Use of 5'-AMP monohydrate in the preparation of a medicament for the treatment of cold preservation injury of a donor heart for cardiac transplantation

Abstract

The application discloses application of 5'-AMP monohydrate in preparation of a medicine for treating cold preservation injury of a heart transplant donor heart, and provides a technical scheme, in which a core mechanism of cold preservation time-dependent myocardial injury is revealed through cross-species multi-omics joint analysis, and a cascade network of "metabolic imbalance-lactate modification disorder and inflammation activation-mitochondrial injury" is constructed. Metabolomics analysis shows that glycolysis-TCA cycle coupling imbalance is characterized by decrease of lactic acid and accumulation of NADH, and up-regulation of LDHB expression drives the change of metabolic preference from lactic acid generation to consumption. The metabolic reconstruction not only leads to depletion of the lactic acid pool, but also forms a vicious cycle through imbalance of NADH / NAD+ redox ratio. Based on the above mechanism, the research successfully screens out a high-efficiency small-molecule inhibitor Adenosine 5'-monophosphate monohydrate (5'-AMP monohydrate) targeting LDHB for the first time. The compound plays a protective effect by inhibiting LDHB activity: can effectively reduce NADH accumulation while maintaining lactic acid metabolic homeostasis, and enhance the stability of mitochondrial structure.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of 5'-AMP monohydrate in the preparation of a medicine for treating cold storage injury of a heart transplant donor. Background Art

[0002] The global incidence and mortality of heart failure continue to rise, and it has become a major public health issue threatening human health. Heart transplantation, as one of the most effective treatments for end-stage heart failure, can provide patients with hope for a new life. However, despite continuous advances in medical technology, heart transplantation still faces many challenges. According to the latest statistics from the International Society for Heart and Lung Transplantation (ISHLT), more than 5,000 heart transplants are performed worldwide each year, but the 10-year survival rate of patients after surgery is still less than 60%. As one of the key factors affecting transplant prognosis, the quality of the donor heart has become a focus of current research.

[0003] Currently, static cold storage (SCS) of donor hearts remains the standard preservation method in most transplant centers worldwide. This technique involves perfusing the donor heart with a specific preservation solution (such as UW preservation solution) and then rapidly transferring and storing it in cold saline or preservation solution at 4°C to ensure donor heart quality. SCS, a classic donor heart preservation technique, is economical and reliable under short-term donor heart transport conditions. However, increased storage time can lead to severe cold ischemia injury. Studies have shown that cold ischemia time (CIT) is closely associated with post-transplant complications (including primary graft dysfunction (PGD)) and mortality. Cold preservation primarily reduces ischemia-reperfusion injury by lowering the metabolic demands of the donor heart through hypothermia. However, studies have shown that cold ischemia times exceeding three hours significantly increase the risk of mortality within 90 days after heart transplantation and lead to an increased incidence of perioperative complications (such as stroke, dialysis requirements, and PGD). According to the 2022 "China Cardiovascular Health and Disease Report," approximately 50% of donor hearts in my country experience transport times exceeding four hours. Hypothermia cannot completely suppress metabolic processes within tissues. As cold ischemia duration increases, toxic metabolites accumulate, energy resources are depleted, and tissue damage worsens. Heart transplant outcomes are suboptimal when the graft ischemia duration exceeds 6 hours. Related studies have shown that for every additional hour of donor heart ischemia, the risk of death in the first year after transplantation increases by 25%. Therefore, the development of targeted drugs to mitigate cold storage injury in heart transplant recipients is of great significance. Summary of the Invention

[0004] The main purpose of the present invention is to propose the use of 5'-AMP monohydrate in the preparation of a drug for treating cold storage injury of heart transplant donors, aiming to provide a drug that can reduce cold storage injury of heart transplant donors.

[0005] To achieve the above-mentioned object, the present invention proposes the use of 5'-AMP monohydrate in the preparation of a drug for treating cold storage injury of a heart transplant donor.

[0006] Heart transplantation is one of the most effective surgical treatments for patients with end-stage heart failure. Postoperative survival is highly dependent on the quality of the donor heart's preservation. Although static cold storage (SCS) can delay ischemic injury by suppressing the donor heart's metabolic demands through hypothermia, ischemia duration exceeding the clinical safety window (4-6 hours) can still lead to irreversible myocardial damage, manifested by mitochondrial dysfunction, metabolic disturbances, and inflammatory cascades, severely impacting post-transplant cardiac function. Currently, the specific biological mechanisms underlying the development and progression of donor heart cold storage injury remain unclear, making it difficult to develop targeted drugs to mitigate this injury.

[0007] Preferably, the dosage of 5'-AMP monohydrate in the drug is 0.8-1.2 μM.

[0008] Preferably, the dosage of 5'-AMP monohydrate in the drug is 1 μM.

[0009] Preferably, the 5'-AMP monohydrate reduces cold storage injury of the heart transplant donor by stabilizing mitochondrial function.

[0010] Preferably, the 5'-AMP monohydrate reduces cold storage injury of heart transplant donors by inhibiting inflammatory signals.

[0011] Preferably, the 5'-AMP monohydrate reduces cold storage injury of heart transplant donors by regulating the NADH / NAD+ metabolic balance.

[0012] Preferably, the 5'-AMP monohydrate reduces cold storage injury of heart transplant donors by regulating the lactic acid modification of key mitochondrial chaperone proteins.

[0013] The 5'-AMP monohydrate (CAS: 18422-05-4) is a monohydrate of adenosine 5'-monophosphate and has no clear clinical indications.

[0014] Preferably, the drug is used to improve the contractile function of a transplanted heart.

[0015] Preferably, the drug is used to increase the heart rate of a transplanted heart.

[0016] Preferably, the drug is used to inhibit excessive generation of reactive oxygen species in transplanted hearts.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) In the technical solution provided by the present invention, the core mechanism of cold storage time-dependent myocardial injury was revealed through cross-species multi-omics joint analysis, and a cascade network of "metabolic imbalance-lactate modification disorder and inflammatory activation-mitochondrial damage" was constructed. In the static cold storage model, extending the cold ischemia time to 8 hours significantly aggravated the release of myocardial enzymes, mitochondrial vacuolation and depletion of glycolipid reserves. Therefore, 8 hours of cold storage was used as the unified time point for constructing a long-term static cold storage model. Metabolomics analysis showed that the imbalance of glycolysis-TCA cycle coupling was characterized by decreased lactate and accumulation of NADH, and the upregulation of LDHB expression drove the shift of metabolic preference from lactate production to consumption. This metabolic remodeling not only led to the depletion of the lactate pool, but also formed a vicious cycle through the imbalance of the NADH / NAD+ redox ratio.

[0019] (2) The present invention further studies the mechanism of action and shows that LDHB-mediated NADH accumulation induces mitochondrial membrane potential depolarization and DNA leakage by activating the phosphorylation of p65-Ser529, a core molecule of the NF-κB pathway, and genetic knockout of LDHB or pharmacological inhibition of NF-κB can significantly reverse the above damage. More importantly, this study innovatively reveals the synergistic regulatory role of lactate metabolism and lactylation modification: the decrease in lactate levels caused by cold storage weakens the lactylation modification of the mitochondrial molecular chaperone TRAP1, disrupts its interaction with the inner membrane scaffold protein IMMT, and thus triggers the collapse of mitochondrial cristae structure. Exogenous lactate intervention or LDHB knockout can stabilize the TRAP1-IMMT complex by restoring the lactylation modification level, confirming that lactate metabolism dynamics is a key factor in maintaining the integrity of mitochondria in cold-preserved cardiomyocytes.

[0020] (3) Based on the above mechanism, this study successfully screened for the first time adenosine 5'-monophosphate monohydrate (5'-AMP monohydrate), a highly effective small molecule inhibitor targeting LDHB. This compound exerts a protective effect by inhibiting LDHB activity: it can effectively reduce NADH accumulation while maintaining lactate metabolic homeostasis and enhancing mitochondrial structural stability. In an in vivo heart transplant model, 5'-AMP treatment significantly improved the ejection fraction and heart rate of the transplanted heart, reduced oxidative stress and cell apoptosis, confirming its multi-target synergistic protective efficacy in animal models. This discovery not only deepens the understanding of the molecular network of cold storage injury, but also provides new ideas for the development of organ preservation programs that combine metabolic regulation and anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is a diagram showing the research mechanism of cold storage injury in heart transplant donor hearts provided by the present invention.

[0023] Figure 2 Figure 1 is a diagram showing the effect of LDHB upregulation on cold storage damage in heart transplant donor hearts provided by the present invention; Figure A is a flow chart of the metabolic reprogramming experiment of long-term cold-stored donor hearts; Figure B is a diagram showing that LDHB expression is upregulated over time during cold storage by Western-Blot; Figure C is a diagram showing that metabolomics of three species confirms that the content of NADH, a related metabolite catalyzed by LDHB, is significantly increased accompanied by a significant decrease in lactate content; Figure D is a diagram showing the constructed myocardial-specific LDHB knockout mouse model (left) and a diagram showing the NADH accumulation level and lactate level after knockout (right); Figure E is a diagram showing the control group (Ctrl) and Echocardiogram of transplanted hearts of LDHB conditional knockout (CKO) mice (upper left), histograms of ejection fraction and heart rate of transplanted hearts of the control (Ctrl) and LDHB conditional knockout (CKO) groups (lower left), fluorescent staining images of reactive oxygen species (ROS) detection and TUNEL-positive fluorescent staining images of the control (Ctrl) and LDHB conditional knockout (CKO) groups (upper right), histograms of reactive oxygen species (ROS) fluorescence signals and histograms of TUNEL-positive fluorescence signals of the control (Ctrl) and LDHB conditional knockout (CKO) groups (lower right).

[0024] Figure 3Figure 1 is a diagram for screening LDHB-specific small molecule inhibitors provided by the present invention; Figure A is a flow chart for screening LDHB-specific small molecule inhibitors; Figure B is a scoring diagram for 5'-AMP monohydrate; Figure C is a schematic diagram of the docking of 5'-AMP monohydrate and LDHB molecules (left) and a molecular structure diagram (right); Figure D is a diagram of the half-maximal inhibitory concentration of 5'-AMP monohydrate on cardiomyocytes; Figure E is a qualitative Western-Blot result diagram of protein thermal degradation (left) and a quantitative thermal stability analysis curve diagram (right).

[0025] Figure 4 Figure 5 shows that the 5'-AMP monohydrate provided by the present invention alleviates cold storage damage in heart transplant donors; Figure A shows the expression level of NADH after the addition of 5'-AMP monohydrate; Figure B shows the expression level of lactate after the addition of 5'-AMP monohydrate; Figures C and D show the cell apoptosis rate and mitochondrial reactive oxygen species (ROS) production levels detected by the TUNEL method and the MitoSOX™ Red fluorescent probe, respectively; Figure E shows the echocardiogram (left), left ventricular ejection fraction (center), and average heart rate (right) of a mouse heart cold storage-peritoneal heterotopic transplantation model; Figure 4 F is a fluorescent staining image of the transplanted heart using dihydroethidium (DHE) fluorescence staining to detect the level of reactive oxygen species (ROS) (first on the left), a positive fluorescent staining image of the TUNEL method to quantify the degree of myocardial cell apoptosis (second on the left), a bar graph of the transplanted heart using dihydroethidium (DHE) fluorescence staining to detect the level of reactive oxygen species (ROS) (first on the right), and a bar graph of the TUNEL method to quantify the degree of myocardial cell apoptosis (second on the right).

[0026] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is mutually contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0029] Example 1 Exploring the pathophysiological mechanism of cold storage injury of donor hearts and screening LDHB-specific inhibitors

[0030] To investigate the pathophysiological mechanism of cold storage injury in heart transplant recipients ( Figure 1 ), the research team first studied the metabolic reprogramming changes of donor hearts caused by long-term cold storage, and finally clarified the core role of lactate dehydrogenase B subunit (LDHB) in damage; on this basis, they further screened out the small molecule compound 5'-AMPmonohydrate that can specifically inhibit LDHB. The specific process is as follows.

[0031] 1. LDHB upregulation aggravates cold storage injury in heart transplant donors

[0032] The experiment selected three donor heart sample models: human donor left atrial tissue, porcine left ventricular myocardial tissue, and mouse isolated heart. Three time gradients of cold storage were established: 0 hours, 4 hours, and 8 hours. Non-targeted metabolomics technology detection found that the donor hearts stored for a long time showed significant metabolic reprogramming changes ( Figure 2 A). Further experiments confirmed that its core mechanism is:

[0033] (1) Upregulation of LDHB expression and metabolic disorders: Western-Blot results showed that the expression of LDHB in the three species models increased significantly with the extension of cold storage time; at the same time, metabolomics detection confirmed that the content of NADH, a related metabolite catalyzed by LDHB, increased significantly, accompanied by a significant decrease in lactate content. LDHB mediated the accumulation of NADH and the consumption of lactate ( Figure 2 B, C).

[0034] (2) Knockout of LDHB can alleviate damage: In a mouse model with myocardial-specific knockout of LDHB, NADH accumulation decreased and lactate levels rebounded after cold storage, suggesting that LDHB is a key molecule in regulating the above-mentioned metabolic disorders ( Figure 2 D).

[0035] (3) In vivo verification of the pathogenicity of LDHB: A mouse abdominal heterotopic heart transplant model was established (the donor heart was transplanted after 8 hours of cold storage). 24 hours after transplantation, the donor heart function was evaluated by echocardiography. The results showed that the ejection fraction (EF) and heart rate of the transplanted heart in the LDHB conditional knockout group were significantly higher than those in the control group (both p < 0.05). Histological analysis further confirmed that the accumulation of ROS in the myocardial tissue of the knockout group was lower than that in the control group (p < 0.001), and the TUNEL-positive fluorescence signal intensity was also significantly lower than that in the control group (p < 0.0001). ( Figure 2 E). The above results indicate that knocking out LDHB can significantly reduce the damage of transplanted donor hearts after cold storage.

[0036] The above results confirmed that long-term cold storage injury of donor hearts induced increased expression of lactate dehydrogenase B subunit (LDHB). LDHB mediated NADH accumulation and lactate consumption, triggering activation of inflammatory pathways and degradation of key mitochondrial proteins, thereby aggravating mitochondrial damage in myocardial cells and exacerbating long-term cold storage injury of donor hearts.

[0037] 2. Screening of LDHB-specific inhibitor 5'-AMP monohydrate

[0038] Given that our study found that prolonged cold storage-induced upregulation of LDHB expression exacerbates the development of cold storage injury in donor hearts, we conducted virtual screening of two standard compound libraries, the MCE Bioactive Compound Library Plus (containing 19,100 compounds) and the FDA-Approved Drug Library (containing 2,816 compounds), to identify potential LDHB inhibitors. The specific process is as follows.

[0039] (1) Virtual screening and molecular docking: Molecular docking technology was used to match the three-dimensional conformation of the compound with the LDHB active pocket, and the binding free energy was evaluated using the Glide XP scoring function. Finally, the top 50 candidate compounds with the best docking scores were screened out ( Figure 3 A).

[0040] (2) Affinity verification: To further verify the virtual screening results, we co-incubated the above candidate compounds with recombinant LDHB protein and detected the efficiency of compound-protein complex formation by affinity mass spectrometry analysis (Affinity-MS). Based on the screening criteria of relative abundance of complex signal intensity (Binding Ratio% ≥300), it was finally determined that Adenosine 5'-monophosphate monohydrate (5'-AMP monohydrate) had significant binding ability with LDHB ( Figure 3 B, C).

[0041] (3) Safety verification: Cell experiments showed that the half-maximal inhibitory concentration (IC50) of 5'-AMP monohydrate on cardiomyocytes was 1189 μM, indicating that its cytotoxicity was low and its safety was good. Figure 3 D).

[0042] (4) Specific binding verification: CESTA experiments further confirmed that 5'-AMP monohydrate can bind to LDHB and inhibit its thermal degradation, confirming its specific effect on LDHB ( Figure 3 E).

[0043] In summary, this study clarified that LDHB upregulation is the key mechanism of cold storage injury in donor hearts, and identified 5'-AMP monohydrate as a specific LDHB inhibitor through systematic screening, providing potential therapeutic targets and candidate drugs for alleviating cold storage injury in donor hearts.

[0044] Example 2 5'-AMP monohydrate reduces cold storage injury in heart transplant donors

[0045] To evaluate the protective effects of adenosine 5'-monophosphate monohydrate (5'-AMP monohydrate) on cold-preserved donor hearts in vivo, the research team conducted cell-based experiments and animal models, examining three aspects: metabolic regulation, cell protection, and overall functional improvement. The specific experimental process and results are as follows:

[0046] 1. 5'-AMP monohydrate reverses LDHB-mediated metabolic disorders

[0047] In a mouse donor heart cold storage model, the experimental group added 5'-AMPmonohydrte to the donor heart protective solution for 8 hours of cold storage, while the blank control group added the same amount of DMSO. The results showed that compared with the blank control group, the NADH level of the donor heart tissue in the experimental group was significantly reduced, while the lactate level was significantly increased ( Figure 4A, B). This result is consistent with the metabolic changes in the LDHB knockout model, suggesting that 5'-AMP monohydrate can inhibit LDHB activity and reverse the metabolic disorders of "NADH accumulation and lactate consumption" mediated by LDHB, thereby alleviating damage at the source of the mechanism.

[0048] 2. Cellular verification: inhibition of apoptosis and mitochondrial oxidative stress

[0049] Cardiomyocytes were cryopreserved and rewarmed using a cold-preservation-reoxygenation model (preserved in 4°C UW solution for 8 hours followed by 1 hour of reoxygenation). The experimental group received 1 μM 5'-AMP monohydrate and then reoxygenated for 8 hours after 4°C UW solution. A control group without drug administration underwent the same procedure. Cell apoptosis and mitochondrial reactive oxygen species (ROS) production were assessed using the TUNEL assay and MitoSOX™ Red fluorescent probe, respectively.

[0050] The results of cell apoptosis detection showed that the proportion of apoptotic cells in the 5'-AMP monohydrate intervention group was significantly lower than that in the control group after cold storage for 8 hours (p<0.0001), indicating that 5'-AMP monohydrate can significantly inhibit the programmed cell death of cardiomyocytes induced by cold storage ( Figure 4 C). Further detection by mitochondrial superoxide-specific probe revealed that ROS accumulation in the 5'-AMP monohydrate-treated group was significantly reduced (p<0.0001), indicating that 5'-AMP monohydrate can alleviate mitochondrial oxidative stress and thus play a cytoprotective role ( Figure 4 D).

[0051] 3. Animal model verification: improving post-transplant cardiac function and tissue damage

[0052] A mouse heart cold preservation and intraperitoneal heterotopic transplantation model was established. 1 μM 5'-AMPmonohydrate was added to the UW protective solution in the experimental group, while an equal volume of DMSO was added to the UW protective solution in the control group. All donor hearts were cold-preserved in 4°C UW solution for 8 hours before intraperitoneal heterotopic transplantation. 24 hours after transplantation, the donor heart function was evaluated by echocardiography. The results showed that the left ventricular ejection fraction (LVEF) in the 5'-AMP monohydrate intervention group was improved compared with the control group (p<0.05), and the average heart rate also showed an upward trend (p<0.05), suggesting that 5'-AMPmonohydrate can effectively improve the contractile function and heart rate of the transplanted heart ( Figure 4 E).

[0053] To further clarify the inhibitory effect of 5'-AMP monohydrate on myocardial tissue damage, this study used dihydroethidium (DHE) fluorescence staining to detect the level of reactive oxygen species in transplanted hearts, and quantified the degree of myocardial cell apoptosis by TUNEL method. Histological analysis showed that the DHE fluorescence intensity of myocardial tissue in the 5'-AMP monohydrate intervention group was significantly lower than that in the control group (p<0.001), indicating that the excessive production of reactive oxygen species (ROS) was effectively inhibited. At the same time, the TUNEL-positive fluorescence intensity was also significantly reduced in the 5'-AMP intervention group (p<0.01), confirming that the compound can exert a cardioprotective effect by reducing oxidative stress and activation of apoptosis signals ( Figure 4 F).

[0054] In conclusion, 5'-AMP monohydrate can reverse LDHB-mediated metabolic disorders, inhibit cardiomyocyte apoptosis and mitochondrial oxidative stress, ultimately improving the post-transplantation function of cold-preserved donor hearts and significantly reducing cold storage injury of heart transplant donors.

[0055] The above experimental results prove that the present invention can reduce the cold storage damage of heart transplant donors by adding adenosine5'-monophosphate monohydrate to the cold storage organ protection solution of donor hearts. It is expected that the cold storage and transportation time of donor hearts can be extended through simple and economical drugs, thereby alleviating the current shortage of transplant donor hearts and improving the survival rate after heart transplantation.

[0056] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

Application of 1.5'-AMP monohydrate in the preparation of a drug for treating cold storage injury of heart transplant donors.

2. The use according to claim 1, characterized in that The dosage of 5'-AMP monohydrate in the drug is 0.8-1.2 μM.

3. The use according to claim 2, characterized in that The dosage of 5'-AMP monohydrate in the drug is 1 μM.

4. The use according to claim 1, characterized in that The 5'-AMP monohydrate reduces cold storage injury of the heart transplant donor by stabilizing mitochondrial function.

5. The use according to claim 1, characterized in that The 5'-AMP monohydrate reduces cold storage injury of heart transplant donors by inhibiting inflammatory signals.

6. The use according to claim 1, characterized in that The 5'-AMP monohydrate reduces cold storage injury of the heart transplant donor by regulating the NADH / NAD+ metabolic balance.

7. The use according to claim 1, characterized in that The 5'-AMP monohydrate reduces cold storage injury of heart transplant donors by regulating the lactic acid modification of key mitochondrial chaperone proteins.

8. The use according to claim 1, characterized in that The drug is used to improve the contractile function of the transplanted heart.

9. The use according to claim 1, characterized in that The drug is used to increase the heart rate of the transplanted heart.

10. The use according to claim 1, characterized in that The drug is used for inhibiting excessive generation of reactive oxygen species in transplanted hearts.

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