Application of compound AJ-091 in preparation of medicine for treating systemic sclerosis

Abstract

The invention discloses application of a compound AJ-091 in preparation of a medicine for treating systemic sclerosis, and the compound AJ-091 shows a good anti-fibrosis effect in cell and animal models, so that the compound AJ-091 is prompted to be capable of effectively relieving or preventing systemic sclerosis and has a clinical development and application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of compound AJ-091 (CAS No.: 255714-23-9) in the preparation of a therapeutic drug for systemic sclerosis. Background Art

[0002] Systemic sclerosis (SSc), also known as systemic scleroderma, is an autoimmune connective tissue disease mainly characterized by skin fibrosis. Skin fibrosis is considered to be the initial symptom of the disease, followed by visceral organ fibrosis. The core feature of fibrosis is the abnormal activation of fibroblasts, the transdifferentiation of quiescent fibroblasts into myofibroblasts, resulting in the excessive deposition of collagen and extracellular matrix, which in turn destroys tissue structure and causes severe organ dysfunction. Fibrosis is one of the main causes of morbidity and mortality in patients with systemic sclerosis, but there is currently no effective treatment method to reverse or control this pathological process.

[0003] The pathogenesis of systemic sclerosis is complex, involving multiple pathological processes such as immune disorders, vascular lesions, and fibrosis. TGF-β1 is a pleiotropic cytokine that can regulate fibrosis, inflammation, and angiogenesis, and is a key factor in the pathogenesis of SSc and is a recognized profibrotic cytokine.

[0004] Subcutaneous injection of BLM in mice has been shown to induce skin fibrosis very similar to SSc. At the site of BLM injection, mice will show pathological features of skin fibrosis such as skin thickening, reduction of subcutaneous adipose tissue, and massive deposition of collagen, and the production of autoantibodies can also be detected. In addition, in the esophagus and stomach, fibrosis of smooth muscle and submucosa is observed, accompanied by fibrosis around small arteries and in the renal interstitium, and these conditions are very similar to the clinicopathological features of SSc. Summary of the Invention

[0005] In the exploration and research of the treatment method for scleroderma, according to the recommendation of Shanghai Medical College of Fudan University, our research group tried to use the small molecule compound AJ-091 to treat systemic sclerosis. We used a TGF-β1-induced fibroblast model to evaluate the inhibitory effect of AJ-091 on the profibrotic response of TGF-β1 by detecting the expression of fibrosis-related proteins in fibroblasts and the cell migration ability of AJ-091; further, we selected subcutaneous injection of BLM in mice to construct a scleroderma model mouse model to explore whether AJ-091 has anti-fibrotic ability in vivo. We were surprised to find in the experiment that AJ-091 showed good anti-fibrotic effects in both cell and animal models, indicating that AJ-091 can be developed into a therapeutic drug for systemic sclerosis. Based on this research result, the present invention provides the following technical solutions:

[0006] The present invention provides the use of the small molecule compound AJ-091 (CAS No.: 255714-23-9) in the preparation of a medicament for treating systemic sclerosis / scleroderma.

[0007] In one embodiment, the above medicament contains a therapeutically effective amount of the compound AJ-091 as the sole active ingredient.

[0008] In another embodiment, the above medicament is a pharmaceutical composition, which, in addition to containing a therapeutically effective amount of the compound AJ-091, further contains one or more pharmaceutically acceptable carriers.

[0009] Furthermore, the above medicament is a pharmaceutical composition, which, in addition to containing a therapeutically effective amount of the compound AJ-091 as an active ingredient, further contains other pharmaceutical ingredients for treating systemic sclerosis / scleroderma.

[0010] For example, the above other pharmaceutical ingredients for treating systemic sclerosis / scleroderma are drugs commonly used clinically at present, and are selected from vasoactive drugs, anti-fibrotic drugs, glucocorticoids, immunosuppressants, non-steroidal anti-inflammatory drugs, etc.

[0011] Optionally, the vasoactive drug is selected from the following group: stanozolol, captopril, urokinase, agkistrodon acutus enzyme, hydralazine, vitamin E, prazosin, reserpine, dibazol, nifedipine, tolazoline, pentoxifylline;

[0012] The anti-fibrotic drug is selected from the following group: penicillamine, colchicine, isotretinoin, acitretin, aspirin, asiaticoside;

[0013] The glucocorticoid is prednisone acetate;

[0014] The immunosuppressant is selected from the following group: azathioprine, cyclophosphamide, methotrexate, chlorambucil or cyclosporine;

[0015] The non-steroidal anti-inflammatory drug is selected from the following group: indomethacin, ibuprofen, naproxen, human recombinant relaxin, thalidomide, penicillin, erythromycin.

[0016] The dosage form of the above medicament is an oral preparation, an injection or a topical preparation.

[0017] The oral preparation is selected from the following group: tablets, capsules (including but not limited to dispersible capsules and gelatin capsules), granules, powders, solutions, syrups.

[0018] In the oral preparation, the pharmaceutically acceptable carriers include one or more of the following groups: fillers or extenders, binders, wetting agents, disintegrants, absorbents, lubricants, buffers, complexing agents, colorants.

[0019] When the dosage form of the drug is an injection, it is applicable to intravenous injection and intravenous drip.

[0020] When the dosage form of the drug is a topical preparation, it can be selected from the following group: creams, ointments, gels, sprays, film-forming agents, etc., or can also be a novel topical dosage form such as solid lipid nanoparticles.

[0021] The present invention for the first time attempted the possibility of the small molecule compound AJ-091 (CAS No.: 255714-23-9) in the treatment of systemic sclerosis / scleroderma at the cell level and experimental animal level. Experiments found that AJ-091 showed good anti-fibrotic effects in both cells and animal models. The present invention intervened on TGF-β1-induced fibroblasts with AJ-091 at the dose of IC 50 in cell experiments, and AJ-091 could significantly reduce the fibrotic response induced by TGF-β1. In animal experiments, AJ-091 was intraperitoneally injected at the doses of 10 mg / kg and 50 mg / kg to treat scleroderma mice. After treatment with 10 mg / kg and 50 mg / kg, the symptoms were alleviated and effectively relieved, suggesting the application potential of AJ-091 in the treatment of systemic sclerosis and its good safety. Therefore, the present invention provides a new idea for the treatment of systemic sclerosis / scleroderma, brings new choices for such patients, and has broad potential in clinical application prospects. Description of the Drawings

[0022] Figure 1 Shows the molecular structure of the small molecule compound AJ-091 and its effect on cell viability. Among them, A: the molecular structure of compound AJ-091; B: the effect of the small molecule compound AJ-091 on cell viability; C: the dose-response curve of AJ-091 and the IC 50 value. ****P < 0.0001.

[0023] Figure 2Shown is the situation of AJ-091 inhibiting TGF-β1-induced profibrotic responses. Among them, A: Detection of the expression of fibrosis-related proteins by Western blot; B: Quantitative analysis of the bands of COL1A1 and α-SMA; C: Immunofluorescence showing the expression of COL1A1 and α-SMA in cells, scale bar: 40 μm. D: Quantitative analysis of the fluorescence intensities of α-SMA and COL1A1. E: Transwell assay to detect the migration ability of fibroblasts, scale bar: 50 μm; F: Statistical analysis of the number of migrated cells in the Transwell assay; G: Migration of fibroblasts in different groups in the cell scratch assay, scale bar: 200 μm; H: Quantitative analysis of the migration rate in the cell scratch assay. ns indicates no statistical difference, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.

[0024] Figure 3 Shown is the effect of AJ-091 on the general condition of scleroderma model mice. Among them, A: Schematic diagram of the experiment; B: Skin lesions and hair growth on the backs of mice in each group (n = 3); C: Changes in the body weights of mice in each group ((n = 3); D: Effects of AJ-091 on the liver and kidney functions of scleroderma model mice (n = 3). ns indicates no statistical difference, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.

[0025] Figure 4 Shown is the effect of AJ-091 in improving skin and lung fibrosis in scleroderma model mice. Among them, A: HE and Masson staining of mouse skin, scale bar: 200 μm; B: Statistical analysis of the dermal thickness of mice; C: HE and Masson staining of mouse lungs, scale bar: 200 μm; D: Ashcroft scores of mice in each group; E: Detection of the expression of skin and fibrosis-related proteins by Western blot; F: Quantitative analysis of the bands of COL1A1 in skin and lung tissues. ns indicates no statistical difference, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001. Detailed implementation manners

[0026] In our research on exploring new methods for treating systemic sclerosis / scleroderma, we focused on investigating the compound AJ-091. See Figure 1 A therein, the molecular formula of this compound is C 28 H 25NO2S, with a molecular weight of 439.6 g / mol and a CAS number of 255714-23-9 (PubChem CID: 2802455), has a core structure containing a benzene ring, a thioether bond, and a ketone group. The Chinese name of AJ-091 is 3-[(2-aminophenyl)thio]-1-[2-(benzyloxy)phenyl]-3-phenyl-1-propanone, also known as Oprea1_271542 and NS00016382.

[0027] Through cell experiments and animal model experiments, AJ-091 demonstrated good anti-fibrotic effects, suggesting its potential application in the treatment of systemic sclerosis and anti-fibrotic treatment including the treatment of pulmonary fibrosis.

[0028] Moreover, biochemical detection indicators in animal experiments also showed that compound AJ-091 had no obvious effect on liver and kidney functions, suggesting its good safety within the therapeutic dose range.

[0029] As an application mode of the present invention, compound AJ-091 can be prepared into a therapeutic drug for systemic sclerosis. In this article, for the purpose of describing its role in a drug or a pharmaceutical composition, the active ingredient compound AJ-091 can be referred to as the "active compound".

[0030] The drug can be a single-component drug containing a therapeutically effective amount of compound AJ-091, or a pharmaceutical composition further containing other components such as a pharmaceutically acceptable carrier.

[0031] Compound AJ-091 can be the sole active ingredient in the drug, or can be used in combination with a PD-1 inhibitor and / or other drug ingredients for the treatment of systemic sclerosis / scleroderma.

[0032] As a specific implementation mode of using two or more drug ingredients in combination, the drug is a pharmaceutical composition, which contains a therapeutically effective amount of compound AJ-091 and / or other drug ingredients for the treatment of systemic sclerosis / scleroderma.

[0033] It should be understood that the term "and / or" 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" 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).

[0034] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and are 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 injurious to the patient, including, for example, aqueous solutions (such as water or physiological buffer saline) or other solvents or vehicles (such as glycols, glycerin, oils (such as olive oil), or injectable organic esters). Excipients can be selected, for example, to effect delayed release of the medicament or to selectively target one or more cells, tissues, or organs. Pharmaceutical compositions can be in dosage unit form, such as tablets, capsules (including dispersed capsules and gelatin capsules), granules, powders, solutions, syrups, suppositories, injections, etc.

[0035] As used herein, the term "effective amount" refers to the therapeutically effective amount required to alleviate at least one or more symptoms of a disease or condition and relates to an adequate amount of a drug that provides the desired effect. Thus, the term "therapeutically effective amount" refers to the amount of a therapeutic agent that, when administered to a typical subject, is sufficient to cause a particular effect. In various contexts, the effective amount as used herein also includes an amount sufficient to delay the development of a disease condition, alter the course of a disease condition (e.g., but not limited to, slow 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 a therapeutic or prophylactic application, the amount of the 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 status, 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 compound AJ-091 can be determined with reference to its currently safe usage amount when administered to cancer patients for treating cancer and through clinical investigation. The appropriate effective dosage also needs to consider therapeutic factors such as the dosage form of the drug, the constitution, weight, age, disease progression, and administration site of the individual being administered.

[0036] The drug ingredient compound AJ-091 can also be administered in combination with one or more additional therapeutic compounds.

[0037] In addition to containing the main component compound AJ-091, 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 carboxymethyl cellulose, 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 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 used in pharmaceutical preparations.

[0038] The pharmaceutical preparation can be administered to a subject by any one of a number of administration routes, including, for example, oral (e.g., as a drench, tablet, capsule (including dispersed capsules and gelatin capsules), bolus, powder, granule, paste for application to the tongue in an aqueous solution or a non-aqueous solution or suspension); absorption through the oral mucosa (e.g., sublingually); 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 compound AJ-091 can also be formulated for inhalation. In certain embodiments, the compound AJ-091 can simply be dissolved or suspended in a sterile solvent.

[0039] The term "subject" as used above refers to a human or an animal. Generally, the animal is a vertebrate, such as a primate, a rodent, a domestic animal or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys and macaques, such as rhesus monkeys. Rodents include mice, rats, ground squirrels, ferrets, rabbits and hamsters. Domestic animals and game animals include cows, horses, pigs, deer, bison, buffalo, 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. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses or cows, but are not limited to these examples. Advantageously, non-human mammals can be used as subjects for animal models representing type II diabetes with acarbose resistance. The subject can be male or female. Obviously, when the "subject" refers to an animal, the drugs for preventing and treating aortic aneurysm and / or aortic dissection refer to veterinary drugs or animal medications.

[0040] 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 the active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. The amount of the 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. Generally, this amount ranges from about 1% to about 99% by weight of the active ingredient, such as about 5% to about 70%.

[0041] The methods for preparing these preparations or compositions include the step of combining an active compound (such as compound AJ-091) with a carrier and optionally one or more auxiliary ingredients. Generally, the preparation is made by uniformly and intimately combining compound AJ-091 with a liquid carrier or a finely divided solid carrier or both, and then shaping the product if necessary.

[0042] The preparations of the present invention suitable for oral administration can be in the form of: capsules (including dispersed capsules and gelatin capsules), cachets, pills, tablets, troches (using a flavored base, usually sucrose and gum arabic or tragacanth), lyophilisates, 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 lozenge (using an inert matrix such as gelatin and glycerin, or sucrose and gum arabic) and / or as a mouthwash, etc., each containing a predetermined amount of compound AJ-091 as the active ingredient. The composition or compound can also be administered as a bolus, electuary or paste.

[0043] For the preparation of solid dosage forms for oral administration (capsules (including dispersed 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 dibasic calcium 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, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia); (3) wetting agents (such as glycerol); (4) disintegrants (such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate); (5) solution blockers (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 lauryl sulfate, and mixtures thereof); (10) complexing agents (such as modified or unmodified cyclodextrins); and (11) coloring agents. In the case of capsules (including dispersed capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also contain buffering agents. Similar types of solid compositions 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, etc.

[0044] Tablets may be manufactured by compression or molding, optionally using one or more accessory ingredients. Compressed tablets may be prepared using binders (such as, for example, gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (such as sodium starch glycolate, or cross-linked sodium carboxymethylcellulose), surfactants, or dispersing agents. Molded tablets may be manufactured by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0045] Tablets and other solid dosage forms of the pharmaceutical composition (such as dragees, capsules (including dispersed capsules and gelatin capsules), pills, and granules) can optionally be scored, or prepared with coatings and shells (such as enteric coatings and other coatings well-known in the field of pharmaceutical formulation). They can also be formulated to provide slow release or controlled release of the active ingredient therein, using, for example, different proportions of hydroxypropyl methylcellulose to provide the desired release profile, other polymer matrices, liposomes, and / or microspheres. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain emulsifying agents and can be compositions that release one or more active ingredients only in or preferentially in a certain part 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 can also be in microencapsulated form, which, where appropriate, has one or more of the above excipients.

[0046] Liquid dosage forms suitable 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 can contain inert diluents commonly used in the art (such as, for example, water or other solvents, cyclodextrins and their derivatives), solubilizing agents, and emulsifying agents (such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (particularly, cottonseed oil, groundnut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof).

[0047] In addition to the inert diluent, oral compositions can also contain adjuvants (such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives).

[0048] In addition to the active compound, suspensions can contain suspending agents (such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, and tragacanth, and mixtures thereof).

[0049] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier, as well as with any preservatives, buffers, or propellants that may be required.

[0050] In addition to the active compound, ointments, pastes, creams and gels may contain excipients (such as animal and vegetable fats, oils, waxes, paraffin wax, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof).

[0051] In addition to the active compound, powders and sprays may contain excipients (such as lactose, talc, silicic acid, aluminium hydroxide, calcium silicate and polyamide powder, or mixtures of these substances). Sprays may additionally contain conventional propellants (such as chlorofluorocarbons and volatile unsubstituted hydrocarbons (such as butane and propane)).

[0052] Transdermal patches have the additional advantage of providing controlled delivery of the active compound to the body. Such dosage forms may be manufactured by dissolving or dispersing the active compound in a suitable medium. Penetration enhancers may also be used to increase the flux of the compound across the skin. The rate of such flux may be controlled by providing a rate controlling membrane or by dispersing the compound in a polymeric matrix or gel.

[0053] 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, etc.) 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, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). For example, appropriate fluidity may be maintained by using coating materials (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using surfactants. For example, appropriate fluidity may be maintained by using coating materials (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using surfactants.

[0054] These compositions may also contain auxiliaries (such as preservatives, wetting agents, emulsifying agents and dispersing agents). The action of microorganisms may be prevented by including various antibacterial and antifungal agents (for example, methyl paraben, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents (such as sugars, sodium chloride, etc.) in the compositions. In addition, prolonged absorption of injectable drug forms may be caused by including agents that delay absorption (such as aluminium monostearate and gelatin).

[0055] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from a subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oily vehicle.

[0056] Injectable depot forms are manufactured by forming a microencapsulated matrix of the subject compound in a biodegradable polymer such as polylactide - polyglycolide. The drug release rate can be controlled according to the ratio of the drug to the polymer and the nature of the specific polymer employed. 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 that are compatible with body tissues.

[0057] For use in the methods of the present invention, the active compound can be administered itself 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.

[0058] The actual dosage level of the active ingredient in the pharmaceutical composition can vary to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, and that is non - toxic to the patient.

[0059] The chosen dosage level will depend on various factors including the activity of the specific compound or combination of compounds employed, or its esters, salts, or amides, the route of administration, the time of administration, the excretion rate of one or more specific compounds employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with one or more specific 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.

[0060] A physician or veterinarian of ordinary skill in the art can readily determine and prescribe a therapeutically effective amount of the required pharmaceutical composition. For example, a physician or veterinarian can start with a dose of the pharmaceutical composition or compound that is below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. A "therapeutically effective amount" means the concentration of a compound sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of a compound will vary depending on the weight, sex, age, and medical history of the subject. Other factors that can affect the effective amount can include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and (if required) another type of therapeutic agent administered in combination with compound AJ-091. A larger total dose can be delivered by multiple administrations of the medicament. Methods for determining efficacy and dosage are known to those of ordinary skill in the art.

[0061] Generally, the suitable daily dose of the active compound used in the compositions and methods of the present 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.

[0062] If desired, the effective daily dose of the active compound can be administered as one, two, three, four, five, six, or more sub-doses administered separately, optionally in unit dosage form, at appropriate time intervals throughout the day. In certain embodiments of the present invention, the active compound can be administered two or three times a day. In other embodiments, the active compound will be administered once a day.

[0063] The patients receiving this treatment are any animals in need, including primates (especially humans); and other mammals (such as horses, cows, pigs, sheep, cats, and dogs); poultry; and common pets.

[0064] The present invention provides a new drug candidate AJ-091 for treating systemic sclerosis. Through cell experiments, AJ-091 was used to intervene in TGF-β1-induced fibroblasts at the IC50 dose, and AJ-091 could significantly reduce the fibrotic response induced by TGF-β1. Through animal experiments, AJ-091 was intraperitoneally injected at doses of 10 mg / kg and 50 mg / kg to treat scleroderma mice. After treatment with 10 mg / kg and 50 mg / kg, the symptoms were alleviated and effectively relieved, showing good clinical application prospects and finding a new treatment approach for systemic sclerosis patients.

[0065] The following further describes the present invention in detail with specific examples. It should be understood that the following examples are only for illustrating the present invention and not for limiting the scope of the present invention.

[0066] Examples

[0067] In the embodiments of the present invention, if no specific description is made for the experimental operation temperature, the temperature generally refers to room temperature (10 - 30 °C).

[0068] In this article, the addition amounts, contents and concentrations of various substances are involved. Among them, the percentage content, unless otherwise specified, all refers to the mass percentage content.

[0069] Statistical analysis: In this study, numerical variables were all expressed as mean ± standard error. The two-group comparison was performed using a two-tailed Student t-test, and the three-group comparison was performed using an ANOVA test. When P < 0.05, it was considered to have statistical significance.

[0070] Example 1: Cell experiment

[0071] 1. Isolate and culture healthy volunteer skin fibroblasts (passages 3 - 7) collected from Zhongshan Hospital Affiliated to Fudan University, and stimulate them with DMSO solutions of AJ-091 at concentrations of 0 μΜ, 5 μΜ, 10 μΜ, 20 μΜ, 25 μΜ, and 50 μΜ for 24 hours, and perform a CCK-8 experiment to detect the cell viability after different concentration treatments and calculate the half-inhibitory concentration IC 50 .

[0072] 2. Seed healthy skin fibroblasts at 1×10 5 cells / well into a 6-well plate, add 10 ng / ml of TGF-β1 to stimulate the fibroblasts, and collect the cells after intervening with the IC 50 concentration of AJ-091 for 24 h, and perform Western blot and immunofluorescence to detect the effects of AJ-091 on the expression of COL1A1 and α-SMA proteins in TGF-β1-induced fibroblasts.

[0073] 3. Evaluate the effect of the IC 50 concentration of AJ-091 on cell migration ability through Transwell and cell scratch experiments.

[0074] Example 2: Animal experiment

[0075] 1. AJ-091 was purchased from Specs Company, and the solvent was DMSO; the administration method was intraperitoneal injection.

[0076] 2. Preparation of main solutions:

[0077] 1) AJ-091 (10 mg / kg): Weigh 6 mg of AJ-091 and dissolve it in 150 μl of DMSO, vortex until completely dissolved, then add 1200 μl of PEG300 and 150 μl of Tween80 in sequence, and mix well after each solvent addition; finally, make up to 3 ml with 1500 μl of sterile ddH2O, and prepare the drug immediately before use.

[0078] 2) AJ-091 (50 mg / kg): Weigh 30 mg of AJ-091 and dissolve it in 150 μl of DMSO. Vortex until completely dissolved, then sequentially add 1200 μl of PEG300 and 150 μl of Tween80, and mix well after each addition of the solvent. Finally, make up the volume to 3 ml with 1500 μl of sterile ddH2O. The drug should be prepared freshly before use.

[0079] 3) Control solvent: Prepared according to the same formula and steps as above.

[0080] 4) Bleomycin (BLM) stock solution: Dissolve 15 mg of BLM powder in 10 ml of 0.9% normal saline to prepare a bleomycin stock solution with a concentration of 1.5 mg / ml. After aliquoting, store it in a -20°C refrigerator.

[0081] 3. Experimental protocol:

[0082] Select 12 healthy female C57BL / 6 mice (7 weeks old), purchased from Shanghai Jihui Laboratory Animal Co., Ltd., and routinely housed in the SPF-grade animal facility of Zhongshan Hospital Affiliated to Fudan University. Randomly divide the mice into 4 groups according to the random number table method, namely the control group, the model group, the low-dose treatment group (10 mg / kg), and the high-dose treatment group (50 mg / kg), with 3 animals in each group. It was confirmed by statistical test that the initial body weights among groups were balanced (P > 0.05). All mice were adaptively housed in the SPF-grade facility for 7 days. One day before the start of the experiment, use a low-noise electric hair clipper to remove the hair from the designated area on the back of the mice (4 cm 2 ) Avoid damaging the skin during the operation. Observe continuously for 24 hours after the treatment. Start the modeling after excluding adverse reactions such as skin allergies. Put on special ear studs for the mice with sterile ear tags, and record the initial body weight and skin thickness of the mice at the same time. Regularly record the body weight and skin thickness of the mice during the experiment.

[0083]

[0084]

[0085] 1) Control group: Subcutaneously inject normal saline from day 1, 0.1 ml / d for 21 days, and intraperitoneally inject an equal volume of solvent daily from day 7.

[0086] 2) Model group: Subcutaneously inject 1.5 mg / ml bleomycin solution from day 1, 0.1 ml / d for 21 days, and intraperitoneally inject an equal volume of solvent daily from day 7.

[0087] 3) Low-dose treatment group (10 mg / kg): From day 1, inject 1.5 mg / ml bleomycin solution subcutaneously at a dose of 0.1 ml / d for 21 days. From day 7, inject AJ-091 intraperitoneally at a dose of 10 mg / kg·d.

[0088] 4) High-dose treatment group (50 mg / kg): From day 1, inject 1.5 mg / ml bleomycin solution subcutaneously at a dose of 0.1 ml / d for 21 days. From day 7, inject AJ-091 intraperitoneally at a dose of 50 mg / kg·d.

[0089] 4. After modeling, it was confirmed that the systemic sclerosis (SSc) model was successfully constructed through morphological evaluation. The specific manifestations were as follows: The skin at the injection site of the mice showed significant thickening, the tissue texture was hardened, the elasticity was poor, and there was hair regeneration disorder. After 21 days, after the last administration, the mice were fasted for 24 h (free access to water), and then intraperitoneally injected with 2% pentobarbital sodium (3 ml / kg). After confirming the depth of anesthesia, the mice were sacrificed by cervical dislocation and immediately dissected.

[0090] Experimental results

[0091] In the cell experiment, first, the CCK-8 experiment was used to explore the effect of AJ-091 on cell viability, and its IC 50 value was calculated. All subsequent experiments were intervened at this concentration. As Figure 1 shown in B and C below, Figure B shows that AJ-091 inhibits the proliferation of fibroblasts in a dose-dependent manner. Figure C is the dose-response curve of AJ-091, and its IC 50 value of 14.34 μM can be calculated.

[0092] The results of Western blot and immunofluorescence experiments showed that AJ-091 could inhibit the expression of fibroblast fibrosis-related proteins (α-SMA and COL1A1). The down-regulation of both indicated that AJ-091 could effectively inhibit the activation of fibroblasts and the synthesis of type I collagen. As Figure 2 shown, Figure A is the Western blot detection of the expression of fibrosis-related proteins, Figure B is the quantitative analysis of the COL1A1 and α-SMA bands. The experimental results showed that AJ-091 could inhibit the expression of fibrosis-related proteins COL1A1 and α-SMA induced by TGF-β1 in a dose-dependent manner. Figure C used a laser confocal microscope for immunofluorescence detection to observe the imaging of COL1A1 and α-SMA. Figure D is the quantitative analysis of the fluorescence intensity of COL1A1 and α-SMA. The experimental results showed that AJ-091 inhibited the expression of COL1A1 and α-SMA.

[0093] In addition, the results of Transwell assay and wound healing assay showed that AJ-091 significantly inhibited the migratory ability of fibroblasts induced by TGF-β1. This implies that AJ-091 may have an inhibitory effect on the activation and migration of fibroblasts in SSc and has the ability to resist fibrosis. As Figure 2 shown, Figure E is a schematic diagram of the results of the Transwell assay, and Figure F is the statistical analysis of the number of migrated cells in the Transwell assay. The results showed that the number of migrated cells significantly decreased after AJ-091 intervention. Figure G is a schematic diagram of the wound healing assay, and Figure H is the quantitative analysis of the migration rate in the wound healing assay. The results showed that after AJ-091 intervention, the migration rate of fibroblasts significantly decreased and the migratory ability was significantly inhibited.

[0094] In the animal experiment, we established a systemic sclerosis (SSc) mouse model by subcutaneous injection of bleomycin. Starting from the 7th day after modeling, AJ-091 was intraperitoneally injected into the SSc model mice for 14 days to evaluate the therapeutic effect of AJ-091 on the SSc model mice. As Figure 3 shown, Figure A is a schematic diagram of the animal experiment protocol. Figure B shows the skin lesions and hair growth on the backs of mice in each group. Compared with the control group, the skin of the mice in the BLM model group became harder, the elasticity decreased, the hair growth was slow, and the activity level decreased. After AJ-091 intervention, the degree of skin sclerosis in the mice was alleviated, the hair growth improved, and the activity level recovered somewhat. Figure C shows the changes in the body weight of mice in each group. The body weight of the mice in the BLM model group decreased significantly, and the downward trend of body weight was alleviated after AJ-091 intervention.

[0095] The experimental results showed that the HE and Masson staining of the skin and lung tissues presented fibrotic pathological features similar to those of SSc, indicating that bleomycin successfully induced the SSc mouse model.

[0096] Subsequently, we evaluated the safety of AJ-091 in vivo through biochemical tests. The results showed that AJ-091 had no obvious effect on the liver and kidney functions of the SSc model mice, suggesting that it has good safety within the therapeutic dose range. As Figure 3As shown in the figure, Figure D shows the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CREA), and uric acid (UA). The levels of AST, ALT, ALP, BUN, CREA, and UA in the low-dose intervention group (10 mg / kg) did not increase significantly compared with the model group, indicating that low-dose AJ-091 has no obvious hepatotoxicity and nephrotoxicity in SSc model mice. In the high-dose intervention group, the levels of AST, ALT, ALP, and BUN increased slightly compared with the model group, but all indicators were within the normal range, suggesting that high-dose AJ-091 has a minor impact on the liver and kidney functions of model mice and is within a controllable range.

[0097] In addition, pathological analysis of the skin and lung tissues of SSc model mice after AJ-091 intervention showed that the degree of fibrosis was significantly improved, and the expression of type I collagen in the skin and lung tissues was also significantly reduced. As Figure 4 shown, Figure A shows the HE and Masson staining of mouse skin (scale bar: 200 μm), and Figure B shows the statistical analysis of the dermis thickness of mice. The HE and Masson staining results of mouse skin showed that the epidermal layer structure of the skin tissue of the control group mice was clear, and the stratum corneum, spinous cell layer, and basal cell layer were arranged neatly; the collagen in the dermis layer was arranged loosely and evenly, and a small number of skin appendages such as hair follicles were visible. The dermis layer of the model group mice was significantly thickened, the collagen fibers increased, and the arrangement was disordered; fibrosis spread to the deep layer, the fat layer became thinner or even disappeared, the skin appendages were damaged, and a large number of lymphocytes and neutrophils infiltrated the superficial dermis; the blood vessel wall was significantly thickened, and the lumen was narrowed. The dermis layer of the skin of the AJ-091 treatment group mice was thinned to varying degrees compared with the model group, the collagen fibers decreased, and the inflammatory cell infiltration decreased, and the skin fibrosis was improved to a large extent. At the same time, the Image J software was used to quantitatively analyze the thickness of the dermis layer of mouse skin. As shown in Figure B, the dermis layer thickness of the model group was significantly increased compared with the control group. After AJ-091 intervention, the dermis layer thickness of mice was significantly reduced, indicating that AJ-091 intervention has a relatively obvious improvement effect on mouse skin fibrosis.

[0098] Figure C shows the HE and Masson staining of mouse lungs (scale bar: 200 μm), and Figure D shows the Ashcroft scores of mice in each group. As shown in Figure C, the results of HE and Masson staining of mouse lung tissues showed that in the control group, the bronchial wall structure was clear, the alveolar septa were evenly distributed, there was no accumulation of exudate in the alveolar cavity, no inflammatory cell infiltration was seen, and less collagen deposition was observed. In the model group, the lung tissue structure was disordered, the alveolar septa were significantly thickened, the alveolar structure was damaged, red blood cells and inflammatory cell infiltration were visible, collagen fiber hyperplasia was seen around blood vessels, and collagen was diffusely distributed. After AJ-091 intervention, the lung structure of mice was more complete, and collagen deposition decreased, showing an obvious anti-fibrotic effect. As shown in Figure D, the degree of pulmonary fibrosis in each group of mice was analyzed by Ashcroft score. Compared with the control group, the Ashcroft score in the model group was significantly increased. After AJ-091 intervention, the Ashcroft score of mouse lung tissue decreased, indicating that AJ-091 intervention had a significant improvement on pulmonary fibrosis in mice.

[0099] Figure E shows the Western blot detection of the expression of COL1A1 in the skin and lungs, and Figure F shows the quantitative analysis of the expression of COL1A1 in the skin and lung tissues. The results showed that compared with the control group, the protein expression of COL1A1 in the skin and lung tissues of the model group was significantly increased. After AJ-091 intervention, the protein expression level of COL1A1 was significantly decreased, and the difference was statistically significant.

[0100] In summary, AJ-091 not only significantly inhibited the pro-fibrotic response induced by TGF-β1 in cell experiments, but also significantly improved skin and lung fibrosis in SSc model mice within a safe dose range in animal experiments, effectively alleviating the condition of SSc model mice, which suggests the potential of AJ-091 in the anti-fibrotic treatment of systemic sclerosis.

[0101] It should be understood that the above embodiments are only the preferred embodiments of the present invention, and do not limit the present invention in any form and substance. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Application of compound AJ-091 in the preparation of drugs for the treatment of systemic sclerosis.

2. The use according to claim 1, characterized in that The drug contains a therapeutically effective amount of compound AJ-091 as the only active ingredient.

3. The use according to claim 1, characterized in that The drug is a pharmaceutical composition, which contains, in addition to a therapeutically effective amount of compound AJ-091, one or more pharmaceutically acceptable carriers.

4. The use according to claim 1, characterized in that The drug is a pharmaceutical composition, which contains, in addition to a therapeutically effective amount of compound AJ-091 as an active ingredient, other pharmaceutical ingredients for treating systemic sclerosis / scleroderma.

5. The use according to claim 4, characterized in that The other drug ingredients for treating systemic sclerosis / scleroderma are selected from vasoactive drugs, anti-fibrotic drugs, glucocorticoids, immunosuppressants, and non-steroidal anti-inflammatory drugs.

6. The use according to claim 5, characterized in that The vasoactive drug is selected from the group consisting of stanozolol, captopril, urokinase, agkistrodon, hydralazine, vitamin E, prazosin, reserpine, dibazoline, nifedipine, tolazoline, and pentoxifylline; The anti-fibrotic drug is selected from the group consisting of penicillamine, colchicine, isotretinoin, acetylcholine, aspirin, and centella asiatica; The glucocorticoid is prednisone acetate; The immunosuppressant is selected from the group consisting of azathioprine, cyclophosphamide, methotrexate, chlorambucil or cyclosporine; The non-steroidal anti-inflammatory drug is selected from the following group: indomethacin, ibuprofen, naproxen, human recombinant relaxin, thalidomide, penicillin, and erythromycin.

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

8. The use according to claim 7, characterized in that The oral preparation is selected from the group consisting of tablets, capsules, granules, powders, solutions, and syrups.

9. The use according to claim 7, characterized in that The injection is suitable for intravenous injection or intravenous drip.

10. The use according to claim 7, characterized in that The external preparation is selected from the group consisting of creams, ointments, gels, sprays, and film coatings.

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