Small molecule ligand for targeted administration, ligand drug conjugate, rheumatoid arthritis treatment drug and application

Abstract

The invention belongs to the technical field of biological medicines, and discloses a small molecule ligand for targeted administration, a ligand medicine conjugate, a rheumatoid arthritis treatment medicine and application. The small molecule ligand for targeted administration provided by the invention takes a structure as shown in a formula (1) as a linker, and meanwhile, a group M1 derived from a fibroblast activating protein inhibitor is introduced, so that the small molecule ligand can be well applied as a drug delivery platform for RA treatment; the molecular biological activity of the anti-rheumatism medicine is kept, and meanwhile, the anti-rheumatism medicine is fully released in an inflammation joint area, so that a better rheumatoid arthritis treatment effect is achieved, and the anti-rheumatism medicine has a good application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a small molecule ligand for targeted drug delivery, a ligand-drug conjugate, and a drug for treating rheumatoid arthritis and their application. Background Art

[0002] Rheumatoid arthritis (RA) is the most common chronic autoimmune disease, which is mainly manifested by multi-joint erosive arthritis and may be accompanied by symptoms of multiple organ systems such as the heart, lungs, and kidneys. Methotrexate (4-amino-10-methylfolate, MTX for short) is a folic acid analog that competitively inhibits dihydrofolate reductase, hinders the conversion of dihydrofolate into tetrahydrofolate, and thus inhibits DNA synthesis and mitosis in cells. MTX can inhibit overactive immune cells and abnormally proliferating fibroblast-like synoviocytes to alleviate the disease symptoms and joint damage progression of RA patients, and is a traditional first-line drug for RA treatment. However, MTX has poor targeting and may also inhibit normal cells in non-inflammatory joint areas, leading to adverse reactions such as gastrointestinal dysfunction and elevated serum liver enzyme indicators, and may even lead to serious adverse reactions such as cell reduction, severe infection, or respiratory failure.

[0003] Therefore, it is of great significance to develop a safer and more effective drug delivery platform for the treatment of RA to achieve the targeted release of MTX in the inflamed joint area and achieve a good therapeutic effect. Summary of the invention

[0004] The first purpose of the present invention is to provide a small molecule ligand for targeted drug delivery. The small molecule ligand for targeted drug delivery can be well used as a drug delivery platform for RA treatment and has good application prospects.

[0005] The second object of the present invention is to provide a ligand-drug conjugate.

[0006] The third object of the present invention is to provide the use of the above-mentioned small molecule ligand and / or ligand-drug conjugate for targeted drug delivery in the preparation of drugs for the treatment of rheumatoid arthritis.

[0007] The fourth object of the present invention is to provide a drug for treating rheumatoid arthritis.

[0008] Specifically, the structure of the small molecule ligand for targeted drug delivery provided by the present invention is as shown in formula (1):

[0009]

[0010] In the formula (1), M1 is a group derived from a fibroblast activation protein α inhibitor.

[0011] Furthermore, the fibroblast activation protein α inhibitor is selected from one or more of FAPI, NTFAPI, UAMC1110, NH2-UAMC1110 and HYINC-FAPI-4.

[0012] Furthermore, in formula (1), the structure of M1 is as shown in formula (2), (3), (4) or (5):

[0013]

[0014] The ligand-drug conjugate provided by the present invention comprises an anti-rheumatic drug molecule and the above-mentioned small molecule ligand for targeted drug delivery connected by chemical bonding.

[0015] Furthermore, the structure of the ligand-drug conjugate is shown in formula (6):

[0016]

[0017] In formula (6), M1 is a group derived from a fibroblast activation protein α inhibitor, and M2 is a group derived from an anti-rheumatic drug molecule.

[0018] Furthermore, the anti-rheumatic drug molecule is selected from one or more of methotrexate, leflunomide, hydroxychloroquine and cyclophosphamide.

[0019] Furthermore, in formula (6), the structure of M2 is shown in formula (7):

[0020]

[0021] The present invention also provides the use of the above-mentioned small molecule ligand and / or ligand-drug conjugate for targeted drug delivery in the preparation of a drug for treating rheumatoid arthritis.

[0022] The rheumatoid arthritis therapeutic drug provided by the present invention comprises the above-mentioned ligand-drug conjugate.

[0023] Beneficial effects:

[0024] The small molecule ligand for targeted drug delivery provided by the present invention uses the structure shown in formula (1) as a linker, and simultaneously introduces a group M1 derived from a fibroblast activation protein α inhibitor, and the * site in formula (1) is bonded to an anti-rheumatic drug molecule to achieve the loading of the anti-rheumatic drug molecule; in the small molecule ligand for targeted drug delivery, the targeting property of the group M1 to the inflamed joint region where fibroblast activation protein α (FAPα) is highly expressed is utilized to locate the anti-rheumatic drug molecule loaded thereon to the inflamed joint region, and the above-mentioned linker contains a high-activity enzyme cleavage site of FAPα, which can be well cleaved by FAPα in the inflamed joint region, thereby achieving its full release while maintaining the biological activity of the anti-rheumatic drug molecule, thereby enabling the anti-rheumatic drug molecule to better play its role in the inflamed joint region, thereby achieving a better therapeutic effect, and having a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the H NMR spectrum of the ligand-drug conjugate provided in the embodiments of the present invention.

[0026] Figure 2 This is a graph showing the experimental results of the MTX release capacity test provided in the embodiments of the present invention.

[0027] Figure 3 This is one of the experimental result diagrams of the FAPα distribution test provided in the embodiment of the present invention.

[0028] Figure 4 This is the second diagram of the experimental results of the FAPα distribution test provided in the embodiment of the present invention.

[0029] Figure 5 This is one of the experimental result diagrams of the MTX drug distribution test provided in the embodiments of the present invention.

[0030] Figure 6 This is the second experimental result diagram of the MTX drug distribution test provided in the embodiment of the present invention.

[0031] Figure 7 FIG3 is the experimental result of the MTX drug distribution test provided in the embodiment of the present invention.

[0032] Figure 8 FIG4 is the experimental result of the MTX drug distribution test provided in the embodiment of the present invention.

[0033] Fig. 9 This is a graph showing the experimental results of a mouse weight test provided in an embodiment of the present invention.

[0034] Fig.10 This is a graph showing the experimental results of a mouse paw thickness test provided in an embodiment of the present invention.

[0035] Fig.11 This is a graph showing the experimental results of the mouse joint inflammation index scoring provided in the examples of the present invention.

[0036] Fig.12 This is one of the experimental result diagrams of the mouse articular cartilage content test provided in the embodiments of the present invention.

[0037] Fig.13 This is the second figure of the experimental results of the mouse articular cartilage content test provided in the embodiments of the present invention.

[0038] Fig.14 This is one of the experimental result diagrams of the mouse Treg cell percentage test provided in the embodiments of the present invention.

[0039] Fig.15 This is the second figure of the experimental results of the mouse Treg cell percentage test provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0040] Based on a profound understanding of the pathogenesis of rheumatoid arthritis and pathological changes related to the inflamed joint area, the inventors of the present invention have conducted extensive and in-depth research and found that using FAPα highly expressed in the inflamed joint area as a target site and using a fibroblast activation protein α inhibitor with specific recognition and binding ability to FAPα as a targeting molecule to construct a drug delivery platform for RA treatment can theoretically achieve targeted delivery of anti-rheumatic drug molecules to the inflamed joint area; however, the inventors found in a large number of experiments that the ligand-drug conjugate obtained by using conventional linkers to bridge the fibroblast activation protein α inhibitor and the anti-rheumatic drug molecule has poor targeting and cannot well achieve the release of the anti-rheumatic drug molecule, and the drug efficacy is low. Based on the problems found, the inventors creatively obtained the small molecule ligand for targeted drug delivery provided by the present invention after further research and a large number of experiments.

[0041] In the present invention, the structure of the small molecule ligand for targeted drug delivery is shown in formula (1):

[0042]

[0043] In formula (1), M1 is a group derived from a fibroblast activation protein α inhibitor. It should be noted that the * site shown in formula (1) refers to the site where the small molecule ligand for targeted drug delivery is chemically bonded to the anti-rheumatic drug molecule.

[0044] In the present invention, the fibroblast activation protein α inhibitor refers to a class of small molecule compounds that can specifically recognize and bind to FAPα, and specific examples thereof include but are not limited to: FAPI (CB Number: CB15849362), NTFAPI (CB Number: CB28056102), UAMC1110 (CB Number: CB83052176), NH2-UAMC1110 (CBNumber: CB48131098) and HYINC-FAPI-4 (CB Number: CB713117819) One or more.

[0045] In some specific embodiments, in formula (1), the structure of M1 is preferably as shown in formula (2), (3), (4) or (5); in this case, the small molecule ligand for targeted drug delivery can better target and localize in the inflamed joint area.

[0046]

[0047] It should be noted that the * site in formulas (2) to (5) refers to the site where the group is chemically bonded to the linker shown in formula (1).

[0048] Based on the targeted delivery and full release capabilities of the above-mentioned ligand-drug conjugates for anti-rheumatic drug molecules, the present invention also provides a ligand-drug conjugate.

[0049] In the present invention, the ligand-drug conjugate comprises an anti-rheumatic drug molecule and the above-mentioned small molecule ligand for targeted drug delivery connected by chemical bonding, and the specific structure is shown in formula (6):

[0050]

[0051] In formula (6), M1 is a group derived from a fibroblast activation protein α inhibitor, and M2 is a group derived from an anti-rheumatic drug molecule.

[0052] In the present invention, the anti-rheumatic drug molecule refers to a class of compounds that can alleviate or treat rheumatoid arthritis, and specific examples thereof include but are not limited to: one or more of methotrexate, leflunomide, hydroxychloroquine and cyclophosphamide.

[0053] In some specific embodiments, in the ligand-drug conjugate, the structure of M1 is preferably as shown in formula (2), and the structure of M2 is preferably as shown in formula (7); in this case, the group M1 can be better targeted to the inflamed joint area and hydrolyzed by FAPα to better achieve the sustained release of anti-rheumatic drug molecules in the inflamed joint area, and the hydrolysis products of the ligand-drug conjugate can cooperate synergistically to achieve a better effect of alleviating the symptoms of rheumatoid arthritis.

[0054]

[0055] It should be noted that the * site in formula (7) refers to the site where the anti-rheumatic drug molecule and the small molecule ligand for targeted drug delivery are chemically bonded.

[0056] Based on the excellent performance of the above-mentioned small molecule ligands and ligand-drug conjugates for targeted drug delivery, the present invention also provides the use of the above-mentioned small molecule ligands and / or ligand-drug conjugates for targeted drug delivery in the preparation of drugs for the treatment of rheumatoid arthritis.

[0057] Based on the potential of the ligand-drug conjugate in the preparation of a drug for treating rheumatoid arthritis, the present invention also provides a drug for treating rheumatoid arthritis. The drug for treating rheumatoid arthritis specifically includes the ligand-drug conjugate.

[0058] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0059] Example

[0060] This example is used to illustrate a ligand-drug conjugate and its related properties, specifically including:

[0061] 1. Acquisition and structure of ligand-drug conjugates

[0062] The ligand-drug conjugate provided in this embodiment has a structure shown in formula (8). The synthesis of the structure was conducted by Chengdu WuXi AppTec New Drug Development Co., Ltd. to obtain the ligand-drug conjugate and its H-NMR spectrum. Figure 1 shown.

[0063]

[0064] 2. MTX Release Capacity of Ligand-Drug Conjugates

[0065] 50 μmol / L fibroblast activation protein α (FAPα, MedChemExpress, catalog number HY-P77934) was added to 50 μmol / L ligand drug conjugate aqueous solution and incubated at 37°C for 6 h. At 0, 2, 4 and 6 h of incubation, samples were taken for HPLC detection of MTX concentration in the solution. The results were as follows: Figure 2 shown.

[0066] Depend on Figure 2The test results shown show that the ligand-drug conjugate provided in Example 1 of the present invention can be effectively hydrolyzed by FAPα to achieve the effect of sufficient release of MTX.

[0067] 3. Drug Efficacy of Ligand-Drug Conjugates

[0068] 1. Construction of mouse model: 8-week-old DBA-1 male mice weighing 18-22 g were used as experimental animals. They were given a 12-h light cycle and free access to food and water at a temperature of 23-25°C and a humidity of 45%-50% for 5 days. The mice were randomly divided into groups and subjected to the following treatments:

[0069] (1) Normal group: Normal saline and an equal volume of Freund's adjuvant (Chondrex. Inc., catalog number 7001) were fully mixed and emulsified, and 0.1 mL was injected subcutaneously at the base of the mouse tail. 0.1 mL was injected again on the 21st day after the injection. 24 hours after the injection, 100 μL of normal saline was injected into the tail vein of the mouse. This was repeated twice a week for three consecutive weeks.

[0070] (2) Model group: Type II collagen (Chondrex. Inc., catalog number 20021) was dissolved in acetic acid to prepare a solution with a concentration of 2 mg / mL, and then thoroughly mixed and emulsified with an equal volume of Freund's adjuvant. 0.1 mL was injected subcutaneously at the base of the mouse tail. 0.1 mL was injected again on the 21st day after injection. The model was successfully established when the mice showed redness and swelling of the paws, agitation, licking of the paws, and lameness 24 hours later. Then, 100 μL of normal saline was injected into the tail vein of the mice twice a week for three consecutive weeks.

[0071] (3) Experimental group: The mice were treated with the same method as the model group to construct a mouse model of rheumatoid arthritis; then 100 μL of drug solution was injected into the tail vein of the mice twice a week for three consecutive weeks. The drug solution included 3 mg / mL FAPI (Nanchang Tanzhen Biotechnology, catalog number p240925), MTX (Shanghai Yuanye Biotechnology Co., Ltd., catalog number S18026) or ligand-drug conjugates.

[0072] 2. Experimental results: (1) Distribution of FAPα: The mice in the normal group and the model group were killed, and the heart, lung, liver, spleen, kidney and left posterior ankle joint were removed. The fur and muscles were removed, washed, fixed, and decalcified. 5 μm thick sagittal sections were made, and FAPα monoclonal antibody (Invitrogen, catalog number PA5-99313) was used to stain the FAPα in the tissues. The sections were washed and observed under a microscope. The results are as follows: Figure 3 and 4 shown.

[0073] Depend on Figure 3 and 4 The test results shown show that FAPα is highly expressed in the inflamed joint areas of rheumatoid arthritis model mice compared to normal mice.

[0074] (2) Drug distribution: Each experimental group used indocyanine green-labeled FAPI, indocyanine green-labeled MTX or indocyanine green-labeled ligand-drug conjugate for the first administration. The fluorescence signal of the left posterior ankle joint of the mice was tested at 10 minutes, 2 hours, 4 hours, 8 hours, 20 hours, 30 hours and 48 hours after administration, and the MTX content in the blood, serum and joints of the mice was tested at 48 hours. The test results are as follows: Figures 5 to 8 shown.

[0075] Depend on Figures 5 to 8 The test results shown show that, compared with the administration of MTX alone, the administration of MTX by ligand-drug conjugates can better deliver MTX to the inflamed joint area and achieve sustained release of MTX over a certain period of time.

[0076] (3) Body weight of mice: Starting from the time of the first administration of type II collagen stimulation, the body weight of mice was measured on days 21, 30, 34, 36, 40, 43, 46, 49, and 50. The results are shown in Table 2. Fig. 9 shown.

[0077] Depend on Fig. 9 The test results shown in the figure show that the administration of FAPI, MTX and ligand-drug conjugates in the experimental group did not have much effect on the body weight of mice.

[0078] (4) Joint inflammation index score of mice: Starting from the time of the first administration of type II collagen stimulation, the paw thickness was measured on days 21, 30, 34, 36, 40, 43, 46, 49 and 5, and the mice were scored for the arthritis index. The test results are shown in Table 1. Fig.10 and 11 shown.

[0079] The criteria for scoring the arthritis index are as follows: 0 points, no swelling or erythema; 1 point, detectable arthritis with mild swelling or some erythema; 2 points, moderate swelling; 3 points, severe swelling of the entire paw including the fingers; 4 points, stiffness or even deformity of the maximum swollen joint.

[0080] Depend on Fig.10 and 11 The test results shown show that, compared with the single administration of FAPI or MTX, the ligand-drug conjugate provided by the embodiment of the present invention was used to treat the rheumatoid arthritis mouse model, and the paw thickness and arthritis index score of the mice were significantly reduced, which has a better effect of alleviating the symptoms of rheumatoid arthritis.

[0081] (5) Cartilage content of mouse joints: Starting from the time of the first stimulation with type II collagen, the cartilage content of the left posterior ankle joint of the mouse was measured by nuclear magnetic resonance imaging on the 50th day. The results are as follows: Fig.12 and 13 shown.

[0082] Depend on Fig.12 and 13 The test results shown show that, compared with the single administration of FAPI and MTX, the cartilage content in the joints of mice was significantly increased after the ligand-drug conjugate was used for drug administration, indicating that the ligand-drug conjugate provided in the embodiment of the present invention can well improve the bone damage caused by inflammation in the joints of mice.

[0083] (6) Treg cell ratio in mice: Starting from the time of the first collagen II stimulation, the mice were killed on the 50th day, and the popliteal lymph nodes of the mice were washed, ground and purified to obtain a cell suspension. The cell suspension was detected by flow cytometry, and the ratio of Treg cells was calculated. The results are shown in Fig.14 and 15 shown.

[0084] Depend on Fig.14 and 15 The test results shown show that compared with the separate administration of FAPI and MTX, the proportion of Treg cells in mice was significantly increased after the ligand-drug conjugate was treated, which can effectively inhibit the production of proinflammatory cytokines by other CD4+T cells and monocytes, thereby achieving the effect of alleviating the symptoms of rheumatoid arthritis.

[0085] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A small molecule ligand for targeted drug delivery, characterized in that: The structure of the small molecule ligand for targeted drug delivery is shown in formula (1): In the formula (1), M1 is a group derived from a fibroblast activation protein α inhibitor.

2. The small molecule ligand for targeted drug delivery according to claim 1, characterized in that: The fibroblast activation protein α inhibitor is selected from one or more of FAPI, NTFAPI, UAMC1110, NH2-UAMC1110 and HYINC-FAPI-4.

3. The small molecule ligand for targeted drug delivery according to claim 1, characterized in that: In formula (1), the structure of M1 is as shown in formula (2), (3), (4) or (5):

4. A ligand-drug conjugate, characterized in that: The ligand-drug conjugate comprises an anti-rheumatic drug molecule connected by chemical bonding and the small molecule ligand for targeted drug delivery according to any one of claims 1 to 3.

5. The ligand-drug conjugate according to claim 4, characterized in that: The structure of the ligand-drug conjugate is shown in formula (6): In formula (6), M1 is a group derived from a fibroblast activation protein α inhibitor, and M2 is a group derived from an anti-rheumatic drug molecule.

6. The ligand-drug conjugate according to claim 5, characterized in that: The anti-rheumatic drug molecules are selected from one or more of methotrexate, leflunomide, hydroxychloroquine and cyclophosphamide.

7. The ligand-drug conjugate according to claim 5, characterized in that: In formula (6), the structure of M2 is shown in formula (7):

8. Use of the small molecule ligand for targeted drug delivery according to any one of claims 1 to 3 and / or the ligand-drug conjugate according to any one of claims 4 to 7 in the preparation of a drug for treating rheumatoid arthritis.

9. A drug for treating rheumatoid arthritis, characterized in that: The rheumatoid arthritis therapeutic drug comprises the ligand-drug conjugate according to any one of claims 4 to 7.

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