An anti-inflammatory polypeptide compound, its preparation method and its application

Abstract

This invention provides an anti-inflammatory polypeptide compound, its preparation method, and its application, relating to the field of polypeptide compound technology. The anti-inflammatory polypeptide compound is shown in structure I: wherein AA1 is selected from any one of D- or L-type lysine, D- or L-type 2,3-diaminopropionic acid, D- or L-type 2,4-diaminobutyric acid, D- or L-type ornithine, D- or L-type 2,7-diaminoheptanoic acid, or D- or L-type 2,8-diaminooctanoic acid; AA2 is selected from any one of (lysine)6 or not present; AA3 is selected from any one of amino and hydroxyl groups; 0≤m1≤10, 0≤m2≤6, 0≤m3≤6; 0≤n1≤10, 0≤n2≤6 0 ≤ n3 ≤ 6; when R1 is selected as Cyclo(Cys-AA4-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys), R2 is selected as Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val; when R1 is selected as Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val, R2 is selected as Cyclo(Cys-AA4-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys). The anti-inflammatory polypeptide compound shown in Structure I provided by this invention is stable and is a novel anti-inflammatory polypeptide compound with anti-inflammatory, repair, anti-wrinkle, and anti-aging effects on the skin.

Description

Technical Field

[0001] This invention relates to the field of polypeptide compound technology, and in particular to an anti-inflammatory polypeptide compound, its preparation method, and its application. Background Technology

[0002] Peptides possess moisturizing, nourishing, anti-aging, wrinkle-reducing, and whitening properties. They can directly penetrate the dermis, replenishing lost collagen, restoring skin elasticity, promoting cell metabolism, and delaying cell aging. They can smooth fine lines and leave skin soft and smooth. Furthermore, small molecule peptides contain pigment-lightening factors that reduce melanin formation and also have anti-inflammatory effects on the skin.

[0003] The purpose of this invention is to provide a new anti-inflammatory polypeptide compound for use in the medical aesthetics field, such as skin anti-inflammatory, repair, anti-wrinkle, and anti-aging. Summary of the Invention

[0004] This invention provides an anti-inflammatory polypeptide compound, its preparation method, and its application, giving consumers a new option.

[0005] In a first aspect, this invention provides an anti-inflammatory polypeptide compound, the anti-inflammatory polypeptide compound being as shown in structure I:

[0006]

[0007] AA1 is selected from any one of D-type or L-type lysine, D-type or L-type 2,3-diaminopropionic acid, D-type or L-type 2,4-diaminobutyric acid, D-type or L-type ornithine, D-type or L-type 2,7-diaminoheptanoic acid, and D-type or L-type 2,8-diaminooctanoic acid.

[0008] AA2 is selected from either (lysine)6 or none of the following;

[0009] AA3 is selected from either amino or hydroxyl groups;

[0010] 0≤m1≤10, 0≤m2≤6, 0≤m3≤6;

[0011] 0≤n1≤10, 0≤n2≤6, 0≤n3≤6;

[0012] When R1 is selected as Cyclo(Cys-AA4-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys), R2 is selected as Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val, where AA4 is selected from either tyrosine or not present.

[0013] When R1 is selected as Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val, R2 is selected as Cyclo(Cys-AA4-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys), where AA4 is selected from either tyrosine or not present.

[0014] Optionally, the AA1 is selected from any one of D- or L-type lysine, D- or L-type 2,3-diaminopropionic acid, or D- or L-type 2,4-diaminobutyric acid.

[0015] Optionally, the AA2 is selected as (lysine)6.

[0016] Optionally, the AA3 is selected as an amino group.

[0017] Optionally, the AA4 is selected as tyrosine.

[0018] Optionally, AA1 is selected as lysine, AA2 is selected as absent, AA3 is selected as amino, R1 is selected as Cyclo(Cys-AA4-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys), m1=m2=m3=0, R2 is selected as Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val, n1=5, n2=1, n3=1.

[0019] Optionally, the anti-inflammatory polypeptide compound comprises a pharmaceutical salt, chelate, or non-covalent complex formed from the anti-inflammatory polypeptide compound, as well as a precursor of the anti-inflammatory polypeptide compound, or any mixture thereof.

[0020] A second aspect of the present invention provides a method for preparing the anti-inflammatory polypeptide compound as described in any one of the first aspects, comprising: preparing a peptide resin using a solid-phase polypeptide synthesis method, acid hydrolyzing the peptide resin to obtain a crude product, and purifying the crude product to obtain a purified anti-inflammatory polypeptide compound.

[0021] The third aspect of this invention provides the application of the anti-inflammatory polypeptide compound described in any one of the first aspects above in the preparation of anti-inflammatory polypeptide compositions in the field of medical aesthetics.

[0022] Optionally, the anti-inflammatory peptide composition is used for skin anti-inflammatory, repair, anti-wrinkle, and anti-aging purposes. Detailed Implementation

[0023] This invention discloses an anti-inflammatory polypeptide compound, its preparation method, and its applications. Those skilled in the art can refer to the content of this invention and appropriately modify the relevant parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method of this invention has been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention. The chemical reagents used in the embodiments of this invention were purchased from Chengdu Huirong Biotechnology Co., Ltd., the biological reagents were purchased from Chengdu Vanke Industrial Co., Ltd., and the chromatograph was a GL6000-100 model from Chengdu Glei Precision Instruments Co., Ltd.

[0024] The Chinese names corresponding to the English abbreviations involved in this invention are shown in Table 1:

[0025] Table 1. English Abbreviations and Their Corresponding Chinese Names

[0026]

[0027]

[0028] In a first aspect of the present invention, an anti-inflammatory polypeptide compound is provided, the anti-inflammatory polypeptide compound being as shown in structure I:

[0029]

[0030] AA1 is selected from any one of D-type or L-type lysine, D-type or L-type 2,3-diaminopropionic acid, D-type or L-type 2,4-diaminobutyric acid, D-type or L-type ornithine, D-type or L-type 2,7-diaminoheptanoic acid, and D-type or L-type 2,8-diaminooctanoic acid.

[0031] AA2 is selected from either (lysine)6 or none of the following;

[0032] AA3 is selected from either amino or hydroxyl groups;

[0033] 0≤m1≤10, 0≤m2≤6, 0≤m3≤6;

[0034] 0≤n1≤10, 0≤n2≤6, 0≤n3≤6;

[0035] When R1 is selected as Cyclo(Cys-AA4-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-

[0036] When Cys), R2 is selected as Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val, where AA4 is selected from either tyrosine or not present.

[0037] When R1 is selected as Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val, R2 is selected as Cyclo(Cys-AA4-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys), where AA4 is selected from either tyrosine or not present.

[0038] In an optional embodiment of the present invention, the AA1 may be selected from any one of D-type or L-type Lys, D-type or L-type Dap, or D-type or L-type Dab.

[0039] In an optional embodiment of the present invention, the AA1 is selected from any one of D-type or L-type lysine, D-type or L-type 2,3-diaminopropionic acid, or D-type or L-type 2,4-diaminobutyric acid.

[0040] In an optional embodiment of the present invention, the AA2 is selected as (lysine)6.

[0041] In an optional embodiment of the present invention, the AA3 is selected as an amino group.

[0042] In an optional embodiment of the present invention, AA4 is selected as tyrosine.

[0043] In an optional embodiment of the present invention, AA1 is selected as lysine, AA2 is selected as absent, AA3 is selected as amino, R1 is selected as Cyclo(Cys-AA4-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys), m1=m2=m3=0, R2 is selected as Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val, n1=5, n2=1, n3=1.

[0044] In an optional embodiment of the present invention, the anti-inflammatory polypeptide compound comprises a pharmaceutical salt, chelate, or non-covalent complex formed from the anti-inflammatory polypeptide compound, as well as a precursor of the anti-inflammatory polypeptide compound, or any mixture thereof.

[0045] This invention also provides a method for preparing the anti-inflammatory polypeptide compound of the first aspect above, comprising: preparing a peptide resin by solid-phase polypeptide synthesis, obtaining a crude product by acid hydrolysis of the peptide resin, and obtaining a purified anti-inflammatory polypeptide compound by purifying the crude product.

[0046] The step of preparing peptide resin by solid-phase polypeptide synthesis involves sequentially inserting the corresponding protected amino acids from the following sequences onto a carrier resin via solid-phase coupling synthesis to prepare peptide resin.

[0047] In the above preparation method, the amount of Fmoc (9-fluorenylmethoxycarbonyl protecting group, 9-fluorenylmethyloxycarbonyl)-protected amino acid is 1.2 to 6 times the total molar amount of the resin added, and preferably the amount of Fmoc-protected amino acid is 2.5 to 3.5 times.

[0048] In the above preparation method, the substitution value of the carrier resin is 0.3 to 1.5 mmol / g resin, and preferably the substitution value of the carrier resin is 0.6 to 1.0 mmol / g resin.

[0049] In an optional embodiment of the present invention, the solid-phase coupling synthesis method specifically involves: removing the Fmoc protecting group from the protected amino acid-resin obtained from the reaction and then coupling it with the next protected amino acid. The deprotection time for removing the Fmoc protecting group is 10–60 minutes, preferably 15–25 minutes. The coupling reaction time is 60–300 minutes, preferably 100–140 minutes.

[0050] The coupling reaction requires the addition of a condensing agent, which is selected from any one of DIC (N,N'-Diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolylphosphide hexafluorophosphate, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, benzotriazol-N,N,N',N'-tetramethylurea hexafluorophosphate, or O-benzotriazol-N,N,N',N'-tetramethylurea tetrafluoroborate; preferably, the condensing agent is N,N-diisopropylcarbodiimide. The molar amount of the condensing agent is 1.2 to 6 times the total molar amount of amino groups in the amino resin, and preferably 2.5 to 3.5 times.

[0051] The coupling reaction requires the addition of an activating agent, which is selected from either 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole. Preferably, the activating agent is 1-hydroxybenzotriazole. The amount of activating agent used is 1.2 to 6 times the total molar number of amino groups in the amino resin, and preferably 2.5 to 3.5 times the total molar number of amino groups in the amino resin.

[0052] In an optional embodiment of the present invention, the reagent for removing Fmoc protection is a PIP / DMF (Piperidine / N,N-Dimethylformamide) mixed solution, wherein the piperidine content in the mixed solution is 10-30% (V). The amount of the Fmoc removal reagent used is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

[0053] In one optional embodiment of the present invention, the peptide resin is acid-hydrolyzed to remove the resin and side chain protecting groups, and then oxidatively cyclized to obtain the crude product.

[0054] Furthermore, the acid hydrolysate used during the acid hydrolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethylenedithiol (EDT), and water, with the volume ratio of the mixed solvent being: TFA 80-95%, EDT 1-10%, and the remainder being water.

[0055] Further preferably, the volume ratio of the mixed solvent is: TFA 89-91%, EDT 4-6%, and the balance is water. Most preferably, the volume ratio of the mixed solvent is: TFA 90%, EDT 5%, and the balance is water.

[0056] The amount of acid hydrolysant used is 4 to 15 mL per gram of peptide resin; preferably, the amount of acid hydrolysant used is 7 to 10 mL per gram of peptide resin.

[0057] The pyrolysis time using the acid hydrolysate is 1 to 6 hours at room temperature, preferably 3 to 4 hours at room temperature.

[0058] The oxidant used in the oxidative cyclization is any one of iodine, H2O2 (hydrogen peroxide), or DMSO (dimethyl sulfoxide), with iodine being the preferred oxidant. The oxidant is added by titration until the oxidation endpoint is reached, at which point the addition is stopped.

[0059] Furthermore, the crude product was purified by high performance liquid chromatography and lyophilized to obtain the pure product.

[0060] This invention also provides an application of the anti-inflammatory polypeptide compound in the first aspect above in the preparation of anti-inflammatory polypeptide compositions in the field of medical aesthetics.

[0061] In an optional embodiment of the present invention, the anti-inflammatory polypeptide composition is used for skin anti-inflammatory, repair, anti-wrinkle and anti-aging purposes.

[0062] The anti-inflammatory polypeptide compounds in the embodiments of the present invention further comprise pharmaceutical salts, chelates or non-covalent complexes formed from the anti-inflammatory polypeptide compounds, based on the precursors of the anti-inflammatory polypeptide compounds, or any mixtures thereof.

[0063] This invention also provides an application of the anti-inflammatory polypeptide compound described in the first aspect above in the preparation of anti-inflammatory polypeptide compositions in the field of medical aesthetics.

[0064] Optionally, the anti-inflammatory peptide composition is used for skin anti-inflammatory, repair, anti-wrinkle, and anti-aging purposes.

[0065] Unless otherwise specified, the quantities of different components and reaction conditions used in this invention are to be interpreted as "approximately" or "about". Accordingly, unless otherwise specified, the numerical parameters referred to below and in the claims are approximate parameters, and different numerical parameters may be obtained under their respective experimental conditions due to different standard errors.

[0066] In this invention, when there is disagreement or ambiguity regarding the chemical structure and chemical name of a compound, the compound is precisely defined by its chemical structure. The anti-inflammatory peptide compounds described herein may contain one or more chiral centers, and / or double bonds and similar structures, and may also exist as stereoisomers, including isomers of double bonds (e.g., geometric isomers), optical enantiomers, or diastereomers. Accordingly, any chemical structure within the scope of this description, whether partially or entirely containing similar structures, includes all possible enantiomers and diastereomers of this anti-inflammatory peptide compound, including any single stereoisomer (e.g., a single geometric isomer, a single enantiomer, or a single diastereomer) and any mixture of these isomers. These mixtures of racemic and stereoisomers can be further separated into enantiomers or stereoisomers of their constituent components by those skilled in the art using continuous separation techniques or chiral molecule synthesis methods.

[0067] The anti-inflammatory peptide compounds of Formula I include, but are not limited to, the optical isomers, racemates, and / or other mixtures of the aforementioned anti-inflammatory peptide compounds. In the above cases, the single enantiomer or diastereomer, such as the optically active isomer, can be obtained by asymmetric synthesis or by racemic resolution. Racemic resolution can be achieved by various methods, such as conventional recrystallization with a resolving agent or by chromatographic methods. Additionally, the anti-inflammatory peptide compounds of Formula I also include cis and / or trans isomers with double bonds.

[0068] The anti-inflammatory polypeptide compounds of this invention include, but are not limited to, the anti-inflammatory polypeptide compounds shown in Formula I and all pharmaceutically usable forms of said anti-inflammatory polypeptide compounds. These pharmaceutically usable forms of the aforementioned anti-inflammatory polypeptide compounds include various pharmaceutically usable salts, solvates, complexes, chelates, non-covalent complexes, drug prodrugs based on the aforementioned substances, and any mixtures of these forms.

[0069] The aforementioned drug prodrugs include esters or amide derivatives of polypeptide compounds, such as those shown in structural formula I, contained within the compounds.

[0070] The polypeptide compound shown in Structure I provided by this invention is stable and is a novel anti-inflammatory polypeptide compound with anti-inflammatory, repairing, anti-wrinkle, and anti-aging effects on the skin.

[0071] The following describes, with reference to specific embodiments, an anti-inflammatory polypeptide compound, its preparation method, and its application provided by the present invention.

[0072] Example 1: Preparation of anti-inflammatory polypeptide compounds

[0073] A: Synthesis of peptide resins

[0074] The carrier resin was taken, and the corresponding protective amino acids were sequentially added through Fmoc deprotection and coupling reactions to obtain peptide resin. The specific operation is as follows:

[0075] (1) Access the first protected amino acid in the main chain

[0076] Take 0.03 mol of the first protected amino acid and 0.03 mol of HOBt (1-Hydroxybenzotriazole), and dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid while stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution for later use.

[0077] Take 0.01 mol of Rink amide MBHA resin (Rink amide-MBHA Resin, Rink amide MBHA resin) (substitution value approximately 0.4 mmol / g), protect it with 20% PIP / DMF solution for 25 minutes, wash and filter to obtain Fmoc-free resin.

[0078] The activated solution of the first protected amino acid was added to the Fmoc-free resin, and the coupling reaction was carried out for 60–300 minutes. After filtration and washing, a resin containing one protected amino acid was obtained.

[0079] (2) Incorporate other protected amino acids into the main chain

[0080] Using the same method as described above for adding the first protected amino acid to the main chain, other corresponding protected amino acids are sequentially added to the main chain to obtain a resin containing main chain amino acids.

[0081] (3) Add the first protected amino acid to the side chain

[0082] Take 0.03 mol of the first protected amino acid on the side chain and 0.03 mol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid while stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution.

[0083] Take 2.5 mmol of tetrakis(triphenylphosphine)palladium and 25 mmol of phenylsilane, dissolve them in an appropriate amount of dichloromethane, remove the protection for 4 hours, filter and wash to obtain a resin with alloc (allyloxycarbonyl) removed for later use.

[0084] The activated side-chain first protected amino acid solution was added to the de-Alloc resin, and the coupling reaction was carried out for 60-300 minutes. After filtration and washing, a resin containing the first protected amino acid in the side chain was obtained.

[0085] (4) Add other protected amino acids to the side chain

[0086] Using the same method as described above for adding the first protected amino acid to the main chain, the corresponding protected amino acids of the side chains are added sequentially, and finally the protection is removed to obtain peptide resin.

[0087] B: Preparation of crude product

[0088] Take the above peptide resin and add a lysis reagent with a volume ratio of TFA:water:EDT = 95:5:5 (the ratio of lysis reagent to peptide resin is 10 mL / g resin). Stir well and react at room temperature for 3 hours. Filter the reaction mixture using a sintered funnel and collect the filtrate. Wash the lysed peptide resin three times with a small amount of TFA. Combine the filtrates and concentrate under reduced pressure. Add anhydrous diethyl ether to precipitate the precipitate and wash it three times with anhydrous diethyl ether. Dry the precipitate to obtain an off-white powder.

[0089] The off-white powder was dissolved in a 20% acetic acid aqueous solution, and a saturated iodine / ethanol solution was added dropwise with stirring until complete cyclization. The solution was then concentrated under reduced pressure at 35–40°C to obtain a crude concentrated solution.

[0090] C: Preparation of pure product

[0091] Take the above crude product concentrate solution, filter it through a 0.45μm mixed microporous membrane, and purify it for later use;

[0092] Purification was performed using high performance liquid chromatography (HPLC). The chromatographic packing material was a 10 μm reversed-phase C18 column, and the mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution. The flow rate of the 30 mm * 250 mm column was 20 mL / min. Gradient elution was used, and the sample was injected repeatedly for purification. The crude product concentrate was loaded into the column, the mobile phase was started for elution, and the main peak was collected. After acetonitrile was removed, the purified intermediate concentrate was obtained.

[0093] The purified intermediate concentrated solution was filtered through a 0.45 μm filter membrane for later use. High-performance liquid chromatography (HPLC) was used for salt replacement. The mobile phase system was 1% acetic acid / water solution-acetonitrile. The chromatographic packing material was a 10 μm reversed-phase C18 column (30 mm * 250 mm) with a flow rate of 20 mL / min (the flow rate can be adjusted according to different column specifications). Gradient elution and cyclic loading were used. The sample was loaded into the column, the mobile phase was started for elution, the chromatogram was collected, the absorbance change was observed, the main peak of the salt replacement was collected, and the solution was concentrated under reduced pressure to obtain pure acetic acid aqueous solution. The solution was then freeze-dried to obtain the pure product.

[0094] The above method can be used to prepare the following polypeptide compounds with structure I, as detailed in Table 2.

[0095] Table 2. Structures of polypeptide compounds prepared in the embodiments of the present invention.

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Example 2: Determination of bioactivity

[0103] Macrophages were stimulated with 50 μg / mL LPS (Lipopolysaccharide) solution. The anti-inflammatory efficacy of the test samples (positive control sample and peptide compound 1-peptide compound 60) was evaluated by detecting the changes in the content of pro-inflammatory factor (IL-1β) after treatment of macrophages with peptide compound 1-peptide compound 60 prepared in the embodiments of the present invention. The positive control sample was dexamethasone 0.01% (m / v).

[0104] The bioactivity assay results of polypeptide compound 1-polypeptide compound 60 prepared in the embodiments of the present invention are shown in Table 3.

[0105] Table 3. Anti-inflammatory effects of the polypeptide compounds prepared in this invention and positive control samples.

[0106]

[0107]

[0108]

[0109]

[0110]

[0111] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above provides a detailed description of an anti-inflammatory polypeptide compound, its preparation method, and its applications. Specific examples have been used to illustrate the principles and implementation methods of the invention. The descriptions of the embodiments are merely for the purpose of helping to understand the method and core ideas of the invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the invention.

Claims

1. An anti-inflammatory polypeptide compound or a pharmaceutically acceptable salt thereof, characterized in that, The anti-inflammatory polypeptide compound is shown in the following table: 。 2. A method for the preparation of an anti-inflammatory polypeptide compound according to claim 1 and pharmaceutically acceptable salts thereof, characterized by, Comprising: The peptide resin is prepared by solid-phase polypeptide synthesis method, the crude product is obtained by acidolysis of the peptide resin, and the purified anti-inflammatory polypeptide compound is obtained by purification of the crude product.

3. Use of the anti-inflammatory polypeptide compound and the pharmaceutical salt formed by the anti-inflammatory polypeptide compound in claim 1 in the preparation of an anti-inflammatory polypeptide composition in the field of medical cosmetology.

4. Use according to claim 3, characterized in that, The anti-inflammatory polypeptide composition is used for skin anti-inflammation.

Images