WO2023050037A1 - 一种胞外亲环素抑制剂及其应用 - Google Patents

一种胞外亲环素抑制剂及其应用 Download PDF

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WO2023050037A1
WO2023050037A1 PCT/CN2021/121201 CN2021121201W WO2023050037A1 WO 2023050037 A1 WO2023050037 A1 WO 2023050037A1 CN 2021121201 W CN2021121201 W CN 2021121201W WO 2023050037 A1 WO2023050037 A1 WO 2023050037A1
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4mcsa
csa
mice
extracellular
cypa
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French (fr)
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全军民
杨震
刘思宇
张庆舟
胡敏强
李凤霞
付佳苗
朱振东
李勤凯
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北京大学深圳研究生院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to an extracellular cyclophilin inhibitor and application thereof.
  • Cyclophilins are a family of highly conserved multifunctional proteins that are widely distributed in nature and have been identified as the main intracellular binding protein of the immunosuppressive drug cyclosporin A (CsA). Cyclophilin has the activity of peptidyl-prolyl cis-trans isomerase (PPIase) and the function of molecular chaperone, which can assist in the correct folding of intracellular proteins, participate in immunosuppression, and mediate inflammatory reactions and participate in various biological functions such as oxidative stress response. It is mainly located in the cytoplasm and plays an important role in many life processes.
  • PPIase peptidyl-prolyl cis-trans isomerase
  • Cyclophilin A (Cyclophilin A, CypA) can be secreted to the outside of the cell through vesicle transport to form extracellular CypA (eCypA), then binds to membrane receptors, initiates signal transduction reactions in target cells, chemoattracts immune cells such as neutrophils, eosinophils, and T cells, mediates immune responses and inflammatory responses, and interacts with multiple Inflammation-related diseases are closely related. Different from the distribution inside and outside the cell, CypA plays different roles.
  • Extracellular cyclophilins are thought to play key roles in a variety of serious diseases.
  • monocytes, vascular smooth muscle cells and endothelial cells can secrete a large amount of extracellular cyclophilin through vesicles when stimulated by ROS and inflammation.
  • cyclophilin secreted extracellularly can also interact with membrane proteins, stimulate the activation of ERK1/2, AKT, JAK, NF-KB and JNK, and then further amplify ROS and inflammation, and induce monocytes and macrophages
  • the cells secrete IL-1 ⁇ , IL-6, and IL-8; it can activate matrix metalloproteinases MMP-2 and MMP-9, and promote the proliferation and migration of vascular smooth muscle cells.
  • extracellular cyclophilin in these pathways mediates the proliferation and migration of tumors, insulin resistance, cardiovascular disease, neurodegeneration and many other disease processes; extracellular cyclophilin has been confirmed in the above chronic inflammatory conditions It is up-regulated and plays a key role in the development of these diseases, such as cancer, rheumatoid arthritis, respiratory inflammation, diabetes, hypertension, cardiovascular disease, lupus, skin disease, dry eye disease, intestinal disease, lipid Metabolic abnormalities and aging, etc.
  • diseases such as cancer, rheumatoid arthritis, respiratory inflammation, diabetes, hypertension, cardiovascular disease, lupus, skin disease, dry eye disease, intestinal disease, lipid Metabolic abnormalities and aging, etc.
  • Inhibitors of cyclophilins have been reported to have a broad therapeutic range, such as anti-infection, asthma, pneumonia, cardiovascular disease, diabetes, promotion of hair growth, suppression of immune response, arthritis, dermatitis, psoriasis, multiple sclerosis , cancer, Alzheimer's disease, etc. Inhibitors of cyclophilins are therefore an important class of drugs.
  • Cyclosporin as the first discovered natural cyclophilin inhibitor, forms a complex with intracellular CypA to inhibit the dephosphorylation of NFAT by calcineurin, thereby inhibiting the activation of T cells and exerting immunosuppression It is widely used in organ transplantation, autoimmune diseases, etc. CsA can inhibit both extracellular and intracellular cyclophilin function, so it is also used in many inflammatory diseases. However, to achieve specific extracellular effects on extracellular cyclophilins, higher doses often need to be applied to compensate for the "loss" of the molecule's migration into the cell.
  • this kind of purely chemically modified CsA can not enter cells with the help of negatively charged groups, but there is still an obvious problem that the increase in hydrophilicity leads to a decrease in its accumulation in the body, and it is quickly absorbed by the kidneys. Exhausted, the metabolism is faster, and it is difficult to exert a long-term effect.
  • the purpose of the present invention is to provide an extracellular cyclophilin inhibitor 4MCsA.
  • Another object of the present invention is to provide the application of the above-mentioned compound 4MCsA.
  • the extracellular cyclophilin inhibitor has a structure as shown in formula (I):
  • R 2 is H, SR 2 ', CH 2 SR 2 ' or CH 2 OR 2 ', wherein R 2 ' is selected from alkyl, carboxyl, hydroxyl, acetamido or phenyl;
  • the phenyl group is one or more times identically or differently substituted by: -COOCH 3 , -CH 2 NH(C ⁇ O)CH 3 .
  • the cycloalkyl group is preferably cyclopropyl.
  • alkyl is a saturated hydrocarbon group, which is a hydrocarbon group formed by missing a hydrogen atom in an alkane molecule, and is a chain organic group containing only two atoms of carbon and hydrogen.
  • the alkyl group applicable in the present invention Including but limited to methyl CH 3 -, ethyl CH 3 CH 2 -, CH 3 CH 2 CH 2 -, etc.
  • cycloalkyl in the present invention includes saturated monocyclic, bicyclic, tricyclic or polycyclic hydrocarbon groups having 3 to 12 carbon atoms, wherein any substitutable ring atoms may be substituted by substituents.
  • Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclohexyl.
  • R 2 is H
  • R 2 is H
  • R 2 is H
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an extracellular cyclophilin inhibitor and a non-toxic pharmaceutically acceptable salt thereof as an active ingredient.
  • pharmaceutically acceptable salt refers to salts that retain the biological effectiveness and properties of the compounds of this invention and are not biologically or otherwise undesirable.
  • the compounds of the present invention may form acid and/or base salts by virtue of the presence of amino and/or carboxyl or similar groups.
  • Pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids
  • pharmaceutically acceptable base addition salts can be prepared from inorganic or organic bases.
  • non-toxic pharmaceutically acceptable salt refers to a non-toxic salt formed with a non-toxic pharmaceutically acceptable inorganic or organic acid or inorganic or organic base.
  • such salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, and from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, Malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, fumaric acid, methanesulfonic acid, and toluenesulfonic acid Acid etc.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like
  • organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, Malic acid, tartaric acid, citric acid, ascorbic acid, pamoi
  • the compounds of formula (I) according to the invention may be administered alone, preferably they are presented as pharmaceutical compositions.
  • the aforementioned pharmaceutical composition useful according to the present invention comprises at least one compound of formula (I) as defined above, and one or more pharmaceutically acceptable carriers and any other therapeutic ingredients of choice.
  • the active ingredients required in combination therapy may be combined into a single pharmaceutical composition for simultaneous administration.
  • compositions, carriers, diluents, and agents are used interchangeably when referring to compositions, carriers, diluents, and agents and mean that the substance can be administered to a mammal without adverse effects.
  • Physiological effects such as nausea, dizziness, heart noise, etc.
  • compositions in which the active ingredient is dissolved or dispersed are prepared as injectables, either as liquid solutions or suspensions; however, solid forms suitable for solution in, or suspension in, liquid prior to use can also be prepared.
  • the formulation may also be emulsified.
  • the above-mentioned pharmaceutical composition can be formulated into solid dosage forms, such as capsules, tablets, pills, powders, dragees or granules.
  • excipients and the content of active ingredients in the excipients are usually determined according to the solubility and chemical properties of the active compound, the specific mode of administration and the regulations observed in pharmaceutical practice.
  • excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate
  • disintegrants such as starch, alginic acid, and certain complex silicates
  • lubricants such as magnesium stearate, lauryl A combination of sodium alkyl sulfate and talc
  • aqueous suspensions When aqueous suspensions are used, they may contain emulsifying agents or suspension enhancers. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.
  • the above-mentioned pharmaceutical composition can be administered to humans and animals in a suitable dosage form, by local or systemic administration, including oral administration, rectal administration, nasal cavity administration, buccal administration, eye administration, sublingual administration, transdermal administration, etc. Administration, rectal administration, topical administration, vaginal administration, parenteral administration (including subcutaneous injection, arterial injection, intramuscular injection, intravenous injection, intradermal injection, intrathecal injection, epidural injection), intracisternal injection Administration and intraperitoneal administration. It is to be understood that the preferred route may vary with, for example, the circumstances of the recipient.
  • the formulations may be prepared in unit doses by any methods well known in the art of pharmacy. Such methods include bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the above formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the total daily dose of a compound of the invention is administered to a subject in single or multiple doses.
  • the present invention also provides the application of the above-mentioned extracellular cyclophilin inhibitors in the preparation of drugs for the prevention or treatment of diseases mediated by cyclophilins, wherein the diseases mediated by cyclophilins include but not limited to a) viral infection, b) metabolic diseases, c) acute and chronic inflammatory diseases, d) cancer, e) neurodegenerative diseases, f) degenerative muscle diseases, g) cardiovascular diseases, h) obesity, i) diabetes.
  • diseases mediated by cyclophilins include but not limited to a) viral infection, b) metabolic diseases, c) acute and chronic inflammatory diseases, d) cancer, e) neurodegenerative diseases, f) degenerative muscle diseases, g) cardiovascular diseases, h) obesity, i) diabetes.
  • the present invention obtains a brand-new 4MCsA compound, which can limit the effect of the compound to the extracellular space after efficiently reacting with Albumin, increase the metabolic time of the compound, and improve Specific, potent inhibitor of extracellular cyclophilins.
  • the compound or its corresponding pharmaceutically acceptable salt form can bind extracellular cyclophilin, thereby inhibiting the amplification of chronic inflammation. In various chronic inflammatory diseases, such as tumors, type 2 diabetes and atherosclerosis, etc., exhibit good therapeutic effect.
  • Figure 1 shows the mass spectrum of albumin modified by 4MCsA, wherein, the upper figure is BSA, and the lower figure is BSA-4MCsA conjugate;
  • Figure 2 shows the identification results of Cysteine modification sites on BSA by 4MCsA, in which, the upper figure shows the 2+ ions of the corresponding peptides of the BSA sample, and the lower figure shows the 3+ ions of the corresponding peptides of BSA-4MCsA;
  • FIG. 3 shows the EIC diagram of CsA, 4MCsA and thiophenol-blocked 4MCsA (Ar-S-4MCsA),
  • I.F. represents the intracellular extract
  • E.S. is the curve of the standard, and the area under the curve is used to quantitatively calculate the intracellular concentration, Fig. The shaded part is the area under the I.F. curve;
  • Fig. 4 is the competitive flow cytometry figure of CsA-FITC fluorescently labeled Jurkat cells
  • Figure 5 shows the in vitro activity verification of 4MCsA molecules, wherein, A is the binding ability of CsA ( ⁇ ), 4MCsA ( ⁇ ) and BSA-4MCsA ( ⁇ ) to CypA measured by competitive fluorescence polarization; B is CsA ( ⁇ ), 4MCsA ( ⁇ ) and BSA-4MCsA ( ⁇ ) inhibit the cis-trans isomerase activity of CypA in a dose-dependent manner; C is the corresponding K i and IC50 of each inhibitor;
  • Figure 6 shows that the 4MCsA molecule has no immunosuppressive activity; wherein, A is the Western Blot detection molecule to the dephosphorylation inhibition of NFAT1 induced by 25nM PMA and 1mg/mL ionomycin in Jurkat cells (the lower molecular weight band), B To detect the secretion of IL-2 after Jurkat cell activation by ELISA;
  • Figure 7 shows that 4MCsA inhibits inflammation and monocyte migration stimulated by extracellular CypA; wherein, A is the expression of IL-8 in THP-1 cells induced by CypA, and after THP-1 cells are treated with 100nM CypA for 12h, the mRNA of IL-8 The level was significantly increased, and 4MCsA and CsA can inhibit this effect at the same time; B is the migration coefficient of THP-1 cells induced by CypA; C is the representative figure of THP-1 cells stained with crystal violet in the cell migration experiment;
  • Figure 8 shows the cytotoxic effect of CsA and 4MCsA on human kidney cell HEK293;
  • Figure 9 shows the disease state in the T2DM mouse model and the serum CypA content after treatment with CypA inhibitors
  • Figure 10 shows the control of blood sugar in DB mice and HFD mice by tail vein injection of 4MCsA and CSA, wherein, a is the fasting blood sugar of DB mice; b is the fasting blood sugar of C57 mice; c is the blood sugar of DB mice Body weight after administration; d is the body weight of the corresponding C57 control of HFD and DB mice; e is the fasting blood sugar of HFD mice; f is the control of 4MCsA and CsA 5mg/kg/week to the HbA1c of HFD;
  • Figure 11 shows the impact on GTT under different drugs and doses in DB mice
  • Figure 12 shows 4MCsA and CsA and its impact on GTT at different time intervals after administration in HFD mice
  • Figure 13 shows the impact of CsA and 4MCsA on insulin, wherein, a is the basal level of insulin in DB mice after administration, and b is the insulin secretion of DB mice in response to glucose stimulation after administration;
  • Figure 14 shows the effects of CsA and 4MCsA on ITT experiments in mice, where a, c are the ITT of DB mice after administration; b, d are the ITT responses in C57 mice; e, f are the ITT of HFD mice after administration;
  • Figure 15 shows the levels of inflammatory factors in the blood of DB mice after administration; a is the level of IL1 ⁇ ; b is the level of CRP; c is the level of TNF- ⁇ ; d is the level of IL-6;
  • Figure 16 shows the evaluation results of hepatic and renal toxicity in DB mice after medication; wherein, a is the level of aspartate aminotransferase AST; b is the level of alanine aminotransferase ALT; C is the level of urea; d is the level of creatinine;
  • Figure 17 shows the changes in tumor volume in tumor model mice
  • Figure 18 shows the effect of 4MCsA on CD8 + T cells in the tumor microenvironment
  • Figure 19 shows the influence of 4MCsA on the degree of aortic lesion in high-fat diet-induced atherosclerotic mice
  • Figure 20 shows the influence of 4MCsA on the CRP content in serum of atherosclerotic mice induced by high-fat diet
  • Figure 21 shows the influence of 4MCsA on routine blood lipid indexes in the serum of atherosclerotic mice induced by high-fat diet; wherein, a is serum total cholesterol (TC) content; b is serum triglyceride (TG) content; c is serum triglyceride (TG) content; Low-density lipoprotein cholesterol (LDL-C) content; d is high-density lipoprotein cholesterol (HDL-C) content; e is the ratio of total cholesterol to high-density lipoprotein cholesterol (TC/HDL-C);
  • Figure 22 shows the effect of long-term injection of 4MCsA on the contents of ALT and AST in serum of mice with atherosclerosis induced by high-fat diet.
  • the present invention takes 4MCsA modified by maleimide (maleimide) at the 4th position as an example, by introducing a maleimide reactive group at the 4th position of CsA to covalently react with BSA, the molecular confinement can be realized. Extracellular effects.
  • MM284 To selectively target extracellular CypA, a previous modification (MM284) introduced a negatively charged group at the double bond at the 1-position of cyclosporine. Due to the existence of the double bond at the 1-position of cyclosporin, it is relatively easy to modify this position, but this position is also the binding site of cyclosporin and cyclophilin, which is not suitable for introducing larger groups. Moreover, such CsA analogs are usually rapidly cleared by the kidneys in blood circulation, which hinders further clinical applications. In the binding form of cyclosporine and cyclophilin, the P4 position is located away from the cyclophilin, which is an ideal modification site. However, the modification of this site is relatively difficult and requires a relatively complicated synthetic route.
  • cyclosporine As a complex natural product molecule, cyclosporine has a molecular weight greater than 1000Da, so it is challenging to separate, purify and control impurities in the reaction. Therefore, there is no use of cyclosporine derivatives and protein coupling for pharmaceutical applications.
  • the present invention introduces derivative molecules 3 and 8 of the Michael acceptor at the 4-position of cyclosporine, and uses it for cyclosporine-albumin coupling and activity verification Work.
  • the synthesis process of 4MCsA of the present invention only uses two-step column chromatography purification, which greatly reduces the difficulty of operation and greatly improves the yield
  • the synthetic scheme of the present invention comprises two main steps:
  • the first one is the synthesis process of decapeptide 4 which is opened from the CsA(1) P3 and P4 positions and removes the P4 amino acid.
  • a key challenge in the synthesis of 4 is the Edman degradation step, which in most cases generates a mixture of undecapeptide (1b), decapeptide (4) and nonapeptides with valine deletion.
  • an excess (2.0-5.0 equivalent) of PhNCS is fully reacted with 1a, and then unreacted PhNCS is captured with DMAPA. In this way, excess DMAPA after Edman degradation can be washed away to obtain high-purity intermediate 4.
  • the second one is the coupling of decapeptide 4 with the amino acid Fmoc-N-Me-Lys(Boc)-OH(5), then use NMe4OH to remove the Fmoc, acetyl group, and hydrolyze the methyl ester, and finally use HATU for macrocyclization to obtain the ring Peptide [MeLys(Boc)4]CsA(6).
  • [MeLys(Boc)4]CsA(6) was deprotected from Boc under the condition of trifluoroacetic acid (TFA) to obtain [MeLys4]-CsA, and then reacted with 6-maleimidocaproic acid-N-succinimide ester (7) Coupling produces 4MCsA (3).
  • TFA trifluoroacetic acid
  • Embodiment 1 The synthetic technique of linear decapeptide
  • 4MCsA(3):6 (89mg, 66.4mol, 1.0eq) was dissolved in 2mL of DCM, then 650L of TFA was added and stirred for 30 minutes. The solution was then concentrated and the residue was dissolved in DCM, washed with water, saturated NaHCO 3 and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was dissolved in 1.0 mL of anhydrous DCM, then N-succinimidyl-6-maleimide hexanoate (7) (31 mg, 0.1 mmol, 1.5 equiv) and Et 3 N (28 ⁇ L , 0.2 mmol, 3.0 equivalents). The solution was stirred overnight.
  • the reactivity of the molecule with albumin was determined using the intact protein method by a Q Exactive HFX mass spectrometer (Thermo Fisher) mass spectrometer after in vitro binding.
  • 4MCsA increases the molecular weight of albumin by 1410Da, and 4MCsA can effectively covalently bind albumin, with a stoichiometric ratio of 1:1 and good reactivity.
  • C 18 Spin Columns Thermo Fisher
  • the bottom-up peptide was identified to have a peptide fingerprint of the 4MCsA modified peptide [K].GLVLIAFSQYLQQcPFDEHVK.[L], showing the 4MCsA molecule modified by Cys34.
  • the rapid and specific reactivity of 4MCsA molecules with BSA provides support for its cell non-penetrating properties.
  • Example 7 Determination of the membrane penetration of molecules in Jurkat cells by high-resolution mass spectrometry
  • the concentration of compounds in cells was quantified by external standard method using QE-Focus mass spectrometer.
  • Example 8 Fluorescence competition method to detect the determination of the membrane penetrability of molecules in Jurkat cells
  • CsA-FITC fluorescent probe combined with intracellular CypA was used to detect the effect of the compound on the intracellular fluorescent signal by flow cytometry using a competition method.
  • CsA-FITC can effectively bind intracellular CypA to make the cells labeled with fluorescence, and after CsA enters the cells, it competes with the CsA-FITC probe to bind intracellular CypA, thereby greatly reducing the proportion of fluorescent cells (from 95.30% to 6.75% level). In contrast, however, 4MCsA had very little effect on the intracellular fluorescence signal.
  • 4MCsA achieves the effect of localizing the molecule outside the cell, and is an effective molecule for subsequent targeting of extracellular eCypA.
  • Fluorescence polarization is a widely used technique for measuring ligand-receptor binding in solution.
  • the free fluorescent probes in the free state will rotate rapidly in the system, and most of the emitted fluorescence is depolarized light. Once the probe is bound to a larger molecule, the molecular rotation becomes constrained, and the emission of polarized light increases.
  • the present invention synthesizes a CsA analogue labeled with fluorescein isothiocyanate as a fluorescent tracer probe (CsA-FITC), and uses a competitive fluorescence polarization assay to measure the affinity of the ligand to CypA.
  • CsA-FITC fluorescent tracer probe
  • the binding constant K d of CsA-FITC and CypA was determined to be 14.81 ⁇ 0.70nM.
  • 50nM CypA was combined with different concentrations of inhibitors, and then 20nM CsA-FITC probe was added to measure the competition fluorescence polarization curve.
  • Embodiment 10 4MCsA and the inhibition of compound to peptidyl-prolyl-cis/trans-isomerase (PPI enzyme) activity
  • Chymotrypsin was used to hydrolyze N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide substrate peptide, and the effect of inhibitors on CypA enzyme activity was determined.
  • the proline of the substrate peptide is mainly in the cis form in a specific component system, and the cleavage efficiency of chymotrypsin is low at this time.
  • proline cis-trans isomerase After being catalyzed by proline cis-trans isomerase, its configuration changes from cis to In trans, it can be specifically cleaved by chymotrypsin and release p-nitroanilide, which has a maximum absorption near 390nm.
  • the immunosuppressive effect of CsA is mainly due to the formation of CypA/CsA complex with intracellular CypA after the molecule enters the cell, and then binds to calcineurin, preventing the enzyme from dephosphorylating and activating NFAT protein.
  • the present invention studies the effect of the molecule on NFAT signal transduction during the activation of Jurkat T cells induced by ionomycin and PMA.
  • 1 ⁇ 10 6 Jurkat cells were seeded in RPMI1640 medium (containing 10% serum) pre-added with CsA and 4MCsA and incubated for 1.5 h, then treated with 25 nM phorbol ester PMA and 1 ⁇ g/mL ionomycin for 2 h to induce the growth of Jurkat cells After activation, cells are collected, extracted, electrophoresis, transferred, and NFAT antibody is used to detect the dephosphorylation level of NFAT protein. After NFAT is dephosphorylated, two bands, phosphorylated and non-phosphorylated, appear.
  • the present invention further detects the secretion of IL-2 protein downstream of T cell activation involved in NFAT, and uses the ELISA method to measure the content of IL-2 protein in the culture medium after being stimulated by PMA and ionomycin for 24 hours. Consistent with the results of Western blot, 0.2 ⁇ M CsA can significantly inhibit the secretion of IL-2, and the concentration of 4MCsA up to 5 ⁇ M has almost no effect on the secretion of IL-2. The above results show that 4MCsA has no immunosuppressive effect, which is consistent with its effective restriction outside the cell, so that it cannot form a complex with intracellular CypA to interfere with the activation of calcineurin and NFAT.
  • the present invention firstly verifies the secretion of pro-inflammatory factor IL-8 promoted by CypA by 4MCsA.
  • the method of stimulating cells with CypA protein after pre-incubation of drug molecules with the medium was used to detect the transcriptional changes of CypA-stimulated pro-inflammatory factor IL-8 and the migration of inflammatory cells by 4MCsA and CsA.
  • 4MCsA and CsA were pre-incubated with RPMI1640 medium containing 0.1% BSA for 1 hour, and then CypA was used to stimulate THP1 cells for 12 hours at the same time.
  • RT-PCR method was used to detect the changes in the transcription level of IL-8; in the Transwell migration experiment, in the lower chamber Pre-incubate 4MCsA and CsA with RPMI1640 medium containing 0.1% BSA for 1 hour, then add 200ng/mL CypA, and add 1.0 ⁇ 105 THP1 cells to the upper chamber, incubate at 37°C for 1 hour, and then crystallize the lower membrane of the chamber After violet staining, photographs were taken to record the number of migrated cells.
  • This example compares the cytotoxic effects of CsA and non-cell-penetrating 4MCsA on normal HEK293 human kidney cells.
  • the killing effect of cells treated with 4MCsA and CsA was detected by MTT assay.
  • 4MCsA has almost no cytotoxicity when the concentration reaches 50 ⁇ M.
  • CsA significantly inhibits the proliferation of HEK293 cells, and exhibits concentration-dependent cytotoxicity as the concentration increases. This is consistent with the liver and kidney toxicity of CsA in clinical use, and 4MCsA has shown great advantages in this regard.
  • Example 14 4MCsA medication can reduce CypA in diabetes model
  • mice leptin receptor-deficient db/db genetically engineered mice
  • CsA or 4MCsA and 1 mg/kg weekly dose of 4MCsA were administered to DB and C57 mice for 3 weeks, then the serum of the mice was collected, and the content of CypA in blood was detected by ELISA.
  • Example 15 4MCsA reduces fasting blood glucose in T2DM mouse model
  • this embodiment selects DB mice and high-fat diet-induced obesity mouse models (HFD mice), as two types of type 2 diabetes mouse models with different inducements as research objects.
  • the DB model was set up with control group Ctrl, 4MCsA 5mg/kg/week group DB 4MC5, 4MCsA 1mg/kg/week group DB 4MC1, CsA 5mg/kg/week group DB CsA5); in the HFD model, the doses of 4MCsA and CsA were both 5mg/kg, both models were administered by tail vein injection once a week.
  • 4MCsA can significantly reduce the fasting blood sugar and the content of glycosylated hemoglobin in DB and HFD mice after administration, indicating that 4MCsA can effectively control blood sugar in diabetes for a long time. It is also worth noting that after 5 weeks of administration to normal C57 mice, the fasting blood glucose of the CsA 5mg/kg/week experimental group was slightly higher than that of the C57ctrl group, while the 4MCsA 5mg/kg/week was comparable to the normal group.
  • Embodiment 16 4MCsA improves T2DM mouse model glucose tolerance
  • Glucose Tolerance Test was performed on the mice administered with the two models in Example 15 to detect the ability of the mice to control glucose. After 5 weeks of administration, the mice were fasted for 16 hours, and then the mice were injected with glucose (1 g/kg for DB mice and 2 g/kg for HFD mice) by intraperitoneal injection, and then at different times Take blood and check blood sugar level. The blood samples corresponding to the time points were also saved for the insulin detection of the subsequent glucose stimulation.
  • both 4MCsA and CsA 5mg/kg can significantly improve the glucose tolerance state of DB mice ( Figure a-b).
  • Figure e compared with the Ctrl group, the area under the curve (AUC) of the 5 mg/kg drug group was significantly reduced, and the glucose clearance rate in the body was faster.
  • AUC area under the curve
  • FIG. d and f it can be observed that CsA significantly increases the AUC of C57 healthy mice, while the AUC of the 4MCsA group increases less than that of the CsA group, indicating that CsA medication has no effect on healthy mice.
  • Glucose tolerance of rats may be damaged to some extent, and the effect of 4MCsA is relatively smaller.
  • Embodiment 17 The influence of 4MCsA on the insulin secretion stimulated by glucose
  • the basal level of insulin in DB mice was significantly higher than that in the C57 healthy group. Insulin was slightly higher than CsA group ( Figure a right). After glucose stimulation, there was no significant difference between 4MCsA and CsA on the insulin secretion of DB mice at different stages compared with DB ctrl group (Fig. b left). The use of 4MCsA and CsA 5mg/kg/day did not change the insulin secretion of DB mice, so the improvement of GTT in DB mice was caused by affecting the insulin signal response.
  • Embodiment 18 The influence of 4MCsA on insulin sensitivity
  • Insulin sensitivity test (insulin tolerance test, ITT) was performed on the two kinds of mice in Example 15 to further measure the insulin signal response. After the mice were fasted for 6 hours, insulin was injected intraperitoneally (the dose of DB mice was 2 U/kg, and the dose of HFD mice was 1 U/kg), and then the blood glucose levels at different time points were detected.
  • both 4MCsA and CsA can significantly improve DB insulin sensitivity, and it was also observed in healthy C57 that both of them improved ITT after administration.
  • 4MCsA has a certain dose effect on ITT in DB mice ( Figure a-d).
  • 4MCsA and CsA can also weaken insulin resistance caused by obesity and improve ITT, and the AUC of 4MCsA is lower than that of CsA, and the effect is more significant ( Figure e-f).
  • Embodiment 19 The influence of 4MCsA on inflammatory factors in DB mice
  • 4MCsA can significantly reduce the levels of inflammatory factors TNF-a and IL1 ⁇ in DB mice after 42 days of administration, while CsA can only improve IL1 ⁇ . It can be seen that 4MCsA can reduce inflammation in mice, and its potential is higher than that of CSA, which can reduce the damage of insulin signal caused by inflammation, improve insulin sensitivity, and improve the diabetic state of mice.
  • Example 20 Investigate the effects of 4MCsA and CSA on the hepatic and renal toxicity in DB mice
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • the ALT and AST of DB mice were significantly increased compared with C57 healthy mice. There was no further increase in hepatic and renal toxicity in DB mice (c and d in Figure 16). Similarly, 4MCsA and CSA did not produce significant renal toxicity, and the levels of urea and creatinine had no difference between the groups. However, it can still be seen that in C57 healthy mice, after 50 days of long-term administration, the levels of AST, ALT and urea in the CsA administration group were significantly increased, while 4MCsA remained close to the level of the healthy control. This result more directly shows the advantages of the 4MCsA of the present invention in reducing toxicity.
  • CsA has been reported to be metabolized faster in diabetic subjects, it has potential harm to healthy subjects, which has also been proved many times in the invention's experiments: healthy Long-term administration of C57 mice will damage their GTT, reduce insulin secretion, increase blood sugar levels, and increase liver and kidney toxicity. However, these adverse reactions in healthy C57 were significantly reduced by our designed 4MCsA.
  • Example 21 4MCsA plays an anti-tumor effect in a mouse colon cancer tumor model
  • the inoculation amount is 106/mouse
  • the tumor size is about 100mm 3 after six days, start to measure the administration with a vernier caliper at the same time
  • the administration of 4MCsA The dosage is 5mg/kg, administered every day, the tumor is measured every two days, and the tumor size is counted. The experiment was terminated after three weeks.
  • the thickness of the frozen section is required to be 4-8 ⁇ m, take it out from -80°C and place it at room temperature for 30 minutes, fix it with acetone at 4°C for 10 minutes, wash with PBS, 5 minutes ⁇ 3; block with 5-10% normal goat serum (diluted in PBS) , and incubate at room temperature for 10 minutes. Pour away the serum, do not wash, add the primary antibody or primary antibody working solution diluted in appropriate proportion dropwise, and incubate at 37°C for 1-2 hours or overnight at 4°C.
  • 4MCsA can promote the infiltration of CD8 T cells in the tumor microenvironment, thereby achieving a tumor suppressive effect.
  • the 5-week-old ApoE gene-deficient C57BL/6J strain mice were randomly divided into groups and then high-fat induced by Western diet. From the seventh week, 4MCsA was administered to the experimental group by tail vein injection. It is 5mg/kg/week, and the administration cycle is 9 weeks. After the experiment, the experimental animals were euthanized. After dissection, the right atrial appendage was first perfused with 10 mL of PBS, and then 10 mL of tissue fixative (4% paraformaldehyde). The aorta was completely separated under a stereoscope, and the blood vessels were peeled off with fat forceps. Adhesive adipose tissue. The isolated aorta was stained grossly by Oil Red O staining, and photographed and recorded. The lesion area of the aortic arch in the experimental group and the control group was counted using ImageJ, and the difference was statistically significant.
  • mice After the atherosclerotic mice were successfully modeled, the experimental mice were injected with 5 mg/kg 4MCsA into the tail vein once a week by using the method described in Example 23, and the administration was continued for 9 weeks.
  • the CRP in the mouse serum Level By detecting the CRP in the mouse serum Level, to assess the risk of cardiovascular disease in the body, so as to evaluate the impact of the compound on the occurrence of cardiovascular disease in the body.
  • 4MCsA can significantly reduce the content of CRP in the serum of mice in the experimental group, indicating that 4MCsA has a very good predictive effect on the occurrence of cardiovascular diseases in the body.
  • Example 25 Effect of 4MCsA on conventional blood lipid indexes in serum of atherosclerotic mice induced by high-fat diet
  • Example 23 The method described in Example 23 was used to induce the mouse model of atherosclerosis with a high-fat diet, and administration was carried out in groups, and 5 mg/kg of 4MCsA or placebo was injected into the tail vein once a week. After 9 consecutive weeks of administration, The mice were euthanized, and serum was collected for routine blood lipid detection.
  • Example 26 Effect of long-term injection of 4MCsA on ALT and AST levels in serum of mice with atherosclerosis induced by high-fat diet
  • Example 25 The method described in Example 25 was used to induce and administer the mouse atherosclerosis model. After the animal experiment period ended, the mice were euthanized, and the serum was taken to detect alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum. ) content.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase

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Abstract

本发明属于生物医药领域，具体涉及一种胞外亲环素抑制剂及其应用。本发明的胞外亲环素抑制剂的结构通式如式（I）所示。本发明通过新型环孢菌素衍生物侧链引出能与血液白蛋白上特异位点半胱氨酸巯基反应的基团，进入体内快速形成药物偶合物，能够有效将药物限制于细胞外，形成针对胞外亲环素的靶向抑制，从而实现治疗与此相关疾病的目的。

Description

一种胞外亲环素抑制剂及其应用 技术领域
本发明属于生物医药领域，具体涉及一种胞外亲环素抑制剂及其应用。
背景技术
亲环素(cyclophilins，CyPs)是一类广泛分布于自然界、具有高度保守性的多功能蛋白家族，被鉴定为免疫抑制药物环孢霉素(Cyclosporin A,CsA)的主要胞内结合蛋白。亲环素具有肽基脯氨酰顺反异构酶(peptidyl-prolyl cis-trans isomerase，PPIase)的活性和分子伴侣功能，能辅助细胞内蛋白质的正确折叠、参与免疫抑制、介导炎性反应和参与氧化应激应答等多种生物学功能。它主要定位于细胞质中，在众多生命过程中发挥重要作用，而在氧化应激状态或炎症环境下，亲环素A(Cyclophilin A，CypA)可通过囊泡运输形式分泌到细胞外形成胞外CypA(eCypA)，随后与膜受体结合,在靶细胞中启动信号传导反应，趋化嗜中性粒细胞、嗜酸性粒细胞和T细胞等免疫细胞，介导免疫应答及炎症反应，与多种炎症相关疾病密切相关。区别于细胞内外分布，CypA发挥着不同的作用。
胞外亲环素被认为在多种严重的疾病中起到了关键的作用。在慢性炎症状态下，单核细胞、血管平滑肌细胞和内皮细胞，在被ROS和炎症刺激时可通过囊泡分泌大量胞外亲环素到胞外。同时分泌到胞外的亲环素也能够与膜蛋白相互作用，刺激ERK1/2、AKT、JAK、NF-KB和JNK的活化，进而再对ROS和炎症进一步放大，诱导单核细胞和巨噬细胞分泌IL-1β、IL-6和IL-8；它可以激活基质金属蛋白酶MMP-2和MMP-9，促进血管平滑肌细胞的增殖和迁移。胞外亲环素在这些通路的作用介导了肿瘤的增殖迁移、胰岛素耐受、心血管疾病、神经退行性病变等诸多疾病过程；胞外亲环素已被证实在以上多种慢性炎症条件下处于上调状态，在这些疾病的发生发展中起到了关键作用，比如癌症、类风湿关节炎、呼吸道炎症、糖尿病、高血压、心血管疾病、狼疮、皮肤疾病、 干眼病、肠道疾病、脂代谢异常和衰老等。亲环素的抑制剂被报道具有很广泛的治疗范围，比如抗感染、哮喘、肺炎、心血管疾病、糖尿病、促进头发生长、抑制免疫应答、关节炎、皮炎、银屑病、多发性硬化症、肿瘤、阿兹海默病等。因此亲环素的抑制剂是一种重要的药物类别。
环孢霉素(CsA)作为最早发现的天然亲环素抑制剂，其通过与胞内CypA形成复合物，抑制钙调磷酸酶对NFAT的去磷酸化，从而抑制T细胞的激活，发挥免疫抑制作用，被广泛应用于器官移植、自身免疫疾病等。CsA能同时抑制细胞外和细胞内的亲环素功能，因此也被应用于多种炎症疾病。然而在获得针对胞外亲环素特定的细胞外作用时，往往需要应用更高的剂量弥补分子迁移到细胞内的“损失”。但是由于胞内亲环素的诸多重要功能，高剂量下CsA产生的毒性尤其是胰岛损伤、肝肾毒性，极大限制了其发展。因此，如果能够开发抑制胞外亲环素类的化合物，在产生期待的治疗效果的同时，不进入细胞，那么就可能避免这些不期待的副作用。
现有技术通过靶向分泌型的胞外亲环素能够有效减少如呼吸道和心血管疾病的炎症。通过对CsA加上羧基苯并咪唑改性后的分子MM284，能够提高分子亲水性，而不穿透细胞，把其作用限制胞外部分的亲环素，有效抑制免疫细胞的迁移，从而能够降低炎症。类似的分子还有MM218，给CsA加上罗丹明侧链后能阻止其进入细胞，进而减少肺部的炎症，改善哮喘，但是由于这类基团能够和多耐药转运***作用，因此可能会造成一些不良反应。另外这类单纯化学改造的CsA借助带负电的基团，虽然能够实现不进细胞，但是还有一个明显的问题就是亲水性的提升导致其在体内的积累也随之减少，很快被肾脏排掉，代谢较快，难以发挥长效的作用。
发明内容
本发明的目的在于提供一种胞外亲环素抑制剂4MCsA。
本发明的再一目的在于提供上述化合物4MCsA的应用。
根据本发明具体实施方式的胞外亲环素抑制剂，其结构如式(I)所示：
Figure PCTCN2021121201-appb-000001
其中，R 1为-CH＝CHR 1’或-CH 2CH 2R 1’；R 1’选自烷基、羧基、乙酰氨基或苯基；
R 2为H、SR 2’、CH 2SR 2’或CH 2OR 2’，其中，R 2’选自烷基、羧基、羟基、乙酰氨基或苯基；
linker选自-(CH 2) x-NH(C＝O)-(CH 2) y-或-(CH 2) x-NH(C＝O)-(CH 2CH 2O) y-，其中，x为1-4的整数，y为1-6的整数。
根据本发明具体实施方式的胞外亲环素抑制剂，R 1’选自-CH 3、-(CH 2) n-COOH、-(CH 2)n-NH(C＝O)CH 3、-苯基或环烷基，其中，n为1-6的整数，即n为1、2、3、4、5或6；
所述苯基被以下基团一次或多次相同或不同地取代：-COOCH 3、-CH 2NH(C＝O)CH 3。
所述环烷基优选为环丙基。
根据本发明具体实施方式的胞外亲环素抑制剂，R 2’选自-CH 3、-(CH 2) n-COOH、-(CH 2) n-OH、-(CH 2)n-NH(C＝O)CH 3、-苯基或环烷基，n为1-6的整数；所述苯基所述苯基被以下基团一次或多次相同或不同地取代：-COOCH 3、-CH 2NH(C＝O)CH 3。
本发明中“烷基”即饱和烃基，是烷烃分子中少掉一个氢原子而成的烃基，是一类仅含有碳、氢两种原子的链状有机基团，本发明中适用的烷基包括但限于甲基CH 3-，乙基CH 3CH 2-、CH 3CH 2CH 2-等。
本发明中的术语“环烷基”包括具有3到12个碳原子的饱和的单环、双 环、三环或多环烃基，其中任何能发生取代的环原子可被取代基取代。环烷基的例子包括但不限于环丙基、环丁基、环己基。
根据本发明具体实施方式的胞外亲环素抑制剂，R 1为-CH＝CHCH 3，R 2为H，linker选自-(CH 2) x-NH(C＝O)-(CH 2) y-或-(CH 2) x-NH(C＝O)-(CH 2CH 2O) y-，其中，x为1-4的整数，y为1-6的整数。
优选的，R 1为-CH＝CHCH 3，R 2为H，linker为-(CH 2) x-NH(C＝O)-(CH 2) y-，其中，x为2或3，y为3、4或5；
更优选的，R 1为-CH＝CHCH 3，R 2为H，linker为-(CH 2) 3-NH(C＝O)-(CH 2) 5-。
本发明还提供胞外亲环素抑制剂及其非毒性药学上可接受的盐作为活性成分的药物组合物。
术语“药学上可接受的盐”指保留了本发明的化合物的生物有效性和特性的盐，且不在生物方面或其他方面是不理想的。在许多实例中，本发明的化合物利用氨基和/或羧基或类似的基团的存在可形成酸式和/或碱盐。药学上可接受的酸式加成盐可由无机或有机酸制备，而药学上可接受的碱式加成盐可由无机或有机碱制备。药学上可接受的盐的综述见Berge等((1977)J.Pharm.Sd，vol.66，1)。“非毒性药学上可接受的盐”指与非毒性的药学上可接受的无机或有机酸或无机或有机碱形成的非毒性的盐。例如，上述盐包括那些衍生自无机酸例如盐酸、氢溴酸、硫酸、氨基磺酸、磷酸、硝酸等，以及由有机酸例如乙酸、丙酸、琥珀酸、乙醇酸、硬脂酸、乳酸、苹果酸、酒石酸、枸橼酸、抗坏血酸、扑酸、马来酸、羟基马来酸、苯乙酸、谷氨酸、苯甲酸、水杨酸、磺胺酸、富马酸、甲磺酸和甲苯磺酸等。
本发明的式(I)的化合物可单独施用，优选将它们作为药物组合物提供。根据本发明有用的上述药物组合物，不管是用于兽药还是用于人类用途，包含至少一种具有上述定义的式(I)的化合物、和一种或多种药学上可接受的载体和任选的其它治疗成分。
在某些优选实施方案中，联合治疗中所需的活性成分可合并到单个药物组 合物中以同时给药。
当述及组合物、载体、稀释剂和试剂时，本文使用的术语“药学上可接受的”和它们语法上的变化可相互交换使用且表示该物质可施用于哺乳动物而不会产生不良的生理效果例如恶心、头晕、心嘈等。
其中溶解或分散有活性成分的药物组合物的制备方法是现有技术中已知的，且不需要基于配方对其进行限定。通常这种组合物以液体溶液或混悬液制备成注射剂；然而，也可制备适于在使用前溶解或混悬在液体中的固体形式。制剂也可为乳化的。特别地，上述药物组合物可配置成固体剂型，例如胶囊、片剂、丸剂、粉剂、糖丸剂或颗粒剂。
通常根据活性化合物的溶解性和化学性质、给药的具体模式和药学实践中遵守的规定来确定赋形剂的选择和赋形剂中活性成分的含量。例如，赋形剂(如乳糖、柠檬酸钠、碳酸钙、磷酸二钙)和崩解剂(如淀粉、褐藻酸和某些复合硅酸盐)与润滑剂(如硬脂酸镁、十二烷基硫酸钠和滑石粉)结合可用于制备片剂。为了制备胶囊，使用乳糖和高分子量聚乙二醇是有利的。当采用水混悬液时，其可包含乳化剂或混悬促进剂。也可以使用稀释剂如蔗糖、乙醇、聚乙二醇、丙二醇、甘油和氯仿或其混合物。
上述药物组合物可以适合的剂型施用于人和动物，通过局部或全身给药，包括口服给药、直肠给药、鼻腔给药、口腔给药、眼部给药、舌下给药、透皮给药、直肠给药、局部给药、***给药、肠胃外给药(包括皮下记注射、动脉注射、肌肉注射、静脉注射、皮内注射、鞘内注射、硬膜外注射)、脑池内给药和腹腔给药。应当理解的是优选的途径可随着例如接受者的情况而变化。
配方可按照药剂领域公知的任何方法制备成单位剂量。这种方法包括使活性成分和由一种或多种助剂组成的载体相结合。通常上述配方通过将活性成分与液体载体或极细固体载体或两者均匀且密切地结合而制备，然后如果需要对产品成形。以单次或多次剂量施用于对象的本发明的化合物的每日总剂。
本发明还提供上述胞外亲环素抑制剂在制备预防或治疗由亲环素介导的疾病的药物方面的应用，其中，由亲环素介导的疾病包括但不限于a)病毒感染、 b)代谢类疾病、c)急性和慢性炎症疾病、d)癌症、e)神经变性疾病、f)退化性肌肉疾病、g)心血管疾病、h)肥胖症、i)糖尿病。
本发明的有益效果：
本发明基于亲环素的活性口袋和已知的CsA构效关系，得到全新的一种4MCsA化合物，能够通过与Albumin高效反应后，将化合物作用限制在胞外，同时增加化合物的代谢时间，提高特异性，是胞外亲环素的有效抑制剂。该化合物或其相应的药用的盐的形式，可结合胞外亲环素，从而抑制慢性炎症的放大，在多种慢性炎症疾病中，如肿瘤、二型糖尿病和动脉粥样硬化等，展现出良好的治疗效果。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1显示4MCsA修饰的白蛋白的质谱图，其中，上图为BSA，下图是BSA-4MCsA偶合物；
图2显示4MCsA对BSA上Cysteine修饰位点的鉴定结果，其中，上图为BSA样品对应肽段的2+离子，下面为BSA-4MCsA对应肽段的3+离子；
图3显示CsA，4MCsA和硫酚封闭的4MCsA(Ar-S-4MCsA)的EIC图，I.F.代表细胞内提取部分，E.S.的是标准品的曲线，曲线下面积用于定量计算胞内浓度,图中阴影部分为I.F.曲线下面积；
图4为CsA-FITC荧光标记Jurkat细胞的竞争性流式细胞术图；
图5显示4MCsA分子的体外活性验证，其中，A为竞争性荧光偏振测定CsA(●)，4MCsA(■)和BSA-4MCsA(▲)对CypA的结合能力；B为CsA(●)，4MCsA(■)和BSA-4MCsA(▲)以剂量依赖的方式抑制CypA的顺反异构酶活；C为各抑制剂对应的K i和IC50；
图6显示4MCsA分子没有免疫抑制活性；其中，A为Western Blot检测分子对Jurkat细胞中由25nM PMA和1mg/mL离子霉素诱导的NFAT1的去磷酸化抑制(分子量偏下的条带)，B为ELISA检测Jurkat细胞活化后IL-2的分泌；
图7显示4MCsA抑制由胞外CypA刺激的炎症和单核细胞迁移；其中，A为CypA诱导THP-1细胞的IL-8表达，THP-1细胞被100nM CypA处理12h后，IL-8的mRNA水平显著提高，而4MCsA与CsA能同时抑制此效应；B为由CypA诱导的THP-1细胞迁移系数；C为细胞迁移实验中结晶紫染色的THP-1细胞代表图；
图8显示CsA和4MCsA对人肾细胞HEK293的细胞毒性作用；
图9显示T2DM小鼠模型中疾病状态及给与CypA抑制剂治疗后的血清CypA含量；
图10显示4MCsA和CSA以尾静脉注射的给药方式在DB鼠和HFD鼠中对血糖的控制，其中，a为DB鼠的禁食血糖；b为C57鼠的禁食血糖；c为DB鼠给药后的体重；d为HFD和DB鼠相对对应的C57对照的体重；e为HFD鼠的禁食血糖；f为4MCsA和CsA 5mg/kg/week对HFD的HbA1c的控制；
图11显示DB鼠中不同药物和剂量下对GTT的影响；
图12显示HFD鼠中4MCsA和CsA及其在给药后不同时间间隔对GTT的影响；
图13显示CsA和4MCsA对胰岛素的影响，其中，a为用药后DB鼠胰岛素基础水平，b为用药后DB鼠响应葡萄糖刺激的胰岛素分泌；
图14显示CsA和4MCsA对小鼠ITT实验的影响，其中a,c为DB鼠用药后的ITT；b,d为C57鼠中的ITT响应；e,f为HFD小鼠用药后的ITT；
图15显示DB鼠用药后血液中炎症因子的水平；a为IL1β的水平；b为CRP的水平；c为TNF-α的水平；d为IL-6的水平；
图16显示DB鼠在用药后肝肾毒性的评估结果；其中，a为谷草转氨酶AST的水平；b为谷丙转氨酶ALT的水平；C为尿素的水平；d为肌酐的水平；
图17显示肿瘤模型小鼠体内肿瘤体积的变化情况；
图18显示在肿瘤微环境中4MCsA对CD8 +T细胞的影响；
图19显示4MCsA对高脂饮食诱导的动脉粥样硬化小鼠主动脉病变程度的影响情况；
图20显示4MCsA对高脂饮食诱导的动脉粥样硬化小鼠血清中CRP含量的影响情况；
图21显示4MCsA对高脂饮食诱导的动脉粥样硬化小鼠血清中常规血脂指标的影响情况；其中，a为血清总胆固醇(TC)含量；b为血清甘油三酯(TG)含量；c为低密度脂蛋白胆固醇(LDL-C)含量；d为高密度脂蛋白胆固醇(HDL-C)含量；e为总胆固醇与高密度脂蛋白胆固醇含量比值(TC/HDL-C)；
图22显示长期注射4MCsA对高脂饮食诱导的动脉粥样硬化小鼠血清中ALT、AST含量的影响。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
本发明以4号位修饰maleimide(顺丁烯二酰亚胺)修饰的4MCsA为例，通过在CsA的4号位引入马来酰亚胺反应基团与BSA共价反应，能够实现将分子限制于胞外之功效。
为了选择性靶向胞外CypA，以往的修饰(MM284)是在环孢素的1位双键处引入负电荷基团。由于环孢素1位双键的存在，该位置修饰较为容易，然而该位置也是环孢素与亲环素结合的部位，不适合引入较大的基团。而且，这类CsA类似物通常在血液循环中肾脏快速清除，从而阻碍了进一步的临床应用。环孢素与亲环素的结合形式中P4位是位于远离亲环素的方向，是一个理想的修饰位点，然而该位点的修饰较为困难，需要较为复杂的合成路线。环孢素作为复杂的天然产物分子，分子量大于1000Da，分离纯化及反应中的杂质控制均具有较高的挑战。所以目前尚未有利用环孢素衍生物与蛋白偶联应用于药物应用。
本发明为获得环孢素4位与白蛋白偶联的活性产物，在环孢素4位引入迈克尔受体的衍生物分子3和8，并用于环孢素-白蛋白偶联，及活性验证工作。
本发明4MCsA的合成过程仅使用两步柱层析纯化，大大降低了操作难度，且极大的提高了收率
反应的合成路线如下：
Figure PCTCN2021121201-appb-000002
本发明的合成方案包括两个主要步骤：
第一个是从CsA(1)P3及P4位开环，并脱除P4位氨基酸的十肽4的合成过程。合成4的关键挑战是Edman降解步骤，大多数情况下会生成十一肽(1b)、十肽(4)和缬氨酸缺失的九肽的混合物。本发明使用过量(2.0-5.0当量)的PhNCS与1a充分反应，然后用DMAPA捕获未反应的PhNCS。通过这种方式，Edman降解后过量的DMAPA可以被洗掉，获得高纯度的中间体4。
第二个是十肽4与氨基酸Fmoc-N-Me-Lys(Boc)-OH(5)偶联，然后用NMe4OH去除Fmoc、乙酰基、并水解甲酯，最后用HATU进行大环化得到环肽[MeLys(Boc)4]CsA(6)。
[MeLys(Boc)4]CsA(6)在三氟乙酸(TFA)条件下脱除Boc保护得到[MeLys4]-CsA，然后与6-马来酰亚胺己酸-N-琥珀酰亚胺酯(7)偶联生成4MCsA(3)。
最终从CsA(1)出发经过10步反应，两步柱层析纯化，以30％的总产率合成4MCsA(3)。
实施例1 线性十肽的合成工艺
Figure PCTCN2021121201-appb-000003
将CsA(24.0g，20mmol，1.0当量)溶解在乙酸酐(80mL)和吡啶(80mL)的混合物中。在0℃下加入DMAP(0.8g，6.5mmol，0.33当量)并将反应搅拌过夜。LC/MS检测CsA完全消耗后，将反应液置于冰浴中，滴加冰水(100mL)，同时有白色固体沉淀。然后通过过滤分离出白色固体，将粗产物进一步溶解在DMF(72mL)中并通过加入水(72mL)沉淀出来。洗涤、过滤和干燥后，得到白色固体1a(23.2g，18.6mmol，93％)。C 64H 113N 11NaO 13 +([M+Na] +)的HRMS(ESI)m/z计算值：1266.8412；找到：1266.8446。
将1a(37.4g,30mmol,1.0equiv.)溶解在DCM(300mL)中并加入三甲氧基 四氟硼酸盐(13.3g,90mmol,3.0当量)。反应20小时后，加入乙腈和水，再反应3小时。将有机相分层，水洗3次，每次100mL。然后，将反应溶液浓缩并真空干燥。然后将白色结晶粉末在2-甲基四氢呋喃(100mL)和甲基叔丁基醚(50mL)的混合物中结晶以形成1b(29.9g,21.9mmol,73％)。C 65H 118N 11O 14 +([M+H] +)的HRMS(ESI)m/z计算值：1276.8854；找到：1276.8882。
Figure PCTCN2021121201-appb-000004
将1b(13.7g,10.0mmol)溶解在DCM(100mL)中，加入DIPEA(6.6mL,40mmol,4.0equiv.)和异硫氰酸苯酯(4.1g,30mmol,3.0equiv.)，反应搅拌2.0小时，加入N，N-二甲基-1，3-丙二胺(3.2mL，25mmol，2.5当量)，继续搅拌60分钟。然后加入甲醇(60mL)和50％氟硼酸水溶液(23g，130mmol)，LCMS检测直至反应完成。用DCM稀释反应，用1N HCl和盐水洗涤。合并有机相，浓缩，在2-甲基四氢呋喃(50mL)和甲基叔丁基醚(25mL)的混合溶液中重结晶，得到白色泡沫状产物4(11.2g，91％)。C 58H 105N 10O 13 +([M+H] +)的HRMS(ESI)m/z计算值：1149.7857；找到：1149.7902。
实施例2 从十肽到4MCsA的合成步骤
Figure PCTCN2021121201-appb-000005
4a：将线性十肽4(1.20g,0.97mmol,1.0equiv.)溶于10mL无水DMF中，搅拌下继续加入氨基酸Fmoc-N-Me-Lys(Boc)-OH(5)(468mg,0.97mmol,1.0当量).)和HATU(737mg,1.94mmol,2.0当量)。反应用冰浴将溶液冷却至0℃后，加入DIPEA(0.68mL，3.88mmol，4.0当量)并在室温下搅拌溶液过夜。通过LCMS检测反应完成后，通过加入1N HCl(10mL)溶液淬灭反应混合物，所得混合物用DCM(2×30mL)萃取。合并的有机层用盐水洗涤，并用硫酸钠干燥。真空浓缩萃取液，残留物溶于15mL甲醇。4a的表征数据：C 83H 135N 12O 17 +[M+H] +的MS(ESI)m/z计算值：1572.0，实测值1572.0。
6：向4a的甲醇溶液中加入5mL NMe 4OH(25％甲醇溶液)，并在室温下搅拌反应。在LCMS检测到P1位的乙酰基和N端Fmoc去除后，将0.5mL水加入溶液中并搅拌30分钟以水解P3位甲酯。反应完成后，通过加入1N HCl将pH调节至5.0来淬灭反应，并用DCM萃取所得混合物。合并的有机层用盐水洗涤，并经硫酸钠干燥。在真空下浓缩萃取液的溶剂并重新溶解在200mL无水DCM中。然后将HATU(1.32克，3.47毫摩尔，3.85当量)加入到搅拌的溶液中。HATU溶解后，冰浴冷却至0℃，滴加N-甲基吗啉(0.76mL，6.94mmol，7.7当量)。通过LCMS检测反应完成后，反应混合物用1N HCl、水和盐水洗涤。有机层用硫酸钠干燥，真空浓缩，残余物通过硅胶快速色谱纯化(DCM:MeOH＝20:1)，得到白色形式的6(992mg，78％产率)。
6的表征数据：1H NMR(500MHz,CDCl 3)δ7.88(d,J＝9.6Hz,1H),7.75(d,J＝7.3Hz,1H),7.43(d,J＝8.4Hz,1H),7.15(d,J＝7.8Hz,1H),3.49(s,3H),3.36(s,3H),3.22(s,3H),3.12(s,3H),3.07(s,3H),2.72(s),3H),2.69(s,3H)。HRMS(ESI)m/z：C 67H 120N 12NaO 14 +([M+Na] +)的计算值：1339.8939；找到：1339.8937。
4MCsA(3):6(89mg,66.4mol,1.0当量)溶解在2mL DCM中，然后加入650L TFA，搅拌30分钟。然后将溶液浓缩并将残余物溶解在DCM中，用水、饱和NaHCO 3和盐水洗涤。有机层用无水硫酸钠干燥并真空浓缩。将残余物溶解在1.0mL无水DCM中，然后加入N-琥珀酰亚胺基-6-马来酰亚胺己酸酯 (7)(31mg，0.1mmol，1.5当量)和Et 3N(28μL，0.2mmol，3.0当量)。将溶液搅拌过夜。然后用10mL DCM稀释反应混合物并用1N HCl、水和盐水洗涤。有机层经硫酸钠干燥，真空浓缩，残余物经HPLC纯化，得到白色形式的4MCsA(3)(58mg，41.1μmol，62％产率)。
4MCsA(3)的表征数据：HPLC纯化方法，梯度(70％MeOH 10min，70％-100％10-25min，100％25-35min)，Rt＝32.6min。1H NMR(500MHz,CDCl 3)δ7.91(d,J＝9.4Hz,1H),7.72(d,J＝7.3Hz,1H),7.52(d,J＝8.4Hz,1H),7.20(d,J＝7.8Hz,1H),6.68(s,3H),3.49(m,6H),3.36(s,3H),3.21(s,6H),3.12(s,3H),3.09(s,3H),2.71(s,3H),2.69(s,3H)。C 72H 123N 13NaO 15 +[M+Na] +的HRMS(ESI)m/z计算值：1432.9154，实测值1432.9165。
实施例3 化合物8的合成
Figure PCTCN2021121201-appb-000006
8a：将化合物6在TFA条件下脱除Boc保护所得产物50mg,41.06μmol)，9(28.03mg,82.13μmol)以及HATU(31.23mg,82.13μmol)溶于3ml DCM中，随后在搅拌下加入DIPEA(21.23mg,164.25μmol,28.61μL)并反应过夜。使用柱层析纯化得淡黄色固体产物8a(50mg,32.45μmol,79％产率)。
8：将8a(12mg,7.79μmol)溶于MeOH(2mL)并随后加入碳酸钾(2.15mg,15.57μmol,0.94μL)，室温下反应至原料完全转化。旋转蒸发除去溶剂 后，产物直接溶于DCM。随后加入10(1.37mg,15.17μmol,1.25μL)及DIPEA(1.96mg,15.17μmol,2.64μL)。反应完成后，萃取并柱层析得到产物9(10.3mg，7.4μmol，95％)。
4MCsA-2(8)的表征数据：1H NMR(500MHz,CDCl3)δ7.97(d,J＝9.4Hz,1H),7.67(d,J＝7.3Hz,1H),7.48(d,J＝8.4Hz,1H),7.15(d,J＝7.8Hz,1H)。C 69H 121N 13NaO 15 +[M+Na]+的HRMS(ESI)m/z计算值：1394.8997，实测值1394.9006。
实施例4 CsA-FITC探针的合成路线
Figure PCTCN2021121201-appb-000007
CsA-FITC的表征数据：HPLC法，梯度(30％-100％ACN，0-25min)，柱温70℃，R t＝16.9分钟。C 93H 134N 14NaO 19S +[M+Na] +的HRMS(ESI)m/z计算值：1805.9563，实测值1805.9536。
实施例5 验证4MCsA与Albumin的反应性
分子与albumin的反应性经体外结合后通过Q Exactive HFX mass spectrometer(Thermo Fisher)质谱仪使用完整蛋白方法测定。
在体外以4MCsA：BSA＝2:1的比例在室温反应1小时，随后使用超滤管离心去除过量的分子和盐分，将样品加载到反相苯基柱(MAbPac，Thermo Fisher，088648)上，经过LC-ESI-MS采集数据，使用BioPharma Finder软件(Thermo Fisher)通过原始质谱的解卷积解析完整蛋白质质量。
如图1所示，4MCsA使得白蛋白的分子量增加1410Da，4MCsA能够有效 共价结合白蛋白，化学计量比为1:1，有良好的反应性。
实施例6 验证4MCsA与Albumin半胱氨酸反应位点
取实施例4中的复合物，经过尿素变性后，以胰酶：蛋白＝1:50比例加入稀释后的蛋白样品，37℃酶解18h后，使用
Figure PCTCN2021121201-appb-000008
C 18Spin Columns(Thermo Fisher)脱盐小柱对肽段样品除盐，洗脱物使用70％乙腈洗脱后，冻干，使用0.1％甲酸水溶解，LC-ESI-MS分析肽段，使用BioPharmaFinder软件(Thermo Fisher)分析修饰位点。
如图2所示，bottom-up肽段鉴定到具有4MCsA修饰肽段[K].GLVLIAFSQYLQQcPFDEHVK.[L]的肽指纹图谱，显示Cys34修饰上4MCsA分子。4MCsA分子与BSA快速且特异的反应性为其细胞非穿膜性提供了支撑。
实施例7 高分辨质谱测定分子在Jurkat细胞的穿膜性
为了直接检测分子的穿透性，采用QE-Focus质谱仪对细胞内的化合物浓度进行了外标法定量。
将1×10 8Jurkat细胞加入到含有5μM CsA、4MCsA和硫酚封闭的4MCsA的RPMI1640培养基中，37℃孵育2小时，随后使用甲醇提取胞内的化合物组分进行分析，使用5μM对应分子的甲醇溶液作为外标，提取液进行质谱分析。选择对应分子的m/z信号对色谱图进行峰提取，得到对应分子提取的离子色谱图(EIC)，随后用标准品和细胞提取物的EIC图的曲线下峰面积对提取物浓度进行定量。结果见图3、表1。
表1 Jurkat细胞内化合物含量
Figure PCTCN2021121201-appb-000009
结果如表1、图3所示，CsA由于与细胞内丰富的CypA结合，在胞内存 在富集作用，胞内CsA的浓度比胞外更高(7.2μM vs 5μM)。4MCsA在胞内没有检出，然而经过疏水的硫酚封闭的4MCsA能够更容易的进入细胞内。硫酚封闭的4MCsA分子中的马来酰亚胺基团和硫酚反应后，不能再结合白蛋白，同时细胞穿膜性甚至比CsA更高，说明4MCsA中的马来酰亚胺基团对于分子的细胞非穿膜性至关重要。4MCsA被有效的限制在了细胞外。
实施例8 荧光竞争法检测分子在Jurkat细胞中穿膜性的测定
为了进一步比较CsA和4MCsA的细胞穿膜性，本实施例采用一种结合胞内CypA的CsA-FITC荧光探针，通过流式细胞术，使用竞争法检测了化合物对胞内荧光信号的影响。
结果如图4所示，CsA-FITC能有效结合胞内的CypA使得细胞标记上荧光，而CsA进入细胞后与CsA-FITC探针竞争结合胞内CypA，进而大幅度减少了荧光细胞比例(从95.30％到6.75％的水平)。然而相比之下，4MCsA对胞内荧光信号的影响非常小。
结合实施例6的结果，4MCsA实现了将分子局限于细胞外的作用，是后续靶向胞外eCypA的有效分子。
实施例9 测定4MCsA及复合物对肽酰-脯氨酰-顺式/反式-异构酶(PPI酶)结合能力
荧光偏振(FP)是一种广泛使用的测定溶液体系下配体受体结合的技术。自由状态的游离荧光探针在体系中会快速转动，所发射的荧光大多为去偏振光(depolarized light)。而一旦探针结合更大的分子，分子转动就会受到限制，此时偏振光的发射会增加。
本发明合成了一种用异硫氰酸荧光素标记的CsA类似物作为荧光示踪剂探针(CsA-FITC)，使用竞争荧光偏振测定法测定配体对CypA的亲和力。首先确定CsA-FITC与CypA的结合常数K d＝14.81±0.70nM。进一步使用50nM CypA和不同浓度抑制剂结合，随后加入20nM CsA-FITC探针，测得竞争荧光 偏振曲线。
结果如图5所示，CsA和4MCsA与重组CypA的K i分别为195.8±25.7nM和197.8±17nM。同时BSA-4MCsA复合物对CypA的K i为86.9±6.2nM，三者与CypA的结合能力接近，说明4MCsA及4MCsA与BSA的偶联不影响抑制剂与CypA的结合，活性和CsA相当。
实施例10 4MCsA及复合物对肽酰-脯氨酰-顺式/反式-异构酶(PPI酶)活性的抑制
使用糜蛋白水解N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide底物肽，测定抑制剂对CypA酶活力影响。底物肽的脯氨酸在特定的组分体系中主要为顺式，而糜蛋白酶此时切割效率较低，当经过脯氨酸顺反异构酶催化后，其构型由顺式转为反式，随后能够被糜蛋白酶特异性地切割，并释放出p-nitroanilide，在390nm附近有最大吸收。因此CypA酶活越强则底物降解速度越快，390nm的吸光值增加越快。使用50nM CypA与不同浓度的抑制剂结合后，加入250μg/mL糜蛋白酶，最后使用带自动进样器的酶标仪加入80μM超干的底物肽溶液(470mM LiCL的三氟乙醇溶解)启动反应，进行动力学监测。
如图5所示，CypA与不同浓度的抑制剂分子孵育后，其酶活被显著抑制，拟合曲线得4MCsA、CsA和BSA-4MCsA的IC50分别为22.55±4.30nM,17.16±4.09nM和21.87±3.16nM，进一步说明了4MCsA及其与BSA的偶联物能有效抑制CypA酶活，并与CsA的效率相当，这和结合活力的测定一致，证明4MCsA偶联BSA后能正常发挥靶向CypA的抑制作用
实施例11 检测NFAT信号转导中的非免疫抑制作用
CsA的免疫抑制作用主要是由于分子进入胞内后与胞内CypA形成CypA/CsA复合物，随后结合钙调磷酸酶，阻碍了该酶对NFAT蛋白的去磷酸化的活化。
基于此，本发明研究了分子对于由离子霉素和PMA引起的Jurkat T细胞活 化过程中NFAT信号转导的影响。将1×10 6的Jurkat细胞种于预先加入CsA,4MCsA的RPMI1640培养基(含10％血清)中孵育1.5h,随后使用25nM佛波酯PMA和1μg/mL离子霉素处理2h诱导Jurkat细胞的活化，收集细胞后，经过提取，电泳，转印，NFAT抗体检测NFAT蛋白的去磷酸化水平。NFAT去磷酸化后会出现磷酸化和非磷酸化两条带。
如图6所示，CsA显著地抑制了由PMA和离子霉素诱导的NFAT的去磷酸化，而细胞非穿透性的4MCsA没有明显的效应。
同时，本发明进一步检测了NFAT参与的T细胞活化的下游IL-2蛋白的分泌，在PMA和离子霉素刺激24h后使用ELISA方法测定培养基中IL-2蛋白的含量。与Westernblot结果一致，0.2μM CsA即可显著抑制IL-2的分泌，而4MCsA浓度达到5μM也几乎不影响IL-2的分泌。以上结果说明，4MCsA没有免疫抑制作用，这一点和其被有效限制于细胞外一致，从而不能与胞内CypA形成复合物干扰钙调磷酸酶和NFAT的活化。
实施例12 4MCsA对炎性细胞的影响
本发明首先验证4MCsA对由CypA促进的促炎因子IL-8的分泌。
采用药物分子与培养基预孵后使用CypA蛋白刺激细胞的方法，检测4MCsA和CsA对CypA刺激的促炎因子IL-8的转录变化，以及炎症细胞的迁移。将4MCsA和CsA与含0.1％BSA的RPMI1640培养基预孵1小时，然后用CypA同时刺激THP1细胞12小时，RT-PCR方法检测IL-8的转录水平变化；在Transwell迁移实验中，在下室中将4MCsA和CsA与含0.1％BSA的RPMI1640培养基预孵1小时，然后添加200ng/mL CypA，并向上室添加1.0×10 5的THP1细胞，37℃培养1小时，然后将小室下层膜进行结晶紫染色后，拍照记录迁移细胞数量。
如图7所示，胞外CypA显著提高了IL-8的mRNA的转录水平，此效应能被4MCsA和CsA显著抑制。随后本发明使用Transwell验证4MCsA对CypA诱导的THP-1细胞迁移的抑制。CypA促进的迁移活性，能显著地被4MCsA和 CsA消除。以上结果表明，4MCsA和CsA都能靶向CypA的酶活口袋，4MCsA能有效干预由胞外CypA酶活口袋介导的炎症效应和白细胞迁移。
实施例13 4MCsA与天然环孢菌素的细胞毒性比较
本实施例对比了CsA和非细胞穿透性的4MCsA对正常HEK293人肾脏细胞的细胞毒性作用。
为了检验药物的细胞毒性，采用MTT法检测4MCsA和CsA处理后细胞的杀伤效果。将HEK293细胞以1.0×10 4/孔的密度接种到96孔板，待细胞贴壁生长至适宜密度；同时，配置4MCsA和CsA的梯度浓度药物，与含4％BSA的培养基预孵1小时，随后将96孔板培养基替换为药物预孵培养基，继续培养30小时，MTT法检测细胞存活率。如图8所示，4MCsA浓度到达50μM也几乎没有细胞毒性，相比之下CsA显著抑制了HEK293细胞的增殖，随着浓度的增加呈现了浓度依赖的细胞毒性作用。这与CsA在临床使用中所出现的肝肾脏毒性作用一致，而4MCsA在此方面体现了巨大的优势。
实施例14 4MCsA用药能够降低糖尿病模型中CypA
本实施例选用瘦素受体缺失的db/db基因工程鼠(DB鼠)作为T2DM模型检测了药物对小鼠血清CypA的含量影响(C57小鼠作为健康对照组)。采用尾静脉注射方式，以5mg/kg每周剂量的CsA或4MCsA和1mg/kg每周的4MCsA对DB和C57小鼠用药3周后，采集小鼠血清，使用ELISA检测血液CypA的含量。
如图9所示，糖尿病模型小鼠在用药后血液CypA含量降低，表明4MCsA能够阻断胞外亲环素在炎症和ROS循环放大过程中的作用，同时减少由该放大过程中CypA的分泌。
实施例15 4MCsA减少T2DM小鼠模型的禁食血糖
为有效评估药物在T2DM中的作用效果，本实施例选择DB鼠和高脂饮食 诱导的肥胖小鼠模型(HFD鼠)，作为2种不同诱因的2型糖尿病小鼠模型为研究对象。DB模型分别设置对照组Ctrl，4MCsA 5mg/kg/week组DB 4MC5，4MCsA 1mg/kg/week组DB 4MC1，CsA 5mg/kg/week组DB CsA5)；HFD模型中4MCsA和CsA给药剂量都为5mg/kg，两种模型都采用每周1次尾静脉注射的方式给药。
如图10所示，4MCsA用药后，相比于CsA，能够显著降低DB和HFD鼠的禁食血糖，降低糖化血红蛋白含量，表明4MCsA能长期有效的控制糖尿病的血糖。另外值得注意的是，在对正常C57小鼠用药5周后，CsA 5mg/kg/week实验组的禁食血糖略高于C57ctrl组，而4MCsA 5mg/kg/week和正常组相当。
实施例16 4MCsA改善T2DM小鼠模型葡萄糖耐受
对实施例15中两种模型给药鼠，进行葡萄糖耐受实验(GTT)，检测小鼠对葡萄糖的控制能力。给药5周后，将小鼠禁食16小时，随后给小鼠以腹腔注射的方式注射葡萄糖(DB小鼠剂量为1g/kg，HFD小鼠剂量为2g/kg)，随后在不同的时间点采血，检测血糖含量。对应时间点的血样同时保存用于后续葡糖糖刺激的胰岛素检测。
如图11所示，4MCsA和CsA 5mg/kg都能明显的改善DB鼠葡萄糖耐受的状态(图a-b)。如图e所示，相比于Ctrl组，5mg/kg的用药组曲线下面积(AUC)显著降低，其葡萄糖在体内的清除速度更快。在C57组的GTT曲线和其AUC(图d和f)中，可观察到CsA使C57健康小鼠的AUC显著增加，而4MCsA组的AUC相对于CsA组增加较少，说明CsA用药对健康小鼠的葡萄糖耐受可能有一定损害，4MCsA影响相对更小。
在HFD模型中，针对CsA和4MCsA对T2DM小鼠GTT的改善做了更进一步的研究。
如图12所示，在HFD模型中，在给药第23天的36hrs内测得4MCsA和CsA对HFD的GTT都有明显的改善，AUC显著下降。在给药第17天的60小时内测得结果为CsA对HFD的GTT没有作用，而4MCsA仍然明显改善，显著降低AUC。上述结果表明，4MCsA有更长的代谢时间，能够通过对胞外 eCypA的持续控制，从而长时间改善炎症和ROS状态，始终发挥药效。
实施例17 4MCsA对葡萄糖刺激的胰岛素分泌的影响
取实施例16中葡糖糖注射后的对应时间点的血样，使用ELISA的方式检测其中胰岛素的含量，得到葡萄糖刺激的胰岛素分泌结果。
如图13所示，DB鼠体内的胰岛素基础水平显著高于C57健康组，用药后CsA和4MCsA对DB鼠基础的胰岛素水平影响不大(图a左)，但是在C57健康组中4MCsA组的胰岛素略高于CsA组(图a右)。在给与葡萄糖刺激后，4MCsA和CsA对DB鼠不同阶段刺激的胰岛素分泌与DB ctrl组比无显著差别(图b左)。4MCsA和CsA 5mg/kg/day的使用不改变DB鼠的胰岛素分泌，所以DB鼠GTT的改善是通过影响胰岛素信号响应造成的。但是在健康C57小鼠中(图b右)，仍然能观察到4MCsA相比C57ctrl能够更多的增加葡萄糖刺激的胰岛素分泌，而CsA组则减少了了葡萄糖刺激的胰岛素产生，说明在GTT实验中C57用CsA后的影响。从而，进一步证明本发明的4MCsA优于CsA。
实施例18 4MCsA对胰岛素敏感性的影响
对实施例15中两种小鼠分别进行胰岛素敏感性实验(胰岛素耐受实验，ITT)，进一步测定胰岛素信号响应。小鼠禁食6小时后，采用腹腔注射的方式注射胰岛素(DB小鼠剂量为2U/kg,HFD小鼠剂量为1U/kg)，随后检测不同时间点的血糖含量。
如图14所示，4MCsA和CsA都能明显改善DB胰岛素敏感性，同时在健康C57中也观察到用药后二者对ITT有改善。4MCsA在DB鼠中对ITT的影响有一定的剂量效应(如图a-d)。在HFD模型的小鼠中，4MCsA和CsA同样能削弱由肥胖引起的胰岛素耐受，改善ITT，并且4MCsA相较于CsA的AUC更低，效果更加显著(如图e-f)。
实施例19 4MCsA对DB鼠炎症因子的影响
实施例15中的DB模型鼠(对照组Ctrl，4MCsA 5mg/kg/week组DB 4MC5，4MCsA 1mg/kg/week组DB 4MC1，CsA 5mg/kg/week组DB CsA5)给药42天后，采集血液，通过ELISA测定血清炎症因子水平。
如图15所示，4MCsA用药42天后能显著降低DB鼠中炎症因子TNF-a和IL1β的水平，而CsA仅改善IL1β。可以看到的是4MCsA能够降低小鼠炎症，且潜能高于CSA，由此可以减少炎症导致的胰岛素信号受损，提高胰岛素敏感性，从而改善小鼠糖尿病状态。
实施例20 考察4MCsA和CSA对DB鼠肝肾毒性的影响
用药50天后，取实施例15中两种模型小鼠对应给药组的血清，使用南京建成生化试剂盒，检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)的含量(肝脏毒性指标)和肾毒指标(肌酐和尿素)。
如图16中a和b所示，DB鼠相较于C57健康鼠的ALT和AST都显著增加，这可能是肥胖小鼠容易得非酒精性脂肪肝导致这些肝脏指标上升，而用4MCsA和CsA没有进一步提升DB鼠的肝肾毒性(图16中c和d)。同样4MCsA和CSA也未产生明显的肾脏毒性，其尿素和肌酐水平在组间没有差异。然而仍然可以看到，这里在C57健康小鼠中，50天长期用药后，CsA用药组的AST、ALT和尿素水平均显著提高，而4MCsA均保持接近健康对照的水平。该结果更加直接的表明本发明的4MCsA在降低毒性方面的优点。
在DB鼠和HFD两种T2DM模型鼠中，对比4MCsA和CsA在改善T2DM的效果。在疾病模型中，以低剂量长周期给药的方式，二者均能够降低禁食血糖，改善葡萄糖耐受，提高胰岛素敏感性，同时不影响胰岛素分泌和其他肝肾毒性的改变。在这些指标中，4MCsA均表现出更大的潜力，相对CsA更长期持续的控制，改善效果更明显，起效时间更早，毒性更低。
另外值得注意的是，由于CsA已被报道在糖尿病对象中代谢速度会更快，所以对健康状态的对象来说，它有着潜在的危害，这一点也在发明的实验中多次得以证明：健康C57小鼠长期用药后会损害其GTT，减少胰岛素的分泌，提高血糖水平，增加肝肾毒性。而经过我们设计的4MCsA对健康C57中的这些 不良反应显著降低。
实施例21 4MCsA在小鼠结肠癌肿瘤模型中发挥抗肿瘤效应
本实施例建立了小鼠肿瘤模型，通过测量肿瘤的大小来反映药物的作用效果。
在Balb/C小鼠的背侧边皮下接种CT26-LUC小鼠结肠癌细胞，接种量为106/只，六天之后肿瘤大小约为100mm 3，开始用游标卡尺测量同时给药，4MCsA的给药剂量为5mg/kg，每天给药，两天测量一次肿瘤，统计肿瘤大小。三周之后结束实验。
实验结果如图17所示，4MCsA表现出一定的抗肿瘤效应，具有作为抗肿瘤药物的潜力。
实施例22 考察4MCsA提高肿瘤微环境中CD8 T细胞的浸润
冰冻切片的厚度要求在4～8μm，从-80℃拿出后室温放置30分钟，4℃下丙酮固定10分钟，PBS洗，5分钟×3；5～10％正常山羊血清(PBS稀释)封闭，室温孵育10分钟。倾去血清，勿洗，滴加适当比例稀释的一抗或一抗工作液，37℃孵育1～2小时或4℃过夜。PBS冲洗，5分钟×3次；滴加适当比例稀释的生物素标记二抗(1％BSA-PBS稀释)，37℃孵育90-120分钟；或第二代辣根酶标记链霉卵白素工作液，37℃或室温孵育10～30分钟；PBS冲洗，5分钟×3次,封片后用倒置荧光显微镜拍照。最后用imageJ进行统计分析。
结果如图18所示，4MCsA能够促进肿瘤微环境中CD8 T细胞的浸润，从而达到肿瘤抑制效果。
实施例23 4MCsA对高脂饮食诱导的动脉粥样硬化小鼠主动脉病变程度的影响
选取5周龄的ApoE基因缺陷的C57BL/6J品系小鼠，随机分组后采用Western diet饮食进行高脂诱导，从第7周开始，采取尾静脉注射的方式对实验 组进行4MCsA的给药，剂量为5mg/kg/week，给药周期为9周。实验结束后，将实验动物安乐死，解剖后，从右心耳先灌注10mLPBS，再灌注10mL组织固定液(4％多聚甲醛)，在体视镜下将主动脉完整分离出来，并用脂肪镊剥离血管外黏附脂肪组织。采用油红O染色的方法对分离出的主动脉进行大体染色，拍照记录，使用ImageJ统计实验组和对照组动物主动脉弓病变面积，并统计差异显著性。
如图19所示，在对建模成功的动脉粥样硬化小鼠进行周期性间歇给药后，相对于对照组(ApoE-/-),给药组(4MCsA)主动脉弓的病变面积显著减少，说明4MCsA能够直接改善血管斑块病变的发生。
实施例24 4MCsA对高脂饮食诱导的动脉粥样硬化小鼠血清中CRP含量的影响
在动脉粥样硬化小鼠建模成功后，采用如实施例23中描述方法，对实验小鼠进行每周一次尾静脉注射5mg/kg 4MCsA,连续给药9周，通过检测小鼠血清中CRP水平，评估机体心血管疾病的发生风险，从而评价化合物对机体心血管疾病发生的影响。
如图20所示，与对照组(ApoE-/-)相比，4MCsA能够极显著降低实验组小鼠血清中CRP的含量，说明4MCsA对机体心血管疾病的发生具有非常好的预测改善作用。
实施例25 4MCsA对高脂饮食诱导的动脉粥样硬化小鼠血清中常规血脂指标的影响
采用如实施例23所述方法进行高脂饮食诱导小鼠动脉粥样硬化模型，并按照分组进行给药，每周一次尾静脉注射5mg/kg的4MCsA或安慰剂，连续9周给药后，对小鼠进行安乐死，并取血清，进行常规血脂含量的检测。
结果如图21所示，与对照组相比，4MCsA可以显著降低机体血清中LDL-C含量，一定程度上增加HDL-C的含量，但无显著性，而对TC和TG的含量无 明显影响；除此之外，计算结果显示，4MCsA可以显著降低机体TC/HDL-C，从而对动脉粥样硬化、冠心病等心血管疾病具有一定的预测改善作用。
实施例26 长期注射4MCsA对高脂饮食诱导的动脉粥样硬化小鼠血清中ALT、AST含量的影响
采用如实施例25所描述方法进行小鼠动脉粥样硬化模型诱导和给药，在动物实验周期结束后，将小鼠安乐死，并取血清，检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)的含量。
如图22所示，与对照组相比，实验组的血清中ALT、AST活性无显著变化，说明周期性间歇尾静脉注射4MCsA未对机体造成明显的肝脏毒性。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (10)

  1. 一种胞外亲环素抑制剂，其特征在于，其结构如式(I)所示：
    Figure PCTCN2021121201-appb-100001
    其中，R 1为-CH＝CHR 1’或-CH 2CH 2R 1’；R 1’选自烷基、羧基、乙酰氨基或苯基；
    R 2为H、SR 2’、CH 2SR 2’或CH 2OR 2’，其中，R 2’选自烷基、羧基、羟基、乙酰氨基或苯基；
    linker选自-(CH 2) x-NH(C＝O)-(CH 2) y-或-(CH 2) x-NH(C＝O)-(CH 2CH 2O) y-，其中，x为1-4的整数，y为1-6的整数。
  2. 根据权利要求1所述的胞外亲环素抑制剂，其特征在于，R 1’选自-CH 3、-(CH 2) n-COOH、-(CH 2) n-NH(C＝O)CH 3、-苯基或环烷基，其中，n为1-6的整数。
  3. 根据权利要求2所述的胞外亲环素抑制剂，其特征在于，所述苯基被以下基团一次或多次相同或不同地取代：-COOCH 3、-CH 2NH(C＝O)CH 3。
  4. 根据权利要求2或3所述的胞外亲环素抑制剂，其特征在于，所述环烷基为环丙基。
  5. 根据权利要求1所述的胞外亲环素抑制剂，其特征在于，R 2’选自-CH 3、-(CH 2) n-COOH、-(CH 2) n-OH、-(CH 2) n-NH(C＝O)CH 3、-苯基或环烷基，n为1-6的整数。
  6. 根据权利要求5所述的胞外亲环素抑制剂，其特征在于，所述苯基被以下基团一次或多次相同或不同地取代：-COOCH 3、-CH 2NH(C＝O)CH 3。
  7. 根据权利要求1所述的胞外亲环素抑制剂，其特征在于，R 1为-CH＝CHCH 3，R 2为H，linker选自-(CH 2) x-NH(C＝O)-(CH 2) y-或-(CH 2) x-NH(C＝O)-(CH 2CH 2O) y-，其中，x为1-4的整数，y为1-6的整数。
  8. 根据权利要求1或7所述的胞外亲环素抑制剂，其特征在于，R 1为-CH＝CHCH 3，R 2为H，linker为-(CH 2) x-NH(C＝O)-(CH 2) y-，其中，x为2或3，y为3、4或5。
  9. 权利要求1～8任一项所述的胞外亲环素抑制剂及其非毒性药学上可接受的盐作为活性成分的药物组合物。
  10. 权利要求1～8任一项所述的胞外亲环素抑制剂在制备预防或治疗亲环素介导的疾病的药物方面的应用。
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