Field of the invention

The invention refers to the use of an amidoxime derivative of formula I

Ar— A— C— N— Ri

II I

N R ₂

I

OR ₃

wherein

a) Ar represents a pyridyl group,

A is a valence bond,

Ri stands for a hydrogen atom,

R ₂ represents a hydrogen atom,

R ₃ stands for a group of formula

wherein R ₄ represents a hydrogen atom, R ₅ is a 5-7-membered saturated heterocyclic group containing a nitrogen atom, or b) Ar represents a phenyl group,

A stands for a -CH=CH- group and

bi) R ₁ is a hydrogen atom,

R ₂ represents a group of formula

OR ₄

I

— CH2— CH— CH2— R5 wherein R ₄ represents a hydrogen atom, R ₅ stands for a 5-7- membered saturated heterocyclic group containing a nitrogen atom,

R ₃ is hydrogen atom, or

b ₂ ) R ₂ represents a group of formula

OR ₄ wherein R ₄ represents a hydrogen atom, R ₅ stands for a 5-7- membered saturated heterocyclic group containing a nitrogen atom,

Ri represents a carbonyl group, R ₃ is a valence bond between the carbon atom of the carbonyl group and the oxygen atom adjacent to R ₃ ,

or a pharmaceutically suitable acid addition salt thereof for the preparation of a composition or pharmaceutical composition that ameliorates the tissue regeneration effect of adult stem cells and/or facilitates the survival and adherence of adult stem cells and/or promotes the regulation of adult stem cell differentiation.

Background of the invention

Stem cells are biological cells found in the multicellular organisms. They can divide through mitosis and differentiate into diverse specialized cell types. In mammals, there are two broad types of stem cells: embryonic stem cells and adult stem cells. In a developing embryo, stem cells can differentiate into all the specialized cells, while in adult tissues stem cells and progenitor cells act as a repair system for the body: they refresh the specialized cells and contribute to the normal renewal of the continuously renewed organs (such as blood, skin, tissues of digesting system etc.).

Recently, biotechnology is able to transform stem cells into various specialized cells by growing the stem cells in a cell culture.

Various types of stem cells can be distinguished:

• The totipotent stem cell is essentially a fertilized ovum that is able to produce all tissues and organs.

• The pluripotent stem cell is not able to produce an extraembryonic tissue, however, it is suitable to form all the three germ layers and gametes. Such a pluripotent stem cell is the embryonic stem cell.

• The multipotent stem cell is not able to produce gametes, however, they can differentiate into any other cell types. The tissue stem cells of the organism are miltipotent stem cells.

• The unipotent stem cell can produce only one cell type, its own, but has the property of self-renewal which distinguishes it from non-stem cells.

[Hans R. Scholer (2007): „The Potential of Stem Cells: An inventory". In Nikolaus Knoepffler, Dagmar Schipanski, and Stefan Lorenz Sorgner: Humanbiotechnology as Social Challange. Ashgate Publishing Ltd. P. 28. ISBN 0754657558.]

Tissue stem cells can be found in numerous tissues of the organism. The well known and therapeutically employed stem cell sources include bone marrow, peripheral blood and umbilical cord blood. The bone marrow contains mainly blood-forming (hematopoietic) stem cells which give rise to the three classes of blood cells that are found in the circulation: red blood cells (erythrocytes), white blood cells (leukocytes) and platelets (thrombocytes). From the bone marrow pretreated with a high dosage of colony-stimulating factor (CSF), a high amount of stem cell and progenitor cell get into the peripherial blood from which stem cells suitable for transplantation can be obtained.

An intensive research is directed to the utilization of adult stem cells in medical therapy by using the ability of stem cells according to which they can differentiate into various tissues. Based on this property, stem cells will be applied in many sorts of diseases. Scientific publications appear continuously on the promising possibilities of stem cell applications in the future. In case of diseases that cannot be treated presently or that can be treated only with a limited efficacy such as Alzheimer's disease, Parkinson's disease, stroke, some kinds of muscle atrophy etc. researchers expect the progress from stem cell therapy.

A further promising field of biotechnology includes the preparation of tissues and organs from stem cells. Thereby tissues could be prepared that would be suitable for producing cardiac valves, joints, intervertebral disks for transplantation purposes to take over the function of the ill tissues and organs.

Stem cells may have a role also in gene therapy: the ^meliorated" stem cell obtained by the correction or change of a mutated gene in the stem cell can be implanted to cure the disease. It is expected to effect progress in case of especially the following diseases: stroke, traumatic cerebral injury, learning defects, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), deafness, blindness, baldness, wound-healing, prosthetic dentistry, myocardial infarction, vasoconstriction, bone marrow transplantation, muscle dystrophy, spinal injury, diabetes, tumours, osteoarthritis, rheumatoid arthritis, Crohn's disease.

Most adult stem cells are multipotent and their designation refers, in general, to the tissue origin thereof e.g. mesenchymal stem cell, adipose derived stem cell, endothelial stem cell, dental pulp stem cell etc. [Barilleaux B., Phinney D.G., Prockop D.J. , O'Connor K.C. (2006): „Review ex vivo engineering of living tissues with adult stem cells", Tissue Eng. 12 (1 1), 3007-19. doi:10.1089/ten.2006.12.3007]. Adult stem cell treatments have been succesfully employed for many years to treat leukemia and bone tumours by bone marrow transplantation. Unlike the use of embryonic stem cells, the use of adult stem cells in research and therapy does not include ethical problems since the production of adult stem cells does not require the destruction of an embryo.

Since the adult stem cells are able to differentiate into numerous cell types, they are potentially suitable to promote the regeneration of several types of tissue. Thus, stem cell therapy offers extraordinary possibilities in many degenerative diseases accompanied by tissue destruction through the reformation of the destructed tissues. However, the efficacy of stem cell transplantation is reduced by the fact that out of the stem cells introduced into the damaged tissue environment only a low ratio thereof is survivable. When, for example, adult mesenchymal stem cells obtained from human bone marrow were introduced into mouse cardiac muscle, after 4 days only 0.44 % of the stem cells were living [Toma, C. et al.: Human Mesenchymal Stem Cells Differentiate to a Cardiomyocyte Phenotype in the Adult Murine Heart. Circulation (2002); 105; 93-98].

Therefore, the aim of the invention is to enhance the survival of the adult stem cells introduced into the damaged tissue environment.

The amidoxime derivatives of formula I are known compounds. Amidoxime derivatives of formula I, wherein R-i , R ₂ and R ₃ are as defined in section a) above, can be prepared using the process described in US Patent No. 4, 187,220. The other amidoxime derivatives of formula I can be prepared by the processes described in the PCT application published as WO 01/70674.

In general, the amidoxime derivatives of formula I inhibit the PARP enzyme. Several pharmacological effects of one of them, BGP-15, the dihydrochloride of which can be characterized by formula II, are known.

The use of BGP-15 for the treatment of diabetic angiopathy is known from US Patent No. 4, 187,220 mentioned above.

US Patent No. 6,306,878 refers to a method for the protection of the mitochondrial genome and/or mitochondrion from damage leading to myopathies and neurodegenerative diseases which comprises administering an effective non-toxic dose to a patient susceptible to such a damage of an amidoxime acid derivative including BGP-15.

US Patent No. 6,458,371 refers to a composition comprising 0.1-30 % of a hydroximic acid derivative such as BGP-15 and a carrier. The composition is suitable for reducing the incidence of photodamage by radiation with UV-B.

US Patent No. 6,884,424 refers to a method for preventing actinic keratosis by applying a hydroximic acid derivative e.g. BGP-15 to the affected skin surface.

US Patent No. 6,451 ,851 refers to a method of treating a patient suffering from a viral infection comprising administering to the patient a pharmaceutically effective amount of a known antivirally active agent together with a hydroximic acid derivative e.g. BGP-15.

US Patent No. 6,440,998 refers to a pharmaceutical composition having antitumour activity with reduced side effect comprising cisplatin or carboplatin and a hydroximic acid derivative such as BGP-15. US Patent No. 6,656,955 refers to a pharmaceutical composition having antitumour activity with reduced side effect comprising paclitaxel or docetaxel and a hydroximic acid derivative such as BGP-15. US Patent No. 6,720,337 refers to a pharmaceutical composition having antitumour activity with reduced side effect comprising oxaliplatin and a hydroximic acid derivative such as BGP-15. US Patent No. 6,838,469 refers to a pharmaceutical composition having antitumour activity with reduced side effect comprising pyrimidine derivatives and BGP-15. PCT Application published under No. WO 00/07580 disclosed experimental data for the antidiabetic effect of BGP- 15 in the treatment of type 1 diabetes mellitus.

PCT Application published under No. WO 03/007951 refers to a pharmaceutical combination of hydroximic acid derivatives such as BGP-15 and an antidiabetic or anti- hyperlipidemic active agent for the prevention or treatment of a prediabetic state, metabolic X-syndrome or diabetes mellitus as well as disorders wich are associated with the states listed above, namely endogenic metabolic disorders, insulin resistance, dislipidemia, alopecia, diffuse effluvium and/or female endocrine disorders based on androgenic preponderance.

PCT Application published under No. WO 2005/122678 refers to the use of BGP-15 in a pharmaceutical composition having prokinetic effect (i.e. inducing activity in the stomach and intestines).

PCT Application published under No. WO 2005/123049 refers to the use of BGP-15 for mitochondrial genesis i.e. to increase the number of mitochondria in the cells resulting in roborating effect.

PCT Application published under No. WO 2006/079910 refers to the use of BGP-15 for the treatment of lesions in the oral cavity, especially periodontal disease.

According to European Patent No. 2 089 031 BGP-15 can be employed for reducing overweight or obesity.

According to European Patent No. 2 089 032 BGP-15 reduces the side effect of known antipsychotics, antidepressants and antiepileptics leading to overweight or obesity.

According to PCT Application published under No. WO 2009/074835 BGP-15 can be used for reducing the unfavourable psychiatric side effect of cannabinoid CBi antagonists such as rimonabant.

Summary of the invention

It was found that the above aim was achieved by treating the adult stem cells with an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof during the growing of the stem cells and/or prior to the growing thereof and/or after the growing thereof and/or by introducing the stem cells and an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof simultaneously or one after the other into the damaged tissue environment needing stem cell treatment and/or by introducing the stem cells into a tissue environment treated systemically or locally with an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof.

Thus, according to the invention an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof is used for the preparation of a composition or pharmaceutical composition that ameliorates the tissue regeneration effect of adult stem cells and/or facilitates the survival and adherence of adult stem cells and/or promotes the regulation of adult stem cell differentiation.

Thus, the invention provides the use of an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof for the preparation of a composition or pharmaceutical composition that ameliorates the tissue regeneration effect of adult stem cells and/or facilitates the survival and adherence of adult stem cells and/or promotes the regulation of adult stem cell differentiation.

Furthermore, the invention provides a method to enhance the tissue regeneration effect of adult stem cells which comprises contacting the stem cells with an effective amount of an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof before the growing of the stem cells and/or during the growing of the stem cells and/or after the growing of the stem cells.

The invention provides also a method to facilitate the survival and adherence of adult stem cells which comprises introducing the stem cells and an effective amount of an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof, simultaneously or one after the other, into the damaged tissue environment needing stem cell treatment.

The invention provides also a method to promote the regulation of adult stem cell differentiation which comprises introducing the stem cells into a tissue environment treated systemically or locally with an effective amount of an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof.

The invention provides also any combinations of the preceding methods.

Description of preferred embodiments As the adult stem cell any adult stem cells suitable for therapeutic purposes such as mezenchymal stem cells, adipose derived stem cells, endothelial stem cells, dental pulp stem cells etc. can be used.

By using the composition of the invention and/or the pharmaceutical composition of the invention combined with stem cell therapy or by using the above one or more methods of the invention a definite progress can be expected especially in the treatment of diseases listed above as well as in genetic therapy and in the production of tissues or organs by biotechnology.

In the definition of R ₅ , under a 5-7-membered saturated heterocyclic group containing a nitrogen atom a pyrrolidyl group, piperidyl group or hexamethyleneimino group is meant.

A pharmaceutically suitable acid addition salt is an acid addition salt formed with a pharmaceutically acceptable inorganic or organic acid such as a hydrochloride, acetate, fumarate, maleate etc.

A preferred subgroup of the amidoxime derivatives of formula I consists of the compounds, wherein in the formula Ri, R ₂ and R3 are as defined in section a) above. An especially preferred compound in this subgroup is 0-(3-piperidino-2- hydroxy-1-propyl)nicotinic amidoxime (abbreviated as BGP- 5) which is suitably used in the form of the dihydrochloride thereof of formula II II

2 HCI

Another preferred subgroup of the amidoxime derivatives of formula I consists of the compounds, wherein in the formula Ri, R ₂ and R ₃ are as defined in sections b) and bi) above. An especially preferred compound in the subgroup is N-[3- (hexamethyleneimino)-2-hydroxypropyl]cinnamic amidoxime (abbreviated as NG-094) of formula III

III

Suitably, the dihydrogen maleate of NG-094 is used.

A further preferred subgroup of the amidoxime derivatives of formula I consists of the compounds, wherein in the formula R-i, R ₂ and R ₃ are as defined in sections b) and b ₂ ) above, thus, the compounds contain an oxadiazolin ring. An especially preferred compound in this subgroup is 3-styryl-4-[3- (hexamethylene-imino)-2-hydroxypropyl]-A ² -1 ,2,4-oxadiazolin- 5-one (abbreviated as NG-50) of formula IV

Suitably, the hydrochloride of NG-50 is used.

According to the invention the adult stem cells are treated with an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof. The adult stem cells can be treated with the amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof used as the active ingredient, however, suitably a solution, suspension or emulsion thereof is employed as a composition that contains the active ingredient. As the medium of the composition preferably water, physiological saline or liquid nutrient media suitable for the increase of stem cells are used.

Alternatively or additionally, the damaged tissue environment needing the stem cell treatment can be treated with an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof used as an active ingredient in itself or in a pharmaceutical composition.

The adult stem cells can be treated with an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof prior to the start of their growing and/or during their growing and/or after their growing. For example, also an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof is added to the liquid nutrient medium used for growing the stem cell, then the stem cells are grown in a manner known in itself. Finally, the grown stem cells are separated from the culture and introduced into the tissue environment that needs the stem cell treatment. Of course, the amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof or the composition containing the same can be added to the culture of the adult stem cell in a later period of the increase, too.

In this connection, under the expression ..composition" a solid or liquid mixture is meant which contains, in addition to an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof, also one or more carrier(s) that is/are not toxic to the cell. A liquid composition is preferred. As a matter of fact, the liquid composition is a solution, suspension or emulsion of an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof which can be suitably added to the adult stem cell or a culture thereof. The composition is prepared by admixing the components thereof.

When the untreated adult stem cells or the adult stem cells treated with an amidoxime derivative of formula I are introduced into the damaged tissue environment needing the stem cell treatment, also a pharmaceutical composition containing an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof as the active ingredient can be introduced into the tissue environment needing the stem cell treatment. Both treatments can be carried out simultaneously or one after the other. Alternatively, the individual having a damaged tissue environment that needs the stem cell treatment can be systemically treated with a pharmaceutical composition containing an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof as the active ingredient, wherein the treatment is carried out once or several times, then the treatment with the stem cell is performed.

As a further alternative, the damaged tissue environment needing the stem cell treatment is treated, locally, with a pharmaceutical composition containing an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof as the active ingredient, wherein the treatment is carried out once or several times, then the treatment with the stem cells is performed. Even after this procedure a systemic and/or local treatment with a pharmaceutical composition containing an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof as the active ingredient can be carried out, too.

Of course, any combinations of the above procedures can be employed. For example, the stem cells are treated with an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof prior to the start of their growing and/or during their growing and/or after their growing, then they are introduced into the tissue environment that needs the stem cell treatment in an individual that was pretreated once or several times, even during weeks, systemically and/or locally, with a pharmaceutical composition containing an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof as the active ingredient. When the stem cells have been introduced into the damaged tissue environment, the individual can be further treated, systemically, and/or the damaged tissue environment can be further treated, locally, with a pharmaceutical composition containing an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof as the active ingredient.

Under a ..pharmaceutical composition" any composition for human or veterinary use is meant, wherein the composition comprises, in addition to the active ingredient i.e. an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof, one or more carrier(s) conventionally employed in such compositions. The pharmaceutical composition may include any dosage form suitable for peroral, parenteral or rectal administration or for local treatment, and can be solid or liquid.

The solid pharmaceutical compositions suitable for peroral administration may be powders, capsules, tablets, film- coated tablets, microcapsules etc., and can comprise binding agents such as gelatine, sorbitol, poly(vinylpyrrolidone) etc.; filling agents such as lactose, glucose, starch, calcium phosphate etc.; auxiliary substances for tabletting such as magnesium stearate, talc, poly(ethylene glycol), silica etc.; wetting agents such as sodium laurylsulfate etc. as the carrier. Capsules may contain the pure active agent without any carrier, other dosage forms contain, in addition to the active agent, one or more carrier(s). The liquid pharmaceutical compositions suitable for peroral administration may be solutions, suspensions or emulsions and can comprise e.g. suspending agents such as gelatine, carboxymethylcellulose etc.; emulsifiers such as sorbitane monooleate etc.; solvents such as water, oils, glycerol, propylene glycol, ethanol etc.; preservatives such as methyl p-hydroxybenzoate etc. as the carrier.

Pharmaceutical compositions suitable for parenteral administration consist of sterile solutions of the active ingredients, in general. The sterile solution may contain, in addition to the active agent, pH control agents and osmolarity control agents, preservatives, surfactants etc.

For local treatment, for example, ointments, solutions, creames, transdermal patches etc. can be used.

Dosage forms listed above as well as other dosage forms are known per se, see e.g. Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, USA (1990).

The pharmaceutical composition contains dosage unit, in general. The daily dose amounting generally to 1-1000 mg of amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof can be administered in one or more portions. The actual dosage depends on many factors and is determined by the doctor.

The pharmaceutical composition is prepared by admixing the active ingredient to one or more carrier(s), and converting the mixture obtained to a pharmaceutical composition in a manner known per se. Useful methods are known from the literature, e.g. Remington's Pharmaceutical Sciences mentioned above.

The invention also refers to a method to enhance the tissue regeneration effect of adult stem cells which comprises contacting the stem cells with an effective amount of an amidoxime derivative of formula I or a pharmaceutically suitable acid addition salt thereof before the growing of the stem cells and/or during the growing of the stem cells and/or after the growing of the stem cells.

The effect of the amidoxime derivatives of formula I on the adult stem cells are disclosed in the following Examples. Example 1

Effect of mesenchymal stem cells on blood vessel reformation

In a rat model of hind limb ischaemia, acute circulation insufficiency was generated by resecting the main nutrient artery. Immediately after the intervention, grown mesenchymal stem cells obtained from the bone marrow were injected into the muscle area having ischaemia. The rate of vessel reformation was determined on day 21 of the test based on the capillary density of the muscle tissue having ischaemia. The mesenchymal stem cells were treated with the tested compounds 24 hours before the injection. Also the recipient animals were subjected to a daily once peroral treatment with the tested compounds for 21 days.

Methods

Rat model of hind limb ischaemia In the experiments inbred Lewis rats (Charles River) having a body mass of 200-250 g were employed. Ischemia of the hind limb was developed according to the method of Takashi Iwase et al. [Takashi Iwase et al.: Comparison of angiogenic potency between mesenchymal stem cells and mononuclear cells in a rat model of hind limb ischemia, Cardiovascular Research 66 (2005) 543- 551 C] as follows:

The left common iliaca artery was resected as well as the saphenus artery and vein were removed together with their networks while the animals were kept in narcosis with pentobarbital (50 mg/kg i.p.). The circulation in the right limb remained intact.

Isolation of the mesenchymal stem cells obtained from the bone marrow

From both femur and tibia bones of male Lewis rats the bone marrow was removed by washing with PBS (physiological saline containing phosphate buffer). From the bone marrow cells a cell culture was started in a 100 mm tissue culture vessel using an A-MEM medium complemented with 10 % fetal bovine serum and antibiotics. The non-adherent haemopoietic cells were removed by the change of the medium. The number of the adhering spindle-shaped mesenchymal stem cells, after 4-5 passages, reached a value of 5x10 ⁷ .

Treatment of the mesenchymal stem cells and the recipient animals

During the last 24 hours of the growing the grown stem cells were treated with the test compounds in a concentration of 10 pm, then the stem cells were harvested and suspended. Prior to the iliaca surgery, a part of the animals were treated with the test compound at a dose of 20 mg/kg, perorally, and this treatment was repeated for three weeks, daily.

Directly after the iliaca surgery a total of 5x10 ⁶ mezenchymal stem cells were injected into the limb muscle at 5 different sites. The control animals obtained only PBS solution instead of the mezenchymal stem cells. Eight animals were used in each treatment group.

Determination of the capillary density

On day 21 after the surgery the animals were kept in narcosis with pentobarbital, then samples taken from the adductor muscle having ischaemia were frozen in liquid nitrogen. Using frozen sections, the endothelial cells of the capillaries were determined based on the alkali phosphatase activity of the cells by the indoxyl tetrazolium method according to Shintani et al. [Shintani S. et al.: Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation 2001 ;103:897- 903]. In case of each sample, the number of capillaries and muscle fibres were determined in 5 fields of view.

Results

The results obtained are summarized in Table 1.

Table 1

Capillary/muscle fibre ratio in the muscle having ischaem

day 21

Statistics: ANOVA, Bonferroni test.

+ p<0.05 compared with the control group.

++ p<0.05 compared with the group treated with the mesenchymal stem cells.

Evaluation

The introduction of mesenchymal stem cells into the muscle could significantly improve the reformation of blood vessels in the ischaemic area. This corresponds to literature data. The treatment with only the compounds tested could also result in a certain effect. However, the combined treatment with a test compound and the mesenchymal stem cells resulted in about a two-fold increase of the blood vessel formation effect of the mesenchymal stem cells. From a biological point of view, an increase of the capillary network is very important since, depending on the metabolic activity of the tissues, the capillaries can supply the oxygen and fulfil the metabolic demand only at a distance of 40-200 pm. The area served by a capillary is rather narrow also in the muscle that uses much energy and oxygen. Due to the two-fold increase, the capillary network is able to double the matter change and gas change of the muscle tissue, thereby facilitating the survival of the muscle fibres, the preservation of the muscle tissue and the amelioration of the muscle function.

Example 2

Effect on the regeneration of the cardiac muscle after an acute myocardial infarction

Mesenchymal stem cells are able to differentiate into both endothelial and muscle cells, thus, they can have an eminent role in facilitating the tissue regeneration after a myocardial infarction.

An acute myocardial infarction was developed in rat by clamping the coronary artery. Immediately after the intervention, an amount of 2x10 ⁵ grown mesenchymal stem cells obtained from bone marrow was injected into the zone of infarction. Both the mesenchymal stem cells (24 hours prior to the injection) and the recipient animals (once daily p.o. for 7 days) were treated with the test compounds. After a week, the proportion of the area of the left ventricle effected by the infarction to the whole area of the left ventricle as well as the capillary density of the zone of infarction were determined. The implanted mesenchymal stem cells reduced the growth of the zone of infarction and increased the capillary density of the area effected by ischaemia. The test compounds enhanced the regenerative effect of the stem cells in a statistically significant manner.

Methods

Isolation of bone marrow-derived mesenchymal stem cells

Isolation and growing of the bone marrow-derived stem cells were carried out principally according to Song's method [Song H, Chang W, Lim S et al. Tissue transglutaminase is essential for integrin-mediated survival of bone marrow-derived mesenchymal stem cells. Stem Cells 2007;25:1431-1438]. The femoral and tibial bone marrow substance of four weeks' old Sprague-Dowley rats was separated in Dulbecco's Eagle nutrient medium containing low glucose, 10 % of fetal calf serum as well as penicillin/streptomycin. The bone marrow cells were centrifuged at 1600 rpm for 5 minutes, then resuspended in a similar nutrient medium and layered to the top of a Percoll gradient. The cells were centrifuged at 1600 rpm for 30 minutes. The mononuclear cells collecting at the layer border were sucked, twice washed, resuspended in 10 % FBS-DMEM nutrient medium, then 10 ⁶ cells were transferred into 100 m ² culture dishes. The cultures were grown for 3 days at 37 °C in a humidified air containing 5 % C0 ₂ . The non-adhering cells were removed, the cultures washed with PBS solution, then growing was continued in 10 % FBS-DMEM nutrient medium. The nutrient medium was changed after every 3 days. After 10 days' growing, the cells were suspended and the cells expressing the CD34 surface antigen were concentrated by means of Dynabed pearls covered with anti-CD34 monoclonal antibody. The cell culture enriched for CD34 antigen (10 ^s cells/100 cm ² dish) was grown for further 10 days.

Producing myocardial infarction

Sprague-Dawley male rats having 250 g body mass were intubated in pentobarbital narcosis and mechanical ventilation (Harward ventillator) was provided. The heart was opened through laparotomy on the left side by a 2 cm incision and 6.0 silk thread was placed under the proximal portion of the left coronary artery. The ends of the thread were led through a plastic tube forming a loop around the coronary artery. The blood flow through the coronary artery was stopped for 60 minutes by tightening the loop. Ischaemia was indicated by the immediate discoloration of the heart muscle. The blood flow in the coronary artery was restarted by loosening the loop. This fact was directly indicated by the change of the tissue colour. The chest was closed and the animals were uncoupled from the mechanical ventilation.

Marking the mesenchymal stem cells with DAPI and injection into the zone of infarction

A portion of the grown bone marrow-derived cells was treated with a 10 μΜ concentration of the test compounds on the last day of growing. The cells were stained with DAPI to allow the follow-up thereof. The cells were contacted with 50 pg/ml of DAPI (4',6-diamidino-2-phenylindole) for 30 minutes, then the fluorescent stain that did not bind to the cells was removed by repeated washing with PBS. The cells were separated from the culturing surface by a treatment with trypsin and suspended in serum-free nutrient medium at a concentration of 2x10 ⁵ cell/10 μΙ. A total of 2x10 ⁵ cells were injected into 3 different areas of the muscle tissue bordering the zone of infarction directly after the occlusion. The animals which obtained stem cells treated with a test compound were treated with a 20 mg/kg oral dose of the test compound once daily for 7 days beginning on the day of producing the infarction. The cells stained with DAPI were injected into 3 animals in each group. The mesenchymal stem cells were injected into 8 animals in each group in order to test the extent of infarction.

Identification of the stem cells stained with DAPI in the heart muscle

3 days after the infarction, hystological sections were prepared from the left ventricle of 3 animals in each group and the number of stem cells stained with DAPI was determined at the border of the zone of infarction in each field of view using a fluorescent microscope.

Determination of the extent of infarction

The extension of the infarction was determined by staining with TTC. 7 days after the infarction, transaxial tissue slices were cut from the heart and the slices were incubated in a 1 % 2,3,5-triphenyltetrazolium chloride (TTC) solution (pH=7.4) at 37 °C for 20 minutes. The tissue was fixed with 10 % formalin puffered with PBS at 4 °C for 12 hours. Photos were taken from both sides of the slices. The proportion of the zone of infarction was expressed as the percentage of the cross- section of the left ventricle.

Results

The effect of the test compounds on the number of mesenchymal stem cells stained with DAPI in the heart muscle after myocardial infarction is shown in Table 2. The average of 5 fields of view is used for the evaluation in case of each sample.

Table 2

Statistical analysis: ANOVA, Bonferroni paired test, n=3

^* referred to the group that was not treated with the test compound p<0,05. Table 3 indicates the effect of the test compounds on the size of the zone of infarction

Table 3

Statistical analysis: ANOVA, Bonferroni paired test, n=8

^* referred to the group without mesenchymal stem cell transplantation p<0,05

^** referred to the group treated only with the mesenchimal stem cells p<0,05 ⁺ In the Sham surgery, the chest of the animal is opened up, however, no myocardial infarction is produced. This surgery is used to determine the effect of a non-specific surgery

Evaluation

Transplantation of the mesenchymal stem cells reduced the tissue necrosis developed by the infarction, significantly. The treatment with only the test compounds could also produce a low reduction of the size of the zone of infarction. The in vitro and in vivo treatments with the test compounds ameliorated the survival of the mesenchimal stem cells injected into the zone of infarction and further reduced the tissue necrosis developed by the infarction, significantly. Both the therapeutical effect of the test compounds and the reduction of the extension of the necrotized zone can be well explained based on the higher survival of the mesenchymal cells. The same mechanism i.e. the higher adherence of the endogenous stem ceils may explain the effect of the test compounds used alone.

Example 3

Effect of BGP-15 on the survival of stem cells in ischemic mouse hind limb.

Male C57BL mice underwent surgery to induce unilateral hind limb ischemia. Animals were anesthetized by pentobarbital and the right femoral artery at the inguinal ligament as well as the branches of the artery in the area were ligated. Wounds were closed. Immediately after the operation, in vitro propagated stem cells were injected into the gastrocnemius (1 ,5x10 ⁵ ) and thigh muscle (1 ,8x10 ⁵ ) of the ischemic leg. Stem cells were originating from bone marrow of C57BI mice carrying green fluorescent protein (GFP) in all cells. Cells were expanded in mesenchyme stem cell specific condition. Harvested stem cells were divided into three portions and two of those were treated with 50 and 300 μΜ concentration of BGP-15 for 6 hours before injection. Immediately before injection stem cells were concentrated by centrifugation and the recipient mice (3 in each group) were treated, orally, with 20 and 100 mg/kg BGP-15, respectively. The same dose of BGP- 15 treatment was repeated daily for 3 more days. One control animal was processed immediately after the injection of the cells. On the 4th day the gastrocnemius muscles were excised and three histological sections were prepared from each tissue. Sections were separated by 0,5 mm distance from each other. The number of GFP fluorescent cells was evaluated in three randomly selected fields of each section by two photon fluorescence microscopy

Results

Table 4

Conclusion

The results prove that the test compounds ameliorate, considerably and in a statistically significant manner, the adherence and function of adult stem cells, consequently, the compounds are suitable for enhancing the efficiency of the stem cell therapy.