CN116236477A - Application of lysophosphatidic acid receptor 5 antagonist in preparation of heart protection medicine - Google Patents

Abstract

The invention discloses application of a lysophosphatidic acid receptor 5 antagonist in preparing a cardioprotective medicament, wherein the lysophosphatidic acid receptor 5 antagonist TC LPA54 can effectively reduce or prevent cardiotoxicity induced by an inhibitor of programmed death protein-1, and has clinical development and application prospects.

Description

Application of lysophosphatidic acid receptor 5 antagonist in preparation of heart protection medicine

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of a lysophosphatidic acid receptor 5 antagonist in preparing a heart protecting medicine, in particular to application of TC LPA54 in preparing a heart protecting medicine.

Background

The inhibitor of apoptosis protein 1 (programmed death protein-1, PD-1) is a main Immune Checkpoint Inhibitor (ICIs) and has become one of the most widely applied malignant tumor immunotherapy drugs in clinic at present. The main mechanism is to block the combination of PD-1 and its inhibitory ligand PD-L1 and restore the killing capacity of T cell tumor. However, blockade of PD-1/PD-L1 binding can also lead to immune tolerance imbalance, inducing immune related adverse events, especially ICIs-related myocarditis (ici am) with mortality rates as high as 27% -67%, often limiting its clinical application. Although current approaches to alleviating ici am by withdrawal and glucocorticoid therapy have made substantial therapeutic advances, there is still no complete prevention of myocardial damage caused by PD-1 inhibitors. Thus, there is a need to explore new targets for the treatment of cardiotoxicity caused by PD-1 inhibitors.

Myocardial lipid metabolism abnormality is an important pathological basis of myocardial injury induced by PD-1 inhibitors, and lysophosphatidic acid (Lysophosphatidic acid, LPA) plays a key role as a phospholipid substance and an important extracellular signal molecule which are mainly accumulated in the pathological process. It has been reported that LPA accumulated during this process binds to its downstream lysophosphatidic acid receptor (LPAR) and can promote increased assembly of myocardial cells NLRP3 inflammatory bodies, activate caspase-1, cleave Gasderm D, wherein N-Gasderm D can induce cell membrane perforation and rupture, release contents, and cause inflammatory reaction; at the same time, activated caspase-1 cleaves IL-1β and IL-18 precursors to form activated IL-1β and IL-18 which are released extracellular, recruit inflammatory cells and amplify inflammatory responses, together resulting in increased myocardial apoptosis. Thus blocking the binding of LPA to its receptor can be a potential target for preventing PD-1 inhibitor-induced cardiac injury.

Disclosure of Invention

LPARs are a generic term for a variety of cell membrane receptors that bind LPA. The subject group found through research experiments that specific binding of LPA receptor 5 (Lysophosphatidic acid receptor, LPAR 5) to LPA in the LPAR family was a key element in inducing PD-1 inhibitor-associated myocardial apoptosis. First, the coupling of LPAR5 to the 2G proteins of G12/13 and Gq plays a key regulatory role in a variety of inflammatory signaling pathways. Secondly, LPAR 5-coupled G protein is also a goatprotein, which can directly bind to NLRP3 inflammatory corpuscles, promote assembly and activation thereof, and further activate downstream pathways. Further studies have found that inhibition of activation of LPAR5, usually accompanied by abnormal activation of LPAR5, can significantly improve coke death, which is verified in related studies of islet inflammation and neuroinflammation in LPAR5 KO mice.

At present, several clinical studies have been developed to treat inflammatory diseases such as psoriasis, neuroinflammation, etc. by limiting the activation of LPAR 5. However, the therapeutic advantage of LPAR5 antagonists against PD-1 inhibitor-induced cardiotoxicity has not been explored. In view of this, the present invention provides the following technical solutions:

the present invention provides the use of a lysophosphatidic acid receptor 5 antagonist (LPAR 5 antagonist) in the manufacture of a cardioprotective medicament.

Preferably, the lysophosphatidic acid receptor 5 antagonist is TC LPA54, cas no: 1393814-38-4. Its molecular formula is C ₂₃ H ₂₃ ClN ₂ O ₃ The chemical structural formula is as follows:

in one embodiment, the above "cardioprotection" refers to reducing or preventing apoptosis protein-1 (PD-1) inhibitor-induced cardiotoxicity; accordingly, the medicament is a medicament for reducing or preventing apoptosis protein-1 (PD-1) inhibitor-induced cardiotoxicity.

Further, the above-mentioned cardiotoxicity refers to cardiac injury; accordingly, the medicament is a medicament for treating or preventing a programmed death protein-1 inhibitor (PD-1 inhibitor) -induced cardiac injury.

For example, the cardiac injury described above may refer to ICIs-related myocarditis (ici am); accordingly, the medicament is a medicament for treating or preventing ICIs-related myocarditis (ici am).

The subject to which the above drugs are administered is in particular a tumor patient receiving treatment with a programmed death protein-1 inhibitor (PD-1 inhibitor).

As a specific mode of the above application, the medicament comprises a therapeutically effective amount of a lysophosphatidic acid receptor 5 antagonist as the sole active ingredient.

As another specific mode of the above application, the medicament is a pharmaceutical composition comprising, in addition to a therapeutically effective amount of a lysophosphatidic acid receptor 5 antagonist as an active ingredient, a programmed death protein-1 inhibitor (PD-1 inhibitor) and/or other pharmaceutical ingredients for preventing cardiac damage.

Those skilled in the art will readily appreciate that the above-described medicaments may further comprise one or more pharmaceutically acceptable carriers.

The dosage form of the medicine can be oral preparation or injection.

The oral formulation is selected from the group consisting of: tablets, capsules (including but not limited to dispersion capsules and gelatin capsules), granules, powders, solutions, syrups.

In the oral formulation, the pharmaceutically acceptable carrier comprises more than one of the following groups: fillers or extenders, binders, wetting agents, disintegrants, absorbents, lubricants, buffers, complexing agents, colorants.

When the dosage form of the medicine is injection, the medicine is suitable for intravenous injection and intravenous drip.

The invention discovers that the lysophosphatidic acid receptor 5 antagonist such as TC LPA54 can effectively relieve the cardiotoxicity induced by the apoptosis protein-1 inhibitor, improve the myocardial cell apoptosis, play a role in protecting heart and delay the occurrence and development of myocardial injury, and has important clinical application value.

Drawings

Figure 1 shows echocardiography, left Ventricular Ejection Fraction (LVEF) and left ventricular short axis shrinkage (LVFS) statistics (ns: no statistical difference:. P < 0.05:. P < 0.01:. P < 0.001) measured on day 17 of Sham + Control, PD-1inhibitor + Control and PD-1inhibitor+TC LPA54 mice modelling.

FIG. 2 shows H-E staining photographs of heart sections of mice.

FIG. 3 shows immunofluorescence photographs of mouse heart sections.

Fig. 4 shows a photograph of a mouse heart tissue Western Immunoblot assay.

FIG. 5 shows immunofluorescence photographs of atrial myocytes of HL-1 mice.

FIG. 6 shows a Western Immunoblot assay of HL-1 mouse atrial myocytes.

Detailed Description

In the study, the subject group found that the lysophosphatidic acid receptor 5 antagonist TC LPA54 inhibits activation of a downstream focal death pathway by specifically antagonizing LPAR5, and improves the reduction of cardiac function and inflammatory infiltration of mice induced by PD-1 inhibitors. It was demonstrated that TC LPA54 can protect PD-1 inhibitor-induced cardiotoxicity by inhibiting cardiac myocyte apoptosis. The related experimental method, detection means, data analysis and the like are proven and widely applied reliable methods, and the result is reliable.

LPAR5 acts as one of the receptors for the critical signaling molecule LPA in cells and is critical for regulating apoptosis of the cell's coke and maintaining normal function of the cell. The study finds that the use of TC LPA54 can down regulate myocardial cell apoptosis, relieve cardiac toxicity caused by PD-1inhibitor, resist side effects caused by PD-1inhibitor use, expand the use range of PD-1inhibitor, increase the use safety of PD-1inhibitor and improve prognosis of tumor patients clinically using PD-1 inhibitor.

TC LPA54 is a specific non-lipid LPA ₅ (GPR 92) antagonists which inhibit LPA-induced aggregation of isolated human platelets (LPA 5-RH7777 cell line) and inhibit proliferation and migration of thyroid cancer cells are currently used clinically for the treatment of tumors. However, TC LPA54 has not been used in clinical practice for the treatment of PD-1 inhibitor-induced cardiotoxicity.

As an application mode of the invention, TC LPA54 can be prepared into heart protecting medicines. For purposes of describing the role in a drug or pharmaceutical composition herein, an active ingredient lysophosphatidic acid receptor 5 antagonist, such as TC LPA54, may be referred to as an "active compound".

The medicament may be a single component medicament comprising a therapeutically effective amount of a lysophosphatidic acid receptor 5 antagonist, such as TC LPA54, or a pharmaceutical composition further comprising other components, such as a pharmaceutically acceptable carrier.

TC LPA54 can be used as the only active ingredient in the medicament, or can be used in combination with PD-1 inhibitors and/or other pharmaceutical ingredients for preventing cardiac damage.

In particular embodiments, the medicament is a pharmaceutical composition comprising a therapeutically effective amount of a lysophosphatidic acid receptor 5 antagonist TC LPA54 and/or a PD-1inhibitor, and/or other pharmaceutical ingredients for preventing cardiac damage.

The pharmaceutical composition containing the lysophosphatidic acid receptor 5 antagonist such as TC LPA54 and the programmed death protein-1 inhibitor has the effect of combined administration of the programmed death protein-1 inhibitor and the lysophosphatidic acid receptor 5 antagonist TC LPA54, and can be used for treating malignant tumors and relieving cardiac toxicity induced by the programmed death protein-1 inhibitor.

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 herein such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass 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).

The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers are well known in the art and include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious 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 may be selected, for example, to achieve delayed release of the agent or to selectively target one or more cells, tissues or organs. The pharmaceutical compositions may be in the form of dosage units, such as tablets, capsules (including dispersible capsules and gelatin capsules), granules, powders, solutions, syrups, suppositories, injections and the like.

The term "effective amount" as used herein refers to the amount of treatment required to alleviate at least one or more symptoms of a disease or condition, and relates to a sufficient amount of a drug to provide the desired effect. Thus, the term "therapeutically effective amount" refers to a therapeutic amount sufficient to cause a particular effect when administered to a typical subject. In various contexts, an effective amount as used herein also includes an amount sufficient to delay the progression of a disease state, alter the course of a disease state (e.g., without limitation, slow the progression of a disease state), or reverse a disease state. It should be appreciated that there are many ways known in the art to determine an effective amount for a given application. For example, pharmacological methods for dose determination may be used in a therapeutic setting. In the context of therapeutic or prophylactic applications, the amount of composition administered to a subject will depend on the type and severity of the disease and the characteristics of the individual, such as general health, age, sex, weight and tolerance to drugs. It also depends on the extent, severity and type of the disease. One skilled in the art will be able to determine the appropriate dosage based on these and other factors. For example, a therapeutically effective amount of TC LPA54 can be determined by clinical investigation with reference to its safe use in the present administration to a tumor patient for the treatment of tumors. Suitable effective dosage amounts also take into account the therapeutic factors such as the dosage form, constitution, weight, age, condition course, site of administration, etc. of the individual to be administered.

The pharmaceutical component lysophosphatidic acid receptor 5 antagonist, e.g. TC LPA54, may also be administered in combination with one or more additional therapeutic compounds.

The pharmaceutical dosage form may comprise a pharmaceutically acceptable carrier in addition to the major component TC LPA5 4. Some examples of materials that may 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate); (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients (such as cocoa butter and suppository waxes); (9) Oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn 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) buffering agents (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 employed in the pharmaceutical formulation.

The pharmaceutical formulation may be administered to the subject by any of a number of routes of administration including, for example, orally (e.g., as a drench in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including dispersion capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingual); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment, or spray applied to the skin). The TC LPA54 compounds may also be formulated for inhalation. In certain embodiments, the TC LPA54 compound may simply be dissolved or suspended in a sterile solvent.

The term "subject" as used above refers to a human or animal. Typically, the animal is a vertebrate, such as a primate, rodent, livestock or hunting animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchuck, ferrets, rabbits, and hamsters. Domestic animals and hunting animals include cattle, horses, pigs, deer, wild cattle, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves). In some embodiments, the subject is a mammal, e.g., a primate such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Advantageously, a non-human mammal may be used as the subject of an animal model representing type II diabetes mellitus having/developing acarbose resistance. The subject may be male or female. It is apparent that when "subject" means an animal, the agent for controlling aortic aneurysm and/or aortic dissection means a veterinary drug or an animal drug.

The pharmaceutical formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will generally be the amount of the compound that produces a therapeutic effect. Typically, this amount ranges from about 1% to about 99% active ingredient, such as from about 5% to about 70% by weight.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound (such as TC LPA5 4) with the carrier and optionally one or more accessory ingredients. Typically, the formulation is prepared by uniformly and intimately bringing the TC LPA54 into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of: capsules (including dispersion capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, typically sucrose and acacia or tragacanth), lyophile, 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 pastilles (using an inert base such as gelatin and glycerin, or sucrose and acacia), and/or as a mouthwash, and the like, each containing a predetermined amount of TC LPA54 as the active ingredient. The composition or compound may also be administered as a bolus, electuary or paste.

To prepare solid dosage forms (capsules (including dispersible and gelatin capsules), tablets, pills, dragees, powders, granules and the like) for oral administration, 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, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia); (3) humectants (such as glycerol); (4) Disintegrants (such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate); (5) solution retarders (such as paraffin); (6) absorption accelerators (such as quaternary ammonium compounds); (7) Wetting agents (such as, for example, cetyl alcohol and glycerol 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) a colorant. In the case of capsules (including dispersion capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. 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, as well as high molecular weight polyethylene glycols and the like.

Tablets may be made by compression or molding, optionally with the use of one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate, or croscarmellose sodium), surfactants or dispersants. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of the pharmaceutical compositions (e.g., dragees, capsules (including dispersible capsules and gelatin capsules), pills and granules) may optionally be scored or otherwise prepared with coatings and shells (e.g., enteric coatings and other coatings well known in the pharmaceutical formulating art). They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized, for example, by filtration through a filter that entraps bacteria, or by incorporation of sterilizing agents in the form of sterile solid compositions that may be dissolved in sterile water or some other sterile injectable medium just prior to use. These compositions may also optionally contain opacifying agents, and may be compositions which release one or more active ingredients only or preferentially in a certain portion of the gastrointestinal tract (optionally in a delayed manner). Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, with one or more of the above excipients, as appropriate.

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 forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions can also include adjuvants (such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents).

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).

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 which may be required.

Ointments, pastes, creams and gels may contain, in addition to an active compound, excipients (e.g., animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof).

Powders and sprays can contain, in addition to the active compound, excipients (such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances). The spray may additionally contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

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

Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). For example, proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. For example, proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants (such as preserving, wetting, emulsifying and dispersing agents). Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents (e.g., methylparaben, chlorobutanol, phenol sorbic acid, and the like). It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption (e.g., aluminum monostearate and gelatin).

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

Injectable depot forms are made by forming a microencapsulated matrix of the subject compound in a biodegradable polymer (e.g., polylactide-polyglycolide). Depending on the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

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

The actual dosage level of the active ingredient in the pharmaceutical composition may be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, but is non-toxic to the patient.

The dosage level selected will depend on a variety of factors including the particular compound or combination of compounds employed, or the activity of the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound or compounds employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular 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.

A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian may begin with a dosage of the 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 a concentration of a compound sufficient to cause a desired therapeutic effect. It is generally understood that the effective amount of the compound will vary depending on the weight, sex, age and medical history of the subject. Other factors that affect an effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent administered with TC LPA5 4. A larger total dose may be delivered by multiple administrations of the agent. Methods of determining efficacy and dosage are known to those skilled in the art.

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

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

The patient receiving this treatment is any animal in need thereof, including primates (particularly humans); and other mammals (e.g., horses, cattle, pigs, sheep, cats, and dogs); poultry; and in general pets.

In certain embodiments of the invention, a lysophosphatidic acid receptor 5 antagonist, such as TC LPA54, is administered in combination with a programmed death protein-1 inhibitor (PD-1 inhibitor).

The subject group was validated for cardioprotection of TC LPA54 at both animal and cellular levels. In terms of animal level, a PD-1inhibitor (PD-1 inhibitor) and troponin I (TnI) are used for constructing a PD-1inhibitor induced myocarditis mouse model, and experiments show that the PD-1inhibitor+TC LPA54 group (treatment group) has obviously improved cardiac function, heart inflammation infiltration and fibrosis compared with the PD-1inhibitor+control group (control group), and the main focal death marker of myocardial tissue is obviously down-regulated. At the cellular level, the experiment found that the cardiomyocyte primary coke death markers were down-regulated in the lps+tc LPA54 group (treatment group) compared to the lps+pbs group (control group). These results indicate that lysophosphatidic acid receptor 5 antagonist TC LPA54 ameliorates myocardial apoptosis, inflammatory infiltrate and myocardial fibrosis caused by PD-1 inhibitors, reducing PD-1 inhibitor-induced cardiotoxicity.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

In the examples of the present invention, if no specific explanation is given for the experimental operating temperature, this temperature is usually referred to as room temperature (10-30 ℃).

The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.

Statistical analysis: in the study, the numerical variables are all expressed by mean ± standard error, the comparison between two groups adopts a double-tail Student t test, and the comparison between three groups adopts an ANOVA test. Statistical differences were considered when P < 0.05.

Example 1: experimental animal grouping and animal model preparation

SPF-class male C57BL/6 mice were purchased from Shanghai Jieshijie laboratory animals Co., ltd, and had a week-old age of 8-10 weeks and a body weight of 20-25g, and were divided into 3 groups of 8: sham+control group (dummy group); PD-1inhibitor+control group (control group); PD-1inhibitor+TC LPA54 group (treatment group).

PD-1inhibitor+control group and PD-1inhibitor+TC LPA54 group were subcutaneously injected with TnI+complete fluoro reagent (TnI 250. Mu.g/mouse, manufactured by Biotechnology (Shanghai) Co., ltd.) on day 1 and day 7, respectively, and TnI sequence HARVDKVDEERYDVEAKVTKNITEIADLTQKIYDLRGKFKRPTLRRVRIS (troponin I); complete fluoro reagent 100. Mu.L/mouse, sigma, F5881) was designed to construct a PD-1 inhibitor-induced myocarditis mouse model.

PD-1inhibitor (15 μg/InVivoMab anti-mouse PD-1, CD 279) was injected intraperitoneally 5 times every two days from day 8 of molding; in addition, PD-1inhibitor+TC LPA54 group was intraperitoneally injected with TC LPA54 (10 mg/kg/d, medchemepress, HY-107615) 10 days after molding day 8; the sham+control group and the PD-1inhibitor+control group were injected with the same amount of physiological saline as TC LPA54 from day 8 of molding as a Control (Control). On the 17 th day of modeling, the color Doppler ultrasound of the mouse heart is detected, heart specimens are collected for H-E staining, the NLRP3 expression of myocardial tissues is detected by immunofluorescence, and the main focal death marker expression of the myocardial tissues is detected by WB.

Example 2: mouse echocardiography detection

On day 17 of modeling, each group of mice echocardiogram was examined and images were acquired using a Vevo2100 ultrasound system (Visual sonic) with a 30MHz high frequency scanning probe. Mice were anesthetized with isoflurane inhalation and lying flat, and the chest was prepared for skin. M-mode images were recorded at the maximum left chamber diameter of the long axis section of the parasternal left chamber while the heart rate of the mice was maintained at 450-500 times/min, and were continuously acquired for 15s. The functional indexes comprise: left Ventricular Ejection Fraction (LVEF), left ventricular short axis shrinkage (LVFS). The mice of each group were compared for cardiac function changes. All measurements were averaged over 5 consecutive cardiac cycles and were made by 3 experienced technicians.

Referring to fig. 1, the experimental results show that: the heart function index of the PD-1inhibitor+control group mice is obviously lower than that of the sham+control group, the heart function of the mice is obviously improved after the PD-1inhibitor is injected with TC LPA54, and the EF% of the PD-1inhibitor+TC LPA54 group mice is 14.80% higher than that of the PD-1inhibitor+control group mice (P < 0.001); group PD-1inhibitor+TC LPA54 mice had an FS% higher than group PD-1inhibitor+control mice by 11.20% (P < 0.05).

Example 3: H-E staining of mouse hearts

After mice were sacrificed, hearts were fixed with 10% paraformaldehyde solution for 24 hours, embedded with paraffin, and then horizontal sections were made 5 μm thick. Representative images under the light glasses (40 x magnification) were selected for analysis with H-E staining and the results are shown in fig. 2.

The results show that: the PD-1inhibitor+control group mice have significantly more heart inflammation infiltration and fibrosis than the sham+control group, the PD-1inhibitor is administrated to the mice after injection of TC LPA54 to improve heart inflammation infiltration and fibrosis, and the PD-1inhibitor+TC LPA54 group mice have less heart inflammation infiltration and fibrosis than the PD-1inhibitor+control group mice.

Example 4: immunofluorescence detection of mouse heart section

Slides of heart sections were deparaffinized, rehydrated and boiled in Tris-EDTA solution (Beyotime Biotechnology, ST 725), antigen retrieval was performed for 20 minutes at 95 ℃. All slides were then blocked with 5% goat serum and incubated with primary antibody overnight at 4 ℃): anti-NLRP3 (1:50, abcam, ab 26389), anti-Cardiac Troponin T (cTnT, 1:200, abcam, ab 209813) and incubation at 37℃for 1 hour. Washed thoroughly and conjugated to anti-rabbit IgG (1:1000, alexa

594Conjugate,Cell Signaling Technology, # 8889) and anti-mouse IgG (1:1000, alexa)

488Conjugate,Cell Signaling Technology, # 4408) were incubated at 37℃for 1 hour and nuclei were counterstained with DAPI. The stained sections were observed by fluorescence microscopy as shown in fig. 3.

The results show that: the expression of the myocardial tissue NLRP3 of the mice in the PD-1inhibitor+control group is obviously higher than that of the mice in the Sram+control group, and compared with the expression of the myocardial tissue NLRP3 of the mice in the PD-1inhibitor+control group, the expression of the myocardial tissue NLRP3 of the mice is obviously down-regulated after the administration of the PD-1inhibitor and the injection of the TC LPA5 4.

Example 5: mouse heart tissue Western Immunoblot (WB) detection

Western Immunoblot (WB) detection: 10. Mu.g of mouse heart tissue was weighed, lysed in a mixture of precooled RIPA lysate (Beyotime Biotechnology, P0013B) and cocktail protease inhibitor (Beyotime Biotechnology, P1112) for 30 minutes, and centrifuged at 12000rpm for 15 minutes at 4 ℃. Then, the gel was electrophoresed through SDS-PAGE, and transferred onto a polyvinylidene fluoride membrane (Bio-Rad). Membranes were cut into different fractions according to the molecular weight of each protein and blocked with 5% skim milk (biological engineering (Shanghai) Co., ltd., A600669) for 2 hours, primary antibody incubated overnight at 4 ℃): anti-NLRP3 (1:1000, abcam, ab26389), anti-Pro-Caspase-1+p10+p12 (1:1000, abcam, ab179515), anti-Gasderm D (1:1000,Cell Signaling Technology, # 39754), anti-Cleaved Gasdermin D (1:1000,Cell Signaling Technology, # 10137), anti-IL-1β (1:1000,Cell Signaling Technology, # 12242), anti-IL-18 (1:1000; ABclonal, A1115), anti-GAPDH (1:5000, protect h,1E6D 9). The next day the membrane was washed and the secondary antibody was incubated at room temperature for 1 hour: dyligtTM 800 4XPEG conjugated goat anti-rabbit IgG (H+L) or goat anti-mouse IgG (H+L) (1:10000,Cell Signaling Technology, #79408, # 59997). Imaging was performed using a solar full-automatic digital gel/chemiluminescent image analysis system (Tanon 4600 SF) and results are shown in fig. 4.

The results show that: the myocardial tissue coke death level of the mice in the PD-1inhibitor+control group is obviously higher than that of the mice in the Sram+control group, and the myocardial tissue coke death level of the mice is obviously down-regulated compared with that of the mice in the PD-1inhibitor+control group after the administration of the PD-1inhibitor and the reinjection of TC LPA5 4.

Example 6: cell grouping and cell model preparation

HL-1 mouse atrial myocytes were purchased from the national academy of sciences typical culture storage committee cell bank and divided into 3 groups: PBS group (sham group), lps+pbs group (control group), lps+tc LPA54 (treatment group).

LPS+TC LPA54 groups were pre-treated with TC LPA54 (5. Mu.M, medchemepress, HY-107615) for 2 hours and then with LPS (5. Mu.g/ml, sigma, L2630) for 24 hours; LPS+PBS groups cells were treated with 5. Mu.g/ml LPS plus equal amounts of PBS (Beyotime Biotechnology, C0221A) for 24 hours; the PBS group treated cells with only an equal amount of PBS for 24 hours. Immunofluorescence detects myocardial cell NLRP3 expression, WB detects myocardial cell main pyro-death marker expression.

Example 7: cell immunofluorescence assay

Immunofluorescence: HL-1 cells were mounted on slides and fixed with 4% paraformaldehyde. Subsequent blocking, primary incubation, secondary incubation and light microscopy procedures were performed as described for animal tissue immunofluorescence method of example 4. The results are shown in FIG. 5.

The results show that: myocardial cells NLRP3 expression in LPS+PBS group is significantly higher than in PBS group; after pretreatment with TC LPA54, cardiomyocyte NLRP3 expression was significantly down-regulated compared to lps+pbs group.

Example 8: cell Western Immunoblot (WB) assay

Referring to the method in example 5, cardiomyocytes after the administration treatment were lysed, centrifuged, electrophoresed, transferred, blocked, primary antibody incubated, secondary antibody incubated, and imaged by an image analysis system using Western Immunoblot (WB), and the results are shown in fig. 6.

The results show that: the myocardial cell pyrosis level of the LPS+PBS group is obviously higher than that of the PBS group; after pretreatment with TC LPA54, myocardial cell scorch levels were down-regulated compared to lps+pbs group.

The invention is illustrated by the above examples, and proves that the lysophosphatidic acid receptor 5 antagonist TC LPA54 treatment is expected to obviously improve myocardial cell apoptosis, inflammatory infiltration and myocardial fibrosis caused by PD-1 inhibitors, improve prognosis of tumor patients, play a role in protecting heart, and provide a new scheme for tumor treatment.

Claims (10)

1. Use of a lysophosphatidic acid receptor 5 antagonist in the manufacture of a cardioprotective medicament.

2. The use according to claim 1, wherein the lysophosphatidic acid receptor 5 antagonist is TC LPA54, cas number: 1393814-38-4.

3. The use according to claim 1 or 2, wherein cardioprotection is reducing or preventing apoptosis protein-1 inhibitor induced cardiotoxicity; accordingly, the medicament is a medicament for reducing or preventing apoptosis protein-1 inhibitor-induced cardiotoxicity.

4. The use of claim 3, wherein the cardiotoxicity is cardiac injury; accordingly, the medicament is a medicament for treating or preventing a cardiac injury induced by an inhibitor of apoptosis protein-1.

5. The use according to claim 3, wherein the cardiac injury is ici-associated myocarditis; accordingly, the medicament is a medicament for treating or preventing ICIs-related myocarditis.

6. The use of claim 1, wherein the subject to which the medicament is administered is a tumor patient receiving treatment with the inhibitor of apoptosis protein-1.

7. The use according to claim 1, wherein the medicament comprises a therapeutically effective amount of a lysophosphatidic acid receptor 5 antagonist as the sole active ingredient.

8. The use according to claim 1, wherein the medicament is a pharmaceutical composition comprising, in addition to a therapeutically effective amount of a lysophosphatidic acid receptor 5 antagonist as active ingredient, an inhibitor of programmed death protein-1 and/or other pharmaceutical ingredients for preventing heart damage.

9. The use according to claim 1, wherein the medicament is in the form of an oral formulation or an injection.

10. The use according to claim 9, wherein the oral formulation is selected from the group consisting of: tablets, capsules, granules, powders, solutions, syrups; the injection is suitable for intravenous injection or intravenous drip.

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