TW202330578A - Use of nerve growth factor in manufacture of medicament for treating or improving reproductive system disease - Google Patents

Abstract

The present invention provides use of nerve growth factor in the manufacture of a medicament for treating a reproductive system disease or improving a reproductive system disease.

Description

Use of nerve growth factor in preparing medicines for treating or improving reproductive system diseases

The invention belongs to the technical field of biomedicine, and specifically relates to the use of nerve growth factor in preparing medicines for treating or improving reproductive system diseases.

Nerve Growth Factor (NGF) 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. effect. Nerve growth factor contains three subunits α, β and γ. The β subunit is an active domain composed of two single strands joined by non-covalent bonds.

For the nervous system, 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. Studies have shown that, in addition to being more distributed in the nervous system, 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. However, there is no report on whether 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, 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, epitesticular tuberculosis, etc.; (4) reproductive tract injury, such as testicular contusion, penile fracture, urethral rupture, etc.; (5) reproductive system deformity, such as hidden penis, webbed penis, cryptotestis, etc.; (6) Sexual dysfunction diseases, such as male erectile dysfunction, premature ejaculation, loss of libido, non-ejaculation, delayed ejaculation, etc.; (7) Related diseases caused by reproductive system diseases, such as varicocele, oligoasthenozoospermia, congenital vas deferens Obstruction, lack of vas deferens, blocked fallopian tubes, immune infertility, premature ovarian failure.

Premature Ovarian Failure (POF) refers to natural amenorrhea before the age of 40 caused by 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%, and it accounts for 2% to 10% in amenorrhea patients. 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 quality of life decline. 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 carotene are mainly used; 2. Surgical treatment: If the patient’s oligoasthenozoospermia is due to varicocele If it is caused by varicocele, surgical treatment of varicocele is required; if it is caused by hidden testes, surgical treatment of hidden testes is required.

There is an urgent need in this field for a drug and method that can effectively treat or improve reproductive system diseases such as premature ovarian failure and oligoasthenospermia.

In the first aspect, 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.

In some embodiments, the diseases of the reproductive system include but are not limited to decreased ovarian reserve, premature ovarian failure (Premature Ovarian Failure, POF), ovarian hyperstimulation syndrome, ovarian remnant syndrome, ovarian follicle dysplasia, and spermatogenesis disorder; preferably Specifically, the spermatogenic disorders include but are not limited to: Oligozoospermia or Oligospermia, asthenospermia, azoospermia, teratospermia, oligoasthenoteratozoospermia (OAT syndrome).

In some embodiments, the improvement of reproductive system diseases includes, but is not limited to, one or more of the following improvements:

(1) promote the proliferation of ovarian granulosa cells and/or estrogen secretion; (2) prevent/reverse the decrease in the number and/or function of follicles, or improve the number and/or function of follicles; (3) improve the atrophy of testicular seminiferous tubules, Seminiferous tubule spermatogenesis disorder and/or epididymal cell fragments; (4) improve the reduction of sperm count and/or motility, or increase the sperm count and/or motility; preferably, the follicles are primitive follicles, primary follicles and/or secondary follicles.

In a second aspect, 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.

In some embodiments, 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, and spermatogenesis disorder ; Preferably, the spermatogenic disorders include but are not limited to: asthenozoospermia, oligospermia, azoospermia, teratospermia child disease, oligoasthenozoospermia.

In some embodiments, the disorders or symptoms include, but are not limited to: decreased proliferation rate of ovarian granulosa cells and/or decreased estrogen secretion, decreased number and/or function of follicles, atrophy of testicular seminiferous tubules, seminiferous tubules Sperm disorders and/or epididymal cell fragments, decreased sperm count and/or reduced motility; preferably, the follicles are primordial follicles, primary follicles and/or secondary follicles.

In a third aspect, 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 epitesticular cell fragments; improve sperm count and/or decrease in motility, or increase sperm count and/or motility, etc. Use in medicine; preferably, the follicles are primordial follicles, primary follicles and/or secondary follicles.

In some embodiments, 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).

In some embodiments, 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).

In some embodiments, the nerve growth factor is mouse wild-type nerve growth factor or human wild-type nerve growth factor.

In some embodiments, 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 Factor glycation products and/or nerve growth factor oxidation products.

In some embodiments, 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. In some embodiments, 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 β-NGF). In some embodiments, the mutant is a variant of wild-type nerve growth factor after deletion, substitution and/or insertion of one or more amino acid residues. In some embodiments, the NGF mutant has reduced side effects (eg, pain) or is painless compared to wild-type NGF.

In some embodiments, the position of the nerve growth factor mutant relative to the human wild-type nerve growth factor sequence (SEQ ID NO: 1 or 2) 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.

In some embodiments, the NGF 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.

In some embodiments, the other proteins or polypeptides include, but are not limited to, the Fc portion, human serum albumin, and the like.

In some embodiments, a nerve growth factor fusion protein of the invention comprises nerve growth factor, an optional peptide linker, and an Fc portion from the N-terminus to the C-terminus.

In some embodiments, 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 NO: 41-45. In some embodiments, the peptide linker comprises an amino acid sequence (GGGGS)n (SEQ ID NO: 43), wherein n is any integer of 1, 2, 3, 4, 5 or 6, preferably n is Any integer from 2 to 6, more preferably n is an integer of 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 part 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. In some embodiments, the Fc portion is from IgG1 Fc, IgG4 Fc, or contains one or more mutations or undergoes glycosylation modification relative to natural IgG1 Fc, IgG4 Fc amino acid sites. In some embodiments, the Fc portion comprises the amino acid sequence shown in SEQ ID NO:46 or 47.

In some embodiments, 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.

The pharmaceutically acceptable carrier is non-toxic to the subject at the dosage and concentration used, and its type is well known in the art, including but not limited to buffer, antioxidant; ascorbic acid, methionine, vitamin E, sodium metabisulfite; Preservatives, isotonic agents (such as sodium chloride), stabilizers, metal complexes (such as zinc-protein complexes); chelating agents such as ethylenediaminetetraacetic acid (EDTA) and/or non- ionic surfactants, etc.

The drugs of the present invention can be administered by various methods, including but not limited to: oral, subcutaneous, intravenous, intracerebral, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, and the like.

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 granular appearance of human ovaries treated with nerve growth factor ( ^Supeisen® mouse NGF or mNGF118) or nerve growth factor fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM) Proliferation rate of tumor cell lines (KGN).

Figure 2 shows the granule-like appearance of human ovaries treated with nerve growth factor ( ^Supeisheng® mouse NGF or mNGF118) or nerve growth factor fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM) Estrogen concentrations secreted by tumor cell lines (KGN).

Figure 3 shows premature ovarian failure in rats treated with nerve growth factor ( ^Supeisheng® mouse NGF or mNGF118) or nerve growth factor fusion protein (2-118-L3Fc10-M3-5 or 2-118-L3G4-BM) Number of follicles at all levels in animal disease models.

In order to make the technical problems to be solved, the technical solutions adopted and the advantages of the present invention clearer, the present invention will be described in detail below with reference to the drawings and specific embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

The reagents used in the following examples, if no special instructions, all adopt normal Prepared by conventional methods or obtained from commercial sources; the experimental methods used, unless otherwise specified, were conventional methods; the materials, instruments, etc. used, were obtained from commercial sources unless otherwise specified.

4-Vinylcyclohexene diepoxide (VCD), item number/specification: 94956-100ml liquid contains 108g VCD, manufacturer: sigma

The source of nerve growth factor used in the embodiment of the present invention is as follows:

Su ^Taisheng® mouse NGF: from Shutaishen (Beijing) Biopharmaceutical Co., Ltd.

The 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.

Example 1: The therapeutic effect of NGF and NGF-Fc fusion protein on premature ovarian failure

Premature ovarian failure (POF) 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. In this embodiment, the human ovarian granulosa tumor cell line (KGN) proliferation test and the KGN estrogen secretion test and POF model in vitro The therapeutic effect of NGF and NGF-Fc fusion protein on POF was studied in rats.

For the 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 the medium was replaced, NGF ( ^{Threuterson®} mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) was added to the wells of the experimental group respectively, so that the The final concentration in the medium was 10 μg/mL, and the negative control group was not treated after replacing the medium. Each group had 4 replicate wells, and after 48 hours, 10 uL of CCK-8 (Dojin Chemical Research Institute, #CK04) was added to each well to determine the number of viable cells. After incubation for 1 h, the absorbance at 450 nm was measured, and the recorded data were analyzed by the Student t test, and the histogram was drawn using GraphPad Prism 8.0.1.

As shown in Figure 1, compared with the PBS negative control group, NGF ( ^{Threupeisen®} mouse NGF or mNGF118) and NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) Both significantly promoted the proliferation of KGN (p<0.05).

For the KGN estrogen secretion test, seed KGN cells in a 24-well plate at 1×10 ⁵ cells/well (the confluence of the cells is about 80%), and replace the serum-free medium. After replacing the medium, add NGF ( ^Threotide® mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) to the wells of the experimental group The final concentration in was 10 μg/ml. The negative control group did not receive any treatment after replacing the culture medium. Four replicate wells per group were incubated for 18 hours, washed twice, and 2.2×10 ^-8 M testosterone (Beijing Suo Laibao Technology Co., Ltd., #IT0110) and 0.01 IU/ml sheep follicle stimulating hormone (NHPP, USA) were used. , Ovine FSH) after 24 hours of treatment, the supernatant was diluted 1.6 times, and the estrogen assay kit (KGE014) produced by R&D Systems was used to measure the absorbance at 450nm, and the secreted estrogen concentration was calculated, and the recorded data were obtained using Student t Tests were analyzed and histograms were drawn using GraphPad Prism 8.0.1.

As shown in Figure 2, compared with the negative control group, NGF ( ^{Threupeisen®} mouse NGF or mNGF118) and 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).

4-Vinylcyclohexenedioxirane (VCD) can selectively destroy primordial follicles 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. In order to further study the in vivo therapeutic effect of NGF and NGF-Fc fusion protein on POF, SD rats were injected intraperitoneally with VCD for two consecutive weeks to construct a POF rat model (for example, see 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.). NGF was administered at the beginning of model establishment, which was recorded as day 1. Subcutaneous injection was adopted, and the injection dose of NGF ( ^{Threupeisen®} mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) experimental group was 10 μg/kg bw, the same volume of sterile saline was used as a negative control. NGF ( ^{Threuterson®} mouse NGF, mNGF118) or sterile saline was administered every other day, and NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) was administered once a week. After 42 days, all the rats were euthanized, the ovarian tissues were fixed, routinely embedded in paraffin, sectioned and stained with H&E (hematoxylin and eosin) and follicles at all levels were counted. The recorded data were analyzed by Student t test, and the histogram was drawn using GraphPad Prism 8.0.1.

As shown in Fig. 3, compared with the negative control group, NGF ( ^Threupei® mouse NGF or mNGF118) and NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) both Significantly increased the number of primary follicles (p<0.05), indicating that they have an excellent effect in improving the reduction in the number of primary follicles caused by POF. Compared with the negative control group, POF rat models treated with NGF ( ^Threotide® mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3G4-BM or 2-118-L3Fc10-M3-5) also Exhibited a higher number 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 proliferating cells. The truth EXAMPLES The therapeutic effect of NGF and NGF-Fc fusion protein on oligoasthenospermia (oligospermia and asthenospermia) was studied in a mouse spermatogenesis disorder model.

C57BL/6JSHjh mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) were used in this experiment. For the experimental group, 20 μg/kg bw/time of NGF (Su ^Taisheng® mouse NGF or mNGF118) or 60 μg/kg bw/time of 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 (NGF, NGF-Fc fusion protein or sodium chloride) was the first day. In order to construct an animal model of spermatogenic disorder caused by heat stress in the testis of mice, the mice were anesthetized 4 hours after the first administration, and after the mouse testis descended to the scrotum, the spermatogenic 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. Su ^Taisheng® mouse NGF or mNGF118 was administered once 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. On the 37th day after administration, the mice were euthanized, and the left epitestis tail was weighed and put into 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 TOXIVOS sperm analyzer to detect sperm count and sperm motility. The recorded data were analyzed by Student's t test.

As shown in Table 2, the sperm count and sperm motility of the spermatogenesis disorder model control group were significantly lower than those of the normal control group, indicating that the animal model was successfully constructed. Compared with the control group of spermatogenesis disorder model, the mice in the experimental group of NGF ( ^Threupei® mouse NGF or mNGF118) or NGF-Fc fusion protein (2-118-L3Fc10-M3-5 and 2-118-L3G4-BM) Sperm count and sperm motility were significantly increased. It can be seen that subcutaneous injection of 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).

In order to further verify the therapeutic effect of NGF and NGF-Fc, the right testis and epididymis of the above-mentioned euthanized mice were taken, weighed, fixed with 10% neutral formalin, embedded, sectioned and H&E stained to evaluate the histopathology medical disease. As shown in Table 3, the effects of NGF (Threuterson® mouse NGF or mNGF118) and NGF-Fc fusion proteins (2-118-L3Fc10-M3-5 and 2-118-L3G4-BM) on heat stress-induced The symptoms of seminiferous tubule atrophy, seminiferous tubule spermatogenic disorder and epitesticular cell debris showed obvious therapeutic effect.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention. Should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

sequence listing

SEQ ID NO: 1 (wild type human β-NGF, 120aa)

SEQ ID NO: 2 (wild type human β-NGF, 118aa)

SEQ ID NO: 3 (F12E mutant, 120aa)

SEQ ID NO: 4 (K32G mutant, 120aa)

SEQ ID NO: 5 (K32L mutant, 120aa)

SEQ ID NO: 6 (K32Y mutant, 120aa)

SEQ ID NO: 7 (R59L mutant, 120aa)

SEQ ID NO: 8 (R59A mutant, 120aa)

SEQ ID NO: 9 (D65A mutant, 120aa)

SEQ ID NO: 10 (D65G mutant, 120aa)

SEQ ID NO: 11 (K74L mutant, 120aa)

SEQ ID NO: 12 (K88F mutant, 120aa)

SEQ ID NO: 13 (K88L mutant, 120aa)

SEQ ID NO: 14 (K88E mutant, 120aa)

SEQ ID NO: 15 (K88G mutant, 120aa)

SEQ ID NO: 16 (Q96E mutant, 120aa)

SEQ ID NO: 17 (R114V mutant, 120aa)

SEQ ID NO: 18 (R114F mutant, 120aa)

SEQ ID NO: 19 (R114G mutant, 120aa)

SEQ ID NO: 20 (R114L mutant, 120aa)

SEQ ID NO: 21 (F101A mutant, 120aa)

SEQ ID NO: 22 (F12E mutant, 118aa)

SEQ ID NO: 23 (K32G mutant, 118aa)

SEQ ID NO: 24 (K32L mutant, 118aa)

SEQ ID NO: 25 (K32Y mutant, 118aa)

SEQ ID NO: 26 (R59L mutant, 118aa)

SEQ ID NO: 27 (R59A mutant, 118aa)

SEQ ID NO: 28 (D65A mutant, 118aa)

SEQ ID NO: 29 (D65G mutant, 118aa)

SEQ ID NO: 30 (K74L mutant, 118aa)

SEQ ID NO: 31 (K88F mutant, 118aa)

SEQ ID NO: 32 (K88L mutant, 118aa)

SEQ ID NO: 33 (K88E mutant, 118aa)

SEQ ID NO: 34 (K88G mutant, 118aa)

SEQ ID NO: 35 (Q96E mutant, 118aa)

SEQ ID NO: 36 (R114V mutant, 118aa)

SEQ ID NO: 37 (R114F mutant, 118aa)

SEQ ID NO: 38 (R114G mutant, 118aa)

SEQ ID NO: 39 (R114L mutant, 118aa)

SEQ ID NO: 40 (F101A mutant, 118aa)

SEQ ID NO: 41 (peptide linker)

GGGGSGGGGSGGGGS

SEQ ID NO: 42 (peptide linker)

GGGGGGSGGGGSGGGGSA

SEQ ID NO: 43 (peptide linker; n is an integer of at least 1)

(GGGGS) _n

SEQ ID NO: 44 (peptide linker)

GSGGGSGGGGSGGGGSGGGGS

SEQ ID NO: 45 (peptide linker)

KTGGGSGGGS

SEQ ID NO: 46 (Modified IgG1 Fc M3-5 [L234A+L235A+P331S truncated relative to IGHG1*03, N' 5aa])

SEQ ID NO: 47 (Modified IgG4 Fc [S228P+F234A+L235A])

SEQ ID NO: 48 fusion protein (amino acid sequence of mature 2-118-L3Fc10-M3-5; β-NGF (mutant 118aa) in bold, linker underlined, modified IgG1 Fc in italics and bold)

SEQ ID NO: 49 fusion protein (amino acid sequence of mature 2-118-L3G4-BM; β-NGF (mutant 118aa) in bold, linker underlined, modified IgG4 Fc in italics and bold)

Claims (18)

Use of nerve growth factor or nerve growth factor fusion protein in preparing medicines for treating reproductive system diseases or improving reproductive system diseases. The use as described in claim 1, wherein the diseases of the reproductive system include but are not limited to: decreased ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian remnant syndrome, ovarian follicle dysplasia, and spermatogenesis disorder; Preferably, the spermatogenesis disorder includes but not limited to: asthenospermia, oligospermia, azoospermia, teratozoospermia, oligoasthenospermia. The use as described in claim 1, wherein the improvement of reproductive system diseases includes but is not limited to one or more of the following improvements: (1) promoting the proliferation of ovarian granulosa cells and/or estrogen secretion; (2) preventing/reversing Decrease in the number and/or function of follicles, or increase the number and/or function of follicles; (3) Improve the atrophy of the testicular seminiferous tubules, the spermatogenesis disorder of the seminiferous tubules and/or the fragmentation of epitesticular duct cells; (4) Improve the decrease in the number of spermatozoa and/or decreased motility, or increased sperm count and/or motility; Preferably, the follicles are primordial follicles, primary follicles and/or secondary follicles. Use of nerve growth factor or nerve growth factor fusion protein in the preparation of a medicament for improving a disease or symptom, the disease or symptom being a disease or symptom related to a reproductive system disease, or a disease or symptom caused by a reproductive system disease . The use as described in claim 4, wherein the diseases of the reproductive system include but are not limited to: decreased ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, ovarian remnant syndrome, ovarian follicle dysplasia, and spermatogenesis disorder; Preferably, the spermatogenesis disorder includes but not limited to: asthenospermia, oligospermia, azoospermia, teratozoospermia, oligoasthenospermia. The use as described in claim 5, wherein the disease or symptoms include but are not limited to: decreased proliferation rate of ovarian granulosa cells and/or decreased estrogen secretion, decreased follicle number and/or decreased function, and atrophy of testicular seminiferous tubules , seminiferous tubule spermatogenesis disorder and/or epididymal cell debris, decreased sperm count and/or motility; Preferably, the follicles are primordial follicles, primary follicles and/or secondary follicles. Nerve growth factor or nerve growth factor fusion protein is being prepared 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 increasing the number and/or function of follicles; Improving testis seminiferous tubule atrophy, seminiferous tubule spermatogenic disorder and/or epitesticular cell fragments; improving sperm count reduction and/or motility reduction, or improving sperm count and/or motility, etc.; preferably , the follicles are primordial follicles, primary follicles and/or secondary follicles. The use as described in claim 7, wherein the patient is a patient suffering from reproductive system diseases or related diseases caused by reproductive system diseases, including but not limited to patients with reduced ovarian reserve, premature ovarian failure, ovarian hyperstimulation syndrome, Patients with ovarian remnant syndrome and/or ovarian follicle dysplasia, or patients with spermatogenesis disorders; preferably, the spermatogenesis disorders include but are not limited to: asthenozoospermia, oligospermia, azoospermia, deformity Sperm disease, oligozoospermia. The use according to any one of claims 1 to 8, characterized in that the nerve growth factor includes wild-type nerve growth factor, recombinant nerve growth factor, and nerve growth factor mutants. The use as described in claim 9, wherein the wild-type nerve growth factor comprises wild-type nerve growth factor isolated from various sources, including livestock, primates, rabbits and rodents; preferably, the The wild-type nerve growth factor is mouse-derived wild-type nerve growth factor or human-derived wild-type nerve growth factor. The use as described in claim 9, wherein said recombinant nerve growth factor comprises recombinant nerve growth factor prepared by recombinant expression or synthetic method; preferably, said recombinant nerve growth factor is mouse-derived recombinant nerve growth factor or human Source recombinant nerve growth factor. The use as described in claim 9, wherein the nerve growth factor mutant is a nerve growth factor having most or all of the biological activities of the wild-type nerve growth factor Long factor mutants, including mutations at one or more amino acid sites of wild-type nerve growth factor; 2, including one or more of the following amino acid mutations: F12E, K32G, K32L, K32Y, R59L, R59A, D65A, D65G, K74L, K88F, K88L, K88E, K88G, Q96E, R114V, R114F, R114G, R114L and F101A. The use according to claim 12, wherein the NGF mutant comprises any amino acid sequence in SEQ ID NO: 3-40. The use according to any one of claims 1 to 8, characterized in that the nerve growth factor fusion protein comprises nerve growth factor, an optional peptide linker and an Fc part from the N-terminal to the C-terminal. The use according to claim 14, wherein the nerve growth factor comprises any amino acid sequence in SEQ ID NO: 1-40; preferably, the peptide linker comprises the amino acid sequence in SEQ ID NO: 41-45 Any amino acid sequence, wherein n in said SEQ ID NO: 43 is an integer of 1, 2, 3, 4, 5 or 6; preferably, said Fc part comprises SEQ ID NO: 46 or 47 amino acid sequence. The use according to claim 14, wherein the nerve growth factor fusion protein comprises the amino acid sequence shown in SEQ ID NO: 48 or 49. The use according to any one of claims 1 to 16, characterized in that the drug contains a therapeutically effective amount of nerve growth factor or nerve growth factor fusion protein as the main active ingredient and a pharmaceutically acceptable carrier. The use according to claim 17, wherein the dosage form of the drug includes injection, capsule, tablet or powder injection.

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