WO2010000784A1

WO2010000784A1 - Treatment of restenosis

Info

Publication number: WO2010000784A1
Application number: PCT/EP2009/058279
Authority: WO
Inventors: Claudio Pisano; Mario Bernardino Guglielmi; Marcella Marcellini
Original assignee: Sigma-Tau Industrie Farmaceutiche Riunite S.P.A.
Priority date: 2008-07-03
Filing date: 2009-07-01
Publication date: 2010-01-07

Abstract

It is described here the use of a compound (ST1926) 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

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, all-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 a decrease of neointimal mass and accelerated reendothelialization with retinoid administration after vascular injury (Miano JM et al. Circulation 98: 1219- 1227, 1998; Wiegman PJ et al. Arteήoscler. Thromb. Vase. Biol. 20: 89-95, 2000). At the 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'-(l-adamantyl)-4'- hydroxy[l , l '-biphenyl]-4-yl]-2-propenoic acid (also named ST1926) is reported.

International patent application WO 2007/071605 relates to the use of STl 898 for the treatment of restenosis. STl 898 is the (2E)-3-[3'-(2- adamantyl)-4'-methoxy[l , l '-biphenyl]-4-yl]-2-propenoic acid, which belongs to the same class of ST1926, is structurally very similar to ST1926 and its synthesis is described in WO03/01 1808. DESCRIPTION OF THE INVENTION It has now surprisingly been found that ST 1926 is more potent than STl 898 in this indication, notwithstanding the structural similarity of these two compounds.

Therefore the main object of the present invention is the use of (2E)-

3-[3'-(l-adamantyl)-4'-hydroxy[l , r-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, N,N'-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'-(l-adamantyl)-4'-hydroxy[l, l'-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 [rat aortic intima-derived smooth muscle cells (SMCIT)I 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. Arteήoscler Thromb. 14: 982-9, 1994).

Comparable experimental models were previously used for molecules as Sirolimus [Dichtl W et al. Atherosclerosis. 186(2): 321-30, 2006] and Paclitaxel (Blagosklonny MV et al. CeZZ Cycle 3: 1050- 1056, 2004), that have been tested and approved for stent-coating. Below we document the in vitro activity of ST 1926 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 ST1926 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: 1 107- 1 1 12, 1990). ST1926 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 STl 926 among the selected cell lines in a nanomolar range. Notably, when comparing the respective IC50, ST1926 resulted more active than STl 898 [(2E)-3-[3'-(2-adamantyl)-4'- methoxy[l , l '-biphenyl]-4-yl]-2-propenoic acid, which belongs to the same class of STl 926, structurally very similar to STl 926 and whose synthesis is described in WO03/01 1808]. Table 1

Example 2 Flow Cytometry and cell cycle analysis [rat aortic intima-derived smooth muscle cells (SMCIT)I

Flow cytometry was used to evaluate cell cycle distribution and apoptosis of SMCIT cells after exposure to ST1926. Paclitaxel was used as reference compound. To this purpose, cells were seeded in 100 mm dishes and exposed the next day to the molecule (2xICso dose) for various times. At the end, cells were harvested with trypsin/ EDTA, pooled with the respective supernatant, rinsed with PBS, and fixed in ice-cold 70% ethanol at 4°C. The day of analysis, ethanol was removed by centrifugation, cells were rinsed twice with PBS, and treated with RNAse (75 KU/ml, Sigma, St Louis, MO, USA) for 30 min at 37°C. Propidium iodide (PI) was finally added (50 μg/ml, Sigma) to stain cellular DNA. Cells (2x10⁴/ sample) were acquired on a FACScan flow cytometer (Becton Dickinson, NJ, USA), and data collected with the CELLQuest software (Becton Dickinson). Apoptosis was evaluated by measuring the percentage of cells with hypodiploid DNA content (sub-Go/ 1 population) with the same CELLQuest program, while cell cycle analysis was performed with the ModFit software (Verity Software House, ME, USA).

Results summarized in Table 2 showed that unexpectedly STl 926, more than STl 898, at lower concentration, strongly and steadily arrested SMCIT cell cycle in early S phase (Gi/ S phase transition) upon exposures of 24 h (97.7 %), 48 h (93.6 %), and 72 h (91.5%), and, noteworthy, the compound resulted devoid of pro-apoptotic activity.

In comparison, Paclitaxel blocked cell cycle of SMCIT in G2/M but inducing apoptosis upon a 72 h exposure (Table 3).

Table 2

Cell line : SMCIT

Go_/i (%) eS (%) S (%) G₂/M (%) Apo (%)

Oh

Untreated 69.0 16.9 14.1 0.2

24h

Untreated

69.9 16. 7 13.4 0.1 ST 1898 (lμM)

28.0 69. 7 2.4 0.1 ST 1898 (2μM)

25.7 74. 2 0.1 0.1 ST 1898 (5μM)

93 .5 6.5 0.1 ST 1926 (0.2μ

NE 97 .7 2.3 0.1 M)

48h

Untreated ST 1898 (lμM) 91.5 4.1 4.4 0.2 ST 1898 (2μM) 57.0 30.2 12.8 0.5 ST 1898 (5μM) 21.9 61.3 16.9 0.6 ST 1926 (0.2μ 0.0 100.0 0.0 0.7 M) NE 93.6 7.4 0.2

72h

Untreated

90.0 5.1 4.9 0.1 ST 1898 (lμM)

59.4 27.0 8.2 0.9 ST 1898 (2μM)

19.4 73.2 19.5 1.0 ST 1898 (5μM)

0.0 100.0 0.0 0.6 ST 1926 (0.2μ

NE 91.5 8.5 1.2 M)

early S phase; Apo= sub Go/i population; NE= non evaluable. Table 3

Cell line : SMCIT

G₂/M

Go/ i (%) S (%) Apo (%) (%)

Oh

Untreated 58 .7 19.7 21.6 0. 3

72h

Untreated 57 .0 23.8 19.3 0. 1

Paclitaxel (0.08μM) 4. 2 9.8 86.0 13 .0

Example 3

Chemiotaxis assay [rat aortic intima-derived smooth muscle cells (SMCIT)]

Further experiments were performed to evaluate the potential of ST 1926

to inhibit the migration of intimal smooth muscle cells with a chronic

treatment. The capability of SMCIT cells to migrate was analyzed by vitro

Boyden chamber test, using polycarbonate filters with 8 μm-pores

(Nuclepore, Clifton, NJ, USA) coated with 1 mg/1 of porcine gelatin (Sigma,

St Louis, MO, USA). Cells (5xlO⁴), after 24h-, 48h- and 72h- exposure

with 0.1 μM (IC50 value) of ST1926, were introduced in the Boyden upper

chamber while medium containing 5% FCS was used as stimuli in the lower chamber. After a four-hour incubation at 37°C, filters were fixed

with methanol and stained by Diff-QuiK dye (Dade Behring, Germany,

EU). Cells from the upper surface were removed by scraping, and the

number of migrating cells attached to the lower surface of the filter was

counted in 20 randomly selected microscopic fields (X400) per experimental condition. Data were expressed as percent of inhibition compared to migration of control cells toward the chemoattractant. As summarized in next table, ST1926, produced the strongest inhibition on SMCIT migration after 24 h of drug pre treatment, in correlation with the cell cycle analysis, and this inhibitory effect partially persisted up to 72 h (see Table 4).

Table 4

Example 4

Cell culture and cytotoxicity assay [human aortic smooth muscle cells

(hAoSMC)l

As experimental model we used human aortic smooth muscle cells

(AoSMC) that were obtained from Lonza (Lonza Group Ltd, Switzerland) and grown in SGM-2 (Lonza).

To test the effect of ST 1926 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. Paclitaxel and Sirolimus were used as reference compounds. 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: 1 107- 1 1 12, 1990). Drug concentration inhibiting 50% of cell growth (ICso ± SD) was evaluated by "ALLFIT" computer program and reported in Table 5. ST 1926 is effective in a nanomolar range with potency comparable to that of Paclitaxel, while Sirolimus affected cell proliferation in a micromolar range.

Table 5

Example 5

Flow Cytometry and cell cycle analysis [human aortic smooth muscle cells (hAoSMC)l

Flow cytometry was used to evaluate cell cycle distribution and apoptosis of hAoSMC cells after exposure to ST1926. Paclitaxel was used as a reference compound. To this purpose, cells were seeded in 100 mm plastic dishes, allowed to attach, and treated for 72h with the molecule (ICso dose). Upon treatment, cells were also allowed to recover in drug-free medium for further 24h and 72h. At the end, cells were harvested with trypsin/ EDTA, pooled with the respective supernatant, rinsed with PBS, and fixed in ice-cold 70% ethanol at 4°C. The day of analysis, ethanol was removed by centrifugation, cells were rinsed twice with PBS, and treated with RNAse (75 KU/mL, Sigma, St Louis, MO, USA) for 30 min at 37°C. Propidium iodide (PI, Sigma) was finally added (50 μg/mL) to stain cellular DNA and cells (2x10⁴/ sample) were processed on a FACScan flow cytometer (Becton Dickinson), using the CELLQuest software (Becton Dickinson) to collect data. Apoptosis was evaluated by measuring the percentage of cells with hypodiploid DNA content (sub-Go/ 1 population) with the same CΕLLQuest software, while cell cycle was analyzed with the ModFit software (Verity Software House, ME, USA).

Results summarized in Table 6 showed that ST 1926 strongly and steadily arrested hAoSMC cell cycle in the early S phase upon exposure of 72 h (95.8 %) and the effect persisted up to 72h after drug removal (93.1 %). Noteworthy, the compound resulted devoid of pro-apoptotic activity. In comparison, Paclitaxel blocked cell cycle of hAoSMC in G2/M but induced some apoptosis upon 72h and after recovering.

Table 6

Cell line: hAoSMC test items: ST1926 (0.06 μM); paclitaxel (0.07 μM)

Go/i (% S (%) G₂/ M (%) Apo

0 h

Untreated 56.4 30.8 12.8 0.5

Go_/i (%) S (%) G₂/ M Apo (%)

72 h

Untreated 94.3 5.7 0.0 0.2

STl 926 0.0 95.8 4.3 1.3 paclitaxel 33.8 18.9 47.3 9.0

Go/i S (%) G₂/ M (%) Apo

72 h + 24 h recovery

Untreated 74.2 21.4 4.5 0.2

STl 926 0.0 90.2 9.8 0.3 paclitaxel 32.4 13.9 53.7 3.9

Go/i (%) S (%) G₂/ M (%) Apo (%)

72 h + 72 h recovery

Untreated 92.5 3.9 3.6 0.2

STl 926 0.0 93.1 6.9 0.5 paclitaxel 24.8 29.2 46.0 8.9

Apo= sub Go/i population. Example 6

Chemiotaxis assay [human aortic smooth muscle cells (AoSMC)] Further experiments were performed to evaluate the potential of ST 1926 to inhibit the migration of aortic smooth muscle cells with a chronic treatment. The capability of human AoSMC cells to migrate was analyzed by in vitro Boyden chamber test, using polycarbonate filters with 8 μm- pores (Nuclepore, Clifton, NJ, USA) coated with 5 mg/1 of porcine gelatin (Sigma, St Louis, MO, USA)CeIIs, after 24h- and 48h- incubation with 0.03 μM (IC50 value) of ST1926, were washed three times with PBS and counted with a hemocytometer. After drug removal, cells (5x10⁴/ well) were introduced in the Boyden upper chamber while medium containing 100ng/ml of PDGF-BB (Sigma) was used as stimuli in the lower chamber. After a four-hour incubation at 37°C, filters were fixed with methanol and stained by Diff-QuiK dye (Dade Behring, Germany, EU). Cells from the upper surface were removed by scraping, and the number of migrating cells attached to the lower surface of the filter was counted in 20 randomly selected microscopic fields (X400) per experimental condition. Data were expressed as percent of inhibition compared to migration of control cells toward the chemoattractant. As summarized in the next table, ST1926 produced a remarkable inhibition of hAoSMC migration after 24h of drug pre treatment and this inhibitory effect persisted for at least 48h (see Table 7). Table 7

ST1926 0.031 μM migration inhibition exposure (%)

24h 64

48h 54

Claims

1. Use of (2E)-3-[3'-(l-adamantyl)-4'-hydroxy[l , l '-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'-(l-adamantyl)-4'-hydroxy[l , l '-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'-(l-adamantyl)-4'- hydroxy[l , l '-biphenyl]-4-yl]-2-propenoic acid or its pharmaceutically acceptable salts.