Small molecule ligands for targeted drug delivery, ligand-drug conjugates, drugs for the treatment of rheumatoid arthritis, and their applications

Abstract

The present invention belongs to the technical field of biomedicine, and discloses a small molecule ligand for targeted drug delivery, a ligand-drug conjugate, a therapeutic drug for rheumatoid arthritis and applications thereof. 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 inhibitor, which can be well applied as a drug delivery platform for the treatment of RA, and can achieve the full release of the anti-rheumatic drug molecule in the inflammatory joint region while maintaining the biological activity of the anti-rheumatic drug molecule, thereby achieving a better therapeutic effect on rheumatoid arthritis and having good application prospects. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a small molecule ligand for targeted drug delivery, a ligand-drug conjugate, a therapeutic drug for rheumatoid arthritis and applications thereof. Background Art

[0002] Rheumatoid arthritis (RA) is one of the most common chronic autoimmune diseases, mainly manifested as polyarticular erosive arthritis, and can be accompanied by systemic symptoms of multiple organs such as the heart, lungs, and kidneys. Methotrexate (4-amino-10-methylfolic acid, abbreviated as MTX) is a folic acid analogue that can competitively inhibit dihydrofolate reductase, hinder the conversion of dihydrofolic acid to tetrahydrofolic acid, and thus inhibit the synthesis of DNA and mitosis in cells. MTX can inhibit overactive immune cells and abnormally proliferating fibroblast-like synoviocytes to achieve the effect of alleviating the disease symptoms and the progression of joint damage in RA patients. It is a traditional first-line drug for RA treatment. However, MTX has poor targeting and may also inhibit normal cells in non-inflammatory joint regions, leading to adverse reactions such as gastrointestinal dysfunction and elevated serum liver enzyme levels, and may even cause serious adverse reactions such as cytopenia, severe infection, or respiratory failure.

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

[0004] The first object of the present invention is to provide a small molecule ligand for targeted drug delivery. This small molecule ligand for targeted drug delivery can be well applied 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 application of the above-mentioned small molecule ligand for targeted drug delivery and / or ligand-drug conjugate in the preparation of a therapeutic drug for rheumatoid arthritis.

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

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

[0009]

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

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

[0012] Further, 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 includes an antirheumatic drug molecule and the above-mentioned small molecule ligand for targeted delivery connected by a chemical bond.

[0015] Further, the structure of the ligand-drug conjugate is as 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 antirheumatic drug molecule.

[0018] Further, the antirheumatic drug molecule is selected from one or more of methotrexate, leflunomide, hydroxychloroquine, and cyclophosphamide.

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

[0020]

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

[0022] The therapeutic drug for rheumatoid arthritis provided by the present invention includes 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 a group M1 derived from a fibroblast activation protein α inhibitor is introduced at the same time. The * site in formula (1) is bonded to an anti-rheumatic drug molecule to achieve the loading of the anti-rheumatic drug molecule. In this small molecule ligand for targeted drug delivery, the targeting property of group M1 to the inflamed joint area with high expression of fibroblast activation protein α (FAPα) is utilized to localize the anti-rheumatic drug molecule loaded thereon to the inflamed joint area. Moreover, the linker contains a high-activity enzyme cleavage site of FAPα and can be well cleaved by FAPα in the inflamed joint area, realizing the full release of the anti-rheumatic drug molecule while maintaining its biological activity, so that the anti-rheumatic drug molecule can better play a role in the inflamed joint area, achieving a better therapeutic effect and having good application prospects. Description of the Drawings

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

[0026] Figure 2 Experimental result graph of the MTX release ability test provided in the examples of the present invention.

[0027] Figure 3 One of the experimental result graphs of the FAPα distribution test provided in the examples of the present invention.

[0028] Figure 4 Another experimental result graph of the FAPα distribution test provided in the examples of the present invention.

[0029] Figure 5 One of the experimental result graphs of the MTX drug distribution test provided in the examples of the present invention.

[0030] Figure 6 Another experimental result graph of the MTX drug distribution test provided in the examples of the present invention.

[0031] Figure 7 Another experimental result graph of the MTX drug distribution test provided in the examples of the present invention.

[0032] Figure 8 Another experimental result graph of the MTX drug distribution test provided in the examples of the present invention.

[0033] Figure 9 Experimental result graph of the mouse body weight test provided in the examples of the present invention.

[0034] Figure 10 Experimental result graph of the mouse paw thickness test provided in the examples of the present invention.

[0035] Figure 11 It is a graph showing the experimental results of the mouse joint inflammation index score provided in the embodiments of the present invention.

[0036] Figure 12 It is one of the graphs showing the experimental results of the mouse joint cartilage content test provided in the embodiments of the present invention.

[0037] Figure 13 It is the second graph showing the experimental results of the mouse joint cartilage content test provided in the embodiments of the present invention.

[0038] Figure 14 It is one of the graphs showing the experimental results of the proportion of mouse Treg cells provided in the embodiments of the present invention.

[0039] Figure 15 It is the second graph showing the experimental results of the proportion of mouse Treg cells provided in the embodiments of the present invention. Detailed implementation manners

[0040] Based on a profound understanding of the pathogenesis of rheumatoid arthritis and the related pathological changes in the inflamed joint area, the inventors of the present invention, through extensive and in-depth research, found that by using FAPα highly expressed in the inflamed joint area as the target site and using fibroblast activation protein α inhibitor with specific recognition and binding ability to FAPα as the targeting molecule to construct a drug delivery platform for RA treatment, theoretically, it is possible to 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 a conventional linker to connect 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, resulting in low drug efficacy. Based on the problems found, the inventors, through further research and a large number of experiments, creatively obtained the small molecule ligand for targeted drug delivery provided by the present invention.

[0041] In the present invention, the structure of the small molecule ligand for targeted drug delivery is shown as 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 undergoes chemical bonding with 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: one or more of FAPI (CB Number: CB15849362), NTFAPI (CB Number: CB28056102), UAMC1110 (CB Number: CB83052176), NH2-UAMC1110 (CB Number: CB48131098), and HYINC-FAPI-4 (CB Number: CB713117819).

[0045] In some specific embodiments, in formula (1), the structure of M1 is preferably as shown in formula (2), (3), (4), or (5); at this time, this 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 undergoes chemical bonding with the linker shown in formula (1).

[0048] Based on the ability of the above-mentioned ligand-drug conjugate to achieve targeted delivery and fully release anti-rheumatic drug molecules, the present invention also provides a ligand-drug conjugate.

[0049] In the present invention, the ligand-drug conjugate includes an anti-rheumatic drug molecule and the above-mentioned small molecule ligand for targeted drug delivery connected by a chemical bond, and the specific structure is as 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 relieve 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); at this time, the group M1 can better target and localize in the inflamed joint area and be hydrolyzed by FAPα, so as to better achieve the continuous 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 relieving 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 undergoes chemical bonding with the small molecule ligand for targeted drug delivery.

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

[0057] Based on the potential of the above-mentioned ligand-drug conjugate in the preparation of drugs for the treatment of rheumatoid arthritis, the present invention also provides a drug for the treatment of rheumatoid arthritis. Specifically, the drug for the treatment of rheumatoid arthritis includes the above-mentioned ligand-drug conjugate.

[0058] The following details the embodiments of the present invention. The examples are intended to explain the present invention and should not be construed as limiting the present invention. For those without specific technical or conditions noted in the examples, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0059] Example

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

[0061] I. Obtaining and Structure of the Ligand-Drug Conjugate

[0062] The ligand-drug conjugate provided in this example has the structure shown in formula (8). The synthesis of this structure was entrusted to WuXi AppTec (Chengdu) Co., Ltd. to obtain the ligand-drug conjugate and its 1H NMR spectrum, specifically as Figure 1 shown.

[0063]

[0064] II. MTX Release Ability of the Ligand-Drug Conjugate

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

[0066] From Figure 2From the test results shown, 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] III. Drug Efficacy of Ligand-Drug Conjugate

[0068] 1. Construction of mouse model: Using 8-week-old male DBA-1 mice weighing 18 - 22 g as experimental animals, under the conditions of temperature 23 - 25°C and humidity 45% - 50%, with a 12-hour light cycle and free access to food and water, they were adaptively raised for 5 days; the mice were randomly grouped and the following treatments were carried out:

[0069] (1) Normal group: Normal saline was fully mixed and emulsified with an equal volume of Freund's adjuvant (Chondrex.Inc., catalog number 7001), and 0.1 mL was subcutaneously injected intradermally at the base of the mouse tail. Another 0.1 mL was injected on the 21st day after the first injection. And 24 hours after the injection, 100 μL of normal saline was injected into the mouse tail vein, and the injection was carried out 2 times 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, fully mixed and emulsified with an equal volume of Freund's adjuvant, and 0.1 mL was subcutaneously injected intradermally at the base of the mouse tail. Another 0.1 mL was injected on the 21st day after the injection. When the mouse's paw became red and swollen, the mouse was restless, licked its paw, and showed lameness 24 hours later, the model was successfully established; then 100 μL of normal saline was injected into the mouse tail vein, and the injection was carried out 2 times a week for three consecutive weeks.

[0071] (3) Experimental group: The mice were treated using the method of the model group to construct a rheumatoid arthritis mouse model; then 100 μL of the drug solution was injected into the mouse tail vein, and the injection was carried out 2 times a week for three consecutive weeks. Among them, the drug solution included 3 mg / mL of FAPI (Nanchang Tenzhen Biotechnology, catalog number p240925), MTX (Shanghai Yuanye Bio-Technology Co., Ltd., catalog number S18026), or the ligand-drug conjugate.

[0072] 2. Experimental results: (1) Distribution of FAPα: After sacrificing the mice in the normal group and the model group, the heart, lungs, liver, spleen, kidneys, and the left posterior ankle joint were taken, the fur and muscle were removed, washed, fixed, decalcified, made into 5-μm thick sagittal sections, and the FAPα in the tissue was stained with an anti-FAPα monoclonal antibody (Invitrogen, catalog number PA5-99313), washed, and observed under a microscope. The results are as Figure 3 and 4 shown.

[0073] FromFigure 3 and 4 As can be seen from the test results shown in 4 , compared with normal mice, FAPα is highly expressed in the inflamed joint regions of rheumatoid arthritis model mice.

[0074] (2) Drug distribution: Each experimental group was first administered indocyanine green-labeled FAPI, indocyanine green-labeled MTX, or indocyanine green-labeled ligand-drug conjugate. The fluorescence signals of the left posterior ankle joints of the mice were measured at 10 min, 2 h, 4 h, 8 h, 20 h, 30 h, and 48 h after administration, and the MTX contents in the blood, serum, and joints of the mice were measured by sampling at 48 h. The test results are as Figures 5 - 8 shown.

[0075] From Figures 5 - 8 the test results shown in Figures 5 - 8 , it can be seen that compared with the administration of MTX alone, the administration of the ligand-drug conjugate can better deliver MTX to the inflamed joint regions and can achieve the sustained release of MTX within 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 weights of the mice were measured on the 21st, 30th, 34th, 36th, 40th, 43rd, 46th, 49th, and 50th days, and the results are as Figure 9 shown.

[0077] From Figure 9 the test results shown in Figure 9 , the administration of FAPI, MTX, and the ligand-drug conjugate in the experimental group did not have much impact on the body weight of the mice.

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

[0079] Among them, the criteria for the arthritis index score are specifically as follows: 0 points, no swelling or erythema; 1 point, arthritis can be detected with mild swelling or some erythema; 2 points, moderate redness and swelling; 3 points, severe redness and swelling of the entire paw including the fingers; 4 points, the largest swollen joint is stiff or deformed.

[0080] From Figure 10 and 11 the test results shown in Figure 10 and 11 , it can be seen that compared with the single administration of FAPI or MTX, the treatment of the rheumatoid arthritis mouse model with the ligand-drug conjugate provided in the embodiment of the present invention significantly reduced the paw thickness and arthritis index score of the mice, and had a better effect of alleviating the symptoms of rheumatoid arthritis.

[0081] (5) Cartilage content of mouse joints: Starting from the time of the first administration of type II collagen stimulation, on the 50th day, the cartilage content of the left posterior ankle joint of the mouse was detected using a nuclear magnetic resonance instrument, and the results are as Figure 12 and 13 shown.

[0082] From Figure 12 and 13 the test results shown, compared with the single administration of FAPI and MTX, when the ligand-drug conjugate was used for administration treatment, the cartilage content at the joints of the mice increased significantly, indicating that the ligand-drug conjugate provided in the embodiments of the present invention can well improve the bone destruction caused by inflammation to the joints of the mice.

[0083] (6) Proportion of Treg cells in mice: Starting from the time of the first administration of type II collagen stimulation, on the 50th day, the mice were sacrificed, and the popliteal lymph nodes of the mice were washed, ground and purified to obtain a cell suspension. The cell suspension was detected using a flow cytometer, and the cell proportion of Treg was calculated. The results are as Figure 14 and 15 shown.

[0084] From Figure 14 and 15 the test results shown, compared with the single administration of FAPI and MTX, when the ligand-drug conjugate was used for administration treatment, the proportion of Treg cells in the mice increased significantly, and it can effectively inhibit other CD4+ T cells and monocytes from producing pro-inflammatory cytokines, 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 can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. A ligand-drug conjugate, characterized in that, The structure of the ligand-drug conjugate is shown in formula (8):

2. Use of the ligand-drug conjugate according to claim 1 in the preparation of a therapeutic drug for rheumatoid arthritis.

3. A therapeutic drug for rheumatoid arthritis, characterized in that, The therapeutic drug for rheumatoid arthritis comprises the ligand-drug conjugate according to claim 1.

Images