CN118453827A - Anti-fibrosis treatment method - Google Patents

Anti-fibrosis treatment method Download PDF

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CN118453827A
CN118453827A CN202310083616.9A CN202310083616A CN118453827A CN 118453827 A CN118453827 A CN 118453827A CN 202310083616 A CN202310083616 A CN 202310083616A CN 118453827 A CN118453827 A CN 118453827A
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cadherin
fibrosis
cardiac fibrosis
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Abstract

The present invention relates to a method of anti-fibrotic treatment, in particular, the present invention provides the use of an N-cadherin inhibitor or antagonist for the preparation of a composition or formulation for the prevention and/or treatment of cardiac fibrosis caused by post-stress loading, which is found for the first time to be effective in preventing and/or treating cardiac fibrosis caused by post-stress loading by inhibiting the expression and/or activity of N-cadherin.

Description

Anti-fibrosis treatment method
Technical Field
The invention relates to the field of biological medicine. In particular, the invention relates to an anti-fibrotic therapeutic method.
Background
Heart failure is the final stage of heart disease caused by various factors, and heart fibrosis is a common pathological feature in various heart diseases, and excessive heart fibrosis seriously affects myocardial contraction and relaxation functions, often accompanied by severe heart failure, and is the key point and difficulty of heart failure prevention and treatment in clinic at present. In ventricular remodeling due to pressure afterload such as hypertension and obstructive cardiomyopathy, cardiac fibrosis is a major pathological change, and a large number of fibroblasts are activated and extracellular matrix deposition causes a decrease in cardiac compliance, resulting in diastolic dysfunction, and a decrease in myocardial function when progressing to the late stage, and at the same time, causes systolic dysfunction.
The energy of systole is stored in the heart muscle as potential energy, and the release of this potential energy causes the heart muscle to lengthen in early diastole, thereby creating diastole suction which draws blood into the heart chamber. Some of the energy is always lost (as heat) due to friction, but the amount of energy lost increases with fibrosis. Furthermore, increased myocardial stiffness can impede subsequent passive inflow and cause the diastolic blood flow to be rearward, further compromising early filling. Thus, most of the filling depends on the final atrial contraction and the total diastole becomes less efficient. The accumulation of large amounts of collagen surrounds the myocardium, particularly under the endocardium, and limits the stretching of diastole cardiomyocytes, thereby reducing the production of length-dependent forces in systole.
At present, no effective treatment means for cardiac fibrosis is available, so that the proliferation of cardiac fibroblasts can be inhibited, the progress of cardiac fibrosis can be inhibited, symptomatic treatment can be only adopted for the type of cardiac diseases of patients, the cardiac function can be maintained, and the effect of delaying the fibrosis can be achieved by delaying the progress of the diseases. Certain treatments for heart failure may act to some extent to inhibit the progression of cardiac fibrosis.
At present, no medicine or treatment means related to fibroblast proliferation and differentiation exists clinically, and the progress of heart fibrosis can be directly reduced.
Thus, there is a great need in the art to develop a new therapeutic approach to anti-cardiac fibrosis.
Disclosure of Invention
The present invention aims to provide a novel anti-cardiac fibrosis treatment method.
It is another object of the present invention to provide an N-cadherin antagonist which inhibits or neutralizes the activity of N-cadherin for use in the treatment or prevention of fibrosis.
In a first aspect of the invention there is provided the use of an N-cadherin inhibitor or antagonist for the preparation of a composition or formulation for use in the prevention and/or treatment of cardiac fibrosis.
In another preferred embodiment, the N-cadherin inhibitor or antagonist is selected from the group consisting of: a small molecule compound, an antibody, a polypeptide, a nucleic acid, or a combination thereof.
In another preferred embodiment, the N-cadherin inhibitor or antagonist comprises Exherin (ADH-1) or an analog thereof.
In another preferred embodiment, the composition or formulation is further used for one or more uses selected from the group consisting of:
(e) Inhibiting formation, aggregation, migration and proliferation of fibroblast links;
(f) Inhibit formation of heterogeneous cell connection of fibroblast and vascular endothelial cell, thereby relieving heart
Visceral interstitial fibrosis;
(g) Inhibiting aggregation, migration and proliferation of fibroblasts co-cultured with vascular endothelial cells;
(h) Improving cardiac dysfunction caused by stress and load, and relieving fibrosis.
In another preferred embodiment, the composition comprises a pharmaceutical composition.
In another preferred embodiment, the pharmaceutical composition contains (a) an N-cadherin inhibitor or antagonist; and (b) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid, solid, or semi-solid.
In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a tablet, a granule, a capsule, an oral liquid, or an injection.
In another preferred embodiment, said component (a) comprises 1 to 99wt%, preferably 10 to 90wt%, more preferably 30 to 70wt% of the total weight of said pharmaceutical composition.
In another preferred embodiment, the composition further comprises other agents for preventing and/or treating cardiac fibrosis.
In another preferred embodiment, the additional agent for preventing and/or treating cardiac fibrosis is selected from the group consisting of: pirfenidone, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, beta receptor antagonists, or combinations thereof.
In another preferred embodiment, the angiotensin converting enzyme inhibitor comprises enalapril maleate.
In another preferred embodiment, the angiotensin II receptor blocker comprises losartan, sha Kuba norvalsartan.
In another preferred embodiment, the beta receptor antagonist comprises metoprolol tartrate.
In another preferred embodiment, the compositions or formulations may be used alone or in combination in the prevention and/or treatment of cardiac fibrosis.
In another preferred embodiment, the combination comprises: used in combination with other drugs for preventing and/or treating cardiac fibrosis.
In a second aspect, the invention provides a pharmaceutical composition comprising:
(a1) A first active ingredient for use in the prevention and/or treatment of cardiac fibrosis, the first active ingredient comprising: n-cadherin inhibitors or antagonists;
(a2) Optionally, a second active ingredient for preventing and/or treating cardiac fibrosis, the second active ingredient comprising: other drugs for preventing and/or treating cardiac fibrosis; and
(B) A pharmaceutically acceptable carrier.
In another preferred embodiment, the component (a 1) comprises 1 to 99wt%, preferably 10 to 90wt%, more preferably 30 to 70wt% of the total weight of the pharmaceutical composition.
In another preferred embodiment, the component (a 2) comprises 1 to 99wt%, preferably 10 to 90wt%, more preferably 30 to 70wt% of the total weight of the pharmaceutical composition.
In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.
In another preferred embodiment, the additional agent for preventing and/or treating cardiac fibrosis is selected from the group consisting of: pirfenidone, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, beta receptor antagonists, or combinations thereof.
In another preferred embodiment, the pharmaceutical composition may be a single compound or a mixture of compounds.
In another preferred embodiment, the pharmaceutical composition is used for the preparation of a medicament or formulation for the treatment or prevention of cardiac fibrosis.
In another preferred embodiment, the pharmaceutical dosage form is an oral or non-oral dosage form.
In another preferred embodiment, the oral administration form is a tablet, powder, granule or capsule, or an emulsion or syrup.
In another preferred embodiment, the non-oral administration form is an injection or an injection.
In another preferred embodiment, the total content of active ingredient (a 1) and active ingredient (a 2) is from 1 to 99% by weight, more preferably from 5 to 90% by weight, based on the total weight of the composition.
In a third aspect the present invention provides a kit comprising:
(i) A first container, and an active ingredient (a 1) an N-cadherin inhibitor or antagonist, or a medicament containing an active ingredient (a), located in the first container; and
(Ii) An optional second container, and an active ingredient (a 2) other medicament for preventing and/or treating cardiac fibrosis or a medicament containing the active ingredient (a 2) located in the second container.
In another preferred embodiment, the first container and the second container are the same or different containers.
In another preferred embodiment, the first container of medicament is a single formulation comprising an N-cadherin inhibitor or antagonist.
In another preferred embodiment, the medicament of the second container is a single formulation containing other medicaments for preventing and/or treating cardiac fibrosis.
In another preferred embodiment, the pharmaceutical is in the form of an oral dosage form or an injectable dosage form.
In another preferred embodiment, the kit further comprises instructions describing the administration of active ingredient (a 1) in combination with active ingredient (a 2) for the prevention and/or treatment of cardiac fibrosis.
In another preferred embodiment, the dosage form of the preparation containing the active ingredient (a 1) N-cadherin inhibitor or antagonist or containing other drugs for preventing and/or treating cardiac fibrosis comprises a capsule, a tablet, a suppository, or an intravenous injection, respectively.
In a fourth aspect, the present invention provides the use of a pharmaceutical composition according to the second aspect of the invention or a kit according to the third aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of cardiac fibrosis.
In a fifth aspect, the present invention provides a method for preventing and/or treating cardiac fibrosis, comprising the steps of:
administering to a subject in need thereof an N-cadherin inhibitor or antagonist, a pharmaceutical composition according to the second aspect of the invention, or a kit according to the third aspect of the invention.
In another preferred embodiment, said administration comprises oral administration.
In another preferred embodiment, the subject comprises a human or non-human mammal.
In another preferred embodiment, the non-human mammal comprises a rodent and primate, preferably a mouse, rat, rabbit, monkey.
In another preferred embodiment, the N-cadherin inhibitor or antagonist is administered at a frequency of 1-7 consecutive days per week, preferably 2-5 consecutive days per week, more preferably 2-3 consecutive days per week.
In another preferred embodiment, the N-cadherin inhibitor or antagonist is administered for a period of time of 1-20 weeks, preferably 2-12 weeks, more preferably 4-8 weeks.
In a sixth aspect, the invention provides a method of screening for a candidate drug for treating cardiac fibrosis, comprising the steps of:
(a) Culturing cells expressing N-cadherin in a culture system in the presence of a test substance for a period of time T1 in a test set, and detecting the expression level E1 and/or activity A1 of N-cadherin in the culture system in the test set;
and detecting the expression level E2 and/or activity A2 of N-cadherin in the culture system of a control group in the absence of the test substance and under otherwise identical conditions;
(b1) Comparing E1 and E2, if E1 is significantly lower than E2, indicating that the test substance is a candidate drug for treating cardiac fibrosis; and/or
(B2) Comparing A1 to A2, if A1 is significantly lower than A2, it indicates that the test substance is a candidate drug for treating cardiac fibrosis.
In another preferred embodiment the cell is a mammalian cell.
In another preferred embodiment, the cell is selected from the group consisting of: cardiac microvascular endothelial cells, cardiac fibroblasts, or a combination thereof.
In another preferred embodiment, the cells are cells cultured in vitro.
In another preferred embodiment, the term "substantially lower" means E1/E2.ltoreq.1/2, preferably.ltoreq.1/3, more preferably.ltoreq.1/4.
In another preferred embodiment, the term "significantly lower" means A1/A2.ltoreq.1/2, preferably.ltoreq.1/3, more preferably.ltoreq.1/4.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In another preferred embodiment, the method comprises step (c): administering the candidate agent determined in step (a) to a non-human mammal, thereby determining its effect on cardiac fibrosis in the non-human mammal.
In another preferred embodiment, the test substance is selected from the group consisting of: a small molecule compound, an antibody, a polypeptide, a nucleic acid, or a combination thereof.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows that endothelial cells form N-Cadherin-based cell links with fibroblasts in a stress post-load induced heart fibrosis model.
FIG. 2 shows the effect of in vitro application of N-cadherin inhibitor (ADH-1) on endothelial cell and fibroblast link formation and fibroblast proliferation.
FIG. 3 shows the in vivo use of N-cadherein inhibitor (ADH-1) to examine its effect on diastolic dysfunction and cardiac fibrosis due to stress afterloading.
Detailed Description
The present inventors have made extensive and intensive studies and have unexpectedly found that an N-cadherin inhibitor or antagonist is effective in preventing and/or treating cardiac fibrosis for the first time. On this basis, the present inventors have completed the present invention.
Cardiac fibrosis
The pharmaceutical composition containing the active ingredient of the invention has remarkable inhibiting activity on cardiac fibrosis.
Cardiac fibrosis refers to the expansion of the matrix due to the accumulation of matrix proteins in the interstitial space of the heart, which is accompanied by most of the pathological processes of the heart, and is closely related to the adverse effects such as reduced compliance of the ventricular wall, diastolic dysfunction, arrhythmia and the like. Systemic hypertension and cardiac pressure afterload lead to progressive deposition of matrix proteins in the cardiac matrix and perivascular space, increasing ventricular wall stiffness and leading to diastolic dysfunction. Cardiac fibrosis is an important link in pathological remodeling of the heart, affecting disease prognosis.
In the present invention, the post-stress compliance with heart fibrosis-related diseases are not particularly limited, and include various diseases known in the art related to post-stress compliance with heart fibrosis. Representative examples of diseases associated with stress post-load cardiac fibrosis include (but are not limited to): hypertension, hypertrophic obstructive cardiomyopathy, aortic valve stenosis, aortic stenosis, pulmonary artery stenosis, pulmonary arterial hypertension, and the like.
N-cadherin
(N-cadherein) is a calcium ion-dependent single channel transmembrane glycoprotein that mediates homotypic and heterotypic intercellular adhesion and plays an important role in the development and functional regulation of the nervous system, brain, heart, skeletal muscle, blood vessels. Upregulation of N-cadherin expression in dermal fibrotic scar formation is necessary for fibroblast-like migration and focal aggregation, blocking N-cadherin cell link formation inhibits fibroblast aggregation, skin contraction and reduces dermal scar formation. There is no study or report on its use in the treatment of cardiac fibrosis, and inhibition of its action may exert an effect of treating or preventing cardiac fibrosis.
N-cadherin inhibitors or antagonists
As used herein, an "N-cadherin inhibitor or antagonist" is a small or large molecule compound that is specific for N-cadherin and inhibits or antagonizes its function of promoting endothelial cell and fibroblast link formation, promoting fibroblast proliferation, migration, and aggregation.
In a preferred embodiment, the N-cadherin inhibitor or antagonist includes, but is not limited to: exherin (ADH-1), anti-N-cadherin blocking antibodies, and other short synthetic linear peptides containing the cadherin CAR sequence of type I.
Exherin (ADH-1) of the formula: c 22H34N8O6S2, the structural formula of which is as follows:
Pharmaceutical compositions and methods of administration
In another aspect, the present invention also provides a pharmaceutical composition comprising (a) a safe and effective amount of an N-cadherin inhibitor or antagonist of the present invention; and (b) a pharmaceutically acceptable carrier or excipient. The severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. In general, satisfactory results are obtained when the active ingredient of the present invention is administered daily at a dose of about 0.00001mg to 50mg/kg of animal body weight (preferably 0.0001mg to 25mg/kg of animal body weight) (25 mg/kg of experimental mice). For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition. In addition, the N-cadherin inhibitors or antagonists of the invention may be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such agent carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and do not have excessive toxicity after administration. Such vectors are well known to those of ordinary skill in the art. A sufficient discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub.Co., N.J.1991). Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
The pharmaceutically acceptable carrier in the therapeutic composition may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
In general, the therapeutic compositions may be formulated as an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for incorporation into a solution or suspension, and a liquid carrier prior to injection.
Once formulated into the compositions of the present invention, they may be administered by conventional routes including, but not limited to: intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or topical administration. The subject to be prevented or treated may be an animal; especially humans.
When the pharmaceutical composition of the present invention is used for actual treatment, various different dosage forms of the pharmaceutical composition can be employed according to the use condition. Preferably a vein the preparation is used for treating the disease.
These pharmaceutical compositions may be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and cosolvents, and the formulation process may be carried out in a conventional manner according to dosage forms.
For example, the ophthalmic solution may be formulated as follows: the N-cadherin inhibitor or antagonist of the present invention is dissolved in sterile water (in which a surfactant is dissolved) together with a base substance, osmotic pressure and ph are adjusted to physiological states, and appropriate pharmaceutical additives such as a preservative, a stabilizer, a buffer, an isotonic agent, an antioxidant and a tackifier are optionally added, and then completely dissolved.
The pharmaceutical compositions of the present invention may also be administered in the form of a slow release formulation. For example, the N-cadherin inhibitors or antagonists of the invention may be incorporated into a pellet or microcapsule that is supported on a slow release polymer, which is then surgically implanted into the tissue to be treated. As examples of the sustained-release polymer, there may be exemplified ethylene-vinyl acetate copolymer, polyhydroxymethacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like, and preferably there may be exemplified biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer.
When the pharmaceutical composition of the present invention is used for actual treatment, the dosage of the N-cadherin inhibitor or antagonist of the present invention as an active ingredient can be appropriately determined according to the weight, age, sex, and symptom degree of each patient to be treated.
The main advantages of the invention include:
(1) The present invention for the first time has found that cardiac fibers can be effectively prevented and/or treated by inhibiting the expression and/or activity of N-cadherin.
(2) The invention discovers for the first time that the invention can inhibit the formation of heterogeneous cell connection of fibroblast by inhibiting N-cadherein of various cells of heart, inhibit proliferation, migration and aggregation of the fibroblast and inhibit active fibrosis reaction.
(3) The present invention provides a novel method for treating cardiac fibrosis with active fibrotic response.
(4) The method has the advantages of simple operation, simple preparation of the medicine and high efficiency.
(5) The invention may be used in other methods of inhibiting N-cadherein such as gene-targeted therapy or cell-targeted therapy.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Unless otherwise specified, materials and reagents used in the examples of the present invention are commercially available products.
Example 1
In a stress afterload induced cardiac fibrosis model, endothelial and fibroblast cells form N-Cadherin-based cell links
1.1 Endothelial cell fibroblast link formation in cardiac pathological fibrosis: aortic arch ligation of mice induced cardiac pathologic fibrosis (ref: DEALMEIDA AC ET al.J Vis Exp.2010;21 (38): 1729.), cardiac tissue sections, immunofluorescent staining. The results showed a significant increase in endothelial cell lineage markers co-labelling with the myofibroblast surface marker Vimentin and the myofibroblast marker a-SMA (fig. 1A). Indicating an increase in endothelial fibroblast link formation during cardiac fibrosis with post-stress loading, endothelial and fibroblast link formation may play a role in cardiac fibrosis.
1.2 Significant upregulation of N-cadherein expression during pathologic fibrosis in the heart: the isolated mouse heart endothelial cells after TAC surgery found a significant increase in cadherin-N (N-cadherin) expression (fig. 1B), while immunofluorescence co-labeling staining showed a significant increase in heart fibrosis site endothelial cell and fibroblast link site N-cadherin expression after TAC modeling (fig. 1C). The results suggest that increased endothelial and fibroblast markers co-labeling in cardiac fibrosis may be due to the formation of N-cadherein-based cellular links between the two.
1.3 In vitro cell experiments. N-cadherein expression was significantly up-regulated in mouse cardiac microvascular endothelial cells (mCMVECs) following TGF-beta 1 stimulation (FIG. 1D). Further co-culture of mCMVECs with Mouse Cardiac Fibroblasts (MCFs) (fig. 1E) revealed that adhesion of the fibroblast MCFs to endothelial cells mCMVECs after tgfβ1 stimulation was significantly increased and that most of the endothelial to fibroblast inter-links were positively stained with N-cadherein fluorescence (fig. 1F).
And (3) result prompting: n-cadherin is involved in endothelial cell fibroblast link formation, fibroblast aggregate migration and focal aggregation, and blocking N-cadherin can block cell link formation, inhibit fibroblast aggregation, and possibly inhibit fibrosis progression.
Example 2
In vitro application of N-cadherin inhibitor (ADH-1) to detect its effect on endothelial cell and fibroblast link formation and fibroblast proliferation
Murine cardiac microvascular endothelial cells (mCMVECs) (from SCIENCELL RESEARCH Laboratories, catalog#6000) were co-cultured with Murine Cardiac Fibroblasts (MCF) (from SCIENCELL RESEARCH Laboratories, catalog#M 6300-57) experiments: after mCMVECs h stimulation with PBS/TGF beta, mCMVECs was mixed with MCF at 1:2 (7 ten thousand: 14 ten thousand cells) are spread in a 6-hole plate in proportion, endothelial cells and fibroblasts are separated by magnetic bead separation after co-culture for 24 hours, and the expression condition of the endothelial cells N-cadherein is detected by western blot. The results showed a significant increase in N-cadherein expression in mCMVECs stimulated with TGF-beta compared to the unstimulated group (FIG. 2A);
2.2 murine cardiac microvascular endothelial cells (mCMVECs) and Murine Cardiac Fibroblasts (MCF) co-culture adhesion experiments: after mCMVECs was stimulated with tgfβ for 24 hours, mCMVECs was combined with MCF at 1:2 (7 ten thousand: 14 ten thousand cells were counted) in 6-well plates, and after adding N-cadherein inhibitor (ADH-1) and co-culturing for 24 hours, the cell slide was taken out for stationary staining. The results show that: the formation of links between endothelial cells (CD 31-Red) and fibroblasts (MCF-Green) after TGF-beta stimulation was found to be reduced by adding N-cadherin inhibitor (ADH-1) during co-culture, and the adhesion of endothelial cells to fibroblasts was effectively reduced by TGF-beta stimulation (FIGS. 2B, C).
2.3 Murine cardiac microvascular endothelial cells (mCMVECs) co-culture with Murine Cardiac Fibroblasts (MCFs) fibroblast proliferation experiments: after mCMVECs was stimulated with tgfβ for 24 hours, mCMVECs was combined with MCFs at 1:2 (7 ten thousand: 14 ten thousand cells were counted) were plated in 6-well plates, and after 24 hours of co-culture with N-cadherein inhibitor (ADH-1), EDU was added to the plates to react for 2 hours, and then the cell slides were taken out to fix and stain. The results show that: endothelial cells (CD 31-Red) and fibroblasts (MCF-Green) after TGF-beta stimulation were able to promote fibroblast proliferation, and by adding N-cadherin inhibitor (ADH-1) during co-culture, it was found that the number of fibroblast proliferation was reduced, and N-cadherin inhibitor (ADH-1) was able to effectively reduce endothelial cell-promoted fibroblast proliferation under TGF-beta stimulation (FIGS. 2D, E).
Example 3
In vivo application of N-cadherein inhibitor Exherin (ADH-1) for relieving diastolic dysfunction and cardiac fibrosis caused by pressure overload
3.1 Mice aortic stenosis pressure overload mouse heart fibrosis model constructed: mouse heart fibrosis model induction by aortic arch constriction surgical model (TAC) establishment: after the mice are anesthetized, the trachea cannula is connected with an upper breathing machine in parallel, and the upper end of the chest is cut off to a second rib along the midsternum; after the thymus was opened with forceps, the aortic arch step was carefully isolated; threading (6-0) silk thread on aortic arch, placing 271/2G needle between the silk thread and artery, rapidly drawing out the needle after ligation, suturing sternum and chest skin, performing heart ultrasonic detection and sampling on 3,6,9,12 days after operation of mice by injecting N-cadherin inhibitor (ADH-1) into tail vein on 14 days;
3.2 in vivo application of N-cadherin inhibitors to alleviate diastolic dysfunction resulting from pressure overload: the mice were examined for changes in cardiac ejection function and diastolic function by imaging and functioning with cardiac ultrasound (Vevo 2100, visual sonics) of the mice before and after molding. The results show that: following Adh-1 administration, TAC-induced diastolic dysfunction was significantly reversed (FIGS. 3A-E).
3.3 In vivo application of N-cadherin inhibitors to relieve cardiac fibrosis due to pressure overload: mice were sacrificed at the corresponding time points after molding, hearts were weighed, pathological sections were performed in parallel, and heart fibrosis was observed by Masson's staining. The results show that Adh-1 was able to significantly reverse TAC-induced cardiac fibrosis (FIGS. 3F-G), indicating that N-cadherein inhibitor (ADH-1) has an inhibitory effect on cardiac fibrosis.
3.4N-cadherein inhibitor (ADH-1) study of the mechanism of inhibiting fibrosis and downstream Signal pathway: fibroblast can promote proliferation and migration of cells through a membrane FGF receptor 1, so that the fibrosis process of the heart is regulated and controlled, rat heart microvascular endothelial cells (mCMVECs) are co-cultured with rat heart fibroblasts (MCFs), endothelial cells and fibroblasts are separated through magnetic bead sorting after 24 hours, and western blot detection of the expression condition of the FGF receptor 1 (FGFR 1) of the fibroblasts shows that the application of ADH-1 can reduce the expression of FGFR1 obviously related to the proliferation of the fibroblasts, and the expression of FGFR1 of the fibroblasts is possibly reduced to participate in regulating and controlling heart fibrosis (figure 3H).
Reference to the literature
1.deAlmeida AC,van Oort RJ,Wehrens XH.Transverse aortic constriction in mice.J Vis Exp.2010Apr 21;(38):1729.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (10)

1. Use of an N-cadherin inhibitor or antagonist for the preparation of a composition or formulation for the prevention and/or treatment of cardiac fibrosis.
2. The use according to claim 1, wherein the N-cadherin inhibitor or antagonist is selected from the group consisting of: a small molecule compound, an antibody, a polypeptide, a nucleic acid, or a combination thereof.
3. The use according to claim 1, wherein the N-cadherin inhibitor or antagonist comprises Exherin (ADH-1) or an analogue thereof.
4. The use according to claim 1, wherein the composition or formulation is further for one or more uses selected from the group consisting of:
(a) Inhibiting formation, aggregation, migration and proliferation of fibroblast links;
(b) Inhibit the formation of heterogeneous cell connection of fibroblast and vascular endothelial cell, thereby alleviating cardiac interstitial fibrosis;
(c) Inhibiting aggregation, migration and proliferation of fibroblasts co-cultured with vascular endothelial cells;
(d) Improving cardiac dysfunction caused by stress and load, and relieving fibrosis.
5. A pharmaceutical composition, in the form of a capsule, characterized by comprising the following steps:
(a1) A first active ingredient for use in the prevention and/or treatment of cardiac fibrosis, the first active ingredient comprising: n-cadherin inhibitors or antagonists;
(a2) Optionally, a second active ingredient for preventing and/or treating cardiac fibrosis, the second active ingredient comprising: other drugs for preventing and/or treating cardiac fibrosis; and
(B) A pharmaceutically acceptable carrier.
6. The pharmaceutical composition according to claim 5, wherein the other drug for preventing and/or treating cardiac fibrosis is selected from the group consisting of: pirfenidone, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, beta receptor antagonists, or combinations thereof.
7. A medicine box, which comprises a medicine box body, characterized by comprising the following steps:
(i) A first container, and an active ingredient (a 1) an N-cadherin inhibitor or antagonist, or a medicament containing an active ingredient (a), located in the first container; and
(Ii) An optional second container, and an active ingredient (a 2) other medicament for preventing and/or treating cardiac fibrosis or a medicament containing the active ingredient (a 2) located in the second container.
8. Use of a pharmaceutical composition according to claim 5 or a kit according to claim 7 for the preparation of a medicament for the prevention and/or treatment of cardiac fibrosis.
9. A method of screening for a candidate agent for treating cardiac fibrosis, comprising the steps of:
(a) Culturing cells expressing N-cadherin in a culture system in the presence of a test substance for a period of time T1 in a test set, and detecting the expression level E1 and/or activity A1 of N-cadherin in the culture system in the test set;
and detecting the expression level E2 and/or activity A2 of N-cadherin in the culture system of a control group in the absence of the test substance and under otherwise identical conditions;
(b1) Comparing E1 and E2, if E1 is significantly lower than E2, indicating that the test substance is a candidate drug for treating cardiac fibrosis; and/or
(B2) Comparing A1 to A2, if A1 is significantly lower than A2, it indicates that the test substance is a candidate drug for treating cardiac fibrosis.
10. The method of claim 9, wherein the cells are selected from the group consisting of: cardiac microvascular endothelial cells, cardiac fibroblasts, or a combination thereof.
CN202310083616.9A 2023-02-08 2023-02-08 Anti-fibrosis treatment method Pending CN118453827A (en)

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CN202310083616.9A CN118453827A (en) 2023-02-08 2023-02-08 Anti-fibrosis treatment method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310083616.9A CN118453827A (en) 2023-02-08 2023-02-08 Anti-fibrosis treatment method

Publications (1)

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CN118453827A true CN118453827A (en) 2024-08-09

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