Use of btk inhibitors for treatment of disease

Abstract

The present disclosure relates to the use of BTK inhibitors for the treatment of disease. Specifically, the invention relates to application of the BTK inhibitor in preparation of drugs for preventing or treating membranous nephropathy, proteinuria, lupus nephritis or lupus renal injury.

Description

Use of BTK inhibitors for treating diseases

[0001] This application claims priority to the Chinese patent application (application number CN202210532478.3) filed on May 10, 2022. Technical Field

[0002] The present disclosure belongs to the field of medicine and relates to the use of BTK inhibitors in treating diseases. Background Art

[0003] Membranous nephropathy (MN) is one of the most common pathological types of nephrotic syndrome in adults. It can be divided into primary membranous nephropathy (PMN) and secondary membranous nephropathy based on the cause. PMN accounts for approximately 75% of MN patients.

[0004] The characteristic pathological changes of PMN are the accumulation of large immune complexes on the epithelial side of the glomerular capillary loops; immunofluorescence reveals primarily IgG4 deposits; and electron microscopy reveals subepithelial electron-dense material, effacement of foot processes, and thickening of the glomerular basement membrane due to matrix secretion between the deposits. Furthermore, researchers have found that approximately 70-80% of PMN patients harbor autoantibodies against the podocyte antigen phospholipase A2 receptor (PLA2R). The most common clinical manifestation of PMN is nephrotic syndrome and its associated symptoms, including varying degrees of edema, hypoalbuminemia, and hyperlipidemia. PMN is also prone to thrombotic and thromboembolic events, increasing the risk of infection and cardiovascular disease. If long-term nephrotic syndrome is untreated, nearly 60% of patients will experience renal impairment, and 30-40% will eventually progress to end-stage renal disease (ESRD) within 10 years.

[0005] Currently, no medications are approved for PMN in any country. According to the "Primary Membranous Nephropathy" section of the 2012 and 2020 KDIGO guidelines, patients with low-risk, non-acutely progressive disease should initially receive adequate non-immunosuppressive supportive care (including antihypertensive and antiproteinuric therapies). Given the significant toxicity and side effects of immunosuppressive drugs, the guidelines recommend only disease-specific immunosuppressive therapy (IST) for patients at high risk of disease progression. These include alkylating agents (cyclophosphamide and hormones administered alternately for 6 months), calcineurin inhibitors (cyclosporine, tacrolimus), and rituximab. Toxic side effects of immunosuppressants primarily include infection with alkylating agents, relapse of viral hepatitis, alopecia, gonadal damage, hemorrhagic cystitis, carcinogenesis, bladder and ureteral cancer, toxic hepatitis, and potential nephrotoxicity with cyclosporine. In recent years, the CD20 antibody rituximab has been used to treat PMN. Existing clinical data show that the clinical remission rate at 12 months after treatment with rituximab was significantly higher than that in the control group receiving only supportive care (64.9% vs. 34.2%). Rituximab reduced adverse reactions of grade 3 or higher, but the overall adverse reaction rate did not significantly decrease. Compared with cyclosporine, rituximab also increased the risk of infusion reactions, skin, and upper respiratory tract infections.

[0006] Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase primarily expressed in B cells. It is a key kinase in the B cell receptor (BCR) signaling pathway. When the BCR on the B cell surface binds to a ligand, it induces activation of downstream Src family kinases. Through precise regulation, this activates downstream signaling pathways such as PKCβ and MAPK, NF-κB, and AKT, thereby regulating B cell survival, proliferation, and differentiation, as well as cytokine release. BTK influences B cell activation by mediating signals related to antigen receptor activation, as well as binding to CD40, TLR, Fc, and chemokine receptors. The survival of autoreactive B cells is more dependent on BTK function than that of normal B cells. Furthermore, studies of BTK-deficient cells have demonstrated that BTK plays a role in cytoskeletal reorganization and cell motility, which directly influence B cell antigen presentation and migration.

[0007] WO2016007185 relates to a compound represented by formula (I), namely (R)-4-amino-1-(1-(but-2-ynyl)pyrrolidin-3-yl)-3-(4-(2,6-difluorophenoxy)phenyl)-1,6-dihydro-7H-pyrrolo[2,3-d]pyridazin-7-one. This compound is a novel BTK kinase inhibitor with improvements in kinase selectivity, clinical efficacy or indications, and safety. Its structure is shown below:

[0008] WO2013091539 relates to a compound represented by formula (II), namely (3aR,5s,6aS)-N-(3-methoxy-1,2,4-thiadiazol-5-yl)-5-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)hexahydrocyclopenta[c]pyrrole-2(1H)-carboxamide, which is a class of JAK kinase inhibitor compounds with in vitro and in vivo activity and high absorption.

[0009] The prior art has not yet provided the use of BTK inhibitors alone or in combination with JAK inhibitors in the preparation of a drug for treating or preventing membranous nephropathy, proteinuria, lupus nephritis or lupus renal injury.

[0010] Summary of the Invention

[0011] The present disclosure provides the use of a BTK inhibitor in preparing a drug for preventing or treating membranous nephropathy, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the membranous nephropathy is PLA2R antibody-positive membranous nephropathy.

[0013] In some embodiments, the membranous nephropathy is primary membranous nephropathy.

[0014] In some embodiments, the BTK inhibitor is capable of reducing PLA2R antibody levels in a subject.

[0015] The present disclosure provides the use of a BTK inhibitor in the preparation of a medicament for preventing or treating proteinuria, wherein the BTK inhibitor is selected from any one or a combination of the following: ibrutinib, acalabrutinib, GS-4059, sepretib, BGB-3111, FIM71224, zanubrutinib, ARQ531, BI-BTK1, vecabrutinib, or the compound represented by the aforementioned formula (I) or a pharmaceutically acceptable salt thereof.

[0016] The present disclosure provides use of a BTK inhibitor in the preparation of a medicament for preventing or treating lupus nephritis or lupus renal injury. The BTK inhibitor is a compound represented by the aforementioned formula (I) or a pharmaceutically acceptable salt thereof.

[0017] The present disclosure provides a use of a combination of a BTK inhibitor and a JAK inhibitor in the preparation of a medicament, wherein the medicament is used to treat any one of the following: membranous nephropathy, proteinuria, lupus nephritis, and lupus renal injury; preferably, the membranous nephropathy is PLA2R antibody-positive membranous nephropathy; preferably, the membranous nephropathy is primary membranous nephropathy; the BTK inhibitor and the JAK inhibitor are administered simultaneously or sequentially; the BTK inhibitor and the JAK inhibitor are in the same or different containers; the BTK inhibitor is a compound represented by the aforementioned formula (I) or a pharmaceutically acceptable salt thereof; the JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,

[0018] Preferably, the pharmaceutically acceptable salt of the compound represented by formula (II) is hydrogen sulfate or sulfate.

[0019] In some embodiments, the dosage range of the BTK inhibitor is selected from 1 mg to 1000 mg.

[0020] In some embodiments, the BTK inhibitor is administered at a frequency selected from any one of the following: once a day, twice a day, three times a day, once every two days, once every three days, and once a week.

[0021] In some embodiments, the BTK inhibitor is administered once a day at a dose selected from the following: 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg g, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 30 0mg, 310mg, 320mg, 330mg, 340mg, 350mg, 360mg, 370mg, 380mg, 390mg, 400mg, 410mg, 420mg, 430mg, 440mg, 450mg, 460mg, 470mg, 480mg, 490mg, 500mg.

[0022] In some embodiments, the BTK inhibitor is administered twice a day at a dose selected from the following: 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 12 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 3 0mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185m g, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg.

[0023] In some embodiments, the JAK inhibitor is administered at a frequency selected from any one of the following: once a day, twice a day, or three times a day.

[0024] In some embodiments, the JAK inhibitor is administered at a dose of 0.5 mg to 20 mg, preferably 1 mg to 5 mg, and more preferably any one selected from the following: 1 mg, 2 mg, and 4 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows the effects of Compound A alone or in combination with Compound B on the urinary protein content in MRL / lpr mice;

[0026] FIG2 shows the effects of Compound A alone or in combination with Compound B on the anti-dsDNA IgG content in the kidneys of MRL / lpr mice;

[0027] FIG3 shows the effects of Compound A alone or in combination with Compound B on the serum urea nitrogen (BUN) content in mice;

[0028] FIG4 shows the effects of Compound A alone or in combination with Compound B on the serum creatinine (CRE) level in mice;

[0029] Figure 5 shows the effects of Compound A alone or in combination with Compound B on the spleen coefficient of MRL / lpr mice;

[0030] Figure 6 shows the effects of Compound A alone or in combination with Compound B on serum IL-6 levels in MRL / lpr mice;

[0031] FIG7 shows the effects of compound A alone or in combination with compound B on the serum INF-γ content in MRL / lpr mice.

[0032] In the accompanying figures, *P<0.05, **P<0.01, ***P<0.001 vs. blank (SLE model). DETAILED DESCRIPTION

[0033] In the present disclosure, the term "inhibitor" refers to a natural or synthetic compound that inhibits (or reduces or downregulates) the expression of a gene and / or protein, and / or inhibits (or reduces or downregulates) the activity of a gene and / or protein, and / or regulates the signal transduction pathway associated with the gene and / or protein. As a non-limiting example, the inhibitor can act on any one of the following aspects or combinations of a gene and / or protein: for example, but not limited to, translation, post-translational processing, stability, degradation, nuclear localization, cytoplasmic localization, transcription, post-transcriptional processing, activation, inactivation, modification, signal transduction. Inhibitors are allowed to be competitive, non-competitive, fully antagonistic, or partially antagonistic.

[0034] BTK inhibitors

[0035] According to some embodiments of the present disclosure, a BTK (Bruton's tyrosine kinase) inhibitor is provided.

[0036] Therefore, a BTK inhibitor refers to a compound that has the following effects: inhibiting (or reducing or downregulating) the expression of a BTK encoding gene, and / or the expression of BTK, and / or the activity of BTK, and / or regulating a BTK-related signaling pathway (for example, but not limited to, upstream or downstream of a BTK-related signaling pathway).

[0037] JAK inhibitors

[0038] According to some embodiments of the present disclosure, a JAK (Janus kinase) inhibitor is provided.

[0039] A JAK inhibitor refers to a compound that has the following effects: inhibiting (or reducing or downregulating) the expression of a JAK encoding gene, and / or the expression of JAK, and / or the activity of JAK, and / or regulating a JAK-related signaling pathway (for example, but not limited to, upstream or downstream of a JAK-related signaling pathway).

[0040] Uses of BTK inhibitors

[0041] In one aspect, the present disclosure provides use of a BTK inhibitor in the preparation of a medicament for preventing or treating membranous nephropathy.

[0042] The present disclosure also provides a BTK inhibitor for preventing or treating membranous nephropathy.

[0043] In certain embodiments, the membranous nephropathy is PLA2R antibody-positive membranous nephropathy.

[0044] The term "PLA2R positive" or "PLA2R antibody positive" means that the subject's serum contains autoantibodies against PLA2R.

[0045] In certain embodiments, the PLA2R antibody titer in a subject with the disease (e.g., but not limited to, blood, whole blood, plasma, serum) is greater than 10 RU / ml, for example, greater than 11 RU / ml, greater than 12 RU / ml, greater than 13 RU / ml, greater than 14 RU / ml, greater than 15 RU / ml, greater than 16 RU / ml, greater than 17 RU / ml, greater than 18 RU / ml, greater than 19 RU / ml, or greater than 20 RU / ml. For example, the PLA2R antibody titer is 14-200 RU / ml, preferably 20-150 RU / ml.

[0046] "Titer" or "antibody titer" refers to the amount of a specific antibody in a sample (e.g., a biological sample obtained from a subject). The titer of an antibody can be measured using any method known in the art. In some embodiments, the titer of an antibody is measured by serially diluting a sample containing the antibody until the antibody is no longer detected above background. In such examples, the titer of the antibody is expressed as a multiple dilution at which the concentration of the antibody is reduced to background levels.

[0047] In certain embodiments, the membranous nephropathy is primary membranous nephropathy or secondary membranous nephropathy.

[0048] In certain embodiments, the membranous nephropathy is primary membranous nephropathy.

[0049] Another aspect of the present disclosure provides a use of a BTK inhibitor in the preparation of a medicament for reducing the level of PLA2R antibodies (eg, but not limited to, blood, whole blood, plasma, serum).

[0050] The present disclosure also provides a BTK inhibitor for reducing the level of PLA2R antibodies.

[0051] In certain embodiments, the reduction of PLA2R antibody levels is a reduction of PLA2R antibody titer to no more than 50 RU / ml, such as no more than 45 RU / ml, 40 RU / ml, 30 RU / ml, 20 RU / ml, 19 RU / ml, 18 RU / ml, 17 RU / ml, 16 RU / ml, 15 RU / ml or 14 RU / ml.

[0052] In certain specific embodiments, the initial titer of the PLA2R antibody level is not less than 20 RU / ml.

[0053] In certain embodiments, the reduction in PLA2R antibody levels is at least 20% compared to the initial titer, such as at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%.

[0054] Another aspect of the present disclosure provides a use of a BTK inhibitor in preparing a medicament for preventing or treating proteinuria.

[0055] The present disclosure also provides a BTK inhibitor for preventing or treating proteinuria.

[0056] Another aspect of the present disclosure provides a use of a BTK inhibitor in the preparation of a medicament for preventing or treating lupus nephritis or lupus renal injury.

[0057] The present disclosure also provides a BTK inhibitor for preventing or treating lupus nephritis or lupus renal injury.

[0058] In any of the aforementioned uses, the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0059] The compounds of the present disclosure may exist as pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" means any non-toxic salt that, when administered to a recipient, is capable of providing the compound or a prodrug thereof. Such salts can be prepared during the final isolation and purification of the compound, or separately by reacting an appropriate nitrogen atom with a suitable acid.

[0060] Acids commonly used to form pharmaceutically acceptable salts include: inorganic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, hydrogen disulfide) and organic acids (e.g., p-toluenesulfonic acid, salicylic acid, tartaric acid, acid tartaric acid, ascorbic acid, maleic acid, benzenesulfonic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid).

[0061] The pharmaceutically acceptable salts of the drugs of the present disclosure may be hydrochlorides, phosphates, hydrogen phosphates, sulfates, hydrogen sulfates, sulfites, acetates, oxalates, malonates, valerates, glutamates, oleates, palmitates, stearates, laurates, borates, p-toluenesulfonates, methanesulfonates, isethionates, maleates, malates, tartrates, benzoates, pamoates, salicylates, vanillates, mandelates, succinates, gluconates, lactobionates, or laurylsulfonates, etc.

[0062] Combination of BTK inhibitors and JAK inhibitors

[0063] Another aspect of the present disclosure provides a use of a BTK inhibitor in combination with a JAK inhibitor in the preparation of a medicament for preventing or treating membranous nephropathy.

[0064] The present disclosure also provides a BTK inhibitor for preventing or treating membranous nephropathy, wherein the BTK inhibitor is administered in combination with a JAK inhibitor.

[0065] The present disclosure also provides a JAK inhibitor for preventing or treating membranous nephropathy, wherein the JAK inhibitor is administered in combination with a BTK inhibitor.

[0066] The present disclosure also provides a composition for preventing or treating membranous nephropathy, wherein the composition comprises a JAK inhibitor and a BTK inhibitor; the JAK inhibitor and the BTK inhibitor are in the same container or different containers.

[0067] In some embodiments, the JAK inhibitor and the BTK inhibitor are administered simultaneously or sequentially, and the time interval between administrations is limited to achieve a synergistic effect, and the time interval between administrations can be determined by a skilled person.

[0068] The "combination" described in the present disclosure is a mode of administration, which refers to the administration of at least one dose of a BTK inhibitor (or a pharmaceutically acceptable salt thereof) and a JAK inhibitor (or a pharmaceutically acceptable salt thereof) within a certain time limit, wherein both drugs show a pharmacological effect. The time limit can be within an administration cycle, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours. The BTK modulator (or a pharmaceutically acceptable salt thereof) and the JAK inhibitor (or a pharmaceutically acceptable salt thereof) can be administered simultaneously or sequentially. This deadline includes treatments in which the BTK inhibitor (or a pharmaceutically acceptable salt thereof) and the JAK inhibitor (or a pharmaceutically acceptable salt thereof) are administered by the same route of administration or different routes of administration.

[0069] In some embodiments, the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof:

[0070] Another aspect of the present disclosure provides a use of a BTK inhibitor in combination with a JAK inhibitor in the preparation of a medicament for preventing or treating lupus nephritis or lupus renal injury, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0071] In certain embodiments, the pharmaceutically acceptable salt of the compound represented by formula (II) is a hydrogen sulfate salt or a sulfate salt.

[0072] In certain specific embodiments, the pharmaceutically acceptable salt of the compound represented by formula (II) is a hydrogen sulfate salt.

[0073] Prevention and treatment methods

[0074] In one aspect, the present disclosure provides a method for preventing or treating membranous nephropathy, comprising administering to a subject a preventively or therapeutically effective amount of a BTK inhibitor; the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0075] In the present disclosure, a subject is an individual who is diagnosed with, suspected of having, or susceptible to the disease or symptoms.

[0076] A "prophylactically or therapeutically effective amount" refers to an amount of a therapeutic agent that produces the desired effect.

[0077] In some embodiments, the term refers to an amount sufficient to prevent or treat the disease, disorder and / or condition when applied to a population suffering from or susceptible to the disease, disorder and / or condition according to a regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of the disease, disorder and / or condition, and / or delays its onset. Those of ordinary skill in the art will appreciate that the term "prophylactic or therapeutically effective amount" does not actually require successful prevention or treatment in a particular individual. A prophylactic or therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a large number of subjects when applied to a subject. In some embodiments, mentioning a prophylactic or therapeutically effective amount can refer to an amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a prophylactic or therapeutically effective amount of a specific agent or therapy can be formulated and / or administered in a single dose. In some embodiments, a prophylactic or therapeutically effective amount of an agent can be formulated and / or administered in multiple doses, for example, as part of a regimen.

[0078] The amount of drug required for a single administration to a subject can be conveniently calculated by multiplying the subject's weight by the unit weight dose required for the subject's single administration. For example, in the process of preparing a drug, it is generally believed that the weight of an adult is 50-70 kg, and the drug dosage can be initially determined by the equivalent dose conversion relationship between the unit weight dose of experimental animals and humans. For example, it can be determined based on the guidance provided by drug administration agencies such as the FDA and SFDA, or by reference to ("Conversion of equivalent doses between animals and between animals and humans in pharmacological experiments", Chinese Journal of Clinical Pharmacology and Therapeutics, 2004 Sep; 9(9): 1069-1072).

[0079] In the embodiments of the present disclosure, the dosage for humans and mice can be converted using a conversion factor of 0.0026 based on the body surface area of ​​humans and mice.

[0080] On the other hand, the present disclosure provides a method for preventing or treating membranous nephropathy, comprising administering to a subject a preventive or therapeutically effective amount of a BTK inhibitor and a JAK inhibitor, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK inhibitor and the BTK inhibitor are administered simultaneously or sequentially.

[0081] In one aspect, the present disclosure provides a method for preventing or treating lupus nephritis or lupus renal injury, comprising administering to a subject a preventively or therapeutically effective amount of a BTK inhibitor, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0082] On the other hand, the present disclosure provides a method for preventing or treating lupus nephritis or lupus renal injury, comprising administering to a subject a preventive or therapeutically effective amount of a BTK inhibitor and a JAK inhibitor, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK inhibitor and the BTK inhibitor are administered simultaneously or sequentially.

[0083] In another aspect, the present disclosure provides a BTK inhibitor for preventing or treating membranous nephropathy, which is administered in combination with a JAK inhibitor, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0084] In another aspect of the present disclosure, a BTK inhibitor for preventing or treating lupus nephritis or lupus renal injury is provided, which is administered in combination with a JAK inhibitor, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0085] In another aspect of the present disclosure, a JAK inhibitor for preventing or treating membranous nephropathy is provided, which is administered in combination with a BTK inhibitor, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0086] In another aspect of the present disclosure, a JAK inhibitor for preventing or treating lupus nephritis or lupus renal injury is provided, which is administered in combination with a BTK inhibitor, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0087] In certain embodiments, the BTK inhibitor is selected from ibrutinib, acalabrutinib, GS-4059, septinib, BGB-3111, FIM71224, zanubrutinib, ARQ531, BI-BTK1, vecabrutinib, or a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0088] In certain embodiments, the BTK inhibitors described herein are administered at a dose of 1-1000 mg, for example, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg 470mg, 480mg, 490mg, 500mg, or a range between any two of the above values, which can be an integer or a decimal.

[0089] In certain embodiments, the BTK inhibitors described herein may be administered once a day, twice a day, three times a day, once every two days, once every three days, once a week, etc.

[0090] In certain embodiments, the BTK inhibitor is administered once daily at a dose of 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 10mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg, 310mg, 320mg, 330mg, 340mg, 350mg, 360mg, 370mg, 380mg, 390mg, 400mg, 410mg, 420mg, 430mg, 440mg, 450mg, 460mg, 470mg, 480mg, 490mg, 500mg, or a range between any two of the above values, which can be an integer or a decimal.

[0091] In certain embodiments, the BTK inhibitor is administered once daily at a dose of 50 mg, 100 mg, 150 mg, and 200 mg.

[0092] In certain embodiments, the BTK inhibitor is administered twice a day at a dose of 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, or a range between any two of the above values, which can be an integer or a decimal.

[0093] In certain embodiments, the BTK inhibitor is administered twice daily at a dose of 50 mg, 100 mg, 150 mg, and 200 mg each time.

[0094] In certain embodiments, the unit dose of the BTK inhibitor is 50 mg.

[0095] In certain embodiments, the unit dose of the BTK inhibitor is 100 mg.

[0096] In certain embodiments, the unit dose of the BTK inhibitor is 150 mg.

[0097] In certain embodiments, the unit dose of the BTK inhibitor is 200 mg.

[0098] In certain embodiments, the JAK inhibitor is administered once a day, twice a day, or three times a day at a dose of 0.5-20 mg.

[0099] In certain embodiments, the JAK inhibitor is administered to human subjects at a dose selected from 0.5-20 mg, including but not limited to 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg, 7.5 mg, 8.0 mg, 8.5 mg, 9.0 mg , 9.5mg, 10.0mg, 10.5mg, 11.0mg, 11.5mg, 12.0mg, 12.5mg, 13.0mg, 13.5mg, 14.0mg, 14.5mg, 15.0mg, 15.5mg, 16.0mg, 16.5mg, 17.0mg, 17.5mg, 18.0mg, 18.5mg, 19.0mg, 19.5mg or 20.0mg, or a range between any two of the above values, which can be an integer or a decimal.

[0100] In certain embodiments, the JAK inhibitor is administered once a day or twice a day at a dose of 1-5 mg.

[0101] In certain embodiments, the JAK inhibitor is administered once a day or twice a day at a dose selected from 1 mg, 2 mg, and 4 mg.

[0102] In certain embodiments, the JAK inhibitor is administered once a day at a dose of 1 mg.

[0103] In certain embodiments, the JAK inhibitor is administered once a day at a dose of 2 mg.

[0104] In certain embodiments, the JAK inhibitor is administered once a day at a dose of 4 mg.

[0105] In certain embodiments, the JAK inhibitor is administered twice a day at a dose of 1 mg.

[0106] In an optional embodiment, the BTK inhibitor is administered in a dosage selected from 50 mg, 100 mg, 150 mg and 200 mg, and the administration frequency is once a day or twice a day, and the JAK inhibitor is administered once a day or twice a day, and the administration dosage is selected from 1 mg, 2 mg and 4 mg.

[0107] In an optional embodiment, the BTK inhibitor is administered in a dosage selected from 50 mg, 100 mg, 150 mg and 200 mg, and the administration frequency is once a day or twice a day, and the JAK inhibitor is administered once a day or twice a day, and the administration dosage is 1 mg.

[0108] In an optional embodiment, the BTK inhibitor is administered in a dosage selected from 50 mg, 100 mg, 150 mg and 200 mg, and the administration frequency is once a day or twice a day, and the JAK inhibitor is administered once a day or twice a day, and the administration dosage is 2 mg.

[0109] In an optional embodiment, the BTK inhibitor is administered in a dosage selected from 50 mg, 100 mg, 150 mg and 200 mg, and the administration frequency is once a day or twice a day, and the JAK inhibitor is administered once a day or twice a day, and the administration dosage is 4 mg.

[0110] The BTK inhibitors described herein can also be administered in combination with other drugs, wherein the combined administration is selected from simultaneous administration, independent formulation and co-administration, or independent formulation and sequential administration.

[0111] The administration route of the drug of the present disclosure is selected from oral administration, parenteral administration, and transdermal administration. The parenteral administration includes but is not limited to intravenous injection, subcutaneous injection, and intramuscular injection.

[0112] The present disclosure further relates to a method of reducing PLA2R antibody levels in a subject, comprising administering to the subject an effective amount of a BTK inhibitor.

[0113] The present disclosure further relates to a method for treating proteinuria comprising administering to a subject a therapeutically effective amount of a BTK inhibitor.

[0114] In certain embodiments, the treated subject has a 24-hour urine protein of no more than 3.5 g / d, 3.0 g / d, 2.5 g / d, 2.0 g / d, 1.5 g / d, 1.0 g / d, 0.9 g / d, 0.8 g / d, 0.7 g / d, 0.6 g / d, 0.5 g / d, 0.4 g / d, or 0.3 g / d; or a 24-hour urine protein of less than 3.5 g / d, and is reduced by ≥20% compared to the initial concentration, for example, by ≥25%, by ≥30%, by ≥35%, by ≥40%, by ≥45%, or by ≥50%.

[0115] During the treatment process disclosed herein, non-immunosuppressant supportive therapy (NIST) can also be given simultaneously, including ACE inhibitors and ARBs for reducing proteinuria and blood pressure, and antihypertensive, lipid-lowering, anticoagulant, and diuretic treatments for nephrotic syndrome symptoms.

[0116] Pharmaceutical composition

[0117] The present disclosure also relates to a pharmaceutical composition comprising:

[0118] The compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and

[0119] One or more pharmaceutically acceptable carriers, excipients, and diluents.

[0120] The pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form, for example, tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injections, sterile powders for injection, and concentrated solutions for injection), suppositories, inhalants, or sprays.

[0121] The present disclosure also provides a drug packaging box, comprising one or more containers, each of which independently packages any one selected from the following: a compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present disclosure, a compound of formula (II) or a pharmaceutically acceptable salt thereof according to the present disclosure, or a pharmaceutical composition according to the present disclosure.

[0122] The compounds of the present disclosure may also exist in different tautomeric forms, all of which are within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. An example of a lactam-lactim equilibrium is between A and B as shown below,

[0123] In certain embodiments, the compounds of formula (I) disclosed herein can be drawn as Form A or Form B. The naming of the compounds does not exclude any tautomers.

[0124] Example

[0125] Example 1. Study on the efficacy of compound A alone or in combination with compound B in MRL / lpr spontaneous lupus erythematosus mice

[0126] 1. Test objectives and evaluation indicators

[0127] This example aims to investigate the pharmacodynamic effects of Compound A alone or in combination with Compound B on MRL / lpr spontaneous lupus erythematosus mice. Spontaneous SLE mouse models can be used to simulate the mechanisms and symptoms of membranous nephropathy, proteinuria, lupus nephritis, and lupus renal injury (see: E Garcia-Vives et al., Antibodies to M-type phospholipase A2 receptor (PLA2R). Lupus (2019) 0: 1–10; Pierre Ronco et al., Membranous nephropathy. Nature Reviews, DISEASE PRImERS, Article citation ID: (2021) 7: 69; Anthony D. Marinov et al., The Type II Anti-CD20 Antibody Obinutuzumab (GA101) Is More Effective Than Rituximab at Depleting B Cells and Treating Disease in a Murine Lupus Model. American college of rheumatology, 2020, 826-836; US20230039927A1). Evaluation indicators:

[0128] a: Determination of protein content in urine of mice in each group;

[0129] b: Determination of the content of anti-dsDNA IgG in the kidneys of mice in each group;

[0130] c: Determination of blood urea nitrogen (BUN) and blood creatinine (CRE) levels in the serum of mice in each group;

[0131] d: The levels of IL-6 and IFN-γ in the serum of mice in each group were measured;

[0132] e: Calculate the spleen coefficient of each group of mice;

[0133] f: Pathological observation and scoring of kidney tissues of mice in each group.

[0134] 2. Experimental Design

[0135] 2.1 Experimental drugs and experimental animals

[0136] Compound A: Prepared according to the method in WO2016007185.

[0137] Compound B: The hydrogen sulfate salt of the compound of formula (II) is prepared by referring to the method in WO2013091539.

[0138] Experimental Animals: Female BALB / c mice and female MRL / C mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd. (license number SCXK(Shanghai)2014·0004) and maintained in the SPF animal facility of the School of Pharmacy, Fudan University. The animals were housed at a temperature of 20–24°C and a humidity of 50 ± 5%. Sterile feed and water were provided ad libitum. All animal experiments were conducted in accordance with the Regulations for Laboratory Animal Care of the School of Pharmacy, Fudan University.

[0139] Other reagents:

[0140] 2.2 Experimental methods

[0141] 2.2.1 Experimental Grouping

[0142] Female BALB / c mice served as normal controls, and female MRL / lpr mice were randomly divided into eight groups based on proteinuria and body weight (Table 1). Administration began at 8 weeks of age and continued for 8 consecutive weeks. During the treatment period, mouse body weight was measured weekly, and urine protein levels were measured every two weeks. Clinical observations were also conducted on the mice. The experimental groups are shown in Table 1.

[0143] Table 1. Experimental design and grouping

[0144] 2.2.2 Preparation of test samples

[0145] 2.2.2.1: Preparation and storage of solvent reference substances

[0146] Weigh the required amount of CMC-Na, dissolve it in sterile water for injection, and dilute to a final concentration of 0.5%. Store at 2 to 8°C until ready for use.

[0147] 2.2.2.2: Drug preparation and storage methods

[0148] Weigh a certain amount of Compound A and prepare a 4.8 mg / mL aqueous solution (based on Compound A) with 0.5% CMC-Na aqueous solution as a stock solution, corresponding to an administration dose of 15 mg / kg. Dilute the stock solution 10-fold and 3-fold with 0.5% CMC-Na aqueous solution to obtain 0.48 mg / mL and 1.6 mg / mL aqueous solutions, respectively, corresponding to administration doses of 1.5 and 5 mg / kg. The oral gavage volume is 3.125 mL / kg. Preparation frequency: at least once a week. Post-preparation storage conditions and shelf life: Store at 2-8°C in the dark and use within 7 days.

[0149] Weigh a certain amount of compound B and prepare a 2.4 mg / mL aqueous solution (based on compound B) as the stock solution. Stir magnetically overnight. Dilute the stock solution 7.5 times and 3 times with 0.5% CMC-Na aqueous solution to obtain 0.32 mg / mL and 0.8 mg / mL aqueous solutions, respectively. The oral gavage volume is 3.125 mL / kg, corresponding to the administration doses of 1, 2.5, and 7.5 mg / kg. Preparation frequency: at least once a week; Storage conditions and shelf life after preparation: Store at 15 to 25°C in the dark and use within 7 days.

[0150] 2.2.3 Inspection indicators

[0151] A: Urine protein concentration measurement

[0152] In this experiment, mouse urine was collected using the tail reflex method and the urine protein concentration was measured using a urine protein assay kit (CBB method). The assay was adjusted to zero using double-distilled water. Using a 563 mg / L protein standard solution as the standard, 5 μL of urine sample was mixed thoroughly with 300 μL of CCB (Coomassie Brilliant Blue) reagent. The mixture was allowed to react at room temperature for 5 minutes, and the OD value was read at a wavelength of 595 nm using a multifunctional microplate reader.

[0153] B: Serum creatinine (CRE) test

[0154] This experiment uses a creatinine (CRE) assay kit to detect the serum creatinine content in mouse serum at the end of the experiment. The experimental steps are as follows: (1) Pipette 6 μL of double-distilled water, standard solution, and test sample serum in sequence and add them to a 96-well plate; (2) Add 180 μL of reagent 1 enzyme solution A to each well, incubate at 37°C for 5 minutes, and measure the absorbance value A1 at a wavelength of 546 nm; (3) Add 60 μL of reagent 2 enzyme solution B to each well, incubate at 37°C for 5 minutes, and measure the absorbance value A2 at a wavelength of 546 nm; (4) Calculate the results:

[0155] C: Urea nitrogen (BUN) measurement

[0156] In this experiment, a urea nitrogen (BUN) test kit was used to detect the urea nitrogen content in mouse serum. The experimental steps are as follows: (1) 10 μL of double-distilled water as a blank control, 10 mmol / L BUN standard application solution as a standard sample, and 10 μL of serum from each group of mice were added to a 1.5 mL EP tube, followed by 125 μL of buffered enzyme solution; (2) After mixing, the tube was incubated in a 37°C water bath for 10 minutes; (3) 500 μL of phenol color developer and 500 μL of alkaline sodium hypochlorite were added to each sample in sequence, mixed thoroughly, and then incubated in a 37°C water bath for 10 minutes. Due to the large number of samples, batch operation was adopted, and the number of each batch was controlled at 15; (4) 200 μL of each sample was aspirated and added to a 96-well plate, and one replicate was made for each sample. The absorbance value of each tube was measured at a wavelength of 640 nm using a microplate reader; (5) Calculation results: urea nitrogen content (mmol / L) = (measured OD value - blank OD value) / (standard OD value - blank OD value) × standard concentration (10 mmol / L) × sample dilution multiple before testing.

[0157] D: Renal pathology testing

[0158] The left kidney of the mouse was removed and fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Paraffin sections were prepared and stained with H&E. The sections were observed under a microscope and pathological scores were performed. Pathological damage to glomeruli, renal interstitial tissue, and blood vessels was graded. The scoring criteria are shown in Table 2.

[0159] Table 2. Kidney pathology scoring criteria

[0160] MNC: mononuclear cell.

[0161] E: Autoantibody detection in the kidney

[0162] Preparation of renal tissue homogenate: (1) Remove the kidneys from each group and cut off approximately the upper 1 / 3 of each kidney; (2) Rinse the tissue with pre-cooled PBS to remove residual blood, weigh and mince the tissue; (3) Add the minced tissue and the corresponding volume of PBS containing protease inhibitors (1:9 weight-to-volume ratio, 1 mg tissue sample corresponds to 9 μL PBS) into a 1.5 mL EP tube; (4) Add two zirconium oxide grinding beads to each EP tube and place it in a multi-sample tissue grinder. Grind and lyse the tissue thoroughly at 45 Hz for 30 seconds each time, repeat 3 times; (5) Centrifuge the homogenate at 5000 g for 10 minutes and remove the supernatant for later use. (6) Determine the concentration of each sample by BCA assay; (7) Each sample is diluted to a total protein concentration of 150 μg with PBS containing protease inhibitors to a protein concentration of 5 μg / μL, and use this as the final tissue homogenate for subsequent ELISA assay.

[0163] ELISA method to detect the content of anti-dsDNA IgG in kidney:

[0164] The specific steps of anti-dsDNA IgG detection are as follows: (1) All reagents of the ELISA kit were equilibrated at room temperature for 30 minutes before the experiment, and all experimental operations were completed at room temperature; (2) Initial dilution of the experimental group sample (1 / 5): 30 μL tissue homogenate + 120 μL working sample diluent (WSD) mixed; secondary dilution (1 / 50): 10 μL initial dilution (1 / 5) + 90 μL Low NSB sample diluent (LNSD) mixed; (3) First incubation: 100 μL sample diluent (blank), standard solution and experimental sample were added to the 96-well plate, and the plate was gently tapped to mix the reagents and incubated for 60 minutes; (4) Wash the wells 4 times with washing solution and then dry them on clean filter paper; (5) Second incubation: 100 μL diluted anti-mouse IgG-HRP was added to each well and incubated for 30 minutes; (6) Wash 5 times according to step 4; (7) Substrate incubation: 100 μL was added to each well The TMB substrate was wrapped in aluminum foil and incubated in the dark for 15 min; (8) Terminate the reaction: add 100 μL of stop solution to each well and gently tap to mix; (9) Read the absorbance at a single wavelength of 450 nm using a microplate reader, and calibrate the data variation using the OD value at 630 nm.

[0165] F: Cytokine detection

[0166] The cytokines IFN-γ and IL-6 in serum were detected by ELISA.

[0167] The method for determining serum IFN-γ is as follows: (1) Place all reagents at room temperature for 30 minutes, prepare 1× diluent and washing solution, and prepare the standard solution with gradient concentrations of 600, 300, 150, 75, 37.5, 18.8, and 9.4 pg / mL in sequence according to the instructions; (2) Remove the microplate from the sealed bag that has been equilibrated to room temperature, and add 50 μL of diluent (1×) to each microplate; (3) Add the standard solution and experimental sample of different concentrations to the corresponding wells, 50 μL per well. Seal the reaction wells with sealing tape and incubate at room temperature for 2 hours; (4) Shake off the liquid in the plate, add 300 μL of washing solution to each well, let it stand for 30 seconds, and then blot dry on clean filter paper. Repeat this operation 4 times; (5) Add 100 μL of enzyme-labeled detection antibody to each well. Seal the reaction wells with a sealing film and incubate at room temperature for 2 hours; (6) Repeat step 4; (7) Add 100 μL of colorimetric substrate to each well and incubate at room temperature in the dark for 30 minutes; (8) Add 100 μL of stop solution to each well and tap the microplate to mix the solution evenly. The color of the solution in the well will change from blue to yellow; (9) Within 30 minutes after adding the stop solution, use a microplate reader to measure the absorbance value at 450 nm and set 570 nm as the calibration wavelength; (10) Calculate the results: The horizontal axis of the curve is the OD value of the standard curve point, and the vertical axis is the mouse IFN-γ concentration value of the standard curve point. Use Excel to fit the best standard curve and calculate the corresponding concentration based on the sample OD value.

[0168] The steps for serum IL-6 determination are as follows: (1) Place all reagents at room temperature for 30 minutes, prepare 1× diluent and washing solution, and prepare the standard sample with gradient concentrations of 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 pg / mL in sequence according to the instructions; (2) Subsequent operations are the same as steps 2-10 of serum IFN-γ determination.

[0169] 3. Experimental Results

[0170] 3.1 Effects of Compound A alone or in combination with Compound B on urine protein levels in MRL / lpr mice

[0171] During the development of lupus erythematosus in MRL / lpr mice, the kidneys are damaged by the deposition of dsDNA antibodies, leading to decreased glomerular filtration and reabsorption capacity and increased urinary protein concentration. As lupus nephritis develops in MRL / lpr mice, urine protein levels gradually increase.

[0172] As shown in Figure 1, after 8 weeks of administration, the protein content in the urine of the MRL / lpr blank group (model group) was significantly higher than that of the normal mice of the same strain (Balb / c) (P<0.01) (normal control group), indicating that the MRL / lpr mice developed spontaneous renal damage due to the deposition of autoantibodies.

[0173] As shown in Figure 1, after 8 weeks of administration, the average urine protein levels of mice in the 1 mg / kg and 2.5 mg / kg Compound B (BID) groups were significantly lower than those in the blank group (P<0.01). The average urine protein levels of mice in the 7.5 mg / kg Compound B (BID) group were also lower than those in the blank group. The average urine protein levels of mice in the 1.5, 5, and 15 mg / kg Compound A (QD) groups were all significantly lower than those in the blank group (P<0.001). This suggests that once-daily administration of 1.5 to 15 mg / kg Compound A can significantly reverse spontaneous renal injury caused by antibody deposition in MRL / lpr mice. The average urine protein levels of mice in the combination group (2.5 mg / kg Compound B (BID) + 1.5 mg / kg Compound A (QD)) were significantly lower than those in the blank group (P<0.001). Compared to the corresponding doses of Compound B or Compound A alone, the average urine protein levels of mice in the combination group showed a downward trend.

[0174] 3.2 Effects of Compound A alone or in combination with Compound B on the anti-dsDNA IgG levels in the kidneys of MRL / lpr mice

[0175] MRL / lpr mice have an insertional mutation in the Fas gene, resulting in a functional defect in the Fas protein and blocking lymphocyte apoptosis. Due to the excessive accumulation of lymphocytes that cannot undergo apoptosis, the genomic DNA of the cells acts as an antigen, generating a large amount of dsDNA antibodies in the body, which are deposited in the kidneys and cause functional renal damage.

[0176] As shown in Figure 2, compared with normal mice, the anti-dsDNA IgG content in the kidneys of mice in the MRL / lpr blank group (model group) was significantly increased (P<0.01), indicating that the kidneys of mice in the MRL / lpr model group showed obvious anti-dsDNA antibody deposition;

[0177] As shown in Figure 2, the anti-dsDNA IgG content in the kidneys of mice in the 7.5 mg / kg Compound B (BID) group was significantly lower than that in the blank group (P<0.01), and showed a clear dose-dependent effect within the dose range of 1 to 7.5 mg / kg, indicating that Compound B administered twice daily can dose-dependently reverse the deposition of anti-dsDNA IgG in the kidneys. The anti-dsDNA IgG content in the kidneys of mice in the 1.5, 5, and 15 mg / kg Compound A (QD) groups was significantly lower than that in the blank group (P<0.01), and showed a clear dose-dependent effect, indicating that Compound A administered once daily can also significantly reverse the deposition of anti-dsDNA IgG in the kidneys within the dose range of 1.5 to 15 mg / kg. The anti-dsDNA IgG content in the kidneys of mice in the 2.5 mg / kg compound B (BID) + 1.5 mg / kg compound A (QD) combination group was significantly lower than that in the blank group (P<0.001); compared with the corresponding doses of compound B or compound A single-drug group, the anti-dsDNA IgG content in the kidneys of mice in the combination group was significantly decreased (P<0.05), indicating that the combination of 2.5 mg / kg compound B (BID) and 1.5 mg / kg compound A (QD) has a significant synergistic effect on the deposition of anti-dsDNA IgG in the kidneys.

[0178] 3.3 Effects of Compound A alone or in combination with Compound B on renal function in MRL / lpr mice

[0179] Urea nitrogen and creatinine are metabolic products of human protein and muscle, respectively. The kidneys are their final excretion organs. When renal function is impaired, blood urea nitrogen and creatinine cannot be effectively excreted, and their concentrations gradually increase due to retention. Therefore, serum BUN and CRE levels are the main indicators of renal function in clinical practice.

[0180] As shown in Figure 3, compared with normal mice, the blood urea nitrogen (BUN) in the serum of mice in the MRL / lpr blank group (model group) was significantly increased (P<0.001), further suggesting that functional renal damage occurred in mice in the MRL / lpr model group.

[0181] As shown in Figure 3, the urea nitrogen content in the serum of mice in the 1, 2.5 and 7.5 mg / kg Compound B (BID) groups was significantly lower than that in the blank group (P<0.05, P<0.01, P<0.001), and showed a clear dose-dependent pattern, indicating that within the dose range of 1 to 7.5 mg / kg, Compound B administered twice daily can significantly reverse the spontaneous renal injury in MRL / lpr mice in a dose-dependent manner. The urea nitrogen content in the serum of mice in the 1.5, 5 and 15 mg / kg Compound A (QD) groups was also significantly lower than that in the blank group (P<0.001), and showed a clear dose-dependent pattern, indicating that within the dose range of 1.5 to 15 mg / kg, Compound A administered once daily can also significantly reverse the spontaneous renal injury in MRL / lpr mice in a dose-dependent manner. The urea nitrogen content in the serum of mice in the 2.5 mg / kg compound B (BID) + 1.5 mg / kg compound A (QD) combination group was also significantly lower than that in the blank group (P<0.001); compared with the corresponding doses of compound B or compound A single-drug groups, the urea nitrogen content in the serum of mice in the combination group was significantly reduced (P<0.05), indicating that the combination of 2.5 mg / kg compound B (BID) and 1.5 mg / kg compound A (QD) has a significant synergistic effect on improving the renal function of mice.

[0182] As shown in Figure 4 , compared with normal mice, the serum creatinine (CRE) content of mice in the MRL / lpr blank group (model group) was significantly increased (P<0.001), further suggesting that functional renal damage occurred in mice in the MRL / lpr model group;

[0183] As shown in Figure 4, the creatinine content in the serum of mice in the 1, 2.5 and 7.5 mg / kg Compound B (BID) groups was lower than that in the blank group; the creatinine content in the serum of mice in the 5 and 15 mg / kg Compound A (QD) groups was significantly lower than that in the blank group (P<0.01, P<0.001), and showed obvious dose-dependence within the dose range of 1.5 to 15 mg / kg; the creatinine content in the serum of mice in the 2.5 mg / kg Compound B (BID) + 1.5 mg / kg Compound A (QD) combination group was also significantly lower than that in the blank group (P<0.001); compared with the corresponding doses of Compound B and Compound A single-drug groups, the serum creatinine content of mice in the combination group was significantly reduced (P<0.05), indicating that the combination of 2.5 mg / kg Compound B (BID) and 1.5 mg / kg Compound A (QD) has a significant synergistic effect on improving the renal function of mice.

[0184] 3.4 Effects of Compound A alone or in combination with Compound B on the spleen coefficient of MRL / lpr mice

[0185] Due to the mutation of the Fas gene in MRL / lpr mice, the apoptosis signaling pathway is inhibited, leading to spontaneous lymph node enlargement and abnormal T cell proliferation.

[0186] As shown in Figure 5, compared with normal mice, the spleen weight of mice in the MRL / lpr blank group (model group) was significantly increased (P<0.001); the spleen weight of mice in the 1, 2.5 and 7.5 mg / kg compound B (BID) groups showed a downward trend compared with the blank group; except for the spleen weight of mice in the 15 mg / kg compound A group, which was significantly reduced compared with the blank group (P<0.01), the spleen weight of mice in the 1.5 and 5 mg / kg compound A groups showed a downward trend compared with the blank group; the spleen weight of mice in the 2.5 mg / kg compound B (BID) + 1.5 mg / kg compound A (QD) combination group was significantly lower than that in the blank group (P<0.01, P<0.001), and showed an obvious dose-dependence, indicating that the combination of compound B and compound A can synergistically reduce spleen weight and inhibit T and B cell proliferation.

[0187] 3.5 Effects of Compound A alone or in combination with Compound B on serum IL-6 levels in MRL / lpr mice

[0188] As shown in Figure 6, compared with normal mice, the IL-6 content in the serum of mice in the MRL / lpr blank group (model group) was significantly increased (P<0.001); the IL-6 content in the serum of mice in the 7.5 mg / kg compound B (BID) group was significantly lower than that in the blank group (P<0.01), and showed obvious dose-dependence in the dose range of 1 to 7.5 mg / kg, indicating that compound B monotherapy administered twice a day can dose-dependently reduce the concentration of IL-6 in mouse serum; except for the mice in the 1.5 mg / kg compound A (QD) group, the IL-6 content in the serum of mice in the 5 and 15 mg / kg compound A groups was significantly lower than that in the blank group (P<0.01). The IL-6 content in the serum of mice in the 2.5 mg / kg compound B (BID) + 1.5 mg / kg compound A (QD) combination group was significantly lower than that in the blank group (P<0.001, P<0.01); compared with the corresponding doses of compound B or compound A single-drug group, the IL-6 content in the serum of mice in the combination group was significantly reduced (P<0.05), indicating that the combination of 2.5 mg / kg compound B (BID) and 1.5 mg / kg compound A (QD) has a significant synergistic inhibitory effect on inhibiting IL-6 release.

[0189] 3.6 Effects of Compound A alone or in combination with Compound B on serum INF-γ levels in MRL / lpr mice

[0190] As shown in Figure 7, compared with normal mice, the INF-γ content in the serum of mice in the MRL / lpr blank group (model group) was significantly increased (P<0.001); the INF-γ content in the serum of mice in the 1, 2.5 and 7.5 mg / kg compound B (BID) groups was significantly lower than that in the blank group (P<0.05 or P<0.001), and showed a clear dose-dependency, indicating that within the dose range of 1 to 7.5 mg / kg, compound B administered twice a day can significantly reduce the INF-γ content in the serum of mice in a dose-dependent manner; the INF-γ content in the serum of mice in the 5 and 15 mg / kg compound A groups was also significantly lower than that in the blank group (P<0.01, P<0.01). <0.001), and showed obvious dose dependence in the dose range of 1.5 to 15 mg / kg; the INF-γ content in the serum of mice in the 2.5 mg / kg compound B (BID) + 1.5 mg / kg compound A (QD) combination group was significantly lower than that in the blank group (P<0.01, P<0.05); compared with the 1.5 mg / kg compound A (QD) single-drug group, the INF-γ content in the serum of mice in the combination group was significantly reduced (P<0.05), indicating that the combination of 2.5 mg / kg compound B (BID) and 1.5 mg / kg compound A (QD) has a certain synergistic inhibitory effect on the release of INF-γ in mouse serum.

[0191] 3.7 Effects of Compound A alone or in combination with Compound B on renal lesions in MRL / lpr mice

[0192] At the end of the experiment, the left kidney of the mouse was removed and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned, and then stained with H&E. The pathological damage of the mouse kidney was observed under a microscope.

[0193] The kidneys of mice in the MRL / lpr blank group (model group) showed severe pathological damage to the glomeruli, renal interstitium and blood vessels, with glomeruli sclerosis and crescent formation, a large number of cells infiltrating in the perivascular and interstitial areas, and glomerular atrophy. The pathological damage to the glomeruli, renal interstitium and blood vessels of mice in the compound A alone or combined with compound B groups was significantly alleviated.

[0194] As shown in Table 3, compared with normal mice, the total kidney pathology score of mice in the MRL / lpr blank group (model group) was significantly increased (P<0.001), indicating that the kidneys of mice in the MRL / lpr model group had obvious lesions.

[0195] As shown in Table 3, the total renal pathology scores of mice in the 1, 2.5 and 7.5 mg / kg Compound B (BID) groups were significantly lower than those in the blank group (P<0.01 or P<0.001), and showed a clear dose-dependence, indicating that within the dose range of 1 to 7.5 mg / kg, Compound B monotherapy administered twice daily can significantly improve MRL / lpr spontaneous renal lesions in a dose-dependent manner; the total renal pathology scores of mice in the 1.5, 5 and 15 mg / kg Compound A groups were significantly lower than those in the blank group (P<0.001), and showed a clear dose-dependence, indicating that within the dose range of 1.5 to 15 mg / kg, Compound A administered once daily as a single agent also significantly improved MRL / lpr spontaneous renal lesions in a dose-dependent manner; the total kidney pathology scores of mice in the 2.5 mg / kg compound B (BID) + 1.5 mg / kg compound A (QD) combination group were significantly lower than those in the blank group (P<0.001); compared with the corresponding doses of compound B or compound A single-agent groups, the total kidney pathology scores of mice in the combination group were significantly reduced (P<0.05), indicating that the combination of 2.5 mg / kg compound B (BID) and 1.5 mg / kg compound A (QD) has a significant synergistic improvement effect on renal lesions.

[0196] Table 3. Renal pathology scoring table

[0197] Remark:

[0198] *P<0.05, **P<0.01, ***P<0.001 vs. blank group (SLE model);

[0199] # P<0.05 vs. equal dose of compound B alone,

[0200] $ p<0.05 vs. equal doses of Compound A alone, n=8 to 14.

[0201] 4. Conclusion

[0202] (1) After the administration period (approximately 16 weeks of age), MRL / lpr spontaneous lupus erythematosus mice showed significant increases in urine protein content, serum urea nitrogen (BUN) and creatinine (CRE) concentrations, and kidney anti-dsDNA IgG content compared with normal mice of the same strain, with obvious pathological damage to the kidneys; spleen weight increased significantly; and serum IL-6 and IFN-γ levels increased significantly.

[0203] (2) Oral administration of compound B at doses of 1, 2.5, and 7.5 mg / kg twice daily for 8 weeks significantly reduced the level of anti-dsDNA IgG in the kidneys, significantly improved renal function (reduced serum BUN and CRE levels), and significantly slowed the progression of spontaneous renal tissue lesions in MRL / lpr mice. Compound B also significantly reduced serum IL-6 and IFN-γ levels. These improvements were dose-dependent.

[0204] (3) Oral administration of 1.5, 5, and 15 mg / kg of compound A once daily for 8 weeks also significantly reduced the level of anti-dsDNA IgG in the kidneys, significantly improved the renal function of MRL / lpr mice (reduced the levels of BUN and CRE in the serum), and significantly slowed the spontaneous renal tissue lesions in these mice; significantly inhibited spontaneous spleen swelling in MRL / lpr mice; and significantly reduced the levels of IL-6 and IFN-γ in the serum. The above-mentioned improvements also showed a clear dose-dependent effect.

[0205] (4) Compared with the same dose of compound B or compound A alone, the combination of 2.5 mg / kg compound B (BID) and 1.5 mg / kg compound A (QD) had a significant synergistic effect in reducing the level of anti-dsDNA IgG in the kidneys, improving the renal function of mice (reducing the levels of BUN and CRE in serum), reducing the levels of IL-6 and INF-γ in serum, and improving renal lesions.

[0206] In summary, under the conditions of this example, both Compound A and Compound B alone had a significant improvement effect on MRL / lpr spontaneous lupus erythematosus, and the combined use of the two had a significant synergistic effect.

[0207] Since the main clinical symptoms and observation indicators of primary membranous nephropathy (PMN) are similar to those of the MRL / lpr spontaneous lupus erythematosus model, including urine protein, serum creatinine, and autoantibody levels, it is expected that Compound A, alone or in combination with Compound B, will also be effective in treating PMN.

[0208] Example 2. Randomized, double-blind, placebo-controlled clinical trial of the efficacy and safety of the compound represented by formula (I) for the treatment of primary membranous nephropathy (PMN)

[0209] 1. Experimental Drugs

[0210] The tablets of the compound represented by formula (I) have a specification of 25 mg / tablet.

[0211] 2. Inclusion criteria

[0212] 1) Applicants must be between 18 and 75 years old (inclusive) at the time of screening, regardless of gender;

[0213] 2) Patients diagnosed with primary membranous nephropathy (PMN) before or during screening (confirmed by renal biopsy);

[0214] 3) Patients who have not received immunosuppressive treatments targeting PMNs (cyclophosphamide; calcineurin inhibitors such as cyclosporine, tacrolimus) and B-cell depleting treatments (such as rituximab); or who have relapsed after achieving complete remission or partial remission with the above treatments (determined and recorded by the investigators), and have not received the above treatments after relapse (excluding those who are ineffective or resistant to B-cell depleting drugs), but are required to have discontinued calcineurin inhibitors for at least 3 months, cyclophosphamide for at least 6 months, and B-cell depleting drugs for at least 9 months before enrollment;

[0215] 4) serum PLA2R antibody titer ≥20 RU / ml at screening;

[0216] 5) 24-hour urine protein test value ≥3.5g / d at screening;

[0217] 6) Estimated eGFR at screening (calculated using the CKD-EPI formula) ≥ 60 mL / min / 1.73 m 2 .

[0218] 3. Application plan

[0219] Eligible subjects were randomly stratified in a 2:2:1 ratio (stratification factors included: PLA2R antibody titer <50 RU / ml and ≥50 RU / ml; body weight ≤75 kg and >75 kg) and assigned to either Group A (administered with the compound of Formula (I), Group B (administered with the compound of Formula (I), or a placebo control group). The administration schedule was as follows.

[0220] Table 4

[0221] Conclusion: The main purpose of this example is to verify the preliminary efficacy of the investigational drug in PMN subjects, aiming to observe indicators such as the proportion of subjects achieving clinical remission after 24 weeks of treatment, the proportion of subjects with negative conversion of PLA2R antibodies, the decrease in PLA2R antibody titer, the improvement in 24-hour urine protein, and the relevant safety of the investigational drug.

[0222] In this example, it was observed that the overall urine protein showed a significant and sustained improvement trend from baseline to week 24, and the PLA2R antibody titer showed a significant and sustained downward trend from baseline to week 24. The serum albumin of the subjects continued to increase, indicating that the albumin of the subjects gradually returned to normal and the clinical symptoms gradually improved. From the perspective of safety indicators: the eGFR index was stable, indicating that the renal function of the subjects was stable and no decline in renal function was shown. The B lymphocyte count was stable from baseline to week 24, indicating that the number of B lymphocytes of the subjects was stable and no decline in B cells occurred. The incidence of adverse events was low. In summary, the current experimental drug has shown significant efficacy in the indication of PMN and has good safety.

Claims

1. Use of a BTK inhibitor in the preparation of a drug for preventing or treating membranous nephropathy, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, 2 . The method according to claim 1 , wherein the membranous nephropathy is PLA2R antibody-positive membranous nephropathy.

3. The use according to claim 1 or 2, wherein the membranous nephropathy is primary membranous nephropathy.

4. The use according to any one of claims 1 to 3, wherein the BTK inhibitor is capable of reducing the PLA2R antibody level in the subject.

5. Use of a BTK inhibitor in the preparation of a medicament for preventing or treating proteinuria, wherein: The BTK inhibitor is selected from any one of the following or a combination thereof: ibrutinib, acalabrutinib, GS-4059, sepretinib, BGB-3111, FIM71224, zanubrutinib, ARQ531, BI-BTK1, vecabrutinib, or a compound of formula (I) or a pharmaceutically acceptable salt thereof; 6. Use of a BTK inhibitor in the preparation of a medicament for preventing or treating lupus nephritis or lupus renal injury, wherein the BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, 7. Use of a combination of a BTK inhibitor and a JAK inhibitor in the preparation of a medicament for treating any one of the following: membranous nephropathy, proteinuria, lupus nephritis, and lupus renal injury; Preferably, the membranous nephropathy is PLA2R antibody-positive membranous nephropathy; Preferably, the membranous nephropathy is primary membranous nephropathy; The BTK inhibitor and the JAK inhibitor are administered simultaneously or sequentially; The BTK inhibitor and the JAK inhibitor are in the same or different containers; The BTK inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, The JAK inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof, Preferably, the pharmaceutically acceptable salt of the compound represented by formula (II) is hydrogen sulfate or sulfate.

8. The use according to any one of claims 1 to 7, wherein the dosage range of the BTK inhibitor is selected from 1 mg to 1000 mg.

9. The use according to any one of claims 1 to 8, wherein the administration frequency of the BTK inhibitor is selected from any one of the following: once a day, twice a day, three times a day, once every two days, once every three days, and once a week.

10. The use according to claim 9, wherein: The BTK inhibitor is administered once a day, with a dosage selected from any of the following: 10 mg each time, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120 mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 21 5mg,220mg,225mg,230mg,235mg,240mg,245mg,250mg,255mg,260mg,265mg,270mg,275mg,280mg,285mg,290mg,295mg,300mg,310mg,3 20mg, 330mg, 340mg, 350mg, 360mg, 370mg, 380mg, 390mg, 400mg, 410mg, 420mg, 430mg, 440mg, 450mg, 460mg, 470mg, 480mg, 490mg, 500mg; Alternatively, the BTK inhibitor is administered twice daily at a dose selected from the following: 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg per dose , 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg , 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg.

11. The use according to any one of claims 8 to 10, wherein: The administration frequency of the JAK inhibitor is selected from any one of the following: once a day, twice a day or three times a day, The JAK inhibitor is administered in a dosage of 0.5 mg to 20 mg, preferably 1 mg to 5 mg, and more preferably any one selected from the following: 1 mg, 2 mg, and 4 mg.

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