CN116159061A - Application of pyruvate kinase M2 activator in preparation of medicines for preventing or treating heart failure - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and provides a novel pharmaceutical application of a pyruvate kinase M2 activator TEPP-46. According to the invention, the TEPP-46 can obviously improve the heart function, ventricular remodeling and fibrosis level of a heart failure model mouse induced by pressure load. Therefore, TEPP-46 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer or prodrug thereof is hopefully a therapeutic small molecule drug targeting PKM2 protein for heart failure metabolism, so that the therapeutic small molecule drug is converted from a low-activity dimer form to a high-activity tetramer form, and myocardial cell metabolism level is improved, thereby preventing and/or treating heart failure.

Description

Application of pyruvate kinase M2 activator in preparation of medicines for preventing or treating heart failure

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of a pyruvate kinase M2 activator in preparation of a medicament for preventing or treating heart failure.

Background

Mortality from heart failure has increased year by year due to the complex interactions of environmental and genetic risk factors. Heart failure can be classified as a change in ejection fraction: heart failure with reduced ejection fraction and heart failure with retained ejection fraction. Heart failure is manifested clinically by a systolic/diastolic dysfunction of the left ventricle, an elevated pressure load on the left ventricle, and an impaired local systolic function. The pathophysiological changes are associated with a number of determinants, including abnormalities in extracellular matrix, mitochondrial homeostasis, inflammation, cardiomyocyte stiffness, etc. Although many molecular pathways can lead to changes in the organism at the organ or tissue level, there is still a lack of successful therapies due to the complexity of the gene regulatory network. The mitochondrial metabolism level in heart tissue of heart failure patient is obviously lower than that of normal heart tissue, and some therapeutic means for improving myocardial mitochondrial metabolism and improving mitochondrial morphology have obtained good therapeutic effect in mice heart failure model. It is shown that mitochondrial morphology and metabolic levels may play an important role in the progression of heart failure, however, drugs targeting mitochondrial metabolism are rarely used in clinical treatment of heart failure.

TEPP-46 (ML-265) is a selective activator of pyruvate kinase M2 (pyruvate kinase M; PKM 2) with an AC50 value of 92nM and little or no effect on PKM1, PKL and PKR. TEPP-46 activates PKM2 by a mechanism similar to that of the endogenous activator FBP. TEPP-46 pretreatment of cells prevents pervanadate-induced inhibition of PKM2 activity.

Disclosure of Invention

The invention aims to provide a novel pharmaceutical application of a pyruvate kinase M2 activator (TEPP-46).

Specifically, the invention provides application of a pyruvate kinase M2 activator in preparing a medicament for preventing or treating heart failure, wherein the pyruvate kinase M2 activator is TEPP-46, the CAS number of the TEPP-46 is 1221186-53-3, and the molecular formula of the TEPP-46 is C ₁₇ H ₁₆ N ₄ O ₂ S ₂ The structural formula is

The heart failure refers to the condition that the systolic function and/or the diastolic function of the heart are impaired, venous return blood volume cannot be sufficiently discharged out of the heart, venous system blood is accumulated, and arterial system blood is not filled enough, so that heart circulatory disorder syndrome is caused.

Wherein, the medicine for preventing or treating heart failure takes TEPP-46 as the only active ingredient, or also comprises pharmaceutically acceptable auxiliary materials.

Wherein, the dosage form of the medicine for preventing or treating heart failure is oral preparation, pulmonary inhalation preparation, mucosa administration preparation, ophthalmic preparation or injection; the oral preparation is one or more of granule, powder, pill, tablet, capsule and oral liquid.

In the medicament for preventing or treating heart failure, TEPP-46 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer or prodrug thereof is targeted to myocardial cells.

The invention also provides a medicament for preventing or treating heart failure, which comprises a pyruvate kinase M2 activator, wherein the pyruvate kinase M2 activator is TEPP-46, the CAS number is 1221186-53-3, and the molecular formula is C ₁₇ H ₁₆ N ₄ O ₂ S ₂ The structural formula is

According to the invention, the study on the heart function of the heart failure model mouse induced by the pressure load is carried out by the TEPP-46, and the experiment proves that the heart function, the ventricular remodeling and the fibrosis level of the heart failure model mouse induced by the pressure load can be obviously improved by the TEPP-46. Therefore, TEPP-46 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer or prodrug thereof is hopefully a therapeutic small molecule drug targeting PKM2 protein for heart failure metabolism, so that the therapeutic small molecule drug is converted from a low-activity dimer form to a high-activity tetramer form, and myocardial cell metabolism level is improved, thereby preventing and/or treating heart failure.

Drawings

FIG. 1 is a schematic diagram of ultrasound of a mouse heart;

FIG. 2 is a graph showing the statistical results of the systolic function parameters of ejection fraction EF, shortening fraction FS, left ventricular diastolic inner diameter LVID (d), and left ventricular systolic inner diameter LVID(s) in the cardiac superconductivity of mice;

FIG. 3 is a schematic representation of MASSON fibrosis staining;

FIG. 4 is a graph of statistical results of the proportion of fibrotic regions to total area of the visual field in MASSON fibrosis staining;

FIG. 5 is a graph of statistical results of the ratio of heart weight to body weight of mice;

FIG. 6 is a graph of Western Blot results of monomeric, dimeric, tetrameric forms of PKM 2;

fig. 7 is a statistical plot of the magnitude of increases in PKM2 tetramer form following TEPP46 administration.

Detailed Description

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

TEPP-46 heart failure model mouse heart function experiment for improving pressure load induction

1. Material

1. Experimental animal or material sources and treatments:

the experimental animal was a C57 mouse from velocilla.

2. Medicine and reagent:

the PKM2 protein primary antibody and the donkey anti-rabbit and donkey anti-mouse secondary antibody are Abcam brands.

3. Instrument:

mouse heart ultrasound was purchased from Vevo sonic Inc., and electrophoresis apparatus was purchased from Berle Inc.

4. The experimental steps are as follows:

4.1 establishment of a stress-induced mice heart failure model (TAC model):

1) Male mice were selected for 8-10 weeks, and dehaired for 24h before molding.

2) 100 microliters of 1% pentobarbital is injected into abdominal cavity for anesthesia, and when the mice do not respond to the pain stimulus of lower limbs and breathe (thoracic cavity fluctuation) evenly, the anesthesia is successful, and the molding is prepared.

3) The four limbs of the mice were fixed with tape, the field of view was fully exposed, and the skin of 75% alcohol cotton ball was sterilized.

4) The supraclavicular access is used, the anterior sternum skin and the neck skin are cut off by using common tissues, the neck muscle is separated by vascular forceps in a blunt way, and the upper third of the sternum is cut off.

5) The thymus was blunt separated, exposing the aortic arch.

6) The constriction is ligated between the left supraclavicular artery and the left common carotid artery with a 6-0 wire, a 27 gauge needle.

7) The pad needle is pulled out, the sternum and superficial skin are sutured, and the alcohol cotton ball is wiped and disinfected.

8) The mice were placed on a 37 degree heating pad and returned to the feeder cage after they were anesthetized and awakened.

9) The levofloxacin is diluted and then added into drinking water of mice to prevent wound infection.

10 Checking the wound suture condition three days before molding.

11 2 weeks, 4 weeks, 6 weeks after molding, and collecting the heart super-evaluation molding conditions and animal phenotypes.

Double blindness is not clear to operators and assisted in the mouse molding process, and the third person uncovers the blindness after molding.

4.2TEPP dosing:

gastric lavage administration was started two weeks after TAC molding, once a day for two weeks, and 20mg/kg was metered.

4.3 detection of pharmacodynamic action:

1) Extraction of heart tissue protein: to the centrifuge tube containing heart tissue, 500. Mu.L of RIPA lysate (with protease inhibitor and phosphatase inhibitor added in advance) was added, and the tissue was thoroughly homogenized and crushed by a homogenizer, during which the rotor was washed with PBS. The homogenized tissue was placed on ice for 10 minutes. And placing the centrifuge tube into an ultrasonic crusher for ultrasonic crushing. Ultrasonic procedure: the sonication time was 3 minutes and 30 seconds, the work was 6 seconds, the intervals were 9 seconds, 12000 r/min, and the centrifugation was performed for 20 minutes at 4 ℃. The resulting supernatant was aspirated into another clean centrifuge tube and protein concentration was measured using BCA method. And obtaining a standard curve by using the measured standard concentration, and calculating the protein concentration of the sample.

2) Western Blot: the SDS-PAGE electrophoresis tank was filled with running buffer and the protein samples were added to the SDS-PAGE gel wells. Electrophoresis procedure: 150V,50 minutes. Proteins were transferred to PVDF membranes using wet transfer. The electrophoresis procedure was: 100V,1.5 hours. 5% nonfat milk powder was used as a blocking solution and incubated at 160rpm shaker for 1.5h at room temperature. An anti-4deg.C shaker overnight. The membrane was washed 3 times, 7 minutes each, and developed using PBST.

3) Analysis of systolic function: heart was subjected to M-mode ultrasound testing for different month old mice using a Vevo 2100 sonicator (visual), recording the following parameters: end diastole left ventricular anterior wall thickness (LVAW; d); end-diastole left ventricular post wall thickness (LVPW; d); end diastole left ventricular inner diameter (LVID; d); end-diastole left ventricular volume (LVVol; d); end systole left ventricular anterior wall thickness (LVAW; s); end systole left ventricular posterior wall thickness (LVPW; s); end systole left ventricular inner diameter (LVID; s); end systole left ventricular volume (LVVol; s); left Ventricular Mass (LVM); left ventricular Ejection Fraction (EF); left ventricular contractility index (FS).

4) Paraffin section: after the removed heart tissue was fixed overnight with 4% pfa, it was placed in an embedding machine for serial tissue dehydration. The procedure was as follows: 70% ethanol for 30 min; 80% ethanol for 30 min; 90% ethanol for 30 min; 95% ethanol for 30 min; absolute ethanol, 30 minutes; absolute ethanol, 30 minutes; xylene, 15 minutes; xylene, 15 minutes. The dehydrated tissue is put into paraffin wax for 2 hours, the paraffin wax is replaced for 2 times, and the tissue is put into a constant temperature incubator at 60 ℃. The tissue was placed in a preheated embedding frame, placed in position with forceps, and the hot wax solidified. The paraffin-embedded tissue was fixed on a paraffin microtome to begin sectioning. The slices were 5 μm thick and spread on a water surface at 42℃with a brush pen and attached to a slide glass. Overnight at 37 ℃ for subsequent staining.

5) H & E staining: the prepared paraffin tissue slice is placed in a constant temperature incubator at 60 ℃ for 2h. Paraffin tissue sections were subjected to dewaxed series of ethanol rehydration: xylene, 10 minutes; xylene, 20 minutes; xylene, 20 minutes; absolute ethanol, 15 minutes; absolute ethanol, 15 minutes; 95% ethanol for 5 min; 90% ethanol for 5 min; 80% ethanol for 5 min; 70% ethanol, 5 minutes. The tissue sections were washed with distilled water for 5 minutes. The tissue sections were stained in Harris hematoxylin for 5 minutes, then with tap water for 5 minutes, ethanol hydrochloride differentiated until blue color faded, and then with tap water for 5 minutes to blu, and nuclear staining was observed under a microscope. The tissue slice is put into a mixed dye solution of 95% ethanol and eosin for dyeing for 1 minute, and then tissue dehydration is carried out by 95% ethanol, 100% ethanol and 100% ethanol for one minute each, xylene is transparent for 2 times for 2 minutes each, and the neutral resin is sealed.

6) Masson staining: paraffin tissue sections were deparaffinized by baking (step (ii)) as described above. Tissue sections were placed in Bouin's fixative overnight at room temperature. The sections were placed in distilled water to rinse thoroughly to remove the color of fixative on the sections. Tissue sections were stained in hematoxylin for 5 min. Rinse with tap water for 5 minutes and soak with distilled water for 8 minutes. Tissue sections were stained for 3 min with Biebrich Scarlet-Acid Fuchsin. Distilled water was soaked for 10 minutes (water was changed once), and then the tissue sections were stained in a phosphotungstic acid solution for 5 minutes. The sections were then stained in Aniline Blue solution for 8 minutes. The sections were placed in distilled water for about 5s and immediately removed as seen by the blue fading. Tissue sections were rapidly placed in 95% ethanol for serial dehydration of the tissue and sealed with neutral resin. And (3) dehydration: 95% ethanol for 1 minute; absolute ethanol, 1 minute; absolute ethanol, 1 minute; xylene, 2 minutes; xylene, 2 minutes.

7) Data statistics: two-tailed t-test was used for two-group comparisons and one-factor analysis of variance was used for multiple-group comparisons using Graphpad Prism software (9.4), where the figure indicates that the statistical p-value was less than 0.001.

4.4 detection results:

FIGS. 1 to 7 show the cardiac function improvement effect of TEPP-46 on pressure load-induced heart failure model mice.

Fig. 1 is a schematic diagram of ultrasound of a mouse heart.

Figure 2 shows statistics of systolic function parameters EF (ejection fraction), FS (shortening fraction), structural parameters (left ventricular diastolic inner diameter, LVID (d), left ventricular systolic inner diameter, LVID (s)) in the cardiac supercomput of the mice.

FIG. 3 is a schematic representation of MASSON fibrosis staining; fig. 4 is a statistical result of the proportion of the fibrotic region to the total area of the visual field.

Figure 5 is a statistical result of the heart weight to body weight ratio of mice.

FIG. 6 is a Western Blot results of monomeric, dimeric, tetrameric forms of PKM 2; figure 7 is a statistical plot of the magnitude of tetrameric form elevation after TEPP46 administration.

From the detection results, the TEPP-46 can obviously improve the heart function, ventricular remodeling and fibrosis level of the heart failure model mouse induced by the pressure load. TEPP-46 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer or prodrug thereof is hopefully a therapeutic small molecule drug targeting PKM2 protein for heart failure metabolism, so that the therapeutic small molecule drug is converted from a low-activity dimer form to a high-activity tetramer form, and myocardial cell metabolism level is improved, thereby preventing and/or treating heart failure.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. In accordance with the scope of the claims appended hereto,

some basic feature applications may be made.

Claims (6)

1. Use of a pyruvate kinase M2 activator in the preparation of a medicament for preventing or treating heart failure, wherein the pyruvate kinase M2 activator is TEPP-46, has a CAS number of 1221186-53-3, and has a molecular formula of C ₁₇ H ₁₆ N ₄ O ₂ S ₂ The structural formula is

2. The use according to claim 1, wherein heart failure is a heart failure syndrome caused by insufficient venous blood flow out of the heart due to impaired systolic and/or diastolic function of the heart, resulting in venous blood stasis and arterial blood perfusion.

3. The use according to claim 1, wherein the medicament comprises TEPP-46 as the sole active ingredient, or further comprising pharmaceutically acceptable excipients.

4. The use according to claim 1, wherein the medicament is in the form of an oral formulation, a pulmonary inhalation formulation, a mucosal formulation, an ophthalmic formulation or an injection; the oral preparation is one or more of granule, powder, pill, tablet, capsule and oral liquid.

5. The use according to claim 1, wherein TEPP-46 or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer or prodrug thereof in said medicament is targeted to cardiomyocytes.

6. A medicine for preventing or treating heart failure comprises pyruvate kinase M2 activator, wherein the pyruvate kinase M2 activator is TEPP-46, has CAS number of 1221186-53-3, and has molecular formula of C ₁₇ H ₁₆ N ₄ O ₂ S ₂ The structural formula is

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