperfusion organ damage (such as heart, liver, kidney and gastrointestinal tract) resulting from storage, transplantation or ischemic disease (for example thrombosis and myocardial infarction) bowel diseases such as endotoxin shock, pseudomembranous colitis, and colitis caused by drug or radiation kidney diseases such as acute ischemic kidney failure and chronic kidney failure, lung diseases such as lung oxygen or drug induced toxinosis (for example paracort or bleomycin), lung cancer and emphysema, eye diseases such as cataracts, iron, retinitis, discoloration, senile macular degeneration, vitreous scarring and alkali burns of the cornea, dermatitis such as erythema multiforme linear IgA dermatitis and cementitious dermatitis, and others such as gingivitis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution (e.g. air pollution), aging, cancer formation, cancer metastasis and ailments caused by lowering atmospheric pressure , diseases caused by the release of histamine or leukotriene C4, Behcet's disease such as enteric, vascular or neural Behcet's disease as well as Behcet's disease affecting the oral cavity, skin, eyes, vulva, joints, epididymis, lungs, kidneys and so on. In addition, the compounds are useful in the treatment and prevention of liver diseases such as immunogenic diseases (e.g., chronic autoimmune liver diseases such as autoimmune hepatitis, primary biliary cirrhosis, and sclerosing cholangitis), partial liver removal, acute liver necrosis (e.g. toxin necrosis, viral hepatitis, shock or hypoxia), hepatitis B, nonA / nB hepatitis, cirrhosis (such as alcoholic cirrhosis), and liver failure such as fulminant liver failure, delayed onset liver failure, and acute for chronic liver failure (acute liver failure in chronic liver diseases), and moreover, they are useful in a variety of diseases because of their beneficial effects such as promoting the chemotherapeutic effect, cytomegalovirus infection, especially HCMV infection, anti-inflammatory effect, sclerosis and fibrosis related diseases such as nephrosis, scleroderma, pulmonary fibrosis, and hardening of the arteries , congestive heart failure, ventricular hypertrophy, postoperative adhesions and scarring, stroke, myocardial infarction and damage associated with ischemia and reperfusion and the like.

The compounds also have FK-506 antagonistic properties. The compounds can therefore be used in the treatment of immunosuppression and immunodeficiency disorders. Examples of immunosuppressive disorders include AIDS, cancer, fungal infections, senile dementia, trauma (including wound healing, surgery, and shock), chronic bacterial infections, and certain disorders of the central nervous system. Immunosuppression requiring treatment may be caused by an overdose of an immunosuppressive macrocyclic compound, for example a 12- (2-cyclohexyl-1-methylvinyl) -13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo [22. 3.1.0^4,9] octacose-18-ene such as FK-506 or rapamycin. Patients overdose on such drugs is quite common when they find out that they have forgotten to take their medication in a timely manner, and this can lead to serious side effects.

The ability of the compounds to treat proliferative diseases can be demonstrated according to the method described in Bunchman ET and CA Brookshire, Transplantation Proceed. 23, 967-968 (1991), Yamagishi et al., Biochem. Biophys. Res. Comm. 191, 840-84 6 (1993), and Shichir et al., J. Clin. Invest. 87, 1867-1871 (1991). The proliferative diseases include smooth muscle hyperplasia, generalized sclerosis, liver cirrhosis, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, diabetic or other retinopathies, psoriasis, scleroderma, prostatic enlargement, cardiac hyperplasia, restenosis due to damage to an artery or other pathological narrowing of blood vessels. These compounds further antagonize cellular responses to a number of growth factors and therefore have vascular anti-growth properties which make them regulating or reversing the growth of certain tumors as well as fibrotic diseases of the lungs, liver and kidneys.

PL 209 153 B1

The liquid aqueous compositions are particularly useful in the treatment and prevention of various eye diseases such as autoimmune diseases (including, for example, keratoconus, keratitis, corneal epithelial damage, cataracts, Mooren's cancer, scleritis, and Graves' ophthalmopathy) and corneal graft rejection.

When used in the above and other therapeutic procedures, a therapeutically effective amount of one of the compounds may be used in pure form or, where such form exists, in the form of a pharmaceutically acceptable salt, ester or precursor. The compound can optionally be administered as a pharmaceutical composition containing the compound of interest in combination with one or more pharmaceutically acceptable excipients. The term "therapeutically effective amount of a compound" means an amount of the compound sufficient to treat diseases, with a reasonable benefit / risk ratio found in any medical procedure. However, it will be understood that the total amount of compounds and compositions used in the day will be decided by the attending physician based on a medical judgment. The specific level of a therapeutically effective dose for a given patient will depend on various factors, including the nature of the disorder being treated and the severity of the disorder, the activity of the particular compound used, the particular composition used, the age, weight, general health, sex, and diet of the patient. time of administration, route of administration and rate of excretion of the specific compound employed, duration of treatment, drugs used in combination or concomitantly with a given compound employed, and the like well known in the medical art. For example, one skilled in the art knows well to start dosing the compound at a level below that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired result is obtained.

The total daily dose of the compounds administered to a human or animal may range from about 0.01 to about 10 mg / kg / day. For oral administration, more preferred oral doses are in the range of from about 0.001 to about 3 mg / kg / day. For local delivery from a stent, the daily dose a patient will receive depends on the length of the stent. For example, a 15 mm coronary stent can contain drug in an amount from about 1 to about 120 micrograms and can deliver drug over a period ranging from several hours to several weeks. If necessary, the effective daily dose can be divided into multiple doses for administration. As a result, a single dose composition may contain amounts of submultiples thereof to make up the daily dose. Topical administration may require doses ranging from 0.001 to 3 mg / kg / day, depending on the site of administration.

Pharmaceutical compositions

Pharmaceutical compositions include the compound and a pharmaceutically acceptable carrier or excipient which can be administered orally, rectally, parenterally, intravaginally, vaginally, intraperitoneally, topically (as powders, ointments, drops or patches transdermally), buccally, as an oral or nasal spray, or topically as an oral or nasal spray. a stent placed in the vascular system. The term "pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid or liquid filler, diluent, microencapsulating material or formulation auxiliary of any kind. The term "parenteral" refers to methods of administration including intravenous, intraarterial, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection, infusion and insertion, such as into the vasculature, for example.

The pharmaceutical compositions for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and also sterile powders for reconstitution into sterile injectable solutions or dispersions just before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose, and suitable mixtures thereof, vegetable oils (such as olive oil), and additives. organic esters such as ethyl oleate are injectable. The proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the appropriate particle size in the case of suspensions, and by the use of surfactants.

These compositions may also contain additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be achieved by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenyl sorbic acid, and the like. It may also be desirable to include tonics such as sugars, sodium chloride, and the like. Prolonged absorption of the drug to

For injection purposes, it is possible to incorporate an agent that delays absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow its absorption from subcutaneous or intramuscular injection. This can be achieved by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends on its rate of dissolution, which in turn may depend on the crystal size and crystalline form. Delayed absorption of a parenterally administered drug may otherwise be achieved by dissolving or suspending the drug in an oily vehicle.

Injectable release forms are made by microcapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the type of polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable sustained release formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

Injectable formulations may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents into a sterile solid composition which may be dissolved or dispersed in sterile water or other injection medium immediately before use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable vehicle or carrier, such as sodium citrate or calcium hydrogen phosphate, and / or a) fillers or diluents, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid. , b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) wetting agents such as glycerin, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate, e) dissolution retardants such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) adsorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. For capsules, tablets, and pills, the administration forms may also contain buffering agents.

Solid compositions of a similar type can also be used as fillers in soft, semi-solid and hard capsules or liquid capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

Solid dosage forms: tablets, dragees, capsules, pills, and granules can be made with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical art. They may optionally contain iridescent agents, and their composition may also allow the active ingredient (s) to be released only or mainly in a certain part of the gastrointestinal tract, possibly with a delay. Examples of embedding compositions that can be used include polymeric substances and waxes. Such drug-containing deposition materials can be applied to medical devices such as stents, grafts, catheters, and balloons.

The active substances can also be in micro-encapsulated form, if desired, with one or more of the excipients mentioned.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. As a supplement to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate. , propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cotton seed oils, peanut oils, corn, sprouts, olives, and sesame), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

PL 209 153 B1

The suspensions may contain, in addition to the active compounds, suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitol esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Topical administration includes administration to the skin or mucous membranes, including the lung and eye surfaces. Compositions for topical administration, including those for inhalation, may be made as dry powders, which may or may not be pressurized. In non-pressurized powder compositions, the active ingredient in finely divided form can be used in admixture with a finely divided pharmaceutically acceptable inert carrier consisting of particles with a diameter of, for example, up to 100 microns. Suitable inert carriers include sugars such as lactose. It is desirable that at least 95% by weight of the active ingredient particles have an effective particle size in the range of 0.01 to 10 micrometers. Compositions for topical administration also include ointments, creams, lotions, and gels.

Alternatively, the compositions may be pressurized and contain a pressurized gas such as nitrogen or a liquefied propellant. The liquefied propellant and, of course, the entire composition are preferably such that the active ingredient does not dissolve therein to any significant degree. The pressurized composition may also contain a surfactant. The surfactant can be a liquid or solid nonionic surfactant or a solid anionic surfactant. The use of a solid anionic surfactant in the form of the sodium salt is recommended.

Another form for topical administration is the ocular form, such as for the treatment of immune mediated ocular conditions such as autoimmune diseases, allergic or inflammatory conditions, and corneal transplants. The compound is incorporated into a pharmaceutically acceptable ophthalmic carrier such that the compound remains in contact with the ocular surface for a time sufficient to allow the compound to penetrate the corneal and inner regions of the eye, e.g., the anterior chamber, posterior chamber, vitreous humor, aqueous humor. , vitreous, cornea, iris, lens, choroid / retina, and sclera. The pharmaceutically acceptable ophthalmic carrier can be, for example, an ointment, a vegetable oil or an encapsulating material.

Compositions for rectal or vaginal administration are preferably suppositories or retention enemas which can be made by mixing the compounds with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or suppository wax which are solid at room temperature but liquid at body temperature. and therefore they melt in the rectum or vagina and release the active compound.

The compounds can also be administered in the form of liposomes. As is known to those skilled in the art, liposomes are usually derived from phospholipids or other lipid substances. Liposomes are formed by single or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present liposome composition may contain, in addition to the described compound, stabilizers, preservatives, excipients and the like. The preferred lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods for liposome formation are known to those skilled in the art, Prescott, Ed., Methods in Cell Biology, Vol. XIV, Academic Press, New York, NY (1976), pp. 33 et seq.

These compounds may be administered simultaneously with one or more immunosuppressive agents. Immunosuppressants include IMURAN® azathiprine sodium, brequinar sodium, SPANIDIN © gusperim trihydrochloride (also known as deoxyspergualin), mizoribine (also known as bredynine), CELLCEPT® mycophenolate mofetil, cyclosporin A NEORAL © (marketed as other cyclosporin A formulation as A under the trade name SANDIMMUNE®), tacrolimus PROGRAF © (also known as FK-506), sirolimus and RAPAMUNE © leflunomide (also known as HWA-486), glucocorticoids such as prednisolone and its derivatives, antibody therapies such as ortoclone (OKT3) and Zenapax ©, and anti-thymocytoglobulins such as thymoglobulins.

Example 3

The purpose of this example is to determine the effect of a rapamycin analog on neointima formation in porcine coronary arteries containing stents. This example shows that the rapamycin analog A-179578, when incorporated and administered from the Biocompatibles BiodiviYsio PC Coronary stent, favorably affects neointimal hypertrophy and lumen of a porcine coronary artery. This discovery indicates that the connection

Such a method can bring significant clinical benefits when properly applied to humans by reducing neointimal hyperplasia.

Agent A-179578 is an analog of rapamycin. The aim of the study in this example was to evaluate the ability of rapamycin A-179578 analog to reduce neointimal hyperplasia in a porcine coronary artery model. The effectiveness of A-179578 in this model would indicate its clinical ability to reduce and treat coronary restenosis in stents after percutaneous vascularization is restored. The domestic pig was used because this model appears to have results comparable to other studies conducted to reduce neointimal hyperplasia in humans.

In the example, A-179578 eluted from coronary stents placed in young breeding pigs was tested and the results were compared with control stents. Control stents had struts coated with polymer only. This is important because the polymer itself should not significantly stimulate neointimal hypertrophy. As the eluted drug disappears, the inflammatory reaction to the polymer would lead to a later "catch-up effect" in which the restenosis process is not stopped but slowed down. This phenomenon would cause the stenosis to recur later in humans.

Stents were implanted in each pig in two blood vessels. The pigs used in this model were 2-4 months old and weighed 30-40 kg. Two coronary stents were implanted into each pig, visually establishing a "normal stent: artery ratio 1.1-1.2".

Beginning on the day of surgery, pigs were given orally aspirin (325 mg daily) and continued for the remainder of the time. General anesthesia was obtained by intramuscular injection followed by intravenous injection of ketamine (30 mg / kg) and xylazine (3 mg / kg). Additional drug administration at the time of induction of anesthesia consisted of atropine (1 mg) and flocillin (1 g) administered intramuscularly. A single intra-arterial dose of 10,000 heparin units was administered during stent insertion.

The artery was accessed by cutting the right external carotid artery and placing an 8F sheath. After surgery, the animals were kept on a normal diet with no cholesterol or other special additives.

A BiodivYsio stent with a nominal target vessel size of 3.0 mm was used [Figure 2]. Two coronary arteries were randomly selected for stent placement in each pig. The stent was either a drug eluting stent (polymer and drug only stent) or a polymer only coated stent (polymer only stent). Stents were inserted using standard guide catheters and wires. The stent balloons were inflated to the correct size for less than 30 seconds.

Each pig had one polymer only stent and one polymer and drug stent placed in separate coronary arteries, so each pig had one stent for drug and one for control.

A sample of a total of 20 pigs was selected to detect the predicted difference in neointima thickness of 0.2 mm with a standard deviation of 0.15 mm at a power of 0.95 and a beta of 0.02.

on the day the animals were sacrificed for histopathological examination and quantification. After the heart was disconnected from the perfusion pump system, the atrial appendage was removed to access adjacent coronary arteries. The damaged sections of the coronary arteries were dissected from the epicardium. The sections containing the lesions were isolated, leaving sufficient tissue containing the blood vessel intact at both ends. The above sections, each roughly 2.5 cm long, were embedded and processed using standard plastic embedding techniques. Tissues were then processed and stained using hematoxylin-eosin and the van Gieson technique.

Optical microscopes of low and high magnification with a calibrated grid and a digital microscope system connected to a computer with calibration analysis software were used to carry out length measurements in the plane of the microscope view.

The degree of vessel damage and neointimal response were measured using a calibrated digital microscope. The importance of not breaking the inner elastic layer is well known to the skilled person. It was confirmed that the histopathological assessment of the damage to stent blood vessels is closely related to the thickness of the neointima.

This assessment is related to the depth of the damage and is as follows:

Assessment Description of the damage

Inner elastic layer intact, medulla normally exposed, midline compressed but not torn.

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Ragged inner elastic layer, medium diameter usually compressed but not torn.

Inner elastic layer torn, medium-diameter visibly torn, outer elastic layer intact but compressed.

The outer elastic layer is torn, usually large rags of the medium diameter extending beyond the outer elastic layer, coils of the wire, sometimes in the adjacency.

The quantitative damage measurement described was performed on all stent wires from each stent section. A calibrated digital image was also used to measure the thickness of the neointima at each location with the stent wire. The lumen section, the area with the inner elastic layer and the area within the outer elastic layer were also measured.

The thickness of the neointima at each location with the stent wire in a given segment was averaged to obtain an average damage score for each segment. Measurements of the thickness of the neointima were taken to the illuminant side of the stent wire as the neointima covers this thickness in all cases.

The segment in the center of the stent was used for measurement, analysis and comparison. The results for the proximal and distal sections were also recorded (and included in the data section of the report).

The data analysis methods used in the studies do not need to take into account the arterial lesion variables across the treated / control groups because mild to moderate injury is sensitive enough to detect treatment differences. A pair t-test was performed to compare the changes in cross-section of the polymer-only stents (control group) and the polymer-drug stents (treated group). No animal died during the study before the scheduled time.

Table 3 shows the study results for pigs and arteries. In Table 3, LCX stands for the left coronary artery, LAD stands for the left anterior descending coronary artery, and RCA stands for the right coronary artery.

Table 3

Pigs and blood vessels

<td> 1</td><td>2000-G-693</td><td>RCA-control</td>

<td></td><td>2000-G-693</td><td>LCX-study</td>

<td> 2</td><td>2000-G-698</td><td>RCA study</td>

<td></td><td>2000-G-698</td><td>LAD-control</td>

<td> 3</td><td>2000-G-702</td><td>RCA study</td>

<td></td><td>2000-G-702</td><td>LAD-control</td>

<td> 4</td><td>2000-G-709</td><td>RCA-control</td>

<td></td><td>2000-G-709</td><td>LAD-study</td>

<td> 5</td><td>2000-G-306</td><td>RCA-control</td>

<td></td><td>2000-G-306</td><td>LAD-study</td>

<td></td><td>2000-G-306</td><td>* LCX-study</td>

<td> 6</td><td>2000-G-672</td><td>RCA study</td>

<td></td><td>2000-G-672</td><td>LAD-control</td>

<td> 7</td><td>2000-G-712</td><td>RCA-control</td>

<td></td><td>2000-G-712</td><td>LCX-study</td>

<td> 8</td><td>2000-G-735</td><td>RCA-control</td>

<td></td><td>2000-G-735</td><td>LAD-study</td>

<td> 9</td><td>2000-G-736</td><td>RCA-control</td>

<td></td><td>2000-G-736</td><td>LCX-study</td>

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<td> 10</td><td>2000-G-740</td><td>RCA study</td>

<td></td><td>2000-G-740</td><td>LAD-control</td>

<td> 11</td><td>2000-G-742</td><td>LAD-study</td>

<td></td><td>2000-G-742</td><td>OM (LCX) -control</td>

<td> 12</td><td>2000-G-744</td><td>RCA study</td>

<td></td><td>2000-G-744</td><td>LAD-control</td>

<td> 13</td><td>2000-G-748</td><td>RCA study</td>

<td></td><td>2000-G-748</td><td>LAD-control</td>

<td> 14</td><td>2000-G-749</td><td>RCA-control</td>

<td></td><td>2000-G-749</td><td>LCX-study</td>

<td> 15</td><td>2000-G-753</td><td>RCA-control</td>

<td></td><td>2000-G-753</td><td>LAD-study</td>

<td> 16</td><td>2000-G-754</td><td>RCA study</td>

<td></td><td>2000-G-754</td><td>LCX-control</td>

<td> 17</td><td>2000-G-755</td><td>RCA-control</td>

<td></td><td>2000-G-755</td><td>LAD-study</td>

<td> 18</td><td>2000-G-756</td><td>RCA study</td>

<td></td><td>2000-G-756</td><td>LAD-control</td>

<td> 19</td><td>2000-G-757</td><td>LAD-control</td>

<td></td><td>2000-G-757</td><td>LCX-study</td>

<td> 20</td><td>2000-G-760</td><td>LAD-study</td>

<td></td><td>2000-G-760</td><td>LCX-control</td>

Table 4 summarizes the results including all data on mean damage and neointimal thickness for each stent, including proximal, medial and distal segments. Table 4 also shows the lumen size, percent constriction, and artery size as measured from the inner flexible layer (WWE) and outer flexible layer (ZWE).

Table 4

Summary: All measurements (distant, middle, closer)

<td>Specify.</td><td>position ref.</td><td>distance ref.</td><td>light</td><td>WWE</td><td>ZWE</td><td>medium damage</td><td>strictures</td><td>Surface neointimes</td><td>GNI</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>Control</td><td>Further</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Average</td><td> 4,46</td><td> 3,96</td><td> 4,88</td><td> 7,66</td><td> 9,00</td><td> 0,22</td><td> 36,10</td><td> 2,79</td><td> 0,41</td>

<td>SD</td><td> 1,20</td><td> 1,16</td><td> 1,30</td><td> 1,25</td><td> 1,10</td><td> 0,26</td><td> 15,41</td><td> 1,29</td><td> 0,17</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Control</td><td>Center</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Average</td><td> 4,46</td><td> 3,96</td><td> 4,94</td><td> 7,71</td><td> 9,08</td><td> 0,08</td><td> 36,23</td><td> 2,77</td><td> 0,38</td>

<td>SD</td><td> 1,20</td><td> 1,16</td><td> 1,44</td><td> 1,07</td><td> 1,15</td><td> 0,14</td><td> 14,93</td><td> 1,20</td><td> 0,16</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Control</td><td>Closer</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

PL 209 153 B1 cont. table 4

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td>

<td>Average</td><td> 4,46</td><td> 3,96</td><td> 5,11</td><td> 7,89</td><td> 9,30</td><td> 0,15</td><td> 35,35</td><td> 2,78</td><td> 0,38</td>

<td>SD</td><td> 1,20</td><td> 1,16</td><td> 1,38</td><td> 1,33</td><td> 1,42</td><td> 0,22</td><td> 11,94</td><td> 1,04</td><td> 0,12</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Control</td><td>Further</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Average</td><td> 4,46</td><td> 3,41</td><td> 6,04</td><td> 7,70</td><td> 9,01</td><td> 0,26</td><td> 22,35</td><td> 1,66</td><td> 0,25</td>

<td>SD</td><td> 1,20</td><td> 0,96</td><td> 1,55</td><td> 1,49</td><td> 1,47</td><td> 0,43</td><td> 8,58</td><td> 0,58</td><td> 0,06</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Control</td><td>Center</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Average</td><td> 4,46</td><td> 3,41</td><td> 6,35</td><td> 7,75</td><td> 8,98</td><td> 0,04</td><td> 18,71</td><td> 1,41</td><td> 0,22</td>

<td>SD</td><td> 1,20</td><td> 0,96</td><td> 1,29</td><td> 1,18</td><td> 1,21</td><td> 0,07</td><td>5, f8</td><td> 0,33</td><td> 0,05</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Control</td><td>Closer</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Average</td><td> 2,56</td><td> 2,15</td><td> 3,31</td><td> 4,06</td><td> 4,66</td><td> 0,19</td><td> 16,79</td><td> 1,29</td><td> 0,18</td>

<td>SD</td><td> 1,66</td><td> 1,37</td><td> 2,39</td><td> 3,48</td><td> 4,15</td><td> 0,13</td><td> 9,97</td><td> 0,80</td><td> 0,12</td>

There is no statistically significant difference in neointimal area or thickness between proximal, middle, or distal segments in the test group (polymer-drug stents) or in the control group (polymer-only stents). This observation is fully consistent with previous studies and therefore allows the use of only middle sections for statistical comparison of test devices (polymer and drug stents) with control devices (polymer only stents).

Table 5 shows the comparisons with the statistical t-test in the study and control groups. There was a statistically significant difference in neointima thickness, neointima area, lumen size, and percent lumen taper, obviously in favor of the drug eluting stent. Conversely, there are no statistically significant differences between the test group (polymer-drug stents) and the control group (polymer-only stents) in terms of damage assessment and external or internal elastic surfaces.

Table 5

Statistical comparison of test and control parameters: middle segment data

<td colspan="8">T-test statistics</td>

<td>Parameter</td><td>Difference</td><td>Test t</td><td>DF</td><td>Error st.</td><td>Lower 95%</td><td>Higher 95%</td><td>P.</td>

<td>Light</td><td> -1,17</td><td> -2,28</td><td> 38</td><td> 0,52</td><td> -2,21</td><td> -0,13</td><td> 0,029</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>WWE</td><td> 0,03</td><td> 0,088</td><td> 38</td><td> 0,36</td><td> -0,71</td><td> 0,78</td><td> 0,93</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>ZWE</td><td> 0,2</td><td> 0,499</td><td> 36</td><td> 0,39</td><td> -0,599</td><td> 0,99</td><td> 0,62</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Thickness NI</td><td> 0,18</td><td> 5,153</td><td> 38</td><td> 0,034</td><td> 0,106</td><td> 0,244</td><td> &lt;0,0001</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Pow. NI</td><td> 1,21</td><td> 3,62</td><td> 38</td><td> 0,33</td><td> 0,53</td><td> 1,88</td><td> 0,0008</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Wed damaged</td><td> 0,038</td><td> 1,137</td><td> 38</td><td> 0,033</td><td> -0,02</td><td> 0,106</td><td> 0,26</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>- Strictures</td><td> 14,54</td><td> 2, 97</td><td> 38</td><td> 4,9</td><td> 4,61</td><td> 24,47</td><td> 0,005</td>

PL 209 153 B1

Reference arteries proximal and distal to the stent sections were observed and quantified. These vessels in all cases appeared normal and were undamaged in both the control group (polymer only stents) and the test group (polymer and drug stents). See Figures 3A and 3B. The data below shows that there are no statistically significant differences in size between the stents in the control group and the stents in the test group.

<td>Control</td><td>References proximal diameter (mm)</td><td>Reference distal diameter (mm)</td>

<td>(mean ± ZD)</td><td> 4,46 ± 1,20</td><td> 3,96 ± 1,16</td>

<td>Research</td><td></td><td></td>

<td>(mean ± ZD)</td><td> 4,26 ± 1,26</td><td> 3,41 ± 0,96</td>

The data show that there are statistically significant differences and that these differences speak in favor of a stent that elutes A-179578. The described stent results in a smaller neointima area, a smaller neointima thickness, and a larger lumen area. In the study group (polymer-drug stents) and the control group (polymer-only stent) there were no significant differences in terms of neointimal parameters and damage. There were no significant differences in arterial size (including the stent) between the control and study groups. The latter finding suggests no significant difference in the artery modeling properties of the drug-containing polymeric coating.

For both the polymer-drug stent and the polymer-only stent, at most mild inflammation was found. This finding indicates that the polymer exhibits satisfactory biocompatibility, even without drug loading. Other studies indicate that after the drug is completely released from the polymer, the polymer itself induces enough inflammation to cause neointima. This phenomenon may be responsible for the phenomenon of the recent clinical catch-up of delayed restenosis. Since in this case the polymer was not causing the coronary arteritis, late problems with the polymer after the drug has been exhausted is unlikely.

In conclusion, the stent containing A-179578 compound with the polymer showed a reduction in neointimal hyperplasia in the porcine model when placed in the coronary artery.

Example 4

The purpose of this example is to determine the release rate of A-179578 drug from 316L electrochemically polished stainless steel coupons coated with a biocompatible polymer containing side phosphorylcholine groups.

The rubber septa from the HPLC vial closures were removed from the vials and placed in the glass vials with the "teflon" side up. These septa served as supports for the test samples. The tested samples were coupons made of 316L stainless steel, previously coated with a biocompatible polymer containing side phosphorylcholine groups (PC polymer). Coronary stents are typically 316L stainless steel and can be coated with a PC polymer to create a depot site for drug delivery. The coated coupons, intended to simulate stents, were placed on the septum. A solution of A-179578 and ethanol (10 µl) was applied to the surface of each coupon using a Hamilton syringe. The solution contained A-179578 (30.6 mg) dissolved in 100% ethanol (3.0 ml). Between applications, the syringe was rinsed with ethanol. The lid of the glass vial was placed loosely on the vial, thus ensuring adequate ventilation. The coupons were left to dry for at least 1.5 hours. Twelve (12) coupons were thus charged - six were used to determine the average amount of drug applied to the device and six were used to measure the time required for the release of the drug from the devices.

To determine the total amount of A-179578 applied to the coupon, the coupon was removed from the vial and placed in a 50/50 acetonitrile / 0.01M phosphate buffer (pH 6.0, 5.0 mL) solution. The coupon was placed in a Branson 5210 ultrasonic cleaner for one hour. The coupon was then removed from the solution and the solution analyzed by HPLC.

Release time studies were performed by dipping and removing the individual coupons from fresh aliquots (10.0 ml) of pH 6.0 phosphate buffer at each of the following time intervals - 5, 15, 30 and 60 minutes. For the remaining time points of 120, 180, 240, 300, 360 minutes, a buffer volume of 5.0 ml was used. To facilitate mixing during drug release, the samples were placed on an Eberbach shaker set to low speed. After testing the last sample, all aliquots of the solution were analyzed by HPLC.

PL 209 153 B1

HPLC analyzes were performed using a Hewlett Packard 1100 series instrument with the following settings:

Injection volume = 100 µl

Acquisition time = 40 minutes

Flow = 1.0 ml / min

Column temperature = 40 ° C

Wavelength = 278 nm

Mobile phase = 65% acetonitrile / 35% H2O

Column = YMV ODS-A 55 µm, 4.6 x 250 mm

Part no. A1205254WT

The results of the above experiment showed the following release values:

Table 6

<td>Time (min)</td><td>Release percentage</td><td>Standard deviation</td>

<td> 0,00</td><td> 0,00</td><td> 0,00</td>

<td> 5,00</td><td> 1,87</td><td> 1,12</td>

<td> 15,00</td><td> 2,97</td><td> 1,47</td>

<td> 30,00</td><td> 3,24</td><td> 1,28</td>

<td> 60,00</td><td> 3,29</td><td> 1,29</td>

<td> 120,00</td><td> 3,92</td><td> 1,28</td>

<td> 180,00</td><td> 4,36</td><td> 1,33</td>

<td> 240,00</td><td> 4,37</td><td> 1,35</td>

<td> 300,00</td><td> 6,34</td><td> 2,07</td>

<td> 360,00</td><td> 7,88</td><td> 1,01</td>

Example 5

The purpose of this example was to determine the loading amount and release of A-179578 from BiodivYsio drug delivery stents.

To load the drug into the stents, a 50 mg / ml solution of A-179578 in ethanol was prepared and distributed among twelve vials. Twelve individual polymer-coated stents were placed on supports designed to hold the stents upright and the stents were immersed vertically for five minutes in the drug solution. The stents and supports were removed from the vials, and excess drug solution was removed by contacting the stent with an absorbent material. The stents were then allowed to air dry in an inverted vertical position for 30 minutes.

The stents were removed from the supports and each stent was placed in 50/50 acetonitrile / phosphate buffer (pH 5.1, 2.0 ml) and sonicated for one hour. The stents were removed from the solution and the solutions tested for drug concentration, allowing the initial amount of drug on the stents to be calculated. The presented method independently demonstrated removal of at least 95% of the drug from the stent coating. The stents contained an average of 60 micrograms of drug ± 20 micrograms.

Drug loaded stents were placed on supports and placed in individual vials in 0.01 M phosphate buffer (pH = 6.0, 1.9 ml). These samples were placed on an Eberbach shaker set at low speed for backward and forward mixing. To avoid reaching drug saturation in buffer, stents were periodically transferred to fresh buffer vials at the following times: 15, 30, 45, 60, 120, 135, 150, 165, 180, 240, 390 minutes. At the end of the test period for drug release, vials with buffer solution were tested by HPLC for drug concentration. The results, presented as% cumulative drug release as a function of time, are presented in Table 7.

PL 209 153 B1

Table 7

<td>Time (min)</td><td>% Cumulative Drug Release</td>

<td> 15</td><td> 0,3</td>

<td> 30</td><td> 1,1</td>

<td> 45</td><td> 2,1</td>

<td> 60</td><td> 3,2</td>

<td> 120</td><td> 4,3</td>

<td> 135</td><td> 5,9</td>

<td> 150</td><td> 6,3</td>

<td> 165</td><td> 6,8</td>

<td> 180</td><td> 7,4</td>

<td> 240</td><td> 10,8</td>

<td> 390</td><td> 13,2</td>

Patent claims