Pharmaceutical composition for targeted activation of FSP1 and application thereof

Abstract

The invention discloses a pharmaceutical composition for targeted activation of FSP1 and application of the pharmaceutical composition. The pharmaceutical composition comprises a medicine capable of activating ferroptosis inhibitory protein 1 and a reduced coenzyme I capable of enhancing the ferroptosis inhibitory protein 1 activation effect of the medicine. The medicine capable of activating the ferroptosis inhibition protein 1 comprises a second-generation sulfonylurea medicine; and the second generation sulfonylurea medicine is glimepiride or glibenclamide. The glimepiride, glibenclamide and other second-generation sulfonylurea drugs are combined with NADH, and the resistance of cells to ferroptosis can be remarkably improved by activating or improving the activity of FSP1, so that cell death after ischemia-reperfusion injury is inhibited; meanwhile, the invention provides new application of the glimepiride, the glibenclamide and other hypoglycemic drugs, the adaptation diseases of the glimepiride, the glibenclamide and other hypoglycemic drugs are expanded, and the glimepiride, the glibenclamide and other hypoglycemic drugs are combined with the NADH, so that the glimepiride, the glibenclamide and other hypoglycemic drugs can be used for treating ischemia reperfusion injury related diseases and organ preservation liquid to improve the protection effect on organs.

Description

Technical Field

[0001] This invention relates to the field of ischemia / ischemia-reperfusion injury technology, and more specifically, to a pharmaceutical composition that targets and activates FSP1 and its application. Background Technology

[0002] Ischemia-reperfusion injury (IRI) is a complex pathophysiological process involving the interaction of ischemia and reperfusion phases, ultimately leading to tissue damage and organ dysfunction. Its core mechanisms include oxidative stress, calcium overload, inflammatory response, mitochondrial dysfunction, and ferroptosis.

[0003] During ischemia, tissues or organs experience insufficient oxygen and nutrient supply due to interrupted blood flow, leading to impaired intracellular energy metabolism. Reduced ATP production and impaired cell function, coupled with increased anaerobic metabolism and lactic acid buildup, result in acidosis. Furthermore, decreased ATP production leads to impaired Na⁺ / K⁺-ATPase function, resulting in reduced intracellular Na⁺ and Ca²⁺ levels. 2+ Accumulation, K⁺ efflux, cell swelling, and ion imbalance.

[0004] When blood flow is restored and the reperfusion phase begins, although the supply of oxygen and nutrients is restored, this process actually exacerbates tissue damage. After reperfusion, a large amount of oxygen enters the tissue, generating a large amount of reactive oxygen species (ROS), such as superoxide anions (O2). - Hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH·) trigger oxidative stress, leading to oxidative damage to lipids, proteins, and DNA. Simultaneously, intracellular calcium... 2+ The rapid increase in concentration activates calcium-dependent proteases, phospholipases, and endonucleases, further damaging cell structure and function. Reperfusion also activates the complement system, neutrophils, and monocytes / macrophages, releasing large amounts of inflammatory factors (such as TNF-α, IL-1β, and IL-6), triggering an inflammatory response and exacerbating tissue damage. Furthermore, reperfusion leads to a decrease in mitochondrial membrane potential, opening of the mitochondrial permeability transition pore (mPTP), release of cytochrome C, and triggering apoptosis. Recent studies have found that iron-dependent lipid peroxidation is exacerbated after reperfusion, leading to cell membrane rupture and cell death. This process, known as ferroptosis, is a novel form of cell death, distinct from traditional apoptosis and necrosis.

[0005] Ultimately, ischemia / ischemia-reperfusion injury leads to cell death through various mechanisms, including necrosis, apoptosis, and ferroptosis, which in turn cause tissue damage and organ dysfunction. For example, in cerebral infarction, neuronal death and cerebral edema lead to neurological dysfunction; in myocardial infarction, cardiomyocyte necrosis and arrhythmia lead to heart failure; in renal ischemia-reperfusion injury, renal tubular epithelial cell necrosis leads to renal failure; and in hepatic ischemia-reperfusion injury, hepatocyte necrosis leads to liver failure.

[0006] Currently, treatments for ischemia / ischemia-reperfusion injury are primarily in the preclinical research stage, and standardized clinical treatment protocols have not yet been established. Existing treatments mainly focus on systemic supportive pharmacological therapy. Antioxidants can reduce oxidative stress by scavenging reactive oxygen species, but their scope of action is limited and they cannot completely block the chain reaction of oxidative stress. Anti-inflammatory drugs reduce damage by inhibiting inflammatory responses, but long-term use may cause serious side effects such as gastrointestinal damage, and simple anti-inflammatory treatment cannot address other key mechanisms such as oxidative stress and calcium overload.

[0007] Therefore, it is essential to find more effective and safer drugs with greater clinical application prospects for protecting cells / tissues to reduce ischemia / ischemia-reperfusion injury.

[0008] In view of the above, this application is hereby submitted. Summary of the Invention

[0009] The existing technology has the problem that current drugs for treating ischemia / ischemia-reperfusion injury have significant side effects, and there is currently no effective treatment in clinical practice. Therefore, this invention provides a pharmaceutical composition that targets and activates FSP1 and its application. By screening out pharmaceutical compositions that can target and activate the function of ferroptosis inhibitor 1 (FSP1), the ferroptosis process can be effectively inhibited, thereby alleviating ischemia / ischemia-reperfusion injury, and it can also effectively preserve cells and isolated organs in vitro.

[0010] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a pharmaceutical composition for targeting and activating FSP1, comprising a drug capable of activating ferroptosis inhibitor 1 (FSP1) and reduced coenzyme I (NADH) that enhances its activation of ferroptosis inhibitor 1.

[0011] This invention screens out drug compositions that can target and activate the function of ferroptosis inhibitor protein 1 (FSP1), which can not only inhibit ferroptosis and thus reduce ischemia / ischemia-reperfusion injury, but also effectively protect cells and improve the preservation effect of ex vivo organs.

[0012] Ferroprelation is a novel iron-dependent form of cell death, with core mechanisms involving iron metabolism disorders, lipid peroxidation, and the collapse of antioxidant defense systems. During ischemia-reperfusion injury, ischemia leads to tissue hypoxia and impaired energy metabolism, while the surge of reactive oxygen species (ROS) during reperfusion further exacerbates cell damage. Ferroprelation plays a crucial role in this process, leading to cell membrane structure disruption and cell death through iron-mediated lipid peroxidation. Multiple studies have confirmed that inhibiting ferroprelation can significantly reduce organ and tissue damage in ischemia-reperfusion injury (IRI). For example, in a myocardial ischemia-reperfusion injury model, inhibiting ferroprelation reduces cardiomyocyte death and improves cardiac function; in cerebral ischemia-reperfusion injury, inhibiting ferroprelation protects neurons and reduces neurological dysfunction. Therefore, inhibiting ferroprelation is an important way to mitigate ischemia-reperfusion injury.

[0013] Currently, the main inhibitory targets of ferroptosis include glutathione peroxidase 4 (GPX4) and ferroptosis inhibitor protein 1 (FSP1). GPX4 inhibits ferroptosis by reducing lipid peroxides, but its activity is dependent on glutathione supply and may be limited in ischemia-reperfusion injury. FSP1, independent of the GPX4 inhibitory pathway, can inhibit ferroptosis by clearing lipid peroxides through its oxidoreductase activity. Furthermore, FSP1 expression levels are closely related to cellular sensitivity to ferroptosis; upregulating FSP1 expression or enhancing its activity can significantly improve cellular resistance to ferroptosis, and its function is not affected by glutathione levels, thus making it a more promising therapeutic target.

[0014] Currently, no agonists capable of activating FSP1 have been found. This invention screens existing drugs applicable to other conditions to identify drugs that can activate FSP1. By activating FSP1 function, ferroptosis is inhibited, thereby alleviating ischemia / ischemia-reperfusion injury. Furthermore, this application discovers that adding NADH to this drug further enhances its FSP1-activating effect.

[0015] In one specific embodiment, the drug capable of activating ferroptosis inhibitor protein 1 includes a second-generation sulfonylurea drug, preferably glimepiride or glibenclamide. The left image shows the molecular formula of glimepiride, and the right image shows the molecular formula of glibenclamide.

[0016]

[0017] Glimepiride and glibenclamide are second-generation sulfonylurea oral hypoglycemic agents that lower blood sugar by stimulating pancreatic β-cells to release insulin. Glimepiride is characterized by its long-acting nature, low dosage, and lower risk of hypoglycemia, making it suitable for elderly patients or those with mild renal impairment. Glibenclamide, on the other hand, has a stronger hypoglycemic effect but carries a higher risk of hypoglycemia and should be used with caution.

[0018] This invention discovers a new use for second-generation sulfonylurea drugs such as glimepiride and glibenclamide. Glimepiride and glibenclamide interact with ferroptosis inhibitor protein 1 (FSP1), thereby significantly increasing the activity of FSP1 and inhibiting cell death after ischemia-reperfusion injury. Therefore, this invention obtains a novel pharmaceutical composition that can inhibit ferroptosis from existing drugs for other indications.

[0019] The drugs mentioned, such as glimepiride and glibenclamide, are FDA-approved and have undergone clinical trials, demonstrating clear safety and pharmacokinetic characteristics. This ensures the feasibility of the drug composition in clinical translation. Furthermore, this invention has verified in animal models that glimepiride and glibenclamide can activate FSP1, thereby inhibiting ferroptosis and protecting against ischemia-reperfusion injury.

[0020] In one specific embodiment, the ratio of the drug capable of activating ferroptosis inhibitory protein 1 to reduced coenzyme I is 0.1~10:0.05~40.

[0021] Secondly, the present invention provides the use of the pharmaceutical composition in the preparation of drugs for treating ischemia / ischemia-reperfusion diseases.

[0022] In one specific embodiment, the ischemia / ischemia-reperfusion diseases include myocardial infarction, cerebral infarction, and renal / hepatic ischemia-reperfusion injury.

[0023] Thirdly, the present invention provides the use of the pharmaceutical composition in organ / cell preservation solutions.

[0024] In one specific embodiment, the amount of the pharmaceutical composition added to the organ / cell preservation solution is 5-500 µM of the drug that activates ferroptosis inhibitor 1 and 5-500 µM of NADH.

[0025] In one specific embodiment, the ratio of the drug capable of activating ferroptosis inhibitory protein 1 to reduced coenzyme I is 0.1~10:0.05~40.

[0026] The pharmaceutical composition of the present invention can not only be used to treat ischemia-reperfusion diseases, but also, because it can protect cells from cell death caused by ferroptosis, it can be added as an additive to organ / cell preservation solutions to significantly improve the protective effect on organs / cells.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention provides a drug composition that targets and activates FSP1 and its application. By combining second-generation sulfonylurea drugs such as glimepiride and glibenclamide with NADH, it can significantly improve the cell's resistance to ferroptosis by activating or enhancing the activity of FSP1, thereby inhibiting cell death after ischemia-reperfusion injury. 2. The pharmaceutical composition that targets and activates FSP1 and its application provided in the embodiments of the present invention provide new uses for hypoglycemic drugs such as glimepiride and glibenclamide, expand their indications, and enable them to be used to treat ischemia-reperfusion injury-related diseases. 3. The present invention provides a drug composition that targets and activates FSP1 and its application, and provides the application of hypoglycemic drugs such as glimepiride and glibenclamide in combination with NADH in organ preservation solution to improve the protective effect on organs. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is the FSP1 activity detection method provided in Embodiment 1 of the present invention; Figure 2 The effect of different drug components provided in Example 1 of this invention on FSP1 activity; Figure 3 The effect of different drug components provided in Example 1 of this invention on FSP1 activity; Figure 4 The effect of different drugs on FSP1 activity provided in Example 2 of this invention; Figure 5 The kidney damage score of mice after IRI modeling and administration of different drugs provided in Example 3 of the present invention; Figure 6 The creatinine content of mice after IRI modeling and administration of different drugs provided in Example 3 of the present invention; Figure 7The blood urea nitrogen content in mice after IRI modeling and administration of different drugs, as provided in Example 3 of this invention; Figure 8 This refers to the kidney damage score of mouse kidneys after transplantation following preservation in different organ fluids, as provided in Example 4 of the present invention. Figure 9 The creatinine content of mouse kidneys after transplantation following preservation in different organ fluids, as provided in Example 4 of this invention; Figure 10 The blood urea nitrogen content of mouse kidneys after preservation in different organ fluids and transplantation, as provided in Example 4 of this invention; Figure 11 This describes the cell survival of kidney cells after preservation in different organ fluids, as provided in Example 4 of the present invention. Figure 12 This describes the cell survival of kidney cells after preservation in different organ fluids, as provided in Example 4 of the present invention. Figure 13 The survival status of kidney cells after preservation in different organ fluids, as provided in Example 4 of the present invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0031] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known materials or methods have not been specifically described in order to avoid obscuring the invention.

[0032] Throughout this specification, references to "an embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the present invention. Therefore, the phrases "an embodiment," "an example," "an example," or "an example" appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0033] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0" and "5" have been listed in this document; "0-5" is merely a shortened representation of these numerical combinations. Furthermore, when a parameter is described as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc. Unless otherwise specified, all steps in this application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) can be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0034] Current treatments for ischemia / ischemia-reperfusion injury have significant side effects. To address these issues: In a first aspect, the present invention provides a pharmaceutical composition comprising a drug capable of activating ferroptosis inhibitory protein 1 and reduced coenzyme I (NADH).

[0035] This invention screens out drug compositions that can target and activate the function of ferroptosis inhibitor protein 1 (FSP1), which can not only inhibit ferroptosis and thus reduce ischemia / ischemia-reperfusion injury, but also effectively protect cells and improve the preservation effect of ex vivo organs.

[0036] Ferroprelation is a novel iron-dependent form of cell death, with core mechanisms involving iron metabolism disorders, lipid peroxidation, and the collapse of antioxidant defense systems. During ischemia-reperfusion injury, ischemia leads to tissue hypoxia and impaired energy metabolism, while the surge of reactive oxygen species (ROS) during reperfusion further exacerbates cell damage. Ferroprelation plays a crucial role in this process, leading to cell membrane structure disruption and cell death through iron-mediated lipid peroxidation. Multiple studies have confirmed that inhibiting ferroprelation can significantly reduce organ and tissue damage in ischemia-reperfusion injury (IRI). For example, in a myocardial ischemia-reperfusion injury model, inhibiting ferroprelation reduces cardiomyocyte death and improves cardiac function; in cerebral ischemia-reperfusion injury, inhibiting ferroprelation protects neurons and reduces neurological dysfunction. Therefore, inhibiting ferroprelation is an important way to mitigate ischemia-reperfusion injury.

[0037] Currently, the main inhibitory targets of ferroptosis include glutathione peroxidase 4 (GPX4) and ferroptosis inhibitor protein 1 (FSP1). GPX4 inhibits ferroptosis by reducing lipid peroxides, but its activity is dependent on glutathione supply and may be limited in ischemia-reperfusion injury. FSP1, independent of the GPX4 inhibitory pathway, can inhibit ferroptosis by clearing lipid peroxides through its oxidoreductase activity. Furthermore, FSP1 expression levels are closely related to cellular sensitivity to ferroptosis; upregulating FSP1 expression or enhancing its activity can significantly improve cellular resistance to ferroptosis, and its function is not affected by glutathione levels, thus making it a more promising therapeutic target.

[0038] Currently, no agonists capable of activating FSP1 have been found. This invention screens existing drugs applicable to other conditions to identify drugs that can activate FSP1. By activating FSP1 function, ferroptosis is inhibited, thereby alleviating ischemia / ischemia-reperfusion injury. Furthermore, this application discovers that adding NADH to this drug further enhances its FSP1-activating effect.

[0039] In one specific embodiment, the drug capable of activating ferroptosis inhibitor protein 1 includes a second-generation sulfonylurea drug, preferably glimepiride or glibenclamide. The left image shows the molecular formula of glimepiride, and the right image shows the molecular formula of glibenclamide.

[0040]

[0041] Glimepiride and glibenclamide are second-generation sulfonylurea oral hypoglycemic agents that lower blood sugar by stimulating pancreatic β-cells to release insulin. Glimepiride is characterized by its long-acting nature, low dosage, and lower risk of hypoglycemia, making it suitable for elderly patients or those with mild renal impairment. Glibenclamide, on the other hand, has a stronger hypoglycemic effect but carries a higher risk of hypoglycemia and should be used with caution.

[0042] This invention discovers a new use for second-generation sulfonylurea drugs such as glimepiride and glibenclamide. Glimepiride and glibenclamide interact with ferroptosis inhibitor protein 1 (FSP1), thereby significantly increasing the activity of FSP1 and inhibiting cell death after ischemia-reperfusion injury. Therefore, this invention obtains a novel pharmaceutical composition that can inhibit ferroptosis from existing drugs for other indications.

[0043] The drugs mentioned, such as glimepiride and glibenclamide, are FDA-approved and have undergone clinical trials, demonstrating clear safety and pharmacokinetic characteristics. This ensures the feasibility of the drug composition in clinical translation. Furthermore, this invention has also verified in animal models that glimepiride and glibenclamide can activate FSP1, thereby inhibiting ferroptosis and protecting against ischemia / ischemia-reperfusion injury.

[0044] In one specific embodiment, the ratio of the drug capable of activating ferroptosis inhibitory protein 1 to reduced coenzyme I is 0.1~10:0.05~40.

[0045] Secondly, the present invention provides the use of the pharmaceutical composition in the preparation of drugs for treating ischemia / ischemia-reperfusion diseases.

[0046] In one specific embodiment, the daily dosage of the drug capable of activating ferroptosis inhibitor protein 1 is 0.1-10 mg / kg, preferably 1-5 mg / kg, more preferably 2-3 mg / kg, and the daily dosage of the reduced coenzyme I is 0.05-40 mg / kg, preferably 0.1-15 mg / kg, more preferably 0.5-2 mg / kg.

[0047] In one specific embodiment, the ischemia / ischemia-reperfusion diseases include myocardial infarction, cerebral infarction, and renal / hepatic ischemia-reperfusion injury.

[0048] Thirdly, the present invention provides the use of the pharmaceutical composition in organ / cell preservation solutions.

[0049] In one specific embodiment, the amount of the pharmaceutical composition added to the organ / cell preservation solution is 5-500 µM of the drug that activates ferroptosis inhibitor 1 and 5-500 µM of NADH.

[0050] In one specific embodiment, the ratio of the drug capable of activating ferroptosis inhibitory protein 1 to reduced coenzyme I is 0.1~10:0.05~40.

[0051] The pharmaceutical composition of the present invention can not only be used to treat ischemia / ischemia-reperfusion diseases, but also, because it can protect cells from cell death caused by ferroptosis, it can be added as an additive to organ preservation solution to significantly improve the protective effect on organs.

[0052] Example 1 This invention provides a method for preparing a pharmaceutical composition, comprising glimepiride and NADH.

[0053] To verify the targeted activation effect between the drug composition of this embodiment and FSP1, six solutions were prepared: buffer solvent, buffer + DMSO, 10µM glimepiride, hFSP1 + DMSO, hFSP1, and hFSP1 + 10µM glimepiride.

[0054] like Figure 1 As shown in a, azadirachtin is a redox-sensitive dye, naturally blue-violet in color, and without fluorescence; NADH is oxidized to NAD by FSP1. + When resazurin is reduced to a strongly red fluorescent halogen, the oxidation activity of FSP1 can be determined by measuring the change in the fluorescence value of the halogen and its reaction rate. Therefore, NADH and resazurin were added to all six groups of solutions.

[0055] The results are as follows Figure 1 b and Figure 2 As shown, it can be seen that two of the solutions containing only buffer, buffer + DMSO, or glimepiride could not cause the entire reaction system to react, so the solution color did not change. In the other three solutions, FSP1 was added, and the reaction system reacted. However, it can be seen that the addition of DMSO had no effect on promoting the oxidation activity of FSP1. However, after adding 10µM glimepiride, it can be seen that the enzyme activity of FSP1 was significantly accelerated, indicating that the activity of FSP1 was targeted.

[0056] In addition, in this embodiment, glimepiride in the above solution was replaced with glibenclamide, and the corresponding detection was also performed. The results are as follows: Figure 3 As shown.

[0057] Example 2 To investigate the catalytic activity of FSP1 with or without the promoter glibenclamide and with NADH or NADPH as electron donors, this example utilizes enzyme reaction kinetics experiments for determination.

[0058] In the experimental system, the FSP1 concentration was fixed at 0.2 μM, and the NADH or NADPH concentration gradient was set from 0 to 1200 μM (0, 50, 100, 200, 400, 600, 800, 1200 μM), while the resazurin concentration was kept constant at 500 μM. Based on the reaction principle of resazurin reducing to halogen (Ex / Em = 553 nm / 594 nm), which has characteristic fluorescent properties, the fluorescence intensity change (RFU) of halogen was monitored in real time, and the fluorescence change rate per unit time (ΔRFU / s) was calculated as the initial reaction rate. Nonlinear regression analysis was performed using the initial reaction rate data at different NADH / NADPH concentrations, and enzyme kinetic parameters were obtained by fitting the Michaelis-Menten equation.

[0059] like Figure 4 As shown in the results, glibenclamide can significantly upregulate the activity of FSP1. The addition of NADH, compared with NADPH, can further enhance the promoting effect of glibenclamide on the activity of FSP1, thereby improving the anti-ferroptosis effect of cells.

[0060] Example 3 This embodiment provides the application of the combination drug of glibenclamide and NADH in the treatment of ischemia / ischemia-reperfusion disorders.

[0061] Male C57BL / 6 mice weighing approximately 20g were selected. Bilateral renal incisions were made on the back of the mice, the perirenal fat was freed, and the renal arteries and veins were exposed. Bilateral arteries and veins were clamped with non-invasive vascular clamps to induce ischemic injury. After 30 minutes, the non-invasive vascular clamps were released to induce reperfusion injury. On the third day, mouse serum and kidney tissue were collected for histological pathological scoring and renal function scoring.

[0062] In this study, glibenclamide was administered to each mouse at a dose of 2 mg / kg via gavage, and NADH was administered at a dose of 15 mg / kg via gavage (it should be noted that the dosages mentioned here are for mouse experiments, and the purpose is to verify whether the composition has the efficacy described in this application; the dosage for human administration requires further clinical confirmation).

[0063] The daily dosage of NADH is 0.05~40 mg / kg, preferably 0.1~15 mg / kg, and more preferably 0.5~2 mg / kg.

[0064] The daily dosage of glibenclamide is 0.1-10 mg / kg, preferably 1-5 mg / kg, and more preferably 2-3 mg / kg.

[0065] like Figure 5-7 As shown, Sham represents the sham surgery group, NADH / Glibenclamide represents drug intervention, and IRI represents renal ischemia-reperfusion modeling.

[0066] After modeling, indicators such as kidney injury score, creatinine, and blood urea nitrogen showed that simply adding glibenclamide or NADH to the sham surgery group did not induce kidney damage, demonstrating the good safety of the FSP1-targeted treatment strategy. After IRI modeling, kidney damage significantly worsened. Adding glibenclamide or NADH alleviated kidney damage, with glibenclamide showing slightly better efficacy and NADH showing relatively poorer efficacy. Simultaneous addition of glibenclamide and NADH further alleviated kidney damage, indicating that this drug combination significantly reduced kidney injury. This demonstrates that glibenclamide can significantly reduce kidney damage, and NADH can enhance the damage-reducing effect of glibenclamide.

[0067] Example 4 This embodiment provides the application of the combination drug of glibenclamide and NADH in organ preservation solution.

[0068] Male C57BL / 6 mice weighing approximately 20g were selected, and their bilateral kidneys were harvested. The kidneys were preserved for 12 hours using different component organ preservation solutions (UW, Celsior, HPG) containing a drug combination (NADH 500µM and glibenclamide 500µM). After 12 hours, the mice underwent allogeneic kidney transplantation. Two days later, the original kidneys were surgically removed. Three days later, mouse serum and kidney tissue were collected for histopathological scoring and renal function scoring.

[0069] The results are as follows Figure 8-10 As shown, after 12 hours of organ preservation, the following indicators, including kidney injury score, creatinine, and blood urea nitrogen, indicate that: the saline group, saline + glibenclamide, and saline + NADH group showed no significant organ protection; various organ preservation solutions (UW, Celcior, HPG) had some organ protection effects; organ preservation solutions with added glibenclamide could play a certain role in protecting kidney function, but adding NADH alone had no significant effect; the simultaneous addition of NADH and glibenclamide to various organ preservation solutions provided the best protection for kidney function.

[0070] In addition, proximal tubular epithelial cells of HK2 kidneys were selected and preserved in different preservation solutions containing drug compositions. The cells were placed at 4°C and the cell viability was tested after 1 day.

[0071] The results are as follows Figure 11 As shown, glibenclamide alone significantly reduced cell death, while NADH and NADPH alone had no significant effect. The combined effect of glibenclamide and NADH was better than that of either drug alone, while the combined effect of NADPH and glibenclamide was similar to that of glibenclamide alone, indicating that the addition of NADPH did not significantly enhance the effect of glibenclamide (glibenclamide 500µM, NADH 500µM).

[0072] from Figure 12-13 It can be seen that glibenclamide, as an additive in organ preservation solutions, exhibits good protective effects at 250 µM, and the protective effect improves with increasing concentration, but there is no significant improvement beyond 1 mM. NADH, as an additive in organ preservation solutions, shows improved cell-protective effects with increasing dosage, but there is no significant improvement beyond 1 mM.

[0073] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for targeting activation of FSP1, characterized by, The drug capable of activating ferroptosis inhibitor 1 and reduced coenzyme I enhancing the activation of ferroptosis inhibitor 1.

2. The pharmaceutical composition of claim 1, wherein, The drug capable of activating ferroptosis inhibitor 1 includes a second-generation sulfonylurea drug.

3. The pharmaceutical composition of claim 2, wherein, The second-generation sulfonylurea drug is glimepiride or glibenclamide.

4. The pharmaceutical composition of claim 1, wherein, The ratio of the drug capable of activating ferroptosis inhibitor 1 to reduced coenzyme I is 0.1-10:0.05-40.

5. The use of the pharmaceutical composition of any one of claims 1-4 in the preparation of a drug for treating ischemia / ischemia-reperfusion diseases.

6. The use of the pharmaceutical composition according to claim 5 for the preparation of a medicament for the treatment of ischemia / ischemia-reperfusion related diseases, characterized in that, The ischemia / ischemia-reperfusion diseases include myocardial infarction, cerebral infarction, and renal / liver ischemia-reperfusion injury.

7. The use of the pharmaceutical composition of any one of claims 1-4 in an organ / cell preservation solution.

8. The use of the pharmaceutical composition according to claim 7 in an organ / cell preservation solution, characterized in that, The addition amount of the pharmaceutical composition in the organ preservation solution is 5-500 µM of the drug capable of activating ferroptosis inhibitor 1 and 5-500 µM of NADH.

9. The use of the pharmaceutical composition according to claim 8 in an organ / cell preservation solution, characterized in that, The ratio of the drug capable of activating ferroptosis inhibitor 1 to reduced coenzyme I is 0.1-10:0.05-40.

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