A method of treating systemic lupus erythematosus using BTK inhibitors.

Abstract

Provided herein are methods of treating lupus nephritis, particularly systemic lupus erythematosus, including active proliferative lupus nephritis, in a subject with (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo-[1,5-a]pyrimidine-3-carboxamide or a pharmaceutically acceptable salt thereof.

Description

Technical Field

[0001] A method for treating systemic lupus erythematosus, including lupus nephritis in a subject, particularly active proliferative lupus nephritis, the method comprising administering to a subject in need thereof (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo-[1,5-a]pyrimidine-3-carboxamide or a pharmaceutically acceptable salt thereof, is disclosed herein.

Background Art

[0002] Systemic lupus erythematosus (SLE) is a complex chronic autoimmune disease of unknown etiology that affects almost all organ systems, following a disease course of relapses and remissions. SLE occurs much more frequently in women than in men, with a frequency up to 9 times higher in some studies, and often occurs during the childbearing years (15 - 45 years). In SLE, the immune system attacks the body's cells and tissues, causing inflammation and tissue damage that can damage the heart, joints, skin, lungs, blood vessels, liver, kidneys, and nervous system.

[0003] Lupus nephritis is a common and serious symptom of SLE. It is a type of glomerulonephritis characterized by the accumulation of immune complexes in the glomeruli, often an inflammatory response in all renal compartments. Over time, the inflammation leads to chronic damage to the renal parenchyma and decreased renal function. The incidence and prevalence of lupus nephritis are influenced by age, sex, race, ethnicity, geographical location, and diagnostic criteria. Clinically apparent lupus nephritis is seen in 20%–60% of patients with SLE, and it almost always develops within six months of SLE diagnosis. The standardized mortality ratio for SLE patients is 2 to 5 times higher than that of the general population (Bernatsky S, Boivin JF, Joseph L, et al. Mortality in systemic lupus erythematosus. Arthritis Rheum. 2006;54(8):2550-7.), and this is further increased in patients who develop chronic kidney disease and end-stage renal disease (ESRD) (Mok CC, Kwok RCL, Yip PSF, et al. Effect of renal disease on the standardized mortality ratio and life expectancy of patients with systemic lupus erythematosus. Arthritis Rheum. 2013;65(8):2154-60.).

[0004] Renal biopsy is a key diagnostic criterion for lupus nephritis, and treatment for lupus nephritis should be guided by its pathological classification. According to the International Society of Nephrology / Japanese Society of Renal Pathology classification of lupus nephritis, it is classified into six classes. Patients with Class I and II lupus nephritis are usually treated as determined by the extrarenal clinical manifestations of lupus. Class III lupus nephritis (focal lupus nephritis) and Class IV lupus nephritis should be treated with corticosteroids and immunosuppressants due to their worsening prognosis. Initial treatment with corticosteroids in combination with either cyclophosphamide or mycophenolate mofetil (MMF) lasts for six months. After completion of initial treatment, patients with Class III and Class IV lupus nephritis receive maintenance therapy with azathioprine or mycophenolate mofetil and low-dose oral corticosteroids (≤10 mg / day of prednisone or equivalent). After complete remission is achieved, maintenance therapy should be continued for at least one year before gradually discontinuing immunosuppressants (KDIGO Clinical Practice Guideline for Glomerulonephritis. Kidney Int Suppl. 2012;2(2):259-74.). However, data from several clinical trials suggest that less than 30% of patients achieved complete renal response after 6 months of induction therapy.

[0005] To date, only voclosporine has been approved in the United States for the treatment of lupus nephritis, and mycophenolate mofetil has been approved in China, but complete remission rates remain low. Therefore, there is still a great unmet need for novel alternative treatments that can provide significant benefits to patients without high safety risks. [Overview of the project] [Means for solving the problem]

[0006] International Publication No. 2014 / 173289A discloses a series of BTK inhibitors, in particular (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (hereinafter referred to as Compound 1). Compound 1 may be used to treat cancers with abnormalities in the FcR signaling pathway in which the B cell receptor (BCR) and BTK play important roles, and has been demonstrated to have potent and irreversible inhibitory activity against BTK. [ka]

[0007] Compound 1 is a potent, specific, and irreversible BTK inhibitor with a favorable pharmacological and pharmacokinetic (PK) profile. Based on results from kinase inhibition and cell-based assays, Compound 1 exhibits good selectivity for off-target kinases, including epidermal growth factor receptor (EGFR), Janus kinase 3 (JAK3), human epidermal growth factor receptor-2 (HER2), TEC, and inducible T-cell kinase (ITK). The good selectivity of Compound 1 for BTK may result in lower incidence and milder off-target toxicity associated with the inhibition of the above-mentioned kinases.

[0008] This disclosure describes that (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 1) or a pharmaceutically acceptable salt thereof has shown a response in subjects with systemic lupus erythematosus (SLE).

[0009] This disclosure also states that (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 1) or a pharmaceutically acceptable salt thereof has shown a response in subjects with lupus nephritis.

[0010] This disclosure also states that (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (Compound 1) or a pharmaceutically acceptable salt thereof has shown a response in subjects with active proliferative lupus nephritis.

[0011] The inventors of this disclosure found that compound 1 demonstrated efficacy in the NZBWF1 / J lupus mouse model, particularly when administered orally at 3 and 10 mg / kg BID, and that compound 1 exhibited a more potent anti-lupus effect compared to mycophenolate mofetil (MMF).

[0012] The inventors of this disclosure also found that compound 1 demonstrated efficacy in systemic lupus erythematosus, such as in a mouse model of chronic graft-versus-host disease (SLE-cGVHD). Oral administration of compound 1 at doses of 10 and 20 mg / kg twice daily inhibited serum anti-dsDNA IgG levels, significantly reduced proteinuria levels, and improved splenomegaly.

[0013] Furthermore, the inventors of this disclosure also found that compound 1 showed dose-dependent efficacy in an MRL / lpr mouse lupus model at a dose range of 1.5 mg / kg to 50 mg / kg twice daily.

[0014] Exposure to compound 1 at 40 mg twice daily (BID) in humans was comparable to exposure at 15 mg / kg twice daily in MRL / lpr mice. Furthermore, effective doses of BTK inhibitors should achieve sustained BTK occupancy of >90% in PBMCs and >70% in the spleen. Compound 1 at doses ranging from 80 mg per day (40 mg BID) to 320 mg per day (160 mg BID) is recommended based on preclinical data, PK variability, and PK-PD simulations based on clinical doses in hematological indications, which have been shown to be effective and well-tolerated in patients with B-cell malignancies.

[0015] Compound 1 may be an effective treatment in treating systemic lupus erythematosus including lupus nephritis by significantly reducing proteinuria and increasing the remission rate compared to current standard treatments. Compound 1 may be well tolerated in systemic lupus erythematosus including lupus nephritis patients as a chronic treatment. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for treating systemic lupus erythematosus, comprising administering a therapeutically effective dose of (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo-[1,5-a]pyrimidine-3-carboxamide (compound 1) or a pharmaceutically acceptable salt thereof to the target. (Item 2) A method for treating lupus nephritis, comprising a therapeutically effective dose of (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo-[1,5-a]pyrimidine-3-carboxamide (compound 1) or a pharmaceutically acceptable salt thereof.

Chemical formula

[0016] [Figure 1A-ab] Figures 1A-a and 1A-b show that compound 1 suppressed proteinuria in a dose-dependent manner. As a positive control, mycophenolate mofetil (MMF) was administered once daily at 100 mg / kg; compound 1 at 0.1 and 0.3 mg / kg was less effective than MMF, but compound 1 at 1, 3, and 10 mg / kg did not show a significant difference compared to MMF. (Proportional odds model, p<0.05 relative to vehicle; **p<0.01, ***p<0.001; MMF: mycophenolate mofetil). [Figure 1A-cd] Figures 1A-c and 1A-d show that compound 1 significantly reduced BUN levels and inhibited pathological damage to the kidney. Compound 1 at 3 mg / kg and 10 mg / kg and MMF significantly inhibited BUN levels. Compound 1 inhibited the kidney histopathological score in a dose-dependent manner, while MMF did not significantly inhibit the histopathological score (one-way ANOVA, p<0.05 relative to *vehicle; BUN: serum urea nitrogen). [Figure 1A-ef]Figures 1A-e show that compound 1 significantly improved splenomegaly. Compound 1 at doses of 3 and 10 mg / kg significantly inhibited spleen weight, but MMF did not significantly inhibit spleen mass (p<0.05 against vehicle by one-way ANOVA; MMF: mycophenolate mofetil). Figures 1A-f show that compound 1 significantly reduced serum TNFα levels. Compound 1 at 10 mg / kg significantly inhibited TNFα secretion. In contrast, MMF showed lower levels of TNFα compared to the control group, without statistical significance (p<0.05 against vehicle by one-way ANOVA; MMF: mycophenolate mofetil). [Figure 1A-g] Figure 1A-g shows that compound 1 significantly reduced serum IL-10 levels. Compound 1 at 10 mg / kg significantly inhibited IL-10 secretion. In contrast, MMF showed lower levels of IL-10 compared to the control group, but without statistical significance. (One-way ANOVA, p<0.05 compared to *vehicle; MMF: mycophenolate mofetil). [Figure 1B-a] Figure 1B-a shows the effect of compound 1 on anti-dsDNA IgG levels in an SLE-cGvHd mouse model. Serum anti-dsDNA IgG levels were detected by ELISA on day 15 of treatment. Data are presented as mean anti-DNA ± standard error (SEM) for each group. Statistical analysis was performed using one-way ANOVA (followed by Dunnett's test). **p<0.01 relative to vehicle. [Figure 1B-bc]Figure 1B-b shows the effect of compound 1 on proteinuria levels in a mouse model of SLE-cGvHD. Proteinuria levels were assessed on day 22 of treatment by determining urinary albumin using URIT 1 vp pieces. Data are presented as mean score ± standard error (SEM) for each group. Statistical analysis was performed using one-way ANOVA (followed by Dunnett's test). *p<0.05, **p<0.01, ****p<0.0001 relative to vehicle. Figure 1B-c shows the effect of compound 1 on the spleen index in a mouse model of SLE-cGvHD. Spleen weight and body weight were measured at the end of the study. The spleen index (ratio of spleen weight to body weight) was calculated. Data are presented as mean spleen index ± standard error (SEM) for each group. Statistical analysis was performed using one-way ANOVA (followed by Dunnett's test). **p<0.01 for the vehicle, ***p<0.001. [Figure 1B-d] Figures 1B-d show the effect of compound 1 on body weight in a mouse model of SLE-cGvHD. Body weight was measured twice a week. Data are shown as the average body weight measured twice a week. Data are shown as the average body weight x ± standard error (SEM) for each group. [Figure 1C-a] Figure 1C-a shows the effect of compound 1 on anti-dsDNA IgG in the MRL / MpJ-Faslpr / J (MRL / lpr) mouse model. At the end of the experiment, serum levels of anti-dsDNA IgG were detected by ELISA. Data are presented as mean anti-dsDNA ± standard error (SEM) for each group. Statistical analysis was performed using one-way ANOVA (followed by Dunnett's test). **p<0.01 relative to vehicle. [Figure 1C-bc]Figure 1C-b shows the effect of compound 1 on proteinuria levels in the MRL / MpJ-Faslpr / J (MRL / lpr) mouse model. Proteinuria levels were assessed on day 118 by determining urinary albumin using URTI 1 vp pieces. Data are presented as the mean score ± standard error (SEM) for each group. Statistical analysis was performed using one-way ANOVA (followed by Dunnett's test). p<0.0001 for ****vehicle. Figure 1C-c shows the effect of compound 1 on the spleen index in the MRL / MpJ-Faslpr / J (MRL / lpr) mouse model. Spleen weight and body weight were measured at the end of the study. The spleen index (ratio of spleen weight to body weight) was calculated. Data are presented as the mean spleen index ± standard error (SEM) for each group. Statistical analysis was performed using one-way ANOVA (followed by Dunnett's test). **** p<0.0001 for vehicle;## p<0.01 for vehicle,#### p<0.0001 for prednisone. [Figure 1C-de] Figures 1C-d show the effect of compound 1 on body weight in the MRL / MpJ-Faslpr / J (MRL / lpr) mouse model. Body weight was measured twice a week. Data are shown as mean body weight ± standard error (SEM) for each group. Figures 1C-e show the effect of compound 1 on BUN levels in the MRL / MpJ-Faslpr / J (MRL / lpr) mouse model. Serum urea nitrogen (BUN) was measured at the end of the experiment. Data are shown as mean BUN ± standard error (SEM) for each group. Statistical analysis was performed using one-way ANOVA (followed by Dunnett's test). **p<0.01 relative to vehicle. [Figure 1C-f]Figures 1C-f show the effect of compound 1 on renal histopathology in the MRL / MpJ-Faslpr / J (MRL / lpr) mouse model. Mice were euthanized with carbon dioxide at the end of the experiment. Kidneys were collected, and mortality scores for inflammatory cell infiltration, glomerulopathy, and casts were quantitatively analyzed. Data are presented as the mean score ± standard error (SEM) for each group. Statistical analysis was performed using one-way ANOVA (followed by Dunnett's test). **p<0.01, ***p<0.001, ****p<0.0001 relative to the vehicle. [Modes for carrying out the invention]

[0017] definition Unless otherwise defined elsewhere in this specification, all other technical and scientific terms used herein have meanings that are generally understood by those skilled in the art.

[0018] In this specification, including in the attached claims, the singular forms of terms such as "a," "an," and "the" refer to the corresponding plural objects unless otherwise specified in the context.

[0019] The term "or" is used to mean, and is used synonymously with, the term "and / or" unless otherwise specified in the context.

[0020] In this specification, the terms “administer,” “to administer,” “to treat,” and “to treat” mean, when applied to animals, humans, subjects, cells, tissues, organs, or body fluids, the contact of an external pharmaceutical, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or body fluid. Cellular treatment includes contact of a reagent with cells, and, when body fluids come into contact with cells, the contact of a reagent with body fluids. The terms “administer” and “treat” also mean, for example, in vitro and ex vivo treatment of cells, reagents, diagnostics, conjugates, or other cells. In this specification, the term “subject” includes any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably a human. Treating any disease or disorder means, in one aspect, improving the disease or disorder (i.e., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another embodiment, “to treat,” “to treat,” or “treatment” means to reduce or improve at least one physical parameter, including one that may not be perceived by the patient. In yet another embodiment, “to treat,” “to treat,” or “treatment” means to modulate a disease or disorder physically (e.g., stabilization of recognizable symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet another embodiment, “to treat,” “to treat,” or “treatment” means to prevent or delay the onset, development, or progression of a disease or disorder.

[0021] As used herein, the term “therapeutic dose” means the amount of a Bcl-2 inhibitor sufficient to provide such treatment for a disease, disorder, or symptom when administered to a subject to treat a disease, disorder, or at least one of the clinical symptoms of a disease or disorder. “Therapeutic dose” may vary depending on the drug, the disease, disorder, and / or the symptoms of the disease or disorder, the severity of the symptoms of the disease, disorder, and / or the symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. An appropriate dose in any given case may be obvious to those skilled in the art or may be determined by routine experimentation. In the case of combination therapy, “therapeutic dose” means the total amount of the combination for effective treatment of the disease, disorder, or condition. In some embodiments of this disclosure, the subject is a human.

[0022] This disclosure provides a method for treating lupus nephritis in a subject, comprising administering compound 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0023] Treatment method In one embodiment, the present disclosure provides a method for treating systemic lupus erythematosus (SLE). SLE refers to a chronic, inflammatory, variable autoimmune disease of connective tissue, which primarily occurs in women and is typically characterized by fever, skin rash, malaise, and joint pain, and often by damage to the blood, kidneys, heart, lungs, and brain (such as hemolytic anemia, nephritis, pleurisy, pericarditis, cognitive impairment, or meningitis). Depending on the primary affected organ or tissue, SLE may be further classified into diseases including, but are not limited to, lupus nephritis, neuropsychiatric lupus, lupus pneumonia, lupus myocarditis, and lupus hepatitis.

[0024] In one embodiment, the present disclosure provides a method for treating lupus nephritis in a subject.

[0025] Lupus nephritis refers to glomerulonephritis associated with systemic lupus erythematosus, typically characterized by proteinuria and hematuria, and often leading to renal failure. Lupus nephritis is classified into six histopathological classes. See Table 1.

[0026] In certain embodiments, the method includes administering compound 1 or a pharmaceutically acceptable salt thereof to a subject requiring it.

[0027] Lupus nephritis is classified into six histopathological classes. See Table 1.

[0028] [Table 1]

[0029] In some embodiments of this disclosure, lupus nephritis is active proliferative lupus nephritis.

[0030] Active proliferative lupus nephritis refers to class III / IV lupus nephritis (LN) as classified by the International Society of Nephrology / Japanese Society of Renal Pathology's classification of lupus nephritis (2003).

[0031] Compound 1 may be administered orally, parenterally, intrapulmonaryly, and intranasally, and, if necessary, by any preferred means including topical treatment and intrafocal administration. Administration may be carried out by any preferred route. Various administration schedules, including single or multiple doses, bolus administration, and pulse infusion at various time points, are envisioned herein, but are not limited to these.

[0032] Compound 1 will be formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the physician.

[0033] In one embodiment, compound 1 is administered orally at a dose of 10 mg to 640 mg per day, preferably 10 mg to 320 mg per day, more preferably 40 to 320 mg per day, and most preferably 80 to 320 mg per day. In another embodiment, compound 1 is administered orally at a dose of 40 mg to 160 mg per day, preferably 80 mg to 160 mg per day.

[0034] In one embodiment, compound 1 is administered orally at a dose of 5 mg to 320 mg twice daily (BID), preferably 5 mg to 160 mg BID, more preferably 20 mg to 160 mg BID, and most preferably 40 mg to 160 mg BID.

[0035] In one embodiment, compound 1 is administered orally at a dose of 10 mg to 640 mg once daily (QD), preferably 10 mg to 320 mg QD, more preferably 40 mg to 320 mg QD, and most preferably 40 mg to 160 mg QD. [Examples]

[0036] The present invention is not limited to, but is further illustrated by the following exemplary embodiments.

[0037] Example 1A: Efficacy of Compound 1 in the NZBWF1 / J lupus model method Ninety 26-week-old NZBWF1 female mice were randomly divided into seven groups of 10 mice each, based on proteinuria levels, anti-dsDNA IgG levels, and body weight. Treatment was initiated after randomization and administered once daily (QD) at 100 mg / kg mycophenolate mofetil (MMF) and twice daily (BID) at 0.1, 0.3, 1, 3, and 10 mg / kg of compound 1 (vehicle (0.5%)) for 96 days. Treatment was administered by forced oral administration (po) at a volume of 10 ml / kg body weight.

[0038] Furthermore, mice were monitored daily for clinical signs of toxicity throughout the study period. The body weight of each animal was recorded twice a week, and proteinuria was measured weekly. Proteinuria was evaluated using URIT 1 vp samples and scored on a scale of 0 to 4, as follows: 0 = trace amounts, 1 ≥ 30 mg / dL, 2 ≥ 100 mg / dL, 3 ≥ 300 mg / dL, and 4 ≥ 500 mg / dL.

[0039] On day 96 of treatment, blood was rapidly collected by cardiac puncture under carbon dioxide euthanasia. 300-500 μL of blood was transferred to a coagulation tube, and the upper serum sample was separated by centrifugation at 2,000 g for 10 minutes. The sample was then stored in a refrigerator at -80°C for later use. 110 μL of serum was used for detection of serum urea nitrogen levels, and 25 μL of serum was used for measurement of TNFα and IL-10 levels using Luminex.

[0040] The kidneys were removed and fixed in a 10% neutral buffered formalin solution. Tissue was taken from the modified blocks, dehydrated little by little with alcohol, embedded in paraffin, thinned using a slide slicer (to a thickness of approximately 3 μm), stained with hematoxylin-eosin (HE), and examined by light microscopy for pathological changes in the kidney tissue. The sections were scored in a blinded manner as a sum of three systems, including inflammation (0-4), glomerular injury (0-4), and sclerosis (0-4).

[0041] result The in vivo efficacy of compound 1 was investigated in the NZBWF1 / J lupus model. Mycophenolate mofetil (MMF) was used once daily at a dose of 100 mg / kg as a positive control. Oral treatment twice daily with compound 1 at doses of 0.1 and 0.3 mg / kg was less effective than MMF, but compound 1 at doses of 1, 3, and 10 mg / kg did not show a significant difference compared to MMF (Figures 1A-a and 1A-b).

[0042] Compound 1 and MMF at doses of 3 and 10 mg / kg significantly inhibited serum urea nitrogen (BUN) levels (Figure 1A-c). Compound 1 dose-dependently inhibited renal histopathological scores, while MMF did not significantly inhibit histopathological scores (Figure 1A-d). Compound 1 at doses of 3 and 10 mg / kg significantly inhibited spleen weight, while MMF did not significantly inhibit splenic mass (Figure 1A-e). Compound 1 at 10 mg / kg significantly inhibited TNFα and IL-10 secretion (Figure 1A-f and 1A-g). In contrast, MMF showed lower levels of TNFα and IL-10, which were not statistically significant compared to the control group (Figure 1A-f and 1A-g). All results indicate that compound 1 has a potent anti-lupus effect in the NZBWF1 / J lupus mouse model.

[0043] Example 1B Efficacy of Compound 1 in systemic lupus erythematosus, such as a chronic graft-versus-host disease (SLE-cGVHD) model. method Splenocytes from DBA / 2 mice were intravenously transplanted into 54 B6D2F1 mice on days -7 and 0. 48 animals were randomly divided into four groups of 12 animals each, according to the inoculation order. Treatment was administered from the day after randomization until the end of day 26. Mice were orally administered a vehicle (0.5% methylcellulose) and compound 1 at 10 or 20 mg / kg twice daily. Treatment was administered by forced oral administration (PO) at a volume of 10 ml / kg body weight.

[0044] Mice were monitored daily for clinical signs of toxicity throughout the study period. Body weight of each animal was recorded twice weekly. On day 15 of treatment, blood was collected from the orbital vein under isoflurane / oxygen anesthesia, and serum samples were prepared. Anti-ds DNA IgG levels in the serum of individual mice were quantified using an anti-ds DNA IgG ELISA kit (Chondrex, Inc., Cat. 3031).

[0045] On days 8, 15, and 22 of treatment, proteinuria was evaluated by determining urinary albumin using URIT 1 vp piece (URIT, China), and scored on a scale from 0 to 4 as follows: 0 = trace amounts, 1 ≥ 30 mg / dL, 2 ≥ 100 mg / dL, 3 ≥ 300 mg / dL, and 4 ≥ 500 mg / dL.

[0046] On day 26 of treatment, blood samples were collected from the posterior orbital sinus under isoflurane / oxygen anesthesia at 0, 0.5, 1.5, 4, and 8 hours for each treatment group of compound 1. Plasma was collected by centrifugation at 5,600 rpm for 7 minutes. Bioanalysis was performed at 3D BioOptima Co., Ltd. (Suzhou, China).

[0047] The mean differences in proteinuria levels, spleen index, and anti-ds DNA IgG levels between the treatment group and the vehicle group were analyzed for significance using one-way ANOVA (followed by Dunnett's test). A p<0.05 was considered statistically significant.

[0048] result The in vivo efficacy of compound 1 was investigated in systemic lupus erythematosus, such as in a mouse model of chronic graft-versus-host disease (SLE-cGVHD). Oral administration of compound 1 at 10 and 20 mg / kg twice daily inhibited serum anti-dsDNA IgG levels, significantly reduced proteinuria levels, and improved splenomegaly (Figure 1B-a, 1B-b, and 1B-c). None of the treatment groups had a significant weight loss throughout the study (Figure 1B-d). The steady state of compound 1 (AUC) is shown. 0-8h and C max Drug exposure increased in a dose-dependent manner. All results indicate that compound 1 improved the disease in the SLE-cGvHD mouse model.

[0049] Example 1C: Efficacy of Compound 1 in the MRL / lpr lupus model method MRL / MpJ-Fas purchased from Jackson Laboratories lpr / J(MRL / lpr) mice were randomly divided into six groups of 11 mice each, based on proteinuria levels, anti-dsDNA IgG, and body weight. Treatment was initiated the day after randomization, with 5.0 mg / kg of prednisone once daily (QD) or a vehicle (0.5% MC, methylcellulose) and compound 1 at 1.5, 5, 15, and 50 mg / kg twice daily (BID) for 17 weeks. Treatment was administered by forced oral administration (po) at a volume of 10 ml / kg body weight.

[0050] During the experiment, the condition and mortality rate of the mice were recorded daily. The weight of each animal was recorded twice a week.

[0051] Proteinuria was measured weekly by determining urinary albumin levels using URIT 1 vp strips (URIT, China) and scored on a scale of 0 to 4 as follows: 0 = trace amounts, 1 ≥ 30 mg / dL, 2 ≥ 100 mg / dL, 3 ≥ 300 mg / dL, and 4 ≥ 500 mg / dL.

[0052] Blood samples were collected from the posterior orbital sinus under isoflurane / oxygen anesthesia at 0, 0.5, 1.5, 4, and 8 hours for each treatment group of compound 1. Plasma was collected by centrifugation at 5,600 rpm for 7 minutes and stored frozen at -80°C until analysis. Bioanalysis was performed at 3D BioOptima Co., Ltd. (Suzhou, China).

[0053] At the end of the experiment, blood was collected from the orbital vein under isoflurane / oxygen anesthesia. Serum samples were prepared and analyzed for BUN levels. Anti-dsDNA IgG levels in the serum of individual mice were quantified using the anti-dsDNA IgG ELISA kit (Chondrex, Inc., Cat. 3031).

[0054] Mice were euthanized using carbon dioxide, and spleen weight and body weight were measured at the end of the experiment. The spleen index (ratio of spleen weight to body weight) was calculated.

[0055] Kidney tissue was collected, preserved in 10% NBF, trimmed, dehydrated, embedded in paraffin, thinned (approximately 3 μm), and stained with HE. Histopathological examination was performed.

[0056] The mean differences in proteinuria levels, spleen index, BUN levels, renal histopathology scores, and anti-dsDNA IgG levels between the treatment group and the vehicle group were analyzed for significance using one-way ANOVA (followed by Dunnett's test). A p<0.05 was considered statistically significant.

[0057] result The in vivo efficacy of compound 1 was demonstrated using MRL / MpJ-Fas lpr The study was conducted using the / J(MRL / lpr) model. Prednisone was administered once daily at 5.0 mg / kg as a positive control. Oral administration of compound 1 at 1.5, 5, 15, and 50 mg / kg twice daily significantly protected mice from lupus nephritis, inhibited serum anti-dsDNA IgG levels compared to the vehicle (Figure 1C-a), significantly reduced proteinuria and serum blood urea nitrogen (BUN) levels (Figures 1C-b and 1C-e), dose-dependently suppressed splenomegaly (Figure 1C-c), and reduced renal histopathological scores (Figure 1C-f). No significant effect on body weight was observed in any of the treatment groups throughout the study (Figure 1C-d). Compound 1 (AUC) at steady state. 0-8h and C max Drug exposure to ) increased independently of dose. All results showed that compound 1 was MRL / MpJ-Fas lpr This study demonstrates a strong anti-lupus effect in the / J(MRL / lpr) mouse model.

[0058] Example 2 A phase 2, multicenter, randomized, double-blind, placebo-controlled trial to evaluate the safety and efficacy of compound 1 in patients with active proliferative lupus nephritis. The primary objective of this study is to evaluate the efficacy of compound 1 in addition to standard treatment, as measured by complete renal response, in participants with active proliferative lupus nephritis.

[0059] Test design: Patients will be randomly assigned to one of the treatment groups according to the following arms and interventions.

[0060] [Table 2]

[0061] Outcome scale Primary outcome measure: 1. Percentage of participants with complete renal response [Period: Week 49, Day 1]

[0062] Secondary outcome measures: 1. Percentage of participants achieving full renal response [Period: Week 25, Day 1] 2. Percentage of participants achieving partial renal response [Period: Week 25, Day 1 and Week 49, Day 1] 3. Percentage of participants achieving a complete renal response [Period: Week 25, Day 1 and Week 49, Day 1] 4. Time to first complete renal response: Time from the day of randomization to the day of first complete renal response [Period: up to 73 weeks and 1 day] 5. Time to first partial renal response: Time from the day of randomization to the day of first partial renal response [Period: up to 73 weeks and 1 day] 6. Changes in the total systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) score [Period: Week 25, Day 1 and Week 49, Day 1] The SLEDAI-2K score is a systemic lupus erythematosus (SLE) activity index ranging from 0 to 105, based on the presence of 24 features in nine organ systems. A higher score indicates more severe features in the participant over the past 30 days. 8. Pharmacokinetics of Compound 1 in patients with lupus nephritis, including area under the plasma concentration-time curve (AUC) [Period: Week 1, Day 1 and Week 5, Day 1]

[0063] Eligibility Main eligibility criteria: 1. A woman or man who is between 18 and 70 years old (including the endpoint) at the time of signing the Informed Consent Form (ICF). 2. Clinical diagnosis of SLE according to the International Collaborative Clinic for Systemic Lupus 2012 criteria. 3. ISN / RPS class III / IV lupus nephritis [type III(A), III(A+C), IV(A), and IV(A+C)] with or without class V, as confirmed by renal biopsy. 4. Positive antinuclear antibody, positive anti-dsDNA autoantibody, and / or positive anti-Smith autoantibody at the time of screening. 5. The patient has a 24-hour urinary protein excretion of >1.0 g at the time of screening.

[0064] Main exclusion criteria: Exclusion criteria related to systemic lupus erythematosus and other diseases: 1. Glomerulonephritis caused by reasons other than systemic lupus erythematosus. 2. Sclerosis in >50% of glomeruli observed in renal biopsies. 3. Any other inflammatory disease that may interfere with the evaluation of efficacy, including but not limited to rheumatoid arthritis, myositis, vasculitis, or overlapping syndromes. 4. Severe extrarenal SLE, including but not limited to pulmonary hypertension, severe myocarditis, and severe central nervous system lupus (neuropsychiatric SLE, seizures, psychosis, transverse myelitis, central nervous system vasculitis, and optic neuritis, etc.).

[0065] Compound 1 may be an effective treatment for lupus nephritis by significantly reducing proteinuria and increasing remission rates compared to current standard treatments. Compound 1 is likely to be well-tolerated in patients with lupus nephritis as a chronic treatment.

[0066] The above examples and descriptions of specific embodiments should be construed as illustrative and not as limiting the invention as defined by the claims. For ease of understanding, many variations and combinations of the features described above may be used without departing from the invention as defined in the claims. All such variations are intended to be within the scope of the invention. All references cited herein are incorporated herein by reference in their entirety.

Claims

1. A pharmaceutical composition for treating systemic lupus erythematosus, comprising (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo-[1,5-a]pyrimidine-3-carboxamide (compound 1) or a pharmaceutically acceptable salt thereof, wherein systemic lupus erythematosus comprises lupus nephritis, neuropsychiatric lupus, lupus pneumonia, lupus myocarditis, and lupus hepatitis.

2. A pharmaceutical composition for treating lupus nephritis comprising (S)-7-(1-acryloylpiperidine-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo-[1,5-a]pyrimidine-3-carboxamide (compound 1) or a pharmaceutically acceptable salt thereof. 【Chemistry 1】

3. The pharmaceutical composition according to claim 2, wherein the lupus nephritis is active proliferative lupus nephritis.

4. The aforementioned treatments are for the following clinical diagnoses of SLE according to the International Collaborative Clinic for Systemic Lupus 2012 criteria: a) ISN / RPS class III / IV lupus nephritis [type III(A), III(A+C), IV(A), and IV(A+C)], with or without class V, as confirmed by renal biopsy; b) Positive antinuclear antibodies, positive anti-dsDNA autoantibodies, and / or positive anti-Smith autoantibodies at the time of screening; and c) Having a 24-hour urinary protein excretion of >1.0 g at the time of screening. A pharmaceutical composition according to any one of claims 1 to 3, for a patient who meets one of the eligibility criteria comprising:

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein compound 1 is administered in a dose that achieves a sustained BTK occupancy rate of >90% in PBMC and >70% in the spleen.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein compound 1 is administered in a dose of 80 to 320 mg per day.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein compound 1 is administered in a dose of 80 mg to 160 mg per day.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein compound 1 is administered orally in a dose of 160 mg BID.

9. The pharmaceutical composition according to any one of claims 1 to 3, wherein compound 1 is administered orally in a dose of 320 mg QD.

10. The pharmaceutical composition according to any one of claims 1 to 8, wherein the subject to be treated exhibits a positive level of autoantibodies containing anti-dsDNA IgG in the serum.

11. Lupus nephritis is the following type of lupus nephritis: Micromesangial lupus nephritis; Mesangial proliferative lupus nephritis; Focal lupus nephritis; Diffuse segmental (IV-S) or pansegmental (IV-G) lupus nephritis; Membranous lupus nephritis; or Advanced sclerosing lupus nephritis The pharmaceutical composition according to claim 2, which is any one of the following.

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