WO2023088351A1 - 神经生长因子在制备用于治疗或改善生殖系统疾病的药物中的用途 - Google Patents

神经生长因子在制备用于治疗或改善生殖系统疾病的药物中的用途 Download PDF

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WO2023088351A1
WO2023088351A1 PCT/CN2022/132495 CN2022132495W WO2023088351A1 WO 2023088351 A1 WO2023088351 A1 WO 2023088351A1 CN 2022132495 W CN2022132495 W CN 2022132495W WO 2023088351 A1 WO2023088351 A1 WO 2023088351A1
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growth factor
nerve growth
ovarian
follicles
use according
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French (fr)
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李加斌
朱庆
李佳腊
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舒泰神（北京）生物制药股份有限公司
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Priority to CN202280008244.7A priority Critical patent/CN116916946A/zh
Publication of WO2023088351A1 publication Critical patent/WO2023088351A1/zh

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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
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to the use of nerve growth factor in the preparation of medicines for treating or improving reproductive system diseases.
  • Nerve growth factor is the first discovered member of the neurotrophic factor family. It was first discovered in mouse sarcoma cells by Italian scientist Levi-Montlcini in 1953. Nerve growth factor is a kind of nerve growth regulator, which has dual biological functions of providing nutrition for neurons and promoting neurite growth, and plays an important role in the development, differentiation, growth, regeneration and functional expression of central and peripheral neurons. Regulatory effect. Nerve growth factor contains three subunits ⁇ , ⁇ and ⁇ . The ⁇ subunit is an active domain composed of two single strands joined by non-covalent bonds.
  • nerve growth factor has the functions of promoting neuron development, axon growth, transmitter synthesis, and inhibiting nerve cell apoptosis; while for other systems such as cardiovascular, immune, and reproductive systems, nerve growth factor is mainly manifested in regulating the immune system. function, inhibit the mitosis of some tumor cells, and promote wound healing.
  • nerve growth factor and its receptors also widely exist in testicular tissue; nerve growth factor mRNA transcripts and proteins are expressed in fetal and adult human ovaries.
  • nerve growth factor can be used to treat reproductive system diseases.
  • Reproductive system diseases generally include the following categories: (1) reproductive system tumors, such as testicular cancer, prostate cancer, uterine fibroids, ovarian tumors, endometrial cancer, fallopian tube cancer, ovarian cancer, uterine cervical cancer, etc.; Systemic inflammation, such as testicular epididymitis, seminal vesiculitis, prostatitis, vaginitis, uterine body inflammation, pelvic inflammatory disease, adnexitis, endometritis, vulvitis, etc.; (3) reproductive tract tuberculosis, such as prostate tuberculosis, seminal vesicle tuberculosis, Testicular tuberculosis, epididymis tuberculosis, etc.; (4) Reproductive tract injury, such as testicular contusion, penile fracture, urethral rupture, etc.; (5) Reproductive system abnormalities, such as hidden penis, webbed penis, cryptorchidism, etc.; (6) Sexual
  • Premature ovarian insufficiency refers to natural amenorrhea before the age of 40 due to ovarian failure. It is often accompanied by a decrease in estrogen levels, an increase in follicle-generating hormone levels, and an increase in gonadotropin levels, and its etiology and mechanism are more complicated. According to statistics, the incidence rate in the population is 1% to 3%, accounting for 2% to 10% in amenorrhea patients, and the incidence rate is on the rise in recent years. Premature ovarian failure leads to loss of fertility and low estrogen status, which has become a non-negligible factor affecting women's reproductive health and social stability.
  • Oligospermia and asthenozoospermia is a relatively common disease in men. Oligosasthenozoospermia mainly manifests as a decrease in sperm count or sperm motility, which will seriously affect the patient's physical and mental health. At the same time, it will also be accompanied by endocrine, nervous system and other diseases, which will further lead to The patient's quality of life is reduced.
  • the clinical treatment of oligoasthenospermia is mainly through drugs and surgery: 1. Drug treatment: it is determined according to the specific situation of the patient.
  • Gonadotropin treatment but clinically more common is oligoasthenozoospermia for which no cause can be found, and drugs such as carnitine are mainly used; 2.
  • Surgical treatment if the patient’s oligoasthenospermia is due to the spermatic vein If it is caused by varicocele, surgical treatment of varicocele is required; if it is caused by cryptorchidism, surgical treatment of cryptorchidism is required.
  • the present invention provides the use of nerve growth factor or nerve growth factor fusion protein in the preparation of medicines for treating reproductive system diseases or improving reproductive system diseases.
  • the reproductive system diseases include but are not limited to decreased ovarian reserve, premature ovarian failure (POF), ovarian hyperstimulation syndrome, ovarian remnant syndrome, ovarian follicle dysplasia, and spermatogenesis disorders; preferably, the The spermatogenic disorders include but are not limited to: oligozoospermia or oligospermia, asthenospermia, azoospermia, teratozoospermia, oligoasthenospermia (OAT syndrome).
  • the improvement of reproductive system diseases includes, but is not limited to, one or more of the following improvements:
  • the follicles are primordial follicles, primary follicles and / or secondary follicles.
  • the present invention provides the use of nerve growth factor or nerve growth factor fusion protein in the preparation of medicines for improving diseases or symptoms, the diseases or symptoms are diseases or symptoms related to reproductive system diseases, or caused by A condition or symptom caused by a disorder of the reproductive system.
  • the diseases of the reproductive system include but are not limited to: premature ovarian failure or oligoasthenozoospermia, decreased ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian remnant syndrome, ovarian follicle dysplasia, spermatogenesis disorder ;
  • the spermatogenic disorders include, but are not limited to: asthenospermia, oligospermia, azoospermia, teratospermia, oligoasthenospermia.
  • the disorders or symptoms include, but are not limited to: decreased proliferation rate and/or decreased estrogen secretion of ovarian granulosa cells, decreased follicle number and/or decreased function, atrophy of testicular seminiferous tubules, seminiferous tubules Sperm disorders and/or epididymal duct cell fragments, decreased sperm count and/or decreased motility; preferably, the follicles are primordial follicles, primary follicles and/or secondary follicles.
  • the present invention provides nerve growth factor or nerve growth factor fusion protein for promoting the proliferation of ovarian granulosa cells and/or estrogen secretion in patients; preventing/reversing the decrease in the number and/or function of follicles, or Improve follicle number and/or function; improve testicular seminiferous tubule atrophy, seminiferous tubule spermatogenic disorder and/or epididymal duct cell fragments; improve sperm count and/or decrease in motility, or increase sperm count and/or motility, etc.
  • the follicles are primordial follicles, primary follicles and/or secondary follicles.
  • the patient is a patient suffering from a reproductive system disease or a related disease caused by a reproductive system disease, including but not limited to suffering from reduced ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian remnant syndrome, Patients with ovarian follicular dysplasia and spermatogenesis disorders; preferably, the spermatogenesis disorders include but not limited to: asthenozoospermia, oligospermia, azoospermia, teratozoospermia, oligoasthenospermia.
  • the nerve growth factor described in the present invention includes but not limited to wild-type nerve growth factor (wild-type NGF), recombinant nerve growth factor (recombinant NGF), and nerve growth factor mutant (NGF mutant).
  • wild-type nerve growth factor includes wild-type nerve growth factor isolated from various sources including, but not limited to, livestock (e.g., cattle, sheep, goats, cats, dogs, donkeys, and horses) , primates (eg, humans and non-human primates such as monkeys or chimpanzees), rabbits, and rodents (eg, mice, rats, gerbils, and hamsters).
  • livestock e.g., cattle, sheep, goats, cats, dogs, donkeys, and horses
  • primates eg, humans and non-human primates such as monkeys or chimpanzees
  • rabbits eg, mice, rats, gerbils, and hamsters.
  • the nerve growth factor is mouse wild-type nerve growth factor or human wild-type nerve growth factor.
  • the mouse-derived wild-type nerve growth factor or human-derived wild-type nerve growth factor refers to natural nerve growth factor derived from mice or humans, including but not limited to intact nerve growth factor, C-terminal deletion Nerve growth factor with 1 or 2 amino acids, nerve growth factor with 8 amino acids deleted from the N-terminus, nerve growth factor with 1 amino acid deleted from the C-terminus and 8 amino acids deleted from the N-terminus, and nerve growth factor glycosylated products and/or Oxidation product of nerve growth factor.
  • the human wild-type nerve growth factor comprises the sequence shown in SEQ ID NO: 1 or 2.
  • the recombinant nerve growth factor of the present invention can be a nerve growth factor prepared by recombinant expression or synthetic method.
  • the recombinant nerve growth factor is mouse recombinant nerve growth factor or human recombinant nerve growth factor.
  • the nerve growth factor of the present invention may be a mutant of nerve growth factor.
  • the mutants described in the present invention are nerve growth factor mutants with most or all of the biological activities of wild-type nerve growth factor.
  • Nerve growth factor mutants include mutations at one or more amino acid sites of nerve growth factor (eg, mature ⁇ -NGF).
  • the mutant is a variant of wild-type nerve growth factor after deletion, substitution and/or insertion of one or more amino acid residues.
  • the mutant nerve growth factor has reduced side effects (eg, pain), or is painless, compared to wild-type nerve growth factor.
  • the position of the nerve growth factor mutant relative to the human wild-type nerve growth factor sequence includes one or more amino acid site mutations as follows: F12E, K32G, K32L, K32Y, R59L, R59A, D65A, D65G, K74L, K88F, K88L, K88E, K88G, Q96E, R114V, R114F, R114G, R114L, and F101A.
  • the nerve growth factor mutant comprises any amino acid sequence as shown in SEQ ID NO: 3-40.
  • the nerve growth factor fusion protein (NGF fusion protein) in the present invention refers to a fusion protein including nerve growth factor and other proteins or polypeptides.
  • the other proteins or polypeptides include, but are not limited to, the Fc portion, human serum albumin, and the like.
  • a nerve growth factor fusion protein of the invention comprises, from N-terminus to C-terminus, nerve growth factor, an optional peptide linker, and an Fc portion.
  • the optional peptide linker can be of any length. In some embodiments, the linker is no longer than necessary to prevent domain interaction and/or optimize biological function and/or stability. In some embodiments, the peptide linker is the hinge region of human IgGl, IgG2, IgG3 or IgG4. In some embodiments, the peptide linker is a mutated human IgGl, IgG2, IgG3 or IgG4 hinge region. In some embodiments, the peptide linker comprises any amino acid sequence of SEQ ID NOs: 41-45.
  • the peptide linker comprises the amino acid sequence (GGGGS) n (SEQ ID NO: 43), said n is any integer in 1, 2, 3, 4, 5 or 6, preferably n is 2 to Any integer in 6, more preferably n is integer 3 or 4. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 41 or 42.
  • the Fc part may be from human, including but not limited to any one of IgA, IgD, IgE, IgG and IgM and their subclasses.
  • the Fc portion may contain one or more mutations or glycosylation modifications relative to the amino acid positions of the natural Fc structure, and the mutations include insertions, deletions and/or substitutions, etc.
  • the Fc part is from IgG1Fc, IgG4Fc or contains one or more mutations or undergoes glycosylation modification relative to natural IgG1Fc, IgG4Fc amino acid positions.
  • the Fc portion comprises the amino acid sequence shown in SEQ ID NO: 46 or 47.
  • the nerve growth factor fusion protein comprises the amino acid sequence shown in SEQ ID NO: 48 or 49.
  • the medicine of the present invention contains a therapeutically effective dose of nerve growth factor or nerve growth factor fusion protein as the main active ingredient and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are nontoxic to the subject at the dosages and concentrations employed and are of a type well known in the art and include, but are not limited to, buffers, antioxidants; ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives Agents, isotonic agents (such as sodium chloride), stabilizers, metal complexes (for example, zinc-protein complexes); chelating agents, such as EDTA and/or nonionic surfactants, etc.
  • the drug of the present invention can be administered by a variety of methods, including but not limited to: oral, subcutaneous, intravenous, intracerebral, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, etc. .
  • the dosage forms of the medicine include but are not limited to injections, capsules, tablets or powder injections and the like.
  • Figure 1 shows the nerve growth factor ( The proliferation rate of human ovarian granulosa cells (KGN) treated with mouse NGF or mNGF118) or nerve growth factor fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM).
  • KGN human ovarian granulosa cells
  • mNGF118 nerve growth factor fusion protein
  • Figure 2 shows the nerve growth factor ( Estrogen concentration secreted by human ovarian granulosa cells (KGN) under treatment with mouse NGF or mNGF118) or nerve growth factor fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM).
  • KGN ovarian granulosa cells
  • mNGF118 nerve growth factor fusion protein
  • Figure 3 shows the nerve growth factor ( Mouse NGF or mNGF118) or nerve growth factor fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM) treatment, the number of follicles at all levels of rat premature ovarian failure animal disease model.
  • nerve growth factor Mouse NGF or mNGF118
  • nerve growth factor fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM) treatment
  • reagents used in the following examples are prepared by conventional methods or obtained from commercial sources; the experimental methods used, if not specified, are conventional methods; the materials, instruments, etc. used, Unless otherwise specified, all were obtained from commercial sources.
  • VCD 4-vinylcyclohexene diepoxide
  • Mouse NGF from Shutaishen (Beijing) Biopharmaceutical Co., Ltd.
  • compositions and amino acid sequences of mNGF118, NGF-Fc fusion protein 2-118-L3G4-BM and NGF-Fc fusion protein 2-118-L3Fc10-M3-5 are shown in Table 1.
  • composition and sequence of table 1 exemplary nerve growth factor and its fusion protein
  • Example 1 The therapeutic effect of NGF and NGF-Fc fusion protein on premature ovarian failure
  • Premature ovarian failure refers to spontaneous amenorrhea before the age of 40 due to ovarian failure. It is often accompanied by a decrease in estrogen levels, an increase in follicle-generating hormone levels, and an increase in gonadotropin levels, and its etiology and mechanism are more complicated.
  • NGF and NGF-Fc fusion protein were studied by in vitro human ovarian granulosa tumor cell line (KGN) proliferation test and KGN estrogen secretion test and POF model rats.
  • KGN proliferation assay 100 ul of KGN suspension (1 ⁇ 10 4 cells/mL) was added to each well of a 96-well plate the day before the assay. Before the experiment, replace the serum-free DMEM medium. After replacing the medium, NGF was added to the wells of the experimental group ( Mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5), so that the final concentration in the culture medium was 10 ⁇ g/mL, and the negative control group was replaced by culture No processing is done after the base.
  • the experimental group Mouse NGF or mNGF118
  • NGF-Fc fusion protein 2-118-L3G4-BM or 2-118-L3Fc10-M3-5
  • CCK-8 Dojin Chemical Research Institute, #CK04
  • NGF Mouse NGF or mNGF118
  • NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) significantly promoted KGN proliferation (p ⁇ 0.05).
  • KGN cells were seeded in a 24-well plate at 1 ⁇ 10 5 cells/well (the confluence of the cells was about 80%), and the serum-free medium was replaced. After replacing the medium, the wells of the experimental group were added with NGF ( mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5), so that the final concentration in the culture medium was 10 ⁇ g/ml. The negative control group did not receive any treatment after replacing the culture medium.
  • NGF mouse NGF or mNGF118
  • NGF-Fc fusion protein 2-118-L3G4-BM or 2-118-L3Fc10-M3-5
  • NGF Mouse NGF or mNGF118
  • NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) both significantly promoted the secretion of estrogen in KGN (p ⁇ 0.05).
  • VCD can selectively destroy primordial and primary follicles in the ovaries of female mice, but has no effect on secondary follicles and antral follicles, resulting in POF in female mice.
  • NGF and NGF-Fc fusion protein were injected intraperitoneally with VCD for two consecutive weeks to construct a POF rat model (for example, see Notice FS et al., Effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague-Dawley rats: ovarian, clinical, and pathologic outcomes [J].
  • Comp Med, 2009, 59(1): 46-59. Effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague-Dawley rats: ovarian, clinical, and pathologic outcomes [J]. Comp Med, 2009, 59(1): 46-59.).
  • NGF was administered at the beginning of model establishment, which was recorded as day 1.
  • Administration by subcutaneous injection NGF ( The injection dose of mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) experimental group was 10 ⁇ g/kg bw, and the same volume of sterile saline was used as negative control.
  • NGF Mouse NGF, mNGF118 or sterile normal saline were administered every other day, and NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) was administered once a week.
  • NGF Both mouse NGF or mNGF118
  • NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) significantly increased the number of primary follicles (p ⁇ 0.05), indicating that they played a role in improving POF-induced It has an excellent effect on reducing the number of primary follicles.
  • NGF Rat models of POF treated with murine NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) also exhibited higher numbers of primordial and secondary follicles.
  • Example 2 The therapeutic effect of NGF and NGF-Fc fusion protein on oligoasthenospermia
  • Oligospermozoospermia is mainly manifested by decreased sperm count and/or decreased sperm motility.
  • Sperm is formed by a series of divisions and differentiation of germ cells with proliferative ability in the seminiferous tubules of the testis. Heat stress can affect the division, differentiation and formation of spermatozoa in the proliferating cells.
  • the therapeutic effect of NGF and NGF-Fc fusion protein on oligoasthenospermia was studied in a mouse spermatogenesis disorder model.
  • mice purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.
  • 20 ⁇ g/kg bw/time of NGF was injected respectively ( mouse NGF or mNGF118) or 60 ⁇ g/kg bw/time NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5).
  • the normal control group or the spermatogenesis disorder model control group were injected with an equal volume of 0.9% sodium chloride injection.
  • the day of the first administration was the first day.
  • mice were anesthetized 4 hours after the first administration, and after the mouse testis descended to the scrotum, the spermatogenesis disorder model control group, NGF experimental group and NGF
  • the lower abdomen (hind limbs, tail and scrotum) of mice in the -Fc fusion protein experimental group was immersed in a 42°C constant temperature water bath for 30 minutes; the lower abdomen (hind limbs, tail and scrotum) of mice in the normal control group was immersed in a 25°C constant temperature water bath for 30 minutes.
  • Mouse NGF or mNGF118 was administered every other day, and 2-118-L3G4-BM or 2-118-L3Fc10-M3-5 was administered twice a week.
  • the normal control group or the model control group were injected with an equal volume of 0.9% sodium chloride injection, once every other day. A total of 5 weeks of administration.
  • the mice were euthanized, and the left epididymis tail was weighed and placed in 37°C preheated M199 culture medium, cut into pieces and placed in an incubator for 5 minutes at 37°C, and the sperm suspension was drawn. Dilute with M199 culture medium at 1:6, mix well, take the diluted solution, and use TOX IVOS sperm analyzer to detect sperm count and sperm motility. The recorded data were analyzed using Student's t-test.
  • NGF The sperm count and motility of the mice in the experimental groups of mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3Fc10-M3-5 and 2-118-L3G4-BM) were significantly increased.
  • NGF or NGF-Fc fusion protein can effectively improve the decrease of sperm count and sperm motility in spermatogenesis disorders (such as asthenospermia, oligospermia and oligoasthenospermia).
  • NGF Mouse NGF or mNGF118
  • NGF-Fc fusion proteins (2-118-L3Fc10-M3-5 and 2-118-L3G4-BM) have effects on testicular seminiferous tubule atrophy, seminiferous tubule dysfunction and The symptoms of epididymis duct cell fragments showed obvious therapeutic effect.

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Abstract

提供了神经生长因子在制备用于治疗生殖系统疾病或改善生殖系统疾病的药物中的用途。

Description

神经生长因子在制备用于治疗或改善生殖系统疾病的药物中的用途 技术领域
本发明属于生物医药技术领域，具体涉及神经生长因子在制备用于治疗或改善生殖系统疾病的药物中的用途。
背景技术
神经生长因子(NGF)是神经营养因子家族中首个被发现的成员，由意大利科学家Levi-Montlcini于1953年在小鼠肉瘤细胞中首次发现。神经生长因子是一种神经生长调节剂，具有为神经元提供营养和促进神经突生长的双重生物学功能，在中枢和外周神经元的发育、分化、生长、再生和功能表达过程中发挥重要的调节作用。神经生长因子包含α、β和γ三个亚基。β亚基是一个活性区域，由两条单链通过非共价键结合而成。
对于神经系统，神经生长因子具有促进神经元发育、轴突生长、递质合成及抑制神经细胞凋亡等功能；而对于心血管、免疫、生殖等其他系统，神经生长因子主要表现在调节免疫系统功能、抑制部分肿瘤细胞的有丝分裂以及促进创口愈合等。有研究表明，除了在神经系统分布较多以外，神经生长因子及其受体也广泛存在于睾丸组织中；神经生长因子mRNA转录物和蛋白质在胎儿和成年人类卵巢中都有表达。然而，对于神经生长因子是否可用于治疗生殖系统疾病并未见报道。
生殖系统疾病一般包含以下几类：(1)生殖系统肿瘤，如睾丸癌、前列腺癌、子宫肌瘤、卵巢肿瘤、子宫内膜癌、输卵管癌、卵巢癌、子宫宫颈癌等；(2)生殖系统炎症，如睾丸附睾炎、精囊炎、前列腺炎、阴道炎、宫体炎、盆腔炎、附件炎、子宫内膜炎、外阴炎等；(3)生殖道结核，如前列腺结核、精囊结核、睾丸结核、附睾结核等；(4)生殖道损伤，如睾丸挫伤、阴茎折断伤、尿道断裂等；(5)生殖系统畸形，如隐匿阴茎、蹼状阴茎、隐睾等；(6)性功能障碍性疾病，如男性勃起功能障碍、早泄、性欲减退、不射精、延迟射精等；(7)生殖系统疾病造成的相关性疾病，如精索静脉曲张、少弱精子症、先天性输精管梗阻、输精管缺、输卵管阻塞、免疫性不孕、卵巢早衰。
卵巢早衰(premature ovarian insufficiency，POI)指卵巢功能衰竭所致的40岁前自然闭经。常伴随雌激素水平下降、卵泡生成素水平升高及促性腺激素水平升高，其病因和机 理较为复杂。据统计人群中发病率1％～3％，在闭经患者中占2％～10％，近年来发病率呈上升趋势。卵巢早衰导致生育力丧失及低雌激素状态，成为影响女性生殖健康和社会稳定不可忽视的因素。
少弱精子症是男性一种较为常见的疾病，少弱精子症主要表现为精子数量减少或精子活力下降，会严重影响患者的身心健康，同时还会伴发内分泌、神经系统等疾病，进一步导致患者的生活质量下降。临床上少弱精子症的治疗主要是通过药物和手术两种方式：1、药物治疗：根据患者的具体情况来决定，如果患者是由于促性腺激素水平低下导致的少弱精子症，则可以通过促性腺激素的治疗；但临床上更常见的是找不到原因的少弱精子症，主要使用的是肉碱一类药物；2、手术治疗：如果病人的少弱精子症是由于精索静脉曲张导致的，要进行精索静脉曲张的手术治疗；如果是隐睾原因导致的，要进行隐睾的手术治疗。
本领域迫切需要一种能有效治疗或改善卵巢早衰和少弱精子症等生殖系统疾病的药物和方法。
发明内容
第一方面，本发明提供了神经生长因子或神经生长因子融合蛋白在制备用于治疗生殖系统疾病或改善生殖系统疾病的药物中的用途。
在一些实施方式中，所述生殖系统疾病包括但不限于卵巢储备减少、卵巢早衰(POF)、卵巢过度刺激综合征、卵巢残余综合征、卵巢卵泡发育不良、生精障碍；优选地，所述生精障碍为包括但不限于：精子症(oligozoospermia或oligospermia)、弱精子症、无精子症、畸形精子症、少弱畸精子症(OAT综合征)。
在一些实施方式中，所述改善生殖系统疾病包括但不限于如下的一种或多种改善：
(1)促进卵巢颗粒细胞的增殖和/或雌激素分泌；(2)预防/逆转卵泡数量和/或功能的降低，或提高卵泡数量和/或功能；(3)改善睾丸生精小管萎缩、生精小管生精障碍和/或附睾管细胞碎片；(4)改善精子数量减少和/或活力的降低，或提高精子数量和/或活力；优选地，所述卵泡为原始卵泡、初级卵泡和/或次级卵泡。
第二方面，本发明提供了神经生长因子或神经生长因子融合蛋白在制备用于改善病症或症状的药物中的用途，所述病症或症状是与生殖系统疾病有关的病症或症状，或是由生殖系统疾病导致的病症或症状。
在一些实施方式中，所述生殖系统疾病包括但不限于：卵巢早衰或少弱精子症，卵巢储备减少、卵巢早衰、卵巢过度刺激综合征、卵巢残余综合征、卵巢卵泡发育不良、生精障 碍；优选地，所述生精障碍包括但不限于：弱精子症、少精子症、无精子症、畸形精子症、少弱精子症。
在一些实施方式中，所述病症或症状包括但不限于：卵巢颗粒细胞的增殖率降低和/或雌激素分泌降低，卵泡数量减少和/或功能降低，睾丸生精小管萎缩、生精小管生精障碍和/或附睾管细胞碎片，精子数量减少和/或活力的降低；优选地，所述卵泡为原始卵泡、初级卵泡和/或次级卵泡。
第三方面，本发明提供了神经生长因子或神经生长因子融合蛋白在制备用于在患者中促进卵巢颗粒细胞的增殖和/或雌激素分泌；预防/逆转卵泡数量和/或功能的降低，或提高卵泡数量和/或功能；改善睾丸生精小管萎缩、生精小管生精障碍和/或附睾管细胞碎片；改善精子数量减少和/或活力的降低，或增加精子数量和/或活力等的药物中的用途；优选地，所述卵泡为原始卵泡、初级卵泡和/或次级卵泡。
在一些实施方式中，所述患者为患有生殖系统疾病或由生殖系统疾病导致的相关疾病的患者，包括但不限于患有卵巢储备减少、卵巢早衰、卵巢过度刺激综合征、卵巢残余综合征、卵巢卵泡发育不良、生精障碍的患者；优选地，所述生精障碍包括但不限于：弱精子症、少精子症、无精子症、畸形精子症、少弱精子症。
本发明所述的神经生长因子包括但不限于野生型神经生长因子(野生型NGF)、重组神经生长因子(重组NGF)、神经生长因子突变体(NGF突变体)。
在一些实施方式中，野生型神经生长因子包括从各种来源分离出的野生型神经生长因子，所述来源包括但不限于家畜(例如，牛、绵羊、山羊、猫、狗、驴和马)、灵长类动物(例如，人类和非人类灵长类动物，如猴子或黑猩猩)、兔子和啮齿动物(例如，小鼠、大鼠、沙鼠和仓鼠)。
在一些实施方式中，神经生长因子为鼠源野生型神经生长因子或人源野生型神经生长因子。
在一些实施方式中，所述鼠源野生型神经生长因子或人源野生型神经生长因子是指来源于小鼠或人类的天然神经生长因子，包括但不限于完整型神经生长因子、C端缺失1或2个氨基酸的神经生长因子、N端缺失8个氨基酸的神经生长因子、C端缺失1个氨基酸且N端缺失8个氨基酸的神经生长因子，以及神经生长因子糖基化产物和/或神经生长因子氧化产物。
在一些实施方式中，所述人源野生型神经生长因子包含如SEQ ID NO:1或2所示的序列。
本发明所述的重组神经生长因子可以通过重组表达或合成方法制备获得的神经生长因子。在一些实施方式中，重组神经生长因子为鼠源重组神经生长因子或人源重组神经生长因子。
本发明所述的神经生长因子可以是神经生长因子突变体。本发明所述的突变体是具有野生型神经生长因子的大部分或全部生物活性的神经生长因子突变体。神经生长因子突变体包括在神经生长因子(例如，成熟β-NGF)的一个或多个氨基酸位点的突变。在一些实施方式中，所述突变体是野生型神经生长因子经缺失、取代和/或插入一个或多个氨基酸残基后得到的变体。在一些实施方式中，与野生型神经生长因子相比，所述神经生长因子突变体的副作用(例如，疼痛)降低，或为无痛的。
在一些实施方式中，所述神经生长因子突变体相对于人源野生型神经生长因子序列(SEQ ID NO:1或2)的位置，包括如下一个或多个氨基酸位点突变：F12E、K32G、K32L、K32Y、R59L、R59A、D65A、D65G、K74L、K88F、K88L、K88E、K88G、Q96E、R114V、R114F、R114G、R114L和F101A。
在一些实施方式中，所述神经生长因子突变体包含如SEQ ID NO:3-40所示的任一氨基酸序列。
本发明所述的神经生长因子融合蛋白(NGF融合蛋白)是指包括神经生长因子和其他蛋白或多肽的融合蛋白。
在一些实施方式中，所述其他蛋白或多肽包括但不限于Fc部分，人血清白蛋白等。
在一些实施方式中，本发明的神经生长因子融合蛋白从N端到C端包含神经生长因子、可选的肽接头和Fc部分。
在一些实施方式中，可选的肽接头可以是任意长度。在一些实施方式中，接头长度不超过防止结构域相互作用和/或优化生物功能和/或稳定性所必需的长度。在一些实施方式中，肽接头为人类IgG1、IgG2、IgG3或IgG4的铰链区。在一些实施方式中，肽接头为突变的人类IgG1、IgG2、IgG3或IgG4铰链区。在一些实施方式中，所述肽接头包含SEQ ID NO:41-45中的任一氨基酸序列。在一些实施方式中，肽接头包含氨基酸序列(GGGGS)n(SEQ ID NO:43)，所述n为1、2、3、4、5或6中的任一整数，优选地n为2到6中的任一整数，更优选地n为整数3或4。在一些实施方式中，肽接头包含氨基酸序列SEQ ID NO:41或42。
所述Fc部分可来自于人，包括但不限于IgA、IgD、IgE、IgG和IgM及其亚类中的任何一种。Fc部分可以相对于天然Fc结构氨基酸位点包含一个或多个突变或进行糖基化修 饰，所述突变包括插入、缺失和/或取代等。在一些实施方式中，所述Fc部分来自IgG1Fc、IgG4Fc或相对于天然IgG1Fc、IgG4Fc氨基酸位点包含一个或多个突变或进行糖基化修饰。在一些实施方式中，所述Fc部分包含SEQ ID NO:46或47所示的氨基酸序列。
在一些实施方式中，所述神经生长因子融合蛋白包含如SEQ ID NO:48或49中所示的基酸序列。
本发明所述药物中包含治疗有效量的神经生长因子或神经生长因子融合蛋白作为主要活性成分以及药学上可接受的载体。
药学上可接受的载体在所用的剂量和浓度下对受试者无毒，其类型是本领域所熟知的，包括但不限于缓冲液、抗氧化剂；抗坏血酸、蛋氨酸、维生素E、焦亚硫酸钠；防腐剂、等渗剂(如氯化钠)、稳定剂、金属络合物(例如，锌-蛋白质络合物)；螯合剂，如EDTA和/或非离子表面活性剂等。
本发明的药物可采用多种方法进行施用，包括但不限于：口服、皮下、静脉、脑内、鼻内、透过皮肤、腹膜内、肌内、肺内、阴道给药、直肠给药等。
所述药物的剂型包括但不限于注射剂、胶囊、片剂或粉针剂等。
附图说明
图1所示为神经生长因子(
Figure PCTCN2022132495-appb-000001
鼠NGF或mNGF118)或神经生长因子融合蛋白(2-118-L3Fc10-M3-5或2-118-L3G4-BM)处理下，人卵巢颗粒细胞(KGN)的增殖率。
图2所示为神经生长因子(
Figure PCTCN2022132495-appb-000002
鼠NGF或mNGF118)或神经生长因子融合蛋白(2-118-L3Fc10-M3-5或2-118-L3G4-BM)处理下，人卵巢颗粒细胞(KGN)分泌的雌激素浓度。
图3所示为神经生长因子(
Figure PCTCN2022132495-appb-000003
鼠NGF或mNGF118)或神经生长因子融合蛋白(2-118-L3Fc10-M3-5或2-118-L3G4-BM)处理下，大鼠卵巢早衰动物疾病模型各级卵泡的数量。
具体实施方式
为使本发明要解决的技术问题、采用的技术方案和优点更加清楚，下面将结合附图及具体实施例对本发明进行详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
下述实施例中所使用的试剂，如无特别说明，均采用常规方法配制或者由商业途径得 到；所使用的实验方法，如无特别说明，均为常规方法；所使用的材料、仪器等，如无特别说明，均由商业途径得到。
4-乙烯基环己烯二环氧乙烷(4-vinylcyclohexene diepoxide，VCD)，货号/规格：94956-100ml液体里含108g VCD，厂家：sigma公司
本发明实施例所用的神经生长因子来源如下：
Figure PCTCN2022132495-appb-000004
小鼠NGF：来自舒泰神(北京)生物制药股份有限公司
mNGF118、NGF-Fc融合蛋白2-118-L3G4-BM和NGF-Fc融合蛋白2-118-L3Fc10-M3-5的组成和氨基酸序列如表1所示。
表1示例性神经生长因子及其融合蛋白的组成和序列
Figure PCTCN2022132495-appb-000005
实施例1：NGF和NGF-Fc融合蛋白对卵巢早衰的治疗效果
卵巢早衰(POF)是指卵巢功能衰竭所致的40岁前自然闭经。常伴随雌激素水平下降、卵泡生成素水平升高及促性腺激素水平升高，其病因和机理较为复杂。本实施例通过体外人卵巢颗粒样肿瘤细胞系(KGN)增殖试验和KGN雌激素分泌试验以及POF模型大鼠研究了NGF和NGF-Fc融合蛋白治疗POF的治疗效果。
对于KGN增殖试验，在试验前一天向96孔板中每孔加入100ul的KGN悬液(1×10 4细胞/mL)。试验前，更换不含血清的DMEM培养基。更换培养基后，实验组孔中分别加入NGF(
Figure PCTCN2022132495-appb-000006
鼠NGF或mNGF118)或NGF-Fc融合蛋白(2-118-L3G4-BM或2-118-L3Fc10-M3-5)，使其在培养基中的终浓度为10μg/mL，阴性对照组更换培养基后不做任何处理。每组4个复孔，48h后，每孔加入10uL CCK-8(日本同仁化学研究所，#CK04)以测定活细胞数量。孵育1h后，测量450nm处的吸光值，记录的数据采用Student t检验进行分析，使用GraphPad Prism 8.0.1绘制柱状图。
如图1所示，与PBS阴性对照组相比，NGF(
Figure PCTCN2022132495-appb-000007
鼠NGF或mNGF118)及NGF-Fc融合蛋白(2-118-L3G4-BM或2-118-L3Fc10-M3-5)均明显促进了KGN增殖(p<0.05)。
对于KGN雌激素分泌试验，在24孔板中接种KGN细胞，1×10 5细胞/孔(细胞的汇合 度约为80％)，更换无血清培养基。更换培养基后，实验组孔分别加入NGF(
Figure PCTCN2022132495-appb-000008
鼠NGF或mNGF118)或NGF-Fc融合蛋白(2-118-L3G4-BM或2-118-L3Fc10-M3-5)，使其在培养基中的终浓度为10μg/ml。阴性对照组更换培养基后不做任何处理。每组4个复孔，孵育18h后，洗两遍，使用2.2×10 -8M的睾酮(北京索莱宝科技有限公司，#IT0110)和0.01IU/ml的绵羊卵泡刺激素(美国NHPP公司，Ovine FSH)处理24h后，取上清稀释1.6倍，使用R&D Systems生产的雌激素测定试剂盒(KGE014)，测量450nm处的吸光值，并计算分泌的雌激素浓度，记录的数据采用Student t检验进行分析，使用GraphPad Prism 8.0.1绘制柱状图。
如图2所示，与阴性对照组相比，NGF(
Figure PCTCN2022132495-appb-000009
鼠NGF或mNGF118)及NGF-Fc融合蛋白(2-118-L3G4-BM或2-118-L3Fc10-M3-5)均明显促进了KGN雌激素分泌(p<0.05)。
去氧乙烯基环己烯(VCD)可选择性破坏雌鼠卵巢内的原始卵泡和初级卵泡，而对次级卵泡和窦卵泡无影响，导致雌鼠发生POF。为进一步研究NGF和NGF-Fc融合蛋白对POF的体内治疗效果，对SD大鼠连续两周腹腔注射VCD以构建POF大鼠模型(例如，参见Muhammad FS et al.,Effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague-Dawley rats:ovarian,clinical,and pathologic outcomes[J].Comp Med,2009,59(1):46-59.)。开始建立模型时即进行NGF给药，记为第1天。采用皮下注射的给药方式，NGF(
Figure PCTCN2022132495-appb-000010
Figure PCTCN2022132495-appb-000011
鼠NGF或mNGF118)或NGF-Fc融合蛋白(2-118-L3G4-BM或2-118-L3Fc10-M3-5)实验组的注射剂量为10μg/kg bw，相同体积的无菌生理盐水作为阴性对照。NGF(
Figure PCTCN2022132495-appb-000012
鼠NGF、mNGF118)或无菌生理盐水隔天给药一次，NGF-Fc融合蛋白(2-118-L3G4-BM或2-118-L3Fc10-M3-5)每周给药一次。42天后，将所有大鼠安乐死，对卵巢组织固定后进行常规石蜡包埋、切片并进行H&E(苏木精和伊红)染色并对各级卵泡计数。记录的数据采用Student t检验进行分析，使用GraphPad Prism 8.0.1绘制柱状图。
如图3所示，与阴性对照组相比，NGF(
Figure PCTCN2022132495-appb-000013
鼠NGF或mNGF118)和NGF-Fc融合蛋白(2-118-L3G4-BM或2-118-L3Fc10-M3-5)均明显提高了初级卵泡的数量(p<0.05)，说明它们在改善POF引起的初级卵泡数量减少方面具有优异的效果。与阴性对照组相比，NGF(
Figure PCTCN2022132495-appb-000014
鼠NGF或mNGF118)或NGF-Fc融合蛋白(2-118-L3G4-BM或2-118-L3Fc10-M3-5)处理的POF大鼠模型也表现出更高的原始卵泡和次级卵泡数量。
实施例2：NGF和NGF-Fc融合蛋白对少弱精子症的治疗效果
少弱精子症主要表现为精子数量减少和/或精子活力下降。精子是由睾丸曲细精管内具有增殖能力的生殖细胞经过一系列分裂分化形成的，热应激可影响增殖细胞分裂、分化和 形成精子。本实施例在小鼠生精障碍模型中研究了NGF和NGF-Fc融合蛋白对少弱精子症(少精子症和弱精子症)的治疗效果。
本实验采用C57BL/6JSHjh小鼠(购自上海吉辉实验动物饲养有限公司)。对于实验组，采用腹股沟皮下注射的给药方式，分别注射20μg/kg bw/次NGF(
Figure PCTCN2022132495-appb-000015
鼠NGF或mNGF118)或60μg/kg bw/次NGF-Fc融合蛋白(2-118-L3G4-BM或2-118-L3Fc10-M3-5)。正常对照组或生精障碍模型对照组注射等体积0.9％氯化钠注射液。首次给药(NGF、NGF-Fc融合蛋白或氯化钠)当天为第1天。为构建小鼠睾丸热应激所致的生精障碍动物模型，在首次给药4小时后麻醉小鼠，待小鼠睾丸下降到阴囊后，将生精障碍模型对照组、NGF实验组和NGF-Fc融合蛋白实验组小鼠下腹(后肢、尾和阴囊)浸入42℃恒温水浴30分钟；正常对照组小鼠下腹(后肢、尾和阴囊)则浸入25℃恒温水浴30分钟。
Figure PCTCN2022132495-appb-000016
鼠NGF或mNGF118隔天给药一次，2-118-L3G4-BM或2-118-L3Fc10-M3-5每周给药2次。正常对照组或模型对照组注射等体积0.9％氯化钠注射液，隔天给药1次。共计给药5周。在给药后第37天安乐死小鼠，取左侧附睾尾称重并放入37℃预热的M199培养液中，剪碎并放入培养箱中37℃孵育5分钟，吸取精子悬液，用M199培养液按1:6稀释，混匀后，取稀释液，采用TOX IVOS精子分析仪检测精子数和精子活力。记录的数据采用Student t检验进行分析。
如表2所示，生精障碍模型对照组的精子数和精子活力均显著低于正常对照组，表明动物模型构建成功。与生精障碍模型对照组相比，NGF(
Figure PCTCN2022132495-appb-000017
鼠NGF或mNGF118)或NGF-Fc融合蛋白(2-118-L3Fc10-M3-5和2-118-L3G4-BM)实验组小鼠的精子数量和精子活力均显著升高。由此可见，皮下注射NGF或NGF-Fc融合蛋白可有效改善生精障碍(如弱精子症、少精子症和少弱精子症)中的精子数量减少和精子活力降低。
表2 NGF及NGF-Fc融合蛋白对生精障碍小鼠精子数和精子活力的影响
Figure PCTCN2022132495-appb-000018
Figure PCTCN2022132495-appb-000019
为进一步验证NGF和NGF-Fc的治疗效果，取上述安乐死小鼠的右侧睾丸和附睾，称重后用10％中性福尔马林固定、包埋、切片并进行H&E染色，以评价其组织病理学病变。如表3所示，NGF(
Figure PCTCN2022132495-appb-000020
鼠NGF或mNGF118)和NGF-Fc融合蛋白(2-118-L3Fc10-M3-5和2-118-L3G4-BM)对热应激所致的睾丸生精小管萎缩、生精小管生精障碍及附睾管细胞碎片症状表现出明显的治疗作用。
表3生精障碍小鼠皮下注射NGF或NGF-Fc融合蛋白的组织病理学统计表(只)
Figure PCTCN2022132495-appb-000021
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
Figure PCTCN2022132495-appb-000022
Figure PCTCN2022132495-appb-000023
Figure PCTCN2022132495-appb-000024
Figure PCTCN2022132495-appb-000025
Figure PCTCN2022132495-appb-000026
Figure PCTCN2022132495-appb-000027
Figure PCTCN2022132495-appb-000028
Figure PCTCN2022132495-appb-000029
Figure PCTCN2022132495-appb-000030

Claims (18)

  1. 神经生长因子或神经生长因子融合蛋白在制备用于治疗生殖系统疾病或改善生殖系统疾病的药物中的用途。
  2. 根据权利要求1所述的用途，其特征在于，所述生殖系统疾病包括但不限于：卵巢储备减少、卵巢早衰、卵巢过度刺激综合征、卵巢残余综合征、卵巢卵泡发育不良、生精障碍；
    优选地，所述生精障碍包括但不限于：弱精子症、少精子症、无精子症、畸形精子症、少弱精子症。
  3. 根据权利要求1所述的用途，其特征在于，所述改善生殖系统疾病包括但不限于如下的一种或多种改善：(1)促进卵巢颗粒细胞的增殖和/或雌激素分泌；(2)预防/逆转卵泡数量和/或功能的降低，或提高卵泡数量和/或功能；(3)改善睾丸生精小管萎缩、生精小管生精障碍和/或附睾管细胞碎片；(4)改善精子数量减少和/或活力降低，或提高精子数量和/或活力；
    优选地，所述卵泡为原始卵泡、初级卵泡和/或次级卵泡。
  4. 神经生长因子或神经生长因子融合蛋白在制备用于改善病症或症状的药物中的用途，所述病症或症状是与生殖系统疾病有关的病症或症状，或是由生殖系统疾病导致的病症或症状。
  5. 根据权利要求4所述的用途，其特征在于，所述生殖系统疾病包括但不限于：卵巢储备减少、卵巢早衰、卵巢过度刺激综合征、卵巢残余综合征、卵巢卵泡发育不良、生精障碍；
    优选地，所述生精障碍包括但不限于：弱精子症、少精子症、无精子症、畸形精子症、少弱精子症。
  6. 根据权利要求5所述的用途，其特征在于，所述病症或症状包括但不限于：卵巢颗粒细胞的增殖率降低和/或雌激素分泌降低，卵泡数量减少和/或功能降低，睾丸生精小管萎缩、生精小管生精障碍和/或附睾管细胞碎片，精子数量减少和/或活力的降低；
    优选地，所述卵泡为原始卵泡、初级卵泡和/或次级卵泡。
  7. 神经生长因子或神经生长因子融合蛋白在制备用于在患者中促进卵巢颗粒细胞的增殖和/或雌激素分泌；预防/逆转卵泡数量和/或功能的降低，或提高卵泡数量和/或功能；改善睾丸生精小管萎缩、生精小管生精障碍和/或附睾管细胞碎片；改善精子数量减少和/或活 力的降低，或提高精子数量和/或活力等的药物中的用途；优选地，所述卵泡为原始卵泡、初级卵泡和/或次级卵泡。
  8. 根据权利要求7所述的用途，其特征在于，所述患者为患有生殖系统疾病或由生殖系统疾病导致的相关疾病的患者，包括但不限于患有卵巢储备减少、卵巢早衰、卵巢过度刺激综合征、卵巢残余综合征、和/或卵巢卵泡发育不良的患者，或者患有生精障碍的患者；优选地，所述生精障碍包括但不限于：弱精子症、少精子症、无精子症、畸形精子症、少弱精子症。
  9. 根据权利要求1-8任一项所述的用途，其特征在于，所述神经生长因子包括野生型神经生长因子、重组神经生长因子、神经生长因子突变体。
  10. 根据权利要求9所述的用途，其特征在于，所述野生型神经生长因子包括从各种来源分离的野生型神经生长因子，所述来源包括家畜、灵长类动物、兔子和啮齿动物；优选地，所述野生型神经生长因子为鼠源野生型神经生长因子或人源野生型神经生长因子。
  11. 根据权利要求9所述的用途，其特征在于，所述重组神经生长因子包括通过重组表达或合成方法制备获得的重组神经生长因子；优选地，所述重组神经生长因子为鼠源重组神经生长因子或人源重组神经生长因子。
  12. 根据权利要求9所述的用途，其特征在于，所述神经生长因子突变体是具有野生型神经生长因子的大部分或全部生物活性的神经生长因子突变体，包括在野生型神经生长因子的一个或多个氨基酸位点突变；优选地，所述神经生长因子突变体相对于人源野生型神经生长因子序列SEQ ID NO:1或2的位置，包括如下一个或多个氨基酸突变：F12E、K32G、K32L、K32Y、R59L、R59A、D65A、D65G、K74L、K88F、K88L、K88E、K88G、Q96E、R114V、R114F、R114G、R114L和F101A。
  13. 根据权利要求12所述的用途，其特征在于，所述神经生长因子突变体包含SEQ ID NO:3-40中的任一氨基酸序列。
  14. 根据权利要求1-8中任一项所述的用途，其特征在于，所述神经生长因子融合蛋白从N端到C端包含神经生长因子、可选的肽接头和Fc部分。
  15. 根据权利要求14所述的用途，其特征在于，所述神经生长因子包含SEQ ID NO:1-40中的任一氨基酸序列；优选地，所述肽接头包含SEQ ID NO:41-45中的任一氨基酸序列，所述SEQ ID NO:43中n为1、2、3、4、5或6中的一个整数；优选地，所述Fc部分包含SEQ ID NO:46或47所示的氨基酸序列。
  16. 根据权利要求14所述的用途，其特征在于，所述神经生长因子融合蛋白包含SEQ  ID NO:48或49所示的氨基酸序列。
  17. 根据权利要求1-16任一项所述的用途，其特征在于，所述药物中包含治疗有效量的神经生长因子或神经生长因子融合蛋白作为主要活性成分以及药学上可接受的载体。
  18. 根据权利要求17所述的用途，其特征在于，所述药物的剂型包括注射剂、胶囊、片剂或粉针剂。
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