HK1112848B - Pharmaceutical composition for regenerating myofibers in the treatment of muscle injuries - Google Patents

Description

Pharmaceutical composition for regenerating muscle fibers in the treatment of muscle injuries

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application No.60/791,462 filed on 13.4.2006, the contents of which are incorporated herein by reference. The present application further claims priority from PCT application nos. PCT/IB2005/003202 and PCT/IB2005/003191, filed on 8.11.2005, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to a pharmaceutical composition and method for regenerating muscle cells and myocardium, which is used for treating muscle injury. In particular, it relates to a pharmaceutical composition and a method for regenerating cardiomyocytes in the treatment or repair of myocardial damage or injury caused by ischemic diseases.

Background

Myocardial Infarction (MI) or heart attack is a disease caused by damage or death of myocardial cells due to interruption of blood supply to a part of the heart. The disease is the leading cause of death in men and women worldwide. After a myocardial infarction, there does not appear to be any natural repair process that produces new cardiomyocytes to replace the lost myocytes, and instead, scar tissue may replace the necrotic myocardium leading to further deterioration of cardiac function.

Therapeutic replacement of necrotic heart tissue with newly regenerating functional cardiomyocytes has been an ideal therapeutic approach until recently not practical, as cardiomyocytes are considered terminally differentiated or, in other words, the heart is a post-mitotic non-regenerating organ (postmitotic non-regenerating organ). However, this view has recently been objected to by Beltrami et al and others who report that some myocytes resident in the myocardium are able to and do replicate after an infarction. Transplantation of cardiomyocytes or skeletal myoblasts was attempted in order to promote and enhance repair of infarcted myocardium, but was not very successful in reconstructing functional myocardium and coronary vessels. Transplantation of adult bone marrow-derived Mesenchymal Stem Cells (MSCs) for cardiac repair following myocardial infarction allows some angiogenesis and myogenesis, but newly-born regenerative cardiomyocytes mostly appear at locations along the limbic zone where blood supply is relatively less affected.

Since acute Myocardial Infarction (MI) can rapidly lead to damage or death of myocytes (cardiomyocytes), vascular structures and non-vascular components in the blood supply area of the ventricle, without local blood system reconstruction by growth of a subpopulation of cardiomyocytes alone^4-8Or MSC^1-3It appears impossible to replace infarcted myocardium (myocardial tissue) in the central infarcted area (absolute ischemic area) with new cardiomyocytes by transplantation. This may explain why regeneration of cardiomyocytes after MSC-only transplantation occurs mostly along the adjacent marginal zone of infarcts that maintain a large blood supply^1-11. Thus, the loss of myocardium, arterioles and capillaries in the central region of infarction appears to be irreversible, ultimately leading to scar formation.

More recent research¹²In vitro heart transplantation of MSCs pre-modified with exogenous Akt was shown to have better results. However, regenerated cardiomyocytes were able to enter scar tissue only from the marginal zone, suggesting that overexpression of exogenous Akt, while increasing the viability of transplanted MSCs, was insufficient to render them viable in central ischemic areas. Furthermore, even in the less ischemic border zone, MSC-derived regenerating cardiomyocytes were noticed to be scattered and seemed to be difficult to bunch up and form regenerating myocardium. This may be due to the low cardiogenic differentiation efficiency (cardiac differentiation efficiency) of the surviving transplanted MSCs. Natural regeneration of cardiomyocytes, including resident progenitor cells or the likeThe differentiation of stem cells of his origin (e.g., endothelial cells or niches in bone marrow) is insufficient to balance the myocardial cell death that occurs in acutely or chronically damaged hearts, a recognition that frustrates researchers who believe that myocardial regeneration will be a promising approach to treating heart disease.

The prior art shows that there are three key requirements in regenerating functional myocytes in the entire area of infarcted myocardium: 1) enhanced viability of the transplanted cells, such that they survive the absolute ischemic phase, i.e., the period from injection of donor cells to neovascularization; 2) early reconstruction of the impaired blood supply network in infarcted myocardium to maintain survival and efficient transport of transplanted cells and to maintain oxygenation and nutrient delivery; and 3) enhanced cardiogenic differentiation efficiency (cardiac differentiation efficiency) of the transplanted cells to differentiate more viable donor cells into cardiac lineages.

Therefore, in order to achieve the therapeutic goal of replacing necrotic heart tissue with new regenerative-functioning cardiomyocytes, new therapeutic approaches are needed to suit the needs of myocardial infarction treatment, e.g., using compounds with biological properties that fully satisfy the needs of the three above aspects.

Summary of The Invention

An object of the present invention is to provide a pharmaceutical composition comprising a compound selected from a class of compounds collectively having a backbone structure represented by the general formula (I). The compounds have potent therapeutic effects on the survival potential of MSCs in vitro and the efficiency of cardiogenic differentiation, as well as MI repair in vivo. These compounds are known per se in the art, but the above-mentioned biological activities and therapeutic effects are not known. They can be isolated from natural sources, in particular from plants, or can also be obtained by total or semi-chemical synthesis using current or future developed synthetic techniques. The backbone structure itself has the myogenic (myogenic) effect described above, and many variations can be formed by the substitution of one or more hydrogen atoms at different positions of the backbone structure. These variants have a common backbone structure and myogenic effects. Of course, their myogenic abilities may vary.

Formula (I)

The backbone structure of formula (I) may have one or more attached substituents. A substituent is a substituent atom or group of atoms at a position in place of a hydrogen atom. Substitution may be achieved by methods known in the art of organic chemistry. For example, by appropriate design, a large number of variants or derivatives having various substituents attached at different positions of the backbone structure can be produced using high throughput combinatorial synthesis methods. The selection of these variants or derivatives of formula (I) is based on Mesenchymal Stem Cell (MSC) activity assays that can rapidly determine whether a particular variant promotes proliferation and cardiogenic differentiation of cultured MSCs. The term "compound of formula (I)" as used herein includes the backbone compound itself and substituted variants thereof having similar biological activity. Examples of such variants are given below, all of which have a similar effect on regenerating functional myocytes as the backbone structure (i.e. the backbone compound itself):

furthermore, as a therapeutic agent, the compound of formula (I) may be in the form of a "functional derivative" as defined below.

It is known to those skilled in the art that the above compounds can be prepared in various racemic isomer, enantiomeric or diastereomeric forms, salts with inorganic and organic acids, and derivatives, such as N-oxides, prodrugs, bioisosteres. "prodrug" refers to an inactive form of a compound that is capable of being metabolized or converted to the active compound within the body (in vivo) after administration due to the attachment of one or more specific protecting groups that are used in a transient manner to alter or eliminate the undesirable properties of the parent molecule. "bioisosteres" refers to compounds produced by exchanging an atom or group of atoms with another, substantially similar atom or group of atoms. The purpose of bioisosteric group replacement is to create new compounds with similar biological properties to the parent compound. Bioisosteric group replacement can be physicochemical or topologically based. The preparation of suitable prodrugs, bioisosteres, N-oxides, pharmaceutically acceptable salts or various isomers from known compounds, such as those disclosed in the specification, is within the knowledge of one skilled in the art. The present invention therefore relates to all suitable isomeric forms, salts and derivatives of the above disclosed compounds.

The term "functional derivative" as used herein refers to a prodrug, bioisostere, N-oxide, pharmaceutically acceptable salt, or various isomers of the particular compounds disclosed above, which may be advantageous in one or more respects as compared to the parent compound. The preparation of functional derivatives can be difficult, but certain related techniques are well known to those skilled in the art. A variety of high-throughput chemical synthesis methods can be used. For example, combinatorial chemistry has led to the rapid expansion of compound libraries, which when combined with a variety of high-performance biological screening techniques, can efficiently find and isolate useful functional derivatives.

The pharmaceutical composition of the present invention is used for treating myocardial damage or necrosis caused by diseases, particularly MI, by regenerating heart tissue. The pharmaceutical composition may be prepared into a suitable dosage form, such as tablets, capsules, injections, solutions, suspensions, powders, syrups, and the like, by conventional methods known to those skilled in the art, and administered to a mammalian subject suffering from myocardial injury or necrosis. Formulation techniques are not within the context of the present invention and therefore do not limit the scope of the present invention.

The pharmaceutical compositions of the present invention may be formulated in the form of body part implants suitable for oral administration, systemic injection, and direct local injection in the heart or for long term sustained release.

In another aspect, the present invention provides a method of treating or ameliorating a pathological condition in a mammal, wherein the pathological condition can be alleviated, treated or cured by regenerating functional myocardium as determined by one of ordinary skill in the medical arts, the method comprising administering to the mammal in the pathological condition an effective amount of a compound of formula (I) or a functional derivative thereof.

In another aspect, the invention provides a method of regenerating functional myocardium in a mammal in need of replacement of dead or damaged cardiac tissue resulting from heart disease, such as Myocardial Infarction (MI). This is a treatment modality based on cell transplantation, comprising the following steps: (a) obtaining stem cells, such as MSCs; (b) contacting the stem cells with a compound of formula (I) or a functional derivative thereof to activate the differentiation pathway of the cardiogen prior to transplantation, and (c) then implanting the activated cells into infarcted cardiac tissue of the mammal. This treatment regime can achieve the following goals: 1) enhancing the survival potential of the implanted cells; 2) early reconstitution of the blood supply network, and 3) enhancement of the efficiency of cardiogenic differentiation of transplanted cells by ex vivo activation of MSC-forming cardiogenic progenitor cells (MSCs) prior to transplantation.

In another aspect, the present invention provides a method of treating ischemic heart disease, particularly MI, in a mammal, comprising the steps of: (a) culturing MSCs or endothelial cells with a compound of formula (I) or a functional derivative thereof, (b) collecting conditioned medium of the treated cells, which contains a secretagogue protein having activity for promoting myocardial infarction repair or cardiogenic differentiation of MSCs, and (c) administering or delivering the conditioned medium to heart tissue in the infarct zone.

In another aspect, the present invention provides a research reagent for cardiogenic transdifferentiation (cardiac differentiation) studies of stem cells (e.g., MSCs). The agent comprises one or more compounds of formula (I) or functional derivatives thereof. It may be in solid or liquid form. For example, it may be a solution of dimethyl sulfoxide.

It can be seen that the present invention is primarily directed to the following aspects:

1. use of an effective amount of a compound of the formula in the preparation of a composition for regenerating myocytes or myocardium in the heart of a mammalian subject suffering from myocardial injury.

2. The use of item 1, wherein the damaged myocardium is caused by an ischemic event.

3. The use of item 2, wherein the ischemic event is myocardial infarction.

4. The use of item 1, wherein the myocyte or myocardium is regenerated by use comprising one or more of the following steps: (a) increasing the viability of myogenic precursor cells to enable said precursor cells to survive an absolute ischemic period; (b) (ii) reconstructing a disrupted blood supply network in the region of the heart in which the damaged muscle is located; and (c) enhancing the efficiency of cardiogenic differentiation of said precursor cells along a cardiac lineage, either simultaneously or in any particular order.

5. The use of item 4, wherein the myogenic precursor cells are mesenchymal stem cells derived from bone marrow through blood circulation.

6. The use of item 1, wherein the composition is contacted with a plurality of stem cells to be implanted into infarcted or damaged cardiac tissue of the mammalian subject.

7. The use of item 1, wherein the composition is prepared as a dosage form that can be systemically administered to the mammalian subject.

8. The use of item 7, wherein said dosage form is selected from the group consisting of tablets, capsules, injections, syrups, suspensions, and powders.

9. The use of item 1, wherein said composition is comprised in a culture medium for culturing a plurality of MSCs or endothelial cells for a period of time, thereby obtaining a culture medium containing secreted proteins from said MSCs or endothelial cells, wherein said culture medium containing secreted proteins from said MSCs or endothelial cells is administered or delivered to cardiac tissue in an infarct area.

The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and specific objects attained by its uses, reference is made to the accompanying drawings and the following description in which there are illustrated and described preferred embodiments of the invention.

Brief Description of Drawings

FIG. 1 outlines the isolation of Niga-ichigoside F1 (referred to as "CMF") from the plant Geum Japonicum (Geum Japonicum) as an example for the preparation of the compounds of the present invention.

FIG. 2 shows the effect of CMF on cardiogenic differentiation of MSCs and the up-regulation of ex vivo phospho-Akt 1.

Figure 3 shows the efficacy of treatment based on CMF-pretreated MSC transplantation.

FIG. 4 shows the distribution of ejection fraction and shortening fraction of three groups of rats 2 days and 2 weeks after cell transplantation (A: normal group; B: MI group transplanted with MSC treated with CMF; C: MI group transplanted with MSC not treated with CMF).

Figure 5 shows the efficacy of CMF in an animal model of myocardial infarction.

Figure 6 shows conditioned medium-induced enhanced proliferation and myocardial regeneration of MSC cultures.

Figure 7 shows the source of cells for CMF-induced myocardial regeneration in animal MI models.

Detailed description of the embodiments

I. Experimental procedure

All experimental designs used in the present invention were in accordance with the guidelines for laboratory animal management and use published by the national institutes of health, and approved by the ethical committee for animal experiments at the university of hong kong, china.

For the following discussion, CMF refers to the base compound (or backbone compound) of the present invention. Its chemical structure is described by the above chemical formula (I).

Obtaining of the compounds of the invention: the compounds can be prepared from plants or by chemical synthesis.

As an example to illustrate the process of preparing compounds from natural sources, details concerning the isolation and purification of CMF from a plant Geum Japonicum (Geum Japonicum) are provided below. Other plants which may contain CMF or variants thereof include, for example, Acaena pinnatifida R.et P, Agrimonia pilosa Ledeb, Asparagus filicinus (Asparagus filicinus), Ardisiajaponica (Ardisiajaponica), Campsis grandiflora (Campsis grandiflora), Chrysanthes pilosa (Franch. Schindl.), Sargentodoxa cuneata (Caulifera), Cedrela sinensis (Cedrela sinensis), Chaenomeles speciosa (Chaetomes sinensis EHNE), Himalayana salicifolia (Debregiella salicifolia), Eriobotrya japonica (Rosemophila japonica), Rosaceae, and the like, Rubus imperialis, Rubus imperialis Chum.Schl. (Rosaceae), Rubus sieboldii, Rumex japonicus (Rumex japonica), Salvia pratense (Salvia triguga Diels), Strasburgria robusta, Strawberry cv. Houkuowase, Tiarella polyphylla (Tiarella polyphenlla), Vochysia pacifica Cuatrec, Zanthoxylum bungeanum (Zanthoxylum piperitum), etc.

Isolation of intermediate myocardial factor (CMF) of ludwigia japonica: referring to fig. 1, july, jujuba juba collected from kukou, guizhou province, china, was dried (10kg) and diafiltered twice with 70% ethanol (100L) at room temperature for three days each. The extracts were combined and spray dried to give a solid residue (1 kg). The solid residue was in 10L H₂Suspended in O, and extracted twice with chloroform (10L) and twice with n-butanol (10L) in this order to obtain the corresponding fractions. The n-butanol (GJ-B) soluble fraction was filtered and spray dried to obtain a powder fraction, and the specific ability of this fraction to stimulate cardiogenic differentiation of MSCs in cell culture was confirmed by the following method. The n-butanol soluble fraction (GJ-B) has been shown to enhance proliferation and cardiogenic differentiation of cultured MSCs in cell culture systems. The GJ-B fraction was then applied to a SephadexLH-20 column equilibrated with 10% methanol and eluted with increasing concentrations of aqueous methanol to dissolve seven fractions GJ-B-1 to GJ-B-7. All eluted fractions were tested for activity using the MSC culture system. The activity assay showed that fraction 6 was most active in enhancing cardiogenic differentiation of cultured MSCs. The pure active substance, which is referred to as CMF in this application, was further isolated from GJ-B-6. The structure of CMF was determined by NMR analysis and compared to literature, shown as formula (I).

Preparation of MSCs for transplantation: MSCs were cultured with CMF (10. mu.g/ml in growth medium) for 6 days. Meanwhile, control MSCs were cultured in growth medium containing an equal volume of 5% DMSO. On day 2, expression of endogenous phospho-Akt1 was assessed by immunocytochemistry and Western blotting. Myogenic differentiation was assessed by immunocytochemistry and Western blotting against MEF2 on day 4 and further confirmed by immunocytochemistry and Western blotting with MHC-specific antibodies on day 6. On day 3, CMF pretreated and control MSCs were labeled with CM-DiI in culture and prepared for transplantation.

Preparing bone marrow mesenchymal stem cells: tibia/femur were removed from Sprague-dawley (sd) rats and Bone Marrow (BM) was washed out of bone with IMDM medium containing 10% heat-inactivated fbs (gibco) and 1% penicillin/streptomycin. BM were mixed thoroughly and centrifuged at 1500rpm for 5 minutes. The cell pellet was suspended in 5ml of growth medium. The cell suspension was carefully placed in 5ml of Ficoll solution and centrifuged at 200rpm for 30 minutes. The second layer containing bone marrow cells was transferred to a tube and washed twice with PBS to remove Ficoll (1200rpm, 5 min). The cell pellet was resuspended in IMDM medium containing 10% heat-inactivated fbs (gibco) and 1% penicillin/streptomycin antibiotic mixture. 5% CO in 37 deg.C incubator₂After 24 hours of culture, the non-adherent cells were discarded and the adherent cells were cultured by changing the medium every 3 days, and the cells became almost full after 14 days of culture. This is the bone marrow cells, hereinafter called MSC, which are used for in vitro and in vivo assays in the present application.

Western blot analysis: whole cell extracts of CMF-treated cells or control cells were prepared by lysing the cells with 3-fold compacted cell volume of lysis buffer (50mM Tris, pH 7.5, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Nonidet P-40, 10% glycerol, 200mM NaF, 20mM sodium pyrophosphate, 10mg/ml leupeptin, 10mg/ml phthalein, 200mM phenylmethanesulfonyl fluoride, and 1mM sodium orthovanadate) on ice for 30 minutes. The protein yield was quantified by the Bio-Rad DC protein assay kit (Bio-Rad). An equal amount (30. mu.g) of total protein was size fractionated by SDS-PAGE and transferred to PVDM membrane (Millipore). Blots were blocked with phosphate buffered saline plus 0.1% (vol/vol) tween 20(PBST) containing 5% (wt/vol) milk Powder (PBSTM) for 30 minutes at room temperature and probed with specific primary antibody (diluted 1: 1000 in PBSTM) against rat phospho-Akt1 (mouse) or rat MHC (mouse, Sigma-Aldrich) for 60 minutes. After extensive washing in PBST, blots were probed with horseradish peroxidase-conjugated anti-mouse igg (amersham bioscience) (1/1000 dilution in PBSTM, 60min), extensively washed with PBST, and developed by chemiluminescence.

CMF pretreated MSC were transplanted to heart tissue: Sprague-Dawley (SD) rats were used and all Animal procedures were approved by the University Animal Committee on Animal Welfare. Each rat was anesthetized with an intraperitoneal injection of pentobarbital (50mg/kg), intubated, and mechanically ventilated with room air using a Harvard ventilator (model 683). Following left thoracotomy, myocardial infarction was induced by permanent ligation of the Left Anterior Descending (LAD) coronary artery. 5X 10 suspended in saline⁵DiI-labeled CMF-pretreated MSCs were injected into three sites (32 rats, test group) of the terminal myocardium (ischemic region) of the ligated artery, respectively, immediately after ligation. Control rats (32 rats) were injected at the same site and time with equal amounts of DiI-labeled untreated control MSCs suspended in saline, and pseudo-ischemic groups (32 rats) were thoracotomy and not LAD ligated. 16 untreated rats were set as a normal control group.

After assessment of cardiac function by echocardiography, half of the experimental rats in the different groups were sacrificed on days 7 and 14 post-infarction according to the test schedule. The heart of the sacrificed rat was removed and washed with PBS and photographed separately. All samples harvested were embedded in paraffin and sectioned for follow-up of DiI signaling and examination of revascularization, infarct size and myocardial regeneration. If the rejuvenated cells were DiI positive, further MHC immunohistochemical staining was performed to confirm their cardiogenic differentiation.

Co-localization of DiI marker and heart-specific marker expression was detected with confocal microscopy (ZEISS, LSM 510 META). Briefly, sections were immunohistochemically stained with rat-specific troponin I antibody. Confirmation of cardiogenic differentiation of DiI-labeled transplanted MSCs (which form regenerated myocardium) was performed by the following method: DiI positive cells were pooled, their donor cell origin was shown, and specific positive staining of cardiac end differentiation marker troponin I was detected using confocal microscopy, suggesting cardiogenic differentiation of these transplanted cells.

CMF direct treatment in MI model: 32 SD rats were randomly divided into four groups: normal, pseudo-ischemic, CMF-treated and untreated controls (8 per group). Rats were anesthetized with intraperitoneal injection of pentobarbital (50mg/kg), intubated, and mechanically ventilated with room air using a Harvard ventilator (model 683). Myocardial infarction was induced by permanent ligation of the Left Anterior Descending (LAD) coronary artery following left thoracotomy. Immediately after ligation of 8 of the rats (CMF-treated group), CMF (0.1ml, containing 0.1mg of CMF) in 5% DMSO solution was injected into the ligated terminal myocardium of the artery (ischemic region). Another 8 rats were injected with an equal amount of 5% DMSO at the same site and time as a non-treated control group. The 8 rats in the pseudo-ischemic group underwent thoracotomy without ligation of LAD. In addition, 8 rats without any treatment were set as a normal group.

Conditioned medium containing secreted proteins derived from MSCs or other cells induced by CMF: MSCs were treated with 10. mu.g/ml CMF for 24 hours to activate/up-regulate gene expression, and then rinsed thoroughly to remove CMF debris. Then 5ml of fresh growth medium was added to the culture and collected after 3 days of culture. The collected medium is called conditioned medium. 5ml of conditioned medium was concentrated to a volume of 1ml and used as a therapeutic agent in the above-described myocardial infarction animal model. Briefly, 0.2ml of conditioned medium was injected into the ligated tip section immediately after left thoracotomy and LAD ligation. Fresh growth medium was used as a control.

Replacement of bone marrow with DiI-labeled MSCs: 16 SD rats five weeks old were used for bone marrow transplantation. Recipient rats were stimulated with 9.5Gy gamma irradiation of a 137Cs source (Elite Grammacell 1000) at a dose of 1.140Gy/min to completely destroy bone marrow-derived stem cells of the rats. When in useDiI-labeled MSC (2X 10) was injected via tail vein within 2 hours after needle stick stimulation with number 27⁸Cells were suspended in 0.3ml pbs). One week after stimulation and transplantation, rats with DiI-labeled bone marrow were divided into two groups: one group was treated directly with CMF and the other group was not treated as a control. Myocardial infarction surgery and treatment protocols were performed as described above. For further evaluation, the trial was terminated on day 14 after surgery and treatment. Heart samples of the sacrificed rats were obtained. All samples were followed for DiI positive cells and their cardiogenic differentiation by immunohistochemical staining with antibodies specific for cardiac troponin i (santa cruz) and pcna (dako). Specific secondary antibodies conjugated to alkaline phosphatase (Santa Cruz) were used to visualize positively stained cells. The DiI positive signal was observed with a fluorescence microscope (Laica).

Evaluation of infarct size: the left ventricle of the test rats sacrificed on day 14 was removed and divided into 3 cross-sections from the tip to the bottom. Sections were fixed with formalin and embedded in paraffin. Sections of the left ventricle (20 μm thick) were stained with Masson's trichrome, which labeled collagen blue and myocardium red. These sections were digitized and all blue staining quantified according to morphometry. Calculating infarct volume (mm) for a particular slice based on slice thickness³). The volumes of infarcted tissue in all sections were added to obtain the total volume for each particular cardiac infarct. All studies were performed by a pathologist in a blind manner.

Assessment of angiogenesis in the infarct site: vascular density was determined from histological sections on day 7 post-infarction by counting the number of vessels within the infarct zone using light microscopy at High Power Field (HPF) (× 400). Six random, non-overlapping HPFs within the infarct were used to count all newly formed vessels in each slice of all tested hearts. The number of vessels in each HPF was averaged and expressed as the number of vessels per HPF.

Assessment of regenerating cardiomyocytes and myocardium: sections of CMF-pretreated MSC-grafts and untreated MSC-graftsThe sections were stained with Ki67 or Myosin Heavy Chain (MHC) antibody at day 7 after ligation to identify regenerated myocardium. Positive staining was visualized with a specific secondary antibody that binds alkaline phosphatase (Santa Cruz). Briefly, paraffin-embedded sections were microwaved in 0.1M EDTA buffer and stained with a 1: 3,000 dilution of a specific polyclonal rabbit antibody against rat Ki67 (Sant Cruz Biotechnology) and incubated overnight at 4 ℃. After washing the sections, they were incubated with goat anti-rabbit IgG secondary antibody (Sigma) conjugated to alkaline phosphatase diluted 1: 200 for 30 minutes and developed with 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine-nitro blue tetrazolium substrate kit (Dako) and positive nuclei were shown to be dark blue. Immediately adjacent sections of the corresponding paraffin tissue blocks were incubated overnight at 4 ℃ in 1: 50 diluted rabbit anti-rat MHC (MF20, university of development Hybridoma Bank, Iowa) antibody and further incubated for 30 minutes at room temperature in 1: 100 diluted peroxidase-conjugated goat anti-rabbit IgG (Sigma). Using 1mg/ml3, 3' -diaminobenzidine (DAB; 0.02% H)₂O₂) After incubation, the slides were observed by microscopic analysis. The regenerated myocardial region is depicted in the projection field by a grid containing 42 sampling points. Approximately, 30-60 calculation points are selected along the edge of a particular regenerating myocardium in each slice. The lattice defines 62,500 μm²For measuring 30-60 calculation points selected in each slice. The shape and volume of the regenerated myocardium in the central region of infarction were determined by measuring the shape of each slice (50 μm apart) of nearly 70 slices and the area occupied by the regenerated myocardium and slice thickness. Using the integral sum of these variables, the spatial structure can be generated and the volume of the specific regenerated myocardium infarcted in the central region in each slice can be obtained. The values and the spatial structure of all slices of a particular tissue mass are added and calculated to obtain the total volume and the entire spatial structure of the regenerated myocardium.

Echocardiographic evaluation of myocardial function: echocardiography studies were performed using a sequoia c256 system (Siemens Medical) with a 15-MHz line array transducer. The chest of the test rat was opened, the animal was placed in a supine position on a hot pad, the ECG limb electrodes were placed, and echocardiograms were recorded under controlled anesthesia. Before the experimental procedure, the echocardiographic baseline of each experimental rat was measured. Two-dimensionally guided M-mode and two-dimensional (2D) echocardiography images are recorded from the parasternal long and short axis views. The size of the Left Ventricle (LV) at the end systole and end diastole, and the wall thickness at systole and diastole were measured from the M-type track using the leading-edge meeting protocol of the American society of Echocardiography. The area of the left ventricle at end diastole (LVDA) and end systole (LVSA) was measured from the parasternal long axis and the volume of the left ventricle at end diastole and end systole (LVEDV and LVESV) was calculated by the M-type method. Left Ventricular Ejection Fraction (LVEF) and shortening Fraction (FS) are derived from the cross-sectional area of the left ventricle in a 2D short axis view: EF ═ LVEDV/LVEDV ] × 100% and FS ═ LVDA-LVSA/LVDA ] × 100%. Standard formulas are used for echocardiography calculations. At the end of the study, all data was analyzed offline with the software in the ultrasound system. All measurements and calculated indicators were averaged over three to five consecutive measurements.

Statistics: all morphometric data were collected blindly and blinded at the end of the experiment. Results are expressed as mean ± SD calculated from mean measurements obtained from each heart. The statistical significance of the comparison between the two measurements was determined using the unpaired two-tailed Student's t test. Values of P < 0.05 were considered significant.

II.CMF-induced increased survival potential and cardiogenic differentiation of isolated MSCs

Referring to FIG. 2, immunocytochemical staining of cells with phospho-Akt 1-specific antibody showed that after two days of CMF (10. mu.g/ml) treatment in culture, the expression of phospho-Akt1 was significantly up-regulated compared to the untreated control, and positive cells stained red were predominantly cytoplasmic (FIG. 2 a: 1). Western blotting confirmed that increased expression of phospho-Akt1 was 3-4 fold greater than in untreated cells (FIG. 2 b: 5A). In MSCs upregulated in phospho-Akt1, more than 90% of them stained positive when cultured for an additional 2 days with myocyte enhancer factor (MEF2) -specific antibodies, one of the early markers of the cardiogenic lineage (cardiac linking), which stained orange in the nucleus (FIG. 2 a: 2) and confirmed by western blot (FIG. 2 b: 6A), indicating the commitment of cardiogenic differentiation. It was noted that cultured MSCs were not all positively stained with anti-MEF 2 antibody (fig. 2 a: 2), as shown by blue nuclei, probably because not all cultured MSCs were converted by CMF via the cardiogenic differentiation pathway, or because there were small amounts of certain impurities in the preparation of MSCs. Similarly, most cultured MSCs were positive when stained with cardiac myosin heavy chain-specific antibody, positive cells stained red in the cytoplasm when cultured in the presence of CMF for 6 days (FIG. 2 a: 3) and confirmed by Western blotting (FIG. 2 b: 7A), whereas control cells were negative when stained with all three specific antibodies (FIG. 2 a: 4&2 b: Bs). The continuous induction of MEF2 and MHC expression confirmed the CMF-induced development of cardiogenic differentiation of ex vivo MSCs. In FIG. 2B, A represents the CMF treated sample and B represents the untreated control.

III.Curative effect of MSC transplantation by CMF pretreatment

To determine whether MSCs treated with CMF in vitro show increased survival potential and cardiogenic differentiation efficiency prior to transplantation that could lead to significant improvement in MI repair in vivo, or in other words, whether the ex vivo CMF effect has any therapeutic value, cell transplantation experiments were performed with an animal model of MI, with MSCs pretreated with CMF implanted into the infarct zone. Homing, survival, proliferation, cardiogenic differentiation and maturation of transplanted cells was followed by immunohistochemical staining of Ki67 and MHC with any DiI fluorescence positive signal in sections from hearts at day 7 and day 14 post-infarction and cell transplantation. As shown in FIG. 3, on day 7, DiI-positive cells having a phenotype characteristic of cardiomyocytes were observed throughout the infarct zone in the test myocardial group (FIG. 3: 1), indicating the source of donor cells and the distribution throughout the infarct zone. In the control group (not treated with CMF), scattered DiI signals were only visible around the infarct border (FIG. 3: 2). Positional overlap of the DiI signal (red) and cardiac-specific troponin I expression (green) was observed throughout the infarct zone using confocal microscopy (FIGS. 3: 3-5). Overlapping images of DiI positive (red) and heart specific marker troponin I expression (green) resulted in overlapping colors of yellow-red-green in the same cells, thus confirming cardiogenic differentiation and maturation of transplanted CMF ex vivo pretreated MSCs in vivo. It was also noted that a few troponin I positive cells (green) were not DiI positive (fig. 3: 3&5), probably because some regenerating myocytes were not produced by the transplanted DiI-labeled MSC. Similarly, a few cells that showed DiI positive in the light blue circle were negative in troponin I immunostaining, indicating that a small fraction of the transplanted cells did not undergo cardiogenic differentiation in vivo, or impurities were present in the preparation of MSCs.

In the experimental group, neovascularization was detected as early as 12 hours post-transplantation, and many newly formed blood vessels and capillaries filled with blood cells were observed throughout the infarct zone within 24 hours post-infarction (before any regenerating cardiomyocytes were visible) and 7 days post-infarction (FIG. 3: 1, yellow circles). On day 7, the density of newly formed vessels in the infarcted area of CMF pretreated MSC-transplanted myocardium was 8 ± 2 per high power field (40 ×) (HPF) on average. However, the nascent blood vessels were not DiI positive, indicating that the cellular source of the blood vessels may not be from the donor cells. It is estimated that donor MSCs may activate by CMF pretreatment to stimulate and upregulate specific signaling pathways inducing angiogenesis of the expression of certain angiogenic factors, which directly enhances the process of early revascularization in infarcted myocardium. In contrast, on day 7, approximately 3. + -.2 vessels per HPF were observed in infarcted myocardium of control group transplanted with non-pretreated MSCs (FIG. 3: 2, yellow circles).

As shown in FIG. 3, a number of myocytes produced by the donor cells were clustered in the infarct zone and composed of myocardium-like tissue, which was positively stained by MHC (FIG. 3: 6, blue circle) and Ki67 (FIG. 3: 7, blue circle) specific antibodies, indicating that these transplanted CMF-pretreated MSCs retain dividing capacity and can undergo cardiogenic differentiation after in vivo transplantation. In high power fields, these myocardium-like tissues showed typical myocardial morphology except that the size was smaller than the intact, pre-existing myocytes (FIG. 3: 9, blue circles). In the test myocardium group, these highly organized regenerated myocardium-like tissues averaged 70. + -.8% of the total infarct volume at day 7 post-infarction, and replaced infarct myocardium areas averaged 80. + -.8.5% of the total infarct volume at day 14 post-infarction (FIG. 3: 8, R, regenerated cardiomyocytes; N, pre-existing normal cardiomyocytes). As shown by echocardiography measurements (figure 4 and table 1), function was significantly improved with replacement of infarcted heart tissue. Ejection Fraction (EF) of MI hearts transplanted with pretreated MSCs was significantly higher at day 2 (59.79 ± 2.33 versus 52.1 ± 2.54, P ═ 0.03) and significantly increased at day 14 (67.13 ± 2.53 versus 53.3 ± 2.31, P ═ 0.001) compared to MI groups transplanted with non-pretreated MSCs at day 2 and day 14 post-infarction. Similarly, the Fractional Shortening (FS) of transplanted MI hearts was significantly high on day 2 (29.43 ± 1.35 versus 24.07 ± 1.47, P ═ 0.01) and increased significantly on day 14 (31.72 ± 2.57vs 23.49 ± 1.99, P ═ 0.002). The significant improvement in EP and FS is a strong reflection of the recovery of cardiomyocyte function (table 1).

Table 1: distribution of Ejection Fraction (EF) and Fractional Shortening (FS). (mean. + -. SE):

16 rats in the normal group, 32 rats in each of the pseudo-ischemic group, CMF pretreatment group and MSC control group.

Phi, 8 rats in the normal group, 16 rats in each of the pseudo-ischemic group, CMF pretreatment group and MSC control group.

EF, P ═ 0.03 on day 2; p-0.001 on day 14, and FS, 0.01 on day 2, and 0.002 on day 14.

Direct efficacy in MI model without pretreated MSC and transplantation

Referring to fig. 5, after direct local injection of CMF in MI model, it was found that the terminal myocardium at the ligation site of the control group (without CMF treatment) became substantially white in visual examination after 2 weeks of infarction (fig. 5: 2) due to ischemic necrosis. In contrast, the same portion of the CMF-treated heart was relatively redder in appearance, probably due to neovascularization (FIG. 5: 1), which was similar to the non-ischemic portion of the heart, and had infarct size significantly smaller than the control heart (FIG. 5: 2). Furthermore, the left ventricular wall of the CMF-treated heart (FIG. 5: 3) was significantly thicker than the control heart (FIG. 5: 4) in the transverse section of the infarct zone. Histological observations showed that the infarct size of CMF-treated hearts (n-8) was on average about 1/3-1/2 times smaller than control hearts (n-8), which was calculated by measuring the infarct volume at the free wall of the left ventricle on day 14 after ligation. In CMF treated hearts, myocyte-like cell clusters, aligned in nearly the same direction as the infarcted myocardium or nearby surviving myocardium, were found to be distributed throughout most of the infarcted area by Masson's trichrome staining (FIG. 5: 5). In contrast, the infarcted area of the control hearts was almost completely occupied by fibrous tissue substitutes, leaving no sites for any possible regeneration of cardiomyocytes (fig. 5: 6). In contrast to the total blue-stained fibrous scar in the control group (FIG. 5: 8), the entire infarcted area in the CMF-treated group was filled with clusters of regenerated myocytes, well-formed, forming a myocardial morphology with little fibrous tissue therein (FIG. 5: 7), in high power fields, although these regenerated myocytes were smaller in size than the adjacent existing myocytes, probably because they were maturing.

Moreover, echocardiography showed that the function of CMF-treated hearts was significantly improved the second day of infarction as structurally integrated regenerated myocardium and reconstituted vasculature replaced infarcted heart tissue, and further improved on day 14, as compared to control hearts, probably due to the growth and maturation of regenerated myocardium and vasculature to repair the infarct.

Efficacy of conditioned Medium induced by CMF-treated MSCs

To determine whether conditioned media containing certain induced production proteins secreted by CMF-activated MSCs would produce a similar effect to MSCs pretreated directly with CMF or by transplantation of CMF to the infarct zone, the conditioned media were tested with MSC cultures and cardiac infarct animal models. Referring to fig. 6a, after 24 hours of treatment with conditioned medium, the proliferation rate of MSCs increased to 120% compared to control medium (fresh growth medium). Referring to FIG. 6b, myocardial regeneration was observed by local injection of conditioned medium into the ischemic region of the MI model. Briefly, many regenerated myocardium and newly formed blood vessel filled with blood cells were visible throughout the infarct zone (FIG. 6 b: 2) compared to the fibrosis replacement in the control group (FIG. 6 b: 1).

V.Regeneration of cellular origin of cardiac muscle after direct CMF treatment

Referring to fig. 7, a study of the MI model with replacement of bone marrow by DiI labeled MSCs provides direct evidence that the cellular origin of the regenerating myocardium is bone marrow-derived MSCs. One week after bone marrow replacement, the above myocardial infarction surgery was performed. At 14 days post-infarction, the entire infarct zone of the CMF-treated heart was found to be fully occupied by DiI-labeled cells, which were mostly absent from any non-infarct zone with pre-existing viable cardiomyocytes. These DiI positive cells clustered together with a myocardium-like morphology but were smaller in size compared to pre-existing myocardium (fig. 7: 1). In contrast, only a few scattered DiI-positive cells were observed along the infarct border zone in control infarcted hearts (fig. 7: 2). To confirm that these well organized DiI positive cells are regenerating myocardium, immunohistochemical experiments were performed using antibodies specific for troponin I and PCNA. It was found that a large number of DiI-positive cells distributed throughout the infarct zone were positively stained by antibodies specific for troponin I (fig. 7: 3) or PCNA (fig. 7: 4). In CMF-treated hearts, these DiI and troponin I or PCNA positive stained cells were organized as myocardium-like tissue throughout the infarct zone. (FIG. 7: 3& 4). At higher fold visual field, these regenerated myocardium-like tissues showed typical myocardial morphology with significant intercalated disk connections between the regenerated myocardium, indicating superstructure maturation of the single regenerated myocardium into integrated myocardium (FIG. 7: 5). Without structural integration between the regenerating myocardium and the pre-existing active myocardium, functional integration and synchronized mechanical activity would not be guaranteed. These regenerated myocardium accounted for an average of 69.3% of the total infarct volume at 14 days post-infarction. In contrast, in the six control hearts, only a few cells were positive for both troponin I and DiI and scattered around the vessels, while the infarcted area was mostly occupied by fibrous scar (FIG. 7: 6). These results demonstrate that CMF-induced regeneration of myocardium is functional and produced by bone marrow MSCs.

IVPreparation of pharmaceutical composition and its use in treating ischemic heart disease in mammals

Once an effective chemical is identified and a partial or substantially pure preparation of the compound is isolated from a natural source (e.g., plant) or chemically synthesized, various pharmaceutical compositions or preparations can be prepared from the partial or substantially pure compound using commercially available or future developed methods. The particular method of preparing pharmaceutical formulations and dosage forms (including, but not limited to, tablets, capsules, injections, syrups) from the compounds is not part of the invention, and one of ordinary skill in the art of the pharmaceutical industry can practice the invention using one or more methods that are already established in the industry. In other words, one of ordinary skill in the art can modify existing conventional methods to make them more suitable for the compounds of the present invention. For example, the patent or patent application databases provided by the USPTO official website contain abundant resources for the preparation of pharmaceutical formulations and products from effective compounds. Another useful information resource is the Handbook of pharmaceutical manufacturing Formulations, compiled by Sarfaraz K.Niazi and sold by Culinary & Hospital Industry Publications Services.

The term "plant extract" as used in the present description and claims refers to a mixture of naturally occurring compounds obtained from plant parts, wherein at least 10% of the compounds of the total dry mass are unidentified compounds. In other words, the plant extract does not include the identified substantially pure compounds from the plant.

The term "pharmaceutical excipient" refers to a non-pharmaceutically active ingredient contained in a pharmaceutical formulation. The term "effective amount" refers to an amount sufficient to produce a therapeutic effect on the subject being treated. As will be appreciated by those skilled in the art, the effective dosage may vary depending on the type of condition being treated, the route of administration, the use of excipients, and the possibility of co-use with other therapeutic treatments. One skilled in the art can determine the effective amount in a particular situation.

While there have been described and pointed out fundamental novel features of the invention as applied to a preferred embodiment thereof, it will be understood that various omissions and substitutions and changes in the form and details of the embodiments illustrated, and in their operation, may be made by those skilled in the art without departing from the spirit of the invention. The invention is not limited to the above-described embodiments described by way of example, but can be modified in various ways within the scope of the appended patent claims.

Reference to the literature

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Claims (9)

1. Use of an effective amount of a compound of the formula in the preparation of a composition for regenerating myocytes or myocardium in the heart of a mammalian subject suffering from myocardial injury.

2. The use of claim 1, wherein the damaged myocardium is caused by an ischemic event.

3. The use of claim 2, wherein the ischemic event is myocardial infarction.

4. The use of claim 1, wherein the myocyte or myocardium is regenerated by use comprising one or more of the following steps: (a) increasing the viability of myogenic precursor cells to enable said precursor cells to survive an absolute ischemic period; (b) (ii) reconstructing a disrupted blood supply network in the region of the heart in which the damaged muscle is located; and (c) enhancing the efficiency of cardiogenic differentiation of said precursor cells along a cardiac lineage, either simultaneously or in any particular order.

5. The use of claim 4, wherein said myogenic precursor cells are mesenchymal stem cells from bone marrow via blood circulation.

6. The use of claim 1, wherein the composition is contacted with a plurality of stem cells to be implanted into infarcted or damaged cardiac tissue of the mammalian subject.

7. The use of claim 1, wherein said composition is prepared in a dosage form that can be systemically administered to said mammalian subject.

8. The use of claim 7, wherein said dosage form is selected from the group consisting of tablets, capsules, injections, syrups, suspensions, and powders.

9. The use of claim 1, wherein said composition is comprised in a culture medium for culturing a plurality of MSCs or endothelial cells for a period of time, thereby obtaining a culture medium containing secreted proteins from said MSCs or endothelial cells, wherein said culture medium containing secreted proteins from said MSCs or endothelial cells is administered or delivered to cardiac tissue in an infarct area.