Application of RBC8 in preparation of heart protection medicine

Abstract

The invention discloses an application of an RAL GTPase small-molecule inhibitor in preparation of a heart protection drug, RBC8 can effectively relieve or prevent myocarditis caused by an immune checkpoint inhibitor, and has a clinical development and application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the application of RBC8, a small molecule inhibitor of RAL GTPase, in the preparation of cardioprotective drugs, and in particular to the application of RBC8 in the preparation of K399 Arg- Application of RALBP1 acylation modification in the treatment of ICIs-related myocarditis. Background Art

[0002] Immune checkpoint inhibitor (ICI)-associated myocarditis is a cardiotoxic reaction caused by immune checkpoint inhibitors, with a median onset of 30-65 days and a mortality rate of over 50%. There is no specific treatment option [1]. Modern cancer therapeutics have shown that drug combinations can lead to synergistic side effects, especially cardiovascular disease [2]. Studies have shown that mitochondrial targeting is the main determinant of cardiotoxic effects caused by chemotherapeutic agents for solid and hematological tumors [3]. This anti-tumor therapy-induced mitochondrial toxicity is due to different mechanisms, usually altering the mitochondrial respiratory chain, energy production and mitochondrial dynamics, or inducing mitochondrial oxidative / nitrosative stress, ultimately leading to cell death [4].

[0003] Programmed cell death protein 1 (PD-1) inhibitors are a major immune checkpoint inhibitor (ICIs) and have become one of the most widely used immunotherapy drugs for malignant tumors. Its main mechanism is to block the binding of PD-1 to its inhibitory ligand PD-L1 and restore the tumor-killing ability of T cells. However, the blockade of PD-1 / PD-L1 binding can also lead to immune tolerance disorders and induce immune-related adverse events, especially ICIs-related myocarditis (ICIAM) with a mortality rate of up to 27% to 67%, which often limits its clinical application. Necrotic tissue has been observed in the pathological tissues of patients with myocarditis caused by PD-1 inhibitors, but it is not clear whether programmed necrosis occurs and how it occurs. Although the current regimen of alleviating ICIAM through drug withdrawal and glucocorticoid treatment has achieved substantial progress in efficacy, it is still unable to completely prevent myocardial damage caused by PD-1 inhibitors. Therefore, it is urgent to explore new targets for the treatment of cardiotoxicity caused by PD-1 inhibitors.

[0004] Amide bond post-translational modification (PTMs), also known as acylation, is a regulation of protein levels by sensing metabolite levels through substrate proteins [5]. Acylation mainly catalyzes the formation of lysine aminoacyl bonds (K-AA) in substrate proteins. Acylation changes the physicochemical properties of the modified proteins and participates in key cellular processes related to physiology and disease, such as protein stability, protein subcellular localization, enzyme activity, transcriptional activity, protein-protein interaction, and protein-DNA interaction, thereby finely regulating the physiological and pathological processes of the body [6]. Under enzyme-dependent conditions, the "writer" acyltransferase is responsible for adding the acyl group in the "donor" acyl CoA to the side chain of lysine, glycine, cysteine, serine or other amino acid residues, resulting in protein acylation. In addition, the "eraser" deacylase can catalyze the removal of the acyl groups on the above amino acid residues. The acylation modification mark is usually read by a specific protein domain, also known as the "reader" [7].

[0005] RalA binding protein 1, also known as RALBP1, plays a role in receptor-mediated endocytosis and is a downstream effector of the small GTP-binding protein RAL[8]. Ral GTPases have emerged as important drivers of tumor growth and metastasis in lung, colon, pancreatic, and other cancers. Inhibition of Ral GTPases exhibits antitumor activity by binding to the GDP-binding allosteric site of RAL[9]. As a downstream effector of RALA and RALB, it can activate CDC42 and RAC1 by stimulating their GTPase activity. During mitosis, RALBP1 acts as a scaffold protein to phosphorylate EPSIN / EPN1 through the mitotic kinase B-CDK1, thereby preventing endocytosis

[10] . As an effector of RALA, it is recruited to mitochondria by RALA and acts as a scaffold to promote the phosphorylation of the mitotic kinase cyclin B-CDK1 and the activation of DNM1L, namely DRP1, which controls mitochondrial fission

[11] . At the same time, it has been reported that RALBP1 is overexpressed in a variety of cancers and is resistant to chemotherapeutic drugs

[12] . Mitochondrial fission can be a key event in regulating the development of programmed necrosis. Mitochondrial fission involving DRP1 can form a series network with the key programmed necrosis pathway RIP3-CAMKII / MLKL, namely the MAMs-CAMKIIδ pathway, to jointly regulate programmed necrosis. Programmed necrosis is the terminal event of myocardial tissue disease

[13] . Summary of the invention

[0006] Our research group has found in mass spectrometry that arginine acylation modification may occur at the 399th lysine (K399) site of RALBP1 in ICIs-related myocarditis. Through sequencing analysis, we found that the K399 site mutation of RALBP1 is associated with the progression of ICIs-related myocarditis, but the pathogenic mechanism and clinical application are still not well understood. Based on this, we proposed a scientific hypothesis: mutations in cardiac GTP-binding proteins lead to abnormal mitochondrial fusion and fission, causing mitochondrial dysfunction in cells, thereby leading to myocardial structural damage and dysfunction.

[0007] The selective inhibitor of GTPases RalA and RalB, RBC8, can effectively inhibit the activation of RALGTPases. RBC8 inhibits the oncogenic function of sorafenib in a dose-dependent manner and sensitizes HCC cells to sorafenib treatment, potentially enhancing the inhibition of mammalian rapamycin signaling targets

[14] . In order to provide a preliminary basis for clinical transformation, we used RALBP1 knockout mice, specific inhibitors RBC8, and point mutation viruses of RALBP1 to reveal from both positive and negative perspectives that the RALBP1 mutation-related pathway is an important mechanism for the occurrence of ICIs-related myocarditis. The development of related inhibitor drugs targeting this pathway is expected to bring new possibilities for the treatment of ICIs-related myocarditis in the future.

[0008] We used RBC8, a small molecule inhibitor of RAL GTPase, and a myocardial-specific promoter point mutation virus to inhibit the abnormal activation of the MAMs-CAMKIIδ pathway caused by RALBP1 gene mutation, thereby improving the cardiac function prognosis of patients with ICIs-related myocarditis with RALBP1 gene mutation. The experiment found that the MAMs-CAMKIIδ pathway and K399 in the myocardial tissue of patients with myocarditis were significantly upregulated. Arg- RALBP1 is activated. An ICIs-related myocarditis mouse model was constructed, and the therapeutic effect of RBC8 on ICIs-related myocarditis was detected by indicators such as changes in cardiac function and activation of related signaling molecules, providing a theoretical basis for the application of RBC8 in the clinical treatment of ICIs-related myocarditis. Based on the above research results, the present invention provides the following technical solutions:

[0009] The present invention provides the use of a RAL GTPase small molecule inhibitor in the preparation of a cardioprotective drug.

[0010] Preferably, the above RAL GTPase small molecule inhibitor is RBC8, CAS No.: 361185-42-4. Its chemical name is 6-amino-4-(2,5-dimethoxyphenyl)-3-naphthalen-2-yl-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile, and its molecular formula is C 25 H 20 N4O3, chemical structure is as follows:

[0011]

[0012] In one embodiment, the above-mentioned "cardioprotection" refers to reducing or preventing cardiotoxicity caused by immune checkpoint inhibitors (ICIs); accordingly, the drug is a drug for reducing or preventing cardiotoxicity caused by immune checkpoint inhibitors (ICIs);

[0013] For example, the cardiotoxicity refers to myocardial damage caused by immune checkpoint inhibitors (ICIs); accordingly, the drug is a drug used to treat or prevent myocardial damage caused by immune checkpoint inhibitors (ICIs).

[0014] Furthermore, the myocardial injury refers to ICIs-associated myocarditis (ICIAM); accordingly, the drug is a drug for treating or preventing ICIs-associated myocarditis (ICIAM).

[0015] For example, the ICIs-related myocarditis refers to K399 Arg- ICIs-related myocarditis caused by acylation modification of RALBP1; accordingly, the drug is used to treat or prevent K399 Arg- Drugs for ICIs-related myocarditis caused by RALBP1 acylation modification.

[0016] The subjects for administration of the above-mentioned drugs are especially tumor patients who are receiving immune checkpoint inhibitors (ICIs) such as programmed cell death protein 1 (PD-1) inhibitors, namely PD-1 inhibitors.

[0017] In one embodiment, the medicament comprises a therapeutically effective amount of a small molecule inhibitor of RAL GTPase, such as RBC8, as the only active ingredient.

[0018] In another embodiment, the drug is a pharmaceutical composition which, in addition to a therapeutically effective amount of a RALGTPase small molecule inhibitor such as RBC8 as an active ingredient, also contains an immune checkpoint inhibitor such as a PD-1 inhibitor and / or other pharmaceutical ingredients for preventing cardiac damage such as glucocorticoids.

[0019] Optionally, the above-mentioned medicine may further contain one or more pharmaceutically acceptable carriers.

[0020] The dosage form of the above-mentioned medicine can be an oral preparation or an injection.

[0021] The oral preparation is selected from the group consisting of tablets, capsules (including but not limited to dispersible capsules and gelatin capsules), granules, powders, solutions, and syrups.

[0022] In the oral preparation, the pharmaceutically acceptable carrier includes one or more of the following groups: filler or extender, binder, wetting agent, disintegrant, absorbent, lubricant, buffer, complexing agent, colorant.

[0023] When the dosage form of the drug is an injection, it is suitable for intravenous injection or intravenous drip.

[0024] This paper first proposed to use the post-translational modification of RALBP1 protein as an entry point. The results of the previous mass spectrometry test showed that the 399th lysine site of RALBP1 may undergo amino acid acylation modification. The experiment found that RBC8 can effectively treat K399 Arg- ICIs-related myocarditis caused by acylation modification of RALBP1 provides a new treatment for ICIs-related myocarditis caused by RALBP1 mutations, bringing new options for this type of patients and has broad potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The immunofluorescence detection photos of myocardial tissue samples of myocarditis patients and normal adult myocardial tissue sample slices in Example 1 are shown. Among them, the upper row is the group without immune-related toxicity and / or immune checkpoint inhibitors (non-irAEs / ICIs), and the lower row is the ICIs-Myocarditis group; MLKL, RIP3, and CANKIIδ are all key proteins of programmed necrosis, MLKL is a mixed lineage kinase domain-like protein that is positively correlated with inflammation, CAMKIIδ is a calcium ion / calmodulin-dependent protein kinase 2 that is positively correlated with inflammation, and RIP3 is a receptor interacting protein 3 that is positively correlated with inflammation; DAPI is a photo of cell nucleus staining; Merge is a picture of several staining images synthesized together.

[0026] Figure 2The immunofluorescence detection photos of myocardial tissue samples of myocarditis patients and normal adult myocardial tissue samples in Example 1 are shown. Among them, the upper row is the non-irAEs / ICIs group, and the lower row is the ICIs-Myocarditis group; K399 Arg- RALBP1 is K399 Arg- RALBP1 is acylated, DRP1 is dynamin-related protein 1, which is positively correlated with inflammation, and RARS is arginyl-tRNA synthetase, which is positively correlated with inflammation.

[0027] Figure 3 The echocardiograms of the mice in the Control group, ICIs group and ICIs+Arg (arginine) group after modeling in Examples 2 and 3 are shown.

[0028] Figure 4 HE staining photos of heart sections of mice in the Control group, ICIs group and ICIs+Arg group after modeling in Examples 2 and 3 are shown.

[0029] Figure 5 The echocardiograms of the Vector (empty adeno-associated virus vector) + ICIs + Arg group, i.e., the control group, the RALBP1-Vector + ICIs + Arg group, the K399A-Vector + ICIs + Arg group, and the K399R-Vector + ICIs + Arg group in Example 4, detected 7 days after the mice were modeled.

[0030] Figure 6 HE staining photographs of heart sections of mice in the ICIs+Arg+Vector group, i.e., the control group, ICIs+Arg+RALBP1-Vector group, ICIs+Arg+K399A-Vector group, and ICIs+Arg+K399R-Vector group after modeling in Example 2 are shown.

[0031] Figure 7 The immunofluorescence detection photos of heart sections of mice in the ICIs+Arg+Vector group, i.e., the control group, the ICIs+Arg+RALBP1-Vector group, the ICIs+Arg+K399A-Vector group, and the ICIs+Arg+K399R-Vector group after modeling in Example 2 are shown.

[0032] Figure 8 The Western Immunoblot detection photos of cardiomyocytes in the ICIs group, Arg group, K399R-AVV group, K399A-AVV group and RALBP1-AVV group mice after modeling in Example 2 are shown.

[0033] Fig. 9 The echocardiograms of mice in the ICIs+Arg+Saline group (control group) and the ICIs+Arg+RBC8 group on the 30th day of treatment in Example 4 are shown.

[0034] Fig.10 HE staining photos of heart sections of mice in the ICIs+Arg+Saline group (control group) and the ICIs+Arg+RBC8 group on day 30 of treatment in Example 5 are shown.

[0035] Fig.11 The immunofluorescence detection photos of myocardial cells of mice in the ICIs+Arg+Saline group (control group) and the ICIs+Arg+RBC8 group on the 30th day of treatment in Example 7 are shown.

[0036] Fig.12 The photos of Western Immunoblot detection of heart tissues of mice in the ICIs+Arg group (control group) and the ICIs+Arg+RBC8 group on the 30th day of treatment in Example 6 are shown. DETAILED DESCRIPTION

[0037] This research topic is a National Natural Science Foundation project, project name: Molecular mechanism of AGPAT2 / LPA-mediated cardiomyocyte pyroptosis promoting ICIs-related myocarditis, project number 82170359.

[0038] Arginine and related metabolic pathways are associated with cancer therapeutics-related cardiac dysfunction (CTRCD). In our metabolomics study of ICIs-related myocarditis patients and mice, we found that arginine is a non-essential amino acid that is highly expressed in both. Combined with previous studies, it was found that arginine may participate in the occurrence and development of ICIs-related myocarditis as an important metabolite through acylation modification. When arginine was exogenously administered to mice with ICIs-related myocarditis, it was found that after exogenous arginine supplementation, the cardiac function of mice significantly deteriorated and inflammatory infiltration increased. For the first time, we found that RALBP1 has a teratogenic effect on ICIs-related myocarditis. RALBP1 promotes the occurrence and development of ICIs-related myocarditis by acylation modification of arginine at the 399th lysine site. The application of RALBP1-specific inhibitor RBC8 improved the progression of cardiac function and prognosis of mice with ICIs-related myocarditis, which provides a theoretical basis for the clinical application of RBC8 in the treatment of ICIs-related myocarditis.

[0039] As an application mode of the present invention, RBC8 can be prepared into a cardioprotective drug. Herein, in order to describe the effect in a drug or a pharmaceutical composition, the active ingredient RAL GTPase small molecule inhibitor such as RBC8 can be referred to as an "active compound".

[0040] The drug may be a single-component drug comprising a therapeutically effective amount of a small molecule inhibitor of RAL GTPase, such as RBC8, or a pharmaceutical composition further comprising other components, such as a pharmaceutically acceptable carrier.

[0041] RBC8 can be used as the sole active ingredient in the drug or in combination with a PD-1 inhibitor and / or other drug ingredients used to prevent heart damage, such as glucocorticoids.

[0042] As a specific embodiment of the combined use of two or more drug ingredients, the drug is a pharmaceutical composition comprising a therapeutically effective amount of a RAL GTPase small molecule inhibitor RBC8 and / or a PD-1 inhibitor, and / or other drug ingredients for preventing cardiac damage such as glucocorticoids.

[0043] The above-mentioned pharmaceutical composition comprising a RAL GTPase small molecule inhibitor such as RBC8 and an immune checkpoint inhibitor (ICIs) such as a PD-1 inhibitor has the effect of combined administration of an immune checkpoint inhibitor (ICIs) and a RAL GTPase small molecule inhibitor RBC8, and can be used to treat malignant tumors and reduce the cardiac toxicity caused by immune checkpoint inhibitors (ICIs).

[0044] It should be understood that the term "or" as used herein sometimes means "and / or", and the term "or" sometimes means "and / or". The term "and / or" as used in phrases such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0045] The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. Pharmaceutically acceptable carriers are well known in the art, and these carriers include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and harmless to the patient, including, for example, aqueous solutions (such as water or physiological buffered saline) or other solvents or vehicles (such as glycols, glycerol, oils (such as olive oil) or injectable organic esters). Excipients can be selected, for example, to achieve delayed release of the medicament or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in the form of dosage units, such as tablets, capsules (including dispersed capsules and gelatin capsules), granules, powders, solutions, syrups, suppositories, injections, etc.

[0046] As used herein, the term "effective amount" refers to the therapeutic amount required to alleviate at least one or more symptoms of a disease or condition, and relates to a sufficient amount of a drug that provides a desired effect. Therefore, the term "therapeutically effective amount" refers to a therapeutic amount sufficient to cause a specific effect when applied to a typical subject. In various contexts, the effective amount as used herein also includes an amount sufficient to delay the development of a disease condition, change the process of a disease condition (for example, but not limited to, slowing down the progression of a disease condition), or reverse a disease condition. It should be understood that there are many ways known in the art to determine the effective amount for a given application. For example, pharmacological methods for dose determination can be used in a therapeutic context. In the context of therapeutic or preventive applications, the amount of the composition applied to the subject will depend on the type and severity of the disease and the characteristics of the individual, such as overall health, age, sex, weight, and tolerance to the drug. It also depends on the degree, severity, and type of the disease. Those skilled in the art will be able to determine the appropriate dose based on these and other factors. For example, the therapeutically effective amount of RBC8 can be determined by referring to its current safe use amount for the treatment of tumors in tumor patients, and by clinical investigation. The appropriate effective dosage also needs to take into account therapeutic factors such as the dosage form of the drug, the constitution, weight, age, disease progression, and administration site of the individual being administered.

[0047] Pharmaceutical Agents Small molecule inhibitors of RAL GTPases, such as RBC8, may also be administered in combination with one or more additional therapeutic compounds.

[0048] In addition to the main component RBC8, the pharmaceutical dosage form may also contain a pharmaceutically acceptable carrier. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars (such as lactose, glucose and sucrose); (2) starches (such as corn starch and potato starch); (3) cellulose and its derivatives (such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate); (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients (such as cocoa butter and suppository wax); (9) oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, etc.); oil and soybean oil); (10) glycols (such as propylene glycol); (11) polyols (such as glycerol, sorbitol, mannitol and polyethylene glycol); (12) esters (such as ethyl oleate and ethyl laurate); (13) agar; (14) buffers (such as magnesium hydroxide and aluminum hydroxide); (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0049] The pharmaceutical preparation can be administered to a subject by any of a number of routes of administration, including, for example, oral (e.g., as a drench, tablet, capsule (including dispersible capsules and gelatin capsules) in an aqueous or non-aqueous solution or suspension, bolus, powder, granules, paste for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment, or spray applied to the skin). The RBC8 compound can also be formulated for inhalation. In certain embodiments, the RBC8 compound can simply be dissolved or suspended in a sterile solvent.

[0050] The above-mentioned term "subject" means a person or an animal. Generally, an animal is a vertebrate, such as a primate, a rodent, a livestock or a game animal. Primates include chimpanzees, crab-eating monkeys, spider monkeys and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffaloes, feline species (such as domestic cats), canine species (such as dogs, foxes, wolves). In some embodiments, the subject is a mammal, such as a primate such as a human. The terms "individual", "patient" and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow, but is not limited to these examples. Advantageously, a non-human mammal can be used as a subject representing an animal model having / producing ICIs-related myocarditis. The subject can be male or female. Obviously, when "subject" refers to animals, drugs for preventing and treating ICIs-related myocarditis refer to veterinary drugs or animal drugs.

[0051] The pharmaceutical preparation can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary according to the host being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces the therapeutic effect. Typically, by weight, this amount ranges from about 1% to about 99% active ingredient, for example, from about 5% to about 70%.

[0052] The method for preparing these preparations or compositions comprises the step of combining active compound (such as RBC8) with carrier and optionally one or more auxiliary ingredients.Usually, the preparation is prepared by uniformly and intimately combining RBC8 with liquid carrier or finely ground solid carrier or both, and then shaping the product (if necessary).

[0053] Formulations of the invention suitable for oral administration may be in the form of capsules (including dispersible capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophiles, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base such as gelatin and glycerin, or sucrose and acacia) and / or as a mouthwash, etc., each containing a predetermined amount of RBC8 as the active ingredient. The composition or compound may also be administered as a bolus, electuary or paste.

[0054] To prepare solid dosage forms for oral administration (capsules (including dispersible capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers (such as sodium citrate or dicalcium phosphate) and / or any of the following: (1) fillers or extenders (such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid); (2) binders (such as, for example, carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia); (3) wetting agents (such as glycerol); (4) disintegrants (such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate); (5) solution retardants (such as paraffin); (6) absorption accelerators (such as quaternary ammonium compounds); (7) wetting agents (such as, for example, cetyl alcohol and glyceryl monostearate); (8) absorbents (such as kaolin and bentonite); (9) lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium laurate sulfate, and mixtures thereof); (10) complexing agents (such as modified or unmodified cyclodextrins); and (11) coloring agents. In the case of capsules (including dispersible capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also contain a buffer. Solid compositions of a similar type may also be used as fillers in soft-filled and hard-filled gelatin capsules using such excipients as lactose and high molecular weight polyethylene glycols and the like.

[0055] Tablets can be made by compression or molding, optionally using one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate, or cross-linked sodium carboxymethyl cellulose), a surfactant or a dispersant. Molded tablets can be made by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine.

[0056] Tablets and other solid dosage forms of pharmaceutical compositions (such as dragees, capsules (including dispersible capsules and gelatin capsules), pills and granules) may optionally be scored, or prepared with coatings and shells (such as enteric coatings and other coatings well known in the art of pharmaceutical formulation). They may also be formulated to provide slow release or controlled release of the active ingredient therein, using, for example, hydroxypropylmethylcellulose in different proportions to provide the desired release curve, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtering through a filter that retains bacteria, or by incorporating a sterilizing agent in the form of a sterile solid composition, which may be dissolved in sterile water or some other sterile injectable medium just before use. These compositions may also optionally contain an emulsifier, and may be a composition that releases one or more active ingredients only or preferentially in a certain part of the gastrointestinal tract (optionally, in a delayed manner). Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, with one or more of the above-mentioned excipients when appropriate.

[0057] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art (such as, for example, water or other solvents, cyclodextrins and derivatives thereof), solubilizers and emulsifiers (such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, groundnut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof).

[0058] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0059] Suspensions, in addition to the active compounds, may contain suspending agents (such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof).

[0060] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers or propellants that may be required.

[0061] Ointments, pastes, creams and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0062] In addition to the active compound, powders and sprays may contain excipients (such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances). Sprays may additionally contain customary propellants (such as chlorofluorocarbons and volatile unsubstituted hydrocarbons (such as butane and propane)).

[0063] Transdermal patches have the additional advantage of providing controlled delivery of the active compound to the body. Such dosage forms can be manufactured by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or a gel.

[0064] The example of suitable aqueous carrier and non-aqueous carrier that can be used in the pharmaceutical composition of the present invention includes water, ethanol, polyol (such as glycerol, propylene glycol, polyethylene glycol, etc.) and its suitable mixture, vegetable oil (such as olive oil) and injectable organic ester (such as ethyl oleate). The example of suitable aqueous carrier and non-aqueous carrier that can be used in the pharmaceutical composition of the present invention includes water, ethanol, polyol (such as glycerol, propylene glycol, polyethylene glycol, etc.) and its suitable mixture, vegetable oil (such as olive oil) and injectable organic ester (such as ethyl oleate). For example, by using coating material (such as lecithin), by keeping required particle size in the case of dispersion, and by using surfactant, keep suitable fluidity. For example, by using coating material (such as lecithin), by keeping required particle size in the case of dispersion, and by using surfactant, keep suitable fluidity.

[0065] These compositions can also contain adjuvants (such as preservatives, wetting agents, emulsifiers and dispersants). It is possible to ensure the prevention of microbial effects by including various antibacterial and antifungal agents (for example, methylparaben, chlorobutanol, phenol sorbic acid, etc.). It is also desirable to include isotonic agents (such as sugar, sodium chloride, etc.) in the composition. In addition, extended absorption of injectable drug forms can be caused by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0066] In some cases, in order to prolong the effect of the drug, it is desirable to slow down the absorption of the drug from subcutaneous injection or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline material or amorphous material with poor water solubility. The absorption rate of the drug depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, the delayed absorption of the drug form administered parenterally is achieved by dissolving or suspending the drug in an oil vehicle.

[0067] Injectable reservoir forms are made by forming a microencapsulated matrix of the subject compound in a biodegradable polymer (such as polylactide-polyglycolide). Depending on the ratio of the drug to the polymer and the properties of the specific polymer employed, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions compatible with body tissues.

[0068] For use in the methods of the invention, the active compound may be administered per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0069] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0070] The selected dosage level will depend upon a variety of factors including the activity of the specific compound or combination of compounds employed, or the esters, salts or amides thereof, the route of administration, the time of administration, the rate of excretion of the specific compound or compounds employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the specific compound or compounds employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0071] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the therapeutically effective amount of the required pharmaceutical composition. For example, a physician or veterinarian can start with a dosage of a pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved." Therapeutically effective amount" means the concentration of the compound sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age and medical history of the subject. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's condition, the disease treated, the stability of the compound, and (if necessary) another type of therapeutic agent administered together with RBC8. A larger total dose can be delivered by multiple administrations of the medicament. The method for determining efficacy and dosage is known to those skilled in the art.

[0072] In general, a suitable daily dose of the active compound used in the compositions and methods of the invention will be the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0073] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses, optionally in unit dosage form, at appropriate intervals throughout the day. In certain embodiments of the invention, the active compound may be administered twice or three times a day. In other embodiments, the active compound will be administered once a day.

[0074] Patients receiving such treatment are any animal in need thereof, including primates (particularly humans); and other mammals (such as horses, cattle, pigs, sheep, cats, and dogs); poultry; and pets in general.

[0075] In certain embodiments of the invention, a small molecule inhibitor of RAL GTPase, such as RBC8, is administered in combination with an immune checkpoint inhibitor (PD-1 inhibitor).

[0076] The present invention provides a new drug for treating ICIs-related myocarditis caused by acylation modification of RALBP1. By discovering that RALBP1 undergoes arginine acylation modification at the 399th lysine site, point mutations are performed on the corresponding sites to simulate modification (RALBP1-K399R) and demodification (RALBP1-K399A), respectively, to verify the application of acylation modification drugs in ICIs-related myocarditis. Arg-Under the premise that all RALBP1 acylation modifications are activated, the RALBP1 specific inhibitor RBC8 is used to treat ICIs-related myocarditis caused by RALBP1 acylation modification. The present invention provides a theoretical basis for the clinical application of RBC8 to treat ICIs-related myocarditis, and also provides a new target for the treatment of ICIs-related myocarditis in tumor patients, and also finds a new method for the treatment of such patients, and has a good clinical application prospect.

[0077] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0078] In the embodiments of the present invention, if there is no specific description on the experimental operation temperature, the temperature generally refers to room temperature (10-30° C.).

[0079] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0080] Example 1: Preparation of myocardial tissue samples from patients with myocarditis

[0081] Myocardial tissue samples from patients who met the myocardial biopsy criteria and had a confirmed diagnosis of myocarditis were collected at the Heart Institute of Zhongshan Hospital Affiliated to Fudan University and stored in a tissue fixative at 4°C. Due to the limitations of normal adult myocardial tissue sample collection, we collected myocardial tissue from patients with hypertrophic cardiomyopathy who did not undergo inflammatory changes after surgery as a control, i.e., the control group. Detection of K399 in myocardial tissue of patients with myocarditis Arg- The acylation level of RALBP1 is related to the activation of the MAMs-CAMKIIδ pathway. Figure 1 and Figure 2 In. From Figure 1 It can be seen that compared with the control group, the expression of MLKL (red), CAMKIIδ (green) and RIP3 (orange) in the myocarditis tissue of ICIs patients was significantly increased, that is, the fluorescence intensity of MAMs-CAMKIIδ pathway-related proteins in the myocardial tissue of patients with myocarditis after Merge was higher; Figure 2 :Compared with the control group, DRP1 (red) and K399 in myocarditis tissues of ICIs patients Arg The expression of RALBP1 (green) was significantly increased, while the expression of RARS (orange) did not change significantly, that is, K399 in the myocardial tissue of patients with myocarditis after Merge Arg- The fluorescence intensity of proteins related to RALBP1 acylation modification is higher.

[0082] Example 2: Experimental animals and animal model preparation

[0083] SPF male Balb / c mice (purchased from the Experimental Animal Research Center of Fudan University) aged 6-8 weeks and weighing 20-22g were selected. 250μg cTnI was subcutaneously injected on the 0th and 7th days at the beginning of modeling, and the corresponding control group was subcutaneously injected with an equal amount of saline (0.1mL). Starting from the 7th day, 0.1mL 5mg / kg PD-1 inhibitor was intraperitoneally injected every 1 day, that is, on the 7th, 9th, 11th, 13th and 15th days, for a total of 5 times; ICIs-related myocarditis mouse animal model was established.

[0084] (1) Before modeling, mice were gavaged with 10 mg / kg arginine (Arg) for one week.

[0085] (2) RALBP1 wild-type mice (control), RALBP1-K399A mutant mice and RALBP1-K399R mutant mice were constructed using RALBP1 cardiomyocyte-specific promoter virus adeno-associated virus vector (RALBP1-Vector, also known as RALBP1-AVV, and provided by Meta Biotechnology (Shanghai) Co., Ltd.), RALBP1-K399A mutant cardiomyocyte-specific promoter virus adeno-associated virus vector (K399A-Vector, also known as RALBP1-K399A-Vector, K399A-AVV, and provided by Meta Biotechnology (Shanghai) Co., Ltd.) and RALBP1-K399R mutant cardiomyocyte-specific promoter virus adeno-associated virus vector (K399R-Vector, also known as RALBP1-K399R-Vector, K399R-AVV, and provided by Meta Biotechnology (Shanghai) Co., Ltd.). After the myocardial-specific promoter virus was overexpressed in mice, an ICIs-related myocarditis mouse model was constructed, and the cardiac function of the mice was tested after completion. Figures 3 to 8 shown.

[0086] (3) Before modeling, the GTPases RalA and RalB inhibitor RBC8 (50 mg / kg, MCE, HY-12873) was intraperitoneally injected for 14 days. After the model was established, the cardiac function of the mice was tested. Figures 3 to 8 This experimental protocol was approved by the Animal Care and Ethics Committee of Fudan University.

[0087] See also Figures 3 to 8 Animal experiments have shown that: (1) the cardiac function of mice gavaged with arginine was significantly reduced, and the inflammatory infiltration in the myocardial tissue was significantly increased.

[0088] (2) The left ventricular ejection fractions (LVEF) and fraction shortening (FS) of mice in the Vector+ICIs group (empty adeno-associated virus vector control group) were decreased, and the LVEF and FS of mice injected with RALBP1-Vector or RALBP1-K399R-Vector were further decreased; while the LVEF and FS of mice injected with RALBP1-K399A-Vector were increased. Western blot results showed that RALBP1-Vector or RALBP1-K399R-Vector activated the MAMs-CAMKIIδ pathway, while RALBP1-K399A-Vector inhibited the MAMs-CAMKIIδ pathway. In the Vector+ICIs group, RALBP1-Vector+ICIs group and K399R-Vector+ICIs group, there was obvious inflammatory cell infiltration and fibrosis in the myocardial tissue of mice, and the degree of inflammatory cell infiltration in the myocardial tissue of mice in the K399A-Vector+ICIs group was significantly improved, and the degree of fibrosis was significantly reduced.

[0089] (3) The cardiac function of mice in the ICIs group was significantly impaired. However, after the administration of RBC8 to mice with myocarditis, LVEF and FS increased significantly, while the left ventricular end-diastolic diameter (LVEDd) and left ventricular end-systolic diameter (LVEDs) decreased significantly, indicating that the cardiac function was significantly improved. The cardiomyocytes of mice in each group were further isolated, and the results of Western blot showed that the MAMs-CAMKIIδ pathway was activated in the ICIs group, while the MAMs-CAMKIIδ pathway was inhibited in the RBC+ICIs group. After the administration of RBC8, the degree of inflammatory cell infiltration and fibrosis in the myocardial tissue of mice decreased significantly.

[0090] Example 3: Mouse model grouping experiment

[0091] The experimental steps for mouse model grouping are as follows:

[0092] (1) After echocardiography, the mice were divided into three groups: control group, ICIs group and ICIs+Arg group. The control group and ICIs group were intragastrically administered with the same volume of saline.

[0093] (2) After echocardiography, the mice were divided into four groups: Vector+ICIs+Arg group, RALBP1-Vector+ICIs+Arg group, K399A-Vector+ICIs+Arg group, and K399R-Vector+ICIs+Arg group.

[0094] (3) The mice were divided into two groups: ICIs+Arg+Saline group and ICIs+Arg+RBC8 group. The ICIs+Arg group was intragastrically administered with the same volume of saline.

[0095] Example 4: Echocardiography to measure cardiac function in mice

[0096] The mice were subjected to echocardiography 7 days after the model was established. Transthoracic echocardiography was performed on the mice using a 30MHz high-frequency scanning probe. Anesthesia was induced by inhalation of 2% isoflurane at a flow rate of 2L / min for 1-2min. After the mice were anesthetized, they were placed in a supine position on an isothermal experimental table. M-mode echocardiogram images were captured when the heart rate (HR) of the mice was maintained at 350-550bpm. Subsequently, the left ventricular ejection fraction (LVEF), shortening fraction (FS), left ventricular end-diastolic dimension (LVEDd) and left ventricular end-systolic dimension (LVEDs) of the mice were quantified. The echocardiograms of the mice were measured continuously for 3-5 cardiac cycles and in a double-blind condition. The results are shown in Figure 3 , Figure 5 , Fig. 9 .

[0097] Example 5: HE staining of heart tissue

[0098] After echocardiography, mouse samples were collected. The peritoneal paralyzed mice were anesthetized with 10% chloral hydrate, and the heart was removed after inducing paralysis. After the residual blood in the cardiac cavity was pumped out, the heart was fixed in tissue fixative for 7 days. The tissue was transparent after the action of ethanol and xylene. Subsequently, the transparent tissue was embedded in molten paraffin, cooled and solidified in sequence, and finally cut into 5μM slices. The slices were flattened in hot water, mounted on slides, and dried in an incubator at 45°C. Before staining, the paraffin was removed, and the tissue was stained with hemoglobin and eosin, and the cytoplasm was stained pink. Hematoxylin stains the nucleus and intracellular ribosomes blue and purple. The results are shown in Figure 4 , Figure 6 , Fig.10 .

[0099] Example 6: Western blot detection of MAMs-CAMKIIδ pathway

[0100] Myocardial tissue proteins were extracted from each group and tested by western blot. Figure 8 , Fig.12 .

[0101] Example 7: Immunofluorescence detection of myocardial tissue

[0102] Immunofluorescence staining was performed on frozen heart sections. Heart sections were permeabilized in 0.1% Triton X-100 / PBS for 2 min and incubated in blocking buffer (PBS, 5% goat serum, 2% bovine serum) for 1 h at room temperature. The primary antibody was incubated overnight at 4°C and washed 3 times with PBS for 5 min each. Then, the sections were incubated with Alexa Fluor 488 or 568-conjugated secondary antibodies at room temperature in the dark for 1 h, followed by staining of cell nuclei with DAPI for 10 min and then washed 3 times with PBS for 5 min each. The slides were rinsed with deionized water, dried, and covered with antifade agent. Immunoreactivity was detected by fluorescence microscopy the next day. The results are shown in Figure 1-2 , Figure 7 , Fig.11 .

[0103] Experimental Results

[0104] The experimental results of the above embodiments are summarized as follows.

[0105] 1. See Figure 1 The results showed that the fluorescence expression of MLKL protein was red, the fluorescence expression of CAMKIIδ protein was green, and the fluorescence expression of RIP3 protein was orange. The expression of MLKL (red), CAMKIIδ (green), and RIP3 (orange) in myocarditis tissue of ICIs patients was significantly increased, that is, the fluorescence intensity of MAMs-CAMKIIδ pathway-related proteins in myocardial tissue of patients with myocarditis after Merge was higher. This shows that compared with the control group, the programmed necrosis pathway RIP3-CAMKIIδ / MLKL pathway in myocardial tissue of patients in the myocarditis group was activated.

[0106] 2. See Figure 2 The results showed that DRP1 protein fluorescence expression was red and K399 Arg- The expression of RALBP1 protein fluorescence was green, while the expression of RARS protein fluorescence was orange. Compared with the control group, the expression of DRP1 (red) and K399 in myocarditis tissues of ICIs patients was significantly increased. Arg- The expression of RALBP1 (green) was significantly increased, while the expression of RARS (orange) did not change significantly, that is, K399 in the myocardial tissue of patients with myocarditis after Merge Arg- The fluorescence intensity of RALBP1 acylation-related proteins was higher, indicating that compared with the control group, K399 in the myocarditis tissue of patients with myocarditis Arg- Both RALBP1 and DRP1 were significantly increased.

[0107] 3. See Figure 3 The results showed that the left ventricular ejection fraction (LVEF), shortening fraction (FS), and left ventricular posterior wall (LVPW) of mice in the ICIs group were significantly reduced, and the LVEF, FS, and LVPW of mice in the ICIs+Arg group further decreased. This shows that the cardiac function indicators of mice in the ICIs group were significantly lower than those in the control group, and the cardiac function of mice in the ICIs+Arg group further deteriorated.

[0108] 4. See Figure 4 The results showed that the cytoplasm (red) of the right ventricular myocardial tissue in the ICIs group was significantly reduced and replaced by inflammatory cells (purple), indicating that the infiltration of inflammatory cells in the ICIs group was significantly increased, and arginine further promoted the infiltration of inflammatory cells.

[0109] 5. See Figure 5 The results showed that the LVEF and FS of mice in the Vector+ICIs+Arg group were decreased, and the LVEF and FS of mice injected with wild-type RALBP1-AAV or RALBP1-K399R-AAV virus further decreased; while the LVEF and FS of mice injected with RALBP1-K399A-AAV increased, indicating that compared with the Vector+ICIs+Arg group, the cardiac function indicators of mice in the RALBP1-Vector+ICIs+Arg group and the K399R-Vector+ICIs+Arg group were significantly decreased, while the cardiac function indicators of mice in the K399A-Vector+ICIs+Arg group were significantly improved.

[0110] 6. See Figure 6 The results showed that compared with the Vector+ICIs+Arg group, the infiltration of inflammatory cells in the myocardial tissue of mice in the RALBP1-Vector+ICIs+Arg group and the K399R-Vector+ICIs+Arg group was significantly increased, while the degree of inflammatory cell infiltration in the myocardial tissue of mice in the K399A-Vector+ICIs+Arg group was significantly improved.

[0111] 7. See Figure 7 The results showed that compared with the Vector+ICIs+Arg group, the myocardial tissue MLKL (red) and K399 in the RALBP1-Vector+ICIs+Arg group and the K399R-Vector+ICIs+Arg group were significantly higher than those in the Arg- The expression of RALBP1 (green) in the myocardial tissue of mice in the K399A-Vector+ICIs+Arg group was significantly increased, while cTnT is a specific marker for locating myocardial cells and has no difference. Arg-The expression of RALBP1 (green) was decreased, indicating that MLKL and K399 in myocardial tissue of mice in the RALBP1-Vector+ICIs+Arg group and the K399R-Vector+ICIs+Arg group Arg- The levels of RALBP1 in the myocardium of mice in the K399A-Vector+ICIs+Arg group were significantly increased, while the levels of MLKL and K399 Arg- RALBP1 was inhibited.

[0112] 8. See Figure 8 The results showed that compared with the Vector+ICIs+Arg group, the expressions of programmed necrosis-related proteins p-MLKL, p-CAMKIIδ and p-RIP3 in the myocardial tissue of mice in the RALBP1-Vector+ICIs+Arg group and the K399R-Vector+ICIs+Arg group were significantly increased, the expressions of mitochondrial fission proteins p-DRP1 and FIS1 were increased, and the mitochondrial fusion protein MFN2 was decreased, indicating that the MAMs-CAMKIIδ pathway was significantly activated. In the K399A-Vector+ICIs+Arg group, the expressions of p-MLKL, p-CAMKIIδ, p-RIP3, p-DRP1 and FIS1 in the myocardial cells of mice were decreased, and MFN2 was increased, indicating that the MAMs-CAMKIIδ pathway was significantly inhibited.

[0113] 9. See Fig. 9 The results showed that compared with the ICIs+Arg+Saline group, the LVEF and FS of mice in the ICIs+Arg+RBC8 group were increased, indicating that the cardiac function indicators were significantly improved.

[0114] 10. See Fig.10 The results showed that compared with the ICIs+Arg+Saline group, the infiltration of inflammatory cells in the myocardial tissue of mice in the ICIs+Arg+RBC8 group was significantly reduced.

[0115] 11. See Fig.11 The results showed that compared with the ICIs+Arg+Saline group, the myocardial tissue MLKL (red) and K399 in the ICIs+Arg+RBC8 group were significantly higher than those in the Arg- The expression of RALBP1 (green) was decreased, indicating that MLKL and K399 in myocardial tissue of mice in the ICIs+Arg+RBC8 group Arg- RALBP1 levels were significantly suppressed.

[0116] 12. See Fig.12The results showed that compared with the ICIs+Arg+Saline group, the expression of programmed necrosis-related proteins p-MLKL, p-RIP3, and p-CAMKIIδ in the ICIs+Arg+RBC8 group decreased. At the same time, the expression of mitochondrial fission proteins FIS1 and p-DRP1 decreased, and the expression of mitochondrial fusion protein MFN2 increased, indicating that the activation of the MAMs-CAMKIIδ pathway in myocardial cells of mice in the ICIs+Arg+RBC8 group was inhibited.

[0117] Conclusions

[0118] Arginine and related metabolic pathways are associated with cancer therapy-related cardiac dysfunction (CTRCD). In previous metabolomics studies of ICIs-related myocarditis patients and mice, our research group found that arginine is an amino acid that is highly expressed in both conditions. This suggests that arginine may be involved in the development of ICIs-related myocarditis through acylation modification as an important metabolite. After giving exogenous arginine to mice with ICIs-related myocarditis, it was found that the mice's cardiac function deteriorated significantly and inflammatory infiltration increased.

[0119] In the myocardial tissue of patients with ICIs-related myocarditis, it was found that the MAMs-CAMKIIδ pathway was activated, and K399 Arg- The acylation level of RALBP1 was significantly increased.

[0120] RALBP1 is involved in cell mitosis and mitochondrial fission. RALBP1 is associated with the development of cardiovascular diseases caused by metabolic syndrome. Based on our research, the deletion point mutation of the 399th lysine (K) site of RALBP1 to glycine (A) can significantly improve the cardiac function of mice, reduce inflammatory cell infiltration and inhibit the MAMs-CAMKIIδ pathway. However, the point mutation to arginine (R) can worsen the cardiac function of mice, increase inflammatory cell infiltration and activate the MAMs-CAMKIIδ pathway.

[0121] As a novel RALBP1-specific biological effect inhibitor, RBC8 can not only inhibit tumor proliferation, but also significantly improve ICIs-induced myocardial damage in mice, providing a theoretical basis for the application of RBC8 in the clinical treatment of tumor patients with ICIs-related myocarditis. RBC8 is an important supplement to traditional ICIs-related myocarditis treatments such as hormone shock therapy, cardiotonic therapy, diuresis, and vasodilation, which can delay the progression of myocardial damage and improve the quality of life of patients.

[0122] It should be noted that the listing and discussion of previously disclosed documents in this specification should not be regarded as an admission that the document is prior art or common knowledge.

[0123] References

[0124] [1]Wang DY,Salem JE,Cohen JV,Chandra S,Menzer C,Ye F,Zhao S,Das S,Beckermann KE,Ha L,Rathmell WK,Ancell KK,Balko JM,Bowman C,Davis EJ,Chism DD,Horn L,Long GV,Carlino MS,Lebrun-Vignes B,Eroglu Z,Hassel JC,Menzies AM,Sosman JA,Sullivan RJ,Moslehi JJ,Johnson DB.Fatal Toxic Effects AssociatedWith Immune Checkpoint Inhibitors:A Systematic Review and Meta-analysis.JAMAOncol.2018 Dec 1;4(12):1721-1728.

[0125] [2]Herrmann J.Adverse cardiac effects of cancer therapies:cardiotoxicity and arrhythmia.Nat Rev Cardiol.2020Aug;17(8):474-502.

[0126] [3]Jannuzzi AT,Korkmaz NS,Gunaydin Akyildiz A,Arslan Eseryel S,Karademir Yilmaz B,Alpertunga B.Molecular Cardiotoxic Effects of ProteasomeInhibitors Carfilzomib and Ixazomib and Their Combination with DexamethasoneInvolve Mitochondrial Dysregulation.Cardiovasc Toxicol.2023 Apr;23(3-4):121-131.

[0127] [4]Zhang D,Man D,Lu J,Jiang Y,Ding B,Su R,Tong R,Chen J,Yang B,ZhengS,Chen D,Wu J.Mitochondrial TSPO Promotes Hepatocellular CarcinomaProgression through Ferroptosis Inhibition and Immune Evasion.Adv Sci(Weinh).2023May10(15):e2206669.

[0128] [5]Geffen Y,Anand S,Akiyama Y,Yaron TM,Song Y,Johnson JL,Govindan A, Li Y,Huntsman E,Wang LB,Birger C,Heiman DI,Zhang Q,Miller M,MaruvkaYE,Haradhvala NJ,Calinawan A,Belkin S,Kerelsky A,Clauser KR,Krug K,SatpathyS,Payne SH,Mani DR,Gillette MA,Dhanasekaran SM,Thiagarajan M,Mesri M,Rodriguez H,Robles AI,Carr SA,Lazar AJ,Aguet F,Cantley LC,Ding L,Getz G andClinical Proteomic Tumor Analysis Consortium.Pan-cancer analysis of post-translational modifications reveals shared patterns of proteinregulation.Cell.2023Aug 31186(18):3945–3967.e26.

[0129] [6]Wang M,Lin H.Understanding the Function of Mammalian Sirtuins and Protein Lysine Acylation.Annu Rev Biochem.2021Jun 20:90:245-2

[0130] [7]Xie Y,Du S,Liu Z,Liu M,Xu Z,Wang X,Kee JX,Yi F,Sun H,YaoSQ.Chemical Biology Tools for Protein Lysine Acylation.Angew Chem Int EdEngl.2022 May 16;61(21):e202200303.

[0131] [8]Wang S,Chen X,Crisman L,Dou X,Winborn CS,Wan C,Puscher H,Yin Q,Kennedy MJ,Shen J.Regulation of cargo exocytosis by a Reps1-Ralbp1-RalAmodule.Sci Adv.2023Feb 22;9(8):eade2540.

[0132] [9]Jullien-Flores V,Dorseuil O,Romero F,Letourneur F,Saragosti S,Berger R,Tavitian A,Gacon G,Camonis JH.Bridging Ral GTPase to Rhopathways.RLIP76,a Ral effector with CDC42 / Rac GTPase-activating proteinactivity.J Biol Chem.1995 Sep 22;270(38):22473-7.

[0133]

[10] Singhal SS,Salgia R,Verma N,Horne D,Awasthi S.RLIP controlsreceptor-ligand signaling by regulating clathrin-dependentendocytosis.Biochim Biophys Acta Rev Cancer.2020 Jan;1873(1):188337.

[0134]

[11] Awasthi S, Hindle A, Sawant NA, George M, Vijayan M, Kshirsagar S, Morton H, Bunquin LE, Palade PT, Lawrence JJ, Khan H, Bose C, Reddy PH, SinghSP.RALBP1 in Oxidative Stress and Mitochondrial Dysfunction in Alzheimer's Disease.Cells.2021 Nov 10:10(11):3

[0135]

[12] Singhal SS,Salgia R,Singhal S,Horne D,Awasthi S.RLIP:Anexistential requirement for breast carcinogenesis.Biochim Biophys Acta RevCancer.2019 Apr1871(2):281-288.

[0136]

[13] Wang P,Zheng SY,Jiang RL,Wu HD,Li YA,Lu JL,Xiong Y,Han B,LinL.Necroptosis signaling and mitochondrial dysfunction cross-talking facilitate cell death mediated by chelerythrine in glioma.Free Radic BiolMed.2023 Jun6202:76-96.

[0137] Potor L,Nagy P,Mehes G,Hendrik Z,Jeney V, D,Vasas A,Palinkas Z,Balogh E, Whiteman M,Torregrossa R,Wood ME,OlvasztóS,Nagy P,Balla G,Balla J.Hydrogen Sulfide Abrogates Hemoglobin-Lipid Interaction inAtherosclerotic Lesion.Oxid Med Cell Longev.2018 Jan 21;2018:3812568。

Claims

1. Application of small molecule inhibitors of RAL GTPase in the preparation of cardioprotective drugs.

2. The use according to claim 1, characterized in that The RAL GTPase small molecule inhibitor is RBC8, CAS No.: 361185-42-4.

3. The use according to claim 1 or 2, characterized in that: The cardioprotection refers to reducing or preventing the cardiotoxicity caused by immune checkpoint inhibitors; accordingly, the drug is a drug used to reduce or prevent the cardiotoxicity caused by immune checkpoint inhibitors; For example, the cardiotoxicity refers to myocardial damage caused by immune checkpoint inhibitors; accordingly, the drug is a drug used to treat or prevent myocardial damage caused by immune checkpoint inhibitors.

4. The use according to claim 3, characterized in that The myocardial injury refers to ICIs-related myocarditis; accordingly, the drug is a drug used to treat or prevent ICIs-related myocarditis.

5. The use according to claim 4, characterized in that The ICIs-related myocarditis refers to K399 Arg- RALBP1 acylation modification caused by ICIs-related myocarditis; accordingly, the drug is used to treat or prevent K399 Arg- Drugs for ICIs-related myocarditis caused by RALBP1 acylation modification.

6. The use according to claim 1, characterized in that The drug is administered to tumor patients who are receiving immune checkpoint inhibitor treatment.

7. The use according to claim 1, characterized in that The drug contains a therapeutically effective amount of a small molecule inhibitor of RAL GTPase, such as RBC8, as the only active ingredient.

8. The use according to claim 1, characterized in that The drug is a pharmaceutical composition, which, in addition to containing a therapeutically effective amount of a RAL GTPase small molecule inhibitor such as RBC8 as an active ingredient, also contains an immune checkpoint inhibitor such as a PD-1 inhibitor and / or other pharmaceutical ingredients for preventing cardiac damage such as glucocorticoids.

9. The use according to claim 1, characterized in that The dosage form of the drug is an oral preparation or an injection.

10. The use according to claim 9, characterized in that The oral preparation is selected from the following group: tablets, capsules, granules, powders, solutions, syrups; the injection is suitable for intravenous injection or intravenous drip.

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