WO2007071605A1

WO2007071605A1 - The use of st1898 for the treatment of restenosis

Info

Publication number: WO2007071605A1
Application number: PCT/EP2006/069670
Authority: WO
Inventors: Claudio Pisano; Marcella Marcellini; Mario Bernardino Guglielmi
Original assignee: Sigma-Tau Industrie Farmaceutiche Riunite S.P.A.
Priority date: 2005-12-19
Filing date: 2006-12-13
Publication date: 2007-06-28

Abstract

The present invention relates to the use of a compound belonging to the class of atypical retinoids for the preparation of a medicament for treating pathological states, which arise from a complex series of cellular responses to vascular injury, for example after some surgery procedures, such as angioplasty, bypass grafting, endartherectomy and stent implantation.

Description

THE USE OF ST1898 FOR THE TREATMENT OF RESTENOSIS

FIELD OF THE INVENTION

The present invention relates to the use of a compound belonging to the class of atypical retinoids for the preparation of a medicament for treating pathological states, which arise from a complex series of cellular responses to vascular injury, for example after some surgery procedures, such as angioplasty, bypass grafting, endartherectomy and stent implantation. BACKGROUND OF THE INVENTION

Restenosis is an obstructive lesion of the vessel that frequently occurs following surgery with mechanical angioplasty balloon (PTCA) performed for artery disease.

This process has been considered a result of a succession of events: a) endothelial cells damage b) elastic recoil after the stretching of the artery c) neointimal hyperplasia due to proliferation and migration of vascular smooth muscle cells (SMC) d) remodelling and contraction of artery (Austin GE et al. J. Am. Coll.

Cardiol. 6: 369-75, 1985).

In particular the neointimal hyperplasia leads to the re-obstruction of injured artery and is the result of platelet and leukocyte activation besides the proliferation of SMC. Pharmacological inhibition of restenosis often failed when drugs were administered by systemic delivery (Karthikeyan G and Bhargava B Curr. Opin. Cardiol. 19: 500-9. 2004). Moreover, in the therapy of restenosis, the induction of smooth muscle cell apoptosis is considered an unfavorable event because it determines the recruitment of macrophages and T cells contributing to the preexistent local inflammation.

Consequently, an "ideal" therapeutic drug should control smooth muscle cell replication without affecting cell survival.

A significant improvement in the prevention of restenosis has been realized with the implantation of a polymer stent at the site of surgery (Sigwart U et al. N. Engl. J. Med. 316: 701-716, 1987). In addition the implantation of a stent offers the opportunity to vehicle drugs locally through a slow release (Laroia ST and Laroia AT Cardiol. Rev. 12: 37-43, 2004). Retinoids are natural and synthetic derivates of vitamin A that acts on various cellular processes such as growth, differentiation, migration and apoptosis. In recent years, the prototypic natural retinoid, a\\-trans retinoic acid (atRA), and other retinoids have been examined for their effects on vascular smooth muscle cell proliferation and differentiation (Gardner DG and Chen S Life Sci. 65: 1607-1613, 1999; Miano JM and Berk BC Circ. Res. 87: 355-362, 2000). Remarkably, all in vivo studies to date have documented decrease of neintimal mass and accelerated reendothelialization with retinoid administration after vascular injury (Miano JM et al. Circulation 98: 1219-1227, 1998; Wiegman PJ et al. Arterioscler. Thromb. Vase. Biol. 20: 89-95, 2000). At same time, recent studies have documented that atypical retinoids had antiproliferative and pro-apoptotic properties on tumor cells (Cincinelli R et al. J. Med. Chem. 48: 4931-46, 2005; Garattini E et al. Curr. Pharm. Des. 10: 433-448, 2004).

International patent application WO03/011808 discloses a new class of compounds defined as atypical retinoid acids, which show an anti-tumoral activity. Among the tested compounds (2E)-3-[3'-(2-adamantyl)-4'-methoxy[1 ,1 '-biphenyl]-4- yl]-2-propenoic acid (also named ST1898) is reported. DESCRIPTION OF THE INVENTION

On the basis of the results coming from our study we found out that (2E)-3-[3'-

(2-adamantyl)-4'-methoxy[1 ,1 '-biphenyl]-4-yl]-2-propenoic acid (ST1898) presents the characteristics of a molecule able to inhibit smooth muscle cells proliferation without induction of apoptosis or cell death and therefore useful for the systemic and/or local treatment of intimal hyperplasia following vascular surgery.

Therefore the main object of the present invention is the use of (2E)-3-[3'-(2- adamantyl)-4'-methoxy[1 ,1 '-biphenyl]-4-yl]-2-propenoic acid or its pharmaceutically acceptable salts for the preparation of a medicament for the treatment of obstructive vascular lesions following vascular surgery, such as for example after angioplasty, percutaneous transluminal coronary angioplasty (PTCA), bypass grafting, endartherectomy or stent implantation. Preferably the pathological conditions to be treated according to the invention is restenosis. Suitable pharmaceutically acceptable base addition salts for the compound of the present invention include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N₁N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Sodium salts are particularly preferred.

Another object of the present invention is a method of treating a mammal suffering from obstructive vascular lesions following vascular surgery, comprising administering a therapeutically effective amount of (2E)-3-[3'-(2-adamantyl)-4'- methoxy[1 ,1 '-biphenyl]-4-yl]-2-propenoic acid or its pharmaceutically acceptable salts. The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate a targeted disease or condition, or to exhibit a detectable therapeutic effect. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.

The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The precise effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination (s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 100 mg/kg, preferably 0.05 mg/kg to 50 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

The medicament may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent. Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co. , N. J.1991 ).

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated. The medicament of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see W098/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, rectal means or locally on the diseased tissue after surgical operation. The compound of the invention may also be applied (coated) on the stent even incorporated into a controlled-release matrix.

Dosage treatment may be a single dose schedule or a multiple dose schedule.

The invention will now be illustrated in greater detail by means of non-limiting examples.

EXAMPLES Example 1

Cell culture and cytotoxicity assay As experimental model we used rat aortic intima-derived smooth muscle cells

(SMCIT), that were isolated from intimal thickening developing after endothelial denudation of artery by mechanical ballooning. These cells were characterized by epithelial shape, high proliferation rate and low expression and/or organization of α- smooth muscle (SM) actin compared to normal smooth muscle cells of aortic media (SMC) (Orlandi A et al. Arterioscler Thromb. 14: 982-9, 1994).

Comparable experimental models were previously used for molecules as Sirolimus [Dichtl W et al. Atherosclerosis. 22: in press (available on line) 2005] and Paclitaxel (Blagosklonny MV et al. Ce// Cycle 3: 1050-1056, 2004), that have been tested for stent-coating, and are actually under clinical investigation. Below we document the in vitro activity of ST1898 on SMCIT and SMC proliferation in comparison with human endothelial cells.

Primary cultures of rat aortic media- (SMC) and intima-derived (SMCIT) smooth muscle cells (gently supplied by Prof. Augusto Orlandi, Tor Vergata University, Rome, Italy) were maintained in DMEM supplemented with 10% fetal calf serum (FCS) and 50 units/ml gentamycin sulfate. HUVEC (Human umbilical vein endothelial cells) were obtained from BioWhittaker (Walkersville, MD, USA) and grown in EGM-2 (BioWhittaker).

To test the effect of ST1898 on cell growth, cells were seeded in 96-well tissue culture plates (Corning) at approximately 10% confluence and were allowed to attach and recover for at least 24 h. Varying concentrations of the compound were then added to each well. The plates were incubated for 72 h at 37°C. The number of surviving cells was then determined by staining with sulforhodamine B as described by Skehan et al. (J. Natl. Cancer Inst. 82: 1107-1112, 1990). ST1898 inhibitory concentration 50 (IC₅₀) ± SD on different cell lines was evaluated by "ALLFIT" computer program and reported in Table 1. Results showed comparable activity of ST1898 among the cell lines tested.

Table 1

Example2

Flow Cytometry and cell cycle analysis

Flow cytometry was used to evaluate proliferation rate and apoptosis of SMCIT cell line after exposure to ST1898. To this purpose, cells were seeded in 100 mm dishes and exposed the next day to the molecule for various period of time. Upon treatment, cells were also allowed to recover in fresh drug-free medium for different times. After this, cells were harvested with trypsin/EDTA, pooled with the respective supernatant and after two washing with PBS, they were fixed in ice-cold 70% ethanol at 4°C. The day of analysis, ethanol was removed by centrifugation, cells were washed twice with PBS and treated with RNAse (75 KU/ml, Sigma, St Luois, MO, USA) for 30 min at 37°C. Propidium iodide (Pl) was finally added (50 μg/ml, Sigma) to stain cellular DNA and samples were processed on a FACScan flow cytometer (BD Biosciences, Bedford, MA, USA). Twenty thousand cells were acquired for each sample using the CELLQuest software (BD Biosciences) and recording propidium iodide (DNA) FL-2 fluorescence. Apoptosis was evaluated by measuring the percentage of cells with hypodiploid DNA content (sub-Gi/o population) with the same CELLQuest program, while cell cycle analysis was performed with the ModFit software (BD Biosciences).

Results summarized in Table 2 showed that ST1898 resulted able to arrest cell cycle of SMCIT in an early S phase in a dose-response manner, and showed at the same time no sign of pro-apoptotic activity. At a dose corresponding to ICβo (2 μM), the ST1898-treated cells resulted partially arrested in S already upon 24h (74.2 %) and were completely blocked (100%) in this phase upon 72h. The same pattern of activity was observed as the molecule was used at a higher dose (5 μM). Cells were steadily arrested in an early S phase both after treatment (93.5% at 24h; 100% at 48h, 72h, and 6d) and also at this dose no evidence of apoptosis was observed. As shown in Table 3, as cells were allowed to recover following a 72h exposure to ST1898 (at a concentration of 2 μM), cell cycle appeared still blocked in S (100% upon 24h and 48h; 96.2% upon 72h; 71 % upon 8d) and no sign of apoptosis induction was detected at any time-point. Table 2

Treatment G_o/1 (%) S (%) G₂/M (%) Apoptosis (%)

Untreated 53.4 24.7 21.9 0.3

24h

ST 1898 (1 μM) 28.0 69.7 2.4 0.1

ST 1898 (2 μM) 25.7 74.2 0.1 0.1

ST 1898 (5 μM) 93.5 6.5 0.1

48h

ST1898 (1 μM) 57.0 30.2 12.8 0.5

ST 1898 (2 μM) 21.9 61.3 16.9 0.6

ST 1898 (5 μM) 0.0 100.0 0.0 0.7

72h

ST1898 (1 μM) 59.4 27.0 13.6 0.9

ST 1898 (2 μM) 19.4 73.2 7.5 1.0

ST 1898 (5 μM) 0.0 100.0 0.0 0.6

6 days

ST 1898 (1 μM) 79.1 12.7 8.2 0.9

ST 1898 (2 μM) 24.9 55.6 19.5 0.7

ST 1898 (5 μM) 0.0 100.0 0.0 0.9

Table 3

Treatment G_n,, (% S (% G₂/M (%) Apoptosis (%

Untreated 40.9 36.8 22.3 0.3

72h

ST1898 (2 μM) 0.0 100.0 0.0 0.8

72h + 24h recovery

ST1898 (2 μM) 0.0 100.0 0.0 0.5

72h + 48h recovery

ST1898 (2 μM) 0.0 100.0 0.0 0.7

72h + 72h recovery

ST1898 (2 μM) 3.8 96.2 0.0 1.0

72h + 8 days recovery

ST 1898 (2 μM) 24.1 71.0 4.9 7.9

Claims

1. Use of (2E)-3-[3'-(2-adamantyl)-4'-methoxy[1 , 1 '-biphenyl]-4-yl]-2-propenoic acid or its pharmaceutically acceptable salts for the preparation of a medicament for the treatment of obstructive vascular lesions following vascular surgery.

2. The use according to claim 1 , wherein the vascular surgery is selected from the group consisting of angioplasty, percutaneous transluminal coronary angioplasty (PTCA), bypass grafting, endartherectomy and stent implantation.

3. The use according to claims 1 or 2, wherein the obstructive vascular lesion is restenosis.

4. A process for preparing a pharmaceutical composition for the treatment of obstructive vascular lesions following vascular surgery comprising mixing (2E)-3- [3'-(2-adamantyl)-4'-methoxy[1 ,1 '-biphenyl]-4-yl]-2-propenoic acid or its pharmaceutically acceptable salts with pharmaceutically acceptable carriers and/or excipients.

5. Method of treating a mammal suffering from obstructive vascular lesions following vascular surgery, comprising administering a therapeutically effective amount of (2E)-3-[3'-(2-adamantyl)-4'-methoxy[1 ,1 '-biphenyl]-4-yl]-2-propenoic acid or its pharmaceutically acceptable salts.