WO2020243895A1 - 促进毛发生长和/或再生的疫苗、其制备方法及其应用 - Google Patents

促进毛发生长和/或再生的疫苗、其制备方法及其应用 Download PDF

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WO2020243895A1
WO2020243895A1 PCT/CN2019/089955 CN2019089955W WO2020243895A1 WO 2020243895 A1 WO2020243895 A1 WO 2020243895A1 CN 2019089955 W CN2019089955 W CN 2019089955W WO 2020243895 A1 WO2020243895 A1 WO 2020243895A1
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ferritin
vaccine
hair
regeneration
adjuvant
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PCT/CN2019/089955
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English (en)
French (fr)
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徐建青
张晓燕
丁相卿
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上海鑫湾生物科技有限公司
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Priority to PCT/CN2019/089955 priority Critical patent/WO2020243895A1/zh
Publication of WO2020243895A1 publication Critical patent/WO2020243895A1/zh

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    • 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
    • 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

Definitions

  • the present invention belongs to the field of biotechnology, and relates to a ferritin, and more specifically to the use of ferritin for promoting hair growth and/or regeneration.
  • Hair loss refers to the phenomenon of hair loss.
  • Physiological alopecia refers to hairs that are in the anagen phase and telogen phase. The hair entering the anagen phase and newly entering the growth phase will constantly be in dynamic balance, so a normal amount of hair can be maintained. But when the hair falls out abnormally or excessively, it is pathological hair loss.
  • pathological alopecia Although pathological alopecia is not fatal, it can affect the appearance. The mild one can be used as a sub-health state, and the severe one is an external reflection of premature aging or abnormal physiological indicators in the body. There are many reasons for pathological alopecia, but the specific mechanism is not fully understood. The possible causes of alopecia are as follows: 1. Androgenetic alopecia: also known as seborrheic alopecia, which is an autosomal dominant inheritance. Features need to be manifested under the action of androgens. It is usually affected by men, but there are also women. It is characterized by a lot of oil, and obvious hair loss on the top of the head and sideburns. 2. Nervous alopecia: also known as alopecia areata.
  • Endocrine alopecia Hair growth is affected by a variety of endocrine hormones, so when endocrine abnormalities occur, it often causes alopecia diseases, such as postpartum and menopausal alopecia, which are more common in women.
  • Nutritional alopecia Hair is an external manifestation of physical conditions. Malnutrition and abnormal metabolism can cause changes in hair quality and hair color.
  • Physical hair loss Common physical factors that cause hair loss include mechanical stimulation and exposure to radioactive materials.
  • Chemical alopecia chemical factors can cause hair color changes and even hair loss, which is common in patients with perm.
  • Infectious alopecia Infection of various pathogens is an important factor in hair diseases, including infections such as bacteria, viruses, fungi, spirochetes, and parasites. It is common in patients with tinea capitis.
  • Symptomatic alopecia some systemic or local diseases can be accompanied by alopecia, common systemic lupus erythematosus, syphilis, anemia and other diseases.
  • Congenital alopecia complete loss or thinning of hair caused by developmental defects. Patients often have thin and thin hair, or hair is normal at birth, and soon falls off without regenerating. It can be divided into isolated defects and other deformities.
  • hair loss There are many ways to treat hair loss on the market. For pathological hair loss, the underlying disease should be treated. Hair will grow back after the body recovers. For chemical hair loss, stop irritating hair dyes, perming agents, and low-quality shampoos, which can help hair recover. For physical hair loss, reduce the use of plastic combs and plastic head brushes that are prone to static electricity, and wear protective caps in environments with serious air dust pollution, and timely hair washing can effectively slow down hair loss.
  • Modern medical treatment methods for alopecia, seborrheic alopecia are mainly hormone modulators, such as androgen blockers and estrogen mediators or growth factor intervention therapy; and biological response modifiers, such as minoxidil , Tretinoin, etc.
  • the treatment of alopecia areata is mainly immunomodulators, such as corticosteroids, and biological response modifiers, mainly 2% minoxidil solution treatment.
  • Western medicine lacks specific treatments for alopecia. The current treatments have some clinical effects. However, it is difficult to promote and use due to obvious side effects. For example, taking hormones may cause erectile dysfunction and embarrassing symptoms such as breast development.
  • Scalp hair implantation is a method to treat hair loss that has emerged in recent years.
  • the main principle is to use the principle of autologous transplantation of human organs to transplant healthy hair follicles to areas with sparse or no hair, in order to grow hair from the transplanted follicles.
  • Hair transplantation is only suitable for the later stage of severe hair loss, because the hair falls off a large area and the hair follicles can no longer grow hair through the stimulation of drugs. This method is effective quickly and is chosen by more and more people with hair loss.
  • hair transplants also have unsatisfactory disadvantages such as high price and low acceptance by the general public, and the survival rate of new hair transplants is usually less than 100%, which causes damage to the hair follicles that cannot be regenerated.
  • Ferritin (Ferritin) is a type of iron-storing protein widely found in animals and plants. It is the most abundant in the liver and spleen of mammals. Although studies have shown that low levels of ferritin or iron deficiency in the human body may be the main cause of hair loss. The causes of hair loss are complex, and lack of ferritin may be a related factor. However, in the prior art, iron supplementation is used to increase the level of ferritin in the blood to relieve the symptoms of hair loss, with little effect. It may be due to the complex causes of hair loss. It is difficult to treat hair loss through a single factor; and there is currently no evidence that ferritin supplementation alone can restore hair regrowth.
  • the purpose of the present invention is to provide the use of ferritin for promoting hair growth and/or regeneration, and a vaccine containing the ferritin.
  • Another object of the present invention is to provide a method for preparing the above ferritin and the above vaccine.
  • the present invention provides a use of ferritin in preparing a medicine for promoting hair growth and/or regeneration.
  • ferritin is derived from but not limited to prokaryotes, including bacteria, archaea and cyanobacteria; or fungi; or eukaryotes, including plants and animals, including human sources.
  • ferritin is a large family that exists widely, the ferritin gene or ferritin sequence of different species may be different, but as long as the ferritin gene or protein functions as an immune antigen remain unchanged, or can induce the production of antibodies against ferritin , Are all within the protection scope of the present invention.
  • ferritin includes modified ferritin, a fusion protein formed by fusion expression of a ferritin molecule or a partial sequence thereof with other molecules, and a polypeptide fragment derived from ferritin;
  • Methods include, but are not limited to, fusion with the Fc sequence of an antibody, fusion with different signal peptides, fusion with cytokines, and the like.
  • the ferritin comprises an amino acid sequence selected from:
  • amino acid sequence shown in SEQ ID NO:1 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , At least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the nucleotide sequence encoding the ferritin is shown in SEQ ID NO: 2.
  • ferritin may be formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO:1 without affecting its immunogenicity, or induce the production of antibodies against ferritin. Amino acid sequence.
  • the nucleotide sequence encoding the ferritin may be formed by replacing, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO: 2 without affecting the immunogenicity of the encoded protein , Or induce the production of nucleotide sequences against ferritin antibodies.
  • the ferritin itself assembles into nanoparticles, or is wrapped by lipid molecules to form nanoparticles.
  • the present invention provides a vaccine comprising the ferritin.
  • the vaccine according to the present invention wherein the vaccine further comprises an adjuvant.
  • the adjuvant includes but not limited to aluminum salt adjuvant, CpG adjuvant, manganese adjuvant, MF59 adjuvant, QS21 adjuvant, TLR ligand adjuvant, NOD pathway adjuvant, interferon adjuvant, cGAS/ STING pathway adjuvant, cytokine adjuvant (including but not limited to IL-12, GM-CSF, IL-18, IL-21, etc.), PD-1 blocking antibody, CTLA4 blocking antibody, TIGIT blocking antibody, ⁇ Galcer And its derivative adjuvants.
  • the vaccine according to the present invention wherein the ferritin itself forms nanoparticles or is wrapped by lipid molecules to form nanoparticles.
  • the vaccine further comprises a vector; preferably, the vector is selected from a plasmid vector, a poxvirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a CMV vector, a cell Carrier or bacterial carrier.
  • the vector is selected from a plasmid vector, a poxvirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a CMV vector, a cell Carrier or bacterial carrier.
  • the present invention also provides a method for preparing the vaccine, the method comprising:
  • the step 1) includes expressing the ferritin gene in a plasmid vector, a poxvirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a CMV vector, a cell vector or a bacterial vector.
  • the vaccine according to the present invention is used to promote hair growth and/or regeneration.
  • a method for promoting hair growth and/or regeneration comprising administering to a subject in need a therapeutically effective amount of the vaccine according to the present invention.
  • administration mode is selected from subcutaneous injection, intradermal injection, intramuscular injection, oral administration, nasal spray administration, and skin scratch administration.
  • ferritin vaccination to induce an antibody response against ferritin to play a role in the treatment and prevention of hair loss
  • ferritin adjuvants or different carrier forms of ferritin vaccines.
  • the vaccine can be vaccinated to humans or pets to promote hair growth and/or regeneration.
  • the serum induced after the ferritin vaccine immunization plays a role in hair growth and regeneration, and those who have not been vaccinated with ferritin vaccine can be promoted by inputting purified or unpurified serum Hair growth and/or regeneration.
  • the role of antibodies induced after ferritin vaccine immunization in hair growth and regeneration, those who have not been vaccinated with ferritin vaccine can promote hair growth and/or by importing purified antibodies regeneration.
  • the serum of a ferritin vaccination person, or a purified immunoglobulin, or a purified antibody is applied to the affected area to promote hair growth and/or regeneration; or the purified immunoglobulin , Antibody is mixed with other skin medications and applied to the affected area to promote hair growth and/or regeneration.
  • the use or method according to the present invention wherein the promotion of hair growth and/or regeneration includes promotion of hair regeneration in persons with excessive hair loss, and causes of excessive hair loss include, but are not limited to, androgenic alopecia, neurogenic alopecia, and endocrine alopecia , Nutritional alopecia, physical alopecia, chemical alopecia, infectious alopecia, symptomatic alopecia, and congenital alopecia.
  • the use or method according to the present invention, wherein the promotion of hair growth and/or regeneration includes promotion of hair growth in persons with abnormal hair, which includes gray hair, yellow hair, thinning hair and the like.
  • the use or method according to the present invention, wherein the promotion of hair growth and/or regeneration includes the use of ferritin to prepare a vaccine that can be administered alone or in combination with other treatment techniques when treating and preventing hair loss.
  • the ferritin vaccine can be used for preventive administration before pathological alopecia to prevent the occurrence of alopecia or reduce the severity of subsequent alopecia; it can also be administered as a therapeutic drug after the occurrence of pathological alopecia to reduce the pathology The severity of alopecia; it can also be administered continuously or at intervals from before to after the occurrence of pathological alopecia to obtain better curative effects.
  • the beneficial effect of the present invention is that compared with the existing technology for the treatment of hair loss, the present invention finds a new use of ferritin, and the present invention finds that the antibodies induced by ferritin can regenerate the hair of the hair loss mice. Simply supplementing ferritin may have little effect on the treatment of hair loss, but by immunizing ferritin, it can induce the production of antibodies against ferritin, which can promote hair repair.
  • ferritin vaccine in the treatment of pathological alopecia may involve a variety of causes of pathological alopecia, including androgenic alopecia, neurological alopecia, endocrine alopecia, nutritional alopecia, physical alopecia, chemical alopecia, and infectious alopecia , Symptomatic alopecia, congenital alopecia. It has a wider application value for people with pathological hair loss. Compared with the existing technology for the treatment of hair loss, the ferritin of the present invention has wide sources, simple preparation, obvious effect in treating hair loss, low side effects, and has important clinical application value in the treatment of hair loss.
  • the present invention mainly finds that when C57BL/6 mice are immunized with the ferritin of the present invention, the hair of C57BL/6 mice with depilatory phenomenon is repaired and regenerated in a short time. More importantly, the serum after passive adoptive ferritin immunization with C57BL/6 mice can also quickly promote hair repair and regeneration in mice with hair loss. Therefore, ferritin can be used as a more effective vaccine to treat and prevent alopecia symptoms.
  • the present invention provides a vaccine for the treatment of alopecia.
  • the ferritin vaccine is immunized to induce the production of antibodies against ferritin, which has obvious effects in the treatment of alopecia, is low in cost, and has no side effects.
  • Figure 1 shows the construction of the ferritin eukaryotic expression vector of the present invention and SDS-Page gel identification after protein purification.
  • Figure 1a is a map of pSV1.0-ferritin eukaryotic expression vector plasmid construction
  • Figure 1b is the successful expression of ferritin in the supernatant of 293T cells.
  • Figure 2 shows the hair regeneration of mice after immunization with ferritin vaccine according to Example 2 of the present invention.
  • Figure 2a is the PBS control group, and the hair loss phenomenon of mice has not improved;
  • Figure 2b is the ferritin vaccine treatment group.
  • As the immunization progresses 7 days after the immunization, 4 out of 6 mice have the hair loss phenomenon.
  • Significant improvement new hair regeneration in the area of alopecia areata.
  • the hair loss of 6 mice basically disappeared.
  • Fig. 3 shows that after immunization with ferritin vaccine, a high-titer IgG response to ferritin is induced in mice according to Example 3 of the present invention. After immunizing mice with ferritin, it can induce a high titer specific IgG response to ferritin.
  • FIG. 4 shows that the mouse serum immunized with ferritin was injected intravenously according to Example 4 of the present invention, and the mouse hair was regenerated.
  • Figure 4a is the collection of serum from mice immunized with the PBS control group. After intravenous infusion, the hair loss phenomenon of the mice did not disappear;
  • Figure 4b is the serum of mice in the ferritin vaccine treatment group, on the 7th day after the infusion , The hair loss of all mice was improved. After 14 days of reinfusion, all four depilated mice were cured. A single dose of serum was transfused once, and the hair growth effect could be maintained for up to 42 days in 4 depilated mice without recurrence.
  • Fig. 5 shows the intravenous infusion of ferritin-immunized mouse serum according to Example 5 of the present invention, in which IgG has been removed, and no effect of promoting hair regeneration in mice is seen.
  • Protein G was used to treat the serum of ferritin-immunized mice, and after removing the IgG in the serum, the serum was intravenously infused to the depilated mice. The depilation phenomenon of the mice did not improve.
  • the present invention obtains the ferritin gene from Pyrococcus furiosus. Its amino acid sequence is shown in SEQ ID NO:1. After mammalian codon optimization, the nucleotide sequence of the encoding gene is shown in SEQ ID NO. As shown in :2, the recombinant pSV1.0-ferritin particles were transfected into 293T cells, and then the eukaryotic expression of ferritin was detected by WB, and eluted with imidazole with different concentration gradients through a nickel column. After collecting the target protein Filter and wash to obtain high-purity ferritin. The specific steps are as follows:
  • the plasmid pSV1.0-ferritin was transfected into 293T cells, the transfection reagent was TurboFect (Thermo Scientific, catalog number: R0531), the medium was DMEM complete medium (10% FBS and 1% PS), 37°C incubator After 72 hours of incubation, the cells were taken out, collected in a pre-cooled EP tube, and lysed with RIPA lysis buffer, and 5 ⁇ SDS loading buffer was added to the supernatant, and heated in a boiling water bath for 10 minutes to denature the protein , After a brief centrifugation, the supernatant was used as a spot sample.
  • the transfection reagent was TurboFect (Thermo Scientific, catalog number: R0531)
  • the medium was DMEM complete medium (10% FBS and 1% PS)
  • 37°C incubator 37°C incubator
  • the cells were taken out, collected in a pre-cooled EP tube, and lysed with RIPA lysis buffer, and 5 ⁇ SDS loading buffer
  • the protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration of the separation gel was 10%.
  • the electrophoresis voltage is 70V, and the time is 30-40 minutes (marked by the start of the marker separation).
  • the gel was dyed with Coomassie Brilliant Blue, dyed at room temperature for 1 hour, and then eluted and developed with a decolorizing solution.
  • the present invention uses C57BL/6 mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.). C57BL/6 mice will experience hair loss during the breeding process, which is very similar to the clinical manifestations of human alopecia areata. Therefore, it is used as an animal model for hair loss to verify the effect of ferritin on hair regeneration.
  • mice with only hair loss were selected and randomly divided into two groups, which were labeled as the PBS control group and the ferritin vaccine treatment group, respectively.
  • the two groups of mice were immunized once in week 0 and week 2.
  • mice in the PBS control group were injected intramuscularly with 100 ⁇ L of vaccine-free solution (the volume ratio of PBS and aluminum adjuvant is 1:1); mice in the ferritin vaccine treatment group were injected intramuscularly with an equal volume of 10 ⁇ g ferritin vaccine solution (iron The volume ratio of protein to aluminum adjuvant is 1:1).
  • mice After ferritin immunization, mice produced high titer specific antibody responses against ferritin
  • mice After the mice were immunized with the second shot of ferritin vaccine. Collect mouse peripheral blood in a 1.5mL tube, let it stand at room temperature for 2 hours, centrifuge at 7000g for 15 min, draw serum into a new tube, and store at 4°C.
  • the ELISA method was used to detect the response to ferritin-specific IgG in the mouse serum.
  • a 96-well ELISA plate was taken, and 200 ⁇ L of ferritin (final concentration 0.5 ⁇ g/mL) was added to each well and coated at 4°C. overnight.
  • the next day take out the ELISA plate and discard the coating solution, wash the plate 3 times with 0.05% PBST buffer (220 ⁇ L per well); after washing, add 200 ⁇ L ELISA blocking solution to each well and block at room temperature for 2 hours; then use 0.05 Wash the plate 3 times with %PBST (220 ⁇ L per well).
  • Example 4 Infusion of ferritin-immunized mouse serum can quickly promote hair growth and/or regeneration
  • the peripheral blood of the mice in the PBS group and the ferritin vaccine immunization group in Example 2 was collected 2 weeks after the end of immunization, and the serum was separated.
  • mice Prepare 8 C57BL/6 mice with only hair loss and randomly divide them into 2 groups, labeled as PBS control group and ferritin treatment group.
  • PBS control group came from mice inoculated with PBS
  • ferritin treatment group came from mice two weeks after the ferritin immunization.
  • Example 5 The serum of mice immunized with ferritin cannot promote hair growth and/or after removing IgG Or regenerate
  • PBS buffer Protein G agarose beads
  • mice Prepare 5 depilated mice. On the 0th day and the 5th day, 50 ⁇ L of the above-mentioned serum was reinfused into the orbital vein (excluding IgG), and the observation was continued for 21 days.
  • the orbital vein excluding IgG

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Abstract

本发明提供了一种铁蛋白在制备用于促进毛发生长和/或再生的药物中的用途。本发明还提供一种疫苗，所述疫苗包含所述铁蛋白。所述疫苗还包含佐剂。本发明提供的疫苗，通过免疫接种铁蛋白疫苗，诱导产生针对铁蛋白的抗体，在用于脱发治疗上有明显的效果，成本低，并且没有副作用。

Description

促进毛发生长和/或再生的疫苗、其制备方法及其应用 技术领域
本发明属于生物技术领域，涉及一种铁蛋白，更具体地涉及铁蛋白用于促进毛发生长和/或再生的用途。
背景技术
脱发是指头发脱落的现象。生理性脱发是指正由于头发都是处于退行期及休止期的毛发，进入退行期与新进入生长期的毛发会不断处于动态平衡中，因此可以维持正常数量的头发。但是当头发异常或过度的脱落时，便是病理性脱发。
病理性脱发虽然不会致命，但会影响美观，轻者可作为亚健康状态的表现，重者则是早衰或者体内生理指标发生异常的外在反映。引起病理性脱发的原因很多，但是具体机制还不是完全清楚，可能引起的脱发原因由如下几个：1、雄激素性脱发：又称为脂溢性脱发，为常染色体显性遗传，其遗传特征需在雄激素作用下才表现出来。一般是男性发病，但也有女性发病。特点是出油多，头顶和两侧鬓角头发脱落明显。2、神经性脱发：又称为斑秃。精神压力过大时常常出现脱发增多。在精神压力的作用下，人体立毛肌收缩、头发直立，植物神经或中枢神经机能发生紊乱，毛囊毛乳头发生改变和营养不良，从而导致毛发生长功能抑制，毛发进入休止期而出现脱发。3、内分泌脱发：毛发生长受多种内分泌激素的影响，所以当发生内分泌异常时多引起脱发疾病，如产后、更年期脱发，多见于女性。4、营养性脱发：毛发是身体状况的外在表现，机体营养不良和新陈代谢异常可引起发质和发色的改变，严重营养不良甚至导致弥漫性脱发。5、物理性脱发：常见的引起脱发的物理性因素包括机械性刺激和接触放射性物质。6、化学性脱发：化学因素可以导致毛发颜色改变甚至脱发，常见染发烫发患者。7、感染性脱发：各种病原体的感染是毛发疾病中一类重要因素，主要包括细菌、病毒、真菌、螺旋体、寄生虫等感染，常见头癣患者。8、症状性脱发：某些系统性或局部疾病都可伴发脱发，常见系统性红斑狼疮，梅毒，贫血等疾病。9、先天性脱发：发育缺陷所引起的头发完全缺失或稀疏，患者常见头发稀疏细小， 或出生时头发正常，不久就脱落不再生，可分为孤立缺陷和其他畸形。
中国健康促进与教育协会2011年11月公布的“中国脱发人群调查”数据表明，我国脱发人群约达2亿，其中男性脱发人数约1.3亿；2015年预计总脱发人群将接近2.4亿。几乎平均6个中国人当中就有一个脱发问题。这个人群数量是相当惊人的，已经超过糖尿病和高血压人群，成为影响当代健康的第一难题。而且脱发已经呈现了低龄化的趋势，90后也逐渐成为脱发的人群。在中国男性脱发人群中，60％的男性在25岁前就开始脱发，30岁以前开始脱发的则达到84％。
市场上治疗脱发的手段多种多样，对于病理性脱发应治疗基础疾病，身体康复后头发会重新长出。而对化学性脱发停用刺激性强的染发剂、烫发剂及劣质洗发用品，可帮助头发的恢复。针对物理性脱发，减少使用易产生静电的塑料梳子和塑料头刷，在空气粉尘污染严重的环境中要佩戴防护帽，并及时进行头发清洗，可有效减缓脱发。
现代医学对脱发的治疗方法，对脂溢性脱发主要是激素调节剂，如雄激素阻断剂与雌激素介导剂或者生长因子干预治疗；还有生物学反应调节剂，如米诺地尔、维A酸类等。对斑秃的治疗，主要是免疫调节剂，如皮质类固醇等；以及生物反应调节剂，主要是2％的米诺地尔溶液治疗。西医治疗脱发缺乏特效的治疗手段，目前的治疗措施在临床上有一定的效果，但是由于副作用明显导致推广使用比较困难。如服用激素等可能会造成性功能勃起障碍，还有胸部发育等让人颇为难堪的症状出现。近年来有通过间充质干细胞诱导毛发再生或者受损的毛发再生，但效果不够稳定，多数处于临床前的研究。头皮毛发种植术是近年兴起的一种治疗脱发的办法，主要原理是利用人体器官自体移植的原理，将健康的毛囊移植到头发稀少或没有头发的区域，以期移植后的毛囊重新长出头发。植发术只适用于重度脱发的后期，由于头发大面积脱落且已无法通过药物刺激使毛囊重新长出头发的状况。该法显效迅速，为越来越多的脱发人士所选择。但植发术也存在诸如价格昂贵因而一般民众可接受性较低、新植发存活率通常低于100％而造成损害的毛囊不可再生等不尽人意之处。
此外，也有采用中药如防风、连翘、茯苓等应对脱发的策略。根据脱发原因与类型不同而需要适当调整用药的成分和剂量。中医治疗脱发的效果几千年来一直处在参差不齐的状态，且该治疗为循序渐进的过程，希望快速见效的现代人选择中医治疗脱发者有但数量不多。
铁蛋白(Ferritin)，是动植物体内广泛存在的一类贮存铁的蛋白，在哺乳类动物的肝和脾中含量最多。虽然有研究表明，人体内铁蛋白水平低或缺 铁可能是导致脱发的主要原因。脱发诱因复杂，缺乏铁蛋白有可能是其中相关的因素，然而现有技术中有通过补充铁剂来提高血液中铁蛋白的含量来缓解脱发症状，收效甚微，可能是由于引起脱发的原因复杂，很难通过单一因素治疗脱发；而且目前没有证据证实单纯通过补充铁蛋白便可以恢复毛发的再生。
综上，当前对简单有效的促进毛发生长和/或再生的方法存在需求。
发明内容
本发明的目的在于提供铁蛋白用于促进毛发生长和/或再生的用途，以及含有该铁蛋白的疫苗。
本发明的另一个目的是提供一种上述铁蛋白以及上述疫苗的制备方法。
为实现上述目的，本发明采用了以下技术方案：
一方面，本发明提供了一种铁蛋白在制备用于促进毛发生长和/或再生的药物中的用途。
其中，所述铁蛋白来源于但不限于原核生物，包括细菌、古细菌与蓝细菌；或真菌；或真核生物，包括植物以及动物类，包括人类来源。
虽然铁蛋白是广泛存在的大家族，不同物种的铁蛋白基因或者铁蛋白序列可能会有差异，但是只要其铁蛋白基因或蛋白作为免疫抗原的功能不变，或能够诱导产生针对铁蛋白的抗体，均在本发明保护范围内。
根据本发明所述的铁蛋白，其中，所述铁蛋白包括经过修饰的铁蛋白、铁蛋白分子或其部分序列与其他分子融合表达形成的融合蛋白、以及铁蛋白来源的多肽片段；融合表达的方式包括但不限于与抗体的Fc序列融合、与不同信号肽的融合、与细胞因子融合等。
优选地，所述铁蛋白包含选自以下的氨基酸序列：
(i)SEQ ID NO:1所示的氨基酸序列；
(ii)与SEQ ID NO:1所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:1所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
优选地，所述铁蛋白的编码核苷酸序列如SEQ ID NO:2所示。
另外，所述铁蛋白可以是将SEQ ID NO:1所示氨基酸序列经过一个或多个氨基酸残疾的取代，缺失或添加而形成的不影响其免疫原性，亦或者诱导产生针对铁蛋白抗体的氨基酸序列。
所述铁蛋白的编码核苷酸序列可以是将SEQ ID NO:2所示的核苷酸序列经过一个或多个核苷酸的取代，缺失或添加而形成的不影响其编码蛋白质免疫原性，亦或者诱导产生针对铁蛋白抗体的核苷酸序列。
根据本发明所述的用途，其中铁蛋白自身装配成纳米颗粒、或被脂质分子包裹形成纳米颗粒等。
另一方面，本发明提供一种疫苗，所述疫苗包含所述铁蛋白。
根据本发明所述的疫苗，其中，所述疫苗还包含佐剂。
优选地，所述佐剂包括但不限于铝盐佐剂、CpG佐剂、锰佐剂、MF59佐剂、QS21佐剂、TLR配体佐剂、NOD通路佐剂、干扰素佐剂、cGAS/STING通路佐剂、细胞因子佐剂(包括但不限于IL-12、GM-CSF、IL-18、IL-21等)、PD-1阻断抗体、CTLA4阻断抗体、TIGIT阻断抗体、αGalcer及其衍生物佐剂等。
根据本发明所述的疫苗，其中，所述铁蛋白自身形成纳米颗粒或被脂质分子包裹形成纳米颗粒。
根据本发明所述的疫苗，其中，所述疫苗还包含载体；优选地，所述载体选自质粒载体、痘病毒载体、腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体、CMV载体、细胞载体或细菌载体。
再一方面，本发明还提供了所述疫苗的制备方法，所述方法包括：
1)制备铁蛋白并纯化；
2)混合纯化后的铁蛋白和佐剂，即得。
优选地，所述步骤1)包括将铁蛋白基因表达于质粒载体、痘病毒载体、腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体、CMV载体、细胞载体或细菌载体。
根据本发明所述的疫苗用于促进毛发生长和/或再生的用途。
一种促进毛发生长和/或再生的方法，所述方法包括给予有需要的受试者治疗有效量的根据本发明所述的疫苗。
根据本发明所述的用途或方法，其中所述给药方式选自皮下注射、皮内注射、肌肉注射、口服、滴鼻喷雾给药、皮肤划痕给药。
根据本发明所述的用途或方法，其中，利用铁蛋白疫苗接种诱导针对铁蛋白的抗体应答发挥治疗及预防脱发的作用，可以是铁蛋白联合佐剂或者不 同载体形式的铁蛋白疫苗同种多次免疫或者不同疫苗序贯免疫或者不同疫苗混合免疫，以获得更佳疗效。
根据本发明所述的用途或方法，其中，所述疫苗可接种于人或宠物以促进毛发生长和/或再生。
根据本发明所述的用途或方法，其中，所述铁蛋白疫苗免疫接种后所诱导的血清在毛发生长与再生中起的作用，未接种铁蛋白疫苗者可通过输入提纯或未提纯血清而促进毛发生长和/或再生。
根据本发明所述的用途或方法，其中，提纯铁蛋白疫苗接种者血清中的免疫球蛋白并输注给未接种铁蛋白疫苗者以促进毛发生长和/或再生。
根据本发明所述的用途或方法，其中，铁蛋白疫苗免疫接种后所诱导的抗体在毛发生长与再生中的作用，未接种铁蛋白疫苗者可通过输入提纯的抗体而促进毛发生长和/或再生。
根据本发明所述的用途或方法，其中，将铁蛋白疫苗接种者血清、或提纯的免疫球蛋白、或纯化的抗体涂于患处以促进毛发生长和/或再生；或将提纯的免疫球蛋白、抗体与其他皮肤用药混合涂于患处以促进毛发生长和/或再生。
根据本发明所述的用途或方法，其中，所述促进毛发生长和/或再生包括促进毛发过度脱落者的毛发再生，过度脱落的原因包括但不限于雄激素性脱发、神经性脱发、内分泌脱发、营养性脱发、物理性脱发、化学性脱发、感染性脱发、症状性脱发、先天性脱发。
根据本发明所述的用途或方法，其中，所述促进毛发生长和/或再生包括包括促进毛发异常者的毛发生长，毛发异常包括白发、黄发、毛发稀疏等。
根据本发明所述的用途或方法，其中，所述促进毛发生长和/或再生包括利用铁蛋白所制备的疫苗在治疗及预防脱发时可单独给药或与其他治疗技术联合应用。
上述用途或方法中，利用铁蛋白疫苗可用于病理性脱发前预防性给药用以预防脱发发生或降低后续脱发的严重程度；也可在病理性脱发发生后作为治疗性药物给药以降低病理性脱发的严重程度；也可自病理性脱发发生前至发生后连续或间隔给药，以获得更佳疗效。
本发明的有益效果在于：与现有治疗脱发技术相比，本发明发现了铁蛋白的新用途，本发明发现利用铁蛋白诱导产生的抗体可以使脱发的小鼠毛发再生。单纯补充铁蛋白可能对于治疗毛发脱落作用甚微，但是通过免疫接种铁蛋白的方式，诱导针对铁蛋白抗体的产生，可促进毛发的修复。铁蛋白疫 苗在治疗病理性脱发中的作用可涉及到病理性脱发的多种诱因，包括雄激素性脱发、神经性脱发、内分泌脱发、营养性脱发、物理性脱发、化学性脱发、感染性脱发、症状性脱发、先天性脱发。对有病理性脱发的人群有较为广泛的应用价值。与现有治疗脱发技术相比，本发明所述铁蛋白来源广泛，制备简单，治疗脱发效果显著，副作用低，在脱发治疗方面有重要的临床应用价值。
本发明主要发现用本发明所述的铁蛋白免疫接种C57BL/6小鼠时，有脱毛现象的C57BL/6小鼠毛发在较短时间内就得到了修复和再生。更为重要的是，通过被动过继铁蛋白免疫接种C57BL/6小鼠后的血清，同样能够快速促使有脱毛现象小鼠的毛发修复和再生。因此，铁蛋白可作为较为有效的治疗及预防脱发症状的疫苗。
本发明提供了一种治疗脱发的疫苗，通过免疫接种铁蛋白疫苗，诱导产生针对铁蛋白的抗体，在用于脱发治疗上有明显的效果，成本低，并且没有副作用。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为本发明的铁蛋白(Ferritin)真核表达载体的构建与蛋白纯化后SDS-Page胶鉴定。其中，图1a为pSV1.0-铁蛋白真核表达载体质粒构建图谱；图1b为铁蛋白在293T细胞上清中成功表达。
图2为根据本发明的实施例2，免疫接种铁蛋白疫苗后，小鼠毛发得以再生。其中，图2a为PBS对照组，小鼠脱毛现象没有任何改善；图2b为铁蛋白疫苗治疗组，随着免疫进行，免疫接种7天后，6只小鼠中有4只小鼠脱毛现象得到了显著的改善，斑秃区域有新毛再生。在第21天时，6只小鼠脱毛现象基本消失。
图3为根据本发明的实施例3，免疫接种铁蛋白疫苗后，在小鼠体内诱导产生高滴度针对铁蛋白的IgG应答。铁蛋白免疫小鼠后，可以诱导高滴度的针对铁蛋白特异性的IgG应答。
图4为根据本发明的实施例4，静脉回输铁蛋白免疫后的小鼠血清，小鼠毛发得以再生。其中，图4a为采集PBS对照组免疫小鼠的血清，静脉回输后，小鼠脱毛现象未见消失；图4b为回输铁蛋白疫苗治疗组的小鼠血清，在回输后第7天，所有小鼠的脱毛现象均得到改善，回输14天后，所有四 只脱毛的小鼠均得到治愈。单剂量回输血清一次，生发效果在4只脱毛小鼠身上均可维持长达42天而未见复发。
图5为根据本发明的实施例5，静脉回输铁蛋白免疫小鼠血清，其中IgG已经被去除，则未见促进小鼠毛发再生效果。采用Protein G处理铁蛋白免疫的小鼠血清，去除血清中的IgG后，将该血清静脉回输给脱毛小鼠，小鼠脱毛现象没有任何改善。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
在以下的实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。所用试剂的来源、商品名以及有必要列出其组成成分者，均在首次出现时标明，其后所用相同试剂如无特殊说明，均以首次标明的内容相同。
实施例1铁蛋白的纯化鉴定
本发明从强烈火球菌(Pyrococcus furiosus)中获得了铁蛋白基因，其氨基酸序列如SEQ ID NO:1所示，将其进行哺乳动物密码子优化后，编码基因的核苷酸序列如SEQ ID NO:2所示,将重组的pSV1.0-铁蛋白质粒转染进入293T细胞后，再通过WB检测铁蛋白的真核表达，并通过镍柱采用不同浓度梯度的咪唑洗脱，收集目的蛋白后过滤，洗涤，得到高纯度的铁蛋白，其具体步骤如下：
将质粒pSV1.0-铁蛋白转染进入293T细胞，转染试剂为TurboFect(Thermo Scientific公司，货号：R0531)，培养基为DMEM完全培养基(10％FBS和1％P.S.)，37℃孵箱培养72小时后将细胞取出，收集细胞到预冷的EP管中，并以RIPA裂解缓冲液裂解细胞，并在上清中加入5×SDS上样缓冲液，沸水浴中加热10分钟使蛋白变性，瞬时离心后将上清作为点样样品。后通过SDS-聚丙烯酸胺凝胶电泳(SDS-PAGE)分离蛋白，分离胶浓度为10％。电泳的电压为70V，时间为30-40分钟(以marker开始分离为标志)，待溴酚蓝迁移出浓缩胶位置后，将电压调至110V，1小时30分钟后关闭电源，取出SDS-PAGE胶，用考马斯亮蓝进行染色，室温染色1小时，后用脱色液进行洗脱显影。
SEQ ID NO:1
Figure PCTCN2019089955-appb-000001
SEQ ID NO:2
Figure PCTCN2019089955-appb-000002
实施例2铁蛋白免疫接种促进小鼠毛发再生
本发明采用C57BL/6小鼠(购自上海斯莱克实验动物有限责任公司)。C57BL/6小鼠在饲养过程中会有脱毛的现象发生，与人类斑秃的临床表现极为相似，因此，作为脱发的动物模型以验证铁蛋白对毛发再生的作用。
本实施例选取了12只有脱毛现象的小鼠，随机分成两组，分别标记为PBS对照组和铁蛋白疫苗治疗组，在第0周和第2周对两组小鼠各进行一次免疫接种。其中，PBS对照组小鼠肌肉注射100μL不含疫苗的溶液(PBS与铝佐剂体积比为1：1)；铁蛋白疫苗治疗组的小鼠则肌肉注射等体积含有10μg铁蛋白疫苗溶液(铁蛋白与铝佐剂体积比为1：1)。
结果显示，PBS对照组小鼠，从免疫第一针开始到第3周，毛发脱落的面积有扩大的趋势(图2a)。而铁蛋白疫苗治疗组小鼠，免疫第一针结束后6只小鼠中有4只出现毛发脱落面积减小的趋势，第二针免疫结束1周后，6 只小鼠脱落的毛发基本全部得到了恢复，显示了铁蛋白具有促进毛发生长和/或再生的效果(图2b)。
实施例3铁蛋白免疫后小鼠产生高滴度针对铁蛋白特异性的抗体应答
小鼠免疫接种铁蛋白疫苗第二针后。采集小鼠外周血于1.5mL管中，室温静置2小时，7000g离心15min，吸取血清至新的管中，4℃保存。
随后，采用ELISA方法检测小鼠血清中针对铁蛋白特异性IgG的应答，实验前一天，取96孔ELISA板，每孔加入200μL的铁蛋白(终浓度为0.5μg/mL)，4℃包被过夜。第二天，取出ELISA板并弃掉包被液，用0.05％的PBST缓冲液洗板3次(每孔220μL)；洗涤完毕后，每孔加入200μL ELISA封闭液，室温封闭2小时；然后用0.05％的PBST洗板3次(每孔220μL)。
取前述4℃保存的血清，从1:100起始，用稀释液进行2倍的倍比稀释。在上述封闭的ELISA板上设置空白孔(只加稀释液)为阴性对照，其余每孔加入100μL倍比稀释的血清，每个样品设置2个复孔，室温孵育3小时。
样品孵育结束后，用PBST洗板3次(同前)；加入HRP标记的山羊抗鼠的二抗(中杉金桥公司，货号zb-2305)，每孔加入100μL，室温孵育1.5小时；用0.05％的PBST洗板后，每孔加入100μL金银片OPD底物溶液(Sigma公司，货号P9187-50SET)，避光反应5分钟。
用50μL 2nM H 2SO 4终止反应，在酶标仪上读取OD值(范围492-630)；以最后一个稀释度OD492大于2倍的(negative mean+SD)值对应的血清稀释比的倒数作为抗体滴度。结果显示，相比于PBS组，铁蛋白疫苗治疗组产生了高滴度针对铁蛋白的IgG应答反应(图3)。
实施例4回输铁蛋白免疫的小鼠血清能快速促进毛发生长和/或再生
为验证铁蛋白诱导产生的抗体在治疗脱发中的作用，采集实施例2中PBS组和铁蛋白疫苗免疫组免疫结束后2周的小鼠外周血，分离血清。
准备8只有毛发脱落现象的C57BL/6小鼠，随机分成2组，标记为PBS对照组和铁蛋白治疗组。在第0天与第5天，分别进行眼眶静脉回输50μL小鼠的血清。PBS对照组小鼠回输的血清来自PBS接种的小鼠；而铁蛋白治疗组回输的血清则来自于铁蛋白免疫接种两周后的小鼠。
结果发现，PBS对照组回输血清后，小鼠脱毛现象无改善，且继续脱毛进展，且脱毛的部位随着时间有扩大的趋势(图4a)；而铁蛋白治疗组小鼠在第5天，4只小鼠脱毛现象有明显改善，斑秃区域有新毛长出。第10天， 4只小鼠脱毛的区域已经完全被毛，且停止回输血清后，生发效果持续至42天(图4b)。
实施例5铁蛋白免疫后的小鼠血清去除IgG后不能促进毛发生长和/ 或再生
收集实施例2中铁蛋白疫苗治疗组免疫结束后2周的小鼠外周血，分离血清，并去除铁蛋白免疫小鼠血清中IgG：取铁蛋白免疫后小鼠血清300μL，加100μL的PBS缓冲液后，再与200μL Protein G琼脂糖珠子(GE公司，货号28-9031-34)混合，在四维摇床上4℃进行孵育，孵育12小时左右；将孵育好的柱子100g离心30s；然后再用500μL PBS缓冲液洗涤遍，100g离心30s，收集收集管的液体。
准备5只脱毛小鼠。在第0天与第5天，分别进行眼眶静脉回输上述血清50μL(去除IgG)，并持续观察21天。
结果发现，去除IgG的铁蛋白免疫小鼠血清回输后，受体小鼠脱毛现象无改善且持续进展；脱毛的部位随着时间有扩大的趋势(图5)。
上述结果证实，去除了铁蛋白免疫血清中的IgG，不能有效治疗小鼠的脱发状况，说明铁蛋白诱导出的IgG在治疗小鼠脱毛中发挥了重要作用。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。

Claims (20)

  1. 一种铁蛋白在制备用于促进毛发生长和/或再生的药物中的用途。
  2. 根据权利要求1所述的用途，其中，所述铁蛋白包含选自以下的氨基酸序列：
    (i)SEQ ID NO:1所示的氨基酸序列；
    (ii)与SEQ ID NO:1所示的氨基酸序列相比，具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:1所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
  3. 一种铁蛋白，所述铁蛋白包含选自以下的氨基酸序列：
    (i)SEQ ID NO:1所示的氨基酸序列；
    (ii)与SEQ ID NO:1所示的氨基酸序列相比，具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:1所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
    优选地，所述铁蛋白的编码核苷酸序列如SEQ ID NO:2所示。
  4. 根据权利要求3所述的铁蛋白，其特征在于，所述铁蛋白包括经过修饰的铁蛋白、铁蛋白分子或其部分序列与其他分子融合表达形成的融合蛋白、或者铁蛋白来源的多肽片段；优选地，所述融合表达的方式包括与抗体的Fc序列融合、与不同信号肽的融合或与细胞因子融合。
  5. 一种促进毛发生长和/或再生的疫苗，所述疫苗包含如权利要求3或4所示的铁蛋白。
  6. 根据权利要求5所述的疫苗，其中，所述疫苗还包含佐剂；
    优选地，所述佐剂为铝盐佐剂、CpG佐剂、锰佐剂、MF59佐剂、QS21佐剂、TLR配体佐剂、NOD通路佐剂、干扰素佐剂、cGAS/STING通路佐剂、细胞因子佐剂、PD-1阻断抗体、CTLA4阻断抗体、TIGIT阻断抗体、αGalcer及其衍生物佐剂；
    优选地，所述细胞因子佐剂选自IL-12、GM-CSF、IL-18或IL-21。
  7. 根据权利要求5或6所述的疫苗，其特征在于，所述铁蛋白自身形成纳米颗粒或被脂质分子包裹形成纳米颗粒。
  8. 根据权利要求5-7中任一项所述的疫苗的制备方法，所述方法包括：
    1)制备铁蛋白并纯化；
    2)混合纯化后的铁蛋白和佐剂，即得。
  9. 据权利要求8所述的制备方法，其特征在于，所述步骤1)包括将铁蛋白基因表达于质粒载体、痘病毒载体、腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体、CMV载体、细胞载体或细菌载体。
  10. 一种促进毛发生长和/或再生的方法，所述方法包括给予有需要的受试者治疗有效量的如权利要求5-7中任一项所述的疫苗。
  11. 根据权利要求10所述的方法，其特征在于，所述疫苗的给药方式选自皮下注射、皮内注射、肌肉注射、口服、滴鼻喷雾给药和皮肤划痕给药。
  12. 根据权利要求10所述的方法，其特征在于：所述疫苗接种诱导针对铁蛋白的抗体应答，以发挥治疗及预防脱发的作用；
    优选地，所述疫苗为铁蛋白联合佐剂或不同载体形式的铁蛋白疫苗；
    优选地，所述疫苗为同种疫苗多次免疫或者不同疫苗序贯免疫或者不同疫苗混合免疫。
  13. 根据权利要求10所述的方法，其特征在于，所述疫苗接种于人或 宠物，以促进毛发生长和/或再生。
  14. 根据权利要求10所述的方法，其特征在于，将所述疫苗免疫接种后所诱导的血清用于毛发生长与再生；
    优选地，所述血清为提纯或未提纯的血清生长和/或再生。
  15. 根据权利要求10所述的方法，其特征在于，将所述疫苗接种者血清中的免疫球蛋白用于毛发生长与再生生长和/或再生。
  16. 根据权利要求10所述的方法，其特征在于，将所述疫苗免疫接种后所诱导的抗体用于毛发生长与再生，未接种铁蛋白疫苗者可通过输入提纯的抗体而促进毛发生长和/或再生。
  17. 根据权利要求10所述的方法，其特征在于，将铁蛋白疫苗接种者血清、或提纯的免疫球蛋白、或纯化的抗体涂于患处以促进毛发生长和/或再生；或将提纯的免疫球蛋白、抗体与其他皮肤用药混合涂于患处以促进毛发生长和/或再生。
  18. 根据权利要求10所述的方法，其特征在于，所述毛发再生包括促进毛发过度脱落者的毛发再生；
    优选地，所述过度脱落的原因包括雄激素性脱发、神经性脱发、内分泌脱发、营养性脱发、物理性脱发、化学性脱发、感染性脱发、症状性脱发、先天性脱发。
  19. 根据权利要求10所述的方法，其特征在于，所述爆发生长包括促进毛发异常者的毛发生长；
    优选地，所述毛发异常包括白发、黄发、毛发稀疏。
  20. 根据权利要求10所述的方法，其特征在于，所述疫苗单独给药或与其他治疗技术联合应用。
PCT/CN2019/089955 2019-06-04 2019-06-04 促进毛发生长和/或再生的疫苗、其制备方法及其应用 WO2020243895A1 (zh)

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