WO2018120277A1 - 包含f1、f3多肽的药物组合物及其在治疗hpv感染疾病中的应用 - Google Patents

包含f1、f3多肽的药物组合物及其在治疗hpv感染疾病中的应用 Download PDF

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WO2018120277A1
WO2018120277A1 PCT/CN2017/000744 CN2017000744W WO2018120277A1 WO 2018120277 A1 WO2018120277 A1 WO 2018120277A1 CN 2017000744 W CN2017000744 W CN 2017000744W WO 2018120277 A1 WO2018120277 A1 WO 2018120277A1
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cancer
pharmaceutical composition
polypeptide
amino acid
group
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王天放
倪国颖
刘晓松
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王天放
倪国颖
刘晓松
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Priority to AU2017385913A priority Critical patent/AU2017385913B2/en
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Priority to US16/455,609 priority patent/US11040085B2/en

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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
    • A61K38/16—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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the technical field of polypeptide pharmaceutical preparations, in particular to pharmaceutical compositions comprising F1 and F3 polypeptides and their use in the treatment of HPV infection diseases.
  • Cervical cancer is one of the common malignant tumors, and the incidence rate is the second in female tumors. Its occurrence is closely related to the persistent infection of HPV, especially type 16 and 18 HPV.
  • vaccines for the prevention of HPV infection have been used since 2006, such vaccines are only suitable for healthy individuals who have never been infected, and are ineffective for the current HPV infection and its associated diseases.
  • Vulvar cancer, penile cancer, anal cancer and oral cancer are also common clinical malignant tumors.
  • HPV infection can also cause respiratory, genital lesions, condyloma acuminata and recurrent respiratory papillomatosis, the former is a common sexually transmitted disease; the latter is a rare disease, more common in infants and young children, sometimes life-threatening, These two diseases are often caused by infection with HPV type 6,11.
  • Condyloma acuminata is a sexually transmitted disease that is mainly transmitted through unclean sexual intercourse. About 1% of sexually active people with genital warts are one of the most common sexually transmitted diseases. The clinical manifestations are genital, neoplastics in the anus, and some patients continue to infect or relapse after treatment. The cellular immunity of the human body to HPV is an important immune mechanism for controlling genital warts. Patients with immunodeficiency often have multiple genital warts, which are stubborn and difficult to treat. There are many patients with genital warts, which seriously invade medical resources and affect social stability.
  • Topical application of 5% Imiquimod (5% imiquimod) emulsion is currently the most effective method for the treatment of condyloma acuminata.
  • the effective rate is 50%-70%, and the recurrence rate is about 10%.
  • the polypeptide F1, F3 sequences were first published in the literature ST Steinborner, et al., J. Pept. Res., 1998, 51, 121, see page 4, where the F1 sequence is: GLLSVLGSVAKHVLPHVVPVIAEHL-NH2; the F3 sequence is; GLFGVLGSIAKHLLPHVVPVIAEKL-NH2 .
  • the object of the present invention is to overcome the defects that the existing treatment methods can not cure HPV infection related to diseases such as condyloma acuminatum or solid tumor, low efficacy, high recurrence rate, many side effects, long course of treatment and high cost, and provide an inclusion of F1.
  • the tree frog secretes a viscous liquid with a white appearance by means of a direct current stimulation of 9 volts.
  • the carboxyl group of the second amino acid blocked by the amino group is activated by N,N'-dicyclohexylcarbodiimide (DCC, the second amino acid activated by DCC and then attached to the solid phase carrier)
  • DCC N,N'-dicyclohexylcarbodiimide
  • the amino group of the first amino acid reacts to form a peptide bond, such that a dipeptide with a protecting group is formed on the solid support;
  • TFA is the abbreviation of trifluoroacetic acid.
  • the polypeptide prepared by the method has high purity, and the preparation method is simple and easy to operate, and the cost is low.
  • the present invention provides a first pharmaceutical composition comprising a mixture of any one or both of F1, F3 polypeptides and pharmaceutically acceptable excipients.
  • the pharmaceutical composition is prepared as an injection for intravenous, intramuscular or subcutaneous injection suitable for parenteral administration: or an emulsion or ointment formulation for transdermal penetration, more preferably an emulsion or ointment preparation .
  • the F1, F3 polypeptide or a mixture of the two is contained in each pharmaceutical composition in an amount of 3 to 30 ⁇ g, preferably 5 to 20 ⁇ g.
  • the present invention also provides a second pharmaceutical composition, further comprising Imiquimod, the pharmaceutical composition being prepared as an injection for intravenous, intramuscular or subcutaneous injection suitable for parenteral administration; or transdermal penetration An emulsion or ointment formulation, preferably an emulsion or ointment formulation.
  • the mechanism of the second pharmaceutical composition is to induce apoptosis of HPV-infected cells by F1, F3 combination, release more viral antigens for antigen-presenting cells, and then induce stronger by Tmiquimod's non-specific immunostimulatory action. Specific immune response to achieve Imiquimod, or the efficacy of F1 and F3 alone can not be achieved.
  • Imiquimod is a non-specific immunomodulator that activates non-specific immune responses by stimulating Toll-like receptor 7/8, thereby activating specific T cell responses to kill virus-infected cells and directly inducing HPV6,11-infected cells. Apoptosis to achieve therapeutic effects.
  • a disease caused by HPV infection which is a sputum or a solid tumor
  • said sputum preferably genital warts
  • said solid tumor include, but are not limited to, cervical cancer, vulvar cancer, penile cancer, anal cancer, and oral cancer.
  • the use of the second pharmaceutical composition provided by the present invention for the preparation of a medicament for preventing and/or treating a disease caused by HPV infection which is a sputum or a solid tumor, said sputum preferably genital warts; said solid tumor
  • a disease caused by HPV infection which is a sputum or a solid tumor
  • said sputum preferably genital warts
  • said solid tumor include, but are not limited to, cervical cancer, vulvar cancer, penile cancer, anal cancer, and oral cancer.
  • the use of the second pharmaceutical composition provided by the present invention in combination with any one or more of radiotherapy, chemotherapy, and therapeutic vaccine for the preparation of a medicament for preventing and/or treating a disease caused by HPV infection is an entity Tumor
  • the solid tumor includes, but is not limited to, cervical cancer, vulvar cancer, penile cancer, anal cancer, and oral cancer.
  • F1 and F3 significantly activate the TEC kinase signaling pathway compared with untreated, and one of the results is the apoptosis of tumor cells; in addition, F3, the control peptide P3 is labeled with fluorescent FITC, and TC-1 was co-cultured for different times, and after repeated washing, the FITC fluorescence intensity of TC-1 cells was measured by flow cytometry (Fig. 5A).
  • confocal microscopy was used to detect the location of FITC-labeled F3 in TC-1 cells (Fig. 5B); understanding the interaction of F1, F3 and TC-1 cells.
  • F3 was co-cultured with TC-1 for 15 minutes, TC-1 cells could detect FITC.
  • the fluorescence intensity of FITC reached a peak at 2 hours, and then the fluorescence intensity of FITC gradually decreased, maintaining the intensity level at 15 minutes.
  • F3 was co-cultured with TC-1 for one hour. Confocal microscopy showed that F3 could penetrate the TC-1 cell membrane and was unevenly distributed in the cytoplasm.
  • F1 and F3 may act on the cell membrane because it largely alters cell migration, adhesion, and actin cytoskeletal remodeling.
  • the two pharmaceutical compositions provided by the invention have the advantages of unique drug action mechanism, short treatment period, less effective ingredient dosage, exact curative effect, low recurrence rate, small side effects and low treatment cost, and bring very good treatment to patients. way.
  • Figure 1 is a graph showing the inhibition of growth of TC-1 tumor cells in vitro by F1 and F3 of the present invention.
  • Fig. 2 is a diagram showing the inhibitory effect of F1 and F3 on the growth of TC-1 tumor cells in vitro according to the present invention.
  • Figure 3 is a graph showing the inhibitory effect of F1 and F3 on the growth of human cervical cancer cells (HELA) in vitro.
  • Figure 4 is a high throughput quantitative proteome analysis of F1 and F3 of the present invention.
  • Figure 5 is a diagram showing the interaction of F3 and TC-1 of the present invention.
  • Figure 6 is a graph showing the inhibition of growth of TC-1 tumor cells in vitro by F1, F3, and Imiquimod alone or in combination.
  • Figure 7 is a graph showing the inhibitory effect of different doses of Imiquimod on human cervical cancer cells (HELA) in vitro.
  • Figure 8 is a graph showing the inhibitory effect of human, cervical cancer cells (HELA) alone or in combination on F1, F3, and Tmiquimod in vitro.
  • High-throughput high performance liquid chromatography coupled with mass spectrometry Shimadzu Prominance nano reversed-phase high performance liquid chromatography coupled with ABSCIEX triple time flight mass spectrometer equipped with nano electrospray ion source.
  • the tree frog secretes a viscous liquid with a white appearance through a 9 volt DC stimulation.
  • the tree frog is distributed in the rainforest region along the eastern coast of Australia.
  • the white solid powder was dissolved in 0.1% trifluoroacetic acid, and the polypeptide sequence and the post-translational modification were analyzed by high-throughput high performance liquid chromatography and mass spectrometry.
  • chloromethyl polystyrene resin is used as the insoluble solid phase carrier, and the amino acid protected by the C-terminal amino acid of F1 or F3 (amino blocked gene) is covalently linked to the solid phase carrier. ;
  • the carboxyl group of the second amino acid blocked by the amino group is activated by N,N'-dicyclohexylcarbodiimide (DCC, the second amino acid activated by DCC and then attached to the solid phase carrier)
  • DCC N,N'-dicyclohexylcarbodiimide
  • the amino group of the first amino acid reacts to form a peptide bond, such that a dipeptide with a protecting group is formed on the solid support;
  • the glutamic acid and serine side chain carboxyl groups are protected with L-Bu, and to prevent racemization of histidine, the side chain is protected with a Boc group.
  • Example 2 Preparation of an ointment composition comprising F1, F3 polypeptide (formulated in 10 g amount)
  • F1 polypeptide 10 mg
  • F3 polypeptide 10 mg
  • PEG4000 100mg
  • PEG400 Appropriate amount, added to 10g.
  • the above composition is dispensed into an aluminum tube for ointment packaging, and each tube is 2 mg, 5 mg or 10 mg, which is an ointment.
  • Example 3 Preparation of an ointment composition comprising F1, F3 polypeptide and Imiquimod (formulated in 10 g amount)
  • F1 polypeptide 2.5 mg
  • F3 polypeptide 2.5 mg
  • PEG3000 100mg
  • Lysine 5mg
  • PEG200 Appropriate amount, added to 10g.
  • the above composition is dispensed into an aluminum tube for ointment packaging, and each tube is 2 mg, 5 mg or 10 mg, which is an ointment.
  • Example 4 Preparation of a cream composition comprising an F1, F3 polypeptide (formulated in an amount of 10 g)
  • F1 polypeptide 10 mg
  • F3 polypeptide 10 mg
  • the amount of the substrate was increased to 10 g.
  • the above composition is dispensed into an aluminum tube for cream packaging, and 2 mg, 5 mg or 10 mg per tube is a cream.
  • Example 5 Preparation of a cream composition comprising F1, F3 polypeptide and Imiquimod (formulated in 10 g amount)
  • F1 polypeptide 5 mg
  • F3 polypeptide 5 mg
  • the amount of the substrate was increased to 10 g.
  • the above composition is dispensed into an aluminum tube for cream packaging, and 2 mg, 5 mg or 10 mg per tube is a cream.
  • Figure 1 Inhibition of in vitro growth of HPV-transformed cells (TC-1 cells) by F1, F3 and F1+F3.
  • TC-1 cells cultured in 10% fetal calf serum RPMI medium were added to 96-well cell culture plates, and different concentrations of F1 (Fig. 1A) and F3 (Fig. 1B) were added.
  • the control polypeptide P3 (Fig. 1C) was cultured overnight at 37 ° C with 5% CO 2 , and the MTT assay confirmed cell proliferation.
  • Another experiment used immortalized normal cell NP69, specifically 100 ⁇ l of 5 ⁇ 10 3 NP69 cells cultured in 10% calf serum RPMI medium, and added to 96-well cell culture plates, respectively, with different concentrations of F1, F3 and The control polypeptide P3 (Fig. 1D) was cultured overnight at 37 ° C with 5% CO 2 , and the cell proliferation was confirmed by MTT assay.
  • Figure 2 Inhibition of TC-1 tumor cell growth in vitro by F1 and F3.
  • Imiquimod combined with F1, F3 peptide can better kill HPV18E7 transformed human cervical cancer cells (HELA)
  • Figure 3 Inhibition of superimposed action of F1 and F3 on human cervical cancer cells (HELA) growth in vitro.
  • FIG. 4 High-throughput quantitative proteomic analysis of F1 and F3 showed superposition to activate the TEC kinase signaling pathway.
  • Figure 5 Interaction diagram of F3 and TC-1.
  • Figure 6 Inhibition of TC-1 tumor cell growth by F1, F3, Imiquimod alone or in combination.
  • F1, F3, and Imiquimod alone or in combination inhibit the growth of HeLa tumor cells in vitro.
  • Imiquimod and F1 and/or F3 have a superimposed effect on the growth inhibition of HeLa cells in vitro, see Figure 8.

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Abstract

包含F1、F3多肽的药物组合物及其在治疗HPV感染疾病中的应用。药物组合物制备成乳剂、软膏制剂等多种常规剂型，用于治疗HPV感染引起的尖锐湿疣、宫颈癌、外阴癌、阴茎癌、肛门癌和口腔癌等易复发的疾病。

Description

包含F1、F3多肽的药物组合物及其在治疗HPV感染疾病中的应用 技术领域
本发明涉及多肽药物制剂技术领域，具体是包含F1、F3多肽的药物组合物及其在治疗HPV感染疾病中的应用。
背景技术
慢性人乳头瘤病毒(human papillomavirus，HPV)感染可以引起多种疾病。宫颈癌、外阴癌、阴茎癌、肛门癌和口腔癌均可由感染HPV引起。宫颈癌是常见恶性肿瘤之一，发病率占女性肿瘤第二位，其发生与HPV、特别是16、18型HPV的持续感染密切相关。尽管用于预防HPV感染的疫苗已于2006年开始应用，但此类疫苗仅适用于从未感染的健康个体，对现症HPV感染及其所致疾病无效。目前全球每年仍有约50万新发宫颈癌病例，约20万妇女死于该病。我国已有宫颈癌患者40万人，死亡率达11.3％。外阴癌、阴茎癌、肛门癌和口腔癌亦是临床常见恶性肿瘤。
此外，HPV感染还可以导致呼吸道，生殖器官的病变，尖锐湿疣和复发性呼吸道乳头瘤病，前者是一种常见的性传播疾病；后者是少见病，多见于婴幼儿，有时会危及生命，这两种疾病常因感染HPV6，11型引起。
尖锐湿疣是一种性传播疾病，主要通过不洁性交传播。约有1％的性活跃者患有尖锐湿疣，是最常见的性传播疾病之一。临床表现为生殖器，肛门部位疣状新生物，部分患者持续感染或治疗后反复发作。人体对HPV的细胞免疫是控制尖锐湿疣重要免疫机制。免疫缺陷患者尖锐湿疣常常多发，顽固，不易治疗。尖锐湿疣患者众多，严重侵占医疗资源，且影响社会稳定。
目前尚无可以根治尖锐湿疣的方法。通常采用物理或者化学方法去除疣体，如冷冻，激光切除等，此类疗法不易完全去除湿疣体，患者极易复发。免疫疗法治疗尖锐湿疣，如干扰素α湿疣体局部注射，非特异性的免疫治疗也部分有效。
5％Imiquimod(5％咪喹莫特)乳剂局部涂抹是目前国际通行的治疗尖锐湿疣的最有效方法，有效率50％-70％，复发率在10％左右。
多肽F1，F3序列首次发表于文献S.T.Steinborner，et al.，J.Pept.Res.，1998，51，121，见第4页，其中F1序列为：GLLSVLGSVAKHVLPHVVPVIAEHL-NH2；F3序列为；GLFGVLGSIAKHLLPHVVPVIAEKL-NH2。
文献M.A.Apponyi，et al.，Peptides，2004，25，1035中还公开了F1有体外抑制肿瘤作用；F3对肿瘤的作用未见报道，而且目前尚未有F1，F3治疗尖锐湿疣以及二者叠加效应的相关报道。
因此，目前急需开发一种疗程短，有效成分用量较少、疗效确切、复发率低、副作用小， 治疗费用低的药物。
发明内容
本发明的目的就是为了克服现有治疗方法无法根治HPV感染的相关疣如尖锐湿疣或实体肿瘤的疾病，疗效低、复发率高、副作用多、疗程长、费用高等缺陷，提供一种包含F1、F3多肽的药物组合物，用于治疗HPV感染的相关疣或实体肿瘤的疾病。
为解决上述问题，本发明通过以下技术方案得以解决；
首先提供一种F1或F3多肽的制备方法，包括如下步骤：
1)首先通过9伏特的直流电刺激的方式促使树蛙分泌出外观呈白色的粘稠状液体。
2)将液体收集并转移至含有15％～30％醇的水溶液中，优选20％甲醇或乙醇水溶液，震荡再通过0.45μm的PVDF膜过滤。
3)滤出清液在真空低温离心干燥剂中干燥，形成白色固体粉末。
4)将自色固体粉末溶解于0.1％的三氟乙酸中，通过高通量高效液相色谱和质谱联用分析其中的多肽序列及具有的翻译后修饰。
5)利用固相多肽合成方法分别合成多肽F1或F3，具体步骤如下：
a)在CS336X的多肽合成仪上，以氯甲基聚苯乙烯树脂作为不溶性的固相载体，将F1或F3的C端氨基酸(氨基被封闭基团)保护的氨基酸共价连接在固相载体上；
b)在三氟乙酸的作用下，脱掉氨基的保护基，这样第一个氨基酸就接到了固相载体上了；
c)然后氨基被封闭的第二个氨基酸的羧基通过N，N′-二环己基碳二亚胺(DCC，Dicyclohexylcarbodiimide)活化，羧基被DCC活化的第二个氨基酸再与已接在固相载体的第一个氨基酸的氨基反应形成肽键，这样在固相载体上就生成了一个带有保护基的二肽；
d)重复上述肽键形成反应，使肽链从C端向N端生长，直至达到所需要的肽链长度。最后脱去保护基，在TFA/DCM/H 2O/TIPS的体积比为90∶5∶2.5∶2.5的溶液中自发进行树脂的去保护和解聚；
d)通过10倍当量的冷乙醚对其沉淀，并用反相高效液相色谱纯化后获得多肽F1或F3。
特别地，在合成过程中，谷氨酸和丝氨酸侧链羧基用t-Bu保护，而为防止组氨酸的消旋化，其侧链用Boc基团保护。其中TFA为三氟乙酸的英文缩写。
该方法制备的多肽纯度高，而且制备方法简单易操作，成本低。
本发明提供了第一种药物组合物，包含F1、F3多肽中任意一种或二者任意比例的混合物和药学上可接受的辅料。
作为优选，所述的药物组合物制备成适合胃肠外给药的静脉注射、肌内注射或皮下注射的注射液：或经皮肤穿透给药的乳剂或软膏制剂，更优选乳剂或软膏制剂。
进一步的，每份药物组合物中含F1、F3多肽或者二者的混合物为3～30μg，优选5～20μg。
本发明还提供了第二种药物组合物，进一步包含Imiquimod，所述的药物组合物制备成适合胃肠外给药的静脉注射、肌内注射或皮下注射的注射液；或经皮肤穿透给药的乳剂或软膏制剂，优选乳剂或软膏制剂。
第二种药物组合物的机理是通过F1，F3组合诱导HPV感染细胞的凋亡，释放更多的病毒抗原供抗原提呈细胞摄取，然后通过Tmiquimod的非特异性的免疫刺激作用，诱导更强的特异性免疫反应，从而达到Imiquimod，或者F1和F3单独使用不可能达到的疗效。
其中Imiquimod是一种非特异性的免疫调节剂，通过刺激Toll样受体7/8激活非特异性免疫反应，进而激活特异性的T细胞反应来杀死病毒感染细胞，并且直接诱导HPV6，11感染细胞凋亡而达到治疗效果。
本发明提供的第一种药物组合物在制备预防和/或治疗HPV感染导致的疾病的药物中的用途，所述疾病为疣或实体肿瘤，所述的疣优选尖锐湿疣；所述的实体肿瘤包括并不限于宫颈癌、外阴癌、阴茎癌、肛门癌和口腔癌。
本发明提供的第二种药物组合物在制备预防和/或治疗HPV感染导致的疾病的药物中的用途，所述疾病为疣或实体肿瘤，所述的疣优选尖锐湿疣；所述的实体肿瘤包括并不限于宫颈癌、外阴癌、阴茎癌、肛门癌和口腔癌。
本发明提供的第二种药物组合物联合放射性治疗、化学治疗、治疗性疫苗中任意一种或多种在制备预防和/或治疗HPV感染导致的疾病的药物中的用途，所述疾病为实体肿瘤，所述的实体肿瘤包括并不限于宫颈癌、外阴癌、阴茎癌、肛门癌和口腔癌。
为了从机理上探讨其抑制作用，高通量定量蛋白组分析被用于对于分析多肽作用前后的细胞内和上清中蛋白组的变化情况。用胰蛋白酶将分离出的蛋白进行了水解，并用iTRAQ对水解产生的多肽进行了标记，进而应用纳米高效液相色谱及三重时间飞行质谱联用系统对标记多肽进行了定量分析。结果显示，若干与细胞免疫相关的关键信号通路被激活或抑制。以图4为例，与未处理相比较，F1、F3明显激活了TEC激酶信号通路，而导致的结果之一即是肿瘤细胞的凋亡；此外，将F3，对照多肽P3标记荧光FITC，与TC-1共同培养不同时间，反复洗涤后，采用流式细胞仪检测TC-1细胞的FITC荧光强度(图5A)。
在另一个实验，使用共聚焦显微镜检测FITC标记的F3在TC-1细胞中的位置(图5B)；了解F1，F3与TC-1细胞的相互作用。F3与TC-1共同培养15分钟后，TC-1细胞既可以检测可以检出FITC，2小时时FITC荧光强度达到峰值，随后FITC荧光强度逐渐减弱，维持在15分钟时的强度水平。此外，F3与TC-1共同培养一小时，共聚焦显微镜结果表明，F3可以穿透TC-1细胞膜，并且不均匀分布在细胞质中。F1、F3可能作用于细胞膜，因为其很大程度上改变了细胞迁移、附着和肌动蛋白细胞骨架重构。
本发明提供的两种药物组合物，具有药物作用机理独特，使用疗程短，有效成分用量较少、疗效确切、复发率低、副作用小，治疗费用低等优势，给患者带来非常好的治疗途径。
附图说明
图1为本发明F1、F3对TC-1肿瘤细胞生长在体外抑制作用图。
图2为本发明F1、F3对TC-1肿瘤细胞生长在体外抑制叠加作用图。
图3为本发明F1、F3对人宫颈癌细胞(HELA)生长在体外抑制叠加作用图。
图4为本发明F1、F3的高通量定量蛋白质组分析图。
图5为本发明F3与TC-1的相互作用图。
图6为本发明F1、F3、Imiquimod中单独或联合用药对TC-1肿瘤细胞生长在体外抑制作用图。
图7为不同剂量的Imiquimod对人宫颈癌细胞(HELA)在体外抑制作用图。
图8为本发明F1、F3、Tmiquimod中单独或联合用药对人宫颈癌细胞(HELA)在体外抑制作用图。
具体实施方式
本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。
本发明所用仪器型号：
高通量高效液相色谱和质谱联用：Shimadzu Prominance纳米反相高效液相色谱联用配置有纳米电喷射离子源的ABSCIEX三重时间飞行质谱。
实施例1：F1、F3多肽的制备
1)首先通过9伏特的直流电刺激的方式促使树蛙分泌出外观呈白色的粘稠状液体，该树蛙分布于澳大利亚东部沿海雨林地区。
2)将液体收集并转移至含有20％甲醇的超纯水中，震荡再通过0.45μm的PVDF膜过滤。
3)滤出清液在真空低温离心干燥剂中干燥，形成白色固体粉末。
4)将白色固体粉末溶解于0.1％的三氟乙酸中，通过高通量高效液相色谱和质谱联用分析其中的多肽序列及具有的翻译后修饰。
5)利用固相多肽合成方法分别合成多肽F1或F3，具体步骤如下：
a)在CS336X的多肽合成仪上，以氯甲基聚苯乙烯树脂作为不溶性的固相载体，将F1或F3的C端氨基酸(氨基被封闭基因)保护的氨基酸共价连接在固相载体上；
b)在三氟乙酸的作用下，脱掉氨基的保护基，这样第一个氨基酸就接到了固相载体上了；
c)然后氨基被封闭的第二个氨基酸的羧基通过N，N′-二环己基碳二亚胺(DCC，Dicyclohexylcarbodiimide)活化，羧基被DCC活化的第二个氨基酸再与已接在固相载体的第一个氨基酸的氨基反应形成肽键，这样在固相载体上就生成了一个带有保护基的二肽；
d)重复上述肽键形成反应，使肽链从C端向N端生长，直至达到所需要的肽链长度。最后脱去保护基，在TFA/DCM/H2O/TIPS的体积比为90∶5∶2.5∶2.5的溶液中自发进行树脂 的去保护和解聚；
d)通过10倍当量的冷乙醚对其沉淀，并用反相高效液相色谱纯化后获得多肽F1或F3。
特别地，在合成过程中，谷氨酸和丝氨酸侧链羧基用L-Bu保护，而为防止组氨酸的消旋化，其侧链用Boc基团保护。
实施例2：制备包含F1、F3多肽的软膏组合物(以10g量配制)
配方：
F1多肽：10mg；
F3多肽：10mg；
PEG4000：100mg；
精氨酸：20mg；
PEG400：适量，加至10g。
按照常规制备软膏组合物的方法制备，将上述组合物分装到软膏包装用铝管中，每管2mg、5mg或10mg，为软膏剂。
实施例3：制备包含F1、F3多肽和Imiquimod的软膏组合物(以10g量配制)
配方：
F1多肽：2.5mg；
F3多肽：2.5mg；
Imiquimod：30mg
PEG3000：100mg；
赖氨酸：5mg；
PEG200：适量，加至10g。
按照常规制备软膏组合物的方法制备，将上述组合物分装到软膏包装用铝管中，每管2mg、5mg或10mg，为软膏剂。
实施例4：制备包含F1、F3多肽的乳膏组合物(以10g量配制)
基质的制备：
取硬脂酸20g、羟苯乙酯1g、白凡士林2g、液体石蜡16g、甘油5g、三乙醇胺1g、十八醇8g、羊毛脂6g、蒸馏水适量，配制成100g基质备用。
配方：
F1多肽：10mg；
F3多肽：10mg；
基质适量，加至10g。
按照常规制备软膏组合物的方法制备，将上述组合物分装到乳膏包装用铝管中，每管2mg、5mg或10mg，为乳膏剂。
实施例5：制备包含F1、F3多肽和Imiquimod的乳膏组合物(以10g量配制)
配方：
F1多肽：5mg；
F3多肽：5mg；
Imiquimod：60mg
基质适量，加至10g。
按照常规制备软膏组合物的方法制备，将上述组合物分装到乳膏包装用铝管中，每管2mg、5mg或10mg，为乳膏剂。
临床实验
1、F1、F3和F1+F3对HPV转化细胞(TC-1细胞)体外生长的抑制作用
图1：F1、F3和F1+F3对HPV转化细胞(TC-1细胞)体外生长的抑制作用进行了研究。
具体方法是：培养在含10％小牛血清RPMI培养基中的100微升5X10 3TC-1细胞加入96孔细胞培养板中，分别加入不同浓度的F1(图1A)，F3(图1B)和对照多肽P3(图1C)，经37℃含5％CO 2培养过夜，MTT实验明确细胞增殖情况。
另一实验使用永生化的正常细胞NP69，具体为培养在含10％小牛血清RPMI培养基中的100微升5X10 3NP69细胞加入96孔细胞培养板中，分别加入不同浓度的F1，F3和对照多肽P3(图1D)，经37℃含5％ CO 2培养过夜，MTT实验明确细胞增殖情况。
结果表明：F1和F3对TC-1细胞生长具有明显的抑制作用，P3无抑制作用。F1，F3，P3对NP69细胞无抑制作用。
2、Imiquimod与F1，F3多肽联合使用能更好杀死HPV16E7转化的小鼠TC-1肿瘤细胞
图2：F1、F3对TC-1肿瘤细胞生长在体外抑制叠加作用图。
培养在含10％小牛血清RPMI培养基中的100微升5X10 3TC-1细胞加入96孔细胞培养板中，分别加入5微克每毫升的F1或/和F3和对照多肽P3，F4，经37℃含5％CO 2培养过夜，MTT实验明确细胞增殖情况。
结果表明，F1，F3对TC-1生长抑制作用有协同作用。
3、Imiquimod与F1，F3多肽联合使用可以更好杀死HPV18E7转化的人宫颈癌细胞(HELA)
图3：F1、F3对人宫颈癌细胞(HELA)生长在体外抑制叠加作用图。
培养在含10％小牛血清DMEM培养基中的100微升5X10 3 HeLa细胞加入96孔细胞培养板中，分别加入不同浓度的F1或/和F3和对照多肽P3，经37℃含5％ CO 2培养过夜，MTT实验明确细胞增殖情况。
结果表明，F1，F3对TC-1生长抑制作用有协同作用。
图4：F1与F3的高通量定量蛋白质组分析显示叠加作用激活TEC激酶信号通路。
培养在含10％小牛血清RPMI培养基中的1000微升5X10 5TC-1细胞加入24孔细胞培养板 中，加入1微克每毫升的F1和F3混合物(1∶1比例)，培养24小时。收集细胞，提取蛋白并进行iTRAQ标记，之后应用反相高效液相色谱和质谱联用技术定量分析细胞中蛋白的含量，并与未经F1、F3共同培养的细胞做比较，得出蛋白表达的变化，进而分析出若干细胞信号通路的变化。
图5：F3与TC-1的相互作用图。
培养在含10％小牛血清RPMI培养基中的1000微升5X10 5TC-1细胞加入24孔细胞培养板中，加入2微克每毫升的经FITC标记的F3到培养的细胞中，在培养不同的时间点，收集细胞，用PBS缓冲液洗涤，然后用流式细胞仪(A)，或者共聚焦显微镜(B)观察F3与TC-1的关系。
表明F3能与TC-1结合，2小时达到峰值，并且不均匀分布在细胞膜和细胞质中。
图6：F1、F3、Imiquimod单独或联合用药对TC-1肿瘤细胞生长在体外抑制作用图。
培养在含10％小牛血清RPMI培养基中的100微升5X10 3TC-1细胞加入96孔细胞培养板中，分别加入5微克每毫升的F1或/和F3，Imiquimod，经37℃含5％CO 2培养过夜，MTT实验明确细胞增殖情况。
结果表明Imiuqimod与F1，F3对TC-1细胞生长抑制有叠加作用。
F1、F3、Imiquimod单独或联合用药对HeLa肿瘤细胞生长在体外抑制作用。
培养在含10％小牛血清DMEM培养基中的100微升5X10 3HeLa细胞加入96孔细胞培养板中，分别加入不同浓度的F1或/和F3和Imiquimod，经37℃含5％CO 2培养过夜，MTT实验明确细胞增殖情况。
结果表明，Imiquimod对Hela细胞体外生长有抑制作用，见图7；
Imiquimod和F1和/或F3对HeLa细胞体外生长抑制作用有叠加效果，见图8。

Claims (8)

  1. F1或F3多肽的制备方法，包括如下步骤：
    1)首先通过9伏特的直流电刺激的方式促使树蛙分泌出外观呈白色的粘稠状液体；
    2)将液体收集并转移至含有15％～30％醇的水溶液中，优选20％甲醇或乙醇水溶液，震荡再通过0.45μm的PVDF膜过滤；
    3)滤出清液在真空低温离心干燥剂中干燥，形成白色固体粉末；
    4)将白色固体粉末溶解于0.1％的三氟乙酸中，通过高通量高效液相色谱和质谱联用分析其中的多肽序列及具有的翻译后修饰；
    5)利用固相多肽合成方法分别合成多肽F1或F3，具体步骤如下：
    a)在CS336X的多肽合成仪上，以氯甲基聚苯乙烯树脂作为不溶性的固相载体，将F1或F3的C端氨基酸(氨基被封闭基团)保护的氨基酸共价连接在固相载体上；
    b)在三氟乙酸的作用下，脱掉氨基的保护基，这样第一个氨基酸就接到了固相载体上了；
    c)然后氨基被封闭的第二个氨基酸的羧基通过N，N′-二环己基碳二亚胺(DCC，Dicyclohcxylcarbodiimide)活化，羧基被DCC活化的第二个氨基酸再与已接在固相载体的第一个氨基酸的氨基反应形成肽键，这样在固相载体上就生成了一个带有保护基的二肽；
    d)重复上述肽键形成反应，使肽链从C端向N端生长，直至达到所需要的肽链长度；最后脱去保护基，在TFA/DCM/H20/TIPS的体积比为90∶5∶2.5∶2.5的溶液中白发进行树脂的去保护和解聚；
    e)通过10倍当量的冷乙醚对其沉淀，并用反相高效液相色谱纯化后获得多肽F1或F3；特别地，在合成过程中，谷氨酸和丝氨酸侧链羧基用t-Bu保护，而为防止组氨酸的消旋化，其侧链用Boc基团保护。
  2. 药物组合物，包含F1、F3多肽中任意一种或二者任意比例的混合物和药学上可接受的辅料。
  3. 根据权利要求2所述的药物组合物，其特征在于，所述的药物组合物制备成适合胃肠外给药的静脉注射、肌内注射或皮下注射的注射液；或经皮肤穿透给药的乳剂或软膏制剂，优选乳剂或软膏制剂。
  4. 根据权利要求2或3所述的药物组合物，其特征在于，每份药物组合物中含F1、F3多肽或者二者的混合物为3～30μg，优选5～20μg。
  5. 权利要求2所述的药物组合物，进一步包含Imiquimod，所述的药物组合物制备成适合胃肠外给药的静脉注射，肌内注射或皮下注射的注射液；或经皮肤穿透给药的乳剂或软膏制剂，优选乳剂或软膏制剂。
  6. 权利要求2或3所述的药物组合物在制备预防和/或治疗HPV感染导致的疾病的药物中的用途，所述疾病为疣或实体肿瘤，所述的疣优选尖锐湿疣；所述的实体肿瘤包括并不限于宫颈癌、外阴癌、阴茎癌、肛门癌和口腔癌。
  7. 权利要求5所述的药物组合物在制备预防和/或治疗HPV感染导致的疾病的药物中的用途，所述疾病为疣或实体肿瘤，所述的疣优选尖锐湿疣；所述的实体肿瘤包括并不限于宫颈癌、外阴癌、阴茎癌、肛门癌和口腔癌。
  8. 权利要求5所述的药物组合物联合放射性治疗、化学治疗、治疗性疫苗中任意一种或多种在制备预防和/或治疗HPV感染导致的疾病的药物中的用途，所述疾病为实体肿瘤，所述的实体肿瘤包括并不限于宫颈癌、外阴癌、阴茎癌、肛门癌和口腔癌。
PCT/CN2017/000744 2016-12-27 2017-12-22 包含f1、f3多肽的药物组合物及其在治疗hpv感染疾病中的应用 WO2018120277A1 (zh)

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CN107973846B (zh) * 2017-11-21 2019-02-19 Wnl生物医学科技公司 125I标记的Caerin多肽及其应用
CN109200270B (zh) * 2018-09-30 2022-04-29 中奥生物医药技术(广州)有限公司 一种能提高多肽疫苗对hpv感染肿瘤治疗效果的联合用药物及其应用
CN109675018A (zh) * 2019-01-29 2019-04-26 倪国颖 一种用于涂覆皮肤表面的药物及其应用
CN115957306B (zh) * 2022-11-22 2023-08-22 中奥生物医药技术(广东)有限公司 一种Caerin1.1/1.9联合抗CD47抗体在制备治疗黑色素瘤药物中的应用
CN116789768B (zh) * 2023-07-31 2024-03-15 中奥生物医药技术(广东)有限公司 一种f1多肽的制备方法
CN116789769B (zh) * 2023-07-31 2024-02-23 中奥生物医药技术(广东)有限公司 一种f3多肽的制备方法

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