Use of a polypeptide in the preparation of a medicament for improving male sexual function

Abstract

The application discloses application of a polypeptide in preparation of a medicine for improving male sexual function, and the polypeptide comprises an amino acid sequence Thr-Tyr-Val-Pro-Lys-Ala-Phe. The polypeptide can improve sexual organ index and promote synthesis of testosterone, thereby improving male sexual function. The application further provides a medicine composition containing the polypeptide and a preparation method of the medicine composition.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a polypeptide in the pharmaceutical field, and more particularly to the application of an active polypeptide derived from deer antler in the preparation of drugs that improve male sexual function. Background Technology

[0002] Male sexual dysfunction is a common male health problem in clinical practice, and its incidence increases significantly with age. Epidemiological studies show that after the age of 30, men's serum testosterone levels show a downward trend year by year, with an average annual decrease of more than 1%, accompanied by symptoms such as decreased libido and erectile dysfunction.

[0003] Currently, the first-line drugs used clinically to treat erectile dysfunction in men are mainly phosphodiesterase type 5 (PDE5) inhibitors. These drugs work by inhibiting PDE5 activity, increasing cGMP levels in the corpora cavernosa of the penis, thus achieving a short-term enhancement of erectile function. However, these drugs have significant limitations: firstly, they only improve erectile function in the short term, and the effect disappears after discontinuation, failing to reverse low testosterone levels or sexual organ dysfunction at the underlying cause; secondly, they are contraindicated in certain populations, such as patients currently taking nitrates; and thirdly, long-term use is accompanied by adverse reactions such as headaches and facial flushing, leading to poor patient compliance.

[0004] Deer antler (Cervus elaphus) has been used as a tonic to warm the kidneys and strengthen yang since ancient times. Modern pharmacological studies have shown that it contains a variety of active polypeptide components, but the specific material basis for its effects is still unclear.

[0005] Therefore, there is an urgent need in this field to develop a new type of drug that can fundamentally improve sexual function, is highly safe, and is suitable for a wide range of people. Summary of the Invention

[0006] Existing drugs used to treat male sexual dysfunction (such as phosphodiesterase type 5 inhibitors) can only provide short-term symptomatic relief by dilating blood vessels, and cannot fundamentally improve sex organ indices and testosterone levels. Furthermore, they have drawbacks such as contraindications and adverse reactions. To address these shortcomings, this invention provides a new technical solution, aiming to provide an active polypeptide that can fundamentally improve male sexual function, along with its pharmaceutical uses and pharmaceutical composition.

[0007] In a first aspect, the present invention provides the use of a polypeptide in the preparation of a drug for improving male sexual function, the polypeptide comprising the following amino acid sequence: Thr-Tyr-Val-Pro-Lys-Ala-Phe (SEQ ID NO:1).

[0008] In some embodiments of the present invention, the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO:1.

[0009] In some embodiments of the present invention, the polypeptide is a conserved variant of the sequence shown in SEQ ID NO:1, and the conserved variant has activity that improves male sexual function.

[0010] Preferably, the conserved variant is a variant obtained by conserved substitution of 1 to 3 amino acids in the sequence shown in SEQ ID NO:1.

[0011] In some embodiments of the present invention, the C-terminus of the polypeptide is in the form of a free carboxyl group.

[0012] In some embodiments of the present invention, the C-terminus of the polypeptide is in an amidated form.

[0013] Preferably, the C-terminus of the polypeptide is in an amidated form.

[0014] In some embodiments of the present invention, the polypeptide is derived from deer antler.

[0015] In some embodiments of the present invention, the polypeptide is a synthetically produced polypeptide.

[0016] Preferably, the polypeptide is a polypeptide synthesized using a solid-phase synthesis method.

[0017] In some embodiments of the present invention, the improvement of male sexual function includes, but is not limited to, any one or a combination of the following:

[0018] (i) Increase the sex organ index;

[0019] (ii) Increase testosterone levels;

[0020] (iii) Prolong the duration of sexual intercourse;

[0021] (iv) Enhance libido;

[0022] (v) Improves erectile function.

[0023] Preferably, the enhancement of the sex organ index is the enhancement of the testicular index.

[0024] Preferably, the increase in testosterone level refers to the increase in serum testosterone level.

[0025] In some embodiments of the present invention, the drug is an oral preparation.

[0026] Preferably, the oral preparation is selected from any one of oral liquid, tablets, capsules, granules or oral instant films.

[0027] In a second aspect, the present invention provides a method for improving male sexual function, the method comprising administering an effective amount of the polypeptide described in the first aspect to a subject in need.

[0028] In some embodiments of the present invention, the subject is a mammal.

[0029] Preferably, the subject is a human being.

[0030] In some embodiments of the present invention, the administration is oral.

[0031] In some embodiments of the present invention, the dosage of the polypeptide is 5-100 mg / kg body weight / day.

[0032] Preferably, the dosage of the polypeptide is 10-50 mg / kg body weight / day.

[0033] In some embodiments of the present invention, the polypeptide is administered once daily or in divided doses.

[0034] In some embodiments of the present invention, the polypeptide is administered continuously for at least 7 days.

[0035] Preferably, the polypeptide is administered continuously for more than 4 weeks.

[0036] In some embodiments of the present invention, the method further includes the step of administering a second therapeutic agent to the subject.

[0037] Preferably, the second therapeutic agent is a type 5 phosphodiesterase inhibitor.

[0038] More preferably, the type 5 phosphodiesterase inhibitor is selected from at least one of sildenafil, tadalafil, or vardenafil.

[0039] In some embodiments of the present invention, the polypeptide is administered simultaneously with the second therapeutic agent.

[0040] In some embodiments of the present invention, the polypeptide and the second therapeutic agent are administered separately.

[0041] In some embodiments of the present invention, the polypeptide is administered sequentially with the second therapeutic agent.

[0042] In some embodiments of the present invention, the improvement of male sexual function includes any one or a combination of the following:

[0043] (i) Increase the sex organ index;

[0044] (ii) Increase testosterone levels;

[0045] (iii) Prolong the duration of sexual intercourse;

[0046] (iv) Enhance libido;

[0047] (v) Improves erectile function.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. This invention provides a novel mechanism for improving male sexual function that differs from existing PDE5i drugs.

[0050] Existing PDE5i drugs maintain the relaxation of corpus cavernosum smooth muscle by inhibiting PDE5 activity and reducing cGMP hydrolysis, targeting downstream effects of vasodilation. This invention, however, works by increasing sex organ indices and promoting testosterone synthesis, differing from existing technologies in its mechanism of action and providing a new technical pathway for improving male sexual function.

[0051] 2. This invention has achieved significant improvements in pharmacodynamics.

[0052] Animal experiments confirmed that after administering TYVPKAF peptide at 100 mg / kg / day for 7 days, the sexual interaction time in male mice in the treatment group was significantly prolonged compared to the control group (P < 0.05); after administering the same dose to 56-week-old male mice for 32 weeks, the sex organ index and serum testosterone levels in the treatment group were significantly increased compared to the control group (P < 0.01). This indicates that the present invention has clear efficacy in both short-term behavioral improvement and long-term organ / hormone level improvement.

[0053] 3. This invention has good safety.

[0054] In a 32-week long-term dosing experiment, no adverse reactions such as death, abnormal behavior, reduced food intake, or abnormal weight loss were observed in the treated animals, indicating that the present invention has good safety within the effective dosage range.

[0055] 4. This invention is convenient for administration and suitable for long-term use.

[0056] The polypeptides described in this invention can be administered orally without injection, resulting in high patient compliance and making them particularly suitable for clinical scenarios requiring long-term medication.

[0057] 5. The raw materials for this invention are readily available, making it suitable for industrial production.

[0058] The polypeptides described in this invention can be obtained by extraction from deer antler resources or prepared on a large scale by solid-phase chemical synthesis. The source of raw materials is not limited and the production cost is controllable. Attached Figure Description

[0059] Figure 1is the high performance liquid chromatography diagram for determining the purity of linear peptide TYVPKAF in Example 1 of the present invention.

[0060] Figure 2 is the mass spectrogram for identifying the linear peptide TYVPKAF in Example 1 of the present invention.

[0061] Figure 3 is the bar statistical diagram of the interaction time of male mice in Example 2 of the present invention.

[0062] Figure 4 is the bar statistical diagram of the male mouse sexual organ index in Example 3 of the present invention.

[0063] Figure 5 is the bar statistical diagram of serum testosterone level of male mice in Example 3 of the present invention. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is explained in more detail in combination with specific examples and the accompanying drawings. In the process of implementing the present invention, except for the experimental steps, conditions and methods described in detail below, the rest adopt the technical means and common general knowledge commonly used in the art, without additional special limitations.

[0065] It should be noted that the examples described in this text are only some typical examples of the present invention, not all implementations. The following description of the exemplary examples is only used to illustrate the technical solution, and does not constitute any limitation on the present invention and its application scenarios.

[0066] In the process of presenting and discussing each example, the specific values, parameters, etc. involved are only for reference as examples and do not have limiting effect. Therefore, other implementations based on the inventive concept can adjust the relevant values according to actual needs.

[0067] In addition, the test materials used in the examples are conventional biochemical reagents on the market unless otherwise specified, and the experimental operations also follow the standard procedures in the art.

[0068] Materials and sources

[0069] The experimental animals are SPF grade C57BL / 6j mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal production license number: SCXK (Beijing) 2021-0006. The mice are kept in the SPF grade barrier system of the Laboratory Animal Center of Shanghai Sixth People's Hospital, with a temperature of (22±2) °C, a humidity of (50±10)%, a 12 h / 12 h light-dark alternating illumination, and free access to food and water. All animal experimental protocols have been reviewed and approved by the Animal Ethics Committee of Shanghai Sixth People's Hospital (approval number: SYXK-2023-0058), and are strictly implemented in accordance with the "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T 35892-2018).

[0070] The main reagents and instruments involved in the embodiments of this invention are as follows:

[0071] The mouse testosterone (T) enzyme-linked immunosorbent assay kit (CSB-E05101m) was purchased from Wuhan Huamei Biotechnology Co., Ltd.; the multi-functional microplate reader (model: BioTek Epoch) was purchased from BioTek Inc., USA; the low-temperature high-speed centrifuge (model: Eppendorf 5424R) was purchased from Eppendorf GmbH, Germany; and the precision electronic balance (accuracy 0.1 mg) was purchased from Mettler Toledo, Switzerland.

[0072] In the following examples, all quantitative data are expressed as mean ± standard deviation (Mean ± SD). The two-tailed unpaired Student's t test was used to compare the two groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0073] Example 1: Method for preparing polypeptides

[0074] Using Wang resin as a carrier, Fmoc-Phe (F), Ala (A), Lys (K), Pro (P), Val (V), Tyr (Y), and Thr (T) were sequentially added from the C-terminus to the N-terminus, with stepwise coupling using an HBTU / HOBt system. The coupling time was controlled at 30-45 minutes. After each step of Fmoc removal, the reaction was confirmed to be complete by ninhydrin detection. After synthesis, a lysis reagent of TFA:TIS:H2O = 95:2.5:2.5 was used, and the reaction was carried out at 20°C in the dark for 2 hours. The resin was separated by filtration, and the lysis buffer was collected. Ten volumes of pre-cooled anhydrous diethyl ether were added to the lysis buffer, and the mixture was precipitated at -20°C for 3 hours. The precipitate was collected by centrifugation at 2500×g for 10 minutes, washed repeatedly with anhydrous diethyl ether three times, and dried under vacuum to obtain the crude peptide.

[0075] The crude peptides were dissolved in ultrapure water using ultrasound to prepare a solution with a concentration of approximately 20-30 mg / mL. The solution was then filtered through a 0.22 μm aqueous microporous membrane to completely remove insoluble solids, yielding a clear crude peptide solution. A two-step chromatography combined with nanofiltration or a desalting column process simultaneously achieved purity enhancement and complete desalting, resulting in a final product with a stable purity exceeding 95%.

[0076] An Inertsil ODS-SP C18 (4.6×250mm×5um) column was used. Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA acetonitrile solution. A gradient elution of 25%-40% in phase B was set, and the flow rate was controlled at 1 mL / min. Detection was performed at 214 nm, and the main peak component of TYVPKAF was collected. A hydrophobic cation exchange resin column with a high aspect ratio of 5:1~20:1 was used to adsorb the collected HPLC main peak solution onto the column. Residual hydrophobic impurities and short peptide byproducts were washed away with a low-concentration salt buffer, and the target peptide was collected by elution with a high-salt buffer to further remove impurities with different charge properties. A C18 desalting column was used. Column equilibration was achieved sequentially with 80% acetonitrile and 3% ACN / H2O / 0.1% TFA. The peptide solution was then loaded onto the column for adsorption. After washing with 3% ACN / H2O / 0.1% TFA for 5 column volumes to completely remove salts, the peptides were collected by elution with 80% ACN / H2O / 0.1% TFA. A 500 Da nanofiltration membrane was used to retain TYVPKAF. The ion-exchange eluent was concentrated by nanofiltration and repeatedly replaced with water to completely remove small molecule salts and residual TFA. The desalted peptide solution was pre-frozen to -40°C and then freeze-dried under vacuum to obtain a white lyophilized peptide powder. The final results were verified by both HPLC and LC-MS. Figure 1 As shown, HPLC confirmed that the purity of TYVPKAF was 98.941%. Figure 2 As shown, the molecular weight is approximately 825, consistent with the theoretical value, and there is no obvious salt residue.

[0077] Example 2: Effects of peptides on sexual interaction ability in adult male rats

[0078] 1. Laboratory animals and grouping

[0079] Twelve healthy, 8-month-old SPF-grade male C57BL / 6j mice, weighing 30–35 g, were selected. The twelve male mice were randomly divided into two groups of six each using a random number table: a blank control group (Veh group) and a drug-treated group (PP group). Twelve 10-week-old, inexperienced C57BL / 6j mice were also selected for a mating experiment.

[0080] 2. Administration method

[0081] PP group: The linear heptapeptide TYVPKAF of the present invention was dissolved in sterile drinking water and administered by gavage at a dose of 100 mg / kg body weight (gavage volume calculated as 0.1 mL / 10 g body weight) once a day for 7 consecutive days.

[0082] Veh group: Administered an equal volume of sterile drinking water, with the same frequency and volume of gavage as the PP group.

[0083] 3. Behavioral testing

[0084] 0.5 hours after the 7th administration, each male mouse was placed in a transparent mating observation box (length × width × height = 40 cm × 30 cm × 30 cm) with a corresponding number and no mating experience. The entire process was recorded using an infrared camera for 20 minutes. The experimental environment was kept quiet with a light intensity ≤20 Lux.

[0085] 4. Behavioral indicators for judgment

[0086] Following the criteria for judging sexual behavior in mice, two observers unaware of the experimental groupings independently reviewed the video recordings and recorded the interaction time between each male mouse and the female mouse within 20 minutes. Interaction time was defined as the cumulative time (in seconds) of mounting, insertion, and ejaculation. The average of the two observers' records was used for statistical analysis.

[0087] 5. Experimental Results

[0088] Experimental results are as follows Figure 3 As shown, the average interaction time within 20 minutes was (15.33±20.83) seconds in the Veh group and (74.33±43.03) seconds in the PP group. Compared with the Veh group, the interaction time in the PP group was significantly prolonged (t = 3.023, P<0.05). The results indicate that continuous gavage administration of the linear heptapeptide TYVPKAF of this invention for 7 days can significantly prolong the sexual interaction time in male mice, suggesting that this peptide has a pharmacological effect of enhancing the sexual performance of adult males.

[0089] Example 3: Effects of the present invention's polypeptide on the sex organ index and serum testosterone levels in aged male rats

[0090] 1. Laboratory animals and grouping

[0091] Twenty-four healthy 56-week-old SPF-grade male C57BL / 6j mice, weighing 38–45 g, were selected. They were randomly divided into two groups of 12 mice each using a random number table: a blank control group (Veh group) and a drug-treated group (PP group).

[0092] 2. Administration method

[0093] PP group: The linear heptapeptide TYVPKAF of this invention was dissolved in sterile drinking water and administered to mice at a dose of 100 mg / kg / day. Fresh drug solution was changed daily to ensure that the actual daily intake of each mouse reached the target dose. The administration was continued for 32 weeks (224 days).

[0094] Veh group: Sterile drinking water was given, and the feeding method and cycle were the same as those of the PP group.

[0095] 3. Sample Collection and Processing

[0096] Mice were fasted for 12 hours after the last administration, but allowed free access to water. They were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and their final body weight was measured. Immediately afterward, approximately 1.0 mL of whole blood was collected from the orbital venous plexus into an EDTA-K2 anticoagulant tube, centrifuged at 3000 rpm for 20 minutes at 4°C, and the supernatant plasma was separated and stored at -80°C for later analysis.

[0097] After blood collection, both testes were quickly dissected and removed. The epididymis and surrounding adipose tissue were carefully dissected, rinsed with pre-cooled PBS buffer, blotted dry with filter paper, and weighed wet. The sex organ index was calculated as: bilateral testicular weight / body weight.

[0098] 4. Serum testosterone level detection

[0099] Serum testosterone concentration was measured using a mouse testosterone (T) enzyme-linked immunosorbent assay kit (CSB-E05101m). The procedure was strictly followed according to the kit instructions. The specific steps are as follows:

[0100] (1) Reagent equilibration: Remove the kit components from 2~8℃ and equilibrate at room temperature for 15~30 minutes. Dilute the 30× concentrated washing solution with distilled water to 1× working concentration.

[0101] (2) Preparation of standard: Prepare five different concentration gradients (S1~S5) of standard by serial dilution, and set up a zero concentration standard well (S0).

[0102] (3) Sample addition: Add 50 μL of standard or plasma sample to be tested to each well, followed by 100 μL of horseradish peroxidase-labeled detection antibody (except for blank wells), and seal with sealing film.

[0103] (4) Incubation: Incubate at room temperature in the dark for 120 minutes with the shaker speed at 300 rpm.

[0104] (5) Washing: Discard the liquid in the hole, add 1× washing liquid to each hole, let stand for 30 seconds and then discard, repeat 3 times, and pat dry on thick absorbent paper for the last time.

[0105] (6) Color development: Add 100 μL of TMB color development solution to each well and incubate at room temperature in the dark for 15 minutes.

[0106] (7) Termination and reading: Add 50 μL of stop solution to each well, mix well, and the reaction system will change from blue to yellow. Within 15 minutes after adding the stop solution, measure the optical density (OD value) of each well at a wavelength of 450 nm using a microplate reader.

[0107] 5. Concentration Calculation

[0108] A standard curve was plotted using a four-parameter logistic curve fitting method, with the concentration of each standard as the x-axis and the corrected OD value (OD value of each standard minus the OD value of well S0) as the y-axis. The corrected OD value of the test sample was substituted into the standard curve equation to calculate the serum testosterone concentration (ng / mL).

[0109] 6. Experimental Results

[0110] (1) Sex organ index: such as Figure 4 As shown, the sex organ index of the Veh group was 0.0053±0.0007, and that of the PP group was 0.0071±0.0013. The PP group was significantly higher than that of the Veh group (t = 4.087, P<0.01).

[0111] (2) Serum testosterone levels: such as Figure 5 As shown, serum testosterone levels in the Veh group were (1.4016±0.5541) ng / mL, while those in the PP group were (3.2322±1.2948) ng / mL. The PP group was significantly higher than the Veh group (t = 3.184, P < 0.01).

[0112] The results of this embodiment show that long-term oral administration of the linear heptapeptide TYVPKAF of the present invention can significantly increase the sex organ index and serum testosterone level in aged male mice, suggesting that this polypeptide can improve male sexual function from the upstream through a dual pathway of promoting sex organ development / delaying decline and promoting testosterone synthesis.

[0113] The embodiments described in this specification are only intended to help those skilled in the art better understand and implement the present invention. For those skilled in the art, appropriate adjustments, substitutions, or extensions can be made to these embodiments based on the content disclosed in this invention without requiring creative effort, and the basic principles of the invention can be applied to other similar scenarios. Therefore, the disclosed embodiments should not be considered as limiting the scope of protection of this invention. Any equivalent modifications, improvements, or alternatives made based on this invention without departing from its essential spirit should fall within the scope of protection of this invention.

Claims

1. The use of a polypeptide in the preparation of a drug for improving male sexual function, characterized in that, The polypeptide contains the amino acid sequence shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The polypeptide is a conserved variant of the sequence shown in SEQ ID NO:1, and the conserved variant has the activity of improving male sexual function; preferably, the conserved variant is a variant obtained by conserved substitution of 1 to 3 amino acids in the sequence shown in SEQ ID NO:

1.

4. The application according to any one of claims 1 to 3, characterized in that, The C-terminus of the polypeptide is either a free carboxyl group or an amidated group, preferably an amidated group.

5. The application according to any one of claims 1 to 4, characterized in that, The polypeptide is derived from deer antler, or the polypeptide is a synthetically produced polypeptide, preferably a polypeptide synthesized using a solid-phase synthesis method.

6. The application according to any one of claims 1 to 5, characterized in that, The improvement of male sexual function includes any one or a combination of the following: (i) Improve the sex organ index, preferably improve the testicular index; (ii) Increase testosterone levels, preferably increase serum testosterone levels; (iii) Prolong the duration of sexual intercourse; (iv) Enhance libido; (v) Improves erectile function.

7. The application according to any one of claims 1 to 6, characterized in that, The drug is an oral preparation; Preferably, the oral preparation is selected from any one of oral liquid, tablets, capsules, granules or oral instant films.

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