JP2002529408A - Use of propionyl L-carnitine for the preparation of a medicament capable of inducing apoptosis - Google Patents

Abstract

  (57) [Summary] Propionyl L-carnitine and a pharmaceutically acceptable agent for the preparation of a medicament useful for the treatment of diseases in which apoptosis induction provides therapeutic benefit, in particular restenosis after vascularization, for example angioplasty or coronary stenting, or especially tumors The use of such salts.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to diseases in which a therapeutic benefit is obtained by inducing apoptosis,
For example, the use of propionyl L-carnitine and pharmaceutically acceptable salts thereof for the preparation of a medicament useful for the treatment of restenosis after angioplasty or coronary stenting, or especially tumors.

BACKGROUND OF THE INVENTION Cell Proliferation in Circulatory Disease Many studies have demonstrated that cell proliferation plays a pivotal role in atherosclerosis, hypertension, and restenosis following angioplasty or coronary stenting. (Ross, 1976; Schwartz, 1990). Numerous experimental studies performed on human atherosclerotic plaques have demonstrated that cell proliferation is a crucial phenomenon both in the early and developmental stages of plaque. In addition, the proliferation of smooth muscle cells migrating from the tunica media to the intima explains the cellular basis of coronary restenosis following a revascularization procedure by angioplasty or dilatation using a stent.

[0003] This drawback accounts for about 40% of postoperative disorders and is a major limitation to the application of percutaneous revascularization in patients suffering from acute coronary syndrome (Holmes et a.
l., 1984). Therefore, since the phenomenon of proliferation of smooth muscle cells occurs within a limited period of time corresponding to the first week after treatment, creating a useful substance that can control the proliferation of smooth muscle cells in the vascular wall involves angiogenesis. It is the primary goal of prevention of later restenosis. Growth control in experimental atherosclerotic disorders includes cytostatic drugs such as etoposide (Llera-Moya et al., 1992), steroid hormones (Cavall
ero et al .; 1971; 1973; 1975; 1976), progestin (Spagnoli et al., 1990),
This has been achieved using dexamethasone (Asai et al., 1993).

[0004] Proliferation of smooth muscle cells also reduces DNA synthesis as in the case of verapamil (Stein
et al., 1987) and interference in the second messenger system (cAMP) as demonstrated for nifedipine (Cheung et al., 1987).
It is also inhibited by calcium antagonist substances. Treatment with ACE-inhibitors results in controlled growth of intimal hyperplasia (Powell, 1989).

[0005] Other in vitro studies have demonstrated the antiproliferative effect in cultured rat aortic smooth muscle cells provided by simvastatin, an HMG-CoA reductase inhibitor, used as a hypolipidemic agent (hypolipidemic agent) ( Corsi
ni et al., 1991). In addition, it has been shown that some substances with triglyceridhaemia reducing effects, such as fibrate, can prevent the progression of atherosclerotic disorders in humans (Olsson et., 1990). With behavior similar to that observed in tumorigenesis (Kerr, 1994), it has been observed that a phenomenon of population decline occurs with cell proliferation in atherosclerotic populations and / or vascular endothelial hyperplasia ( Gabbiani, 1995), thus, highly proliferating cells go toward apoptosis, suggesting that changes in apoptosis play an important role in the development of atherosclerotic dysfunction. Apoptosis has been demonstrated in various forms of human and experimental cardiovascular disease (Sharifi AM, Schriffin EL; Am. J. Hypertens. 1998, Sep; 11 (9): 110
8-16).

Angiogenesis causes a number of reactions in the vessel wall, including cell migration, proliferation and matrix accumulation, all of which contribute to neointimal formation and restenosis (Malik N et al; Circulation 1998 Oct 20; 98 (16): 1657-65). Apoptosis induction may also be beneficial for vascular disorders such as pulmonary vascular disorders (Cowan K.
N. et al. Circ. Res. 1999 May 28; 84 (10): 1223-33). Restenosis after angioplasty is due to damaged intimal cells. For example, the use of apoptosis inducers to treat tumors risks stimulating systemic effects with the potential for side effects, which can be very severe, as in stem cells.

[0007] There is a need to find pro-apoptotic drugs without systemic effects. Proposals for preventing restenosis are described, for example, in US Pat.
5835935.

Apoptosis in Tumors It is well known that the use of anticancer drugs in the treatment of humans causes a number of potentially life-threatening toxicities or side effects for patients. These complications actually lead to lower doses of the drug, and sometimes to the treatment itself. In many cases, dose reduction or treatment cessation develops relapses with consequences that are sometimes fatal to the patient, causing worsening of the individual's systemic symptoms. The ever increasing number and importance of anticancer drugs used in human therapy, and the main limitation still being the development of toxicity or side effects, indicate that this problem is still of considerable interest. means.

[0009] That is, there is a strong desire to find new drugs or novel suitable combinations of different drugs that can substantially reduce the toxicity or side effects caused by anticancer drugs used in human therapy. One of the common problems of pharmacological treatment is the therapeutic index of the drug, that is, the ratio of the therapeutically effective dose to the toxic dose or, in any case, the dose that causes the onset of side effects. .

The medical community, in the case of anti-cancer chemotherapy, still recognizes the need for treatment management, particularly in confronting patients with treatments that are difficult to sustain, while seeking to maintain an acceptable quality of life. These considerations also apply to therapeutic treatment of animals, for example so-called pets. Naturally, the tendency to reduce the dosage and the use of pharmaceutical preparations suitable for therapeutically useful administration without forcing the patient to take the drug frequently are unique to each anticancer drug, In contrast to the minimum effective dose.

[0011] That is, substances that can work with anti-cancer agents without true cytotoxic effects, eg, induce apoptosis in tumor cells, and that interfere with tumor cells would be very beneficial. Propionyl L-carnitine is used for its unexpected proapoptosis (proapopt
otic) effect has now been found to have specific activity in the control of vascular smooth muscle cells and tumor cells.

[0012] disease therapeutic benefit by Summary apoptosis induction invention can be obtained, in particular a blood vessel, for example, angioplasty or coronary stenting restenosis or especially for the preparation of a medicament useful for the treatment of tumors, propionyl L The use of carnitine and its pharmaceutically acceptable salts is an object of the present invention.

[0013] The most important and surprising advantage of the present invention is that the administration of propionyl L-carnitine has a significant effect on bone marrow and intestine, which respectively produce good blood cell components and turn over intestinal mucosal cells very well. It has no toxic effect. This and other aspects of the invention will be described in more detail using examples.

[0014] Propionyl L-carnitine has been disclosed as inhibiting the progression of atherosclerotic dysfunction in aged hyperlipidemic rats (Spagnoli LG, Orlandi A., Ma.
rino B., Mauriello A., De Angelis C., Ramacci MT, Atherosclerosis 1995
; 114, 29-44). In this study, the authors reported that propionyl L-carnitine (PL
C (also referred to as C). This effect was actually demonstrated in rabbits, a model that is not suitable for humans, but was explained by a lipid-lowering effect. In fact, the authors state, "The number of animals is not very high, while total triglycerides, IDL- and VLDL-triglycerides [...] are very drastically reduced, while changes in plasma cholesterol levels are slight, and It is enough to show that it was temporary. "(Page 40, left column, 16-1
(9th line). The author describes macrophages and SMCs that make up plaques.
Also show low levels of proliferative activity (Id., Last 5 lines). The author then asserts that "at present, PLC cannot be assumed to have any therapeutic application" (ibid., Right column, first three lines). However, the results of this study show that fibrous tissue atherosclerotic plaque progresses with age-related myointimal thickening and / or
Alternatively, the role of β-VLDL in causing atherogenesis in alteration was confirmed (ibid., Lines 5-10). The authors suggest that plasma triglyceride levels are directly related to the proliferative effects of plaque cell populations, and that pharmacological regulation of these two factors may be associated with a marked reduction in plaque progression in aged hypercholesterolemic rabbits The hypothesis is provided. It also states that some experimental data supports the hypothesis of the relationship between triglycerides and cell proliferation (Id., 41).
Page, right column, lines 20-29). The authors state that "PLC does not appear to act by altering growth factor expression" (ibid., Last two lines) and that "PLC exerts direct control on cell proliferation in atherosclerotic plaques." Further in vitro testing is needed to answer the question of whether or not to do so "(ibid., P. 42, last four lines).

This study teaches that in aged hyperlipidemic rabbits, PLC prevents the progression of atherosclerotic dysfunction by acting as a hypolipidemic agent. That is, PLC acts indirectly as an antiatherogenic drug by lowering the lipids responsible for the cell proliferation of atherosclerotic plaques. No antiproliferative action has been demonstrated for PLC.

[0016] DETAILED DESCRIPTION OF THE INVENTION The present invention, propionyl L- carnitine (hereinafter, also referred to as a PLC for convenience)
Is based on the application of the finding that induces the phenomenon of scheduled death (apoptosis) in cells. This effect allows the treatment of blood vascular diseases caused by the proliferation of smooth muscle cells in the vascular wall, such as pulmonary hypertension, hypertension, angioplasty or restenosis after coronary stenting. Advantageously, PLC is a well-known drug with very limited side effects. Examples of the use of propionyl L-carnitine are described in US Pat. No. 4,415,589, US Pat.
, IT1155772, EP0793962, EP0811376, WO99 /
17623, PCT / IT97 / 00113.

Accordingly, the first aspect of the present invention is useful in the treatment of diseases in which a therapeutic benefit is obtained by inducing apoptosis, in particular restenosis after blood vessels, such as angioplasty or coronary stenting, or especially tumors. It relates to the use of propionyl L-carnitine and a pharmaceutically acceptable salt thereof for the preparation of a medicament.

[0018] Another object of the present invention is the use of propionyl L-carnitine and a pharmaceutically acceptable salt thereof for the preparation of a medicament useful for treating hypertension. Another object of the present invention is the use of propionyl L-carnitine and a pharmaceutically acceptable salt thereof for the preparation of a medicament useful for treating pulmonary hypertension. Yet another embodiment of the present invention is the use of propionyl L-carnitine and a pharmaceutically acceptable salt thereof for the preparation of a medicament useful for preventing restenosis after angioplasty or coronary stenting. .

A further object of the present invention described herein is the coordinated use of propionyl L-carnitine, which has an auxiliary effect with anticancer agents. As an adjunct effect, the combination treatment of an antitumor drug and propionyl L-carnitine is intended, with which PLC exerts an apoptotic effect on tumor cells, thus helping the cytotoxic effect of the antitumor drug. In this way, an improvement in the therapeutic index of the antitumor drug is expected.

[0020] A further object of the invention described herein is the use of propionyl L-carnitine in the preparation of a medicament useful for treating tumors. Yet another object of the invention described herein is the combination of propionyl L-carnitine with an anti-cancer agent and related pharmaceutical composition. In the context of the present invention described herein, "cooperative use" of said compound is defined as (i) co-administration, i.e., propionyl L-carnitine or one of its pharmacologically acceptable salts and the substance of an anticancer agent. Both simultaneous or sequential administration above or (ii) administration of a composition comprising the active ingredient in combination and admixture in addition to any pharmaceutically acceptable excipients and / or vehicles means.

[0021] The invention disclosed herein thus provides a delayed release formulation capable of co-administration of propionyl L-carnitine or one of its pharmaceutically acceptable salts and an anti-cancer agent, and oral, parenteral or nasal administration. And both pharmaceutical compositions comprising the two active ingredients in admixture.

As will be apparent from the following detailed description of the invention, it is also possible to envisage the coordinated use of anti-cancer agents such as, for example, taxol, bleomycin, carboplatin, vincristine, camptothecin. In all these embodiments, propionyl L-carnitine can be used for synergistic use. Co-administration is also a package or product having propionyl L-carnitine or one of its pharmacologically acceptable salts and an anti-cancer agent as separate dosage forms, wherein the administration regimen established by the attending physician based on the patient's circumstances. Means a package or product with instructions for the coordinated simultaneous or regular consumption of the active ingredients according to

Embodiments of the invention disclosed herein stop treatment due to contraindications because the ability to maintain a routine treatment protocol or to increase the dose of chemotherapeutic agents increases therapeutic outcomes Healing the patient without having to do so and prolonging his life. Due to the auxiliary effect of propionyl L-carnitine, it is also possible to anticipate a reduction in the dose of the anticancer drug. The medicament according to the invention contemplates the active ingredient (propionyl L-carnitine or a pharmacologically acceptable salt thereof) enterally (especially orally) or parenterally (especially by intramuscular or intravenous route). It can be obtained by mixing with excipients suitable for the formulation of the composition. All such excipients are well known to those skilled in the art.

As a pharmaceutically acceptable salt of propionyl L-carnitine, any salt thereof with an acid that does not cause undesirable side effects is intended. These acids are well known to pharmacologists and pharmaceutical technology experts. Non-limiting examples of such salts include chloride, bromide, orotate, acid aspartate, acid citrate, acid phosphate, fumarate and acid fumarate, lactate, maleate and acid maleate, acid oxalate, acid sulfate, glucose phosphate, tartrate and acid tartrate. Rate.

Some examples of formulations in unit dosage form are given. (a) Formulation for tablets Tablets are: active ingredient propionyl L-carnitine HCl 500 mg excipient microcrystalline cellulose 54.0 mg polyvinylpyrrolidone 18.0 mg crospovidone 30.0 mg magnesium stearate 15.0 mg fumed silicda 3.0 mg hydroxypropyl methylcellulose 10.0 mg polyethylene glycol 6000 2.5 mg titanium dioxide 1.8 mg methacrylic acid copolymer 8.3 mg (triventilated) 2.4 mg talcum.

(B) Formulation of intravenous injectable bottle The bottle contains: active ingredient propionyl L-carnitine HCl 300 mg excipient mannitol 300 mg; solvent vial; sodium acetate 3.H ₂ O 390 mg U. of sufficient water for up to 5 ml.

The medicament prepared according to the present invention is administered in the form of a pharmaceutical composition which can be prepared according to the common general knowledge of the person skilled in the art. Depending on the route of administration chosen, oral, parenteral or intravenous, the pharmaceutical composition will take an appropriate form. Examples of pharmaceutical compositions which include the agents according to the invention include tablets, capsules of all types, pills, solutions, suspensions, emulsions in unit or divided dose, syrups,
Solid or liquid oral preparations, such as ready-to-use or ready-to-drink unit dosage forms. Other examples are parenteral formulations, injectable formulations for intramuscular, subcutaneous or intravenous administration. Controlled or planned release formulations are also suitable.

The dosage, dosage and general treatment regimen will be determined by the attending physician, depending on the knowledge of the attending physician, the condition of the patient and the condition to be treated. Combinations of the PLC with other active ingredients, whether co-administered with the same drug or separately (simultaneously or sequentially), are also included in the invention. In a first preferred embodiment, the invention relates to restenosis after angioplasty. According to this first preferred embodiment, the pharmacological dose of the PLC is
It does not exceed 0 mM.

The following examples further illustrate the present invention. Example 1 Male Wistar rats weighing 270-290 mg were used for the experiments. Rats were anesthetized by intravenous (ip) administration of Nembutal (35 mg / kg body weight),
The endothelium of the throat portion of the aorta was mechanically modified using a Fogarty 2F balloon probe (Baxter USA) according to the slightly modified (Orlandi 1994) Baugartner and Studer method (1996). Removed. The animals were randomly divided into five groups, each group being shown in Table 1. Two groups were subjected to pharmacological treatment with propionyl L-carnitine (PLC, 120 mg / kg pc. Dead) and one group was using an ACE-inhibitor (Enalapril, 1 mg / Kgp. Dead). The remaining two groups were treated as controls. In addition, some non-ballooned animals were used as blanks.

[0030]

[Table 1] Table 1

The animals were sacrificed 3 and 15 days after the endothelial removal procedure. Two hours before sacrifice, bromodeoxyuridine solution (BrDU) (45 mg) was added to all rats to confirm cell proliferation.
/ Kg body weight) was administered intravenously. One hour before sacrifice, 1 ml of Blue Evans (0.9%) was added to randomly selected animals to assess the extent of aortic destruction.
(1% in NaCl solution) was administered.

At the time of sacrifice, the animals were washed with isotonic saline containing 3% Dextran 70 and buffered formalin for 20 minutes before nembutal was injected intraperitoneally (i.
p. ) Was used to anesthetize and perfuse. The aorta was isolated, washed slightly in saline and cut longitudinally. The carotid artery, heart and small intestine were further removed. All organs were post-fixed in the same formalin solution for 24 hours at room temperature. Some aortic fragments were observed under an electron microscope. Roll up the aorta,
Wrapped in paraffin. Serial sections of 5 μm thickness were prepared using Hematoxylin-Eosin.
Lin-Eosine), Verhoeff-Van Gieson and Movat's pentachromic were used for morphological and morphometric studies. Some non-perfused aortic tissue fragments were frozen in liquid nitrogen for measurement of tissue carnitine and subsequent molecular biological testing.

Immunohistochemical staining To demonstrate proliferating cells in the injured aorta, serial sections in paraffin of the aorta were deparaffinized, hydrated, immersed in a 3% H ₂ O ₂ solution for 20 minutes, and treated with trypsin ( Incubation at 37 ° C. with 0.05 M in Tris-HCl, pH 7.6). The sections were then treated with 2N HCl at 37 ° C. for 30 minutes, washed with 0.1 M sodium tetraborate for 10 minutes, and with standard horse serum (vector),
And subsequently incubated with BrDU monoclonal antibody (Jerm) for 1 hour. The preparation was then reacted with a biotilinated anti-mouse IgG (vector) and a streptavidin-ABC-POD complex (Jerm).

The reaction was demonstrated by using diaminobenzidine (DAB) as the final chromogen. Positive nuclei of BrDU were counted against the total number of nuclei. Such counting was blind-made separately by two researchers. The difference between the two counts was always below 0.5%. All data was analyzed using the T Student test. The difference between groups is p <
It was considered significant for what was 0.05.

Morphometric analysis The presence of intimal hyperplasia 15 days later was evaluated on Verhoeff-Van Gieson stained sections using a grid of 400 points, 1 cm from each section, which was overlapped with an image consisting of 1 cm. did. Histological specimens were analyzed with a Hamamatsu C3077 camera controlled by a Hamamatsu DVS 3000 image analyzer and connected to a Polyvar-Reichert microscope. Morphological evaluation is × 1
Performed at 16 magnifications. The following parameters were evaluated by counting the overlap point for the intima and media. a) relative volume of the intima to the arterial wall; b) relative volume of the media to the aortic wall.

[0036] 3-12 aortic sections were used for each animal at different levels. This number is
That expression (S) indicates the number of fields required to obtain a statistically significant sample.
According to ach's formula), it is determined by the tissue size.

Ultrastructural Study A small aortic sample was selected for electron microscopy. The aorta was double-fixed in osmium tetraoxide and embedded in EPON812. Ultrathin sections were stained with uranyl acetate, then with lead citrate, and evaluated using a Hitachi H-7100FA transmission electron microscope.

Tissue and Plasma Carnitine Assay A 2-3 ml blood sample was collected from each animal before mechanical endothelial removal and at sacrifice. Plasma was separated by centrifugation (300 rpm) for 20 minutes and frozen according to the method of Pace et al. For plasma carnitine assay. Aortic wall samples were collected from some non-perfused material randomly selected from each group, frozen in liquid nitrogen according to the method of Pace et al. Cited above and stored at -80 ° C for the carnitine assay.

Results Ultrastructural Study A small aortic sample was selected for electron microscopy. The aorta was double-fixed in osmium tetraoxide and embedded in EPOC812. Ultrathin sections were stained with uranyl acetate, then with lead citrate, and evaluated by Hitachi H-7100FA transmission electron microscope.

Tissue and Plasma Carnitine Assay A 2-3 ml blood sample was collected from each animal prior to the mechanical endothelial removal procedure and immediately prior to sacrifice. Plasma was separated by centrifugation (3000 rpm) for 20 minutes and frozen according to the method of Pace et al. For plasma carnitine assay. Aortic wall samples were collected from some non-perfused material randomly selected from each group, frozen in liquid nitrogen according to the method of Pace et al., And stored at -80 ° C for the assay of tissue carnitine.

Results Impaired Tissue Study Three days after mechanical injury, rat aorta showed no significant histological changes except for the absence of endothelial cell coating. After 15 days, remodeling of the aorta could be seen due to the presence of vascular endothelial hyperplasia (or new intima) consisting of round or long cells soaked in sufficient extracellular matrix. Immunohistochemical studies have shown, inter alia, that abundant smooth muscle cells (S
MC) was revealed.

Studies on proliferation a) 3 days after endothelial depletion treatment: Counting of anti-BrDU-stained positive nuclei showed a substantial difference between the two groups tested. Quantitative analysis (Table 2) revealed that the number of BrDU-positive nuclei was significantly lower in the media in PLC-treated animals relative to controls (59.3% reduction relative to controls). , P <0.02). In both groups, the distribution of BrDU-positive nuclei is more concentrated in the luminal part of the media at a 2: 1 ratio relative to the adventitia. b) 15 days after endothelial removal treatment: Table 3 shows that in each group the proliferation index of SMC is significantly higher in the intima than in the media (p <0.001). Comparison of PLC, enalapril and control animals, BrD in intima and media
No significant difference is found in the number of U-positive nuclei.

Morphometric analysis As described in Table 3, 15 days after endothelial injury, relative vascular endothelial volume was higher for PLC-treated animals than control animals (31.11% relative to controls). Decreased, p <0.02) and significantly lower in both ACE antagonist treated animals (26.14% decrease relative to control, p <0.01).

[0044]

TABLE 2 Mechanical endothelial removed propionyl L- carnitine on the growth of rat aortic smooth muscle cells after treatment (PLC) in vivo treatment with: 3 ratio days after proliferating cell nuclear (anti-bromodeoxyuridine positive) ( ± sem)

[0045]

[Table 3] Table 3 mechanical endothelial removal propionyl L- carnitine on the proliferation of rat aortic smooth muscle cells after treatment (PLC) and ACE- antagonist enalapril in vivo treatment with: vascular endothelial volume and the aorta wall after 15 days Percentage ratio between volumes (±
s. e. m) (Preliminary result) (a) Intima vs. media: p <0.0001; (b) Intima vs. media: p <0.000
1; (c) intimal vs. media: p <0.001; (d) intimal volume / wall vs. control: p <0.02; (e) intimal volume / wall vs. control : P <0.01

Effect of propionyl L-carnitine on proliferation / apoptosis control Smooth muscle cells (SMC) isolated from the aorta of spontaneously hypertensive rats (SHR) and from normotensive rats (WKY) as controls An in vitro test was performed to evaluate the effect of propionyl L-carnitine (PLC) on SMC. According to these in vitro tests, when administered during the exponential growth phase of the culture,
PLC was found to reduce cell growth as assessed by cell number / ml as well as DNA synthesis and by tritiated thymidine binding (Tables 4 and 5).

[0047]

Table 4: Cell number / ml on days 2, 3, 4 and 6 of culture

[0048]

Table 5: Binding of tritiated thymidine on day 6 of culture

As a further feature of smooth muscle cells in the presence of PLC, the percentage of apoptotic cells was measured under both basal and oxidative stress conditions. After specific DNA staining using Hoechst33258, apoptosis was evaluated by counting the number of apoptotic cells present in all 1000 cells. According to the result of this experiment, SH
In R cultures, PLC determined a significant increase in the rate of apoptosis in basal conditions, demonstrating that this increase was more pronounced under stress conditions. In WKY cultures, the rate of apoptosis is negligible (Table 6).

[0050]

Table 6: Proportion of apoptotic cells under basic conditions and under oxidative stress

The response observed for SHR smooth muscle cells could be somewhat related to the irregular expression of c-myc characteristic of spontaneously hypertensive rats (Negoro et al.
al., 1988). Furthermore, c-myc was found to be actively involved in inducing apoptosis following growth arrest (Bennet et al., 1993; Bissonette e).
t al., 1933), thus the data presented above suggest that PLC antiproliferative effects may be related to the inhibition of DNA replication.

Example 2 Cell Line Human neoplastic cells obtained from Istituto Zooprofilattico of Brescia were cultured. Cells used in the experiments were U266, multiple myeloma, healer, cervical tumor, K562, chronic myeloid leukemia cells. Healer and K562 RPM
Cultured in I + 10% FCS, while cells of line U266 were RPMI + 15% F
Cultured in CS, both media contain penicillin / streptomycin (50 U / ml and 50 μg / mL). Cells were placed in 6-well plates (Falcon). Analysis was performed on cells at 50% confluence. Each cell line was treated with PLC according to the following scheme. a) 1 mM PLC for 24 hours (FIG. 1-3); 1 mM PLC for 48 hours (FIG. 1-3); b) 1 mM PLC for 24 hours, followed by 24 hours in a molecule-free medium (FIG. FIG.
-6).

At the end of each treatment, cells were counted in a Burker chamber in the presence of 0.5% trypan blue diluted 1: 2. Counts were performed for each experimental group in samples from three walls. Treatment of neoplastic cell lines of human origin with 1 mM propionyl L-carnitine demonstrates the ability to inhibit growth after 24 and 48 hours. For more information,
Inhibition was 25% and 17% at 24 and 48 hours relative to control for healers, 46% and 26% at 24 and 48 hours relative to control for U266, and K562 relative to control, respectively. 37 and 39% after 24 and 48 hours.

The PLC inhibitory effect persists even after the substance has been removed from the culture medium. In fact, in this case, the cells were treated with 1 nM PLC for 24 hours, then the medium with PLC was removed and fresh medium without substance was added. Inhibition values were 20%, 26% and 21% for Healer, U266 and K562, respectively.

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──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 105 A61P 43/00 105 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, E, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Claudio Pisano Italy, I-04011 Aprilia, Via San Michele No. 30 F-term ( Reference) 4C084 AA17 MA02 NA14 ZA362 ZA422 ZB211 ZB262 4C206 AA01 AA02 FA47 MA01 MA02 MA04 NA14 ZA36 ZA42 ZB21 ZB26

Claims (9)

[Claims] 1. Use of propionyl L-carnitine or a pharmaceutically acceptable salt thereof for the preparation of a medicament useful in the treatment of a disease in which a therapeutic benefit is obtained by inducing apoptosis. 2. Use according to claim 1, wherein the medicament is useful for treating hypertension. 3. The use according to claim 1, wherein the medicament is useful for treating pulmonary hypertension. 4. Use according to claim 1, wherein the medicament is useful for preventing restenosis after angioplasty or coronary stenting. 5. Use according to claim 1, wherein the medicament is useful for treating a tumor. 6. The salt of propionyl L-carnitine is chloride, bromide, orotate, acid aspartate, acid citrate, acid phosphate, fumarate and acid fumarate, lactate, maleate and acid maleate, acid oxalate, acid sulfate, glucose phosphate, The use according to any one of claims 1 to 5, wherein the use is selected from the group consisting of tartrate and acid tartrate. 7. A combination comprising an anticancer agent and propionyl L-carnitine. 8. Use of a combination according to claim 7 for the preparation of a medicament having anticancer activity, characterized in that the medicament comprises an effective amount of propionyl L-carnitine which has an auxiliary effect on anticancer activity. . 9. A package or product comprising propionyl L-carnitine or one of its pharmacologically acceptable salts and an anti-cancer agent as a separate dosage form, wherein said active ingredient is used for co-administration or regular ingestion of said active ingredient. Package or product with instructions.