CN113893349B

CN113893349B - Application of dapagliflozin and analogs thereof in preparation of medicines for preventing and treating male reproductive dysfunction

Info

Publication number: CN113893349B
Application number: CN202111513681.8A
Authority: CN
Inventors: 魏蕊; 金滋润; 张哲�; 杨进; 魏天娇; 洪天配; 姜辉; 杨宇卓
Original assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2021-12-13
Filing date: 2021-12-13
Publication date: 2022-07-22
Anticipated expiration: 2041-12-13
Also published as: WO2023109784A1; CN113893349A

Abstract

The invention belongs to the technical field of biological medicines. In particular to application of an SGLT2 inhibitor in preparing a medicament for preventing and treating male reproductive dysfunction. The research result of the invention shows that the dapagliflozin treatment can increase the testis weight and the testis/body weight ratio of a db/db mouse and can obviously improve the sperm quality abnormality, the sperm quantity abnormality and the sperm movement abnormality of the db/db mouse. The SGLT2 inhibitor is used for preventing or treating male reproductive dysfunction for the first time, obtains a relatively ideal treatment effect, provides a new prevention and treatment option for treating male sterility in clinical application, particularly male sterility caused by diabetes or hyperglycemia, and has extremely high application value and social benefit.

Description

Application of dapagliflozin and analogues thereof in preparation of medicines for preventing and treating male reproductive dysfunction

Technical Field

The invention belongs to the technical field of biological medicines. In particular to application of SGLT2 inhibitor (especially dapagliflozin and analogues thereof) in preparing medicine for preventing and treating male reproductive dysfunction.

Background

In recent years, the incidence of male infertility tends to increase year by year with the increase in environmental pollution and the postponed growth age. The most direct consequence of the increase of the incidence rate of infertility is the reduction of fertility rate, which further accelerates the progress of China into the aging society, and particularly, the seventh national census finds that the fertility of the domestic population is gradually reduced, which brings serious challenges to the sustainable development of national economy. The occurrence of infertility is related to a plurality of factors, the individual difference is large, the pathogenic factors are complex, and the problems are brought to the research and treatment of diseases. The onset of male infertility is related to various factors such as environment, heredity, lifestyle habits, endocrine diseases, and the like. The pathological changes mainly include spermatogenesis and maturation disorders, which are clinically manifested as oligospermia, asthenospermia, teratospermia and azoospermia, and oligospermia caused by spermatogenesis and abnormal maturation accounts for more than half of male infertility. The pathogenesis of oligoasthenospermia is complex, the heterogeneity of people is strong, and effective and targeted treatment measures are lacked. Therefore, the research on pathogenic mechanisms of the spermatogenesis dysfunction is deeply explored, and the development of the medicine for effectively preventing or treating the diseases related to the spermatogenesis dysfunction has great strategic research significance for improving the population quality and the life quality of China and supporting the construction of 'healthy China'.

Diabetes is one of the most common metabolic diseases affecting male fertility in clinic, diabetes onset has a tendency of becoming younger in recent years, and epidemiological investigation shows that about 4.2 million diabetes patients exist in the world by 2014, so that the number of diabetes patients in the child bearing age affected by diabetes is large, and particularly the number of diabetes patients in children and teenagers is increased year by year. The male reproductive system is one of the more common serious complications of diabetes, the physiological structure and function of the testis of a diabetic patient are seriously damaged, androgen is low, the quantity, activity, abnormality rate and sperm DNA integrity of sperms are damaged to different degrees, but the pathogenesis of the abnormality of the sperms caused by the diabetes is not completely clarified. At present, the decline of male fertility caused by diabetes mainly refers to antioxidant symptomatic treatment, empirical treatment of traditional Chinese medicine and assisted reproduction technology, the treatment effect of part of patients is poor, and a targeted prevention and treatment scheme is lacked. In view of the huge number of people with diabetes worldwide, there is an urgent need to explore the molecular mechanisms of diabetic testicular spermatogenic dysfunction and male infertility and to develop drugs for effectively preventing or treating male reproductive dysfunction caused by diabetes clinically.

Sodium-glucose cotransporter 2 (SGLT 2) inhibitors are novel hypoglycemic drugs and can exert a hypoglycemic effect by inhibiting the reabsorption of glucose in the proximal tubule of the kidney. The SGLT-2 selective inhibitor serving as a new target of the hypoglycemic drug has no obvious influence on other tissues and organs due to the specific distribution of the SGLT-2 selective inhibitor in the kidney; insulin resistant diabetics can still benefit; and has the advantages of being not easy to cause hypoglycemia risk, not increasing the weight of the diabetics and the like. Currently, 6 SGLT2 inhibitors are on the market globally, which are: canagliflozin (Canagliflozin), Dapagliflozin (Dapagliflozin), Empagliflozin (Empagliflozin), Ipagliflozin (Canagliflozin), Luseogliflozin (luagliflozin), and Tofogliflozin (tuagliflozin). In recent years, large-scale cardiovascular fate studies have shown that various SGLT2 inhibitors, including dapagliflozin, can significantly improve cardiovascular and renal fates in type 2 diabetic patients [ N Engl J Med, 2017, 377(7): 644-. Therefore, the medicine is the first choice for patients with type 2 diabetes mellitus and combined cardiovascular disease and diabetic nephropathy. However, the effect of such drugs on reproductive function has not been reported at present.

Disclosure of Invention

In order to solve the problem of serious shortage of medicaments which can be effectively used for preventing or treating male reproductive dysfunction clinically, the inventor discovers that the SGLT2 inhibitor has a treatment effect on a male sterility animal model for the first time, and provides a new prevention and treatment selection for the male reproductive dysfunction.

Specifically, the invention is realized by the following technical schemes:

the invention provides application of an SGLT2 inhibitor in preparation of a medicament for preventing and treating male reproductive dysfunction.

Preferably, the SGLT2 inhibitor is selected from one or more of the following: dapagliflozin, engagliflozin, eprogliflozin, ruagliflozin, or togagliflozin.

More preferably, the SGLT2 inhibitor is dapagliflozin.

In one embodiment, the male reproductive dysfunction is a spermatogenesis disorder.

In a preferred embodiment, the male reproductive dysfunction is abnormal sperm quality, abnormal sperm count and/or abnormal sperm motility.

In another preferred embodiment, the male reproductive dysfunction is azoospermia, oligospermia, asthenospermia and/or teratospermia.

In yet another preferred embodiment, the male reproductive dysfunction is caused by diabetes or hyperglycemia.

Further, the male refers to a male mammal.

Preferably, the mammal is selected from the following: mouse, rat, rabbit, cat, dog, or primate.

More preferably, the mammal is selected from humans.

Compared with the prior art, the invention has the following beneficial effects:

the SGLT2 inhibitor (especially dapagliflozin and analogues thereof) is used for preventing or treating male reproductive dysfunction for the first time, obtains a relatively ideal treatment effect, provides a new prevention and treatment option for clinical application and treatment of male sterility (especially male sterility caused by diabetes or hyperglycemia), and has extremely high application value and social benefit.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1: results of changes in body weight and blood glucose levels in mice.

FIG. 2: gross mouse and testicle weight ratio.

FIG. 3: and (5) mouse testicle tissue HE staining results.

FIG. 4: and (5) identifying the mouse testicular tissue germ cell marker.

FIG. 5 is a schematic view of: and (5) detecting the sperm density and the survival rate of the mouse.

FIG. 6: and (5) detecting the sperm movement index of the mouse.

FIG. 7: and detecting the intrinsic parameters of the mouse sperm movement.

FIG. 8: TUNEL staining of mouse testis tissue.

FIG. 9: and (3) expressing apoptosis-related protein in mouse testis tissues.

FIG. 10: and (3) expressing apoptosis-related protein in mouse testis tissues.

FIG. 11: results of oxidative stress levels in mouse testis tissue.

Detailed Description

The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of the invention.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.

The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example (b):

1. the experimental method comprises the following steps:

classical type 2 diabetic mice: a leptin receptor double-knockout mouse (db/db mouse, purchased from Jiangsu Jiejiegaokang Biotechnology Co., Ltd.) is selected from 8-week-old mice, and after the mice are fed for 1 week adaptively, the random blood sugar of the mice is detected to be more than or equal to 16.7mmol/L, and then the random blood sugar is included in the experiment. Mice were randomly divided into two groups: in the diabetic group and the intervention group, dapagliflozin (Dapa, 1 mg/kg body weight/day) and water (equal volume) were administered, respectively, and the gavage was performed once a day for 5 weeks. Wild type littermates were also selected as normal controls. 9-10 of them in each group. Determination of fasting body weight, random body weight, fasting blood glucose and random blood glucose in mice before and after intervention

Typical characteristics of leptin receptor deletion model mice (db/db) include metabolic disorders such as hyperleptin syndrome, obesity, hyperglycemia, hyperinsulinemia, a decrease in prolactin in serum and an increase in prolactin in pituitary, and typical infertility [ science 1966 Sep 2;153(3740): 1127-. Humans with mutations in leptin or its receptor also exhibit obesity and infertility [ Endocrine reviews. 2006; 27: 710-718 ]. The classical animal model db/db mice for type 2 diabetes show pathological phenotypes in male reproductive system injury very similar to those of diabetic male patients, including spermatogenic dysfunction and sexual dysfunction, and are one of the most common models for studying male sterility and sexual dysfunction [ Biochemical and Biophysical Research Communications, 2017; 485: 686-; diabetologia 202009, 63(9) ]. We therefore selected this typical type 2 diabetic mouse as a model for male testicular tissue damage for study.

The animal experiment strictly follows the regulations of animal experiment management of Beijing university and is approved by Ethics Committee of animal experiment of Beijing university.

Material draw and subsequent experiments were performed the day after completion of dosing.

(1) Tissue retention: mouse weight, testis tissue weight. And (5) reserving fresh epididymis. Fixing one side of testis tissue by paraformaldehyde, dehydrating by conventional alcohol and embedding in paraffin; and directly extracting protein from the testis tissue on the other side or freezing and storing by using liquid nitrogen.

(2) The influence on the morphology of seminiferous tubules of testis tissue is detected: and (3) carrying out paraffin sectioning on the testis tissues, and carrying out conventional histomorphology staining (hematoxylin-eosin staining method) to observe the morphological changes of seminiferous tubules of the testis tissues of each group of mice, such as the arrangement of the seminiferous tubules, the shape of the lumen, the arrangement and the number of the seminiferous cells at each level and the like. The expression of germ cell marker proteins such as DAZL (spermatogonium), SYCP3 (spermatocyte), TNP1 (spermatid), PGK2 (sperm) and SOX9 (supporting cell) is detected by an immunofluorescence method.

(3) Determination of the effect on semen quality: collecting epididymis sperm by diffusion method, and detecting sperm number and sperm survival rate of mouse by computer-assisted semen analysis system (WLJY-9000, model number); rapid forward movement and forward movement; linear velocity, curvilinear velocity, average path velocity, linearity, head roll displacement, and linearity.

(4) Effects on oxidative stress and apoptosis are clear: paraffin sections of testis tissues are subjected to TUNEL apoptosis detection kit (green fluorescence) by one-step method to detect the apoptosis condition in the paraffin sections of the testis tissues of all groups of mice. Extracting proteins from fresh or frozen testis tissue stored in liquid nitrogen, detecting the expression of apoptosis markers (including pro-apoptotic proteins and anti-apoptotic proteins) by Western blot, and respectively detecting the total antioxidant capacity by using a total antioxidant capacity detection kit (ABTS rapid method), a total SOD activity detection kit (NBT method), a glutathione peroxidase detection kit (NADPH method), a hydrogen peroxide and lipid oxidation (MDA) detection kit and the like; SOD and glutathione peroxidase activity, hydrogen peroxide and Malondialdehyde (MDA) content, and Western blot method for detecting protein expression of 4-HNE (4-Hydroxynonenal ).

(5) Data analysis and mapping were performed on all experimental data using GraphPad Prism 7.0 statistical software. Experimental data are expressed as mean ± standard error (mean ± SEM). Comparison of differences between sets of data using analysis of variance and a Holm-Sidak post test, P <0.05 indicates that the differences are statistically significant. Represents P < 0.05; represents P < 0.01; represents P < 0.001.

2. The experimental results are as follows:

2.1 mouse weight and blood glucose level changes

First, fasting body weights and random body weights of littermate wild type mice (wild type/WT mice), leptin receptor double knockout mice (db/db mice), and db/db dapagliflozin treated (Dapa) mice before and at the end of treatment were examined and found (see fig. 1): both db/db and Dapa mice had significantly increased body weight compared to WT mice; next, blood glucose was measured in each group of mice, and as a result, it was found (see fig. 1): at the start of treatment, fasting plasma glucose and random plasma glucose were significantly elevated in db/db mice as well as in Dapa mice compared to WT mice; at the end of the treatment, the fasting plasma glucose and the random plasma glucose of db/db mice were significantly increased compared to WT mice, while Dapa treatment significantly reduced the fasting plasma glucose and the random plasma glucose of db/db mice.

2.2 weight ratio of mouse gross and testis

The body weight, testicular weight, and testicular/body weight ratio of each group of mice at the end of the treatment were examined. As a result, it was found (see fig. 2): both db/db and Dapa mice had significantly increased body weight compared to WT mice; the testis weight and testis/body weight ratio of db/db mice are significantly reduced, while the testis weight and testis/body weight ratio of db/db mice can be significantly increased by treatment with Dapa.

2.3 mouse testis tissue HE staining results

Hematoxylin-eosin (HE) staining of testis tissue was performed on each group of mice, and as a result, it was found (see fig. 3): all levels of spermatogenic cells in spermatogenic tubules of the WT mice are arranged in order, and a large number of mature sperms can be seen in lumens; only a small amount of spermatogenic cells at all levels are in the spermatogenic tubules of the db/db mouse, the arrangement is disordered, and mature sperms are hardly visible in the lumen; all levels of spermatogenic cells reappear in the spermatogenic tubules of the db/db mice treated by Dapa, the arrangement is regular, and a small amount of sperms can exist in the lumens.

2.4 mouse testis tissue germ cell marker identification results

The expression of various spermatogenic cell marker proteins in the testis tissues of each group of mice is detected by an immunofluorescence method. As a result, it was found (see fig. 4): similar to the HE results, the seminiferous cell markers (DAZL/SYCP 3), the sperm cell marker (TNP1), the sperm marker (PGK 2) and the supporting cell marker (sox 9) were all significantly reduced in the seminiferous tubules of db/db mice compared to WT mice; however, after Dapa treatment, these markers were all significantly increased.

2.5 mouse sperm quality results

Semen quality was measured for each group of mice by computer-assisted semen analysis and the results were found (see fig. 5): compared with WT mice, the sperm density of db/db mice is obviously reduced, while the sperm density of Dapa-treated db/db mice is obviously increased; similarly, it was found that the sperm survival rate was significantly reduced in db/db mice, while the sperm density tended to increase in Dapa-treated mice.

Meanwhile, as shown in fig. 6, the rapid forward movement and forward movement of the db/db mouse sperm were significantly decreased, while the rapid forward movement and forward movement of the Dapa-treated mouse sperm were significantly increased.

As shown in fig. 7, the intrinsic movement indexes of the mouse sperms in db/db include linear movement Velocity (VSL), curvilinear movement Velocity (VCL), average path Velocity (VAP), Linearity (LIN) and straight sperm head side-sway displacement (ALH), while the Dapa treatment can improve the intrinsic movement indexes of the sperms except for Linearity (LIN), and neither the simple db/db nor the Dapa treatment has any influence on the linearity of the mouse sperms.

2.6 mouse testis tissue test results

Apoptosis was detected in the testis tissue of each group of mice by TUNEL staining, and the results were found (see fig. 8): the apoptosis in the testis tissue of db/db mice is significantly increased, while the apoptosis in the testis tissue of db/db mice treated by Dapa is significantly decreased.

Changes of related protein expression in two typical paths influencing cell apoptosis in mouse testis tissues are detected by a Western blot method. As a result, found (see fig. 9): the expression of an anti-apoptotic protein Bcl2 in the testis tissues of db/db mice is obviously reduced, while the expression of an apoptotic protein BAX protein has an increasing trend; meanwhile, the expression of the anti-apoptotic protein Bcl2 in the testis tissues of db/db mice treated by Dapa tends to increase. The protein expression of Bcl2/BAX is significantly reduced in the testis tissue of db/db mice, but is significantly up-regulated in the testis tissue of Dapa-treated db/db mice.

In another apoptotic pathway, it was found that (see fig. 10): the expression of the anti-apoptotic protein XIAP in the testis tissue of the db/db mouse is obviously reduced, the expression of the apoptotic protein caspase8 has an increasing trend, the expression of the caspase9 protein is obviously increased, and meanwhile, the expression of the anti-apoptotic protein XIAP in the testis tissue of the db/db mouse treated by Dapa is obviously up-regulated. The expression of XIAP/caspase3 protein is reduced in the testis tissue of db/db mouse, and is obviously up-regulated in the testis tissue of db/db mouse treated by Dapa; protein expression of XIAP/caspase8 was significantly reduced in the testis tissue of db/db mice, but was significantly up-regulated in the testis tissue of Dapa-treated db/db mice; protein expression of XIAP/caspase9 was significantly reduced in the testis tissue of db/db mice, but was significantly up-regulated in the testis tissue of Dapa-treated db/db mice.

Finally, the change of the oxidative stress level in the testis tissue of each group of mice was detected by the method of the kit, and as a result, it was found (see fig. 11): the total antioxidant capacity in the testis tissues of db/db mice is obviously reduced, the activities of antioxidant stress enzyme superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are obviously reduced, and the protein expression of oxidative stress product dodecahydroxynonavaleraldehyde (4-HNE) is obviously increased; meanwhile, the total antioxidant capacity of the testis tissues of db/db mice treated by Dapa tends to be increased, the activity of antioxidant kinase SOD is obviously increased, the activity of GSH-Px tends to be increased, and the protein expression of the oxidative stress product 4-HNE is obviously reduced.

3. Conclusion of the experiment

The dapagliflozin treatment can increase the testis weight and the testis/body weight ratio of a db/db mouse and can remarkably improve the abnormal sperm quality, the abnormal sperm quantity and the abnormal sperm movement of the db/db mouse.

In conclusion, the SGLT2 inhibitor is used for preventing or treating male reproductive dysfunction for the first time, obtains a relatively ideal treatment effect, provides a new prevention and treatment selection for clinically treating male sterility, particularly male sterility caused by diabetes or hyperglycemia, and has extremely high application value and social benefit.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. The application of dapagliflozin in preparing a medicine for preventing and treating male reproductive dysfunction is characterized in that: the male reproductive dysfunction is sperm motility abnormality.

2. Use according to claim 1, characterized in that: the male reproductive dysfunction is asthenospermia.

3. Use according to claim 1, characterized in that: the sperm motility abnormality is caused by diabetes or hyperglycemia.

4. Use according to claim 1, characterized in that: dapagliflozin treatment improves the following sperm intrinsic motility indicators: linear motion Velocity (VSL), curvilinear motion Velocity (VCL), mean pathway Velocity (VAP), and straight sperm head lateral displacement (ALH).

5. Use according to claim 1, characterized in that: the male refers to a male mammal selected from a human.