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WO2021042732A1 - 成骨生长肽碳端五肽衍生物及其制备方法和应用 - Google Patents

成骨生长肽碳端五肽衍生物及其制备方法和应用 Download PDF

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WO2021042732A1
WO2021042732A1 PCT/CN2020/085363 CN2020085363W WO2021042732A1 WO 2021042732 A1 WO2021042732 A1 WO 2021042732A1 CN 2020085363 W CN2020085363 W CN 2020085363W WO 2021042732 A1 WO2021042732 A1 WO 2021042732A1
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gly
ogp
tyr
phe
fmoc
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PCT/CN2020/085363
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French (fr)
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张腾
张忠旗
郭添
刘少军
李晨召
李乾
王昕�
赵金礼
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陕西慧康生物科技有限责任公司
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Priority to CN202080004195.0A priority Critical patent/CN112789286B/zh
Publication of WO2021042732A1 publication Critical patent/WO2021042732A1/zh

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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
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/52—Cyclic peptides containing at least one abnormal peptide link with only normal peptide links in the ring
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the technical field of polypeptides, and specifically relates to an osteogenic growth peptide carbon-terminal pentapeptide OGP (10-14) derivative, and a preparation method and application thereof.
  • Osteogenic Growth Peptide is a kind of polypeptide growth factor, which has the effect of promoting bone formation and stimulating hematopoiesis.
  • the primary structure is ALKRQGRTLYGFGG.
  • OGP (10-14) is the smallest fragment that maintains the full activity of the osteogenic growth peptide, and the amino acid sequence is H-Tyr10-Gly11-Phe12-Gly13-Gly14-OH.
  • OGP (10-14) not only improves the treatment of bone injury and bone metabolism diseases, but also restores the hematopoietic function of patients with radiotherapy and chemotherapy and bone marrow transplant patients.
  • the Italian pharmaceutical company Abiogen has submitted an application to the European Medicines Agency (EMEA) for OGP (10-14) as an orphan drug for the treatment of chronic idiopathic myelofibrosis (CIMF) disease, and it has been approved.
  • EMEA European Medicines Agency
  • Structure-activity relationship studies have shown that, except for Gly13 and Gly14, the other amino acid side chains are pharmacophores that may affect the activity.
  • adjusting the position and number of active sites and constructing cyclic peptides of different configurations can increase the biological activity of the pentapeptide structure or improve its metabolic stability.
  • OGP(10-14) the only report on the structural modification of OGP(10-14) is the modification study of straight-chain OGP(10-14) with no amino group at the nitrogen end by Bab et al. Because OGP (10-14) itself has homology and high conservation, only by modifying the original peptide structure, it is possible to obtain safer and more effective compounds.
  • An object of the present invention is to provide a new OGP(10-14) pentapeptide derivative, which improves the biological activity, improves the proliferation activity of MC3T3E1 osteoblasts and/or NIH3T3 fibroblasts, and/or improves pepsin and/ Or trypsin stability.
  • Another object of the present invention is to provide a method for preparing the OGP(10-14) pentapeptide derivative.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the OGP(10-14) pentapeptide derivative.
  • Another object of the present invention is to provide the application of the OGP(10-14) pentapeptide derivative and the pharmaceutical composition in the preparation of a medicine for treating and/or preventing bone injury.
  • Another object of the present invention is to provide the application of the OGP(10-14) pentapeptide derivative in the preparation of medicines for the treatment and/or prevention of osteometastatic diseases.
  • Another object of the present invention is to provide the application of the OGP(10-14) pentapeptide derivative in the preparation of drugs for the treatment and/or prevention of chronic idiopathic myelofibrosis.
  • the present invention provides an OGP(10-14) pentapeptide derivative, which includes one or more of the following compounds:
  • osteogenic growth peptide As an active candidate, osteogenic growth peptide has the same characteristics as its polypeptide drugs. On the one hand, it has the advantages of simple structure, significant activity, low immunogenicity and good safety, and has the advantages of new drug development; on the other hand, due to its own conformation, it has the disadvantages of low oral utilization, high enzyme degradation, and short half-life.
  • the main reason for these unstable factors is the conformation of specific amino acids in the structure. For example, the connection sequence of the main chain and the types of side chain residues will affect its biological activity. Since the recognition function of OGP and substrate is not clear, it cannot simulate the receptor for drug screening. Therefore, structural modification and structure-activity relationship study is an effective way to improve its physical and chemical properties and metabolic stability.
  • OGP (10-14) pentapeptide derivatives provided by the present invention
  • 2 kinds of linear OGP derivatives and 5 kinds of cyclic OGP derivatives are respectively modified.
  • the original pharmacophore groups (Tyr10, Phe12) of OGP(10-14) have the structural characteristics of improving metabolic stability.
  • the OGP(10-14) pentapeptide derivative of the present invention has undergone cell activity experiments and enzymatic hydrolysis experiments.
  • OGP (10-14) has the biological activity and metabolic stability of OGP (10-14), has stronger in vitro proliferation activity than OGP (10-14), has higher stability to pepsin and trypsin, and can be used in treatment as an active ingredient Or it has a wide range of applications in preventing fracture damage and adjusting bone metabolism imbalance.
  • the present invention also provides a preparation method of the above-mentioned OGP(10-14) pentapeptide derivative, which includes:
  • the peptide resin obtained after the peptide chain is connected is cleaved with an aqueous solution of trifluoroacetic acid to obtain a crude peptide;
  • the crude peptide was purified by reversed-phase high performance liquid chromatography to obtain OGP(10-14) pentapeptide derivative.
  • 2-CTC Resin (2-Chlorotrityl Chloride Resin) is used as the solid-phase carrier in the solid-phase synthesis method.
  • the present invention also provides a pharmaceutical composition, which comprises the above-mentioned OGP(10-14) pentapeptide derivative and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be a solid excipient or a liquid excipient, for example, an organic or inorganic solid or liquid excipient suitable for gastrointestinal administration.
  • the pharmaceutically acceptable carrier may include stabilizers, wetting agents, solubilizers or other commonly used auxiliary materials and/or additives, such as lactose, talc, cellulose, polyvinylpyrrolidone, starch, pectin, Tween-80, polyvinyl alcohol and the like.
  • the existence form of the pharmaceutical composition is a solid form and/or a liquid form;
  • the solid form includes a tablet, a granule, or a capsule;
  • the liquid form includes a suspension, a syrup Agent or emulsion.
  • the active ingredient OGP (10-14) pentapeptide derivative of the present invention is made into various dosage forms of oral drugs, which are taken orally for adults, and the usual dose is 10 -9 to 10 -11 mg once a day, taken after meals, and for children. Decrease as appropriate. Indications: treatment and prevention of bone injury, bone metabolism disease, chronic idiopathic myelofibrosis disease.
  • the present invention also provides the application of the above-mentioned OGP (10-14) pentapeptide derivative or pharmaceutical composition in the preparation of a medicine for treating and/or preventing bone injury.
  • the present invention also provides the application of the above-mentioned OGP(10-14) pentapeptide derivative or pharmaceutical composition in the preparation of a medicine for the treatment and/or prevention of bone metabolism diseases.
  • the present invention also provides the application of the above-mentioned OGP(10-14) pentapeptide derivative or pharmaceutical composition in the preparation of a medicine for the treatment and/or prevention of chronic idiopathic myelofibrosis.
  • the OGP(10-14) pentapeptide derivative provided by the present invention retains the original pharmacophore groups (Tyr10, Phe12) of OGP(10-14) in structure, and has the structural characteristics of improving metabolic stability.
  • OGP(10-14) 10-14) The pentapeptide derivative has the biological activity and metabolic stability of OGP (10-14) through cell activity experiments and enzymatic hydrolysis experiments. It has stronger in vitro proliferation activity than OGP (10-14), and is resistant to pepsin. It has higher stability with trypsin; it can be used as an active ingredient in the treatment or prevention of fracture damage and adjustment of bone metabolism imbalance. Combined with solid or liquid adjuvants commonly used in pharmacy, it can be administered intragastrically or parenterally The way of administration is used.
  • Figure 1 is a liquid chromatogram of H-Dopa-Gly-Phe-Gly-Gly-OH synthesized in Example 1.
  • Example 2 is a mass spectrum of H-Dopa-Gly-Phe-Gly-Gly-OH synthesized in Example 1.
  • Example 3 is a liquid chromatogram of H-D-Tyr-Pro-D-Phe-Gly-Gly-OH synthesized in Example 2.
  • Example 4 is a mass spectrum of H-D-Tyr-Pro-D-Phe-Gly-Gly-OH synthesized in Example 2.
  • Figure 5 is a liquid chromatogram of Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) synthesized in Example 3.
  • Figure 6 is a mass spectrum of Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) synthesized in Example 3.
  • Figure 7 is a liquid chromatogram of Cyclo (Tyr-Gly-D-Phe-Gly-Gly) synthesized in Example 4.
  • Figure 8 is a mass spectrum of Cyclo (Tyr-Gly-D-Phe-Gly-Gly) synthesized in Example 4.
  • Figure 9 is a liquid chromatogram of Cyclo (D-Tyr-Gly-Phe-Gly-Gly) synthesized in Example 5.
  • Figure 10 is a mass spectrum of Cyclo (D-Tyr-Gly-Phe-Gly-Gly) synthesized in Example 5.
  • Figure 11 is a liquid chromatogram of Cyclo (Gly-Gly-D-Phe-Gly-Tyr) synthesized in Example 6.
  • Figure 12 is a mass spectrum of Cyclo (Gly-Gly-D-Phe-Gly-Tyr) synthesized in Example 6.
  • Figure 13 is a liquid chromatogram of Cyclo (Gly-Gly-Phe-Gly-D-Tyr) synthesized in Example 7.
  • Figure 15 is a graph showing the results of the degradation of pepsin by OGP(10-14) pentapeptide derivatives.
  • Figure 16 is a graph showing the experimental results of trypsin degradation of OGP(10-14) pentapeptide derivatives.
  • the synthesis method is as follows:
  • 2-CTC Resin (1.0mmol/g) is selected as the solid phase carrier, in the presence of N,N-diisopropylethylamine, first connect Fmoc-Gly-OH, 2-CTC Resin and Fmoc to the resin
  • the molar ratio of -Gly-OH and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-Gly-2-CTC Resin, and then connect Fmoc-Gly-OH and Fmoc- to the resin in turn.
  • phase A aqueous solution of trifluoroacetic acid with a concentration of 0.1%)
  • phase B a solution of trifluoroacetic acid with a concentration of 0.1% in acetonitrile
  • gradient elution 30 minutes phase B 10% to 90%
  • phase B a solution of trifluoroacetic acid with a concentration of 0.1% in acetonitrile
  • gradient elution 30 minutes phase B 10% to 90%
  • Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd: 515.59, found: 516.33 ([M+H], 100%); 425.40 ([M-91], 100%).
  • the liquid chromatogram is shown in Figure 1, and the mass spectrum is shown in Figure 2.
  • the synthesis method is as follows:
  • the solid phase carrier is 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, first connect Fmoc-Gly-OH, 2-CTC Resin and Fmoc-Gly-OH to the resin
  • the molar ratio of N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-Gly-2-CTC Resin, and then connect Fmoc-Gly-OH and Fmoc-D-Phe- to the resin in turn.
  • the synthesis method is as follows:
  • the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, first connect Fmoc-D-Tyr-(OAll) to the resin, 2-CTC Resin and Fmoc
  • the molar ratio of -D-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-D-Tyr-(OAll)-2-CTC Resin, and then to Fmoc -D-Tyr-(OAll)-2-CTC Resin connected to Fmoc-Pro-OH, Fmoc-D-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-D-Tyr-(OAll) -2-CTC Resin and Fmoc-Pro-OH, Fmoc-D-Phe-OH, Fmoc-Gly-OH
  • Fmoc is removed with 20% piperidine in N,N-dimethylformamide solution
  • allyl removal is piperidine with tetrakis(triphenylphosphine)palladium under nitrogen protection and ice salt bath conditions In solution.
  • phase A (0.1% trifluoroacetic acid in water
  • phase B (0.1% trifluoroacetic acid in acetonitrile)
  • gradient elution 30 minutes phase B 10% to 90%
  • 17.41 minutes freeze-drying
  • the product has a purity of 87.51%. Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd: 521.65, found: 522.41 ([M+H], 40%); 544.34 ([M+Na]+, 100%).
  • the liquid chromatogram is shown in Figure 5, and the mass spectrum is shown in Figure 6.
  • the synthesis method is as follows:
  • the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, first connect Fmoc-L-Tyr-(OAll) to the resin, where 2-CTC Resin and The molar ratio of Fmoc-L-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-Tyr-(OAll)-2-CTC Resin, and then apply them to the resin in turn.
  • the synthesis method is as follows:
  • the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, first connect Fmoc-D-Tyr-(OAll) to the resin, 2-CTC Resin and Fmoc
  • the molar ratio of -D-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-D-Tyr-(OAll)-2-CTC Resin, which are respectively applied to the resin in turn Connect Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-L-Phe-OH, Fmoc-Gly-OH, Fmoc-D-Tyr-(OAll)-2-CTC Resin and Fmoc-Gly-OH, Fmoc respectively -Gly-OH, Fmoc-L-Phe-OH, Fmoc-Gly-OH have a
  • the synthesis method is as follows:
  • the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, connect Fmoc-L-Tyr-(OAll) to the resin, 2-CTC Resin and Fmoc-
  • 2-CTC Resin 1.mmol/g
  • the molar ratio of L-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-Tyr-(OAll)-2-CTC Resin, and then connect Fmoc- to the resin respectively.
  • Example 3 The detection, cyclization, The cutting and purification steps are the same as in Example 3.
  • the chromatographic conditions are the same as in Example 3, 11.82 minutes, and freeze-dried to obtain the product with a purity of 96.77%. Its structure was characterized by electrospray ionization mass spectrometry, [M]calcd:481.59,found:484.70([M+3H],100%).
  • the liquid chromatogram is shown in Figure 11, and the mass spectrum is shown in Figure 12.
  • the synthesis method is as follows:
  • the solid phase carrier adopts 2-CTC Resin (1.0mmol/g), in the presence of N,N-diisopropylethylamine, connect Fmoc-D-Tyr-(OAll) to the resin, where 2-CTC Resin and Fmoc
  • 2-CTC Resin and Fmoc The molar ratio of -D-Tyr-(OAll) and N,N-diisopropylethylamine is 1:1:4 to obtain Fmoc-D-Tyr-(OAll)-2-CTC Resin, and then apply them to the resin respectively.
  • the raw materials, auxiliary materials and their ratios used are:
  • the raw materials, auxiliary materials and their ratios used are:
  • the raw materials, auxiliary materials and their ratios used are:
  • the raw materials, auxiliary materials and their ratios used are:
  • the raw materials, auxiliary materials and their ratios used are:
  • the raw materials, auxiliary materials and their ratios used are:
  • the raw materials, auxiliary materials and their ratios used are:
  • OGP (10-14) pentapeptide derivatives synthesized in Examples 1 to 7 of the present invention were used for cell viability experiments and enzymatic hydrolysis experiments.
  • the various experimental conditions are as follows:
  • thiazole blue (MTT) solution weigh 50.0 mg of thiazole blue, dissolve it in 10 mL of phosphate buffer, stir with a magnetic stirrer until it is completely dissolved, filter and sterilize with a 0.22 ⁇ m microporous membrane, and dispense into 1.5 mL centrifuge tubes.
  • the aluminum box is sealed with paper and stored at –20°C and protected from light.
  • Sample test solution Take the lyophilized samples of OGP(10-14) pentapeptide derivatives of 7 examples, each 0.01mmol, respectively, and dissolve them in 1mL dimethyl sulfoxide to obtain 0.01mmol/mL sample solution, and dilute them separately Into two concentrations of 10 -7 mol/L and 10 -9 mol/L.
  • Cell resuscitation Take frozen osteoblasts (MC3T3E1) and fibroblasts (NIH3T3) separately, and thaw them in a 37°C water bath for 1 to 2 minutes. Suction the cells to a centrifuge tube at 1500 rpm and centrifuge for 4 minutes. The supernatant, use the preheated cell culture fluid to blow the cells evenly, place them in a 37°C, 5% CO 2 incubator for 24 hours, and check by microscopy. If most of the cells have adhered to the wall and are in normal shape, 2 ⁇ 3 Change the cell culture medium once a day.
  • Passage of cells Passage when the cells grow to 80%-90% confluent state. Passaging steps: Wash twice with 5mL phosphate buffer solution, add 1mL of 0.25% trypsin, observe under the microscope, most of the cells shrink in 2 ⁇ 3min, add 2mL cell culture medium to blow down the adherent cells, centrifuge for 1500 Centrifuge at rpm for 4 minutes to remove trypsin. Use the preheated cell culture fluid to blow the cells evenly, divide the cells into three, and place them in a 37°C, 5% CO 2 incubator for 24 hours.
  • Collect the cells in the exponential growth phase wash them twice with phosphate buffer, digest the cells with 0.25% sodium ethylenediaminetetraacetate trypsin, use MEM medium to repeatedly pipette to obtain a cell suspension, adjust the cell density 2 ⁇ 10 7 cells/L ⁇ 5 ⁇ 10 7 cells/L, seeded on 96-well culture plate, so that the cell density is 5000 cells/L/well ⁇ 10000 cells/L/well, after 24 hours, replace without serum Fresh MEM medium (198 ⁇ L/well), and at the same time, add 2 ⁇ L of the sample test solution of the 7 examples in each well of the experimental group.
  • the concentration of each sample test solution is 10 -9 mol/L and 10 -11 mol/L. .
  • the best test concentration in osteoblasts is 10 -11 mol/L
  • the best test concentration in fibroblasts is 10 -9 mol/L.
  • the negative control well is the culture medium containing 2 ⁇ L of dimethyl sulfoxide; the blank zero adjustment hole is the culture medium without osteoblasts and fibroblasts, and each test solution group is set 3 parallel holes. After adding the sample, continue to incubate for 24 hours.
  • Cell proliferation rate average value of experimental group/average value of positive control group
  • pepsin solution Weigh 0.2g of sodium chloride, 0.32g of pepsin, add 50mL of water to dissolve, then add 0.7mL of concentrated hydrochloric acid, dilute to 100mL with water, adjust pH to 1.2, and prepare pepsin with a concentration of 3.2mg/mL Solution.
  • trypsin solution weigh 0.68g of KH 2 PO 4 , add 25mL of water to dissolve; add 19mL of 0.2mol/L NaOH aqueous solution and 40mL of water, 1.0g trypsin, mix the solution, and adjust with 0.2mol/L NaOH solution
  • the pH is 7.5 ⁇ 0.1, the volume of water is 100mL, and the trypsin solution with a concentration of 10mg/mL is prepared.
  • Pepsin degradation sample Take a 5 mL centrifuge tube, add 1.5 mL of pepsin solution, keep at 37°C for 10 minutes, add 1.5 mL of the peptide sample solution of Examples 1-7, and count the time sequentially at 2 minutes, 4 minutes, 8 minutes, At 16 minutes, 30 minutes, 60 minutes, and 120 minutes, take 200 ⁇ L of 7 kinds of peptide sample reaction solutions, add 50 ⁇ L 0.618mol/L Na 2 CO 3 solution to terminate the enzymatic hydrolysis reaction, prepare the sample to be tested, and detect with high performance liquid chromatograph .
  • trypsin degradation samples Take a 5mL centrifuge tube, add 1.5mL trypsin solution, keep at 37°C for 10 minutes, add 1.5mL of the 7 peptide sample reaction solutions, and count them in sequence at 2 minutes, 4 minutes, 8 minutes, and 16 minutes. , 30 minutes, 60 minutes, and 120 minutes, take 200 ⁇ L of 7 peptide sample reaction solutions, add 50 ⁇ L of 30% acetic acid solution to terminate the enzymatic hydrolysis reaction, prepare the sample to be tested, and detect it with high performance liquid chromatography.
  • Mobile phase A an aqueous solution with a concentration of 0.1% trifluoroacetic acid
  • mobile phase B an acetonitrile solution with a concentration of 0.1% trifluoroacetic acid
  • wavelength 215 nm
  • flow rate 1 mL/min.
  • H-Dopa-Gly-Phe-Gly-Gly-OH, H-D-Tyr-Pro-D-Phe-Gly-Gly-OH detection and analysis program 16% constant flow, running time 10 minutes.
  • Cyclo (Gly-Gly-D-Phe-Pro-D-Tyr) detection and analysis program 30% constant current, running time 10 minutes.
  • OGP(10-14) pentapeptide derivatives of the 7 examples retain the original pharmacophore groups (Tyr10, Phe12) of OGP(10-14) in structure, and have a structure that improves metabolic stability. It has the biological activity and metabolic stability of OGP (10-14). It is used as an active ingredient in the treatment or prevention of fracture damage and adjustment of bone metabolism imbalance, combined with solid or liquid adjuvants commonly used in pharmaceutics, and used in gastrointestinal It is used by way of administration or parenteral administration.

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Abstract

本发明提供一种成骨生长肽碳端五肽衍生物及其制备方法和应用。该成骨生长肽碳端五肽衍生物包括：H-Dopa-Gly-Phe-Gly-Gly-OH，H-D-Tyr-Pro-D-Phe-Gly-Gly-OH，Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)，Cyclo(Tyr-Gly-D-Phe-Gly-Gly)，Cyclo(D-Tyr-Gly-Phe-Gly-Gly)，Cyclo(Gly-Gly-D-Phe-Gly-Tyr)，Cyclo(Gly-Gly-Phe-Gly-D-Tyr)中的一种或多种。其在结构上保留了原有五肽的药效基团Tyr10和Phe12，具备改善代谢稳定性的结构特点。

Description

成骨生长肽碳端五肽衍生物及其制备方法和应用 技术领域
本发明属于多肽技术领域，具体涉及一种成骨生长肽碳端五肽OGP(10-14)衍生物及其制备方法和应用。
背景技术
成骨生长肽(Osteogenic Growth Peptide)是一种多肽类生长因子，具有促进成骨和刺激造血的作用，一级结构为ALKRQGRTLYGFGG。其中，碳端五肽OGP(10-14)是保持成骨生长肽全部活性的最小片段，氨基酸序列为H-Tyr10-Gly11-Phe12-Gly13-Gly14-OH。大量试验表明，OGP(10-14)不仅对骨骼损伤、骨代谢疾病有改善治疗的作用，而且对放化疗患者以及骨髓移植患者的造血机能也有恢复作用。据报道，意大利Abiogen制药公司已向欧洲药品管理局(EMEA)递交了将OGP(10-14)作为治疗慢性特发性骨髓纤维化(CIMF)疾病的孤儿药申请，并且已经通过批准。构效关系研究表明，除Gly13和Gly14外，其余氨基酸侧链均是可能影响活性的药效基团。在此基础上，调节活性位点的位置和数量，构建不同构型的环肽，可以提高五肽结构的生物活性或改善其代谢稳定性。
目前仅有的关于OGP(10-14)结构修饰的报道是Bab等人对氮端无氨基的直链OGP(10-14)进行的改构研究。由于OGP(10-14)本身具有同源性和高度保守性，所以只有对原肽结构进行修饰，才有可能得到更安全、有效的化合物。
在成骨生长肽技术领域，当前需迫切解决的一个技术问题是提供一种具有生物活性和代谢稳定性的化合物。
发明内容
本发明的一个目的在于提供一种新的OGP(10-14)五肽衍生物，提高生物活性、提高MC3T3E1成骨细胞和/或NIH3T3成纤维细胞的增殖活性、和/或改善胃蛋白酶和/或胰蛋白酶稳定性。
本发明的另一目的在于提供所述OGP(10-14)五肽衍生物的制备方法。
本发明的另一目的在于提供包括所述OGP(10-14)五肽衍生物的药物组合物。
本发明的另一目的在于提供所述OGP(10-14)五肽衍生物及所述药物组合物在制备治疗和/或预防骨骼损伤的药物中的应用。
本发明的另一目的在于提供所述OGP(10-14)五肽衍生物在制备治疗和/或预防骨代 谢疾病的药物中的应用。
本发明的另一目的在于提供所述OGP(10-14)五肽衍生物在制备治疗和/或预防慢性特发性骨髓纤维化疾病的药物中的应用。
本发明的目的通过以下技术手段得以实现：
一方面，本发明提供了一种OGP(10-14)五肽衍生物，其包括如下化合物中的一种或多种：
H-Dopa-Gly-Phe-Gly-Gly-OH；
H-D-Tyr-Pro-D-Phe-Gly-Gly-OH；
Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)；
Cyclo(Tyr-Gly-D-Phe-Gly-Gly)；
Cyclo(D-Tyr-Gly-Phe-Gly-Gly)；
Cyclo(Gly-Gly-D-Phe-Gly-Tyr)；
Cyclo(Gly-Gly-Phe-Gly-D-Tyr)。
成骨生长肽作为一种活性候选物具有与其多肽药物相同的特点。一方面，结构简单、活性显著、免疫原性低和安全性好，具备新药开发的优势；另一方面，受自身构象的约束，有口服利用率低、酶降解性高以及半衰期短等不足。而导致这些不稳定因素的主要原因就是结构中特定氨基酸的构象，比如，主链的连接次序和侧链残基的种类均会影响其生物活性。由于OGP与底物的识别作用还不明确，不能模拟受体进行药物筛选，因此进行结构修饰和构效关系研究是改善其理化性质和代谢稳定性的有效途径。本发明提供的上述7种OGP(10-14)五肽衍生物中，分别为经过修饰的2种直链OGP衍生物和5种环OGP衍生物。研究发现，本发明的7种OGP(10-14)五肽衍生物相比未修饰的OGP(10-14)五肽和其它修饰的OGP(10-14)五肽衍生物在结构上保留了OGP(10-14)原有的药效基团(Tyr10、Phe12)，具备了改善代谢稳定性的结构特点，本发明的OGP(10-14)五肽衍生物经细胞活性实验、酶解实验证实具有OGP(10-14)的生物活性和代谢稳定性，比OGP(10-14)有更强的体外增殖活性，对胃蛋白酶和胰蛋白酶有更高的稳定性，作为活性成分能够在治疗或预防骨折损伤、调节骨代谢失衡方面具有广泛的应用。
另一方面，本发明还提供上述OGP(10-14)五肽衍生物的制备方法，其包括：
采用Fmoc-固相合成法进行OGP(10-14)直链肽衍生物的肽链连接，或者采用Fmoc-Tyr-(OAll)为起始的二氯树脂环化法进行OGP(10-14)环肽衍生物的肽链连接；
肽链连接后得到的肽树脂利用三氟乙酸水溶液进行切割，获得粗品肽；
粗品肽经过反相高效液相色谱纯化获得OGP(10-14)五肽衍生物。
现有技术报道获取OGP(10-14)五肽的候选衍生物主要是通过缩短肽链长度，修饰酰胺键，改变侧链基团，替换D-氨基酸，构成环五肽(顺时针或逆时针成环)等得到系列衍生物，进行细胞活性筛选，并与原肽比较；直链肽采用Boc固相法，环肽采用溶液假稀释法或污树脂固相法；由于成骨肽作用的底物蛋白结构并不明确，所以没有具体的针对某一位点的修饰依据。本发明中，依据OGP(10-14)和daTyr10[OGP(10-14)]所共有的残基，除Gly11外均可能是接近于最优的药效基团，通过对原五肽OGP(10-14)直接进行改构，直链采用Fmoc-固相法，环肽采用以Fmoc-Tyr-(OAll)为起始的二氯树脂环化法；在此基础上通过胃蛋白酶和胰蛋白酶的降解试验，以提高代谢稳定性为目的改构获得了本发明的7种OGP(10-14)五肽衍生物，本发明方法获得的OGP(10-14)五肽衍生物的纯度＞97％。
上述的方法中，优选地，固相合成法中采用2-CTC Resin(2-Chlorotrityl Chloride Resin)作为固相载体。
另一方面，本发明还提供一种药物组合物，其中，包括上述的OGP(10-14)五肽衍生物以及药学上可接受的载体。
上述的药物组合物中，药学上可接受的载体可以是固体赋形剂或液体赋形剂，例如可以是适宜于胃肠内给药的有机或无机的固体或液体赋形剂等。具体可以包括稳定剂、润湿剂、增溶剂或其它常用的辅料和/或添加剂，如乳糖、滑石粉、纤维素、聚乙烯吡咯烷酮、淀粉、果胶、吐温-80、聚乙烯醇等。
上述的药物组合物中，优选地，所述药物组合物的存在形态为固体形态和/或液体形态；所述固体形态包括片剂、颗粒剂或胶囊剂；所述液体形态包括悬浮剂、糖浆剂或乳剂。
本发明的活性成分OGP(10-14)五肽衍生物制成各种剂型的口服药物，成人口服，常用剂量一次10 -9～10 -11mg，1日1次，饭后服用，儿童用药酌情减量。适应症：治疗和预防骨骼损伤、骨代谢疾病、慢性特发性骨髓纤维化疾病。
本发明还提供上述OGP(10-14)五肽衍生物或药物组合物在制备治疗和/或预防骨骼损伤的药物中的应用。
本发明还提供上述OGP(10-14)五肽衍生物或药物组合物在制备治疗和/或预防骨代谢疾病的药物中的应用。
本发明还提供上述OGP(10-14)五肽衍生物或药物组合物在制备治疗和/或预防慢性特发性骨髓纤维化疾病的药物中的应用。
本发明的有益效果：
本发明提供的OGP(10-14)五肽衍生物在结构上保留了OGP(10-14)原有的药效基团(Tyr10、Phe12)，具备了改善代谢稳定性的结构特点，OGP(10-14)五肽衍生物经细 胞活性实验、酶解实验，具有OGP(10-14)的生物活性和代谢稳定性，比OGP(10-14)有更强的体外增殖活性，对胃蛋白酶和胰蛋白酶有更高的稳定性；能够作为活性成分在治疗或预防骨折损伤、调节骨代谢失衡方面应用，结合药剂学常用的固体或液体辅型剂，以胃肠内给药或胃肠外给药的方式使用。
附图说明
图1是实施例1合成的H-Dopa-Gly-Phe-Gly-Gly-OH的液相色谱图。
图2是实施例1合成的H-Dopa-Gly-Phe-Gly-Gly-OH的质谱图。
图3是实施例2合成的H-D-Tyr-Pro-D-Phe-Gly-Gly-OH的液相色谱图。
图4是实施例2合成的H-D-Tyr-Pro-D-Phe-Gly-Gly-OH的质谱图。
图5是实施例3合成的Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)的液相色谱图。
图6是实施例3合成的Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)的质谱图。
图7是实施例4合成的Cyclo(Tyr-Gly-D-Phe-Gly-Gly)的液相色谱图。
图8是实施例4合成的Cyclo(Tyr-Gly-D-Phe-Gly-Gly)的质谱图。
图9是实施例5合成的Cyclo(D-Tyr-Gly-Phe-Gly-Gly)的液相色谱图。
图10是实施例5合成的Cyclo(D-Tyr-Gly-Phe-Gly-Gly)的质谱图。
图11是实施例6合成的Cyclo(Gly-Gly-D-Phe-Gly-Tyr)的液相色谱图。
图12是实施例6合成的Cyclo(Gly-Gly-D-Phe-Gly-Tyr)的质谱图。
图13是实施例7合成的Cyclo(Gly-Gly-Phe-Gly-D-Tyr)的液相色谱图。
图14是实施例7合成的Cyclo(Gly-Gly-Phe-Gly-D-Tyr)的质谱图。
图15是OGP(10-14)五肽衍生物对胃蛋白酶降解实验结果图。
图16是OGP(10-14)五肽衍生物对胰蛋白酶降解实验结果图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。实施例中，未注明具体条件的实验方法参照所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件操作。
实施例1
本实施例OGP(10-14)五肽衍生物的结构式如下：
H-Dopa-Gly-Phe-Gly-Gly-OH。
其合成方法如下：
反应与检测：固相载体选用2-CTC Resin(1.0mmol/g)，在N,N-二异丙基乙胺存在下,先向树脂上连接Fmoc-Gly-OH，2-CTC Resin与Fmoc-Gly-OH、N,N-二异丙基乙胺的摩尔比为1：1：4，得Fmoc-Gly-2-CTC Resin，然后依次分别向树脂上连接Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Gly-OH、Fmoc-Dopa(tBu)-OH，每步反应均选用0.05g/mL茚三酮的乙醇溶液进行检测，使得正检或反检通过，2-CTC Resin分别与Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Gly-OH、Fmoc-Dopa(tBu)-OH的摩尔比均为1：2，2-CTC Resin与1-羟基苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸、N,N-二异丙基乙胺的摩尔比均为1：2：2：2，肽链连接中用浓度为20％哌啶的N,N-二甲基甲酰胺溶液脱除Fmoc，经异丙醇洗涤2次，N,N-二甲基甲酰胺洗涤3次，无水甲醇洗涤3次，常温抽滤干燥得肽树脂。
切割：肽树脂用95％的三氟乙酸水溶液切割树脂，反应1.5小时，抽滤，滤液再用冷乙醚析出沉淀，抽滤，常温真空干燥得粗品肽。
纯化：粗品肽经反相高效液相制备色谱纯化，冷冻干燥，得到产品。色谱条件：A相(浓度为0.1％的三氟乙酸水溶液)，B相(浓度为0.1％的三氟乙酸乙腈溶液)，梯度洗脱30分钟(B相10％～90％)，14.69分钟，冻干，得产品，纯度为94.55％。其结构由电喷雾电离质谱表征，[M]calcd:515.59，found:516.33([M+H],100％)；425.40([M-91]，100％)。液相色谱图见图1、质谱图见图2。
实施例2
本实施例OGP(10-14)五肽衍生物的结构式如下：
H-D-Tyr-Pro-D-Phe-Gly-Gly-OH。
其合成方法如下：
固相载体选用2-CTC Resin(1.0mmol/g)，在N,N-二异丙基乙胺存在下,先向树脂上连接Fmoc-Gly-OH，2-CTC Resin与Fmoc-Gly-OH、N,N-二异丙基乙胺的摩尔比为1：1：4，得Fmoc-Gly-2-CTC Resin，然后依次分别向树脂上连接Fmoc-Gly-OH、Fmoc-D-Phe-OH、Fmoc-Pro-OH、Fmoc-D-Tyr(tBu)-OH，反应条件、检测、切割、纯化步骤与实施例1相同，冻干，得产品。色谱条件：A相(浓度为0.1％三氟乙酸的水溶液)，B相(浓度为0.1％三氟乙酸的乙腈溶液)，梯度洗脱30分钟(B相10％～90％)，13.25分钟，纯度99.74％。其结构由电喷雾电离质谱表征，[M]calcd:539.65,found:540.37([M+H],100％)；541.50([M+H],20％)。液相色谱图见图3、质谱图见图4。
实施例3
本实施例OGP(10-14)五肽衍生物的结构式如下：
Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)。
其合成方法如下：
固相载体采用2-CTC Resin(1.0mmol/g)，在N,N-二异丙基乙胺存在下,先向树脂上连接Fmoc-D-Tyr-(OAll)，2-CTC Resin与Fmoc-D-Tyr-(OAll)、N,N-二异丙基乙胺的摩尔比为1：1：4，得Fmoc-D-Tyr-(OAll)-2-CTC Resin，然后依次分别向Fmoc-D-Tyr-(OAll)-2-CTC Resin上连接Fmoc-Pro-OH、Fmoc-D-Phe-OH、Fmoc-Gly-OH、Fmoc-Gly-OH，Fmoc-D-Tyr-(OAll)-2-CTC Resin分别与Fmoc-Pro-OH、Fmoc-D-Phe-OH、Fmoc-Gly-OH、Fmoc-Gly-OH的摩尔比均为1：2，Fmoc-D-Tyr-(OAll)-2-CTC Resin与1-羟基苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸、N,N-二异丙基乙胺的摩尔比均为1：2：2：2，每步反应均选用0.05g/mL茚三酮的乙醇溶液进行检测，确保正检或反检完全通过。肽链连接中脱除Fmoc用20％哌啶的N,N-二甲基甲酰胺溶液，烯丙基脱除在氮气保护和冰盐浴条件下用四(三苯基膦)钯的哌啶溶液中进行。将1-羟基苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸、N,N-二异丙基乙胺的N,N-二甲基甲酰胺溶液加入到肽树脂中环合，肽树脂与1-羟基苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸、N,N-二异丙基乙胺的摩尔比为1：2：2：2。切割、纯化步骤与实施例1相同。色谱条件：A相(0.1％三氟乙酸的水溶液)，B相(0.1％三氟乙酸的乙腈溶液)，梯度洗脱30分钟(B相10％～90％)，17.41分钟，冻干，得产品，纯度为87.51％。其结构由电喷雾电离质谱表征，[M]calcd:521.65,found:522.41([M+H],40％)；544.34([M+Na]+,100％)。液相色谱图见图5、质谱图见图6。
实施例4
本实施例OGP(10-14)五肽衍生物的结构式如下：
Cyclo(Tyr-Gly-D-Phe-Gly-Gly)。
其合成方法如下：
固相载体采用2-CTC Resin(1.0mmol/g)，在N,N-二异丙基乙胺存在下,先向树脂上连接Fmoc-L-Tyr-(OAll)，其中2-CTC Resin与Fmoc-L-Tyr-(OAll)、N,N-二异丙基乙胺的摩尔比为1：1：4，得Fmoc-Tyr-(OAll)-2-CTC Resin，然后依次分别向树脂上连接Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-D-Phe-OH、Fmoc-Gly-OH，其中Fmoc-Tyr-(OAll)-2-CTC Resin分别与Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-D-Phe-OH、Fmoc-Gly-OH的摩尔比均为1：2，Fmoc-Tyr-(OAll)-2-CTC Resin与1-羟基苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸、N,N-二异丙基乙胺的摩尔比为1：2：2：2，反应中的检测、环合、切割、纯化步 骤与实施例3相同。色谱条件与实施例3相同，10.42分钟，冻干，得产品，纯度为93.87％。其结构由电喷雾电离质谱表征，[M]calcd:481.59，found:482.35([M+H],60％)；504.31([M+Na]+,45％)。液相色谱图见图7、质谱图见图8。
实施例5
本实施例OGP(10-14)五肽衍生物的结构式如下：
Cyclo(D-Tyr-Gly-Phe-Gly-Gly)。
其合成方法如下：
固相载体采用2-CTC Resin(1.0mmol/g)，在N,N-二异丙基乙胺存在下,先向树脂上连接Fmoc-D-Tyr-(OAll)，2-CTC Resin与Fmoc-D-Tyr-(OAll)、N,N-二异丙基乙胺的摩尔比为1：1：4，得Fmoc-D-Tyr-(OAll)-2-CTC Resin，依次分别向树脂上连接Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-L-Phe-OH、Fmoc-Gly-OH，Fmoc-D-Tyr-(OAll)-2-CTC Resin分别与Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-L-Phe-OH、Fmoc-Gly-OH的摩尔比均为1：2，Fmoc-D-Tyr-(OAll)-2-CTC Resin与1-羟基苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸、N,N-二异丙基乙胺的摩尔比为1：2：2：2，反应中的检测、环合、切割、纯化步骤与实施例3相同。色谱条件与实施例3相同，10.48分钟，冻干，得产品，纯度为91.69％。其结构由电喷雾电离质谱表征，[M]calcd:481.59,found:482.35([M+H],40％)；504.31([M+Na]+,45％)。液相色谱图见图9、质谱图见图10。
实施例6
本实施例OGP(10-14)五肽衍生物的结构式如下：
Cyclo(Gly-Gly-D-Phe-Gly-Tyr)。
其合成方法如下：
固相载体采用2-CTC Resin(1.0mmol/g)，在N,N-二异丙基乙胺存在下,向树脂上连接Fmoc-L-Tyr-(OAll)，2-CTC Resin与Fmoc-L-Tyr-(OAll)、N,N-二异丙基乙胺的摩尔比为1：1：4，得Fmoc-Tyr-(OAll)-2-CTC Resin，依次分别向树脂上连接Fmoc-Gly-OH、Fmoc-D-Phe-OH、Fmoc-Gly-OH、Fmoc-Gly-OH，Fmoc-Tyr-(OAll)-2-CTC Resin分别与Fmoc-Gly-OH、Fmoc-D-Phe-OH、Fmoc-Gly-OH、Fmoc-Gly-OH的摩尔比均为1：2，Fmoc-Tyr-(OAll)-2-CTC Resin与1-羟基苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸、N,N-二异丙基乙胺的摩尔比为1：2：2：2，连接中的检测、环合、切割、纯化步骤与实施例3相同。色谱条件与实施例3相同，11.82分钟，冻干，得产品，纯度为96.77％。其结构由电喷雾电离质谱表征，[M]calcd:481.59,found:484.70([M+3H],100％)。液相色谱图见图11、质谱图见图12。
实施例7
本实施例OGP(10-14)五肽衍生物的结构式如下：
Cyclo(Gly-Gly-Phe-Gly-D-Tyr)。
其合成方法如下：
固相载体采用2-CTC Resin(1.0mmol/g)，在N,N-二异丙基乙胺存在下,向树脂上连接Fmoc-D-Tyr-(OAll)，其中2-CTC Resin与Fmoc-D-Tyr-(OAll)、N,N-二异丙基乙胺的摩尔比为1：1：4，得Fmoc-D-Tyr-(OAll)-2-CTC Resin，依次分别向树脂上连接Fmoc-Gly-OH、Fmoc-L-Phe-OH、Fmoc-Gly-OH、Fmoc-Gly-OH，Fmoc-D-Tyr-(OAll)-2-CTC Resin分别与Fmoc-Gly-OH、Fmoc-L-Phe-OH、Fmoc-Gly-OH、Fmoc-Gly-OH的摩尔比均为1：2，Fmoc-D-Tyr-(OAll)-2-CTC Resin与1-羟基苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸、N,N-二异丙基乙胺的摩尔比为1：2：2：2，连接中的反应与检测、环合、切割、纯化步骤与实施例3相同。色谱条件与实施例3相同，12.58分钟，冻干，得产品，纯度为94.90％。其结构由电喷雾电离质谱表征，[M]calcd:481.59,found:482.35([M+H],100％)；504.28([M+Na]+,60％)。液相色谱图见图13、质谱图见图14。
实施例8
以生产本发明药物片剂产品1000片为例所用的原料和辅料及其配比为：
取实施例1合成的H-Dopa-Gly-Phe-Gly-Gly-OH1000×10 -11mg，淀粉加至300g，按制剂学常规片剂的制备方法进行。每片重0.3g，每片含药用有效成份10 -11mg。每日早饭后服一次，每次1片。
实施例9
以生产本发明药物片剂产品1000片为例所用的原料和辅料及其配比为：
取实施例2合成的H-D-Tyr-Pro-D-Phe-Gly-Gly-OH1000×10 -11mg，淀粉加至300g，按制剂学常规片剂的制备方法进行。每片重0.3g，每片含药用有效成份10 -11mg。每日早饭后服一次，每次1片。
实施例10
以生产本发明药物片剂产品1000片为例所用的原料和辅料及其配比为：
取实施例3合成的Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)1000×10 -11mg，淀粉加至300g，按制剂学常规片剂的制备方法进行。每片重0.3g，每片含药用有效成份10 -11mg。每日早饭后服一次，每次1片。
实施例11
以生产本发明药物片剂产品1000片为例所用的原料和辅料及其配比为：
取实施例4合成的Cyclo(Tyr-Gly-D-Phe-Gly-Gly)1000×10 -11mg，淀粉加至300g，按制剂学常规片剂的制备方法进行。每片重0.3g，每片含药用有效成份10 -11mg。每日早饭后服一次，每次1片。
实施例12
以生产本发明药物片剂产品1000片为例所用的原料和辅料及其配比为：
取实施例5合成的Cyclo(D-Tyr-Gly-Phe-Gly-Gly)1000×10 -11mg，淀粉加至300g，按剂学常规片剂的制备方法进行。每片重0.3g，每片含药用有效成份10 -11mg。每日早饭后服一次，每次1片。
实施例13
以生产本发明药物片剂产品1000片为例所用的原料和辅料及其配比为：
取实施例6合成的Cyclo(Gly-Gly-D-Phe-Gly-Tyr)1000×10 -11mg，淀粉加至300g，按制剂学常规片剂的制备方法进行。每片重0.3g，每片含药用有效成份10 -11mg。每日早饭后服一次，每次1片。
实施例14
以生产本发明药物片剂产品1000片为例所用的原料和辅料及其配比为：
取实施例7合成的Cyclo(Gly-Gly-Phe-Gly-D-Tyr)1000×10 -11mg，淀粉加至300g，按制剂学常规片剂的制备方法进行。每片重0.3g，每片含药用有效成份10 -11mg。每日早饭后服一次，每次1片。
测试实验
为了验证本发明的有益效果，采用本发明实施例1～7合成的OGP(10-14)五肽衍生物进行了细胞活性实验、酶解实验，各种实验情况如下：
1、细胞活性实验
(1)配制样品液
配制细胞培养液：将α-MEM液体培养基、灭活胎牛血清、青链霉素混合液按体积比为89％：10％：1％比例混合，分装密封，配制成细胞培养液，4℃保存备用。另备不含血清的相同培养液，活性测试时。
按常规方法配制成浓度为5mg/mL的磷酸盐缓冲液，调节pH值至7.2-7.4，高压高温灭菌，分装密封，4℃保存备用。
配制噻唑蓝(MTT)溶液：称取噻唑蓝50.0mg，溶于10mL磷酸盐缓冲液，用磁力搅拌机搅拌至完全溶解，用0.22μm微孔滤膜过滤除菌，分装到1.5mL离心管内，铝箱纸封口，–20℃避光保存。
样品供试液：取7个实施例的成OGP(10-14)五肽衍生物冻干样品各0.01mmol,分别溶于1mL二甲基亚砜中,得到0.01mmol/mL样品液,分别稀释成10 -7mol/L和10 -9mol/L两个浓度。
(2)细胞培养
细胞复苏：分别取冻存的成骨细胞(MC3T3E1)和成纤维细胞(NIH3T3)，于37℃水浴中融化1～2分钟，将细胞吸至离心管1500转r/分钟，离心4分钟，除上清液，用已预热的细胞培养液将细胞吹打均匀，置于37℃、5％CO 2培养箱中培养24小时，镜检，若大部分细胞已贴壁且形态正常，2～3天更换一次细胞培养液。
细胞传代：待细胞生长至80％～90％融合状态时，传代。传代步骤：用5mL磷酸盐缓冲液清洗2次，加入1mL浓度为0.25％胰酶，在显微镜下观察，大部分细胞在2～3min收缩，加入2mL细胞培养液将贴壁细胞吹打下来，离心1500转/分钟离心4分钟，除去胰酶，用已预热的细胞培养液将细胞吹打均匀，细胞1分为3，置于37℃、5％CO 2培养箱中培养24小时。
(3)活性测试
收集指数生长期的细胞，用磷酸盐缓冲液清洗两次，用浓度为0.25％乙二胺四乙酸钠的胰酶对细胞进行消化，用MEM培养基，反复吹打得到细胞悬浮液，调整细胞密度为2×10 7个/L～5×10 7个/L，接种于96孔培养板，使得细胞密度为5000个/L/孔～10000个/L/孔，24小时后，更换不含血清的新鲜MEM培养基(198μL/孔)，同时实验组每孔加2μL的7个实施例的样品试验液，每个样品试验液的浓度分别为10 -9mol/L、10 -11mol/L。其中，在成骨细胞中的最佳测试浓度是10 -11mol/L，在成纤维细胞中的最佳测试浓度是10 -9mol/L。设阴性对照孔和空白调零孔，阴性对照孔为含2μL二甲亚砜的培养液；空白调零孔为不含成骨细胞和成纤维细胞的培养液，每个供试液组均设置3个平行孔。加样完毕后，继续培养24小时。更换不含血清的新鲜培养基(180μL/孔)，加入噻唑蓝溶液20μL/孔，培养4小时，用吸管吸去上清液，每孔加入150μL二甲亚砜，37℃震荡10分钟至甲瓒结晶充分溶解。用酶联免疫标定仪波长490nm处空白孔调零，测定各孔吸光度A。与阳性对照组的吸光度A值进行比较，对增殖活性进行评价。
增值率小于1表示比OGP(10-14)活性低，增值率大于1表示比OGP(10-14)活性高，实验重复3次。数据采用SPPS 13.0软件按软件的使用说明进行统计分析，均以x±s表示，组间比较采用t检验。
细胞增殖率＝实验组平均值/阳性对照组平均值
测试结果见表1。
表1 实施例1～7的肽在成骨细胞和成纤维细胞株中的增殖活性(
Figure PCTCN2020085363-appb-000001
n＝3)
Figure PCTCN2020085363-appb-000002
实验结果用统计学处理，*P<0.05，**P<0.01。由表1可见，上述化合物细胞增殖活性与OGP(10-14)相当或更优，且在两种细胞株中的作用强度呈相关性。这些化合物在结构上保留了五肽原有的药效基团(Tyr10、Phe12)，具备改善代谢稳定性的结构(D-构型、环化)。
2、酶解实验
(1)配制样品液
配制7个实施例的OGP(10-14)五肽衍生物溶液：称量7个实施例的OGP(10-14)五肽衍生物冻干粉5mg，加水溶解，容量瓶定容5mL，配成浓度为1mg/mL的7种水溶液。
配制胃蛋白酶溶液：称取氯化钠0.2g，胃蛋白酶0.32g，加水50mL溶解，再加入0.7mL浓盐酸，用水定容100mL，调pH为1.2，配制成浓度为3.2mg/mL的胃蛋白酶溶液。
配制胰蛋白酶溶液：称取KH 2PO 4 0.68g，加水25mL溶解；补加19mL 0.2mol/L的NaOH水溶液和40mL水，1.0g胰蛋白酶，混匀溶液，用0.2mol/L的NaOH溶液调pH至7.5±0.1，水定容100mL，配成浓度为10mg/mL的胰蛋白酶溶液。
(2)实验方法
胃蛋白酶降解样品：取5mL离心管，加入胃蛋白酶溶液1.5mL，37℃恒温10分钟，分别加入实施例1-7的肽样品溶液1.5mL，计时，依次在2分钟、4分钟、8分钟、16分钟、30分钟、60分钟、120分钟取7种肽样品反应液200μL，分别加入50μL 0.618mol/L Na 2CO 3溶液终止酶解反应，配制成待测样品，用高效液相色谱仪检测。
配制胰蛋白酶降解样品：取5mL离心管，加入胰蛋白酶溶液1.5mL，37℃恒温10分 钟，分别加入7种肽样品反应液1.5mL，计时，依次在2分钟、4分钟、8分钟、16分钟、30分钟、60分钟、120分钟取7种肽样品反应液200μL，分别加入50μL浓度为30％的乙酸溶液终止酶解反应，配制成待测样品，用高效液相色谱仪检测。
(3)检测条件
7种待测样品用日立L-2455全自动分析液相色谱仪分离检测，相关参数如下：
流动相A：浓度为0.1％三氟乙酸的水溶液；流动相B：浓度为0.1％三氟乙酸的乙腈溶液；波长：215nm；流速1mL/min。
H-Dopa-Gly-Phe-Gly-Gly-OH，H-D-Tyr-Pro-D-Phe-Gly-Gly-OH检测分析程序：16％恒流，运行时间10分钟。
Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)检测分析程序：30％恒流，运行时间10分钟。
Cyclo(Tyr-Gly-D-Phe-Gly-Gly)，Cyclo(D-Tyr-Gly-Phe-Gly-Gly)，Cyclo(Gly-Gly-D-Phe-Gly-Tyr)，Cyclo(Gly-Gly-Phe-Gly-D-Tyr)检测分析程序：20％恒流，运行时间10分钟。
(4)检测结果
检测结果见图15、图16。
由图15、16可见，通过对OGP(10-14)及其7个实施例的OGP(10-14)五肽衍生物进行体外活性筛选试验，采用MC3T3E1成骨细胞和NIH3T3成纤维细胞作为测试细胞株，以OGP(10-14)的活性表现为阳性对照，评价OGP(10-14)五肽衍生物对细胞增殖活性的影响。结果表明，7个实施例的OGP(10-14)五肽衍生物的细胞活性均保留了甚至优于原直链肽OGP(10-14)的活性。通过对OGP(10-14)及其7个实施例的OGP(10-14)五肽衍生物进行酶解实验，使用胃蛋白酶及胰蛋白酶分别作为酶解条件，以OGP(10-14)的酶解反应为空白对照，评价各衍生物在两种酶中的代谢稳定性。结果表明，除H-Dopa-Gly-Phe-Gly-Gly-OH相较于OGP(10-14)代谢稳定性总体相似，无显著差异以外，其余6个实施例的OGP(10-14)五肽衍生物的代谢稳定性总体均比OGP(10-14)稳定性好。
以上结果表明7个实施例的OGP(10-14)五肽衍生物在结构上保留了OGP(10-14)原有的药效基团(Tyr10、Phe12)，具备了改善代谢稳定性的结构特点，具有OGP(10-14)的生物活性和代谢稳定性，作为活性成分在治疗或预防骨折损伤、调节骨代谢失衡方面应用，结合药剂学常用的固体或液体辅型剂，以胃肠内给药或胃肠外给药的方式使用。

Claims (13)

  1. 一种OGP(10-14)五肽衍生物，其包括如下化合物中的一种或多种：
    H-Dopa-Gly-Phe-Gly-Gly-OH；
    H-D-Tyr-Pro-D-Phe-Gly-Gly-OH；
    Cyclo(Gly-Gly-D-Phe-Pro-D-Tyr)；
    Cyclo(Tyr-Gly-D-Phe-Gly-Gly)；
    Cyclo(D-Tyr-Gly-Phe-Gly-Gly)；
    Cyclo(Gly-Gly-D-Phe-Gly-Tyr)；
    Cyclo(Gly-Gly-Phe-Gly-D-Tyr)。
  2. 权利要求1所述OGP(10-14)五肽衍生物的制备方法，其包括：
    采用Fmoc-固相合成法进行OGP(10-14)直链肽衍生物的肽链连接，或者采用Fmoc-Tyr-(OAll)为起始的二氯树脂环化法进行OGP(10-14)环肽衍生物的肽链连接；
    肽链连接后得到的肽树脂利用三氟乙酸水溶液进行切割，获得粗品肽；
    粗品肽经过反相高效液相色谱纯化获得OGP(10-14)五肽衍生物。
  3. 根据权利要求2所述的制备方法，其中，固相合成法中采用2-CTC Resin作为固相载体。
  4. 一种药物组合物，其包括权利要求1所述的OGP(10-14)五肽衍生物以及药学上可接受的载体。
  5. 根据权利要求4所述的药物组合物，其中，所述药学上可接受的载体为固体赋形剂或液体赋形剂。
  6. 权利要求1所述OGP(10-14)五肽衍生物或权利要求4-5任一项所述的药物组合物在制备治疗和/或预防骨骼损伤的药物中的应用。
  7. 权利要求1所述OGP(10-14)五肽衍生物或权利要求4-5任一项所述的药物组合物在制备治疗和/或预防骨代谢疾病的药物中的应用。
  8. 权利要求1所述OGP(10-14)五肽衍生物或权利要求4-5任一项所述的药物组合物在制备治疗和/或预防慢性特发性骨髓纤维化疾病的药物中的应用。
  9. 根据权利要求6-8任一项所述的应用，其中，所述药物为片剂、颗粒剂、胶囊剂、悬浮剂、糖浆剂或乳剂。
  10. 根据权利要求6-9任一项所述的应用，其中，权利要求1所述OGP(10-14)五肽衍生物是以10 -9～10 -11mg/日/次的剂量用于制备药物。
  11. 一种治疗骨骼损伤的方法，该方法包括给予受试者有效量的权利要求1所述的OGP(10-14)五肽衍生物或权利要求4-5任一项所述的药物组合物。
  12. 一种治疗骨代谢疾病的方法，该方法包括给予受试者有效量的权利要求1所述的OGP(10-14)五肽衍生物或权利要求4-5任一项所述的药物组合物。
  13. 一种治疗慢性特发性骨髓纤维化的方法，该方法包括给予受试者有效量的权利要求1所述的OGP(10-14)五肽衍生物或权利要求4-5任一项所述的药物组合物。
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