Application of UNC 926 hydrochloride in improving male testosterone

Abstract

The invention discloses an application of UNC 926 hydrochloride in improving male testosterone. According to the application disclosed by the invention, UNC 926 hydrochloride micromolecules can increase the secretion amount of testosterone by stimulating testicular interstitial cells, so that a brand new thought is provided for clinically improving the development of low-testosterone treatment medicines for males.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of UNC 926 hydrochloride in increasing male testosterone. Background Art

[0002] Type 2 diabetes (T2D) is a common chronic disease characterized by metabolic disorders, affecting over 425 million people worldwide. Testosterone deficiency is particularly prevalent among men with T2D. Testosterone deficiency is a clinical condition associated with specific symptoms, such as decreased bone density, muscle atrophy, fat accumulation, and decreased cognitive function, and is also known as hypogonadism syndrome. Exogenous testosterone replacement therapy has shown beneficial effects on these metabolic syndromes, including insulin resistance and high cholesterol levels. However, this therapy also carries significant risks and side effects, such as the potential for stroke, myocardial infarction, or prostate cancer. Therefore, there is an urgent need to find safer, more effective, and more durable treatments for testosterone deficiency to improve men's quality of life and health.

[0003] Leydig cells (also known as Leydig cells) located in the testicular interstitium are the primary source of testosterone production. Enhancing endogenous Leydig cell function offers a promising approach to restoring physiological testosterone levels and circumventing the risks of exogenous hormone replacement therapy. Existing studies have shown that transplantation of stem cells, owing to their ability to differentiate into steroidogenic lineages, holds promise for treating male hypogonadism. However, these therapies also have limitations. For example, in aging models, Leydig cell transplantation only transiently restores testosterone production, failing to achieve therapeutic efficacy. Increasing endogenous Leydig cell function holds promise for increasing testosterone production, but currently, no effective method exists for achieving this. Existing approaches use AAV-mediated gene therapy to restore testosterone production, but this approach is hampered by efficiency limitations and gene safety concerns. Therefore, developing methods or products that can target key regulatory pathways within Leydig cells to stimulate endogenous testosterone production, thereby providing more sustained and safe therapeutic benefits, is crucial for treating male testosterone deficiency. Summary of the Invention

[0004] The present invention aims to address at least one of the aforementioned technical problems existing in the prior art. To this end, the present invention provides the use of UNC 926 hydrochloride for increasing male testosterone levels. The present invention discovers that the small molecule UNC 926 hydrochloride can increase testosterone secretion by stimulating Leydig cells in the testes, thereby providing a novel approach for the development of clinical therapeutics for low testosterone levels in men.

[0005] The first aspect of the present invention provides the use of a compound represented by Formula I in the preparation of a medicament for increasing male testosterone secretion;

[0006] Formula I.

[0007] The first aspect of the present invention provides the use of a compound represented by Formula I in the preparation of a medicament for improving or treating male reproductive dysfunction.

[0008] In some embodiments of the present invention, the males include healthy males and diseased males.

[0009] In some embodiments of the present invention, the diseased male organism includes a male organism suffering from at least one of the following diseases: diabetes, obesity, abnormal hormone secretion, and testosterone deficiency.

[0010] In some embodiments of the invention, the organism includes humans and non-human mammals.

[0011] In some embodiments of the invention, the non-human mammals include rodents, canines, felines, rabbits, horses, and primates.

[0012] In some embodiments of the invention, the rodents include rats, mice, and guinea pigs.

[0013] In some embodiments of the invention, the primates include monkeys and apes.

[0014] In some embodiments of the present invention, the drug further contains a pharmaceutically acceptable adjuvant.

[0015] In some embodiments of the present invention, the pharmaceutically acceptable excipients include but are not limited to diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, cross-linked polyvinylpyrrolidone, etc.), lubricants (such as talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (such as titanium dioxide, methylene blue, etc.), coating materials, solvents, pH regulators, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic regulators (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0016] In some embodiments of the present invention, the drug further contains a second active ingredient.

[0017] In some embodiments of the present invention, the second active ingredient comprises at least one of a diabetes drug, a testosterone replacement preparation, and a gonadotropin drug.

[0018] In some embodiments of the present invention, the second active ingredient includes at least one of insulin, testosterone undecanoate, testosterone propionate and human chorionic gonadotropin.

[0019] In some embodiments of the present invention, the dosage form of the drug includes at least one of an injection, an oral preparation, a patch, and a suppository.

[0020] In some embodiments of the present invention, the drug is administered once every 1-10 days.

[0021] In some embodiments of the present invention, the drug is administered once every 2-6 days.

[0022] In the present invention, it was found that UNC 926 hydrochloride can effectively maintain a long-lasting Leydig cell activation effect for 2-6 days after administration.

[0023] The beneficial effects of the present invention are: This study demonstrates for the first time that the UNC 926 hydrochloride small molecule can effectively activate endogenous testosterone synthesis and increase testosterone secretion by stimulating Leydig cells in the testicles. This approach avoids the safety risks and side effects associated with exogenous hormone supplementation. Furthermore, it can be effectively developed into oral or injectable dosage forms, resulting in strong patient compliance and suitable for large-scale commercialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of different concentrations of UNC 926 hydrochloride on testosterone secretion.

[0025] Figure 2 This is a statistical graph showing the effect of different concentrations of UNC 926 hydrochloride on the apoptosis of mouse Leydig cells.

[0026] Figure 3 Figure 2 shows testosterone secretion from 2 to 6 days after administration of UNC 926 hydrochloride. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0028] Experimental Materials In an embodiment of the present invention, the structural formula of UNC 926 hydrochloride is shown in Formula I.

[0029]

[0030] Formula I.

[0031] Its chemical name is: (3-bromophenyl) [4- (1-pyrrolidinyl) -1-piperidinyl] ketone hydrochloride, the molecular formula is: C 16 H 21 BrN2O·HCl, CAS number is 1782573-49-2.

[0032] Example 1 In this example, primary mouse Leydig cell lines were used as experimental subjects. After exogenous addition of UNC 926 hydrochloride to the cell culture system, the testosterone secretion in the culture supernatant was detected using ELISA to examine the effect of UNC 926 hydrochloride on testosterone secretion.

[0033] The specific experimental steps are: Primary mouse testicular Leydig cells were obtained and cultured in modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS).

[0034] Cultured primary mouse Leydig cells were obtained, counted, and seeded into 12-well plates in equal numbers. After 48 hours of attachment, different concentrations (10-100 μM) of UNC 926 hydrochloride were added and cultured in an incubator for 48 hours. An equal volume of DMSO was added to the control wells and cultured in modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) for 48 hours.

[0035] Testosterone levels were measured using a commercially available ELISA kit. After 48 hours of culture, cell culture supernatant was collected and the standard, detection antibody working solution, and wash solution were prepared using a commercially available ELISA kit (Elabscience® E-OSEL-M0003) according to the manufacturer's instructions. Samples were then added and incubated. For sample addition and incubation, 100 μL of the corresponding solution was added to the standard wells and 100 μL of the corresponding solution was added to the sample wells (duplicate wells were used for samples). The plate was sealed and incubated at room temperature for 1-2 hours. For washing, the liquid in the wells was discarded, the plate was washed 3-5 times with wash solution, and patted dry. For antibody incubation, 100 μL of detection antibody working solution was added to each well, incubated at room temperature for 1 hour, and then the wash step was repeated. For enzyme-conjugated incubation, 100 μL of enzyme-conjugated secondary antibody working solution was added to each well, incubated at room temperature for 30 minutes to 1 hour, and then washed again. For color development and termination, 100 μL of TMB colorimetric reagent was added to each well and incubated in the dark at room temperature for 10-15 minutes. Then, 50 μL of stop solution was added. After incubation, the OD value was read at a wavelength of 450 nm using a microplate reader, and a standard curve was drawn based on the standard to calculate the testosterone concentration of the sample.

[0036] The results are as follows Figure 1 shown.

[0037] It was found that when different concentrations of UNC 926 hydrochloride were added to the culture medium of primary mouse Leydig cells and cultured for 48 hours, the maximum testosterone secretion was achieved when UNC 926 hydrochloride reached 40 μM. Subsequent increase in concentration did not stimulate more testosterone secretion, indicating that 40 μM UNC 926 hydrochloride can promote the secretion of testosterone by primary mouse Leydig cells.

[0038] Example 2 The inventors further tested the apoptosis of primary mouse Leydig cells under different concentrations of treatment in the above examples, and the specific steps were as follows: Primary mouse Leydig cells were obtained and cultured according to the method in the above example and treated with UNC 926 hydrochloride. After culture, the apoptotic rate was detected using the Annexin V-FITC / PI cell apoptosis detection kit (purchased from Yisheng Bio).

[0039] The specific detection steps are as follows: After digesting the cells with EDTA-free trypsin, collect the cells by centrifugation at 300g for 5 minutes at 4°C. Wash the cells twice with pre-chilled PBS, centrifuging again at 300g for 5 minutes at 4°C after each wash. Aspirate the PBS and resuspend the cells in 100 μL of 1× Binding Buffer. Add 5 μL of Annexin V-FITC and 10 μL of PI dye, mix gently, and incubate at room temperature in the dark for 10-15 minutes. Add 400 μL of 1× Binding Buffer, mix thoroughly, and place on ice. The percentage of apoptosis is determined by flow cytometry.

[0040] The results are as follows Figure 2 shown.

[0041] It can be found that treating cells with different concentrations of UNC 926 hydrochloride for 48 h had no effect on cell apoptosis, indicating that UNC926 has no toxicity to cells.

[0042] Example 3 To further verify the long-term activation effect of UNC 926 hydrochloride on primary mouse Leydig cells, the inventors conducted another verification according to the method in Example 1. The difference is that in this example, the concentration of UNC 926 hydrochloride was set to 40 μM and the incubation time was 48-144 h.

[0043] After culturing for 48, 96, and 144 hours, the cell culture supernatant was collected and the testosterone concentration was determined using a commercially available ELISA kit according to the method described in the above example.

[0044] The results are as follows Figure 3 shown.

[0045] It can be found that primary mouse Leydig cells treated once with 40 μM UNC 926 hydrochloride can maintain a high testosterone concentration for 2 to 6 days, indicating that this small molecule has a long-lasting activation effect on testosterone.

[0046] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of the compound represented by formula I in the preparation of a medicament for increasing male testosterone secretion; Formula I.

2. Use of the compound represented by Formula I in the preparation of a medicament for improving or treating male reproductive dysfunction; Formula I.

3. The use according to claim 1 or 2, characterized in that The males include healthy males and diseased males.

4. The use according to claim 3, characterized in that The diseased male organisms include male organisms suffering from at least one of the following diseases: Diabetes, obesity, abnormal hormone secretion and testosterone deficiency.

5. The use according to claim 3, characterized in that The organisms include humans and non-human mammals.

6. The use according to claim 1 or 2, characterized in that The medicine also contains pharmaceutically acceptable adjuvants.

7. The use according to claim 1 or 2, characterized in that The medicine also contains a second active ingredient.

8. The use according to claim 7, characterized in that The second active ingredient includes at least one of a diabetes drug, a testosterone replacement preparation, and a gonadotropin drug; Preferably, the second active ingredient comprises at least one of insulin, testosterone undecanoate, testosterone propionate and human chorionic gonadotropin.

9. The use according to claim 1 or 2, characterized in that: The dosage form of the drug includes at least one of an injection, an oral preparation, a patch and a suppository.

10. The use according to claim 1 or 2, characterized in that: The drug is administered once every 1-10 days, preferably, every 2-6 days.

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