EP2760449A1 - A pharmaceutical composition for the treatment of stem cells - Google Patents

A pharmaceutical composition for the treatment of stem cells

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Publication number
EP2760449A1
EP2760449A1 EP12805749.4A EP12805749A EP2760449A1 EP 2760449 A1 EP2760449 A1 EP 2760449A1 EP 12805749 A EP12805749 A EP 12805749A EP 2760449 A1 EP2760449 A1 EP 2760449A1
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EP
European Patent Office
Prior art keywords
formula
stem cells
compound
acid addition
addition salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP12805749.4A
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German (de)
French (fr)
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Literati Nagy Botond
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Literati Nagy Botond
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Publication date
Application filed by Literati Nagy Botond filed Critical Literati Nagy Botond
Publication of EP2760449A1 publication Critical patent/EP2760449A1/en
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines having two or more nitrogen atoms in the same ring, e.g. oxadiazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/07—Heat shock proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • a pharmaceutical composition for the treatment of stem cells A pharmaceutical composition for the treatment of stem cells
  • the invention refers to the use of a compound of formula
  • Aryl-C N-0-CH 2 -CH-CH 2 -N
  • Aryl represents a phenyl, naphthyl or pyridyl group
  • X stands for a halo atom and then Y represents a hydroxy group
  • X is a nitrogen atom and then Y represents a valence bond between this nitrogen atom and the carbon atom adjacent to Y, thus, forming a six-membered oxadiazine ring,
  • R and Ri form together with the adjacent nitrogen atom a 5-7- membered saturated heterocyclic group
  • compositions 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.
  • Stem cells are biological cells found in most of 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.).
  • biotechnology is able to transform stem cells into various specialized cells by growing the stem cells in a cell culture.
  • stem cells Various types 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.
  • 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).
  • red blood cells erythrocytes
  • white blood cells leukocytes
  • platelets thrombocytes
  • CSF colony-stimulating factor
  • a further promising field of biotechnology includes the preparation of tissues and organs from stem cells.
  • 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.
  • 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.
  • tissue origin e.g. mesenchymal stem cell, adipose derived stem cell, endothelial stem cell, dental pulp stem cell etc.
  • Adult stem cell treatments have been successivefully 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.
  • stem cell therapy offers extraordinary possibilities in many degenerative diseases accompanied by tissue destruction through the reformation of the destructed tissues.
  • 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.
  • 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, 105; 93-98 (2002)].
  • the aim of the invention is to enhance the survival of the adult stem cells introduced into the damaged tissue environment.
  • the compounds of formula I are known compounds.
  • US 5,147,879 discloses compounds of formula I, wherein X represents a halo atom and Y stands for a hydroxy group, as well as acid addition salts and the manufacture thereof.
  • the known compounds have selective beta-blocking action and can be used for the treatment of diabetic angiopathy.
  • the effect of the compounds of formula I on the increase of the activity of the molecular chaperon is discussed in EP 801 649. This effect results in many medical uses such as the treatment of cardiovascular, vascular, cerebral, allergic, immune and autoimmune diseases as well as the skin and mucus diseases.
  • WO 00/50403 discloses the compound N-[2-hydroxy-3- (1 -piperidinyl)propoxy]pyridine-1 -oxide-3-carboximidoyl chloride (arimoclomol), stereoisomers thereof and use of the compound for treating insulin resistance and pathologic states connected with insulin resistance.
  • An industrially applicable preparation of the latter compound and stereoisomers thereof is discussed in WO 01/79174.
  • HU-P 226 206 and HU-P 226 617 describe the (+)- and (-)-enantiomer, respectively, of the 5-(piperidin-1-ylmethyl)-3- pyridyl-5,6-dihydro-2H-1 ,2,4-oxadiazine known from EP 801 649.
  • the enantiomers are suitable for the treatment of vascular diseases.
  • the above aim was achieved by treating the adult stem cells with a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound of formula I or a pharmaceutically suitable acid addition salt thereof.
  • a compound of formula I or an N-oxide of the compound 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.
  • the invention provides the use of a compound of formula I or an N-oxide of the compound 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.
  • 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound of formula I or a pharmaceutically suitable acid addition salt thereof.
  • the invention provides also any combinations of the preceding methods.
  • 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.
  • 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.
  • the N- oxide of the nitrogen atom being in the pyridyl group and/or in the 5-7-membered heterocyclic group is meant.
  • the 5-7-membered heterocyclic group formed by R, Ri and the adjacent nitrogen atom is preferably a pyrrolidinyl or piperidinyl group.
  • a halo atom is a fluoro, chloro, bromo or iodo atom, preferably a chloro atom.
  • 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, lactate, tartrate etc.
  • a preferred compound of formula I is N-[2-hydroxy-3-(1- piperidinyl)propoxy]pyridine-3-carboximidoyl chloride (bimoclomol) of formula II
  • the adult stem cells are treated with a compound of formula I or an N-oxide of the compound of formula I or a pharmaceutically suitable acid addition salt thereof.
  • the adult stem cells can be treated with the compound of formula I or an N-oxide of the compound 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.
  • a solution, suspension or emulsion thereof is employed as a composition that contains the active ingredient.
  • the medium of the composition preferably water, physiological saline or liquid nutrient media suitable for the increase of stem cells are used.
  • the damaged tissue environment needing the stem cell treatment can be treated with a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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.
  • a compound of formula I or an N-oxide of the compound 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.
  • the compound of formula I or an N-oxide of the compound 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.
  • a solid or liquid mixture which contains, in addition to a compound of formula I or an N-oxide of the compound 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.
  • the liquid composition is a solution, suspension or emulsion of a compound of formula I or an N-oxide of the compound 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.
  • a pharmaceutical composition containing a compound of formula I or an N-oxide of the compound 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.
  • the individual having a damaged tissue environment that needs the stem cell treatment can be systemically treated with a pharmaceutical composition containing a compound of formula I or an N-oxide of the compound 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.
  • the damaged tissue environment needing the stem cell treatment is treated, locally, with a pharmaceutical composition containing a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound of formula I or a pharmaceutically suitable acid addition salt thereof as the active ingredient can be carried out, too.
  • the stem cells are treated with a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound of formula I or a pharmaceutically suitable acid addition salt thereof as the active ingredient.
  • the individual can be further treated, systemically, and/or the damaged tissue environment can be further treated, locally, with a pharmaceutical composition containing a compound of formula I or an N-oxide of the compound of formula I or a pharmaceutically suitable acid addition salt thereof as the active ingredient.
  • any composition for human or veterinary use is meant, wherein the composition comprises, in addition to the active ingredient i.e. a compound of formula I or an N-oxide of the compound 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.
  • 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.
  • 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.
  • ointments for example, ointments, solutions, creames, transdermal patches etc. can be used.
  • the pharmaceutical composition contains dosage unit, in general.
  • the daily dose amounting generally to 1-1500 mg, preferably 50-500 mg of a compound of formula I or an N-oxide of the compound or a pharmaceutically suitable acid addition salt thereof can be administered in one or more portions to an adult person of about 70 kg body weight.
  • 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 a compound of formula I or an N-oxide of the compound 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 compounds was investigated in a rat model of hind limb ischaemia.
  • Acute circulation insufficiency was generated by resecting the main nutrient artery.
  • 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 20 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 20 days.
  • 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.
  • 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 a D-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 5 passages, reached a value of about 5x10 7 .
  • the 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 20 days, daily. The control animals were treated with the carrier in a similar manner.
  • the area served by a capillary is rather narrow also in the muscle that uses much energy and oxygen. Due to the twofold 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.
  • 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.
  • 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, 25, 1431-1438 (2007)].
  • 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 % CO 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 5 cells/100 cm 2 dish) was grown for further 10 days.
  • 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.
  • a portion of the grown bone marrow-derived cells was treated with a 10 ⁇ concentration of the test compounds on the last day of growing.
  • the cells were stained with DAPI (4',6- diamidino-2-phenylindole) to allow the follow-up thereof within the heart.
  • the cells were contacted with 50 g/ml of DAPI 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 ⁇ .
  • 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 3 days beginning on the day of producing the infarction.
  • the cells stained with DAPI were injected into 3 animals in each group. Identification of the stem cells stained with DAPI in the heart muscle
  • 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.
  • test compounds 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.
  • Bimoclomol, arimoclomol and iroxanadine increase the survival and function of stem cells in case of ischemia and metabolic disorder, thus, they are suitable for the amelioration of the success of stem cell therapy.
  • mesenchymal stem cells have been used in the above tests, the invention can be employed in case of stem cells of other origin, too.

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Abstract

The invention refers to the use of a compound of formula I or an N-oxide of the compound 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.

Description

A pharmaceutical composition for the treatment of stem cells
Field of the invention
The invention refers to the use of a compound of formula
I
X Y R
/
Aryl-C=N-0-CH2-CH-CH2-N
wherein
Aryl represents a phenyl, naphthyl or pyridyl group,
X stands for a halo atom and then Y represents a hydroxy group or
X is a nitrogen atom and then Y represents a valence bond between this nitrogen atom and the carbon atom adjacent to Y, thus, forming a six-membered oxadiazine ring,
R and Ri form together with the adjacent nitrogen atom a 5-7- membered saturated heterocyclic group,
or an N-oxide of the compound 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.
Background of the invention
Stem cells are biological cells found in most of 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.: „Review ex vivo engineering of living tissues with adult stem cells", Tissue Eng. 12 (11), 3007-19 (2006). doi:10. 089/ten.2006. 2.3007]. 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, 105; 93-98 (2002)].
Therefore, the aim of the invention is to enhance the survival of the adult stem cells introduced into the damaged tissue environment.
The compounds of formula I are known compounds.
US 5,147,879 discloses compounds of formula I, wherein X represents a halo atom and Y stands for a hydroxy group, as well as acid addition salts and the manufacture thereof. The known compounds have selective beta-blocking action and can be used for the treatment of diabetic angiopathy. The effect of the compounds of formula I on the increase of the activity of the molecular chaperon is discussed in EP 801 649. This effect results in many medical uses such as the treatment of cardiovascular, vascular, cerebral, allergic, immune and autoimmune diseases as well as the skin and mucus diseases.
WO 00/50403 discloses the compound N-[2-hydroxy-3- (1 -piperidinyl)propoxy]pyridine-1 -oxide-3-carboximidoyl chloride (arimoclomol), stereoisomers thereof and use of the compound for treating insulin resistance and pathologic states connected with insulin resistance. An industrially applicable preparation of the latter compound and stereoisomers thereof is discussed in WO 01/79174.
HU-P 226 206 and HU-P 226 617 describe the (+)- and (-)-enantiomer, respectively, of the 5-(piperidin-1-ylmethyl)-3- pyridyl-5,6-dihydro-2H-1 ,2,4-oxadiazine known from EP 801 649. The enantiomers are suitable for the treatment of vascular diseases.
Summary of the invention
It was found that the above aim was achieved by treating the adult stem cells with a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound of formula I or a pharmaceutically suitable acid addition salt thereof.
Thus, according to the invention a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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.
Under the N-oxide of a compound of formula I, the N- oxide of the nitrogen atom being in the pyridyl group and/or in the 5-7-membered heterocyclic group is meant.
The 5-7-membered heterocyclic group formed by R, Ri and the adjacent nitrogen atom is preferably a pyrrolidinyl or piperidinyl group.
When X represents a nitrogen atom and Y is a valence bond between this nitrogen atom and the carbon atom being adjacent to Y, then a 6-membered 1 ,2,4-oxadiazine ring is formed having a double bond between the carbon atom in position 3 and one of the nitrogen atoms (either in position 2 or in position 4).
A halo atom is a fluoro, chloro, bromo or iodo atom, preferably a chloro atom.
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, lactate, tartrate etc.
A preferred compound of formula I is N-[2-hydroxy-3-(1- piperidinyl)propoxy]pyridine-3-carboximidoyl chloride (bimoclomol) of formula II
or a pharmaceutically suitable acid addition salt thereof or an N- oxide thereof i.e. N-[(2R)-2-hydroxy-3-(1-piperidinyl)propoxy]- pyridine-1-oxide-3-carboximidoyl chloride of formula III
or a pharmaceutically suitable acid addition salt thereof or 5- (piperidin-1-ylmethyl)-3-pyridyl-5,6-dihydro-2H-1 ,2,4-oxadiazine (iroxanadine) of formula IV
and the optically active enantiomers thereof as well as a pharmaceutically suitable acid addition salt thereof.
According to the invention the adult stem cells are treated with a compound of formula I or an N-oxide of the compound of formula I or a pharmaceutically suitable acid addition salt thereof. The adult stem cells can be treated with the compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound of formula I or a pharmaceutically suitable acid addition salt thereof are introduced into the damaged tissue environment needing the stem cell treatment, also a pharmaceutical composition containing a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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 a compound of formula I or an N-oxide of the compound 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. a compound of formula I or an N-oxide of the compound 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, Mack Publishing Co., Easton, USA (recent edition).
The pharmaceutical composition contains dosage unit, in general. The daily dose amounting generally to 1-1500 mg, preferably 50-500 mg of a compound of formula I or an N-oxide of the compound or a pharmaceutically suitable acid addition salt thereof can be administered in one or more portions to an adult person of about 70 kg body weight. 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 a compound of formula I or an N-oxide of the compound 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 compounds 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
The effect of the compounds was investigated 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 20 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 20 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 a D-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 5 passages, reached a value of about 5x107.
Treatment of the mesenchymal stem cells and the recipient animals
During the last 24 hours of 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 20 days, daily. The control animals were treated with the carrier in a similar manner.
Directly after the iliaca surgery a total of 5x106 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 20 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; 103, 897-903 (2001)]. In case of each sample, the ratio 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 ischaemia on day 20
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 bimoclomol and arimoclomol could also result in a certain effect. However, the combined treatment with bimoclomol or arimoclomol 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 twofold 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 survival of mesenchymal stem cells in 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 2x105 grown mesenchymal stem cells obtained from bone marrow was injected into the zone of infarction. Before the implantation of the stem cells, a part of the mesenchymal stem cells and the recipient animals were treated with the test compounds.
The survival of the mesenchymal stem cells was determined on the third day after the implantation by DAPI fluorescence.
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, 25, 1431-1438 (2007)]. 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 106 cells were transferred into 100 m2 culture dishes. The cultures were grown for 3 days at 37 °C in a humidified air containing 5 % CO2. 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 (105 cells/100 cm2 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 (4',6- diamidino-2-phenylindole) to allow the follow-up thereof within the heart. The cells were contacted with 50 g/ml of DAPI 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 2x105 cell/10 μΙ. A total of 2x105 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 3 days beginning on the day of producing the infarction. The cells stained with DAPI were injected into 3 animals in each group. 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.
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.
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. From Table 2 it is evident that bimoclomol and iroxanadine ameliorate, considerably and in a statistically significant manner, the adherence and survival of the stem cells in the zone of infarction of the heart.
Evaluation
Bimoclomol, arimoclomol and iroxanadine increase the survival and function of stem cells in case of ischemia and metabolic disorder, thus, they are suitable for the amelioration of the success of stem cell therapy.
Although mesenchymal stem cells have been used in the above tests, the invention can be employed in case of stem cells of other origin, too.

Claims

What we claim is:
1. Use of a compound of formula I
X Y R
t I /
Aryl-C=N-0-CH2-CH-CH2-N I
wherein
Aryl represents a phenyl, naphthyl or pyridyl group,
X stands for a halo atom and then Y represents a hydroxy group or
X is a nitrogen atom and then Y represents a valence bond between this nitrogen atom and the carbon atom adjacent to Y, thus, forming a six-membered oxadiazine ring,
R and Ri form together with the adjacent nitrogen atom a 5-7- membered saturated heterocyclic group,
or an N-oxide of the compound 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.
2. A use according to Claim 1 in which N-[2-hydroxy-3-(1- piperidinyl)propoxy]pyridine-3-carboximidoyl chloride of formula
II
or a pharmaceutically suitable acid addition salt thereof is used as the compound of formula I.
3. A use according to claim 1 in which N-[(2R)-2-hydroxy-3-(1- piperidinyl)propoxy]-pyridine-1 -oxide-3-carboximidoyl chloride of formula III
or a pharmaceutically suitable acid addition salt thereof is used as the compound of formula I.
4. A use according to claim 1 in which 5-(piperidin-1-ylmethyl)- 3-pyridyl-5,6-dihydro-2H-1 ,2,4-oxadiazine (iroxanadine) of formula IV
or an optically active enantiomer thereof or a pharmaceutically suitable acid addition salt thereof is used as the compound of formula I.
5. A method to enhance the tissue regeneration effect of adult stem cells which comprises contacting the stem cells with an effective amount of a compound of formula I, wherein Aryl, X, Y, R and Ri are as defined in Claim 1 , or an N-oxide of the compound 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.
6. A method according to Claim 5 in which N-[2-hydroxy-3-(1- piperidinyl)propoxy]pyridine-3-carboximidoyl chloride of formula II or a pharmaceutically suitable acid addition salt thereof is used as the compound of formula I.
7. A method according to Claim 5 in which N-[(2R)-2-hydroxy-3- (1-piperidinyl)propoxy]-pyridine-1-oxide-3-carboximidoyl chloride of formula III or a pharmaceutically suitable acid addition salt thereof is used as the compound of formula I.
8. A method according to Claim 6 in which 5-(piperidin-1- ylmethyl)-3-pyridyl-5,6-dihydro-2H-1 ,2,4-oxadiazine of formula IV or a pharmaceutically suitable acid addition salt thereof is used as the compound of formula I.
EP12805749.4A 2011-09-26 2012-09-25 A pharmaceutical composition for the treatment of stem cells Withdrawn EP2760449A1 (en)

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