WO2023212841A1 - Type d'analogue polypeptidique ayant un effet de croissance capillaire - Google Patents

Type d'analogue polypeptidique ayant un effet de croissance capillaire Download PDF

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Publication number
WO2023212841A1
WO2023212841A1 PCT/CN2022/090865 CN2022090865W WO2023212841A1 WO 2023212841 A1 WO2023212841 A1 WO 2023212841A1 CN 2022090865 W CN2022090865 W CN 2022090865W WO 2023212841 A1 WO2023212841 A1 WO 2023212841A1
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WIPO (PCT)
Prior art keywords
fgf5
ser
leu
inhibitor
tyr
Prior art date
Application number
PCT/CN2022/090865
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English (en)
Inventor
Zhiwei Chen
Lijun Ji
Chushan WEI
Bixin Shen
Dongxue Wang
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Wenzhou Medical University
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Publication date
Application filed by Wenzhou Medical University filed Critical Wenzhou Medical University
Priority to PCT/CN2022/090865 priority Critical patent/WO2023212841A1/fr
Publication of WO2023212841A1 publication Critical patent/WO2023212841A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a polypeptide that inhibits the action of FGF5 receptor and its application for promoting hair growth.
  • Hair growth has a certain periodicity. Hair does not grow at a certain speed, but repeats the growth and resting phase at the same time.
  • fibroblast growth factor 5 FGF5
  • FGF5 fibroblast growth factor 5
  • FGF5S a short-chain peptide that expresses FGF5 in a large amount in hair follicle cells in the anagen phase, has an antagonistic effect on FGF5, delays the hair from the anagen phase to the degenerative phase, and promotes hair growth.
  • the invention relates to a class of FGF5 inhibitor polypeptides. Its structure has the following form:
  • Xaa1 Ala, Leu, Val, Met, Ile, Tyr, Phe, Arg, Asn, Lys, Thr, Asp, His, Trp, Gln, Glu, Ser or Gly;
  • Xaa2 Cys or Ser
  • Xaa3 Cys or Ser
  • the object of the invention is to provide a preparation method of FGF5 inhibitor polypeptides.
  • the present invention adopts microwave-promoted Fmoc/tBu orthogonal protection solid-phase synthesis strategy to synthesize efficiently and rapidly to obtain FGF5 inhibitor polypeptides.
  • the proposed (FGF5) inhibitor polypeptide can effectively inhibit FGFR receptors, inhibit downstream signals, and promote hair growth.
  • the peptide chain of the FGF5) inhibitor polypeptide analog synthesized by microwave-promoted solid-phase greatly improves the coupling reaction rate.
  • the conventional solid-phase synthesis method can fully couple an amino acid to the resin, which often requires 2 hours to 20 hours, or even longer. While, on average, the microwave promotion only takes about 10 minutes. Moreover, to remove Fmoc protecting groups, conventional solid-phase synthesis methods often take 30 minutes to 1 hour while the microwave promotion takes only about 5 minutes on average, which greatly improves peptide synthesis efficiency and shortens the synthesis cycle.
  • the purity of the crude peptide chain obtained from the FGF5 inhibitor polypeptide by microwave-promoted solid-phase synthesis is greater than 60%. Compared with the conventional solid-phase synthesis method, it is greatly improved, which facilitates the subsequent purification work.
  • Microwave-promoted solid-phase synthesis of fibroblast growth factor 5 (FGF-5) inhibitor polypeptide analogs has a low cost. Due to the high coupling efficiency, the average amount of protected amino acids required is only 2 times in excess, which is more than the number of such acids under conventional solid-phase synthesis. Conventional solid-phase synthesis method requires 4 to 5 times excess, this method greatly reduces the amount of amino acids used compared to conventional methods.
  • FGF-5 fibroblast growth factor 5
  • the method of microwave-assisted solid-phase synthesis of fibroblast growth factor 5 (FGF-5) inhibitor polypeptide analogs is easy to realize automation and mass production which makes it more suitable for industrial production.
  • the fibroblast growth factor 5 (FGF-5) inhibitor polypeptide analog prepared by the microwave-promoted solid-phase synthesis technology provided by the present invention has high yield, short synthesis cycle, convenient crude product purification, low production cost and easy industrial automation production.
  • the prepared fibroblast growth factor 5 (FGF-5) inhibitor polypeptide is suitable as an active ingredient for treating hair loss and promoting hair growth.
  • Figure 1 shows the binding ability of FGF5 inhibitor polypeptide (SEQ. ID NO: 2) to FGFR receptor;
  • FIG. 2 shows the WB result of blocking effect of FGF5 inhibitor polypeptide (SEQ. ID NO: 2) downstream signaling pathway;
  • FIG. 3 shows the effect of FGF5 inhibitor polypeptide (SEQ. ID NO: 2) on hair growth promotion
  • Et3N Triethylamine; NMM: N-Methylmorpholine; DIEA: N, N'-Diisopropylethylamine; DMF: Dimethylformamide; DMSO: Dimethyl sulfoxide; DCM: Dichloromethane; Fmoc: N-9-Fluorenemethoxycarbonyl; DIC: N, N'-Diisopropylcarbodiimide; CDI: N, N'-Carbonyldiimidazole; DMAP: 4-Dimethylaminopyridine; HOSU: N-Hydroxysuccinimide; EDC.
  • HCl 1-Ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride; HATU: 2- (7-azobenzotriazole) -N, N, N', N'-Tetramethylurea hexafluorophosphate; HBTU: Benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate; HCTU: 6-Chloro Benzotriazole-1, 1, 3, 3-tetramethylurea hexafluorophosphate; HOAT: 1-Hydroxy-7-azobenzotriazole; HOBT: 1-Hydroxy-benzotriazole ; PyBOP: Benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate; HPLC: High performance liquid chromatography; ESI-MS: Electrospray mass spectrometry; Gly: Glycine; Ser: Serine;
  • Fmoc-Rink amide-MBHA Resin (substitution amount 0.4 mmol/g) was weighed 50 mg ) and swelled with 7 mL of DCM for 30 min. Then the solution was filtered off the DCM and swelled with 10 mL of NMP for 30 min. Finally, The final solution was washed with NMP, DCM, NMP 7 mL respectively.
  • Fmoc-Gly-OH (0.04 mmol) , HBTU (0.04 mmol) , HOBT (0.04 mmol) and DIPEA (0.08 mmol) were dissolved in 10 mL of NMP and this solution was added to the above resin in a microwave reactor.
  • the reaction was carried out for 7 min, with the microwave power as 25 Wand the reaction temperature controlled at 50 °C, and an air compressor was used to compress air for cooling. After the reaction, the solution was filtered off, and the resin was washed for three times with 7 mL of DCM and 7 mL of NMP each.
  • Ninhydrin method wash a small amount of resin particles with ethanol, put them into a transparent vial, add 2 drops each of 5%ninhydrin ethanol, KCN pyridine solution (2ml 0.001M KCN diluted in 98ml pyridine) , 80%phenol ethanol solution, and heat at 100°C for 5 minutes. If the resin turns blue, it is positive.
  • Bromophenol blue method take a small amount of resin particles and wash them with dimethylacetamide, put them into a transparent vial, add 3 drops of 1%bromophenol blue dimethylacetamide solution, and shake at room temperature for 3 minutes. If the resin turns blue, it is positive.
  • FGF5 inhibitor polypeptides have an activating effect on FGFR
  • affinity of FGF5 inhibitor polypeptides to FGFR1c was assessed for surface ion resonance SPR experiments in HBS-EP buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% [v/v] polysorbate 20) at 25°C.
  • FGF5 inhibitor In order to study the inhibitory effect of FGF5 inhibitor on FGF5, epidermal cells Hcat were used to stimulate the relevant downstream signaling molecular mechanism. As shown in Figure 2, Western blot results showed that FGF5 can significantly enhance the downstream p-FRS2, p-The expression of Erk2 and p-AKT, and the addition of inhibitor peptides. The phosphorylation indicators corresponding to these signaling pathways were attenuated and decreased in a dose-dependent manner. It shows that the FGF5 inhibitor polypeptide can effectively inhibit the activity of FGF5.
  • C57BL/6 mice were selected and randomly divided into 3 groups, namely PBS group, FGF5 group (30 mg/ml) , and FGF5+FGF5 inhibitor polypeptide group (50 mg/ml) . After applying the drug for 28 days (twice a day) , and recording representatively the scores of new hair growth in mice at the three groups, the effect of FGF5 inhibitor polypeptide application on new hair growth and new hair weight was detected.
  • the hair of the mice with FGF5 inhibitor peptides has basically regenerated, but the hair of the control and FGF5 smeared mice still fails not grow back, indicating that the FGF5 inhibitor peptides have an effect on the hair growth of the mice. It speeds up hair growth and increases the length and weight of new hair of mice.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Dermatology (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Birds (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur l'effet inhibiteur du facteur de croissance des fibroblastes 5 (FGF5) et son utilisation. L'analogue polypeptidique peut inhiber de manière efficace les récepteurs associés au facteur de croissance des fibroblastes 5 (FGF5) et présente une spécificité élevée. L'agent peut être utilisé pour augmenter la longueur des cheveux et présente une valeur d'application significative pour la croissance des cheveux.
PCT/CN2022/090865 2022-05-05 2022-05-05 Type d'analogue polypeptidique ayant un effet de croissance capillaire WO2023212841A1 (fr)

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PCT/CN2022/090865 WO2023212841A1 (fr) 2022-05-05 2022-05-05 Type d'analogue polypeptidique ayant un effet de croissance capillaire

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Application Number Priority Date Filing Date Title
PCT/CN2022/090865 WO2023212841A1 (fr) 2022-05-05 2022-05-05 Type d'analogue polypeptidique ayant un effet de croissance capillaire

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004080478A1 (fr) * 2003-03-14 2004-09-23 The Walter And Eliza Hall Institute Of Medical Research Compositions therapeutiques et prophylactiques et leurs utilisations
US20120114745A1 (en) * 2009-06-26 2012-05-10 Nam-Mew Pui Method for treating or ameliorating mucocutaneous or ocular toxicities
US20150150771A1 (en) * 2012-06-19 2015-06-04 Debraj Shome Composition and method for an intradermal hair growth solution
CN112062828A (zh) * 2019-06-10 2020-12-11 温州医科大学 Fgf-5抑制剂多肽类及其生发应用
CN112110988A (zh) * 2020-08-21 2020-12-22 温州医科大学 一种具有毛发生长作用的多肽类似物及制备方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004080478A1 (fr) * 2003-03-14 2004-09-23 The Walter And Eliza Hall Institute Of Medical Research Compositions therapeutiques et prophylactiques et leurs utilisations
US20120114745A1 (en) * 2009-06-26 2012-05-10 Nam-Mew Pui Method for treating or ameliorating mucocutaneous or ocular toxicities
US20150150771A1 (en) * 2012-06-19 2015-06-04 Debraj Shome Composition and method for an intradermal hair growth solution
CN112062828A (zh) * 2019-06-10 2020-12-11 温州医科大学 Fgf-5抑制剂多肽类及其生发应用
CN112110988A (zh) * 2020-08-21 2020-12-22 温州医科大学 一种具有毛发生长作用的多肽类似物及制备方法和应用

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