Use of bitopertin as direct activator of nrf2

Abstract

The application discloses application of Bitopertin as an Nrf2 direct activator, and experiments prove that Bitopertin can inhibit the combination of Keap1 and Nrf2, reduce ubiquitination of Nrf2, increase the stability of Nrf2, thereby increasing the protein level of intracellular Nrf2, and treat Nrf2 related chronic diseases by activating Nrf2; the effect of Bitopertin is more obvious than that of other Nrf2 agonists, Bitopertin does not affect liver function, has higher biological safety, is a more potential clinical Nrf2 agonist, has greater advantages in treating various Nrf2 related diseases, and has good clinical application prospect.

Description

Technical fields:

[0001] This invention relates to the field of biomedicine, and in particular to the application of bitopertin as a direct activator of Nrf2. Background technology:

[0002] Nrf2 (Nuclear factor erythroid 2-related factor 2) is a key regulator of oxidative stress and anti-inflammatory responses. It primarily regulates the expression of antioxidant proteins such as quinone oxidoreductase-1 (NQO1), enzymes related to glutathione (GSH) synthesis and metabolism, heme oxygenase 1 (HO-1), and ATP-binding cassette transporters, as well as phase II detoxification enzymes and transporters. This helps to scavenge endogenous / exogenous toxins and reactive oxygen species, thereby protecting cells. Under non-stress conditions, Nrf2 is isolated in its Kelch domain by Keap1 in the cytoplasm and ubiquitinated by Cul3 ubiquitin ligase, thus being degraded by the proteasome. During oxidative stress, Nrf2 dissociates from the Kelch domain of Keap1 and translocates to the cell nucleus, where it acts as a transcription factor to promote the expression of various intracellular antioxidant enzymes, thereby alleviating oxidative stress damage. Due to the potent cytoprotective effects of Nrf2, activating its expression using small molecules holds significant promise for the treatment of various chronic diseases. These chronic diseases encompass multiple systemic disorders, including respiratory, cardiovascular, musculoskeletal, and nervous systems. Small molecules can activate Nrf2 in two ways: 1) by covalently targeting the cysteine ​​residue at position 151 of the BTB domain in Keap1 with electrophilic activity, altering the KEAP1 conformation and inhibiting its degradation of Nrf2; 2) by directly inhibiting the binding of the Kelch domain of Keap1 to Nrf2, thereby preventing the degradation of Nrf2 by Keap1 and activating Nrf2. Currently, several Nrf2 agonists have been identified as belonging to Class I, and two of them are used clinically: dimethyl fumarate (DMF) and omaveloxolone (OMA). These electrophilic Nrf2 agonists have many drawbacks in clinical application, including significant off-target effects, high toxicity, poor patient tolerance, and an extremely high incidence of adverse reactions. Precisely because these agonists can cause serious adverse reactions, DMF and OMA are only used to treat rare diseases: DMF is used to treat relapsing-remitting multiple sclerosis; and OMA is used to treat Friedreich's ataxia. Nrf2 degradation involves various chronic diseases affecting multiple systems, and these two drugs are currently not used to treat these chronic diseases due to their high incidence of adverse reactions. Currently, activating Nrf2 by directly inhibiting the interaction between Keap1 and Nrf2 through a second pathway is a new research direction for Nrf2 agonists.Several Keap1 and Nrf2 interaction inhibitors (referred to as Protein-Protein interaction inhibitors, PPIs) have been identified in the literature to activate Nrf2. However, these PPIs are still in the laboratory development stage, and none have yet entered clinical trials; there is still a long way to go before final clinical application. Furthermore, these PPIs generally have large molecular weights, making it difficult for them to cross the blood-brain barrier and enter the central nervous system to treat Nrf2-related central nervous system diseases. Finding a PPI for Nrf2 and Keap1 that has entered clinical development has become a new and urgent problem to be solved. Summary of the Invention:

[0003] (a) Technical problems to be solved

[0004] To address the shortcomings of existing technologies, this invention provides the application of bitopertin as a direct activator of Nrf2. Experiments have demonstrated that bitopertin can activate Nrf2 to treat Nrf2-related chronic diseases, solving the problem that existing PPI inhibitors are difficult to penetrate the blood-brain barrier and enter the central nervous system to treat Nrf2-related diseases, thus affecting the treatment effect.

[0005] (II) Technical Solution

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] Bitopertin is used as a direct activator of Nrf2. Bitopertin has the CAS number 845614-11-1 and the molecular formula C21H20F7N3O4S.

[0008] Furthermore, the bitopertin activates Nrf2 by inhibiting the binding of the Kelch domain of Keap1 to Nrf2, thereby preventing the degradation of Nrf2 by Keap1.

[0009] Furthermore, Bitopertin increases the intracellular protein level of Nrf2 by inhibiting the binding of Keap1 to Nrf2, reducing Nrf2 ubiquitination, and increasing its stability.

[0010] This invention also provides the use of bitopertin as an Nrf2 activator in the preparation of drugs for treating chronic diseases.

[0011] Furthermore, the drug is used to inhibit oxidative stress and inflammatory responses caused by chronic diseases.

[0012] Furthermore, the chronic disease is a disease related to increased Nrf2 ubiquitination and degradation, and insufficient expression.

[0013] Furthermore, the chronic diseases mentioned are osteoporosis and other chronic diseases caused by insufficient Nrf2 expression, including but not limited to chronic respiratory diseases, chronic cardiovascular diseases, and chronic obstructive pulmonary disease.

[0014] Bitopertin is a potent, non-competitive glycine reuptake inhibitor that inhibits glycine uptake, with an IC50 of 25 nM. Its Chinese name is (S)-[4-(3-fluoro-5-trifluoromethylpyridin-2-yl)piperazin-1-yl][5-(methanesulfonyl)-2-(2,2,2-trifluoro-1-methylethoxy)phenyl] ketone.

[0015] As an Nrf2 agonist, Bitopertin has a more pronounced effect than other Nrf2 agonists, does not affect liver function, and has higher biocompatibility. It is a promising Nrf2 agonist for clinical use and has greater advantages in treating various Nrf2-related diseases, showing good prospects for clinical application.

[0016] (III) Beneficial Effects

[0017] The beneficial effects of this invention are:

[0018] 1. This invention proposes that bitopertin, as a PPI inhibitor, has a low molecular weight and can penetrate the blood-brain barrier, thus having an advantage in the treatment of central nervous system diseases.

[0019] 2. This invention provides a novel PPI inhibitor, bitopertin, that inhibits the binding of Keap1 to Nrf2. Bitopertin can be used as an Nrf2 activator to treat various Nrf2-related chronic diseases. This invention uses osteoporosis as an example, but it is also applicable to other diseases caused by Nrf2 deficiency. This invention discovers a new use for bitopertin as an Nrf2 treatment for these diseases.

[0020] 3. This invention verifies through animal model experiments that Bitpertin can activate Nrf2, and verifies through molecular simulation and surface ion resonance (SPR) technology that Bitpertin can directly bind to Keap1. Its biosafety has been clinically verified in humans, showing good safety, extremely low incidence of adverse reactions, and promising clinical application prospects. Attached image description:

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below.

[0022] Figure 1 The molecular structural formula of bitoeprtin;

[0023] Figure 2 Effects of Bitopertin on bone mass in ovariectomized mice: After ovariectomy (OVX), mice showed significant bone loss, while both low and high concentrations of Bitopertin increased bone mass in OVX mice.

[0024] Figure 3 The effect of Bitopertin on the trabecular bone volume (BV / TV) ratio in ovariectomized mice: BV / TV is the ratio of bone volume to total tissue volume; a higher value indicates more bone mass. After ovariectomy (OVX), the BV / TV ratio in mice decreased significantly, while both low and high concentrations of Bitopertin increased the BV / TV ratio.

[0025] Figure 4 The effect of Bitopertin on trabecular bone parameters (Tb.N) in ovariectomized mice: Tb.N represents the number of trabecular bones, with a higher value indicating more trabecular bone. After ovariectomy (OVX), Tb.N in mice was significantly reduced, while both low and high concentrations of Bitopertin increased Tb.N.

[0026] Figure 5 The effect of Bitopertin on trabecular bone parameters (Tb.Th) in ovariectomized mice: Tb.Th is the thickness of trabecular bone, and a higher value indicates more trabecular bone. After ovariectomy (OVX), Tb.Th in mice was significantly reduced, while both low and high concentrations of Bitopertin increased Tb.Th.

[0027] Figure 6 The effect of Bitopertin on trabecular bone parameters (Tb.Sp) in ovariectomized mice: Tb.Sp represents trabecular bone separation, and a higher value indicates more porous trabecular bone. After ovariectomy (OVX), Tb.Sp in mice increased significantly, while both low and high concentrations of Bitopertin decreased Tb.Sp, indicating increased trabecular bone density.

[0028] Figure 7The effect of Bitopertin on serum bone resorption marker CTX in ovariectomized mice: CTX is a marker of osteoclast osteolysis, and higher serum CTX levels indicate stronger osteoclast bone resorption activity. After ovariectomy (OVX), serum CTX levels in mice increased significantly, while both low and high concentrations of Bitopertin decreased CTX levels, indicating that Bitopertin inhibited osteoclast bone resorption activity.

[0029] Figure 8 Effects of Bitopertin on osteoclast formation in the femoral tissue of ovariectomized mice: TRAP staining showed that osteoclast formation was significantly increased in the OVX-treated femoral tissue compared with the sham group, while both low and high concentrations of Bitopertin reduced osteoclast formation.

[0030] Figure 9 The effect of Bitopertin on the number of osteoclasts in bone tissue: The OsteoMeasure software showed that, compared with the sham group, the number of osteoclasts in the OVX femoral tissue was significantly increased, while both low and high concentrations of Bitopertin could reduce the number of osteoclasts in the tissue, indicating that osteoclasts were inhibited in vivo after drug treatment.

[0031] Figure 10 The effect of Bitopertin on osteoclast surface area in bone tissue: Quantitative analysis using OsteoMeasure software showed that, compared with the sham group, the surface area of ​​osteoclasts in the small number of femoral tissues of OVX was increased, indicating an increase in the bone resorption area. Both low and high concentrations of Bitopertin could reduce the surface area of ​​osteoclasts in the tissue, resulting in a significant decrease in the bone resorption area.

[0032] Figure 11 The effect of Bitopertin on osteoclast differentiation in vitro: After RANKL stimulation, osteoclast differentiation was obvious, while treatment with various concentrations (5-15 μM) of Bitopertin significantly inhibited osteoclast differentiation. The 15 μM concentration of Bitopertin could completely inhibit osteoclast differentiation.

[0033] Figure 12 Effects of Bitopertin on the expression of osteoclast marker genes in vitro: RANKL stimulation significantly increased the expression of osteoclast marker genes such as Mmp9, Ctsk, and Trap, while 15 μM Bitopertin significantly inhibited the expression of these marker genes.

[0034] Figure 13Bitopertin does not affect the mRNA transcription level of Nrf2: After treatment of bone marrow macrophages with osteoclast differentiation factor RANKL, the mRNA expression level of Nrf2 decreased significantly, while the mRNA level after treatment with Bitopertin did not increase significantly, indicating that Bitopertin affects the expression level of Nrf2 at the post-translational level.

[0035] Figure 14 Bitopertin significantly increased Nrf2 protein expression levels: Bitopertin treatment promoted Nrf2 protein expression levels both with and without osteoclast-inducing factor RNAKL, indicating that Bitopertin is a potent Nrf2 agonist.

[0036] Figure 15 Bitopertin significantly reduced Nrf2 ubiquitination levels: Under oxidative stress, Nrf2 is bound in cells by Keap1 and then degraded via ubiquitination. RANKL stimulation resulted in higher Nrf2 ubiquitination levels and increased degradation, while Keap1 treatment significantly reduced Nrf2 ubiquitination levels and inhibited its degradation.

[0037] Figure 16 Bitopertin enhances Nrf2 stability: Under oxidative stress, Nrf2 protein stability decreases, and ubiquitination and degradation increase. Under RANKL stimulation, Nrf2 expression gradually decreases over time, while after Bitopertin treatment, Nrf2 levels increase at the same time point, indicating that Bitopertin can increase Nrf2 protein stability.

[0038] Figure 17 Bitopertin inhibits the binding of Nrf2 and Keap1: Under oxidative stress, Nrf2 is bound to Keap1 in cells and then degraded via ubiquitination. After treatment with Bitopertin, the binding amount of Keap1 and Nrf2 decreased, indicating that the degradation of Nrf2 by Keap1 was inhibited by Bitopertin.

[0039] Figure 18 Molecular simulations show that Bitopertin can bind to the Kelch domain of Keap1: Molecular docking using Autodockvina shows that Bitopertin can form hydrogen bonds with the 420th and 606th valine residues and the 367th glycine residue of Keap1. These three Keap1 residues are located in the Kelch domain of Keap1, which means that Bitopertin may competitively occupy the Nrf2 binding site in Keap1, thereby inhibiting the interaction between the two proteins.

[0040] Figure 19 A three-dimensional simulation of the binding of Bitopertin to the Kelch domain of Keap1: Bitopertin can directly occupy the Kelch binding pocket of Keap1, which is also the binding site of Keap1 and Nrf2. The binding free energy of Keap1 and Bitopertin is 110.09 kcal / mol, exhibiting a very stable binding.

[0041] Figure 20 SPR analysis showed that Bitopertin can directly bind to Keap1: In the SPR reaction curve, after the addition of Bitopertin, the reaction curve with Keap1 showed a peak, and the peak height increased with increasing Bitopertin concentration, indicating that Bitopertin can bind to Keap1 in a dose-dependent manner. After about 120 s of reaction, the curve gradually decreased, indicating that the binding of Keap1 to Bitopertin is reversible, suggesting that Keap1 and Bitopertin are linked by hydrogen bonds.

[0042] Figure 21 SPR analysis showed that Bitopertin can specifically and directly bind to the Kelch domain of Keap1: the interaction curve of Bitopertin with the Kelch domain of Keap1 showed a clear peak, while the reaction curves with the BTB and Back domains did not show a peak, indicating that Bitopertin exhibits specific binding to the Kelch domain of Keap1, but does not bind to the other two domains.

[0043] Figure 22 SPR analysis showed that Bitopertin could inhibit the interaction between Keap1 and Nrf2: after the addition of bitopertin, the peak value of the reaction curve between Nrf2 and Keap1 decreased, and the higher the concentration of bitopertin, the lower the peak value of the curve between Nrf2 and Keap1, indicating that Bitopertin can competitively bind to Keap1 with Nrf2 and prevent Nrf2 from being degraded by Keap1.

[0044] Figure 23 Effects of Bitopertin on osteoclast differentiation in wild-type and Nrf2 knockout mice: Bitopertin effectively inhibited osteoclast differentiation in wild-type mice, but could not inhibit osteoclast differentiation in Nrf2 knockout mice, indicating that the inhibitory effect of Bitopertin on osteoclasts is Nrf2-dependent.

[0045] Figure 24Effects of Bitopertin and other clinical-stage Nrf2 inhibitors on body weight in OVX mice: Mice in the Omaveloxolone group experienced severe weight loss after treatment with three Nrf2 agonists. Mice in the DMF and Bitopertin groups gradually increased their body weight from their initial values.

[0046] Figure 25 Effects of Bitopertin and other clinical-stage Nrf2 inhibitors on bone mass in OVX mice: Omaveloxolone treatment did not significantly improve bone mass in OVX mice, while DMF and Bitopertin treatment significantly increased bone mass, although the increase in bone mass was less pronounced in the DMF group than in the Bitopertin group. Bone mass in the Bitopertin group was comparable to that in the zoledronic acid group.

[0047] Figure 26 The effects of Bitopertin and other clinical-stage Nrf2 inhibitors on alanine aminotransferase (ALT) in mice: ALT levels in mice treated with Omaveloxolone and DMF were significantly higher than those in the control group, while liver function in mice treated with Bitopertin were comparable to those in the control group. Bitopertin had no effect on liver function and had better biosafety.

[0048] Figure 27 The effects of Bitopertin and other clinical-stage Nrf2 inhibitors on aspartate aminotransferase (AST) in mice: The AST levels in mice treated with Omaveloxolone and DMF were significantly higher than those in the control group, while the liver function levels in mice treated with Bitopertin were comparable to those in the control group. Bitopertin had no effect on liver function and had better biosafety.

[0049] Figure 28 Comparison of the incidence of any adverse events in humans between Bitoperitin and other Nrf2 inhibitors in clinical stages: In all clinical trials, the incidence of any adverse events in the Bitoperitin group was lower than that of DMF, Bardoxolone, and Omaveloxolone. These differences were statistically significant after one-way ANOVA.

[0050] Figure 29 Comparison of the incidence of any serious adverse events in humans between Bitoperitin and other Nrf2 inhibitors in clinical stages: In all clinical trials, the incidence of any serious adverse events in the Bitoperitin group was lower than that in DMF, Bardoxolone, and Omaveloxolone. These differences were statistically significant after one-way ANOVA. Detailed implementation method:

[0051] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0052] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0053] Example 1: The alleviating effect of bitopertin on bone loss in ovariectomized mice

[0054] (I) Experimental Materials

[0055] a. C57BL / 6J female mice: These mice were obtained from Beijing Vital River Company and were raised in an SPF environment for 11 weeks before undergoing ovariectomy (OVX) to establish a postmenopausal osteoporosis model.

[0056] b. Bitopertin: Purchased from Selleck, CAS number 845614-11-1. A schematic diagram of its molecular structure is attached. Figure 1 As shown.

[0057] (II) Experimental Methods

[0058] Construction of OVX mice

[0059] Eleven-week-old C57 female mice were anesthetized with sodium pentobarbital and placed in a prone position on a sterile operating table. The fur and skin on the ventral and dorsal sides of the mice were disinfected with povidone-iodine. A small incision was made on the ventral and dorsal side near the spine using ophthalmic scissors. The skin, subcutaneous fascia, and peritoneum were then sequentially cut open to expose the ovary. The ovary was then removed along the blood vessels at the root of the ovary. The skin was subsequently sutured, and the incision surface and fur were disinfected with povidone-iodine. The contralateral ovary was treated in the same way. A sham surgery group was set up as a control group for ovarian removal. The procedure was the same as the OVX surgery, except that the ovary was exposed but not removed; the skin was directly sutured. Mice were in recovery for one week post-surgery until week 12, at which point drug intervention began.

[0060] b. Drug treatment

[0061] Mice were divided into four groups: a sham group, an OVX group, an OVX + low-concentration Bitopertin group (6 mg / kg), and an OVX + high-concentration Bitopertin group (30 mg / kg). The Bitopertin solvent was prepared as follows: 15% dimethyl sulfoxide (DMSO) + 85% sterile water (ddH2O) + 0.03% Tween 80. Administered via gavage, 150 μL each time, 5 times a week, for 6 consecutive weeks. The control group received an equal volume of the drug solvent. Each mouse was numbered and grouped during the treatment period.

[0062] c. Mouse sacrifice and specimen collection

[0063] Following in vivo intervention, mice were euthanized by cervical dislocation, and bilateral femurs were collected from each mouse and fixed with 4% paraformaldehyde. The left femur was used for micro-CT scanning (μCT) for bone morphology measurement and analysis. μCT scan parameters were set to 100 kV and 98 μA, with a resolution of 10 pixels per pixel. Trabecular bone parameters included bone volume / total tissue volume (BV / TV), trabecular bone number (Tb.N), trabecular bone separation (Tb.Sp), and trabecular bone thickness (Tb.Th). Bone parameter analysis and three-dimensional reconstruction were performed using the built-in software of μCT.

[0064] The right femur was used for bone histometry analysis. The right femur of each mouse was decalcified in 10% EDTA solution for 2 weeks. The femur was then embedded in paraffin and sectioned at a thickness of 5 μm. Osteoclast formation within the bone tissue was assessed using TRAP staining. Subsequently, bone histometry analysis of the stained bone sections was performed using OsteoMeasure software, including the number of osteoclasts per square centimeter of bone tissue (N.Oc / B.Pm) and the ratio of osteoclast surface area to bone surface area (Oc.S / BS). Analysis was performed in a bone measurement analysis system (boneometrics). All analyses were performed according to the recommendations of the American Bone and Mineral Research Society Nomenclature Committee.

[0065] d. Detection of CTX, a marker of bone resorption in mouse plasma

[0066] The concentration of CTX in mouse plasma was detected using an enzyme-linked immunosorbent assay (ELISA). Before euthanasia, blood was collected from both orbital veins in EP tubes and allowed to stand at 4°C for 2 hours. The tubes were then centrifuged at 2000 rpm for 30 minutes at 4°C. The supernatant plasma was then collected, and the concentration of CTX was detected using a mouse CTX ELISA kit.

[0067] (III) Experimental Results

[0068] a. Effect of Bitopertin on trabecular bone volume in ovariectomized mice

[0069] As attached Figure 2 As shown, compared to the sham group, the femoral trabecular bone volume in mice in the OVX group was significantly reduced, indicating that mice underwent significant trabecular bone loss after OVX surgery, successfully mimicking the phenotype of postmenopausal osteoporosis. However, after treatment with low and high concentrations of Bitopertin, the bone mass of mice was significantly greater than that in the OVX group, indicating that Bitopertin can alleviate ovariectomy-induced bone loss in mice and has important therapeutic value for postmenopausal osteoporosis.

[0070] b. Effects of Bitopertin on trabecular morphological parameters in ovariectomized mice

[0071] As attached Figures 3-6 As shown, after OVX surgery, the experimental mice had a lower BV / TV ratio compared to the sham group mice. Figure 3 ), Tb.N (attached) Figure 4 ), Tb.Th (attached) Figure 5 The value of ) decreased, while the trabecular separation Tb.Sp value increased (see appendix). Figure 6 These parameters indicate that ovariectomy in mice resulted in decreased trabecular bone mass and number, increased laxity, and a phenotype of osteoporosis. However, treatment with low and high concentrations of Bitopertin significantly increased the values ​​of BV / TV, Tb.N, and Tb.Th in mice, with the high-concentration group showing a more pronounced increase than the low-concentration Bitopertin group. This suggests that Bitopertin can dose-dependently alleviate ovariectomy-induced changes in cancellous bone in mice, reduce OVX-induced bone loss, and has significant therapeutic value for postmenopausal osteoporosis.

[0072] c. Effect of Bitopertin on serum bone resorption marker CTX in ovariectomized mice

[0073] As attached Figure 7 As shown, serum CTX levels in the OVX group mice were significantly higher than those in the sham group, indicating that osteoclast activity in the bone tissue of OVX mice was excessively activated, leading to bone loss. However, after treatment with low and high concentrations of Bitopertin, plasma CTX levels decreased significantly, with statistically significant differences, indicating that Bitopertin can inhibit the excessive activation of osteoclast activity, prevent bone loss, and thus increase bone mass.

[0074] d. Effects of Bitopertin on osteoclast formation in the femur of ovariectomized mice

[0075] As attached Figure 8As shown, bone tissue trap staining revealed a significant increase in osteoclast formation in femoral sections from OVX-treated mice, indicating excessive osteoclast formation in the femur after the loss of estrogen protection in the OVX-treated mice. Treatment with low and high concentrations of Bitopertin reduced osteoclast formation, suggesting that Bitopertin can inhibit osteoclast formation in mouse bone tissue. This result is also supported by osteoclast cytometry data in bone tissue. (See attached image) Figure 9 and attached Figure 10 As shown, the N.Oc / B.Pm and Oc.S / BS values ​​of femoral sections from OVX mice were significantly increased, indicating an increase in the number and volume of osteoclasts. Low and high concentrations of Bitopertin could reduce the N.Oc / B.Pm and Oc.S / BS values, further confirming that Bitopertin can inhibit the excessive formation of osteoclasts in vivo.

[0076] (IV) Experimental Conclusions

[0077] Experimental results showed that Bitopertin treatment increased the number, volume, and density of trabeculae in ovariectomized mice and reduced osteoclast formation in the femoral tissue, thereby preventing bone loss induced by ovariectomy. It demonstrated positive therapeutic value for postmenopausal osteoporosis.

[0078] Example 2: Inhibitory effect of Bitopertin on osteoclast differentiation in vitro

[0079] (I) Experimental Materials

[0080] a. Mouse bone marrow macrophages: obtained from the bone marrow cavity of the femur and tibia of 8-week-old C57 mice;

[0081] b. Cytokines: Macrophage colony-stimulating factor MCSF (purchased from Yiqiao Biotechnology Co., Ltd.), osteoclast differentiation inducing factor RANKL (purchased from R&D Company);

[0082] c. TRAP staining kit: obtained from Sigma-Aldrich;

[0083] d. Reverse transcription kit and polymerase chain reaction SYBR mixture: purchased from ABclonal.

[0084] (II) Experimental Methods

[0085] a. Methods for extraction and culture of mouse bone marrow macrophages

[0086] Eight-week-old female C57 mice were euthanized by cervical dislocation and then immersed in 75% alcohol. The mixture was then sterilized under UV light for 15 minutes in a sterile operating table. The femurs and tibias of both mice were then harvested, and the entire bone marrow was flushed out into a 10cm culture dish using a syringe needle with aMEM medium. The aMEM medium was added to a final volume of 8mL, with 30ng / mL MCSF added per mL of medium. The dish was then incubated in a cell culture incubator. After 14 hours, the supernatant was transferred to a new 10cm dish and cultured for another 3 days until all cells adhered. These adherent cells were identified as bone marrow macrophages. 2–4 mL of fresh medium was added daily to support cell growth and proliferation. Once the cells had completely adhered to the bottom of the culture dish, they were digested with trypsin and used for subsequent experiments.

[0087] b. Osteoclast induction and TRAP staining

[0088] After obtaining bone marrow macrophages in 10cm culture dishes, the cells were digested with trypsin and counted. The cells were then seeded in 96-well plates at a density of 2 × 10⁴ cells / well. Osteoclast differentiation was induced for 5 days using MCSF (30 ng / mL) and RANKL (70 ng / mL). Once large, confluent cells were observed, the supernatant was discarded, and the cells were fixed with paraformaldehyde for 15 min. The cells were then stained with a TRAP staining kit and photographed under a microscope. Multinucleated giant cells stained with TRAP were selected as osteoclasts for counting and photographing. The TRAP staining procedure was strictly followed according to the TRAP staining kit instructions.

[0089] c. PCR detection of osteoclast marker gene expression under Bitopertin treatment

[0090] Bone marrow macrophages were seeded at a density of 1×10⁶ cells / well in 6-well plates. Osteoclast differentiation was induced for 3 days using MCSF (30 ng / mL) and RANKL (70 ng / mL). A control group and a Bitopertin (15 μM) treatment group were included. After the intervention, the culture medium was discarded, and 1 mL of TRiZol was added to each well to lyse and digest the cells. The cells were then collected in 1.5 mL centrifuge tubes, and RNA extraction, reverse transcription, and PCR were performed according to the following steps:

[0091] (1) Add 200uL of chloroform to each tube, shake to mix, let stand for 3min, and then centrifuge at 12000rpm for 15min in a centrifuge at 4℃.

[0092] (2) Transfer 450uL of supernatant to another new centrifuge tube and add an equal volume of isopropanol. Invert the tube 6 to 8 times and let it stand at room temperature for 10 minutes.

[0093] (3) Centrifuge at 12,000 rpm for 10 min in a centrifuge at 4℃. At this time, white RNA precipitates can be seen at the bottom of the centrifuge tube.

[0094] (4) After removing the isopropanol, add 500 μL of 75% ethanol solution to each and invert 6 to 8 times.

[0095] (5) Centrifuge at 12,000 rpm for 6 minutes in a centrifuge at 4°C, then remove the ethanol solution and air dry in a fume hood.

[0096] (6) Add 20 μL of RNase-free ddH2O to each tube, shake to mix, and then use Nanodrop to determine the RNA concentration.

[0097] (7) 1 μg of RNA was taken from each sample and reverse transcribed into cDNA using a reverse transcription premix reagent. Primer sequences for osteoclast marker genes were designed, and real-time quantitative PCR was performed using SYBR premix mix to detect the expression levels of osteoclast marker genes in each sample. The primer sequences for each marker gene are shown below:

[0098]

[0099] (III) Experimental Results

[0100] a. Effects of Bitopertin on osteoclast differentiation

[0101] As attached Figure 11 As shown in the image, the cells with multiple deeply stained nuclei in the center of the circle are the induced osteoclasts. The results show that Bitopertin inhibits osteoclast differentiation at concentrations of 5 μM, 10 μM, and 15 μM, with higher concentrations exhibiting stronger inhibitory effects. At 15 μM, osteoclast differentiation was almost completely inhibited. This indicates that Bitopertin is a potent anti-osteoclast drug.

[0102] b. Effects of Bitoperitn on osteoclast marker gene expression

[0103] As attached Figure 12 As shown in the bar chart, RANKL stimulation of osteoclast differentiation significantly induced the expression of osteoclast marker genes such as Mmp9, Ctsk, and Trap. Treatment with 15 μM Bitopertin significantly inhibited the expression of these genes, and statistical analysis showed a statistically significant difference, indicating that Bitopertin can significantly inhibit osteoclast activity in vitro.

[0104] (IV) Experimental Conclusions

[0105] The above experiments show that Bitopertin can significantly inhibit osteoclast differentiation and the expression of osteoclast marker genes.

[0106] Example 3: Verification of the activation effect of Bitopertin on Nrf2 at the cellular level

[0107] (I) Experimental Materials

[0108] a. Mouse bone marrow macrophages: obtained from the bone marrow cavity of the femur and tibia of 8-week-old C57 mice;

[0109] b. Cytokines: Macrophage colony-stimulating factor MCSF (purchased from Yiqiao Biotechnology Co., Ltd.), osteoclast differentiation inducing factor RANKL (purchased from R&D Company);

[0110] c. Protein A&G immunoprecipitation magnetic beads: purchased from CST Company;

[0111] d. Nrf2 antibody, Keap1 antibody, β-actin antibody and ubiquitin antibody: purchased from Wuhan Sanying Biotechnology Co., Ltd.;

[0112] e. Western blot secondary antibody: purchased from Wuhan Aiboteike Company;

[0113] f. ECL chemiluminescent liquid: purchased from Wuhan Boster Biological Technology Co., Ltd.;

[0114] g. Nrf2 full-knock mouse: bred in our laboratory;

[0115] (II) Experimental Methods

[0116] a. Acquisition, culture, and osteoclast-inducing differentiation of bone marrow macrophages

[0117] The steps are the same as those described in Example 2.

[0118] b. Western blot experiment

[0119] After cell culture intervention in 6-well plates, the supernatant was discarded during protein extraction. The cells were then washed once with pre-cooled PBS solution, followed by total protein extraction from the cell samples using RIPA buffer containing 1% protease inhibitor and 1% phosphotransferase inhibitor. The samples were then sonicated and centrifuged at 12000 rpm for 30 min. The supernatant was collected, and protein concentration was measured using a dioctanoic acid (BCA) kit (Boster Biotechnology, Wuhan, China). For Western blotting experiments, 15 μg of protein from each sample was added to the wells of an electrophoresis gel. Proteins were separated by SDS-PAGE (80 V) and transferred to a PVDF membrane (#IPVH00010, Millipore, Boston, USA). The membrane was then blocked for 1 hour at room temperature in 5% BSA containing TBS (0.1% Tween 20) and incubated overnight with primary antibody at 4°C. The next day, the membrane was washed three times and incubated with secondary antibody for 1 hour. The final protein bands were detected using ECL solution (Yeasen, Shanghai, China) and analyzed using the ChemiDoc XRS system (BioRad, California, USA).

[0120] c. Protein-protein interaction and Nrf2 ubiquitination assay

[0121] To investigate the effects of Bitperotin on Nrf2 ubiquitination and the interaction between Nrf2 and Keap1, cells were cultured for 48 hours in 10 cm culture dishes with or without Bitperotin treatment. Cells were then treated with MG132 (10 μM) for 4 hours, after which the culture medium was discarded, and the cells were washed with pre-chilled PBS and lysed in co-precipitation (IP) assay buffer (Beyotime Biotechnology, Beijing, China). After sonication and centrifugation, the total protein content of the lysate was determined, and a lysate containing 500 μg of protein was incubated with A / G magnetic beads (#HY-K0202A, MedChemExpress) at 4°C for 2 hours. The magnetic beads had been pre-washed and incubated with Nrf2 antibody (2 μg) at 4°C for 2 hours. After incubation with the lysis buffer, the magnetic bead-antibody complex formed an antigen-antibody-microbead complex. The complex was washed five times with IP buffer, resuspended in 1×SDS loading buffer, and boiled at 95°C for 5 min. The samples in the SDS loading buffer were then used for Western blot analysis.

[0122] (III) Experimental Results

[0123] a. Effects of Bitopertin on the mRNA transcriptional expression level of Nrf2 in osteoclasts

[0124] As attached Figure 13 As shown, PCR experiments revealed that during osteoclast differentiation, RANKL stimulation significantly decreased the mRNA expression level of Nrf2. However, Bitpertin stimulation significantly increased the mRNA expression level of Nrf2, indicating that Bitpertin affects Nrf2 levels at the post-transcriptional level.

[0125] b. Effect of Bitopertin on Nrf2 protein expression levels in osteoclasts

[0126] As attached Figure 14 As shown, Western blot experiments revealed a significant decrease in Nrf2 protein expression levels upon RANKL stimulation. Bitpertin stimulation significantly increased Nrf2 protein expression levels, and this effect occurred both before and after RANKL administration, indicating that it is a significant Nrf2 agonist in terms of protein level.

[0127] c. Effects of Bitopertin on the ubiquitination level of Nrf2 in osteoclasts

[0128] After ubiquitination, Nrf2 is degraded via the lysosomal pathway. (See attached image) Figure 15 As shown in the Western blot experiment, without Bitopertin, Nrf2 exhibited a high level of ubiquitination after RANKL treatment, indicating that Nrf2 was at a high level of degradation. Bitopertin treatment significantly reduced Nrf2 ubiquitination, thereby inhibiting Nrf2 degradation and increasing Nrf2 protein levels.

[0129] d. Effects of Bitopertin on the stability of Nrf2 in osteoclasts

[0130] As attached Figure 16 As shown, after pre-treatment with CHX to inhibit Nrf2 translation, the protein level of Nrf2 gradually decreased over time. However, after treatment with Bitopertin, the level of Nrf2 increased at the same time point, and the protein level did not change much at most of the time points detected, indicating that Bitopertin can increase the stability of Nrf2 and inhibit its degradation.

[0131] e. Effects of Bitopertin on the interaction between Keap1 and Nrf2 in osteoclasts

[0132] Under normal conditions, Keap1 binds to Nrf2 in the cytoplasm of osteoclasts, restricting Nrf2 and promoting its ubiquitination and degradation. (See attached image) Figure 17 As shown, after treatment with Bitopertin, we found that the binding of Keap1, which binds to Nrf2, decreased, indicating that Bitopertin can inhibit the ubiquitination and degradation of Nrf2 by Keap1, thereby increasing the protein level of Nrf2.

[0133] (IV) Experimental Conclusions

[0134] The aforementioned experimental data indicate that Bitopertin can reduce Nrf2 ubiquitination and increase its stability by inhibiting the binding of Keap1 to Nrf2, thereby increasing the intracellular protein level of Nrf2.

[0135] Example 4: Verification of the binding of Bitopertin to the Nrf2 repressor protein Keap1 using molecular simulation and surface ion resonance.

[0136] (I) Experimental Materials

[0137] a. SPR analyte Bitopertin: manufactured by Selleck;

[0138] b. Recombinant human Keap1 protein, Keap1 BTB, Kelch, and Back domain protein sequences: purified from E. coli expression;

[0139] c. Recombinant human Nrf2 protein: derived from expression and purification in Escherichia coli;

[0140] d. Biacore T200 Evaluation Software: Provided by Wuhan Yanjin Company;

[0141] e.Nrf2 gene knockout mice: bred by the applicant's laboratory.

[0142] (II) Experimental Methods

[0143] a. Surface plasmon resonance (SPR) analysis

[0144] SPR analysis was performed on a Biacore T200 system (Cytiva) equipped with the S-series sensor chip CM5. In short, ligand proteins or peptides (Keap1 protein, Keap1 Kelch domain, Keap1 BTB domain, or Keap1 Back domain) were covalently immobilized onto the CM5 chip via amino acid residues in immobilization buffer. Subsequently, different concentrations of Bitopertin were diluted in analyte buffer and injected into the flow channel to detect the interaction between Keap1 and Bitopertin. The interaction phases included an association phase of 120 s and a dissociation phase of 300 s. Data were analyzed in the Biacore T200 evaluation software (Cytiva).

[0145] b. Recombinant protein construction

[0146] The various ligands used for SPR analysis (Keap1 protein, Keap1 Kelch domain, Keap1 BTB domain, or Keap1 Back domain) were primarily purified through expression in *E. coli*. The Nrf2 recombinant protein was obtained directly from the supplier.

[0147] c. Molecular docking and visualization software

[0148] Autodock vina, PyMoL, and Maestro 13.5 were all obtained through online downloads or academic channels.

[0149] (III) Experimental Results

[0150] a. Molecular simulations predict that Bitopertin can bind with high affinity to Keap1.

[0151] Molecular simulations of Keap1 and Bitoperitn were performed using Autodock Vina. (See attached image.) Figure 18 As shown, the docking results indicate that Bitopertin can form hydrogen bonds with the 420th and 606th valine residues and the 367th glycine residue of Keap1. These three Keap1 residues are located in the Kelch domain, which directly binds to Nrf2. This suggests that Bitopertin may competitively occupy the Nrf2 binding site in Keap1, thereby inhibiting the interaction between the two proteins. A three-dimensional simulation of Keap1 binding to Bitopertin is attached. Figure 19 As shown, the binding free energy of the two is -10.09 kcal / mol (it is generally believed that a binding energy below -7 kcal / mol is a very stable combination).

[0152] b. SPR experiments confirmed that Bitopertin can bind with high affinity to Keap1.

[0153] To further verify that Bitopertin can bind to Keap1, SPR analysis was performed. Results are attached. Figure 20 As shown in the SPR reaction curve, the addition of Bitopertin resulted in a peak at the Keap1 reaction curve, and the peak height increased with increasing Bitopertin concentration, indicating that Bitopertin can bind to Keap1 in a dose-dependent manner. After approximately 120 seconds of reaction, the curve gradually decreased, indicating that the binding of Keap1 to Bitopertin is reversible. This further confirms the results from molecular simulations, namely that Keap1 and Bitopertin are bound together by hydrogen bonds, a binding method different from traditional covalent binding. This effect on Nrf2 protein levels is achieved by directly inhibiting the binding between Keap1 and Nrf2.

[0154] c. SPR experiments confirmed that Bitopertin can specifically bind to the Kelch domain of Keap1.

[0155] To investigate which domain of Keap1 bitopertin binds to, bitopertin was immobilized on a CM5 chip, and the Kelch, Back, and BTB domains of Keap1 were expressed using *E. coli*. These domain peptides were then used as reactants in a sputter-reduction (SPR) experiment. The results are attached. Figure 21 As shown, the interaction curve between Bitopertin and the Kelch domain of Keap1 shows a clear peak, while the curves with the other two domains do not. This indicates that Bitopertin exhibits specific binding with the Kelch domain of Keap1, but not with the other two domains. The Kelch domain is also the domain in which Keap1 interacts with Nrf2.

[0156] d. SPR experiments confirmed that Bitopertin can interfere with the interaction between Keap1 and Nrf2.

[0157] To further verify that Bitopertin can reduce the binding of Keap1 and Nrf2, Keap1 was immobilized on a CM5 chip, and SPR experiments were performed using recombinant Nrf2 protein as a reactant. Different concentrations of Bitopertin were added to the reaction system. The results are attached. Figure 22 As shown, the binding affinity between Nrf2 and Keap1 decreases with the addition of Bitopertin, and the higher the concentration of Bitopertin, the worse the binding affinity between Nrf2 and Keap1. This indicates that Bitopertin can competitively bind to Keap1, thereby preventing Keap1 from binding to Nrf2 and ubiquitinizing and degrading Nrf2.

[0158] e. Bitopertin inhibits osteoclast differentiation by activating Nrf2.

[0159] To verify that bitoperin inhibits osteoclast differentiation by activating Nrf2, osteoclast differentiation was induced in wild-type mice and Nrf2 knockout mice, respectively, and bitoperin was administered during induction. The effects of bitoperin on osteoclasts in both mouse types were observed. The results are attached. Figure 23 As shown, bitopertin significantly inhibited osteoclast differentiation in wild-type mice. However, in Nrf2 mice, the inhibitory effect of bitopertin on osteoclast differentiation disappeared. This indicates that the inhibitory effect of bitopertin on osteoclast differentiation is Nrf2-dependent.

[0160] (IV) Experimental Conclusions

[0161] The aforementioned experimental data indicate that Bitopertin can specifically bind to the Kelch domain of Keap1, thereby reducing the binding of Nrf2 to Keap1 and preventing Keap1-mediated degradation of Nrf2.

[0162] Example 5: Comparison of the efficacy and biosafety of Bitopertin with other clinical electrophilic Nrf2 agonists in a mouse model

[0163] (I) Experimental Materials

[0164] a. C57BL / 6J female mice: obtained from Beijing Vital River Company, fed in SPF environment for 11 weeks and then underwent ovariectomy (OVX) to establish a postmenopausal osteoporosis model;

[0165] b. Bitopertin, purchased from Selleck, CAS number 845614-11-1, molecular structure diagram is attached. Figure 1 As shown.

[0166] c. Dimethyl fumarate (DMF): purchased from Selleck, CAS number 624-49-7.

[0167] d. Omaveloxolone (RTA-408): Purchased from Selleck, CAS number 1474034-05-3.

[0168] (II) Experimental Methods

[0169] a. Drug treatment

[0170] Mice were divided into a control group, a Bitopertin (6 mg / kg) group, a DMF (50 mg / kg) group, and an Omaveloxolone (30 mg / kg) group. These drug dosages were derived from the most commonly used dosages in clinical trials. All mice underwent ovariectomy. The drug solution for each group was prepared as follows: 15% dimethyl sulfoxide (DMSO) + 85% sterile water (ddH2O) + 0.03% Tween 80. Administration was via gavage, 5 times a week, with a total volume of 150 μL each time, for 6 consecutive weeks. The control group received the same volume of drug solution. Mouse weight was measured and recorded weekly during the treatment period. The results were compared with those of the classic anti-osteoporosis drug zoledronic acid sodium.

[0171] b. Mouse sacrifice and specimen collection

[0172] Following in vivo intervention, mice were euthanized by cervical dislocation, and bilateral femurs were collected from each mouse and fixed with 4% paraformaldehyde. The left femur was used for micro-CT scanning (μCT) for bone morphology measurement and analysis. μCT scan parameters were set to 100 kV and 98 μA, with a resolution of 10 pixels per pixel. Trabecular bone parameters included bone volume / tissue volume (BV / TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th). Bone parameter analysis and three-dimensional reconstruction were performed using the built-in software of μCT.

[0173] c. Mouse blood biochemical liver function tests

[0174] Before euthanizing the mice, bilateral orbital venous blood was collected in EP tubes and allowed to stand at 4°C for 2 hours. The tubes were then centrifuged at 2000 rpm for 30 minutes using a 4°C centrifuge. The supernatant plasma was then collected and sent to Wuhan Saiweier Company for the detection of mouse blood biochemical liver function indicators (ALT, AST).

[0175] (III) Experimental Results

[0176] a. Effects of three clinical-stage Nrf2 inhibitors on body weight in OVX mice

[0177] The results are attached. Figure 24 As shown, after treatment with the three Nrf2 agonists, mice in the Omaveloxolone group experienced severe weight loss, with their weight at various time points even falling below their initial weight, indicating that Omaveloxolone has a significant effect on body weight. Mice in the DMF and Bitopertin groups experienced only a slight decrease in body weight compared to the control group. However, their body weight still increased compared to their initial weight, indicating that these two drugs do not cause excessive weight depletion.

[0178] b. Effects of three clinical-stage Nrf2 inhibitors on femoral bone mass in OVX mice

[0179] The results are attached. Figure 25 As shown, treatment with omeveloxolone did not significantly improve bone mass in OVX mice, while treatment with DMF and bitopertin significantly increased bone mass. However, the degree of bone mass increase in the DMF group was lower than that in the bitopertin group. The bone mass in the bitopertin group was comparable to that in the zoledronic acid sodium group, indicating that bitopertin has a greater advantage than other Nrf2 agonists in improving bone mass in postmenopausal mice.

[0180] c. Effects of three clinical-stage Nrf2 inhibitors on liver function in OVX mice

[0181] The results are attached. Figure 26 and 27 As shown, the liver enzyme levels in mice treated with Omaveloxolone and DMF were significantly higher than those in the control group, while the liver function levels in mice treated with Bitopertin were comparable to those in the control group. This indicates that compared with the other two Nrf2 agonists, Bitopertin has no effect on liver function and has better biocompatibility.

[0182] (IV) Experimental Conclusions

[0183] Bitopertin shows a more significant effect than other Nrf2 agonists in improving bone loss due to postmenopausal osteoporosis, without affecting liver function and exhibiting higher biocompatibility. It is a promising clinical Nrf2 agonist with greater advantages in treating various Nrf2-related diseases.

[0184] Example 6: Comparison of the biosafety of Bitopertin with other clinically electrophilic Nrf2 agonists in human subjects

[0185] (I) Analysis of Materials

[0186] a. Literature related to clinical trials of Bitopertin, DMF, and Omaveloxolone in the PubMed database.

[0187] (II) Analytical Methods

[0188] a. This study primarily summarizes adverse reaction data from published clinical trials of Bitopertin, DMF, and Omaveloxolone, statistically analyzing the incidence of any adverse reaction and the incidence of serious adverse reactions, and comparing the significance of these differences using one-way ANOVA. Bardoxolone is also an oral electrophilic Nrf2 agonist evaluated in multiple clinical trials, but due to its serious adverse reactions, it has not yet been approved for clinical use. Relevant clinical data for Bardoxolone were also included in this comparison.

[0189] (III) Analysis Results

[0190] a. Differences in the incidence of adverse reactions among Bitopertin, DMF, and Omaveloxolone in human trials

[0191] The results are attached. Figure 28 and 29 As shown, in all clinical trials, the incidence of any adverse reactions and any serious adverse reactions caused by Bitoperitn was lower than that caused by DMF, Bardoxolone, and Omaveloxolone. These differences were statistically significant after one-way ANOVA.

[0192] (IV) Analytical Conclusions

[0193] Bitoperitn demonstrated a higher clinical safety profile compared to DMF, Bardoxolone, and Omaveloxolone in human subjects. It also showed advantages in treating Nrf2-related diseases.

[0194] In summary, this invention provides an application of an NRF2 agonist in the preparation of drugs for treating chronic diseases. The Nrf2 agonist is bitopertin. Experiments have shown that bitopertin can increase intracellular Nrf2 protein levels by inhibiting the binding of Keap1 to Nrf2, reducing Nrf2 ubiquitination, and increasing its stability. This activates Nrf2 to treat Nrf2-related chronic diseases. Furthermore, bitopertin exhibits a more pronounced effect than other Nrf2 agonists, does not affect liver function, and has higher biocompatibility. It is a promising clinical Nrf2 agonist with significant advantages in treating various Nrf2-related diseases and shows good prospects for clinical application.

[0195] Finally, it should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Furthermore, after reading the technical content of this invention, those skilled in the art can make various modifications, alterations, or variations to the invention, and all such equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. The application of bitopertin as a direct Nrf2 activator in the preparation of drugs for treating osteoporosis, wherein the CAS number of bitopertin is 845614-11-1 and the molecular formula is C21H20F7N3O4S.

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