Application of a BTK PROTAC compound in the preparation of drugs for treating sclerosis

Abstract

The application relates to the field of biological medicine, and in particular to application of a BTK PROTAC compound in preparation of a medicine for treating sclerosis, wherein the medicine takes a BTK PROTAC compound GXH023 as a main active ingredient and is used for treating sclerosis; the BTK PROTAC compound GXH023 has the structure of formula I or a pharmaceutically acceptable salt thereof: (I). The application can solve the problems of poor water solubility and difficulty in penetrating the blood-brain barrier of the BTK PROTAC compound in the prior art, and can provide an efficient and safe treatment strategy for multiple sclerosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the application of a BTK PROTAC compound in the preparation of drugs for treating sclerosis. Background Technology

[0002] Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system mediated by an autoimmune response, characterized by recurrent episodes of neurological dysfunction. In the pathological process of MS, peripheral immune cells (including T cells, B cells, and dendritic cells) are abnormally activated and cross the blood-brain barrier to infiltrate the CNS, triggering an inflammatory cascade that leads to oligodendrocyte damage and demyelination, ultimately causing axonal degeneration and irreversible neurological impairment. Currently, disease-modifying therapy (DMT) is commonly used for MS. Approved DMT drugs include interferon beta, glatiramer acetate, fingolimod, and oligrizumab. However, these drugs generally have limitations such as increased risk of infection, risk of progressive multifocal leukoencephalopathy (PML), the need for frequent infusions, and uncertain long-term safety.

[0003] Bruton's tyrosine kinase (BTK) is a member of the non-receptor tyrosine kinase family of tyrosine kinases (Tec). Besides playing a crucial regulatory role in the B cell receptor (BCR) signaling pathway, recent studies have found that BTK is also widely expressed in myeloid cells, participating in immune regulation through multiple signaling pathways such as TLR and FcγR. In the pathological environment of MS, BTK plays an important role in the development and progression of the disease by mediating abnormal B cell activation and antibody production, and driving pro-inflammatory polarization of microglia.

[0004] Proteolytic targeting chimeras (PROTACs) are a class of bifunctional small molecules composed of a target protein ligand, an E3 ubiquitin ligase ligand, and a linker. They catalyze the degradation of target proteins via the ubiquitin-proteasome pathway. Unlike the "occupation-driven" mechanism of traditional inhibitors, PROTACs employ an "event-driven" mechanism, offering unique advantages such as catalytic recycling, effectiveness at low doses, and the ability to overcome drug resistance mutations.

[0005] Although several BTK PROTAC molecules have been reported, their indications are mainly focused on B-cell malignancies, and there are currently few reports of BTK PROTAC molecules being used to treat multiple sclerosis.

[0006] Furthermore, due to the large molecular weight and strong lipophilicity of BTK PROTAC molecules, the free drug has extremely low solubility in aqueous media, severely limiting its dosability and clinical translation. Moreover, for central nervous system diseases such as MS, systemic administration faces blood-brain barrier limitations, resulting in insufficient effective drug concentrations in the brain and spinal cord, requiring higher systemic doses, thus increasing the risk of systemic toxicity and limiting drug safety. Summary of the Invention

[0007] The purpose of this invention is to provide an application of BTK PROTAC compound in the preparation of drugs for treating sclerosis, so as to solve the technical problems mentioned in the background art. To achieve the above objectives, the present invention adopts the following technical solution: The use of a BTK PROTAC compound in the preparation of a medicament for treating sclerosis, wherein the medicament has BTK PROTAC compound GXH023 as the main active ingredient and is used to treat sclerosis; wherein the BTK PROTAC compound GXH023 has a structure of formula I or a pharmaceutically acceptable salt thereof: (I).

[0008] Furthermore, the drug comprises the following components: a GXH023 solution prepared from BTK PROTAC compound GXH023, a lecithin solution, and a cholesterol solution.

[0009] Furthermore, in the drug, the drug-lipid mass ratio is 1:(31-92).

[0010] Furthermore, the GXH023 solution is prepared by BTK PROTAC compound GXH023 and chloroform, with a concentration of 3.5 mg / mL; the lecithin solution is prepared by lecithin and chloroform, with a concentration of 50 mg / mL; and the cholesterol solution is prepared by cholesterol and chloroform, with a concentration of 5 mg / mL.

[0011] Furthermore, the sclerosis mentioned is multiple sclerosis.

[0012] Furthermore, the drug treats multiple sclerosis by targeting and degrading the BTK protein in dendritic cells.

[0013] Furthermore, the method of administration of the drug includes nasal administration.

[0014] Furthermore, the method for preparing the drug includes the following steps: S1: Add lecithin solution and cholesterol solution to a flask, then add GXH023 solution, and then transfer the flask to an ultrasonic cleaner. Sonicate at 120 W for 2 minutes to mix evenly. S2: Place the eggplant-shaped flask on a rotary evaporator and evaporate it under reduced pressure at 35 ℃ for 15-30 min to remove the organic solvent and obtain a phospholipid film. S3: Take 2 mL of PBS, preheat it to 55 °C in an ultrasonic cleaner, add it to the phospholipid membrane after preheating, and ultrasonically hydrate it for 5-30 min at 120 W in an ultrasonic cleaner to obtain the hydrated liposome suspension. S4: Transfer the hydrated liposome suspension to a 15 mL centrifuge tube and sonicate it at 300 W for 6 min in an ultrasonic homogenizer to homogenize the particle size. S5: Filter and sterilize the liposome suspension after ultrasound to obtain GXH023 liposome drug.

[0015] The advantages of this invention compared to the prior art are as follows: 1. This invention provides a novel use of the BTK PROTAC compound GXH023 in the preparation of drugs for treating multiple sclerosis. GXH023 remodels the immune microenvironment of the central nervous system from the initiation stage of the immune response by degrading BTK protein in dendritic cells. As a PROTAC molecule, GXH023 catalyzes the cyclic degradation of target proteins, achieving efficient clearance of BTK in dendritic cells, thereby downregulating NLRP3 inflammasome expression, inhibiting dendritic cell maturation and activation, and the secretion of pro-inflammatory factors. Simultaneously, it actively induces the transformation of dendritic cells to a tolerant phenotype and promotes IL-10 release. This effect effectively blocks the polarization and proliferation of pathogenic Th1 and Th17 cells and upregulates the level of protective regulatory T cells, thus significantly delaying disease progression, reducing neuroinflammation, and protecting the integrity of myelin sheath structure in multiple sclerosis.

[0016] 2. This invention significantly improves the water solubility and dosage of BTK PROTAC compounds, overcoming the formulation bottleneck of this type of high molecular weight, polycyclic compound. The GXH023 liposomes prepared using the thin-film hydration method exhibit uniform particle size and good stability. The formulation with a drug-to-liposome ratio of 1:31 increases the apparent solubility of GXH023 in PBS by approximately 40 times compared to the free drug, providing a feasible formulation solution for BTK PROTAC compounds.

[0017] 3. This invention prepares GXH023 into a liposomal formulation, enabling efficient enrichment in brain tissue and spinal cord via nasal administration. The actual dose of GXH023-Lip administered nasally is only about 1 / 42 of the dose of free drug injected intraperitoneally, yet it achieves considerable disease improvement, including reduced clinical scores, recovery of motor function, and improvement in neuropathology. This demonstrates that a nasal brain-targeted delivery strategy can significantly improve drug bioavailability and reduce the required effective dose.

[0018] 4. The GXH023-Lip nasal administration of the present invention achieves significant therapeutic effects while reducing systemic drug exposure and increasing local drug concentration in the central nervous system, thereby reducing toxic side effects and providing a safer option for the long-term treatment of multiple sclerosis. Attached Figure Description

[0019] Figure 1 This is a flow cytometry analysis of the effect of GXH023 of the present invention on the activation phenotype of dendritic cells; where A represents CD86. + CD80 + Proportion of double-positive cells; B represents CD86. + MHC II + Percentage of double-positive cells; **P<0.01, ***P<0.001 Figure 2 This is a bar chart showing the effect of GXH023 of the present invention on the secretion levels of inflammatory factors in dendritic cells; where A represents the IL-1β secretion level; B represents the IL-6 secretion level; C represents the TNF-α secretion level; and D represents the IL-12 secretion level; *P<0.05, **P<0.01, ***P<0.001 Figure 3 The present invention GXH023 is effective against PD-L1 in dendritic cells. + Flow cytometry analysis of the effect of expression levels; ***P<0.001; Figure 4 This is an immunofluorescence analysis diagram of the effect of GXH023 of the present invention on PD-L1 expression in dendritic cells; where A is the immunofluorescence staining diagram of PD-L1 in each group; B is the quantitative analysis diagram of fluorescence intensity; *P<0.05, **P<0.01; Figure 5 This is a bar chart showing the effect of GXH023 of the present invention on the level of IL-10 secreted by dendritic cells; *P<0.05, **P<0.01, ***P<0.001; Figure 6 This is a Western blotting analysis of the effects of GXH023 of the present invention on the expression of BTK protein and NLRP3 in dendritic cells; where A is the protein band diagram; B is the gray-scale quantitative analysis diagram of BTK protein; C is the gray-scale quantitative analysis diagram of NLRP3 protein; ****P<0.0001; Figure 7 This is a CFSE flow cytometry analysis of the effect of GXH023 of this invention on T cell proliferation; ***P<0.001, ****P<0.0001; Figure 8This is a flow cytometry analysis of the effect of GXH023 on T cell subset polarization; where A is the proportion of Th1 cells; B is the proportion of Th17 cells; C is the proportion of Treg cells; **P<0.01, ***P<0.001, ****P<0.0001, Figure 9 This is a bar chart showing the effect of GXH023 of the present invention on the cytokine levels in the co-culture supernatant; where A is the IFN-γ content chart; B is the IL-17A content chart; C is the IL-10 content chart; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 10 This is a schematic diagram of the mouse autoimmune encephalomyelitis modeling and drug administration regimen of the present invention; Figure 11 This is a graph showing the effect of GXH023 of the present invention on the clinical symptoms of mice; where A is the curve of clinical score changes; B is the curve of cumulative morbidity; ***P<0.001; Figure 12 This is a bar chart of the effect of the present invention GXH023 on the motor coordination function of mice in a rotarod fatigue experiment; ***P<0.001; Figure 13 This is a schematic diagram of the gait analysis scheme for mice with autoimmune encephalomyelitis according to the present invention; Figure 14 This is an analysis diagram of the effect of the present invention GXH023 on mouse gait parameters; wherein, A is a representative footprint diagram of gait analysis; B is a stride length measurement result diagram; C is a stride width measurement result diagram; D is a footprint overlap distance measurement result diagram; E is a stride distance measurement result diagram; ***P<0.001; Figure 15 This is an HE staining image showing the effect of GXH023 of this invention on mouse spinal cord inflammatory cells; Figure 16 This is a Locke's Fast Blue staining image showing the effect of GXH023 of this invention on demyelination of the mouse spinal cord; Figure 17 This is an MBP immunofluorescence staining image showing the effect of GXH023 of this invention on the structural integrity of the myelin sheath in mouse spinal cord; Figure 18 This is a flow cytometry analysis of the effect of GXH023 of this invention on dendritic cell subsets of the central nervous system in mice with autoimmune encephalomyelitis; where A represents CNS CD45. + CD11c + Dendritic cell proportion diagram; B represents CD86. + MHC II + Proportion of activated dendritic cells; C represents CD86. + CD80 +Proportion of activated dendritic cells; D represents PD-L1 + Proportion of tolerant dendritic cells; **P<0.01, ***P<0.001, ****P<0.0001 Figure 19 This is a flow cytometry analysis of the effect of GXH023 of this invention on T cell subsets of the central nervous system in mice with autoimmune encephalomyelitis; among which, CNS CD3 + CD4 + A) T cell proportion; B) Th1 cell proportion; C) Th17 cell proportion; D) Treg cell proportion; *P<0.05, **P<0.01, ****P<0.0001 Figure 20 This is a distribution diagram of GXH023-Lip after intranasal administration in the central nervous system of mice; where A is a fluorescence imaging diagram; B is a quantitative analysis diagram of fluorescence intensity; *P<0.05, **P<0.01; Figure 21 This is a graph showing the effect of intranasal administration of GXH023-Lip of the present invention on the clinical symptoms of mice; wherein, A is the curve of clinical score change; and B is the curve of cumulative non-morbidity. Figure 22 This is a graph showing the effect of intranasal administration of GXH023-Lip of the present invention on the body weight of mice; Figure 23 This is an analysis of the effect of intranasal administration of GXH023-Lip of the present invention on gait parameters in mice; wherein, A is a representative footprint map of gait analysis; B is a stride length measurement result map; C is a stride width measurement result map; D is a forelimb and hindlimb footprint overlap distance measurement result map; E is a stride distance measurement result map; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Figure 24 This is a bar chart of the rotarod fatigue experiment showing the effect of intranasal administration of GXH023-Lip of the present invention on motor coordination function in mice with autoimmune encephalomyelitis; ***P<0.001, ****P<0.0001. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the invention, and these aspects of the invention can be achieved even without these specific details.

[0021] Example 1 The structure of BTK PROTAC compound GXH023 is shown in formula (I): (I).

[0022] Its chemical name is: 1-(tert-butyl)-N-(4-(2-((1-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-1H-pyrazol-4-yl)amino)-5-fluoropyrimidin-4-yl)-2-methylbenzyl)-1H-1,2,3-triazol-4-carboxamide. The molecular formula is: C 46 H 52 FN 13 O5, the preparation method of which can be found in Example 16 of the patent document with publication number CN 117777102 A.

[0023] This compound is a non-covalent BTK PROTAC molecule based on Spebrutinib, consisting of three parts: (1) a Spebrutinib fragment, a BTK target protein ligand, which binds to the ATP-binding pocket of the BTK protein non-covalently; (2) a pomalidomide fragment, a CRBN E3 ubiquitin ligase ligand, which is responsible for recruiting the CRBN E3 ubiquitin ligase; and (3) a linker connecting the above two parts. GXH023 can simultaneously bridge the BTK protein and the CRBN E3 ubiquitin ligase to form a ternary complex, catalyzing the degradation of the BTK protein through the ubiquitin-proteasome pathway.

[0024] 1. Single-component experiment of BTK PROTAC compound GXH023 1.1 In vitro experiments 1.1.1 Effects of GXH023 on dendritic cell activation and inflammatory function Bone marrow mononuclear cells were aseptically isolated from C57BL / 6 mice. Immature bone marrow-derived dendritic cells (BMDCs) were obtained by combined induction with granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng / mL) and interleukin-4 (IL-4, 10 ng / mL). After pretreatment with different concentrations of GXH023 (0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM) for 2 h, lipopolysaccharide (LPS) and myelin oligodendrocyte glycoprotein (MOG) were added. 35-55 Co-stimulation for 24 h was used to simulate the immune activation state under MS pathological conditions. A blank control group (no stimulant, no drug), a model group (only stimulant, no drug) and a positive control group 1 (Tolebrutinib, 1 μM) were set up.

[0025] CD11c was detected by flow cytometry (FACS). + Gated CD86 + CD80 + and CD86 + MHC II + The proportion of double-positive activated BMDCs, the results are as follows Figure 1 As shown.

[0026] The secretion levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) in cell culture supernatant were detected using multifactor microsphere array (CBA) technology. The results are as follows: Figure 2 As shown.

[0027] from Figure 1 As can be seen from AB, in LPS and MOG 35-55 Under co-stimulation, activated BMDCs (CD86) + CD80 + Double positive and CD86 + MHC II + Double-positive cell population, gated by CD11c + The proportion of activated dendritic cells (DCs) was significantly higher than that of the blank control group, confirming the successful establishment of the in vitro inflammatory activation model. After treatment with GXH023, the proportions of both types of activated BMDCs decreased significantly, showing a clear dose-dependent trend, indicating that GXH023 can effectively inhibit the phenotypic maturation and antigen presentation ability of BMDCs, thereby weakening the potential of dendritic cells (DCs) to activate T cells.

[0028] from Figure 2 As can be seen from the AD, in LPS and MOG 35-55 Under co-stimulation, the release levels of the core pro-inflammatory cytokines driving Th1 and Th17 differentiation, IL-1β, IL-6, TNF-α, and IL-12, were significantly increased compared to the control group. However, after intervention with GXH023, the release levels of all the above-mentioned factors were significantly and dose-dependently inhibited.

[0029] The above results indicate that GXH023 can not only phenotypically block the maturation of BMDCs, downregulate the expression of co-stimulatory molecules and MHCII molecules, and inhibit the secretion of pro-inflammatory cytokines, but also demonstrate that GXH023 has pharmacological activity by inhibiting DC activation and thus blocking pathogenic T cell polarization and inflammatory cascade reactions.

[0030] 1.1.2 Effects of GXH023 on dendritic cell tolerance phenotype BMDC treatment conditions and grouping were the same as in 1.1.1. CD11c was detected using flow cytometry (FACS). + Gated PD-L1 + The proportion of tolerant dendritic cells, the results are as follows Figure 3 As shown.

[0031] Immunofluorescence (IF) technology was used to visualize and analyze PD-L1 expression in BMDC (red: PD-L1, blue: DAPI, scale bar = 20 μm), and fluorescence intensity was quantitatively analyzed. The results are as follows: Figure 4 As shown.

[0032] The secretion level of interleukin-10 (IL-10) in cell culture supernatant was detected using CBA, and the results are as follows: Figure 5 As shown.

[0033] from Figure 3 As can be seen from this, in LPS and MOG 35-55 Under combined stimulation, CD11c in the model group + The positive rate of PD-L1 on the cell surface was 27.8%; and after treatment with different concentrations of GXH023, the expression level of PD-L1 showed a significant dose-dependent upregulation trend.

[0034] from Figure 4 As can be seen in A, the visualization analysis results are consistent with... Figure 3 Consistent with the data, the intensity of red fluorescence signals in the cell membrane and cytoplasm of the GXH023-treated group was significantly enhanced; from Figure 4 As can be seen from B, quantitative analysis shows that the fluorescence intensity of the GXH023-treated group is significantly higher than that of the model group, indicating that GXH023 has a significant advantage in inducing the expression of co-inhibitory molecules.

[0035] from Figure 5 It can be seen that with the increase of GXH023 concentration, the ability of BMDC to secrete the anti-inflammatory cytokine IL-10 is significantly enhanced.

[0036] The above results indicate that GXH023 can not only block the pro-inflammatory maturation of BMDCs, but also actively reshape the immune phenotype of DCs by upregulating the expression of the key negative regulatory molecule PD-L1 and promoting the secretion of the anti-inflammatory factor IL-10, thereby promoting their differentiation into tolerable dendritic cells (tolDCs). This provides theoretical support for explaining the neuroprotective efficacy of GXH023.

[0037] 1.1.3 Effects of GXH023 on the expression of dendritic cell target proteins and downstream molecules BMDC treatment conditions and grouping were the same as in 1.1.1. Total protein was extracted from cells in each group, and the expression levels of BTK protein and the downstream key inflammatory molecule NLRP3 were detected by Western blot, with β-actin used as an internal control. Gray-scale quantitative analysis was performed, and the results are as follows: Figure 6 As shown.

[0038] from Figure 6 As can be seen from the AB, compared with the blank control group and the model group, the expression level of BTK protein in BMDC treated with GXH023 showed a significant concentration-dependent decrease, especially in the highest concentration 1μM treatment group, where the BTK protein band almost completely disappeared, confirming that GXH023 can efficiently and targetedly degrade BTK protein in pathologically activated dendritic cells.

[0039] from Figure 6 As can be seen in C, in LPS and MOG 35-55 Under co-stimulation, the expression level of NLRP3 protein in the model group cells was sharply upregulated; however, after pretreatment with GXH023, the protein expression level of NLRP3 was significantly downregulated with increasing drug concentration. Since the NLRP3 inflammasome, as a core effector of the innate immune response, plays a crucial role in DC-mediated neuroinflammation, its activation is a direct upstream event driving the cleavage, maturation, and release of IL-1β precursors. This data explains the molecular basis of the pro-inflammatory factors observed in 1.1.1, particularly the abrupt decrease in IL-1β secretion dependent on NLRP3 inflammasome cleavage. It also indicates that the anti-inflammatory activity of GXH023 is not merely phenotypic, but rather achieves its effect through a causal chain of targeting protein degradation and subsequently blocking downstream signaling pathways.

[0040] 1.1.4 Effects of GXH023 on T cell responses BMDC treatment conditions and grouping were the same as in 1.1.1. A DC-Naive T cell co-culture system was established: initial CD4+ cells were obtained from the spleens of C57BL / 6 mice via magnetic bead sorting. + T cells, BMDCs and Naive T cells from each group were placed in a container containing anti-CD3 / CD28 antibody and MOG. 35-55 They were cultured in the culture medium for a total of 7 days.

[0041] Naive T cells were pre-stained with CFSE, and the T cell proliferation rate was detected by CFSE dilution method (flow cytometry). The results are as follows: Figure 7 As shown.

[0042] CD4+ co-cultured cells were analyzed by flow cytometry. + Analysis of T cell subsets, including: Th1 cells (CD4+) + IFN-γ +) ratio, Th17 cells (CD4) + IL-17A + The proportion of regulatory T cells (Treg, CD4) + CD25 + FoxP3 + The proportion, the result is as follows Figure 8 As shown.

[0043] The levels of IFN-γ, IL-17A, and IL-10 in the co-culture supernatant were detected using CBA, and the results are as follows: Figure 9 As shown.

[0044] from Figure 7 As can be seen, T cells co-cultured with BMDCs in the model group exhibited a strong proliferative activity (with a significant generational decrease in CFSE signal); however, after co-culturing with BMDCs pretreated with GXH023, the proliferation of T cells was significantly inhibited, and the proportion of cells with no division or low division number increased significantly, indicating that DCs after GXH023 intervention lost their ability to strongly stimulate T cell expansion and antigen presentation.

[0045] from Figure 8 As can be seen from AB, regarding T cell polarization, after co-culturing BMDCs treated with GXH023, Th1 cells (CD4+) secreting pro-inflammatory factors increased. + IFN-γ + ) and Th17 cells (CD4) + IL-17A + The proportions of these two cell types were significantly lower than those in the model group, and these two cell types are the core effector cells that directly attack myelin in MS. Figure 8 As can be seen from C, regulatory T cells (Treg, CD4) with immunosuppressive function + CD25 + FoxP3 + The significant increase in the proportion of GXH023 indicates that it not only disrupts DC-driven pathogenic polarization but also establishes immune tolerance polarization.

[0046] from Figure 9 As can be seen from the AC, in the co-culture supernatant of the GXH023 group, the contents of IFN-γ and IL-17A, which represent pathogenic immunity, decreased significantly with the drug concentration, while the contents of IL-10, which represents immune tolerance, increased significantly.

[0047] The above results indicate that GXH023, when applied to dendritic cells, can effectively block their abnormal activation of adaptive immunity, significantly inhibit the proliferation of antigen-specific T cells, and induce a fundamental reversal of the polarization balance of T cells from pathogenic pro-inflammatory type (Th1 / Th17) to immunosuppressive type (Treg).

[0048] 1.2 In vivo experiments 1.2.1 Construction of an autoimmune encephalomyelitis (EAE) model and in vivo efficacy evaluation of GXH023 EAE modeling: Female C57BL / 6 mice were selected and subcutaneously injected (sc) with MOG on day 0 (Dpi 0). 35-55 Active immunization was performed using an emulsion of peptides and complete Freund's adjuvant (CFA), followed by intravenous (iv) pertussis toxin (PTX) on days 0 and 2 to disrupt the blood-brain barrier and promote immune cell infiltration into the CNS. The modeling protocol was as follows: Figure 10 As shown.

[0049] After modeling, mice were randomly divided into: a blank control group (Control), an EAE model group (EAE), a low-dose GXH023 group (3 mg / kg, ip), a high-dose GXH023 group (10 mg / kg, ip), and a positive control group 2 (Tolebrutinib 10 mg / kg, ip). Drug administration continued after immunization until the experimental endpoint (Dpi 20).

[0050] (1) Clinical symptom assessment: Mice in each group were scored daily using the internationally accepted 0-5 scoring system in a double-blind manner. Clinical score change curves and cumulative morbidity curves were plotted. The results are as follows: Figure 11 As shown.

[0051] (2) Motor function assessment: The mice in each group underwent a rotarod fatigue test (time to fall was measured), and the results are as follows: Figure 12 As shown.

[0052] Gait analysis (footprint test) was performed on mice in each group to quantitatively measure stride length, stride width, overlap distance between forelimb and hindlimb footprints, and intra-step distance. The gait analysis protocol and results are as follows: Figure 13-14 As shown.

[0053] (3) Histopathology: Mice were sacrificed at Dpi 20, and transverse sections of the lumbar edema of the spinal cord were taken. Hematoxylin-eosin (HE) staining was performed to observe inflammatory cell infiltration, Laugher's fast blue (LFB) staining was performed to assess myelin sheath loss, and myelin basic protein (MBP) immunofluorescence staining was performed to observe the structural integrity of the myelin sheath. The results are as follows: Figure 15-17 As shown.

[0054] from Figure 11As can be seen, mice in the EAE model group began to develop symptoms around day 10 post-immunization, with clinical scores rapidly increasing. The GXH023 intervention group significantly delayed disease onset, increased cumulative morbidity, and dose-dependently reduced clinical symptom scores, inhibiting the onset of disease peak. The high-dose group (10 mg / kg) showed clinical improvement comparable to the positive control group (2) at the same dose.

[0055] from Figure 12 As can be seen, the time to fall of the stick in the EAE model group mice was drastically shortened, reflecting severe damage to motor coordination and anti-fatigue ability, while GXH023 significantly prolonged the time to fall of the stick and restored the exercise endurance of the mice.

[0056] from Figure 14 As can be seen, the EAE model group mice exhibited typical demyelinating ataxia gait, manifested in shortened stride, abnormally increased stride width (mice walked in a splits pattern to maintain balance), and disordered overlap distance of forelimb and hindlimb footprints and stride spacing. After treatment with GXH023, all four motor coordination indicators were significantly corrected, recovering to levels close to those of the blank control group, and the overall performance was comparable to that of the positive control group 2.

[0057] from Figure 15 As can be seen from HE staining, large areas of dense inflammatory cell infiltration foci appeared in the spinal cord white matter region of the EAE model group, while the inflammatory infiltration in the GXH023 group was significantly eliminated.

[0058] from Figure 16-17 As can be seen, the EAE model group had severe myelin sheath desquamation and structural damage; the GXH023 group had deep myelin staining and a dense and continuous structure, and the demyelination area was significantly reduced. Its protective effect on myelin sheath integrity is highly consistent with the recovery of motor function mentioned above.

[0059] The above results demonstrate that GXH023 exhibits significant anti-neuroinflammatory and myelin-protective effects in the EAE model, comprehensively improving demyelinating lesions and neurological dysfunction from macroscopic clinical scores and behavioral function to microscopic histopathology. At the same dose, the overall efficacy of GXH023 is comparable to that of the clinically investigated BTK inhibitor Tolebrutinib. Due to the unique mechanistic advantages of PROTAC molecules, such as catalyzing the cyclic degradation of target proteins and achieving sustained efficacy without relying on continuously high drug concentrations, GXH023 has significant clinical translational potential in the long-term treatment of MS.

[0060] 1.2.2 Effects of GXH023 on the immune microenvironment of the central nervous system in EAE mice Based on the modeling and grouping in 1.2.1, mice in each group were sacrificed at the endpoint (Dpi 20). After cardiac perfusion with PBS, brain tissue and the entire spinal cord were rapidly dissected. CNS mononuclear cells (MNCs) were obtained by mechanical grinding and Percoll density gradient centrifugation, and multicolor flow cytometry staining and analysis were performed.

[0061] The testing indicators include: (1) Dendritic cell subsets: CD45 in CNS + CD11c + Total dendritic cells, CD86 + MHC II + Activated dendritic cells, CD86 + CD80 + Activated dendritic cells, CD11c + PD-L1 + The proportion of tolerant dendritic cells (tolDCs), the results are as follows: Figure 18 As shown.

[0062] (2) T cell subsets: CD3 in the CNS + CD4 + Total T cells, CD4 + IFN-γ + Th1 cells, CD4 + IL-17A + Th17 cells, CD25 + FoxP3 + The proportion of Treg cells, the results are as follows Figure 19 As shown.

[0063] from Figure 18 As can be seen from AD, in terms of dendritic cell subsets, compared with the blank control group, the total number of dendritic cells (CD45) infiltrating the brain and spinal cord of EAE model group mice was higher. + CD11c + The proportion of activated DC (CD86) increased significantly. + MHC II + CD86 + CD80 + The proportion of DCs (dendritic cells) also increased significantly, reflecting the large-scale recruitment and abnormal activation of DCs in the CNS under disease conditions. After GXH023 intervention, the infiltration ratio of total DCs and activated DCs in the CNS decreased significantly; at the same time, tolDCs (CD11c) with immune tolerance characteristics increased. + PD-L1 + The proportion of DCs increased significantly in the GXH023 group, confirming that GXH023 can achieve the phenotypic transformation of DCs from pathogenic to tolerant in disease target organs in vivo.

[0064] from Figure 19 As can be seen from AD, in terms of infiltrative T cell subsets, CD3 in the CNS of the EAE model group + CD4 + The proportion of total T cell infiltration was significantly higher than that in the blank control group, but significantly lower after GXH023 treatment. Large amounts of Th1 (CD4+) cells secreting pro-inflammatory factors accumulated in the brain and spinal cord of the EAE model group. + IFN-γ + ) and Th17 (CD4) + IL-17A + Following GXH023 treatment, the CNS infiltration rates of both Th1 and Th17 cells were significantly reduced compared to the model group. Furthermore, Treg cells (CD25) with immunosuppressive function were also observed. + FoxP3 + The proportion of these cells in the brain and spinal cord has increased significantly.

[0065] The above results indicate that GXH023 can achieve systemic remodeling of the immune microenvironment in the CNS lesions of EAE mice: by degrading BTK protein, it inhibits the abnormal activation of DCs and promotes the generation of tolDCs in vivo, thereby weakening antigen presentation and pro-inflammatory signals in the CNS from the source, thus downregulating the infiltration of pathogenic Th1 / Th17 cells and upregulating the level of protective Treg cells.

[0066] In summary, GXH023 efficiently degrades BTK protein in dendritic cells via the PROTAC mechanism, thereby downregulating NLRP3 inflammasome expression. This compound not only inhibits the maturation and activation of BMDCs and the secretion of pro-inflammatory factors, but also actively induces their transformation into tolerant dendritic cells, manifested by upregulation of PD-L1 and increased IL-10 secretion. Dendritic cells treated with GXH023 lose their potent antigen-presenting ability, significantly inhibit naive T cell proliferation, and reverse T cell polarization balance, resulting in a decrease in the proportion of pathogenic Th1 and Th17 cells and an increase in the proportion of protective regulatory T cells.

[0067] In the EAE mouse model, GXH023 significantly delayed disease onset, reduced clinical scores, and effectively improved motor coordination and anti-fatigue ability. Histopathological examination confirmed that it could reduce spinal cord inflammation infiltration, decrease myelin loss, and protect the structural integrity of the myelin sheath, with overall efficacy comparable to that of the BTK inhibitor tolebrutinib at equivalent doses.

[0068] Further analysis of the central nervous system immune microenvironment showed that GXH023 replicated the above mechanism in target organs in vivo: by degrading BTK, it inhibited the abnormal activation of dendritic cells in the CNS and promoted the generation of tolerant dendritic cells, thereby downregulating the infiltration of pathogenic Th1 and Th17 cells and upregulating the level of regulatory T cells.

[0069] These results collectively demonstrate that GXH023 achieves systemic improvement in neuroinflammation and demyelinating lesions in multiple sclerosis by degrading BTK protein in dendritic cells, remodeling the immune microenvironment, and reversing T cell responses. This suggests that its catalytic cyclic degradation mechanism as a PROTAC molecule has significant clinical potential for long-term treatment of multiple sclerosis.

[0070] Example 2 A liposomal drug containing the BTK PROTAC compound GXH023, the preparation method of which includes the following steps: S1: Prepare a lecithin solution with a concentration of 50 mg / mL using lecithin and chloroform; prepare a cholesterol solution with a concentration of 5 mg / mL using cholesterol and chloroform; prepare a GXH023 solution with a concentration of 3.5 mg / mL using BTK PROTAC compound GXH023 and chloroform; add 0.5 mL of lecithin solution and 1 mL of cholesterol solution to a flask, then add 1 mL of GXH023 solution (corresponding to a drug-lipid mass ratio of 1:31), and then transfer the flask to an ultrasonic cleaner and sonicate at 120 W for 2 min to mix thoroughly. S2: Place the eggplant-shaped flask on a rotary evaporator and evaporate it under reduced pressure at 35 ℃ for 25 min to remove the organic solvent and obtain a phospholipid film. S3: Take 2 mL of PBS, preheat it to 55 °C in an ultrasonic cleaner, add it to the phospholipid membrane after preheating, and ultrasonically hydrate it for 20 min at 120 W in an ultrasonic cleaner to obtain the hydrated liposome suspension. S4: Transfer the hydrated liposome suspension to a 15 mL centrifuge tube and sonicate it for 6 min at 300 W in an ultrasonic homogenizer with a pulse mode of 2 s on and 3 s off to homogenize the particle size of the liposome suspension. S5: The sonicated liposome suspension is filtered and sterilized in a clean bench through a 0.22 μm microporous membrane to obtain GXH023 liposomes (GXH023-LiP).

[0071] Example 3 A liposomal drug containing the BTK PROTAC compound GXH023, the preparation method of which includes the following steps: S1: Prepare a lecithin solution with a concentration of 50 mg / mL using lecithin and chloroform; prepare a cholesterol solution with a concentration of 5 mg / mL using cholesterol and chloroform; prepare a GXH023 solution with a concentration of 3.5 mg / mL using BTK PROTAC compound GXH023 and chloroform; add 1 mL of lecithin solution and 1 mL of cholesterol solution to a flask, then add 0.25 mL of GXH023 solution (corresponding to a drug-lipid mass ratio of 1:63), and then transfer the flask to an ultrasonic cleaner and sonicate at 120 W for 2 min to mix thoroughly. S2: Place the eggplant-shaped flask on a rotary evaporator and evaporate it under reduced pressure at 35 ℃ for 20 min to remove the organic solvent and obtain a phospholipid film. S3: Take 2 mL of PBS, preheat it to 55 °C in an ultrasonic cleaner, add it to the phospholipid membrane after preheating, and ultrasonically hydrate it for 10 min at 120 W in an ultrasonic cleaner to obtain the hydrated liposome suspension. S4: Transfer the hydrated liposome suspension to a 15 mL centrifuge tube and sonicate it for 6 min at 300 W in an ultrasonic homogenizer with a pulse mode of 2 s on and 3 s off to homogenize the particle size of the liposome suspension. S5: The sonicated liposome suspension is filtered and sterilized in a clean bench through a 0.22 μm microporous membrane to obtain GXH023 liposomes (GXH023-LiP).

[0072] Example 4 A liposomal drug containing the BTK PROTAC compound GXH023, the preparation method of which includes the following steps: S1: Prepare a lecithin solution with a concentration of 50 mg / mL using lecithin and chloroform; prepare a cholesterol solution with a concentration of 5 mg / mL using cholesterol and chloroform; prepare a GXH023 solution with a concentration of 3.5 mg / mL using BTK PROTAC compound GXH023 and chloroform; add 1 mL of lecithin solution and 1 mL of cholesterol solution to a flask, then add 0.17 mL of GXH023 solution (corresponding to a drug-lipid mass ratio of 1:92), and then transfer the flask to an ultrasonic cleaner and sonicate at 120 W for 2 min to mix thoroughly. S2: Place the eggplant-shaped flask on a rotary evaporator and rotary evaporate under reduced pressure at 35°C for 15 minutes to remove the organic solvent and obtain a phospholipid film. S3: Take 2 mL of PBS, preheat it to 55 °C in an ultrasonic cleaner, add it to the phospholipid membrane after preheating, and ultrasonically hydrate it for 5 min at 120 W in an ultrasonic cleaner to obtain the hydrated liposome suspension. S4: Transfer the hydrated liposome suspension to a 15 mL centrifuge tube and sonicate it for 6 min at 300 W in an ultrasonic homogenizer with a pulse mode of 2 s on and 3 s off to homogenize the particle size of the liposome suspension. S5: The sonicated liposome suspension is filtered and sterilized in a clean bench through a 0.22 μm microporous membrane to obtain GXH023 liposomes (GXH023-LiP).

[0073] 2. Liposome drug experiments containing the BTK PROTAC compound GXH023 2.1 Liposome Properties Experiment The GXH023 liposomes prepared in Examples 2-4 were divided into three groups: GXH023-Lip (D / L 1:31), GXH023-Lip (D / L 1:63), and GXH023-Lip (D / L 1:92) for experiments.

[0074] 2.1.1 Characterization of GXH023-Lip The particle size, PDI and potential of liposomes in the GXH023-Lip (D / L 1:31), GXH023-Lip (D / L 1:63) and GXH023-Lip (D / L 1:92) groups were measured, and the results are shown in Table 1.

[0075] Table 1 Characterization Results As can be seen from Table 1, the particle size of liposomes in each group is below 100 nm, the PDI is less than 0.2, and the potential is around -22 mV. This indicates that the particle size distribution of liposomes in each group is uniform, the formulation quality is good, and it has excellent stability. Moreover, different drug-liposome ratios have no significant effect on the formation, particle size, and potential of liposomes.

[0076] 2.1.2 Apparent Solubility Test of GXH023-Lip The apparent solubility of liposomes in the GXH023-Lip (D / L 1:31), GXH023-Lip (D / L 1:63), and GXH023-Lip (D / L 1:92) groups was tested, and the results are shown in Table 2.

[0077] Table 2. Results of Apparent Solubility Test As shown in Table 2, compared with free GXH023, preparing GXH023 into liposomes can increase the apparent solubility of GXH023 by more than 10 times. Among them, the GXH023-Lip (D / L 1:31) group increased the apparent solubility of GXH023 in PBS by about 40 times compared with the free drug. This indicates that preparing GXH023 into liposomes significantly improves the drugability of the compound. Therefore, GXH023-Lip (D / L 1:31) is the preferred dosage form, and GXH023-Lip (D / L 1:31) will be used for subsequent experiments.

[0078] 2.2 In vivo experiments 2.2.1 Distribution of GXH023-Lip in EAE mice EAE modeling was performed according to method 1.2.1. After the mice entered the peak period of disease (Dpi 10), the free GXH023 suspension and GXH023-Lip (D / L 1:31) were labeled with the near-infrared fluorescent dye DiR and administered via nasal drip.

[0079] Four hours after drug administration, mice were sacrificed, and brain tissue and the entire spinal cord were rapidly isolated. Near-infrared fluorescence signals from the isolated tissues were acquired and quantitatively analyzed using a small animal in vivo optical imaging system. The distribution of the two formulations in the CNS was compared, and the results are as follows: Figure 20 As shown.

[0080] from Figure 20 As can be seen, only weak fluorescence signals were detected in the brain and spinal cord tissues of the free GXH023 group, while the GXH023-Lip group showed significantly enhanced fluorescence enrichment in both brain and spinal cord tissues, indicating that liposome encapsulation can significantly promote the transport and accumulation of GXH023 to the central nervous system via the nasal route.

[0081] 2.2.2 In vivo pharmacodynamic study of GXH023-Lip intranasal administration for the treatment of EAE EAE modeling was performed according to method 1.2.1. Mice were randomly divided into four groups: control group, EAE model group, GXH023-Lip nasal administration group (D / L 1:31, 40 μL / mouse, intranasal instillation), and free GXH023 intraperitoneal injection group (3 mg / kg, ip). Drug administration continued from post-immunization until the experimental endpoint (Dpi 20). Clinical scores were assessed daily and weight changes were recorded. Results are as follows. Figure 21-22 As shown.

[0082] from Figure 21As shown in Figure A, mice in the EAE model group began to develop symptoms around Dpi 10, with clinical scores continuously rising to approximately 3.5-4.0. Both the GXH023-Lip nasal administration group and the free GXH023 intraperitoneal injection group significantly delayed disease progression and reduced clinical scores, with no statistically significant difference between the two groups (P>0.05). Figure 21 As shown in Figure B, the cumulative morbidity curve also indicates that both administration methods significantly delayed disease onset compared to the EAE model group. However, the actual dose of GXH023-Lip nasal administration (approximately 72 μg / kg) was only about 1 / 42 of the intraperitoneal injection dose of free GXH023 (3 mg / kg), yet it achieved comparable efficacy, demonstrating the superior dose advantage of liposome-mediated nasobrain targeted delivery.

[0083] from Figure 22 As can be seen, the weight change trends of mice in the GXH023-Lip nasal administration group and the free GXH023 intraperitoneal injection group during the entire administration period were basically consistent with those of the blank control group, and no significant weight loss was observed, indicating that both administration methods had good tolerability and safety at the experimental dose.

[0084] 2.2.3 Investigation on the effects of GXH023-Lip on motor function in EAE mice Based on the experimental groupings in 2.1.4, gait analysis and rotarod fatigue experiments were performed on each group of mice according to the method in 1.2.1. The results are as follows: Figure 23-24 As shown.

[0085] from Figure 23 As can be seen from A, the EAE model group mice exhibited typical demyelinating ataxia gait. From Figure 23 The BE (Behavioral Emissions) results showed that, compared with the blank control group, the EAE model group mice had significantly shorter stride length, abnormally increased stride width, increased overlap distance of forelimb and hindlimb footprints, and disordered stride spacing. Both the GXH023-Lip nasal administration group and the free GXH023 intraperitoneal injection group significantly corrected the above gait abnormalities, and all four motor coordination indices were significantly improved compared with the EAE model group. There was no significant difference between the two administration methods.

[0086] from Figure 24 As can be seen, the time for dropping the stick in the EAE model group was drastically shorter than that in the blank control group, reflecting severe impairment of motor coordination. The time for dropping the stick in both the GXH023-Lip nasal administration group and the free GXH023 intraperitoneal injection group was significantly longer than that in the EAE model group (P<0.001), and there was no statistically significant difference between the two groups (P>0.05).

[0087] In summary, the GXH023 liposomes prepared by the thin-film hydration method have uniform particle size and distribution, and the formulation with a drug-liposome ratio of 1:31 improves the apparent solubility of the drug by about 40 times compared with the free drug, which significantly improves the water solubility bottleneck of this compound and lays a pharmaceutical foundation for subsequent drug administration.

[0088] In vivo distribution studies have confirmed that GXH023 liposomes can be significantly enriched in brain tissue and spinal cord after nasal administration. Its transport efficiency to the central nervous system is much better than that of free drugs, indicating that liposome encapsulation combined with the nasal route can effectively cross the blood-brain barrier and achieve brain-targeted delivery.

[0089] Furthermore, in the EAE mouse model, GXH023-Lip was administered via the nose at a dose of only about 72 μg / kg, which is only about 1 / 42 of the intraperitoneal injection dose of free GXH023, yet it achieved efficacy comparable to systemic administration, demonstrating that nasal-brain targeted delivery has a superior dose advantage.

[0090] Therefore, intranasal administration of GXH023 liposomes can achieve highly efficient central nervous system drug exposure with extremely low dosage, significantly improving therapeutic efficacy while reducing the potential risks of systemic exposure, providing a highly effective, low-dose and safe formulation for BTK PROTAC treatment of multiple sclerosis.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a BTK PROTAC compound in the preparation of a drug for treating sclerosis, characterized in that: The drug, with BTK PROTAC compound GXH023 as the main active ingredient, is used to treat sclerosis; the sclerosis is multiple sclerosis; the BTK PROTAC compound GXH023 has a structure of formula I or a pharmaceutically acceptable salt thereof: （I）。 2. The application according to claim 1, characterized in that, The drug comprises the following components: a GXH023 solution prepared from BTK PROTAC compound GXH023, a lecithin solution, and a cholesterol solution.

3. The application according to claim 2, characterized in that: The GXH023 solution is prepared by BTK PROTAC compound GXH023 and chloroform, with a concentration of 3.5 mg / mL; the lecithin solution is prepared by lecithin and chloroform, with a concentration of 50 mg / mL; and the cholesterol solution is prepared by cholesterol and chloroform, with a concentration of 5 mg / mL.

4. The application according to claim 3, characterized in that: In the drug, the drug-lipid mass ratio is 1:(31-92).

5. The application according to claim 1, characterized in that: The drug treats multiple sclerosis by targeting and degrading BTK protein in dendritic cells, inhibiting dendritic cell maturation and activation, and inducing their transformation into a tolerance phenotype. This, in turn, blocks pathogenic T cell polarization and upregulates regulatory T cell levels.

6. The application according to claim 1, characterized in that: The drug can be administered via nasal administration.

7. The application according to any one of claims 2-4, characterized in that, The method for preparing the drug includes the following steps: S1: Add lecithin solution and cholesterol solution to a flask, then add GXH023 solution, and then transfer the flask to an ultrasonic cleaner. Sonicate at 120 W for 2 minutes to mix evenly. S2: Place the eggplant-shaped flask on a rotary evaporator and rotary evaporate under reduced pressure at 35 ℃ for 15-30 min to remove the organic solvent and obtain a phospholipid film. S3: Take 2 mL of PBS, preheat it to 55 °C in an ultrasonic cleaner, add it to the phospholipid membrane after preheating, and ultrasonically hydrate it for 5-30 min at 120 W in an ultrasonic cleaner to obtain the hydrated liposome suspension. S4: Transfer the hydrated liposome suspension to a 15 mL centrifuge tube and sonicate it at 300 W for 6 min in an ultrasonic homogenizer to homogenize the particle size. S5: Filter and sterilize the liposome suspension after ultrasound to obtain GXH023 liposome drug.

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