Application of calcium ion channel, alpha1 adrenergic receptor and alpha2 adrenergic receptor in premature ejaculation treatment

Abstract

The invention discloses application of a calcium ion channel, an alpha 1 adrenergic receptor and an alpha 2 adrenergic receptor in treatment of premature ejaculation. The premature ejaculation can be remarkably improved by independently inhibiting calcium ion channels, alpha 1 adrenergic receptors and alpha 2 adrenergic receptors. Meanwhile, through combined application of the three target spots, premature ejaculation can be further improved. Besides, the calcium ion channel blocker and the alpha1 and alpha2 adrenergic receptor antagonists are prepared into an external preparation, so that the absorption of the medicine can be remarkably improved and the medicine effect can be quickly exerted in a smearing medication mode, and the systemic side effects of oral medication are avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of calcium ion channel and alpha 1 and alpha 2 adrenergic receptors in the treatment of premature ejaculation. BACKGROUND

[0002] Premature ejaculation (PE) is one of the most common sexual dysfunctions in men, mainly manifested as a significant shortening of the ejaculatory latency and a decline in the ability to control ejaculation, often accompanied by psychological distress or interpersonal relationship pressure. According to the definition of the International Society for Sexual Medicine (ISSM), primary PE refers to a condition in which a man has a ejaculatory latency of less than 1 minute since the beginning of the first sexual behavior; secondary PE refers to a condition in which a man who once had normal ejaculatory control has a ejaculatory latency of less than 3 minutes at a certain stage, accompanied by subjective distress or a decline in sexual satisfaction. Epidemiological studies have shown that the prevalence of PE is about 30% to 40% in adult men worldwide. According to a review of literature reported in Nature Reviews Urology, the incidence of PE is highly consistent across cultures and regions, and is one of the most widespread problems affecting the quality of sexual life in clinical practice (Nature Reviews Urology, 2006, 3:381-395). PE has a significant adverse effect on the psychological and physical health of patients and their partners. Patients often have psychological burdens such as anxiety, depression, and a decline in self-esteem, and may also cause or exacerbate emotional alienation and disharmony in sexual life between partners, thereby leading to a decline in sexual life satisfaction and overall quality of life. Long-term PE that has not been effectively treated may even progress to more serious sexual dysfunctions such as erectile dysfunction and hypoactive sexual desire disorder.

[0003] Currently, the drugs on the market for the treatment of PE are mainly divided into two categories:

[0004] The first category is systemic (oral) drugs, mainly represented by selective serotonin reuptake inhibitors (SSRIs), such as Dapoxetine. The mechanism of action of these drugs is to prolong the action time of 5-hydroxytryptamine signals in the central nervous system, delay the ejaculation reflex, and prolong the ejaculatory latency. This type of drug shows good efficacy in some patients, but still has the following shortcomings: systemic administration is likely to cause side effects such as headache, nausea, diarrhea, decreased sexual desire, and erectile dysfunction; the efficacy varies greatly among individuals, and long-term use may affect patient compliance due to metabolism and adjustment of blood drug concentration.

[0005] The second type is a topical (application) drug, common representatives include Lidocaine spray, Benzocaine ointment, Tannic acid ointment, etc., the main mechanism is local anesthesia, which can prolong the ejaculation latency by reducing the sensitivity of the mechanical receptors in the glans penis. The advantages of this type of drug are rapid onset, fewer systemic side effects, and convenient use, so it has been widely used in external spray, delay condom and other products. However, this type of drug only realizes physical inhibition from the sensory input link, and does not deeply regulate the core mechanism of the neural reflex pathway. Clinical observation shows that some patients experience decreased erection intensity and reduced sexual pleasure after using the drug, suggesting that local anesthetics may interfere with the natural conduction of sexual stimulation and sexual pleasure experience. In addition, long-term use may cause dependence or reduced effect, and the improvement space is still significant. SUMMARY

[0006] Based on the transcriptomic analysis of PE rats and normal rats, the present application establishes a deeper understanding of the ejaculation reflex loop, and proposes and verifies a new view that intracellular calcium signals play a key central role in regulating the ejaculation process. Related calcium channels such as mitochondrial calcium uptake channels (MCU) and endoplasmic reticulum calcium release channels (IP3R, RyR2) play a regulatory role in key nodes such as neuronal excitability and smooth muscle contraction. At the same time, the present application further focuses on the fact that the membrane potential-dependent L-type calcium channel (LTCC) as the main channel for extracellular calcium influx also plays an important role in amplifying calcium signals and maintaining muscle cell depolarization, especially in the process of enhancing smooth muscle contraction. There is no systematic research in the current public information and patent literature that uses such calcium channels as local targets for ejaculation regulation. In the preliminary animal experiments, the present application observes that local inhibitors targeting the above calcium channels can effectively prolong the ejaculation latency, but due to the abundance of calcium sources and the existence of multiple regulation mechanisms, the inhibitory effect does not show a linear increase with concentration. This finding suggests that the path of targeting a single calcium signal channel for regulation has complex feedback mechanisms or compensatory regulation, resulting in fluctuating efficacy, further supporting the necessity of multi-target joint intervention.

[0007] In addition, the present application proposes that the alpha 1 adrenergic receptor (alpha 1 receptor) as a key regulatory pathway of the terminal smooth muscle contraction link can also be an effective target for PE treatment. This receptor is widely expressed in the bulbospongiosus, ejaculatory duct, prostate and other parts, and can directly affect the ejaculation propulsion process. Although alpha 1 receptor antagonists are widely used for the treatment of prostate hyperplasia and hypertension in the current public literature, there is no clear drug development targeting this target for local treatment of PE.

[0008] Further, the present application also finds that alpha 2 adrenergic receptor (alpha 2 receptor) also has a delaying ejaculation effect in the regulation of central and peripheral nervous system. Alpha 2 receptor regulates norepinephrine release through a negative feedback mechanism on presynaptic neurons, thereby inhibiting sympathetic nerve excitability and reducing the sensitivity of the ejaculation center. Complementary to the peripheral muscle regulation mechanism of alpha 1 receptor, alpha 2 receptor provides a possible path of inhibition from the source of nerve initiation signal, bringing a new direction of central-peripheral joint control for local intervention strategy, and has a clear synergistic effect.

[0009] In summary, the existing treatment scheme still has obvious improvement space in mechanism depth, target accuracy and individualized regulation. The present application proposes a local drug strategy based on calcium signal regulation and alpha 1 receptor intervention, and expands alpha 2 receptor and other nerve regulation targets, combines multi-path precise inhibition mechanism, optimizes drug concentration and dosage form ratio, provides a new idea and technical path for premature ejaculation treatment, and has a clear clinical transformation potential and patent protection value.

[0010] The action principle of each target of the present application is as follows:

[0011] Alpha 1 adrenergic receptor (alpha 1-AR):

[0012] As a direct driver of the end smooth muscle contraction during ejaculation, local antagonism of alpha 1 receptor can reduce the smooth muscle tension of the ejaculatory duct, the urethral crest and the prostate, thereby delaying the occurrence of the ejaculation reflex.

[0013] Alpha 2 adrenergic receptor (alpha 2-AR):

[0014] Alpha 2 receptor is mainly distributed in the central nervous system and peripheral sympathetic nerve endings, and inhibits the release of norepinephrine in the form of presynaptic feedback. By inhibiting alpha 2 receptor, the excitability and signal discharge frequency of the central sympathetic nerve can be reduced, thereby weakening the central initiation mechanism of the ejaculation reflex from the source of nerve signal. The present application first uses alpha 2 receptor as a new target for premature ejaculation intervention, and combines the terminal muscle control mechanism of alpha 1 receptor to form a central-peripheral double-layer regulation system, thereby enhancing the broad-spectrum and individual adaptability of the intervention effect.

[0015] MCU (Mitochondrial Calcium Uniporter):

[0016] By inhibiting mitochondrial calcium uptake, the calcium buffering capacity in neurons and muscle cells is reduced, the nerve excitability and muscle sustained activation are reduced, and the trigger conditions of the ejaculation reflex link are weakened from the source.

[0017] IP3R (inositol triphosphate receptor) and RyR2 (ryanodine receptor):

[0018] These two endoplasmic reticulum calcium release channels regulate the rapid release of intracellular calcium in smooth muscle and neurons. Inhibition of them can effectively relieve muscle excitement and contraction caused by calcium signal activation.

[0019] L-type calcium ion channel (L-type Calcium Channel, LTCC):

[0020] L-type channels are the main channels for exogenous calcium influx, widely distributed in neurons and smooth muscles of the reproductive system. Under the condition of alpha 1 receptor activation or depolarization, the opening of L-type channels can further enhance the influx of extracellular calcium, and expand the calcium signal amplification effect. Therefore, inhibiting L-type calcium channels can cut off this amplification mechanism, further stabilizing the ejaculation control. The present application introduces this target as a supplementary intervention layer to form a transmembrane-intracellular calcium whole-link regulation closed loop.

[0021] In view of the key deficiencies existing in the current treatment methods of premature ejaculation, including the systemic side effects often accompanied by systemic oral drugs, the lack of molecular targeting mechanism of local topical anesthetics, the instability of the therapeutic effect of single calcium ion channel regulation, and the problem that alpha 1 adrenergic receptor has not been used as a target mechanism for premature ejaculation treatment, the present application carries out systematic experimental research, for the first time verifies the core functional position of alpha 1 adrenergic receptor in the regulation of ejaculation, and further compares the independent and combined inhibition effect differences between the receptor and multiple calcium signal pathways (such as MCU, IP3R, RyR2, L-type).

[0022] At the same time, the present application also explores the role of alpha 2 adrenergic receptor in the neurogenic regulation of ejaculation, and finds that the receptor reduces norepinephrine release through presynaptic feedback mechanism, thereby effectively inhibiting central sympathetic nerve excitability, providing an additional neural level intervention path for delaying ejaculation. The introduction of alpha 2 receptor not only expands the range of adrenergic signal intervention, but also forms a central-peripheral synergy with the peripheral smooth muscle regulation mechanism of alpha 1 receptor, which can achieve more stable and efficient ejaculation regulation effect in combined application.

[0023] Specifically, the present application proposes the following three optional technical routes as potential premature ejaculation intervention strategies: (1) by inhibiting alpha 1 adrenergic receptor, blocking its key signal transduction role in controlling smooth muscle contraction and ejaculation propulsion process; (2) by inhibiting alpha 2 adrenergic receptor, reducing synaptic norepinephrine release, reducing sympathetic nerve excitation trigger, to delay the activation of the ejaculation center; (3) by inhibiting multiple intracellular calcium channels, including mitochondrial calcium uptake channel (MCU), endoplasmic reticulum calcium release channel (IP3R, RyR2), and membrane potential-dependent L-type calcium channel (LTCC), to intervene from multiple links Intracellular calcium dynamic changes. These calcium channels play a key role in neuronal excitability, intracellular calcium storage and release, and smooth muscle depolarization and sustained contraction.

[0024] The above-mentioned targets can be used alone or in combination. Experimental results show that alpha 1 and alpha 2 receptor inhibition can significantly prolong ejaculation latency, and its effect is significantly better than single calcium channel inhibition.

[0025] Therefore, the present application not only establishes the key role of alpha 1 receptor as a core regulatory target in the treatment of premature ejaculation, but also establishes a feasible scheme for combined intervention of multiple calcium channels and alpha 1 receptors. The supplementary intervention of alpha 2 receptor further enriches the regulation level and expands the regulation dimension of the neural signal source. The present application further develops the corresponding local application type drug combination and optimizes the drug concentration and administration method, so that the intervention means has the advantages of precise positioning, stable drug effect and strong individual adaptability.

[0026] The present application provides a new theoretical basis and clinical tool for the treatment of premature ejaculation, especially in the aspects of local external use and multi-target joint control, which realizes three-level intervention from neural signal-calcium channel-muscle reaction, and has significant practical value and popularization potential.

[0027] Specifically, in the first aspect of the present application, one or a combination of calcium ion channels, alpha 1 adrenergic receptors and alpha 2 adrenergic receptors is provided as a target in the treatment of premature ejaculation.

[0028] Further, the calcium ion channel includes MCU, IP3R, RyR2, and L-type calcium ion channel.

[0029] In the second aspect of the present application, one or a combination of calcium ion channel blockers, alpha 1 adrenergic receptor antagonists and alpha 2 adrenergic receptor antagonists is provided for use in the preparation of a drug for treating premature ejaculation.

[0030] Further, the calcium ion channel blocker includes one or a combination of MCU inhibitor, IP3R inhibitor, RyR2 inhibitor, and L-type calcium channel blocker.

[0031] In a third aspect, the present application provides an external preparation comprising at least one of a calcium ion channel blocker, an alpha 1 adrenergic receptor antagonist and an alpha 2 adrenergic receptor antagonist.

[0032] Further, the external preparation is a gel, a cream or a spray.

[0033] Further, the external preparation further comprises Tween 80, Tween 20 or other penetration enhancers.

[0034] It can be understood that the alpha 1 adrenergic receptor antagonist can be selected from phentolamine, phenoxybenzamine, prazosin, terazosin, doxazosin, alfuzosin, tamsulosin, silodosin, bunazosin, indoramin, urapidil or traditional Chinese medicine ingredients having alpha 1 adrenergic receptor antagonistic effect, etc.; the alpha 2 adrenergic receptor antagonist can be selected from yohimbine, midodrine, atipamezole, labetalol, carvedilol or traditional Chinese medicine ingredients having alpha 2 adrenergic receptor antagonistic effect, etc.; the MCU inhibitor can be selected from Ru360, Ru265, mitoxantrone, DS16570511, MCU-i4, MCU-i11, berberine, benzalkonium chloride, amiodarone, KB-R7943, etc.; the IP3R inhibitor can be selected from Halichondrin-C, Halichondrin-A, Halichondrin-B, Halichondrin-D, 2-aminoethoxydiphenyl borate, 7-hydroxyhalichondrin A, etc.; the RyR2 inhibitor can be selected from TMDJ-035, M201-A, dantrolene, Ent-(+)-Verticilide, EL20, chlorodimethylphenol, methyl formate, riluzole, etc.; the L-type calcium channel blocker can be selected from verapamil, diltiazem, nifedipine, amlodipine, felodipine, nitrendipine, nimodipine, lacidipine, lercanidipine, isradipine, cinaldipine, benidipine, manidipine, azelnidipine, barnidipine, benidipine, aranidipine, elgodipine, clevidipine, darodipine, nisoldipine, finidipine, tronidipine, guanoxabenz, flunarizine, etc.

[0035] In summary, the multi-target local intervention premature ejaculation treatment scheme provided by the present application has significant technical advantages and positive effects compared with the existing single-target drug, local anesthetics or systemic oral drugs.

[0036] In terms of drug design and use, the present application innovatively finds that the target is suitable for a local application type, which provides guidance for the development of new drugs of this type, such as gels, creams, sprays, etc. The drug directly acts on the target tissue on the surface of the penis, avoiding the systemic side effects of systemic (oral) drugs, reducing the metabolic burden and improving the local drug concentration.

[0037] The present application has at least the following beneficial effects:

[0038] (1) The application proposes the application of multiple targets in the treatment of premature ejaculation, including alpha 1 adrenergic receptor (alpha 1 receptor), alpha 2 adrenergic receptor, MCU, IP3R, RyR2, L-type calcium channel (LTCC) target. The above-mentioned targets can work alone or in combination to produce synergistic effects. Based on the new alpha 1 adrenergic receptor (alpha 1 receptor) antagonist, alpha 2 adrenergic receptor antagonist, MCU inhibitor, IP3R inhibitor, RyR2 inhibitor, L-type calcium channel (LTCC) inhibitor or multifunctional inhibitor for the treatment of premature ejaculation found by the application.

[0039] (2) The application innovatively finds targets suitable for topical application, providing guidance for the development of new drugs for such dosage forms, such as gels, creams, sprays, etc. The drug directly acts on the target tissue on the surface of the penis, avoiding the systemic side effects of systemic (oral) drug use, reducing metabolic burden, and improving local drug concentration. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The figure is a volcano plot of the differentially expressed genes of normal rats (control group, control, con) and premature ejaculation rats.

[0041] Figure 2 The figure is a GO enrichment bubble chart of differentially expressed genes.

[0042] Figure 3 The figure is a KEGG enrichment column chart of differentially expressed genes.

[0043] Figure 4 The figure is a Reactome enrichment bubble chart of differentially expressed genes

[0044] Figure 5 The figure is the change of ejaculation probability after local application of drugs on the penis.

[0045] Figure 6 The figure is the change of ejaculation time after local application of drugs on the penis.

[0046] Figure 7 The figure is the change of penile erection length before and after local application of drugs on the penis.

[0047] Figure 8 The figure is the change of JAK2 / eNOS signaling pathway protein expression after local application of alpha 1 inhibitor on the penis.

[0048] Figure 9 The figure is the change of ERK1 / 2, p-ERK1 / 2 signaling pathway protein expression after local application of alpha 2 inhibitor on the penis.

[0049] Figure 10 The figure is the change of MCU, PDH, P-PDH protein expression after local application of MCU inhibitor on the penis.

[0050] Figure 11 The effects of different drug concentrations on the expression of IP3R and RyR2 proteins.

[0051] Figure 12 The effects of different drug concentrations on the expression of IP3R and RyR2 proteins after silencing IP3R with siRNA were investigated. Detailed Implementation

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0053] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0054] The following specific embodiments illustrate the solution proposed in this invention:

[0055] Example 1

[0056] 1. Drug preparation

[0057] Drug concentration and ratio:

[0058] The embodiments of the present invention are based on the in vitro IC50 of inhibitors for each target. 50 Based on the drug concentration range suitable for topical penile application, one or more concentrations were selected, considering factors such as IC50, bioavailability, transdermal absorption rate, and local efficacy response curves. In actual use, the concentration used varies depending on the in vitro IC50 of the drug. 50 The efficacy, bioavailability, transdermal absorption rate, local pharmacodynamic response curve, and administration method may vary. To balance the convenience of target research, efficacy, permeability, and tissue safety, the local application concentrations for each drug are as follows:

[0059] α1-adrenergic receptor antagonist (Silodosin): The effects of low concentration (2 μM) and high concentration (10 μM) on ejaculation time were studied here. It blocks the signal at the end of smooth muscle contraction and exerts a delaying effect on ejaculation.

[0060] Alpha2 adrenergic receptor antagonist (Yohimbine): Two local concentrations were set, 25 μM (low concentration) and 100 μM (high concentration), to evaluate the inhibitory effect on ejaculation reflex and local tissue tolerance at different doses;

[0061] MCU inhibitor (MCU-i4): The concentration was 15 μM, which could effectively reduce neuronal excitability and prolong ejaculation latency;

[0062] IP3R inhibitor (Xestospongin C) and RyR2 inhibitor (Ruthenium Red): Different concentrations were used to study the effect on ejaculation time. The concentrations of Xestospongin C were 10, 25, 50, and 80 μM, and the concentration of Ruthenium Red was 25 μM, which was suitable for regulating the calcium signaling pathway related to smooth muscle contraction.

[0063] L-type calcium channel blocker (Verapamil): The concentration was 15 μM, which could inhibit membrane potential-dependent calcium influx and enhance overall calcium regulation.

[0064] The above drugs can be used alone as needed, and the combination of MCU inhibitor (MCU-i4), IP3R inhibitor (Xestospongin C), RyR2 inhibitor (Ruthenium Red), and L-type calcium channel blocker (Verapamil) is used.

[0065] To ensure that the drugs are fully dispersed and absorbed on the surface of the penis skin, all drug solutions are prepared using a water-soluble carrier system containing 2% Tween-80. Tween-80, as a non-ionic surfactant, has good wetting, emulsifying, and enhanced transdermal diffusion ability, which helps to effectively deliver small molecule inhibitors to the target tissue and improve local drug efficacy.

[0066] Dosage form design: prepared as gel, cream, spray, etc. for local application, to ensure that the drugs can be evenly covered and absorbed on the penis.

[0067] 2、Animal experiment

[0068] 2.1 Experimental preparation

[0069] First, the experimental rats were fixed, and their limbs were gently fixed to avoid interference with the drug administration process, and the penis area was fully exposed to ensure that the drugs could be evenly and accurately applied to the target surface. Deep anesthesia should not be performed during the experiment. If anesthesia is required, the rats should be awakened before the experiment begins, otherwise the central nervous system will be affected and the final experimental results will be affected.

[0070] 2.2 Experimental grouping design:

[0071] The present application sets up 6 groups, each of which is treated according to different drug concentrations, and tests the individual effects of MCU inhibitors, IP3R inhibitors + RyR2 inhibitors, alpha 1 receptor antagonists, alpha 2 receptor antagonists and L-type calcium channel blockers, as well as the combined use of drugs, i.e. MCU inhibitors, IP3R inhibitors, RyR2 inhibitors, L-type calcium channel blockers. At the same time, a control group is set up, which only uses a single solvent or a blank matrix to exclude background interference.

[0072] 2.3 Drug administration steps

[0073] The drug is administered in several doses, and the specific operation is as follows:

[0074] (1) 10 μL of the prepared drug solution is taken using a pipette, and is evenly applied to the surface of the rat penis, and is left to stand for 10 minutes to ensure that the drug solution is fully penetrated and initially absorbed;

[0075] (2) 10 μL of the same drug solution is again taken using a pipette and is repeatedly applied to the same site, with a total drug dose of 20 μL, and is left to stand for another 10 minutes to ensure that the drug reaches the set concentration and is fully absorbed.

[0076] 2.4 Animal behavior experiment:

[0077] After the second administration is completed, the drug is left to stand for 10 minutes, and after another 10 minutes (i.e. 30 minutes after the first administration), a vibration rod with a fixed frequency of 30-70 Hz is used to stimulate the middle and upper regions of the rat penis to induce ejaculation. The vibration frequency is fixed for experiments in the same group, and is selected according to the species of the rat and the material and structure of the vibration rod.

[0078] 2.5 Western Blot experiment

[0079] In the examples of the present application, in order to verify the regulatory effect of the drug composition on each molecular target, the related proteins in the penis tissue samples are detected by Western Blot method. The specific operation is as follows: first, the penis tissue of the experimental animal is taken, and a lysis buffer containing protease and phosphatase inhibitors is used for thorough homogenization. After centrifugation to remove the insoluble material, the supernatant is collected and the protein is quantified to ensure that the sample amount of each experimental group is consistent. Then, the same amount of protein sample is separated by SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. After the transfer is completed, a suitable blocking solution is used to block the non-specific binding sites to reduce the background signal interference.

[0080] In the detection phase, specific primary antibodies targeting α1 and α2 adrenergic receptors, MCU, IP3R, RyR2, L-type calcium channels, and downstream signaling molecules (such as JAK2 and eNOS) were used for incubation, followed by detection using corresponding secondary antibodies. Signal visualization was recorded using chemiluminescence (ECL) or a fluorescence imaging system, ensuring clear and analyzable protein bands were obtained within the appropriate exposure range.

[0081] During the results analysis, image analysis software was used to measure the grayscale values ​​of each protein band, and the expression of internal reference proteins (β-actin or GAPDH) was used as a correction to calculate the relative expression level of the target protein. All experiments included negative controls, positive controls, and blank control groups, and were repeated at least three times to ensure the reproducibility and reliability of the results. The obtained protein expression data were compared with animal behavioral experimental results (such as prolonged ejaculation latency and decreased ejaculation probability) to confirm the consistency and effectiveness of multi-target combined intervention at the molecular and functional levels, providing experimental evidence for the treatment strategy proposed in this invention.

[0082] 3. Experimental Results and Effect Evaluation

[0083] (1) Omics results

[0084] In this study, the present invention performed transcriptomic differential analysis on penile tissues from normal rats and premature ejaculation model rats. Volcano plot results showed ( Figure 1 A total of 818 genes were significantly upregulated in the model group, 190 genes were significantly downregulated, and the remaining approximately 19,767 genes showed no significant difference. This suggests that there is extensive transcriptional remodeling at the molecular level in the penile tissue of premature ejaculation rats, with upregulated genes being the main component. This may involve abnormal activation of neural excitation, calcium signaling pathways, and adrenergic receptors.

[0085] Further GO enrichment analysis ( Figure 2) showed that, at the biological process (BP) level, the differential genes were significantly enriched in muscle system process, muscle contraction, striated muscle cell differentiation, actomyosin structure organization, etc., indicating that muscle contraction and related regulation played a core role in the pathological mechanism of PE; at the cellular component (CC) level, the differential genes were concentrated in contractile fiber, sarcomere, Z disc, sarcolemma, sarcoplasmic reticulum, etc., reflecting that the key cell structures involved in excitation-contraction coupling had changed significantly; at the molecular function (MF) level, the differential genes were enriched in actin binding, microtubule binding, cytoskeletal motor activity, structural constituent of muscle, etc., suggesting that the abnormal function of cytoskeleton dynamics and contractile proteins might be an important factor leading to ejaculation control disorder.

[0086] KEGG pathway enrichment results Figure 3 ) further revealed that the differential genes were significantly enriched in pathways closely related to smooth muscle contraction and calcium signaling, including cardiac muscle contraction, calcium signaling pathway, vascular smooth muscle contraction, and cardiac muscle cell adrenergic signaling, and also involved MAPK, PI3K-Akt, cGMP-PKG, and other signal transduction axes. This result suggests that PE is closely related to increased sympathetic nerve excitation, abnormal calcium homeostasis, and smooth muscle excitation-contraction coupling disorder.

[0087] Reactome pathway enrichment analysis Figure 4 ) further proved that the differential genes were significantly concentrated in muscle contraction, smooth muscle contraction, striated muscle contraction, cardiac conduction, and other excitation-contraction related pathways, and also significantly enriched in RHO GTPase effector, PI3K signal cascade, and IRS-mediated signal transduction molecular networks, suggesting that cytoskeleton remodeling and intracellular signal abnormalities also play a role in the mechanism of PE. It is worth noting that the ion homeostasis and neuronal system items were significantly enriched, further confirming the key role of calcium signaling disorder and sympathetic nerve hypersensitivity in the occurrence of PE.

[0088] In summary, based on the above omics evidence, the present application clearly proposes a1 / a2 adrenergic receptors and multiple calcium channels (MCU, IP3R, RyR2, LTCC) as core intervention targets, and accordingly constructs a multi-target linkage regulation strategy, providing a solid theoretical and experimental basis for subsequent local drug experimental design and patent protection.

[0089] (2) Animal experiment results

[0090] In the ejaculation behavior experiment, the research team compared and analyzed the drug effects of a1 adrenergic receptor antagonists, a2 adrenergic receptor antagonists, MCU inhibitors, IP3R+RyR2 inhibitors, and the comprehensive effects of multi-target combination drug use.

[0091] The results are shown in Figure 5 and Figure 6 , showing the change trend of ejaculation probability and ejaculation latency of each group of animals. In terms of ejaculation probability, the blank control group had an ejaculation rate of 83.33%; after a1 and a2 receptor antagonists treatment, the ejaculation rate decreased to 25%; the MCU inhibitor group was 50%; the IP3R / RyR2 inhibitor group was 41.66%; the L-type calcium ion channel blocking group was 41.66%; and the multi-target combination drug use group further decreased to 33.33%. If the rat did not ejaculate for more than 60 minutes, the test was considered to be over. The results showed that each single target intervention could reduce the ejaculation rate, and multi-target synergistic intervention had more significant comprehensive inhibition effect.

[0092] Secondly, in terms of ejaculation latency, the average latency of the control group was 18.24±2.14 minutes. After a1 receptor antagonist intervention, the latency was prolonged to 45.35±1.45 minutes; after a2 receptor antagonist intervention, the latency was prolonged to 47.46±1.70 minutes; the MCU inhibitor group was prolonged to 23.34±1.30 minutes; the IP3R / RyR2 inhibitor group was 24.35±0.93 minutes; the L-type calcium ion channel blocking group was 22.59±2.30 minutes. Notably, in the combination drug group, the ejaculation latency was significantly prolonged to 28.41±2.39 minutes, which was significantly better than any single target group, and the difference was statistically significant (P<0.01).

[0093] (2) Safety observation

[0094] After drug smearing, the general physiological indicators such as activity, food intake, water intake, excretion, and body weight change of each group of animals were observed, and blood was collected for blood routine and blood biochemical analysis.

[0095] General state is good: all rats had no obvious stress behavior during the experimental period, and the activity, diet, and excretion were normal;

[0096] Local observation: No signs of irritation such as redness, swelling, erosion, or hair loss were observed on the penile surface of any group of mice. Furthermore, there was no significant difference in penile erection length before and after treatment. Figure 7 ).

[0097] The conclusion shows that the multi-target drug composition proposed in this invention effectively prolongs the ejaculation latency period while exhibiting good biocompatibility and local tolerability.

[0098] (3) Target protein expression analysis (Western Blot results)

[0099] α1 receptor expression analysis:

[0100] like Figure 8 As shown in the Western blot results, α1 receptor protein expression was significantly decreased in both the low-dose and high-dose groups compared to the control group, exhibiting a stable dose-dependent inhibitory trend. JAK2, a classic intracellular signal transduction kinase, also showed a synchronous decrease in expression with α1 receptor inhibition, indicating that JAK2 may be downstream of the α1 receptor signaling pathway and participate in regulating ejaculation-related smooth muscle responses. In contrast, eNOS protein was significantly upregulated after receptor antagonist intervention, especially in the high-dose group. This phenomenon suggests that α1 receptors may have an indirect inhibitory effect on eNOS expression; antagonism of eNOS can relieve this inhibition, enhance local NO production, and thus promote vasodilation or smooth muscle relaxation, helping to prolong ejaculation latency.

[0101] The grayscale statistical analysis results were consistent with the Western blotting bands, further confirming the broad impact of α1 receptor intervention on downstream signaling pathways and supporting the premature ejaculation intervention strategy proposed in this invention, which uses α1 as the core regulatory node. The entire experimental system exhibited good data consistency and reproducibility, and β-actin expression was stable, demonstrating balanced protein loading and reliable experimental results.

[0102] α2 receptor expression analysis:

[0103] like Figure 9 As shown, Western blotting results indicated that treatment with the α2-adrenergic receptor antagonist (Yohimbine) led to a dose-dependent upregulation of α2-AR protein expression, suggesting a typical feedback compensation mechanism following receptor function blockade. Simultaneously, downstream signaling pathways also showed...

[0104] ERK1 / 2 total protein expression did not change significantly, but phosphorylated ERK1 / 2 (p-ERK1 / 2) decreased significantly at low concentrations and partially recovered at high concentrations, indicating that a2-AR intervention can regulate the nerve excitability and muscle contraction pathway by inhibiting p-ERK1 / 2 activity, especially at low doses, which supports its potential value in delaying ejaculation.

[0105] MCU pathway analysis:

[0106] As Figure 10 The results show that after using the MCU inhibitor, the MCU protein band is significantly weakened, which proves that local administration can effectively reduce the expression level of MCU in the target tissue. Correspondingly, the total PDH (pyruvate dehydrogenase) protein level does not change much after MCU inhibition, showing relative stability. However, the P-PDH (phosphorylated PDH, inactive state) band is significantly enhanced after MCU inhibition, suggesting that MCU inhibition reduces the activation level of PDH, which may affect the energy metabolism capacity of the local tissue. The β-actin band is stable, proving that the loading is uniform and the experimental results are reliable.

[0107] IP3R / RyR2 expression analysis:

[0108] In the single drug experiment, the drug concentration was set to 0, 10, 25, 50, and 80 μM. The results show that the protein expression of IP3R and RyR2 gradually decreases at concentrations of 10-25 μM, and the inhibition effect is strongest at 25 μM, with the shallowest band. However, at high concentrations of 50 μM and 80 μM, the band is deepened, and the inhibition effect is weakened, showing a clear concentration-dependent failure or reverse regulation trend. This indicates that when using the inhibitor alone, the drug effect is best at moderate concentrations, and high concentrations may activate compensatory mechanisms, causing the target expression level to rise. Figure 11

[0109] In the combined drug experiment after siRNA silencing IP3R, 0, 25, and 50 μM drug concentrations were used. The results show that siRNA itself (0 μM drug) can significantly reduce the IP3R protein level, and the inhibition effect is further enhanced after combined with 25 μM drug, with the shallowest band, showing a clear synergistic effect. However, when the drug concentration increases to 50 μM, the band intensity rises, indicating that the inhibition effect is weakened at too high a concentration, and there is also a failure or reverse regulation problem. For RyR2 protein, under the condition of siRNA, 25 μM drug concentration shows good inhibition effect, and the band becomes shallow, while at 50 μM concentration, the inhibition effect is not as expected, and the stability decreases. Figure 12

[0110] ​​In general, the WB experiment results show that the optimal inhibitory concentration of the single drug and the combination of siRNA and the drug is 25 μM. Under high concentration conditions, not only can not bring stronger inhibitory effect, but also may activate the compensatory mechanism or produce a counter-regulatory effect in the cell. Therefore, in the subsequent optimization of the drug regimen, the concentration ratio needs to be focused on to ensure that the optimal protein inhibition effect is achieved within the safe concentration range.

[0111] It should be noted that in this paper, the term "includes", "contains" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or includes elements inherent to such process, method, article or device. Without more limitations, the element defined by the statement "includes a" does not exclude the presence of other identical elements in the process, method, article or device including the element.

[0112] The above-mentioned embodiment number of the application is only for description, not representing the advantages and disadvantages of the embodiments.

[0113] The above embodiments are only used to illustrate the technical solutions of the present application and not limit it. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. Application of calcium ion channels, α1-adrenergic receptors, and α2-adrenergic receptors as targets in the treatment of premature ejaculation.

2. The application according to claim 1, characterized in that, The calcium ion channels include MCU, IP3R, RyR2, and L-type calcium ion channels.

3. The use of one or a combination of calcium channel blockers, α1-adrenergic receptor antagonists, and α2-adrenergic receptor antagonists in the preparation of drugs for treating premature ejaculation.

4. The application according to claim 3, characterized in that, The calcium channel blocker includes one or a combination of MCU inhibitors, IP3R inhibitors, RyR2 inhibitors, and L-type calcium channel blockers.

5. A topical preparation, characterized in that, It includes at least one of calcium channel blockers, α1-adrenergic receptor antagonists, and α2-adrenergic receptor antagonists.

6. The topical preparation according to claim 5, characterized in that, The topical preparation is a gel, cream, or spray.

7. The topical preparation according to claim 5, characterized in that, The topical preparation is mainly applied to the glans and coronal sulcus of the male penis.

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