WO2016192067A1 - 趋化素修饰胜肽 - Google Patents

趋化素修饰胜肽 Download PDF

Info

Publication number
WO2016192067A1
WO2016192067A1 PCT/CN2015/080725 CN2015080725W WO2016192067A1 WO 2016192067 A1 WO2016192067 A1 WO 2016192067A1 CN 2015080725 W CN2015080725 W CN 2015080725W WO 2016192067 A1 WO2016192067 A1 WO 2016192067A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
peptide
chemokine
seq
modified
Prior art date
Application number
PCT/CN2015/080725
Other languages
English (en)
French (fr)
Inventor
郑锡聪
程家维
游辉元
Original Assignee
翔升科技股份有限公司
郑锡聪
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 翔升科技股份有限公司, 郑锡聪 filed Critical 翔升科技股份有限公司
Priority to KR1020177034607A priority Critical patent/KR102088904B1/ko
Priority to JP2017562314A priority patent/JP6901407B2/ja
Priority to PCT/CN2015/080725 priority patent/WO2016192067A1/zh
Priority to US15/569,033 priority patent/US20180125938A1/en
Priority to EP15893723.5A priority patent/EP3315510A4/en
Priority to AU2015397751A priority patent/AU2015397751B2/en
Publication of WO2016192067A1 publication Critical patent/WO2016192067A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5421—IL-8
    • 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 present invention relates to a modified chemokine which is useful as a therapeutic agent.
  • the present invention relates in particular to a modified chemokine for treating cancer and inhibiting tumor growth.
  • Chemokine is a group of small molecules (8-14kd) that can be induced, secreted and structurally similar. Chemokines can be divided into CXC, CC, CX3C and C subgroups; if classified by function, chemokines can be divided into Homeostasis chemokines and Inflammatory chemokines.
  • Chemokines usually have three ⁇ -sheets in their structure, and have an ⁇ -helix at the C-terminus and 4 retained cystines at the N-terminus ( Cyceine). According to the first two cystine sequences contained in the N-terminus, it can be divided into four groups: CXC, CC, C, CX3C, and CC and CXC chemotactic hormones.
  • the chemokines on the cells react with the chemokine receptor, and the chemokine receptor has seven G-protein binding receptors across the cell membrane, depending on the type of ligand on the binding target.
  • CXCR CCR
  • CXR CX 3 CR
  • CXCR1, CXCR2, CCR4 a chemotactic receptor
  • the binding markers of inflamed cell infiltration and the chemotactic receptor are not one-to-one.
  • Some of the chemotactic receptors need to be stimulated and induced in different cells. .
  • an ELR-CXC chemokine containing a glutamic acid (E)-leucine (L)-arginine (R) signature sequence (ELR signature sequence), which has an amino acid at the N-terminus of the protein
  • ELR signature sequence a glutamic acid (E)-leucine (L)-arginine (R) signature sequence
  • X may be a polar and electrically or non-electron amino acid, or X is not present, which regulates oncogene growth, IL-8, type II neutrophil
  • the ball-activated peptide (NAP-2) has a receptor for CXCR1 and CXCR2.
  • the main cells are neutrophils, which promote the accumulation and activation of neutrophils.
  • ELR-CXC chemokines A wide range of acute and chronic inflammatory conditions have a major association and play a major role in these inflammatory reactions including psoriasis and rheumatoid arthritis.
  • ELR-CXC chemokines are also more involved in angiogenesis associated with tumor development, and the mechanism of induction is through such chemokines, especially IL-8, and endothelial cells (EC). Activation by the combination of CXCR1 and CXCR2. It has been demonstrated that many different types of tumors produce ELR-CXC chemokines, and tumors that exhibit this type of chemokine have been thought to be involved in poor prognosis of tumors.
  • Cancer is one of the leading causes of death in developed countries. Although there have been advances in the diagnosis and treatment of cancer, surgery and radiotherapy may cure cancer if cancer is detected early, but most drugs do not have major side effects, or they are not effective. Therefore, the medical community urgently needs a method and composition to treat or prevent cancer.
  • the present invention develops a modified chemotactic peptide (peptide) to inhibit tumor growth and treat cancer.
  • the present invention provides a modified chemotactic peptide which comprises an amino acid sequence having an N-terminus (N') carrying: (a) N'-glutamic acid (E)-bright a characteristic sequence of lysine (L)-arginine (R) located at the N-terminus of the chemokine peptide; (b) N'-valine (P)-alanine (A)- a characteristic sequence of serine (S)-glutamine (Q)-phenylalanine (F)-Cys 3 , this characteristic sequence is located at the N-terminus of the chemotactic peptide, and the third cystine (Cys 3 )
  • the chemokine peptide comprises the characteristic sequence of (a), (b) and has a modified position, and the modification position is located on the chemokine peptide from the N-term, 17th, 12th, 13 amino acids.
  • the first cystine of the N-terminus of the source chemotactic peptide and the second 0-2 amino acids can exist between cystines, and when ammonia When the base acid is 1-2, the amino acid is a polar amino acid which is electrically or not electrically.
  • the source chemokine peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO : 6.
  • a modified chemotactic peptide of the invention is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and combinations thereof.
  • the invention further provides a pharmaceutical composition comprising a modified chemotactic peptide of the invention and a pharmaceutically acceptable excipient.
  • the modified chemotactic peptide is used to treat cancer or inhibit tumor growth.
  • the invention further provides a pharmaceutical composition for treating cancer and inhibiting tumors, comprising the modified chemotactic peptide of the invention and a pharmaceutically acceptable excipient.
  • the cancer comprises prostate cancer, breast cancer, uterine cancer, blood cancer, ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, testicular cancer, lymphoma, rhabdomyosarcoma, neuroblastoma, pancreas Dirty cancer, lung cancer, brain tumor, skin cancer, stomach cancer, oral cancer, liver cancer, laryngeal cancer, biliary cancer, thyroid cancer, liver cancer, kidney cancer, and nasopharyngeal cancer.
  • Figure 1 is a source chemokine peptide sequence of the present invention (SEQ ID NO: 1-SEQ ID NO: 9) is also an amino acid sequence alignment map of ELR-CXC chemokines having high affinity for CXCR1 or/and CXCR2.
  • Figure 2 is a diagram showing the amino acid sequence alignment of the modified chemotactic peptides of the present invention (SEQ ID NOS: 10-12).
  • Figure 3 shows the number of cell migrations with the chemokine CXCL8.
  • Figure 4 shows the black fraction with chemical hormone stimulation, the gray fraction with chemical hormone stimulation and CXCL8-IP10, and the white fraction with chemical hormone stimulation and IL8-17LIP10.
  • Figure 5 shows the tumor volume of the experimental group (IL8-17LIP10) and the control group (saline).
  • Figure 6 shows the tumor weight of the experimental group (IL8-17LIP10) and the control group (saline)
  • Figure 7 shows the microvessel density of the experimental group (IL8-17LIP10) and the control group (saline).
  • Figure 8 is a graph showing the tumor volume after injection of CXCL8-IP10 and physiological saline, wherein:
  • Group A CXCL8-IP10 500ug/kg injected 4 times a week
  • Group B CXCL8-IP10 500ug/kg twice a week
  • Group C CXCL8-IP10 250ug/kg twice a week
  • Group D 100 ⁇ l of saline was injected subcutaneously daily and the tumor volume was observed.
  • Figure 9 shows the mouse lung tumor tissue after sacrifice, wherein A is the lungs of the rats injected with CXCL8-IP10 500ug/kg four times a week after sacrifice on the 24th day; B is the lungs of the mice injected with physiological saline.
  • the white tumor site indicated by the arrow is the lesion that is transferred to the lungs.
  • Figure 10 shows the trend of expression of CXCR1 or CXCL8 in tumor cells.
  • Figure 11 shows the effect of the antagonist of the present invention on chemotaxis.
  • the present invention provides a novel chemotactic peptide.
  • the novel chemokine peptide of the present invention is derived from a receptor belonging to ELR-CXC chemokine or having high affinity for CXCR1 or CXCR2, for example, CXCL1 (SEQ ID NO: 3), CXCL2 (SEQ ID NO: 4) , CXCL3 (SEQ ID NO: 5), CXCL5 (SEQ ID NO: 6), CXCL6 (SEQ ID NO: 7), CXCL7 (SEQ ID NO: 8), CXCL8 (SEQ ID NO: 2), hG31P (SEQ ID NO: 1).
  • the present invention is modified according to this type of chemokine, mainly inserting a PASQF characteristic sequence at its 30s-loop (Fig. 1), this PASQF is originally present in the chemotactic CXCL10, and the CXCL10 is a Non-ELR-CXC chemokines.
  • the modified chemokine of the present invention comprises: (a) a characteristic sequence of N'-glutamic acid (E)-leucine (L)-arginine (R), wherein the characteristic sequence is located in the chemotropin N-terminal of peptide; and (b) characteristics of N'-valine (P)-alanine (A)-serine (S)-glutamine (Q)-phenylalanine (F)-Cys 3 Sequence, this characteristic sequence is located at the N-terminus of the chemotactic peptide and the third cystine (Cys 3 ). Furthermore, it should be noted that the chemokine peptide of the present invention has a more mutation position (i.e., a modification position) which is located at the 17th amino acid from the N-terminus of the chemokine peptide.
  • the 17th amino acid of the chemokine peptide of the present invention is originally phenylalanine (F), and is changed to other non-phenylalanine after mutation substitution. Amino acid.
  • the 17th amino acid of the chemokine peptide of the present invention may be alanine (A), cysteine (C), selenocysteine (U), aspartic acid ( D), asparagine (N), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), leucine (L), isoleucine (I) Lysine (K), pyrrolysine (O), methionine (M), valine (P), arginine (R), serine (S), threonine (T), proline (V), tryptophan (W), tyrosine (Y), preferably leucine (L).
  • the 17th amino acid has
  • the modified chemokine peptide of the invention comprises, but is not limited to, SEQ ID NO: 10, SEQ ID NO: 11 and/or SEQ ID NO: 12 (Fig. 2) .
  • SEQ ID NO: 10 has a N-terminal sequence of ELR-CQC and conforms to the rule of amino acid characteristic sequence of ELR-CX n C, and SEQ ID NO: 10 also has an N-terminal Ol-peptide sequence of Pro-Ala-Ser-Gln-Phe (valine-alanine-serine-glutamine-phenylalanine, PASQF), this PASQF is a modified sequence and inserted at the N-terminus Upstream of the third cysteine (Cysteine, C) (10192), and this third cysteine is immediately after the phenylalanine (F) of the PASQF oligopeptide sequence. More importantly, the amino acid at position 17 is not phenylalanine but leucine.
  • the ELR-CXC chemokine analog sequences of SEQ ID NO: 10 to SEQ ID NO: 12 have the third cysteine before the N-terminus. PASQF modified sequence.
  • this hair The 17th amino acid of the chemokine peptide is mutated to leucine.
  • the modified chemotactic peptides, analogs and/or fragments thereof of the present invention can inhibit diseases associated with angiogenesis.
  • Diseases associated with angiogenesis include, but are not limited to, inflammatory diseases, chronic rheumatoid arthritis and psoriasis, diseases associated with abnormal vascular invasion, and cell proliferative diseases such as diseases associated with tumors or cancer (eg, prostate cancer) , breast cancer, uterine cancer, blood cancer, ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, testicular cancer, lymphoma, rhabdomyosarcoma, neuroblastoma, pancreatic cancer, lung cancer, brain tumor, skin cancer , stomach cancer, oral cancer, liver cancer, laryngeal cancer, biliary cancer, thyroid cancer, liver cancer, kidney cancer, and nasopharyngeal cancer, etc.).
  • chemokine-modified peptide of the present invention and/or the pharmaceutical composition comprising the chemokine-modified peptide can be administered orally, parenterally, by inhalation, rectally, vaginally, intradermally, transdermally or topically, and a pharmaceutical unit can include Traditional non-toxic pharmaceutically acceptable carriers, adjuvants and carriers.
  • the chemokine-modified peptide of the present invention and/or the pharmaceutical composition comprising the chemokine-modified peptide can be administered at one time, multiple times or continuously within 24 hours.
  • a suitable conventional means may be used, including, but not limited to, intravenous drip, intravenous drip, implantable syringe pump or topical administration.
  • the duration of treatment can be adjusted according to different conditions, for example, the course and severity of angiogenesis.
  • the chemotactic peptides modified with the present invention alone or in combination with other agents of the invention are cured, or continue to be treated for life.
  • the invention provides a pharmaceutical composition for treating cancer and inhibiting tumors.
  • the pharmaceutical composition comprises an effective amount of the chemoattractant of the present invention A peptide or an analog thereof, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include solvents, dispersing agents, coatings, antibacterial/antifungal agents, isotonicity, absorption delaying agents, and the like.
  • LMVECs were cultured in HuMedia-EB2 containing 2% FCS for 8 hours, then cells of 12 ⁇ 10 4 cells/cm 2 were flattened to a pore size of 5 ⁇ m and coated with 10 ⁇ g/ml fibronectin. Filter (Sigma-Aldrich). 10 ng/mL of CXCL8, CXCL6, CXCL1, CXCL5, and CXCL8-IP10 or IL8-F17LIP10 were placed in the bottom of the Boyden chamber, respectively.
  • LMVECs were cultured in a Boyden chamber for 4 hours at 37 ° C, after which the filters were fixed and stained using Diff-Quick (Harleco), and the migrated cells were counted using (HPFs) (X200) to calculate the number of cells migrated.
  • Figure 3 shows the largest number of migrations with the chemotactic CXCL8.
  • the black part in Figure 4 has only chemical hormone stimulation, the gray part has chemical hormone stimulation and CXCL8-IP10, and the white part has chemical hormone stimulation and IL8-17LIP10. It can be seen from Fig. 4 that CXCL8-IP10 and IL8-17LIP10 also have the effect of inhibiting cell migration, and the effect of IL8-17LIP10 in inhibiting cell migration is better than that of CXCL8-IP10.
  • Athymic male rats (4-6 weeks BALB/c) were placed in a sterile operating room.
  • the aseptic operating room has sufficient water and feed and the mice are monitored daily.
  • the GFP- labeled PC-3 cells (PC-3-GFP) was injected into the three right abdomen of nude mice, 5x10 6 cells per injection in nude mice. After 2-4 weeks of culture, tumors were taken for transplantation. The tumor tissues of the three nude mice were taken out for sectioning (1 mm 3 sections), and transplanted into the prostate of other mice under anesthesia and disinfection.
  • tumor growth images were photographed on a 12th, 18th, and 24th day using an optical dissecting microscope and a digital camera with a 515 mm filter.
  • tumor GFP fluorescence images were obtained.
  • Figure 5 shows the tumor volume of the experimental group and the control group on days 12, 18 and 24.
  • the sequence of the present invention IL8-17LIP10
  • IL8-17LIP10 can significantly inhibit tumor growth, inhibit tumor volume by more than 5 times, and the inhibitory effect is more pronounced with time.
  • Figure 6 shows the tumor weight of the experimental group and the control group on the 24th day.
  • the sequence of the present invention IL8-17LIP10
  • IL8-17LIP10 can significantly inhibit tumor growth and inhibit tumor weight by more than 2 times.
  • Tumor sections embedded in paraffin were dewaxed and rehydrated with PBS.
  • the sections were wetted 3 times with PBS and applied to 10 mM sodium citrate (pH 6.0). Heat treatment for 15 minutes. After that, it was treated with 3% hydrogen peroxide for 10 minutes to remove endogenous peroxidase activity, washed three times with PBS, and treated with protein blocking solution (PBS solution containing 5% horse serum) at room temperature. After a minute, wash with PBS 3 times, then mouse anti-VEGF monoclonal antibody (1:50), rabbit anti-NF- ⁇ B polyclonal antibody or goat anti-CD3 polyclonal antibody (1:50) at 4 °C The reaction was carried out for 20 hours.
  • Each group was analyzed for transplanted tumors of 5 mice. Five tumor images were randomly selected for each tumor to average the grayscale value of each tumor (Csillik et al, 2005). Immunohistochemical analysis of CD31 can be used to identify microvessel density, counted using Image-Pro 6.0 Microsoft software, and expressed as an average.
  • Figure 7 shows the microvessel density of the control group and the experimental group.
  • the sequence of the present invention (IL8-17LIP10) is effective for inhibiting microangiogenesis of prostate cancer in mice.
  • mice C57BL/6 mice were placed in a sterile operating room.
  • the aseptic operating room has sufficient water and feed and the mice are monitored daily.
  • the LLW2 cells Lewis lung carcinma
  • the LLW2 cells was injected into the three right abdomen of nude mice, 5x10 6 cells per injection in nude mice. After 2-4 weeks of culture, tumors were taken for transplantation. A total of 24 mice received tumor transplantation. On the 5th day after transplantation, the animals were divided into 4 groups (6 in each group). Group A was injected with IL8-IP10 500ug/kg 4 times a week, and group B was injected with IL8-IP10 500ug/kg twice a week.
  • Figure 8 shows that group A is most effective in inhibiting tumor volume. On day 24, all mice were sacrificed before the measured tumor size. It was found that the tumor volume of mice injected with CXCL8-IP10 500ug/kg 4 times per week in group A was 30% smaller than that in group D. The results show that CXCL8-IP10 has a significant effect on the control of tumor development.
  • Figure 9 shows the mouse lung tumor tissue after sacrifice, and it can be seen from Figure A that the distant metastasis of the tumor has been significantly inhibited by CXCL8-IP10.
  • A is the lungs taken after 4 weeks of CXCL8-IP10 500ug/kg mice were sacrificed on the 24th day;
  • Figure B is the lungs of the rats that were given saline, in which the white tumor pointed by the arrow The site is the lesion that is transferred to the lungs.
  • the lungs of mice administered with CXCL8-IP10 are very clean and have no pulmonary metastases. The appearance of the stove.
  • Neutrophil leukocyte chemotaxis was assessed by modified Boyden chamber microchemotaxis assays.
  • White blood cells are obtained by separation of human peripheral blood via a general concentration gradient, while neutrophils are obtained from the bottom of a low-concentration separation and use hypotonic lysis to remove contaminating red blood cells.
  • the purified 5 ⁇ 10 6 /ml neutrophils were suspended in HBSS (400 mg/L potassium chloride (KCl), 60 mg/L potassium hydrogen phosphate (KH 2 PO 4 ), 8000 mg/L sodium chloride (NaCl), 350 mg/L sodium bicarbonate (NaHCO 3 ), 90 mg/L sodium hydrogen phosphate (NaH 2 PO 4 ⁇ 7H 2 O), 1000 mg/L glucose, and 0.5% pH 7.4 bovine placental serum (fetal calf serum; pH) 7.4), then neutrophils were cultured for 30 minutes with Calcein AM (Invitrogen, Sweden) at a culture temperature of 37° C.
  • Calcein AM Invitrogen, Sweden
  • Chemokines eg CXCL8 20 ng/mL
  • other antagonists IL8- IP10F17L, etc.
  • the intensity of antagonist refers to the simultaneous movement of the antagonizing protein and CXCL8 produced by the present invention
  • the intensity CXCL8 means that the cells move only through CXCL8 stimulation
  • the intensity HBSS means that the movement of the cells is caused by gravity .
  • RNA of each cancer cell line of the experiment was extracted by TRIzol reagent (Invitrogen, America) and its operation instructions, and most of the RNA was quantified. ND-1000spectrophotometer. Reverse transcription reaction, using a sample of cDNA reverse-transcription kit (Prime Script TM RT reagent kit, Takara, Japan) were cultured, and placed on ice prior to analysis of gene expression. GAPDH is an internal control group. RT-PCR reaction was completed by SYBR (Premix Ex Taq TM, Takara , Japan), which CXCL the following primers:
  • Figure 10 shows that some tumor cells are highly expressed by RT-PCR for the chemokine receptor CXCR1.
  • many tumor cells express high levels of CXCL8, which are two types of receptors, such as CXCR1/2.
  • CXCR1/2 receptor antagonists to achieve the purpose of preventing tumor cell growth and metastasis.
  • Figure 11 shows more antagonist design, adding amino acid variation 12 or/and 13 or/and 17 in the previously disclosed CXCL8–IP 10, which enhances CXCR1/2 for its receptor.
  • the antagonistic effect of the physiological response is effectively reduced by the expression of the CXCR1/2 receptor expressed by the tumor cells, or because the tumor cells express a high amount of their receptor-dependent receptor cytokines (CXCL1, 2, 3, 5, 6,7,8, etc. are accompanied by inhibition of tumor proliferation, drug resistance, metastasis and angiogenesis.
  • chemokine peptide of the present invention can effectively inhibit tumor growth, angiogenesis, and treat cancer.

Landscapes

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

Abstract

提供一种修饰的趋化素胜肽，其包含如下氨基酸序列：（a）N端具有ELR序列；（b）从N端起的第3个半胱氨酸残基前为PASQF序列；以及（c）从N端起的第17位氨基酸残基被修饰。该趋化素胜肽可用于治疗癌症、抑制肿瘤生长。

Description

趋化素修饰胜肽 技术领域
本发明是关于一种经修饰的趋化素，可作为一种治疗剂，尤其，本发明特别有关于一种用于治疗癌症与抑制肿瘤生长的经修饰趋化素。
背景技术
趋化素(Chemokine，又称趋化因子)是一群可被诱导，可被分泌而且结构类似的小分子(8-14kd)。趋化素可分为CXC、CC、CX3C及C四亚群；若以功能来分类，趋化素可分为恒定性趋化素(Homeostasis chemokines)及发炎性趋化素(Inflammatory chemokines)。
趋化素在其结构上通常具有三个β-褶板(β-sheet)，且在C端具有一个α-螺旋(α-helix)，在N端上会有4个保留的胱氨酸(cysteine)。按照N端所含前两个胱氨酸序列可分成四个族群：CXC、CC、C、CX3C，而以CC和CXC趋化激素为主。细胞上的趋化素与趋化素接受器结合之后才产生反应，而趋化素接受器具有7个跨细胞膜的G蛋白结合接受器，依其结合标的上的配体(ligand)种类不同而分别被命名为CXCR、CCR、CXR、CX3CR，若是再依照数目顺序排列，如CXCR1、CXCR2、CCR4等。但在标的细胞上不只表现一种趋化素接受器，发炎细胞浸润的结合标的与趋化素接受器也并非一对一，部份趋化素接受器在不同细胞需一定刺激诱导 才会表现。
例如，含有谷氨酸(E)-亮氨酸(L)-精氨酸(R)特征序列(ELR特征序列)的ELR-CXC趋化素，该ELR-CXC是指在蛋白质N端具有氨基酸序列为ELR-CXC特征的蛋白，X可为极性且带有电性或不具电性的氨基酸，或者X并不存在，其可调控致癌基因的生长、IL-8、第二型嗜中性球活化胜肽(NAP-2)，其接受器为CXCR1、CXCR2，主要作用细胞为嗜中性球，可促进嗜中性球的蓄积与活化作用，是以此类ELR-CXC趋化素与广范围的急性与慢性发炎病症的发生，素有重大关联并扮演着重大的角色，此等的发炎反应包含牛皮癣与风湿性关节炎。
此外ELR-CXC趋化素还更与肿瘤发展时所伴随的血管新生有关，其诱发机制是为经由此类趋化素，尤指IL-8，与血管内皮细胞(Endothelial cell,EC)上的CXCR1以及CXCR2的结合所产生的活化作用。目前已证实有许多不同类型的肿瘤会产生ELR-CXC趋化素，且会表现此类型趋化素的肿瘤已被认为与肿瘤的不良预后发展有关。
藉由拮抗CXCR1或CXCR2与ELR-CXC趋化素的结合，为一可行的策略，以便抑制由CXCR1或/及CXCR2受体激活后所引发的异常讯号传递，进而治疗带有该两种受体的细胞活化所造成的相关疾病，因此科学家正致力于寻找及制备抑制CXC趋化素的受质蛋白相似物。
本案发明人鉴于习知技术中的不足，经过悉心试验与研究，并一本锲而不舍的精神，终构思出本案发明，能够克服先前 技术的不足，以下为本案之简要说明。
发明内容
癌症为已开发国家的重要死因之一。尽管目前对于癌症的诊断及治疗方面已有进展，若能早期发现癌症则手术及放射性治疗可能治愈癌症，但大部分的药物不是有很大的副作用，就是效果不佳。因此，医学界亟需一种方法及组成物来治疗或防止癌症。
有鉴于此，本发明开发一种经修饰趋化素胜肽(peptide，肽)，以抑制肿瘤生长与治疗癌症。
本发明提供一种经修饰趋化素胜肽，其包含一氨基酸序列，此氨基酸具有一N端(N’)，此氨基酸序列带有：(a)N’-谷氨酸(E)-亮氨酸(L)-精氨酸(R)的特征序列，此特征序列位于所述趋化素胜肽N端；(b)N’-脯氨酸(P)-丙氨酸(A)-丝氨酸(S)-谷氨酰胺(Q)-苯基丙氨酸(F)-Cys3的特征序列，此特征序列位于趋化素胜肽N端算起第3个胱氨酸(Cys3)；其中所述趋化素胜肽是包含该(a)、(b)特征序列并具有一修饰位置，且此修饰位置位于所述趋化素胜肽上由N端算起第17,12,13个氨基酸。
在一实施例中，其中此趋化素胜肽上第17个氨基酸由苯基丙氨酸(F)置换为亮氨酸(L)。
在一实施例中，其中此经修饰趋化素胜肽的未经修饰的前身系来自于来源趋化素胜肽，所述来源趋化素胜肽N端的第1个胱氨酸及第2个胱氨酸之间可存在0-2个氨基酸，且当氨 基酸为1-2个时，此氨基酸为具有电性或不具电性的极性氨基酸。
在一实施例中，其中所述来源趋化素胜肽选自由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9及其组合所组成的群组其中之一。
在一实施例中，本发明经修饰趋化素胜肽选自由SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12及其组合所组成的群组其中之一。
本发明另提供一种医药组合物，包含本发明的经修饰趋化素胜肽及医药可接受性赋形剂。
在一实施例中，其中经修饰趋化素胜肽的用途是用于治疗癌症或抑制肿瘤生长。
本发明另提供一种治疗癌症及抑制肿瘤的医药组合物，包括本发明的经修饰趋化素胜肽及医药可接受性赋形剂。
在一实施例中，其中癌症包括前列腺癌、乳癌、子宫癌、血癌、卵巢癌、子宫内膜癌、子宫颈癌、大肠直肠癌、睪丸癌、淋巴癌、横纹肌肉瘤、神经母细胞瘤、胰脏癌、肺癌、脑部肿瘤、皮肤癌、胃癌、口腔癌、肝癌、喉癌、胆癌、甲状腺癌、肝癌、肾脏癌以及鼻咽癌等。
附图说明
图1为本发明的来源趋化素胜肽序列(SEQ ID NO:1-SEQ  ID NO:9)，亦是对CXCR1或/及CXCR2具有高亲和力的ELR-CXC趋化素的氨基酸序列比对图。
图2为本发明的经修饰趋化素胜肽的氨基酸序列比对图(SEQ ID NO:10-12)。
图3显示具有趋化素CXCL8的细胞迁移数量。
图4分别显示黑色部分为具有化学激素刺激，灰色部分为具有化学激素刺激及CXCL8-IP10，白色部分为具有化学激素刺激及IL8-17LIP10之细胞迁移数量。
图5显示实验组(IL8-17LIP10)与对照组(食盐水)的肿瘤体积。
图6显示实验组(IL8-17LIP10)与对照组(食盐水)的肿瘤重量
图7显示实验组(IL8-17LIP10)与对照组(食盐水)的微血管密度。
图8为注射CXCL8-IP10、生理食盐水后肿瘤体积大小观察图，其中：
A组：每个礼拜注射4次的CXCL8-IP10 500ug/kg，B组：每个礼拜注射2次之CXCL8-IP10 500ug/kg，C组：每个礼拜注射2次之CXCL8-IP10 250ug/kg，D组：每天皮下注射100μl的食盐水并观察肿瘤体积大小。
图9为牺牲后的小鼠肺脏肿瘤组织，其中，A为每个礼拜注射4次CXCL8-IP10 500ug/kg的老鼠在第24天牺牲后,取出的肺脏；B为注射生理食盐水的老鼠肺脏，其中箭头所指的白色肿瘤部位为转移到肺部所产生病灶。
图10显示肿瘤细胞中表达CXCR1或CXCL8的趋势。
图11显示本发明拮抗剂对趋化反应的影响。
具体实施方式
本发明提供一种新颖的趋化素胜肽。本发明的新颖趋化素胜肽源自属于ELR-CXC趋化素或是对CXCR1或CXCR2具有高亲和力的受质，例如，CXCL1(SEQ ID NO:3)、CXCL2(SEQ ID NO:4)、CXCL3(SEQ ID NO:5)、CXCL5(SEQ ID NO:6)、CXCL6(SEQ ID NO:7)、CXCL7(SEQ ID NO:8)、CXCL8(SEQ ID NO:2)、hG31P(SEQ ID NO:1)。本发明系根据此一类型的趋化素进行修饰，主要在其30s-loop处插入一段PASQF特征序列(如图1)，此一PASQF原本是存在于趋化素CXCL10中，且该CXCL10为一种非ELR-CXC趋化素。
本发明的经修饰趋化素，包括：(a)N’-谷氨酸(E)-亮氨酸(L)-精氨酸(R)的特征序列，此特征序列位于该趋化素胜肽N端；以及(b)N’-脯氨酸(P)-丙氨酸(A)-丝氨酸(S)-谷氨酰胺(Q)-苯基丙氨酸(F)-Cys3的特征序列，此特征序列是位于趋化素胜肽N端算起第3个胱氨酸(Cys3)。此外，应注意的是，本发明的趋化素胜肽更具有一突变位置(即修饰位置)，其是位于由趋化素胜肽N端算起第17个氨基酸。
在一实施例中，本发明趋化素胜肽的第17位氨基酸原本为苯基丙氨酸(F)，并经突变置换后变为其它非苯基丙氨酸的 氨基酸。在另一实施例中，本发明趋化素胜肽的第17位氨基酸可为丙氨酸(A)、半胱氨酸(C)、硒半胱氨酸(U)、天冬氨酸(D)、天冬酰胺(N)、谷氨酸(E)、谷氨酰胺(Q)、甘氨酸(G)、组氨酸(H)、亮氨酸(L)、异亮氨酸(I)、赖氨酸(K)、吡咯赖氨酸(O)、蛋氨酸(M)、脯氨酸(P)、精氨酸(R)、丝氨酸(S)、苏氨酸(T)、缬氨酸(V)、色氨酸(W)、酪氨酸(Y)，较佳为亮氨酸(L)。与未置换的趋化素多胜肽相比较，第17位氨基酸在置换后的趋化素多胜肽具有较高亲和性并能够有效抑制癌细胞生长。
在本发明一特定实施例中，本发明的经修饰的趋化素胜肽包括，但不限于，SEQ ID NO:10、SEQ ID NO:11及/或SEQ ID NO:12(如图2)。
以SEQ ID NO:10为例，SEQ ID NO:10除具有ELR-CQC的N端序列而符合ELR-CXnC的氨基酸特征序列之规则外，SEQ ID NO：10还具有一由N端起算的Pro-Ala-Ser-Gln-Phe(脯氨酸-丙氨酸-丝氨酸-谷氨酰胺-苯丙氨酸，PASQF)的寡肽序列，此PASQF为一修饰序列，且被插入于N端起算的第3个半胱氨酸(Cysteine,C)的上游(10192)，并且此第3个半胱氨酸紧接在PASQF寡肽序列的苯丙氨酸(F)之后。更重要的是，第17位的氨基酸不为苯丙氨酸，而为亮氨酸。
由本发明所提供的序列表(如图2)可知，SEQ ID NO:10至SEQ ID NO:12的ELR-CXC趋化素类似物序列均具有由N端起算第3个半胱氨酸之前的PASQF修饰序列。另外，本发 明趋化素胜肽的第17位氨基酸经突变为亮氨酸。
本发明的经修饰趋化素胜肽、其类似物及/片断可抑制血管新生相关的疾病。血管新生相关的疾病包括，但不限于，发炎性疾病、慢性风湿性关节炎及牛皮癣、不正常血管侵犯有关的疾病、以及细胞增生性疾病，如与肿瘤或癌症有关的疾病(例如，前列腺癌、乳癌、子宫癌、血癌、卵巢癌、子宫内膜癌、子宫颈癌、大肠直肠癌、睪丸癌、淋巴癌、横纹肌肉瘤、神经母细胞瘤、胰脏癌、肺癌、脑部肿瘤、皮肤癌、胃癌、口腔癌、肝癌、喉癌、胆癌、甲状腺癌、肝癌、肾脏癌以及鼻咽癌等)。
本发明的趋化素修饰胜肽及/或包含此趋化素修饰胜肽的药学组成物可以口服，非肠胃、吸入、直肠、阴道、皮内、经皮或局部给予，一药剂单位可包括传统无毒的药学上可接受的载体、佐剂及载具。
本发明的趋化素修饰胜肽及/或包含此趋化素修饰胜肽的药学组成物可一次给予，24小时内多次给予或连续给予。当注射的方式为连续给予时，可选用适合的习知方式，包括，但不限于，静脉注射点滴、静脉注射帮浦、埋植式注射泵或局部给予。治疗的时间可依不同的情况做适当的调整，例如，血管新生作用的病程及严重性。在以本发明经修饰的趋化素胜肽单独或合并本发明其他药剂直到治愈，或持续治疗终身。
在另一实施例中，本发明提供一种治疗癌症及抑制肿瘤的医药组合物。此药学组成物包括一有效量的本发明趋化素修 饰胜肽或其类似物，以及一药学上可接受的载体。药学上可接受的载体包括溶剂、分散剂、涂层、抗菌剂/抗真菌剂、等渗压、吸收延迟剂及其类似物。
【实施例】
1.细胞趋化
利用Boyden chamber assay评估LMVECs的迁移状况，将LMVECs培养于含有2％FCS的HuMedia-EB2 8小时，之后将12X104cells/cm2的细胞推平于孔径大小5μm且涂布10μg/ml fibronectin的polycarbonate滤膜(Sigma-Aldrich)。分别将10ng/mL of CXCL8,CXCL6,CXCL1,CXCL5,及CXCL8-IP10或IL8-F17LIP10置入Boyden chamber的底部。LMVECs于Boyden chamber于37℃培养4小时，之后使用Diff-Quick(Harleco)将滤膜固定与染色，而迁移的细胞利用(HPFs)(X200)计算迁移的细胞数量。
图3显示具有趋化素CXCL8的迁移数量最多。
图4中黑色部分仅具有化学激素刺激，灰色部分为具有化学激素刺激及CXCL8-IP10，白色部分为具有化学激素刺激及IL8-17LIP10。由图4可知CXCL8-IP10及IL8-17LIP10同样具有抑制细胞迁移的效用，而IL8-17LIP10抑制细胞迁移的效用相对于CXCL8-IP10较好。
2.裸鼠异种移植分析
将无胸腺雄鼠(4-6周BALB/c)置于无菌操作房中。无菌操作房中具有充分的饮水与饲料，并每天监测小鼠。将GFP- 标记PC-3细胞(PC-3-GFP)注射至3只裸鼠的右腹部，每只裸鼠注射5x106的细胞。培养2-4周后，采取肿瘤以供移植。取出此3只裸鼠的肿瘤组织进行切片(1mm3的切片)，并于麻醉及消毒的状态下移植至其它小鼠的前列腺中。移植后第5天，将动物分为2组(每组12只)，每天皮下注射100μl的食盐水(对照组)或本发明的序列(IL8-17LIP10(SEQ ID NO:13)，0.5mg/kg)(实验组)，持续24天。在第12、18、与24天，利用光学解剖显微镜与数字相机以515mm的滤光片摄影肿瘤生长影像。肿瘤体积的计算方式如下：肿瘤体积＝(长度x宽2)/2。在第24天时，将所有小鼠牺牲，并取得肿瘤GFP荧光影像。微血管密度的计算方式如下：密度＝微血管长度/肿瘤体积。肿瘤样本以4％甲醛固定后，包埋于石蜡中，以供后续的免疫组织化学分析。
图5显示第12、18与24天时，实验组与对照组的肿瘤体积。由图5可知，本发明的序列(IL8-17LIP10)可显著地抑制肿瘤生长，可抑制肿瘤体积达5倍以上，且随着时间增加，抑制效果更明显。
图6显示第24天时，实验组与对照组的肿瘤重量。由图6可知，本发明的序列(IL8-17LIP10)可显著地抑制肿瘤生长，可抑制肿瘤重量达2倍以上。
3.免疫组织化学分析
将包埋于石蜡中的肿瘤切片脱蜡并以PBS再水化。详细地说，将切片经PBS润湿3次，并于10mM柠檬酸钠(pH 6.0) 热处理15分钟。之后，以3％的过氧化氢处理10分钟以去除内源性的过氧化酶活性，再以PBS清洗3次，于室温下以蛋白质封阻溶液(含5％马血清的PBS溶液)处理15分钟后，以PBS清洗3次，再以小鼠抗-VEGF单株抗体(1：50)，兔子抗-NF-κB多株抗体或山羊抗-CD3多株抗体(1：50)于4℃下反应20小时。反应完成后，以PBS清洗3次，并以适当的二级抗体于37℃下反应40分钟。再以PBS清洗3次后，将切片以生物素标记的山羊抗小鼠或抗兔子的抗山羊IgG多株抗体于暗室反应30分钟。在呈色反应中，以PBS清洗3次，二氨基联苯胺溶液染色10分钟，再以苏木精染色1分钟(Kollmar et al,2007)。阴性对照组以PBS取代初级抗体。将影像转为灰阶(0-225)，以积分光密度(IOD)表示，并利用Image-Pro 6.0 Microsoft软件测得密度。每组分析5只小鼠的移植肿瘤。每个肿瘤随机选择5个切片影像，以平均出每个肿瘤的灰阶值(Csillik et al,2005)。可使用CD31的免疫组织化学分析来鉴定微血管的密度，利用Image-Pro 6.0Microsoft软件计数，并以平均值表示。
图7显示对照组与实验组的微血管密度。由图7可知，本发明的序列(IL8-17LIP10)可有效地抑制小鼠体内前列腺癌的微血管新生。
4.C57BL/6小鼠分析
C57BL/6小鼠置于无菌操作房中。无菌操作房中具有充分的饮水与饲料，并每天监测小鼠。将LLW2细胞(Lewis lung  carcinma)注射至3只裸鼠的右腹部，每只裸鼠注射5x106的细胞。培养2-4周后，采取肿瘤以供移植。总共有24只小鼠接受肿瘤的移植。移植后第5天，将动物分为4组(每组6只)，A组每个礼拜注射4次IL8-IP10 500ug/kg，B组每个礼拜注射2次IL8-IP10 500ug/kg，C组每个礼拜注射2次IL8-IP10 250ug/kg，D组每天皮下注射100μl的食盐水。肿瘤体积的计算方式如下：肿瘤体积＝(长度x宽2)/2。长度及宽度利用Image-Pro 6.0 Microsoft测量。在第24天时，将所有小鼠牺牲(处死)，并取得肿瘤GFP荧光影像。微血管密度的计算方式如下：密度＝微血管长度/肿瘤体积。肿瘤样本以4％甲醛固定后，包埋于石蜡中，以供后续的免疫组织化学分析。
图8显示A组抑制肿瘤体积的效果最佳。第24天时，将所有小鼠牺牲前,所量测的肿瘤大小,发现A组每个礼拜注射4次CXCL8-IP10 500ug/kg的老鼠肿瘤体积比D组对照组间的肿瘤小了30％.结果显示CXCL8-IP10对控制肿瘤发展有显著的效果。
图9为牺牲后的小鼠肺脏肿瘤组织，而由A图可得知肿瘤的远程转移已被CXCL8-IP10明显抑制。图9中的A为每个礼拜注射4次CXCL8-IP10 500ug/kg的老鼠在第24天牺牲后,取出的肺脏；图B为施打生理食盐水的老鼠肺脏，其中箭头所指的白色肿瘤部位为转移到肺部所产生病灶。经过CXCL8-IP10投药的老鼠肺部则非常干净，并没有肺部转移病 灶的出现。
5.嗜中性白血球趋化性分析
嗜中性白血球趋化性是藉由改良后的Boyden chamber microchemotaxis assays来评估。白血球是由人类外围血液经由一般浓度梯度分离而取得，而嗜中性白血球是由浓度低梯分离的底部取得并利用hypotonic lysis清除污染的红血球细胞。將纯化后的5x106/ml之嗜中性白血球悬浮于HBSS(400mg/L氯化钾(KCl)，60mg/L磷酸氢钾(KH2PO4)，8000mg/L氯化钠(NaCl)，350mg/L碳酸氢钠(NaHCO3)，90mg/L磷酸氢钠(NaH2PO4·7H2O)，1000mg/L葡萄糖(glucose)，以及0.5％pH 7.4牛胎盘血清(fetal calf serum；pH 7.4)，之后嗜中性白血球以Calcein AM(Invitrogen,Stockholm,Sweden)培养30分钟，培养温度为37℃。趋化因子(例如：CXCL8 20ng/mL)单独或与其他结抗剂结合(IL8-IP10F17L,etc.)放置于Boyden chamber的培养区底部而纯化后的嗜中性白血球置于培养槽上端，而培养槽的上端与底部是由孔径5μm的聚碳酸酯滤膜(pore-size polycarbonate filters)所隔开。于37℃及5％二氧化碳(CO2)中培养30分钟之后移除未移动的细胞及滤膜，并将移动的细胞打破并以VICTOR3(Perkin-Elmer,UK，excitation:485nm；emission:530nm)分析，移动的细胞百分比以趋化指数(Chemotaxis Index(CI)value)表示:
CI＝(intensityantagonist-intensityHBSS)/(intensityCXCL8- intensityHBSS)X100％
其中，intensityantagonist是指同时加入本发明所叙的具有拮抗作用的蛋白和CXCL8所产生细胞移动；intensityCXCL8是指细胞仅经由CXCL8刺激而移动，而intensityHBSS是指细胞的移动是由重力所造成。
6.RT-PCR分析各肿瘤细胞中CXCR1/2及CXCL8的基因表现量。
本实验的各癌症细胞株的RNA均藉由TRIzol reagent(Invitrogen,America)及其操作说明萃取而得，其中大部分的RNA的定量是使用
Figure PCTCN2015080725-appb-000001
ND-1000spectrophotometer。于逆转录反应过程，样本是使用cDNA reverse-transcription kit(Prime ScriptTM RT reagent kit,Takara,Japan)进行培养，并于基因表现分析前置于冰上。GAPDH为内部控制组。RT-PCR的反应是藉由SYBR(Premix Ex TaqTM,Takara,Japan)所完成，其CXCL引物为以下所述：
5′-gagcactccataaggcacaaa-3′(forward)and 5′-atg gttccttccggtggt-3′(reverse)for CXCL8；
5′-gaccaacatcgcagacacat-3′(forward)and 5′-tgcttgtctcgttccacttg-3′(reverse)for CXCR1；
5′-ggctaagcaaaatgtgatatgtacc-3′(forward)and 5′-caaggttcgtccgtgttgta-3′(reverse)for CXCR2。
基因表现的计算方法如算式所表现gene  expression＝2-ΔΔCt
表一、肿瘤细胞株
HCC827 lung adenocarcinoma
HCC827GR HCC827gefitinib-resistant
H1975 lung adenocarcinoma
H2170 lung squamous cell carcinoma
H157 oral squamous cell carcinoma
CT26 coloncarcinomacell
CL1-0 lung adenocarcinoma
CL1-5 lung adenocarcinoma
PC9 lung adenocarcinoma
H3255 Non small cell lung carcinoma
A549 lung adenocarcinoma
H520 lung squamous cell carcinoma
H460 Non small cell lung carcinoma
图10显示利用RT-PCR侦测到部分肿瘤细胞对于趋化素受体CXCR1具有高度的表达，此外许多的肿瘤细胞会表达高量的CXCR1/2等两类受体的受质CXCL8,因此可以利用CXCR1/2受体拮抗剂来达到阻止肿瘤细胞生长和转移的目的。
图11显示更多的结抗剂设计,在先前揭露的CXCL8–IP 10中增加了第12号或/和第13号或/和第17号氨基酸的变异,可以增强对于其受体CXCR1/2所引发生理反应的结抗作用,有效达到降低因为肿瘤细胞所表达CXCR1/2受体,或是因为肿瘤细胞高量表达其受体对应受质细胞激素(CXCL1,2,3,5, 6,7,8等)所伴随引发肿瘤增生,抗药性,转移和血管新生的抑制作用.
由此可知，由于本发明趋化素胜肽可有效地抑制肿瘤生长、血管新生，以及治疗癌症。
所有说明书中所揭示的发明技术特点可以任意方式组合。说明书中揭示的每一技术特点可以提供相同、等同或相似目的的其他方式替换。因此，除非另有特别说明，文中所有揭示的特点均只是等同或相似特点的一般系列的实例。
由上述可知，熟习此技艺者能轻易地了解本发明的必要特征，在不脱离其精神与范围之下能就本发明做许多改变与调整以应用于不同用途与条件。

Claims (12)

  1. 一种趋化素修饰胜肽，其特征是，包含一氨基酸序列，所述氨基酸具有一N端(N’)，所述氨基酸序列带有：
    (a)位于所述趋化素胜肽的N端的特征序列A，所述特征序列A为N’-谷氨酸(E)-亮氨酸(L)-精氨酸(R)；
    (b)位于所述趋化素胜肽N端算起第3个半胱氨酸(Cys3)的特征序列B，所述特征序列B为N’-脯氨酸(P)-丙氨酸(A)-丝氨酸(S)-谷氨酰胺(Q)-苯丙氨酸(F)-Cys3；
    其中所述趋化素胜肽是包含该(a)、(b)特征序列并具有一修饰位置，且该修饰位置位于所述趋化素胜肽上由N端算起第17,12,13个氨基酸。
  2. 如权利要求1所述的趋化素修饰胜肽，其特征是，所述趋化素胜肽上N端算起第17个氨基酸由苯丙氨酸(F)置换为亮氨酸(L)、缬氨酸(V)或异亮氨酸(I)。
  3. 如权利要求1所述的趋化素修饰胜肽，其特征是，所述趋化素胜肽上N端算起第12个氨基酸由苏氨酸(T)置换为丝氨酸(S)。
  4. 如权利要求1所述的趋化素修饰胜肽，其特征是，所述趋化素胜肽上N端算起第13个氨基酸由酪氨酸(Y)置换为亮氨酸(L)、苯丙氨酸(F)、色氨酸(W)或异亮氨酸(I)。
  5. 如权利要求1所述的趋化素修饰胜肽，其特征是，所述经修饰趋化素胜肽的未经修饰的前身来自于来源趋化素胜肽，所述来源趋化素胜肽N端的第1个半胱氨酸及第2个半胱氨酸之间存在0-2个氨基酸，且当所述氨基酸为1-2个时，所述氨基酸为具有电性或不具电性的极性氨基酸。
  6. 如权利要求5所述的趋化素修饰胜肽，其特征是，所述来源趋化素胜肽选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:9中的一种或多种。
  7. 如权利要求1所述的趋化素修饰胜肽，其特征是，所述经修饰趋化素胜肽选自SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12中的一种或多种。
  8. 一种医药组合物，其特征是，包含如权利要求1所述的经修饰趋化素胜肽及医药可接受性赋形剂。
  9. 如权利要求8所述的医药组合物，其特征是，所述经修饰趋化素胜肽用于治疗癌症或抑制肿瘤生长。
  10. 一种治疗癌症及抑制肿瘤的医药组合物，其特征是，包含如权利要求1所述的经修饰趋化素胜肽及医药可接受性赋形剂。
  11. 如权利要求10所述的医药组合物，其特征是，所述癌症包括，前列腺癌、乳癌、子宫癌、血癌、卵巢癌、子宫内膜癌、子宫颈癌、大肠直肠癌、睪丸癌、淋巴癌、横纹肌肉瘤、神经母细胞瘤、胰脏癌、肺癌、脑部肿瘤、皮肤癌、胃癌、口腔癌、肝癌、喉癌、胆癌、甲状腺癌、肝癌、肾脏癌或鼻咽癌。
  12. 如权利要求11所述的医药组合物，其特征是，所述癌症的共同特征为具有CXCR1/2的表达或具有高量CXCL8,CXCL1,CXCL2,CXCL3,CXCL5,CXCL6或CXCL7趋化素或其受体表达的肿瘤细胞。
PCT/CN2015/080725 2015-06-03 2015-06-03 趋化素修饰胜肽 WO2016192067A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
KR1020177034607A KR102088904B1 (ko) 2015-06-03 2015-06-03 변형된 케모카인 펩타이드
JP2017562314A JP6901407B2 (ja) 2015-06-03 2015-06-03 改変されたケモカインペプチド
PCT/CN2015/080725 WO2016192067A1 (zh) 2015-06-03 2015-06-03 趋化素修饰胜肽
US15/569,033 US20180125938A1 (en) 2015-06-03 2015-06-03 Modified chemokine peptide
EP15893723.5A EP3315510A4 (en) 2015-06-03 2015-06-03 MODIFIED CHEMOKINPEPTIDE
AU2015397751A AU2015397751B2 (en) 2015-06-03 2015-06-03 Modified chemokine peptide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/080725 WO2016192067A1 (zh) 2015-06-03 2015-06-03 趋化素修饰胜肽

Publications (1)

Publication Number Publication Date
WO2016192067A1 true WO2016192067A1 (zh) 2016-12-08

Family

ID=57439916

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/080725 WO2016192067A1 (zh) 2015-06-03 2015-06-03 趋化素修饰胜肽

Country Status (6)

Country Link
US (1) US20180125938A1 (zh)
EP (1) EP3315510A4 (zh)
JP (1) JP6901407B2 (zh)
KR (1) KR102088904B1 (zh)
AU (1) AU2015397751B2 (zh)
WO (1) WO2016192067A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107034272A (zh) * 2016-12-14 2017-08-11 河北医科大学第四医院（河北省肿瘤医院） Cxcl2在制备诊断或治疗肺腺癌工具中的应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2020260381A1 (en) * 2019-10-30 2021-05-20 Rise Biopharmaceuticals Inc. Pharmaceutical compositions and use thereof for relieving anticancer drug resistance and enhancing sensitivity of anticancer drug
CN113101362A (zh) * 2020-01-10 2021-07-13 北京锐瑟生物医药科技发展有限公司 用于缓解癌症化疗引起抗药性并对化疗增效的医药组合物及其用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0616615A1 (en) * 1991-12-04 1994-09-28 The Biomedical Research Centre Limited Human interleukin-8 analogs
CN101200502A (zh) * 2006-07-26 2008-06-18 趋化因子治疗剂公司 设计用于治疗人类疾病的干扰素诱导型蛋白-10(ip-10或cxcl10)趋化因子类似物
CN102596227A (zh) * 2009-09-11 2012-07-18 普罗塔芬生物技术股份公司 用于治疗cxcl8介导的肺部炎症的组合物

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5595756A (en) * 1993-12-22 1997-01-21 Inex Pharmaceuticals Corporation Liposomal compositions for enhanced retention of bioactive agents
US20100029562A1 (en) * 2008-05-21 2010-02-04 Gordon John R hG31P Expression System
TWI429451B (zh) * 2011-10-26 2014-03-11 Nat Univ Tsing Hua 對具有ｅｌｒ－ｃｘｃ趨化素高度親和力的受體蛋白細胞所引發疾病之拮抗治療劑

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0616615A1 (en) * 1991-12-04 1994-09-28 The Biomedical Research Centre Limited Human interleukin-8 analogs
CN101200502A (zh) * 2006-07-26 2008-06-18 趋化因子治疗剂公司 设计用于治疗人类疾病的干扰素诱导型蛋白-10(ip-10或cxcl10)趋化因子类似物
CN102596227A (zh) * 2009-09-11 2012-07-18 普罗塔芬生物技术股份公司 用于治疗cxcl8介导的肺部炎症的组合物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LE, Y.Y. ET AL.: "Chemokines and Chemokine Receptors: their Maniold Roles in Homeostasis and Disease", CELLULAR & MOLECULAR IMMUNOLOGY, vol. 1, no. 2, 30 April 2004 (2004-04-30), XP055331932 *
See also references of EP3315510A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107034272A (zh) * 2016-12-14 2017-08-11 河北医科大学第四医院（河北省肿瘤医院） Cxcl2在制备诊断或治疗肺腺癌工具中的应用
CN107034272B (zh) * 2016-12-14 2020-09-29 河北医科大学第四医院（河北省肿瘤医院） Cxcl2在制备诊断或治疗肺腺癌工具中的应用

Also Published As

Publication number Publication date
AU2015397751A1 (en) 2018-01-04
EP3315510A4 (en) 2019-02-20
JP6901407B2 (ja) 2021-07-14
KR20170141239A (ko) 2017-12-22
KR102088904B1 (ko) 2020-03-16
JP2018517701A (ja) 2018-07-05
EP3315510A1 (en) 2018-05-02
US20180125938A1 (en) 2018-05-10
AU2015397751B2 (en) 2019-04-04

Similar Documents

Publication Publication Date Title
JP4605798B2 (ja) Ｉｌ−２３アゴニストおよびｉｌ−２３アンタゴニスト；関連試薬の使用
KR102359396B1 (ko) 혈관 신생 억제 활성을 가지는 펩티드 및 이를 포함하는 조성물
Gordon-Weeks et al. Macrophage migration inhibitory factor: a key cytokine and therapeutic target in colon cancer
US20030186889A1 (en) Diagnostic and medicament for analysing the cell surface proteome of tumour and inflammatory cells and for treating tumorous and inflammatory diseases, preferably using a specific chemokine receptor analysis and the chemokine receptor-ligand interaction
KR20130004568A (ko) 항-전이성 요법에서 ａｘｌ 신호전달의 저해
EP1843789A2 (en) Compositions and methods for treating fibrotic disorders
KR20160089523A (ko) 전립선 암 치료용 조성물
JP6921755B2 (ja) 脳腫瘍幹細胞の成長、遊走および浸潤性を低下させ脳腫瘍患者の生存率を改善する方法および組成物
KR20130019365A (ko) 세포 내 전달용 단백질 복합체 및 그의 용도
CN104211765B (zh) 与trb3蛋白特异性结合的多肽，其筛选方法、鉴定和用途
WO2016192067A1 (zh) 趋化素修饰胜肽
WO2017211317A1 (zh) 一种多肽及其在制备治疗和/或预防肿瘤的药物中的应用
Kiyasu et al. Disruption of CCR1-mediated myeloid cell accumulation suppresses colorectal cancer progression in mice
Shim et al. Chemokine receptor CXCR4 as a therapeutic target for neuroectodermal tumors
WO2022143938A1 (zh) 一种激活nk细胞的肿瘤免疫治疗方法
CN106279399B (zh) 趋化素修饰胜肽
WO2017067510A1 (zh) 一种多肽及其在制备治疗和预防肿瘤的药物中的应用
Liu et al. The chemokine CCL1 facilitates pulmonary fibrosis by promoting macrophage migration and M2 polarization
CN110831962A (zh) 治疗方法
TWI665212B (zh) 趨化素修飾胜肽
CN109200287B (zh) 直接或间接调控yb-1磷酸化的物质在制备治疗炎性因子所致疾病的药物方面的应用
Zhao et al. The oncogenic mechanisms of the Janus kinase-signal transducer and activator of transcription pathway in digestive tract tumors
CN114605501B (zh) 一种可拮抗fus蛋白rna结合活性的多肽fip-21及其应用
CN111035752B (zh) 家蚕抗菌肽Cecropin A在食道癌治疗中的用途
Peitzsch et al. Implications of CXCR4/CXCL12 interaction for cancer stem cell maintenance and cancer progression

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15893723

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 15569033

Country of ref document: US

ENP Entry into the national phase

Ref document number: 20177034607

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2017562314

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2015893723

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2015397751

Country of ref document: AU

Date of ref document: 20150603

Kind code of ref document: A