WO2000027386A1

WO2000027386A1 - Use of propionylcarnitine for the manufacture of a medicament for inhibiting smooth muscle cell proliferation

Info

Publication number: WO2000027386A1
Application number: PCT/IT1998/000318
Authority: WO
Inventors: Menotti Calvani
Original assignee: Sigma-Tau Industrie Farmaceutiche Riunite S.P.A.
Priority date: 1998-11-11
Filing date: 1998-11-11
Publication date: 2000-05-18
Also published as: AU1258699A

Abstract

The use of propionyl L-carnitine for the preparation of a medicament having inhibiting activity of the proliferation of smooth muscular cells of vascular wall is herein disclosed. Said medicament is useful for the treatment of vascular pathologies, such as atherosclerosis, hypertension, pulmonary hypertensions, restenosis after angioplasty.

Description

USE OF PROPIONYLCARNITINE FOR THE MANUFACTURE OF A MEDICAMENT FOR INHIBITING SMOOTH MUSCLE CELL PROLIFERATION

The present invention relates to the use of propionyl L-carnitine

and the pharmaceutically acceptable salts thereof for the preparation

of medicaments useful in the treatment of blood vessel pathologies.

Background of the invention.

A number of studies demonstrated that cell proliferation plays a

pivotal role in atherosclerosis, hypertension pathogenesis and

restenosis after angioplasty or coronary stenting (Ross, 1976;

Schwartz, 1990).

Many experimental studies, carried out on human

atherosclerotic plaques, demonstrated that cell proliferation is a

determining phenomenon both in the early phases and in the

progression of the plaque.

Further, proliferation of smooth muscle cells, which migrated to

intima from vascular tunica media, represents cell basis of coronaric

restenosis after rivascularization processes through angioplasty or

dilatation by means of a stent.

This drawback is the major limit to the application of

percutaneous rivascularization in patients affected by acute coronary

syndromes, since it is responsible for about 40% of post-surgical

failures (Holmes et al., 1984). Therefore, making available substances capable of controlling

the proliferation of smooth muscular cells of vessel wall is a goal of

primary importance in the prevention of restenosis after angioplasty,

as the proliferative phenomenon occurs in a determined timed

corresponding to the first weeks following the intervention.

Proliferation control in experimental atherosclerotic lesions has

been obtained with cytostatic drugs, such as etoposide (Llera-Moya et

al., 1992), with steroid hormones (Cavallero et al.; 1971; 1973; 1975;

1976), progestinic hormones (Spagnoli et al., 1990), dexamethasone

(Asai et al., 1993).

Smooth muscle cell proliferation is also inhibited by calcium

antagonist substances due both to a decrease of DNA synthesis, such

as in case of verapamil (Stein et al., 1987) and to the interference in

second messenger systems (cAMP), as demonstrated for nifedipine

(Cheung et al., 1987).

The treatment wάth ACE-inhibitors resulted in the control of the

growth of intima thickening (Powell, 1989).

Other in-vitro studies evidenced an antiproliferative effect on

cultured smooth muscular cells of rat aorta given by simvastatine, a

HMG-CoA reductase inhibitor, used as hypolipidemic agent (Corsini et

al., 1991). Further, some substances having triglyceridemia lowering

effect, such as fibrates, showed to be able to prevent the progression of

atherosclerotic lesions in the human (Olsson et., 1990). In a manner similar to what observed in neoplasia (Kerr, 1994),

phenomena of population decrease are observed to occur together with

cell proliferation in atherosclerotic population and/ or in intima

thickening (Gabbiani, 1995), thus suggesting that highly proliferative

cells go toward apoptosis and that modulation of the latter plays an

important role in atherosclerotic lesion genesis.

Using apoptosis inducing substances bears the risk to provoke

a generalised phenomenon, with possible side effects, which can be

even very severe, such as in the case of stem cells.

It has now been found that propionyl L-carnitine, thanks to its

unexpected proapoptotic effect, is endowed with a specific action of

control on smooth muscular cells of vessels.

Abstract of the invention.

It is an object of the present invention the use of propionyl L-

carnitine and the pharmacologically acceptable salts thereof for the

preparation of a medicament having inhibiting activity on smooth

muscular cells of blood vessel walls.

The most important and surprising advantage of the present

invention is that the administration of propionyl L-carnitine does not

imply toxic effects on bone marrow and in gut, which have a good

production of blood cellular elements and a very good turnover of

intestinal mucosa cells, respectively. This and other aspects of the present invention will be illustrated in detail, also by means of

examples.

Detailed description of the invention.

The present invention is based on the application of the

discovery that propionyl L-carnitine (hereinafter PLC) induces the

phenomenon of programmed death (apoptosis) in the cells. This effect

allows the treatment of blood vessel pathologies based on the

proliferation of smooth muscular cells of vessel walls, such as

pulmonary hypertension, atheroclerosis, hypertension, restenosis after

angioplasty.

Accordingly, a first aspect of the present invention relates to the

use of propionyl L-carnitine and the pharmacologically acceptable salts

thereof for the preparation of a medicament having inhibiting activity

on the proliferation of smooth muscular cells of vessel walls.

A further object of the present invention relates to the use of

propionyl L-carnitine and the pharmacologically acceptable salts

thereof for the preparation of a medicament useful for the treatment of

atherosclerosis.

Another object of the present invention is the use of propionyl L-

carnitine and the pharmacologically acceptable salts thereof for the

preparation of a medicament useful for the treatment of hypertension.

A fourth object of the present invention is the use of propionyl

L-carnitine and the pharmacologically acceptable salts thereof for the preparation of a medicament useful for the treatment of pulmonary

hypertension.

Still another aspect of the present invention is the use of

propionyl L-carnitine and the pharmacologically acceptable salts

thereof for the preparation of a medicament useful to prevent

restenosis after angioplasty.

The medicament according to the present invention can be obtained

admixing the active ingredient (propionyl L-carnitine or a

pharmacologically acceptable salt thereof) with excipients suitable for

formulation of compositions intended for enteral administration (in

particular the oral one) or parenteral administration (in particular

through intramuscular or intravenous route). All such excipients are

well known to persons skilled in the art.

As pharmaceutically acceptable salt of propionyl L-carnitine, it

is intended any salt thereof with an acid which does not give rise to

unwanted side effects. These acids are well known to the

pharmacologists and to the experts of pharmaceutical technology.

Non-limiting examples of said salts are chloride, bromide,

orotate, acid aspartate, acid citrate, acid phosphate, fumarate and acid

fumarate, lactate, maleate and acid maleate, acid oxalate, acid

sulphate, glucose phosphate, tartrate and acid tartrate.

Some examples of formulations in the form of unitary dosages

are given. (a) Formulation for tablets

A tablet contains:

Active ingredient

propionyl L-carnitine HC1 mg 500

Excipients

Microcrystalline cellulose mg 54.0

Polyvinylpyrrolydone mg 18.0

Crospovidone mg 30.0

Magnesium Stearate mg 15.0

Fumed silica mg 3.0

Hydroxypropylmethylcellulose mg 10.0

Poliethylene glycole 6000 mg 2.5

Titanium dioxide mg 1.8

Methacrylate copolymer mg 8.3

Talcum (triventilated) mg 2.4

(b) Formulation of intravenously injectable bottles

A bottle contains:

Active ingredient

Propionyl L-carnitine HC1 mg 300

Excipient

Mannitol mg 300

A solvent vial contains:

Sodium acetate 3Η₂Q mg 390 Water for injectable F.U. q. s. to ml 5

The medicament prepared according to the present invention

will be administered in the form of pharmaceutical composition, which

can be prepared according to the general common knowledge of the

person skilled in the art.

Depending on the administration route appropriately chosen,

oral, parenteral or intravenous; the pharmaceutical composition will be

in the suitable form.

Examples of pharmaceutical compositions, wherein the

medicament according to the present invention is comprised, are the

solid or liquid oral forms, such as tablets, all types of capsules, pills,

solutions, suspensions, emulsions in the form of unitary or divided

doses, syrups, ready-to-use or extemporary drinkable unit doses.

Other examples are parenteral forms, injectable forms for

intramuscular, subcutaneous or intravenous administration.

Controlled or programmed release forms are also appropriate.

Dosages, posolgy and general therapeutic regimen will be

determined by the physician according to his knowledge, patient's

conditions and the pathology to be treated.

The association, whether co-administered in the same

medicament or separately (at the same time or subsequently) of PLC

with other active ingredients is also comprised in the present

invention. In a first preferred embodiment, the present invention relates to

restenosis after angioplasty.

According to this first preferred embodiment, the

pharmacological dose of PLC is such as not to exceed hematic

concentration of 100 mM.

The following example further illustrate the invention.

EXAMPLE

1

Wistar male rats, weighing between 270 and 290 mg, were used

for the experiments. The rats were anaesthetised with Nembutal i.p.

(35 mg/kg body weight) and the thoracic portion of aorta was

submitted to endothelium mechanical removal with Fogarty 2F balloon

probe (Baxter USA), according to the Baugartner and Studer

method(1966) with minor modification (Orlandi 1994). The animals

were randomized into 5 groups, each group is reported in Table 1.

Two groups were subjected to pharmacological treatment with

propionyl L-carnitine (PLC, 120 mg/Kg p.c. die), one group was treated

with an ACE-inhibitor (Enalapril, 1 mg/Kg p.c. die); the two remaining

groups were the control. Moreover, some non-balloonized animals were

used as blanks. Table 1

The animals were sacrificed 3 and 15 days after de-

endothelialization. Two hours before sacrifice, all the rats received i.v.

a Bromodeoxyuridine solution (BrDU) (45 mg/kg body weight) in order

to verify cell proliferation. One hour before sacrifice, some randomly

selected animals received 1 ml Blue Evans (1% in 0.9% NaCl solution)

in order to evaluate the degree of aorta disruption.

At sacrifice, the animals were anaesthetised with i.p. Nembutal

and perfused, after washing with isotonic saline containing 3%

Dextran 70, with buffered formalin for 20 minutes. Aortae were

isolated, slightly washed in saline and dissected longitudinally.

Carotid, heart and small intestine were also excised. All the organs

were post-fixed in the same fixative for 24 hours at room temperature.

Some aortic fragments were used for electronic microscopy. Aortae

were rolled up and included in paraffin. Serial sections having 5 μm thickness were stained with Hematoxylin-Eosine, Verhoeff-Van Gieson

and Movat's pentachromic and used for morphologic and

morphometric studies.

In some non-perfused animals, fragments of aortic tissue were

frozen in liquid nitrogen for the determination of tissular carnitines

and for subsequent studies of molecular biology.

Immunohystochemical staining

In order to put in evidence proliferating cells in damaged

arteries, serial sections in paraffin of aortae were deparaffined,

rehydrated, immersed in a 3% H2O2 solution for 20 minutes and

incubated with trypsine (0,05 M in Tris-HCl, pH 7.6) at 37°C. After

that, sections were treated with 2N HC1 at 37°C for 30 minutes,

washed with 0.1 M sodium tetraborate for 10 minutes, incubated with

normal horse serum (Vector) and subsequently with and-BrDU

monoclonal antibody (Ylem) for 1 hour. The preparates were then

reacted with biotilinated anti-mouse IgG (Vector) and the

Streptoavidine-ABC-POD complex (Ylem).

The reaction was evidenced by using diaminobenzidine (DAB) as

final chromogen. The count of positive nuclei for BrDU was made on

the total number of nuclei. Such count was blind-made by two

researchers separately. The difference between the two counts was

always lower than 0.5%. All data were analysed with the T Student's test. The differences

between the groups were considered to be significant for P<0.05.

Morphometric analysis

The entity of intima thickening after 15 days was evaluated on

Verhoeff-Van Gieson stained sections, using a grid overlapped on the

image, consisting of 400 points, 1 cm from each other.

The analysis was made on hystological preparates through a

Hamamatsu C3077 camera controlled by a Hamamatsu DVS 3000

image analyser and connected to a Polyvar-Reichert microscope.

Morphometric evaluation was made at XI 16 magnification. The

following parameters were evaluated a) relative volume of intima

referred to arterial wall; b) relative volume of tunica media referred to

arterial wall, by counting the overlapping points on the intima and

mean tunica.

3- 12 aorta sections were used at different level for each animal.

This number was a function of the structure sizes, according to Sach's

formula, showing the number of fields necessary to obtain a

statistically significant sample.

Ultrastructural studies

Small aorta samples were selected for electronic microscopy.

Aortae were post-fixed in osmium tetraoxide and embedded in EPON

812. Ultra thin sections were stained with uranyl acetate followed by lead citrate and examined using a Hitachi H-7100 FA transmission

electronic microscope.

Tissular and plasma carnitine assay

2-3 ml of blood samples were withdrawn from each animal

before mechanical de-endothelialization and at the time of sacrifice.

Plasma was separated by centrifugation (300 rpm) for 20 minutes and

frozen for plasmatic carnitine assay according to the Pace et al.

method.

Aorta wall samples were withdrawn from some non-perfused

animals, randomly selected from each group, frozen in liquid nitrogen

and stored at -80°C for the carnitine assay, according to the above

Pace et al. reference.

RESULTS

Ultrastructural studies

Small aorta samples were selected for electronic microscopy.

Aortae were post-fixed in osmium tetraoxide and embedded into EPOC

812. Ultra thin sections were stained with uranyl acetate followed by

lead citrate and examined through a Hitatchi H-7100 FA transmission

electron microscope.

Tissular and plasmatic carnitine assay

2-3 ml blood samples were withdrawn from each animal before

mechanical de-endothelialization and just before sacrifice. Plasma was separated by centrifugation (3000 rpm) for 20 minutes and frozen for

the plasmatic carnitine assay according to the Pace et al. method.

Samples of aortic wall were taken from some non-perfused

animals, randomly selected from each group, frozen in liquid nitrogen

and kept at -80° C for the assay of tissutal carnitines according to the

above-mentioned Pace et al. method.

RESULTS

Lesion morphology

3 days after the mechanical lesion, rat aortae did not show

significant hystological alterations, except the lack of endothelial cell

coating.

15 days after, remodelling of arteria could be observed for the presence

of an intima thickening (or neointima), consisting in round or

lengthened cells immersed in abundant extracellular matrix. Immune

hystochemical study put in evidence in particular the presence of

abundant smooth muscular cells (SMC) inside neointima.

Studies on proliferation

a) 3 days after de-endothelialization: the count of anti-BrDU

staining positive nuclei showed substantial differences between the

two groups examined. Quantitative analysis (Table 2) puts in

evidence that the number of BrDU-positive nuclei is significantly

lower in the tunica media in the PLC-treated animals, with respect

to controls (59.3% reduction against control, p<0.02). In both groups the distribution of BrDU-positive nuclei is more

concentrated in the lumen portion of mean tunica with respect to

the adventitia portion, with a 2: 1 ratio,

b) 15 days after de-endothelialization: Table 3 shows that in each

group the proliferation index of SMCs is significantly higher

(p<0.001) in the intima with respect to the tunica media. No

significant differences are observed in the number of BrDU-positive

nuclei, in the intima and tunica media, by comparing PLC,

Enalapril and control animals.

Morphometric analysis

As described in Table 3, after 15 days from endothelial lesion, the

intima relative volume is significantly lower, both in the PLC-treated

(31.11% reduction against control, p<0.02) and ACE-antagonist-

treated (26.14% reduction against control, p<0.01) animals against

control animals.

Table 2

In- vivo treatment with propionyl L-carnitine (PLC) on the proliferation

of smooth muscle cells of rat aorta after mechanical de-endothelia¬

lization: percentage of proliferating cell nuclei (anti-bromodeoxyuridine

positive) after 3 days (± s.e.m)

Table 3

In vivo treatment with propionyl L-carnitine (PLC) and with the ACE-

antagonist Enalapril on the proliferation of smooth muscular cells of

rat aorta after mechanical de-endothelialization: percentage of

proliferating cells (anti-bromodeoxyuridine positive) and percentage

ratio between intima volume and aorta wall volume after 15 days (±

s.e.m.) (preliminary results).

^(a) intima vs tunica media: p<0.0001 ; ^(b) intima vs tunica media:

p<0.0001; ^(c) intima vs tunica media: p<0.001 ; ^(d) intima vol. /wall vs

controls: p<0.02; ^(e> intima vol/wall vs controls: p< 0.01 EFFECT OF PROPIONYL L-CARNITINE IN THE CONTROL OF

PROLIFERATION/APOPTOSIS.

In vitro experiments were carried out to evaluate the effect of

propionyl L-carnitine (PLC) on smooth muscular cells (SMC) isolated

from aortae of spontaneously hypertensive rats (SHR) and, as control,

on SMC isolated from normotensive rats (WKY).

These in vitro studies evidenced that PLC, when administered

during culture exponential growth phase, reduces cell growth,

evaluated as cell number/ml, as well as DNA synthesis, evaluated

through incorporation of trititated thymidine (Tab. 4 and 5).

Table 4: cell number/ ml at culture days 2, 3, 4 and 6

Table 5: tritiated thymidine incorporation at culture day 6

As a further characterisation of smooth muscular cells in the

presence of PLC, the percentage of apoptotic cells was measured both

in basal conditions and in oxidative stress conditions. Apoptosis

evaluation was carried out by counting the number of apoptotic cells

present on a total of 1000 cells, after specific DNA staining with

Hoechst 33258. The results of this experiment demonstrated that, in

SHR cultures, PLC determines a significant increase of apoptosis

percentage in basal conditions and that this increase is more evident

under stress conditions.

In WKY cultures, apoptosis percentage is negligible (tab. 6)

Table 6: apoptotic cell percentage in basal conditions and under

oxidative stress.

The behaviour observed in SHR smooth muscular cells might be

in some way related to the deregulated expression of c-myc, which

characterises spontaneously hypertensive rats (Negoro et al., 1988).

Moreover, it was observed that c-myc actively cooperates in inducing apoptosis subsequently to a proliferation stop (Bennet et al., 1993;

Bissonette et al., 1993), accordingly the data shown above suggest that

PLC anti-proliferative effect may be related to an interference with DNA

replication.

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Claims

1. Use of propionyl L-carnitine and pharmaceutically acceptable salts

thereof for the preparation of a medicament having inhibiting

activity of the proliferation of smooth muscular cells of vascular

wall.

2. Use according to claim 1, wherein said medicament is useful for the

treatment of atherosclerosis.

3. Use according to claim 1 , wherein said medicament is useful for the

treatment of hypertension.

4. Use according to claim 1, wherein said medicament is useful for the

treatment of pulmonary hypertension.

5. Use according to claim 1, wherein said medicament is useful for the

prevention of restenosis after angioplasty.

6. Use according to anyone of claims 1 to 5, wherein said salt of

propionyl L-carnitine is selected from the group consisting of

chloride, bromide, orotate, acid aspartate, acid citrate, acid

phosphate, fumarate and acid fumarate, lactate, maleate and acid

maleate, acid oxalate, acid sulphate, glucose phosphate, tartrate

and acid tartrate.