Application of 1, 3-propane diamine or pharmaceutically acceptable salt thereof in preparation of medicine for preventing or treating male dyszoospermia

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of 1, 3-propane diamine or pharmaceutically acceptable salt thereof in preparation of a medicine for preventing or treating male dyszoospermia. In a specific embodiment, the invention relates to application of 1, 3-propane diamine or pharmaceutically acceptable salt thereof in preparation of a medicine for preventing or treating male diabetic dyszoospermia. In experiments, the inventor finds that 1, 3-propane diamine can obviously improve the sperm concentration of diabetic mice and improve the expression quantity of testis spermatogenesis related proteins. According to the application, 1, 3-propane diamine is applied to diabetic dyszoospermia for the first time, an ideal treatment effect is achieved, a new treatment choice is provided for clinical application of 1, 3-propane diamine to treatment of diabetic dyszoospermia, and the application has extremely high application value and social benefits.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of 1,3-propanediamine or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention or treatment of male spermatogenesis disorders. Background Technology

[0002] With rising global obesity rates and changing lifestyles, diabetes has become one of the major chronic metabolic diseases threatening human reproductive health, and the reproductive system is one of the key target organs damaged by diabetes. Diabetic spermatogenesis disorder is a common complication in male diabetic patients, mainly caused by nerve and blood vessel damage due to long-term hyperglycemia, leading to abnormal testicular spermatogenesis. This is primarily manifested as reduced sperm count, decreased sperm motility, abnormal sperm morphology, and reduced ability to fertilize an egg, thus affecting fertility. Its occurrence is closely related to insulin secretion defects or impaired insulin action and may be accompanied by other sexual dysfunctions such as decreased libido and erectile dysfunction.

[0003] Treatments for diabetic spermatogenesis disorders primarily include medications to control blood sugar, drugs to improve sexual function, neurotrophic drugs, and antioxidants. While these medications can improve spermatogenesis to some extent, they all have clear drawbacks. First, oral hypoglycemic agents such as metformin and glimepiride indirectly support testicular spermatogenesis by stabilizing blood sugar, but they often cause gastrointestinal reactions such as nausea and diarrhea. Glimepiride can also easily lead to hypoglycemia and weight gain, and long-term use requires monitoring of liver and kidney function. Insulin is suitable for those with poor blood sugar control and can improve metabolic status, but it is cumbersome to administer, easily leads to hypoglycemia and weight gain, and does not directly promote sperm production. Second, PDE5 inhibitors such as sildenafil and tadalafil, which treat erectile dysfunction, can improve penile blood flow, but they are only effective in some patients and should not be used in combination with nitrates, otherwise it may cause severe hypotension, headaches, facial flushing, and even psychological dependence. In addition, neurotrophic drugs such as mecobalamin and epalrestat can help repair nerve damage caused by diabetes, thereby indirectly improving ejaculation and spermatogenesis. However, they require long-term use for several months to show effects and may cause loss of appetite or skin rashes. Finally, antioxidants such as vitamin E and alpha-lipoic acid can reduce oxidative damage to sperm DNA and improve sperm motility. However, alpha-lipoic acid may enhance insulin sensitivity and increase the risk of hypoglycemia, requiring close monitoring of blood glucose levels. Overall, current treatments are mostly indirect interventions, and their efficacy is limited by the degree of neurovascular damage. Furthermore, there are issues regarding drug interactions and long-term safety. Summary of the Invention

[0004] In order to address the lack of effective drugs for treating diabetic (especially type 2 diabetes) reproductive dysfunction in the existing technology, the inventors have discovered for the first time that 1,3-propanediamine can significantly increase sperm concentration, providing a new option for the prevention and treatment of diabetic spermatogenesis disorders.

[0005] Specifically, the present invention is achieved through the following technical solutions: This invention provides the use of 1,3-propanediamine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention or treatment of male spermatogenesis disorders.

[0006] Alternatively, in the above applications, the spermatogenesis disorder is a decrease in sperm concentration and / or a reduction in the number of spermatogenic cells.

[0007] Alternatively, in the above applications, the spermatogenesis disorder is azoospermia or oligospermia.

[0008] Alternatively, in the above applications, the product increases sperm concentration, increases the number of spermatogenic cells, and / or increases the expression levels of reproductive-related proteins.

[0009] Alternatively, in the above applications, the reproductive-related protein is selected from one or more of the following: DAZL, a marker of spermatogonia; SYCP3, a marker of spermatocytes; or PGK2, a marker of sperm cells.

[0010] Alternatively, in the above application, the spermatogenesis disorder is diabetic spermatogenesis disorder.

[0011] Preferably, the diabetes is type 2 diabetes.

[0012] Alternatively, in the above applications, the daily dose of the drug for each patient ranges from 60 to 90 mg.

[0013] Alternatively, in the above application, "male" refers to a male mammal.

[0014] Alternatively, in the above applications, the mammal is selected from the following: mouse, rat, rabbit, cat, dog, or primate.

[0015] More preferably, the primate is selected from humans.

[0016] Alternatively, in the above applications, the drug may also comprise a pharmaceutically acceptable carrier.

[0017] As an alternative, in the above applications, the "pharmaceutically acceptable carrier" of the present invention refers to a conventional pharmaceutical carrier in the field of pharmaceutical formulations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0018] The fillers described in this invention include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binders include, but are not limited to, water, ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, etc.; the disintegrants include, but are not limited to, starch effervescent mixtures, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agents include, but are not limited to, polysaccharides such as farnesian gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan, etc.; and the solvents include, but are not limited to, water, balanced salt solutions, etc.

[0019] Alternatively, in the above applications, the drug is in oral dosage form.

[0020] Alternatively, in the above applications, the oral dosage form is a tablet, pill, oral liquid, capsule, syrup, drop pill, or granule.

[0021] Alternatively, in the above applications, the formulation of the product described in this invention can be prepared using any conventional pharmaceutical formulation method known in the art.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention investigated the role of 1,3-propanediamine in improving diabetic spermatogenesis disorders using a type 2 diabetic mouse model. The results showed that 1,3-propanediamine can increase sperm concentration. Immunofluorescence and Western blotting methods revealed that after treatment with 1,3-propanediamine, the number of spermatogenic cells at all stages increased significantly.

[0023] This invention is the first to use 1,3-propanediamine for diabetic spermatogenesis disorder and achieves relatively ideal therapeutic effects, providing a new treatment option for the clinical application of 1,3-propanediamine in the treatment of diabetic spermatogenesis disorder, and has extremely high application value and social benefits. Attached Figure Description

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1Mouse sperm density and motility results (ratio of A+B grade sperm, ratio of A+B+C grade sperm). Grade A sperm are rapidly progressively motile, Grade B sperm are slowly progressively motile, and Grade C sperm are non-progressively motile. Figure A shows sperm concentration, Figure B shows sperm progressive motility, and Figure C shows total sperm motility. Vehicle served as the control group, while DAP (1,3-propanediamine) was added to the drinking water for treatment (the same applies below). Comparison between the treatment group (DAP) and the control group (Vehicle). This indicates that P < 0.05.

[0025] Figure 2 Results of intrinsic parameters of sperm motility in mice. Figure A shows the linear velocity of sperm, Figure B shows the curvilinear velocity of sperm, Figure C shows the average path velocity of sperm motility, Figure D shows the proportion of sperm with linear motility, Figure E shows the whiplash frequency of sperm motility, and Figure F shows the unilateral head sway amplitude, reflecting motility stability.

[0026] Figure 3 HE staining results of mouse testes in the control and treatment groups. Scale bars are 50 μm. The upper part shows the Vehicle control group, where the seminiferous tubules are loosely arranged, the number of spermatogenic cells at each stage is small, and the intercellular spaces are large. The lower part shows the DAP treatment group, where the seminiferous tubules are more tightly arranged, the tubular structure is more complete, and the number of spermatogonia, spermatocytes, and spermatids all increase to varying degrees, directly demonstrating the improving effect of DAP on spermatogenic cells.

[0027] Figure 4 Immunofluorescence staining of mouse testicular tissue. DAZL signal is green, SYCP3 signal is purple, WT1 signal is yellow, and S100A9 signal is red. Scale bar is 20 μm. The upper part represents the Vehicle control group, and the lower part represents the DAP treatment group. The comparison showed that the fluorescence signals of SYCP3 and WT1 were increased in the DAP treatment group.

[0028] Figure 5 Immunoblotting of mouse testicular tissue proteins. DAZL / SYCP3 / PGK2 / 3β-HSD are protein markers for spermatogonium, spermatocytes, spermatids, and interstitial cells, respectively. Figure A shows the immunoblotting bands, and Figure B shows the quantitative statistical analysis of proteins. The results show that, compared with the control group, the expression of DAZL / SYCP3 / PGK2 proteins in the testicular tissue of mice treated with DAP was significantly increased. Compared with the control group (Vehicle), the treatment group (DAP) and the control group (Vehicle)... This indicates that P < 0.05. This indicates that P < 0.01. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0032] Example: 1. Laboratory animals: Type 2 diabetic mice, 8 weeks old, C57BL / KsJ-db / db strain, SPF grade, were provided by Jicui Pharmaceutical Co., Ltd. After one week of acclimatization, they were divided into a blank control group (normal drinking water) and a model group (drinking water containing 2 mg / mLDAP and 1,3-propanediamine (DAP). Each mouse drank approximately 8-10 mL of water per day, equivalent to a daily dose of approximately 16-20 mg per mouse. Based on body surface area, the total daily dose for adults is approximately 65.3-81.6 mg). Water was changed daily for 6 consecutive weeks.

[0033] Animal experiments were conducted in strict accordance with the animal experiment management regulations of Peking University First Hospital and were reviewed by the Animal Experiment Ethics Committee.

[0034] Tissue collection: Fresh epididymis was collected. Tissue from one testis was fixed in 4% paraformaldehyde, routinely dehydrated in alcohol, and embedded in paraffin for sectioning; protein was directly extracted from the tissue from the other testis or it was cryopreserved in liquid nitrogen.

[0035] Effects on semen quality were determined: Fresh epididymis was used to collect sperm from the tail of the epididymis using the diffusion method, and the sperm density, forward motility, and total motility of mice were detected using a computer-aided semen analysis system (Beijing Suijia Software Co., Ltd.); straight-line speed (VSL), curvilinear speed (VCL), average path speed (VAP), whiplash frequency (BCF), sperm head lateral displacement (ALH), and forward trajectories (STR).

[0036] HE staining: One testicular tissue was fixed with 4% paraformaldehyde, routinely dehydrated with alcohol, and embedded in paraffin for sectioning, and then stained with HE.

[0037] Immunofluorescence: Mouse testes were rapidly isolated, rinsed with PBS to remove blood, embedded in OCT, and then flash-frozen in liquid nitrogen to prepare frozen sections for routine immunofluorescence staining.

[0038] Western blot analysis of testicular proteins: Proteins were extracted from retained testicular samples and then subjected to SDS-PAGE electrophoresis. Proteins on the gel were transferred to a PVDF membrane, and non-specific binding sites on the membrane were blocked with 5% skim milk. Marker antibodies for spermatogenic cells at various stages were incubated overnight at 4°C. After secondary antibody incubation, the proteins were finally visualized and analyzed using chemiluminescence and other methods. All data represent at least three independent experiments and are expressed as mean ± standard error (Mean ± SEM). GraphPad Prism 9 was used for analysis. t For statistical analysis, a p-value < 0.05 was considered statistically significant. This indicates that P < 0.05. This indicates that P < 0.01.

[0039] 2. Experimental Results: 2.1 Sperm density and sperm motility in mice First, the semen quality of mice in the Vehicle (control group) and 1,3-propanediamine (DAP group) groups at the end of treatment was examined using computer-aided semen analysis. The results showed (see...) Figure 1 Compared with the control group (Vehicle) mice, the sperm density of the treatment group (DAP) mice was significantly increased (P < 0.05), while the sperm forward motility and total motility were not significantly different compared with the Vehicle group.

[0040] 2.2 Intrinsic parameters of mouse sperm motility The intrinsic parameters of sperm motility in two groups of mice were detected using computer-aided semen analysis, such as... Figure 2 A to Figure 2 As shown in F, the intrinsic motility parameters of mouse sperm included linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), whiplash frequency (BCF), sperm head lateral displacement (ALH), and forward trajectories (STR). No significant differences were observed between the two groups.

[0041] 2.3 Results of HE staining of mouse testes HE staining results of mouse testes in the control and treatment groups ( Figure 3The scale bars are 50 μm and 20 μm respectively; the upper part is the Vehicle control group, where the seminiferous tubules are loosely arranged, the number of spermatogenic cells at each stage is small, and the intercellular spaces are large; the lower part is the DAP treatment group, where the seminiferous tubules are more tightly arranged, the tubular structure is more complete, and the number of spermatogonia, spermatocytes and spermatids all increase to varying degrees, which directly confirms the effect of DAP on improving spermatogenic function.

[0042] 2.4 Immunofluorescence results of mouse testes Immunofluorescence staining of mouse testicular tissue ( Figure 4 DAZL signal is green, SYCP3 signal is purple, WT1 signal is yellow, and S100A9 signal is red. The scale bar is 20 μm. The upper part is the vehicle control group, and the lower part is the DAP treatment group. The comparison shows that the fluorescence signals of SYCP3 and WT1 are increased in the DAP treatment group.

[0043] 2.5 Results of Western blot analysis of mouse testis proteins Mouse testicular protein blot results ( Figure 5 As shown in the figure, the protein expression of DAZL (a marker of spermatogonia), SYCP3 (a marker of spermatocytes), and PGK2 (a marker of spermatocytes) in the treatment group (DAP) was significantly higher than that in the control group (Vehicle), indicating that 1,3-propanediamine treatment can improve spermatogenesis in diabetic mice.

[0044] 3. Experimental Conclusions As demonstrated by the above embodiments, this invention, based on the treatment of type 2 diabetic mice with 1,3-propanediamine, revealed through computer-aided semen analysis, HE staining, and Western blotting that 1,3-propanediamine can improve sperm concentration in diabetic mice. Therefore, 1,3-propanediamine can be used to prepare therapeutic drugs for improving diabetic spermatogenesis disorders.

[0045] 4. Safety information regarding 1,3-propanediamine According to the literature (see "Complete Handbook of Safety Technology for Hazardous Chemicals" (3rd Edition), General Volume [J]. Analytical Chemistry, 2017, 45(12):1987.), the median lethal dose (LD50) of 1,3-propanediamine in rats is 312 mg / kg. Using the body surface area equivalent conversion method, the equivalent LD50 for a 60 kg adult is 2471 mg. In this experiment, the dosage of 1,3-propanediamine was equivalent to 16-20 mg per mouse per day, which translates to a total daily dose of 65.3 mg-81.6 mg for a 60 kg adult. This dose is significantly lower than the equivalent LD50 of 2471 mg, therefore, it can be determined that oral administration of 1,3-propanediamine for the prevention or treatment of type 2 diabetic spermatogenesis disorder is very safe.

[0046] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of this invention. If such modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include such modifications and variations.

Claims

1. The use of 1,3-propanediamine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention or treatment of male spermatogenesis disorders.

2. The application according to claim 1, characterized in that: The spermatogenesis disorder is characterized by decreased sperm concentration and / or a reduced number of spermatogenic cells.

3. The application according to claim 2, characterized in that: The spermatogenesis disorder mentioned refers to azoospermia or oligospermia.

4. The application according to claim 1, characterized in that: The product increases sperm concentration, increases the number of spermatogenic cells, and / or increases the expression of reproductive-related proteins.

5. The application according to claim 4, characterized in that: The spermatogenesis-related protein is selected from one or more of the following: DAZL, a marker of spermatogonia; SYCP3, a marker of spermatocytes; or PGK2, a marker of long sperm cells.

6. The application according to claim 1, characterized in that: The spermatogenesis disorder mentioned is diabetic spermatogenesis disorder.

7. The application according to claim 1, characterized in that: The term "male" refers to male mammals.

8. The application according to claim 7, characterized in that: The mammal is selected from the following: mouse, rat, rabbit, cat, dog or primate, more preferably, the primate is selected from human.

9. The application according to claim 1, characterized in that: The drug is an oral dosage form.

10. The application according to claim 9, characterized in that: The oral dosage form is tablet, pill, oral liquid, capsule, syrup, drop pill or granule.

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