CN110882250B

CN110882250B - Application of Scriptaid in preparation of medicine, reagent and apparatus for treating asthenospermia

Info

Publication number: CN110882250B
Application number: CN201811047605.0A
Authority: CN
Inventors: 于合国; 杨依婷; 李玉华; 刁华
Original assignee: Shanghai Institute Of Biomedical Technology
Current assignee: Shanghai Institute Of Biomedical Technology
Priority date: 2018-09-07
Filing date: 2018-09-07
Publication date: 2022-09-13
Anticipated expiration: 2038-09-07
Also published as: CN110882250A

Abstract

The invention discloses an application of a compound in treating asthenospermia, which is an application of the compound shown in a structural formula I or pharmaceutically acceptable salt thereof in preparing a medicine or a medicine box for promoting sperm motility, prolonging sperm motility time and/or prolonging survival time of thawed sperms, or an application in preparing a medicine or a medicine box for assisted reproduction, or an application in preparing a medicine or a medicine box for treating asthenospermia.

Description

Application of Scriptaid in preparation of medicine, reagent and apparatus vessel for treating asthenospermia

Technical Field

The present invention relates to compounds that promote sperm motility, prolong sperm survival time, and aid reproduction, and uses thereof.

Background

Currently, there are many assisted reproductive measures for treating infertility, such as Artificial Insemination (AI), in vitro fertilization-embryo transfer (IVF), and intracytoplasmic sperm injection (ICSI). These techniques involve in vitro culture and manipulation of sperm, and maintaining or even improving sperm fertility plays an important role in improving the success rate of assisted reproduction during the in vitro culture and manipulation. In vivo fertilization, sperm with good motility is required to be able to fertilize the ovum immediately upstream to the oviduct; in the in vitro fertilization, it is also essential that the sperm with good motility have enough power to penetrate the cumulus and zona pellucida of the ovum, so that the sperm can smoothly enter the ovum to complete the fertilization. Has very important significance for improving the sperm motility of the sperm with low motility which is common in clinic. The discovery of compounds that enhance sperm motility is therefore one of the focus of research by reproductive medicine workers.

The clinically common oligospermia and asthenospermia are usually subjected to cryopreservation. When a female needs to be treated by an assisted reproduction technology, the fact that usable sperms exist on the day of taking eggs is an important link in the whole treatment process. Progeny are typically obtained by In Vitro Fertilization (IVF) after reviving the previously cryopreserved sperm. Although the semen cryopreservation technology is widely applied, the sperm viability of the semen subjected to the freezing-unfreezing process is far lower than that of fresh semen, and the fertilization rate is often lower than that of natural mating. Experiments prove that the main reasons of low pregnancy rate of frozen semen are the reduced motility of sperms and the short survival time of the sperms in vitro. In human reproduction, the pregnancy rate of artificial insemination in the uterine cavity can be judged through the in vitro survival time of sperms, and the method has important clinical value. Therefore, if the motility of frozen sperm can be improved and the survival time after thawing can be prolonged, the fertilization rate can be increased.

Researches show that the movement function of the sperms is interfered and influenced by environmental factors, the quality of culture solution and instrument/consumable products which are in direct contact with the sperms in medical instruments for human in-vitro assisted reproduction technology, toxic dissolved substances and the like, the influence can be reduced by changing the chemical components of the container, and the adverse influence can be counteracted or counteracted by the coating and the added components, so that the quality of the sperms is improved and the assisted reproduction outcome is improved.

Therefore, there is a strong need in the art for compounds that promote sperm motility and increase sperm motility.

Disclosure of Invention

The invention aims to provide a novel compound which is used for promoting sperm motility, prolonging and maintaining sperm motility time, treating asthenospermia and the like. The compound has good sperm motility promoting activity, and has effects of increasing fertilization rate and promoting embryo development.

In a first aspect of the invention, there is provided the use of a compound of formula i or a pharmaceutically acceptable salt thereof in the manufacture of a medicament or kit for promoting sperm motility, prolonging sperm motility time, and/or prolonging sperm survival time after thawing, or for use in the manufacture of a medicament or kit for assisted reproduction, or for the manufacture of a medicament or kit for the treatment of asthenospermia;

in another preferred embodiment, the medicament or kit further comprises an additional class I, class IIb and/or class IV deacetylase (HDAC) inhibitor; more preferably, the medicament or kit further comprises an additional inhibitor of HDAC1, HDAC2, HDAC6 and/or HDAC 11.

In another preferred embodiment, the dosage form prepared from the compound shown in the structural formula I or the pharmaceutically acceptable salt thereof is a sustained release agent, a gel or a transdermal patch.

In another preferred embodiment, the compound of formula i or a pharmaceutically acceptable salt thereof is formulated for oral, vaginal, or sperm motility.

In another preferred embodiment, the kit for prolonging the survival time of the thawed sperms further comprises a cryopreservation solution and/or a cryopreservation resuscitation solution.

In another preferred embodiment, the kit comprises a coating; the coating is located on the semen contacting device.

In another preferred embodiment, the kit comprises a device for contacting semen, the device comprising a coating comprising a compound of formula I or a pharmaceutically acceptable salt thereof; the appliance comprises a container, a vessel and an instrument.

In a second aspect of the invention, there is provided a method for enhancing sperm motility, increasing sperm motility, and/or increasing the survival of thawed and revived sperm in vitro, or for enhancing sperm-egg binding and fertilization in vitro, comprising the step of contacting semen or sperm with a compound of formula I or a pharmaceutically acceptable salt thereof;

in a third aspect of the invention, there is provided a method of cryopreservation of sperm, the method comprising the steps of: mixing semen with compound shown in formula I or its pharmaceutically acceptable salt, and freezing.

In a fourth aspect of the invention, there is provided the use of a compound of formula i or a pharmaceutically acceptable salt thereof in cryopreservation of sperm.

In another preferred embodiment, the use comprises mixing the compound of formula i or a pharmaceutically acceptable salt thereof with semen or isolated sperm; more preferably, the compound shown in the structural formula I or the pharmaceutically acceptable salt thereof is mixed with semen or separated semen and then is frozen.

In a fifth aspect of the invention, there is provided a coating for an instrument for contacting semen or sperm, said coating comprising a compound of formula I or a pharmaceutically acceptable salt thereof; the appliance comprises a container, a vessel and an instrument.

In a sixth aspect of the invention, there is provided the use of a compound of formula i or a pharmaceutically acceptable salt thereof in the manufacture of a device for contacting sperm or sperm; the compound shown as the structural formula I or the pharmaceutically acceptable salt thereof is used for manufacturing the coating of the appliance; the appliance comprises a container, a vessel and an instrument.

In a seventh aspect of the invention, there is provided a culture system comprising a basal culture system for sperm culture and a compound of formula I or a pharmaceutically acceptable salt thereof; the culture system comprises a culture medium and a culture solution.

In an eighth aspect of the invention, there is provided a kit for use in promoting sperm motility, increasing sperm motility time, and/or increasing survival time after sperm thawing, or for use in assisting reproduction, or for the treatment of asthenospermia, the kit comprising a compound of formula i or a pharmaceutically acceptable salt thereof and optionally a culture system for sperm culture; preferably, the kit contains the culture system provided by the invention as described above.

Accordingly, the present invention provides a compound that promotes sperm motility and increases sperm motility.

Drawings

FIG. 1 shows the results of evaluation in example 1 with respect to sperm motility, forward movement, linear velocity, average path velocity, sperm head rolling amplitude, linearity, protropism, whipping frequency, etc.; SC represents a deacetylase inhibitor Scriptaid, namely, the compound shown as the structural formula I in the invention.

Wherein a relates to sperm motility SM; b about forward motion PR; c is about the average path velocity VAP; d is with respect to linear velocity VSL; e, regarding sperm head side swing amplitude ALH; f is about the whiplash frequency BCF; g about the forward STR; h for linear LIN.

FIG. 2 shows the effect of a compound of formula I or a pharmaceutically acceptable salt thereof on motility and forward motility of cryopreserved sperm.

FIG. 3 shows the effect of varying concentrations of a compound of formula I or a pharmaceutically acceptable salt thereof on sperm motility when added after thawing after cryopreservation; 0 and 1 hour on the graph represent the time after resuscitation.

FIG. 4 shows the results of a human sperm hyaluronic acid binding experiment; SC represents a deacetylase inhibitor Scriptaid, namely the compound shown as the structural formula I in the invention.

Detailed Description

The inventors have made extensive and intensive studies and have unexpectedly found that a deacetylase inhibitor (HDACi) script (SC for short), i.e., a compound of the present invention having the structure represented by formula I, or a pharmaceutically acceptable salt thereof, has the effects of promoting sperm motility, prolonging sperm motility time, prolonging thawed sperm survival time, and assisting reproduction. On the basis of this, the present invention has been completed.

Specifically, the compound with the structure shown in the formula I has the following structure:

the compound of formula I of the present invention having CAS number 287383-59-9 can be prepared by the method described in Gerova M S, Petrov O I.Chemnform, 2014,45(23):76-79, or can be purchased commercially.

It is to be understood that "pharmaceutically acceptable salts" as described herein include various pharmaceutically acceptable salts known in the art.

As used herein, "asthenospermia" refers to poor or even no motility of the sperm. Is one of the most common causes of male infertility in clinical practice.

Based on the properties of the compounds of the invention, it will be understood by those skilled in the art that their use is not limited to human sperm cells, but can be animal sperm cells.

Based on the properties of the compounds of the present invention, it will be understood by those skilled in the art that their use is not limited to infertility, but may be to further improve/promote sperm fertilization and fertility based on a sub-average or average capacity.

The invention provides application of a compound shown as a structural formula I or pharmaceutically acceptable salt thereof in preparing a medicine or a medicine box for promoting sperm motility, prolonging sperm motility time and/or prolonging survival time after sperm thawing, application in preparing a medicine or a medicine box for assisted reproduction and application in preparing a medicine or a medicine box for treating asthenospermia.

Based on the properties of the compounds of the invention, it will be appreciated by those skilled in the art that their use is also suitable for sperm preference in ICSI technology. That is, the use of the properties of the compounds of the present invention to activate the small number (single or multiple) of non-motile or weakly-motile sperm used in clinical ICSI technology to assist the technician in selecting good quality sperm for subsequent single sperm injection.

Based on the properties of the compounds of the invention, it will be appreciated by those skilled in the art that their use is also suitable for cryopreservation of testicular or epididymal spermatozoa (spermatozoa and/or spermatozoa) of infertile patients and/or subsequent sperm resuscitation and assisted reproductive use.

The medicament or kit provided by the present invention may also contain other class i, class iib and/or class iv deacetylase (HDAC) inhibitors, such as, but not limited to, compounds that act individually or together on HDAC1, HDAC2, HDAC6 and/or HDAC 11; these compounds include, but are not limited to, Entinostat, Mocetinostat, Romidepsin, CUDC-101, Tubatistatin A HCl, Quisinosistat, Pracinostat, Droxinostat, Abexinostat, Ricolinostat, Tacedinaline, CUDC-907, Tubacin, RG2833, Resminostat, Tubatistatin A, WT161, Valproic acid, ACY-738, Tucidinostat, Citarinstat, BRD73954, BG45, 4SC-202, CAY10603, Santacruzate A, Nextastat A, HPOB.

The medicine or the medicine box provided by the invention can be preset in the way of sperm movement in an optional mode of transdermal, vaginal or uterine administration and the like, and can act with the sperm to play a role; such as, but not limited to, oral, vaginal rings, and the like.

The present invention also provides a method of promoting sperm motility and/or extending sperm motility in vitro comprising the step of contacting semen with a compound of formula I or a pharmaceutically acceptable salt thereof as described herein.

In the present invention, sperm motility includes, but is not limited to, one or more of sperm motility, forward movement, linear velocity, mean pathway velocity, amplitude of sperm cephalic rolling, linearity, tropism, and whipping frequency. In certain embodiments, the invention is particularly directed to the improvement or enhancement of one or more of sperm motility, forward motility, mean pathway velocity, and whipping frequency.

Typically, after semen is obtained, the semen is mixed with a conventional sperm cell culture medium, such as BWW medium, centrifuged, resuspended, and then incubated for a suitable time with the addition of a compound of formula I or a pharmaceutically acceptable salt thereof as described herein. Typically, the compound of formula I or a pharmaceutically acceptable salt thereof is added to a final concentration of within 100. mu.M, for example 1-100. mu.M, and a final sperm concentration of 1-50 x 10 x 6/ml. The cultivation may be carried out under conventional culture conditions, e.g., 5% CO at 37 deg.C ₂ Is carried out in an incubator. Semen can be from any individual in need thereof, particularly from patients with oligoasthenospermia. Alternatively, in certain embodiments, the instant invention is usedThe medium described herein containing a compound of formula I or a pharmaceutically acceptable salt as described herein resuspends the sperm cells obtained after the above centrifugation.

The present invention also provides a method for prolonging the survival of thawed and resuscitated sperm comprising the step of contacting semen with a compound of formula I, or a pharmaceutically acceptable salt thereof, as described herein. In one embodiment of the invention, semen is contacted with a compound of formula I or a pharmaceutically acceptable salt thereof as described herein before cryopreservation, for example, but not limited to, adding a compound of formula I or a pharmaceutically acceptable salt thereof as described herein to the mixture of semen and cryopreservation solution, and performing cryopreservation after standing at room temperature. Typically, the final concentration of the compound of formula I or a pharmaceutically acceptable salt thereof added to the above mixture of semen and frozen stock solution is in the range of 5-20. mu.M. In another embodiment of the invention, the mixture of frozen semen and frozen stock solution is thawed at room temperature and then added to a compound of formula I or a pharmaceutically acceptable salt thereof as described herein.

The present invention also provides an in vitro method for increasing sperm-egg binding and fertilization comprising the step of contacting semen with a compound of formula I, or a pharmaceutically acceptable salt thereof, as described herein. In one embodiment of the invention, capacitated sperm may be obtained by the above described method of promoting sperm motility and/or extending sperm motility time in vitro, and the capacitated sperm may be cultured with the oocyte. The cultivation may be carried out under conventional culture conditions, e.g., 5% CO at 37 deg.C ₂ Is carried out in an incubator.

In certain aspects, the invention also provides a culture system comprising a culture medium and a culture broth. The culture system contains a compound of formula I or a pharmaceutically acceptable salt thereof. In one embodiment of the invention, the medium contains a basal medium for sperm culture supplemented with a compound of formula I or a pharmaceutically acceptable salt thereof. The basal medium for sperm culture may be any of various media known in the art suitable for sperm culture, such as BWW. The concentration of the compound of formula I or a pharmaceutically acceptable salt thereof in the basal medium can be in the range of 1-100. mu.M, e.g., the compound of formula I or a pharmaceutically acceptable salt thereof can be in the range of 1-50. mu.M, 1-20. mu.M, or 5-15. mu.M.

In certain aspects, the invention also provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof. The kit can be used for promoting sperm motility, prolonging sperm motility time, and/or prolonging survival time after sperm thawing, or for assisting reproduction. Other suitable reagents may also be included in the kit, such as a medium or broth for sperm culture. The compound of formula I or a pharmaceutically acceptable salt thereof and the culture medium may be packaged separately or provided as a mixture. Thus, in certain embodiments, the kit contains a culture medium as described herein.

In certain aspects, the invention also provides a coating for an instrument for contacting semen or sperm, the coating comprising a compound of formula I or a pharmaceutically acceptable salt thereof; the coating can neutralize any influence that is detrimental to sperm motility maintenance, such as, but not limited to, environment, sperm cup material, and the like. Appliances suitable for such coatings include containers, vessels, instruments; the coating is applied at least where in use sperm will come into contact with.

The features mentioned above, or those mentioned in the embodiments, may be combined in any combination. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

The main advantages of the invention are:

the compound of formula I or the pharmaceutically acceptable salt thereof can significantly enhance the motility of sperm, including the recovery rate of sperm after freezing, and has been shown to have a stabilizing effect on the in vitro and in vivo development of embryo, so the compound has great potential in treating weak sperm in males.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. All percentages, ratios, proportions, or parts are by weight unless otherwise specified. The units in weight volume percent in the present invention are well known to those skilled in the art and refer to the weight of solute in 100 milliliters of solvent. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1

In vitro promotion of sperm motility in asthenospermia

Semen samples are from 30 patients with oligoasthenospermia who visit a hospital clinic, desire for 48 hours to 7 days, semen is taken in a dry and sterile semen taking cup by masturbation, and the semen is completely liquefied within 60min at 37 ℃. Each part of semen is as follows: the culture medium (BWW) is placed in a centrifuge tube at a ratio of 1:1, centrifuged at 300g for 15min, resuspended (final concentration of 5 × 10^ 6/ml), and the resuspended solution is equally divided into a drug group and a control group, wherein the drug group is added with 10 μ M of Scriptaid (purchased from Selleck (Shanghai Bao, Japan), and the control group is added with equal volume of sterilized water. Sperm parameters (specifically, sperm motility SM%, forward movement PR%, linear velocity VSL, mean path velocity VAP, sperm head yaw amplitude ALH, linear LIN, forward STR, whip frequency BCF) were evaluated using a fully automated sperm mass analyzer CASA (IVOS, Hamilton-Thorn Research, inc. beverly, MA, USA) after incubation for 1h, 3h, 5h at 37 ℃ in a 5% carbon dioxide incubator. For each specimen, 6-10 visual fields were selected, and the number of sperm counted was not less than 800.

The detailed results are shown in FIG. 1.

The results show that the Scriptaid has the function of improving the sperm motility of patients with asthenospermia.

Example 2

Increase the survival time (to the time of losing motility) of sperms in the culture solution

The procedure of sample collection and grouping in example 1 was followed, and the mixture was incubated at 37 ℃ in a 5% carbon dioxide incubator, and the percentage of motile sperm in the culture was determined every 5 hours using a fully automated sperm mass analyzer. When less than 5% of the sperm are in forward motion, the sample is considered to be no longer viable. Samples were obtained from three different patients with asthenospermia. The detailed results are shown in Table 1.

TABLE 1

The results show that the sperm survival time of the drug group is significantly higher than that of the normal group, and the Scriptaid can prolong the sperm motility time.

Example 3

Improving sperm-egg binding and fertilization ability of asthenospermia

Injecting prolactin PMSG (60 IU/mouse) into abdominal cavity of immature hamster, injecting chorionic gonadotropin HCG (100 IU/mouse) to promote ovulation after 48 hours, killing ovulation-promoting female mouse after 12-15 hours, opening abdominal cavity, taking out oviduct, placing in balanced BWW, tearing open ampulla of oviduct to make cumulus-oocyte compound flow out, removing oviduct and tissue fragment, adding Hyaluronidase (HY) according to 10%, after cumulus cell is desquamated, washing in BWW for 3 times, transferring into desktop liquid to remove zona pellucida, washing in BWW for 3 times, placing in 37 deg.C 5% carbon dioxide incubator for standby. The tail of epididymis is taken after the male rat dies, the epididymis is squeezed by an ophthalmic forceps and placed in BWW to allow semen to swim out, and a sperm analyzer is used for detecting that the forward movement is less than 50 percent of weak sperm and more than 50 percent of normal sperm. Adding SC into weak sperm to make its final concentration be 10 μ M, placing normal sperm group, weak sperm group and SC group into balanced HTF tube, incubating in 37 deg.C and 5% carbon dioxide incubator for 1h to make them capacitate, and incubating the capacitated sperm and hamster oocyte with zona pellucida in 37 deg.C and 5% carbon dioxide incubator for 3 hr. Staining was performed with acetic acid-orcein stain and the number of sperm penetrating each oocyte and the number of oocytes penetrated were counted microscopically, and the detailed results are shown in Table 2.

TABLE 2

The result shows that the penetration rate and fertilization index of the Scriptaid after acting on weak sperms and penetrating the egg cells can be improved by more than 150 percent compared with those of the weak sperms, and the Scriptaid can be close to the level of normal sperms.

Example 4

In vitro fertilization rate and morula and blastocyst formation rate

Immature female mice are injected with menotropin PMSG (10 IU/mouse) in the abdominal cavity, chorionic gonadotropin HCG (10 IU/mouse) is injected after 48 hours, the ovulation-promoting female mice are killed after 12-15 hours, the abdominal cavity is opened, the oviduct is taken out and placed in H-CZB liquid drops, the ampulla of the oviduct is torn open, and the cumulus-oocyte compound flows out. Fallopian tubes and tissue debris were removed and the pellet complexes were transferred to pre-equilibrated H-CZB, 1-2 pellets per drop. The mixture is placed in a 5% carbon dioxide incubator at 37 ℃ for standby. The tail of epididymis is taken out after the male mouse dies, the epididymis is squeezed by an ophthalmic forceps and placed in BWW to allow seminal fluid to swim out. SC was added to the capacitation solution to a final concentration of 1. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, and placed in an equilibrated HTF tube to allow it to gain energy for 1 h. At fertilization, 10. mu.l of sperm were pipetted into 100. mu.l HTF fertilization drops containing cumulus-oocyte complexes using a pipette gun, with the final concentration of sperm controlled at 1-2X 10^ 6/ml, at which time the SC concentration was 90nM, 180nM, 450nM, 900 nM. After 8-10h of culture, the ova were transferred to KSOM droplets and continued to be cultured at 37 ℃ in 5% carbon dioxide. In this process, the fertilization rate (number of fertilized eggs/total number of eggs) was calculated, the 2-cell rate (2-cell/number of fertilized eggs) was observed 24 hours after fertilization, the 4-cell rate (4-cell/number of fertilized eggs) was observed 48 hours later, the morula rate (number of morula/number of fertilized eggs) was observed 72 hours, the blastocyst rate (number of blastocysts/number of fertilized eggs) was observed 96 hours, and the detailed results are shown in Table 3.

TABLE 3

The result shows that the treatment of the sperm of the normal mouse by the Scriptaid in a certain concentration range (less than 5 mu M) has no adverse effect on the fertilization rate of IVF and the generation rate of morula and blastocyst; while fertilization in vivo had a positive effect on the fertilization process and embryogenesis, it is shown from the table ratios that the Scriptaid treatment resulted in improved fertilization rates and blastocyst rates.

Example 5

Toxicity test

In the same manner as in example 1, the procedure for collecting a sample was carried out in terms of semen: the culture medium (BWW) is put into a centrifuge tube at a ratio of 1:1, 300g, and is resuspended after 15min centrifugation, the resuspension solution is provided with an SC concentration gradient of 100 mu M, 50 mu M, 25 mu M, 12.5 mu M, 6.25 mu M and a control group, and after each group of samples are placed at 25 ℃ for 24h, the percentage of motile sperms in the culture is measured by a full-automatic sperm mass analyzer. Samples were obtained from three different patients with asthenospermia. The results are shown in Table 4.

TABLE 4

100μM

50μM

25μM

12.5μM

6.25μM

0μM

Sample 1

46％

40％

42％

39％

36％

35％

Sample

2

50％

46％

47％

43％

39％

Sample 3

55％

53％

49％

47％

43％

The results show that at concentrations below 100. mu.M, the Scriptaid is not toxic to sperm.

Example 6

Human sperm cryopreservation test

The sperm motility and forward motility before cryopreservation were 40% and 34% respectively in the same sample collection procedure as in example 1. According to the semen: mixing frozen stock solution (origin,1067) at a ratio of 1:1, placing in a centrifuge tube, adding 5 μ M,10 μ M, and 20 μ M SC into the mixture, standing at room temperature for 10min, placing in a wheat tube, fumigating in liquid nitrogen vapor for 30min, and storing in liquid nitrogen. Taking out from liquid nitrogen after 24h, thawing at room temperature for resuscitation, and determining the percentage of motile sperm by using a full-automatic sperm quality analyzer, wherein the result is shown in figure 2.

The results show that 5 μ M of Scriptaid can restore sperm motility and forward motility rate to the state before cryopreservation; the 10. mu.M and 20. mu.M of Scriptaid even enabled sperm motility and forward motility to be better than those before cryopreservation, indicating that Scriptaid has the function of promoting sperm motility.

The result shows that the freezing recovery rate of the sperms can be improved by adding Scriptaid into the sperm freezing solution.

Example 7

Human sperm resuscitation test

In the same manner as in example 1, the procedure for collecting a sample was carried out in terms of semen: mixing frozen stock solution (origin,1067) at a ratio of 1:1, placing in a centrifuge tube, standing at room temperature for 10min, placing in a wheat tube, fumigating in liquid nitrogen vapor for 30min, and storing in liquid nitrogen. After 24h, the sperm cells were removed from the liquid nitrogen, thawed at room temperature for resuscitation, centrifuged, resuspended in sperm medium and aliquoted into 4 portions, one of which served as a normal control, and the remainder was incubated at 37 ℃ for 15 minutes with 5. mu.M, 10. mu.M, 20. mu.M SC, and the percentage of motile sperm was determined using a fully automatic sperm mass analyzer, the results are shown in FIG. 3.

The results show that the Scriptaid has the function of improving the recovery of the sperm motility after thawing.

Example 8

Human sperm hyaluronic acid binding assay

In the same sample collection process as in example 1, sperm samples from patients with asthenospermia were selected, placed in centrifuge tubes at a semen-to-medium (BWW) ratio of 1:1, centrifuged at 300g for 15min and then resuspended, the resuspended solution was set in the control group and 5 μ M SC group was added, and the control group and SC group were incubated at 37 ℃ for 1h and then examined with sperm-hyaluronic acid binding kit (HBA), the results of which are shown in fig. 4.

The result shows that the Scriptaid can restore the HA binding capacity of the sperms of the asthenospermia patients to 60 percent of that of the normal patients, and the HA binding capacity is improved by 5 times compared with the corresponding capacity of the sperms of the asthenospermia patients; the Scriptaid was shown to be effective in improving sperm quality.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the scope of the invention, which is defined by the claims appended hereto, and any other technical entity or method that is encompassed by the claims as broadly defined herein, or equivalent variations thereof, is contemplated as being encompassed by the claims.

Claims

1. The application of a compound shown as a structural formula I or pharmaceutically acceptable salt thereof in preparing a medicament or a kit for assisted reproduction or in preparing a medicament or a kit for treating asthenospermia; the assisted reproduction is a method for improving the sperm-egg combination and fertilization capability in vitro; the treatment of asthenospermia is performed by promoting sperm motility, prolonging sperm motility time, and/or prolonging survival time after sperm thawing;

。

2. the use according to claim 1, wherein the medicament or kit further comprises a further class i, class iib and/or class iv deacetylase (HDAC) inhibitor.

3. The use according to claim 2, wherein the medicament or kit further comprises an additional HDAC1, HDAC2, HDAC6 and/or HDAC11 inhibitor.

4. The use according to any one of claims 1 to 3, wherein the compound of formula I or a pharmaceutically acceptable salt thereof is in the form of a sustained release formulation, a gel or a transdermal patch.

5. The use according to any one of claims 1 to 3, wherein the compound of formula I or a pharmaceutically acceptable salt thereof is in a form suitable for oral, vaginal, or sperm motility.

6. The use of claim 1, wherein the kit for prolonging the survival time of thawed sperm cells further comprises a cryopreservation solution and/or a resuscitation solution.

7. The use of claim 1, wherein the kit comprises a coating.

8. A method for enhancing sperm motility, prolonging sperm motility, and/or prolonging survival of thawed and resuscitated sperm in vitro, not for therapeutic purposes, comprising the step of contacting semen or sperm with a compound of formula i or a pharmaceutically acceptable salt thereof;

。

9. a method of cryopreserving sperm, the method comprising the steps of: mixing the sperms with a compound shown as a structural formula I or a pharmaceutically acceptable salt thereof, and freezing and storing;

。

10. the application of a compound shown as a structural formula I or a pharmaceutically acceptable salt thereof in cryopreservation of sperms;

。

11. the use according to claim 10, wherein the compound of formula i or a pharmaceutically acceptable salt thereof is mixed with semen or isolated sperm.

12. The use according to claim 10, wherein the compound of formula i or a pharmaceutically acceptable salt thereof is mixed with semen or isolated sperm and cryopreserved.

13. A coating for an instrument for contacting semen or sperm, said coating comprising a compound of formula i or a pharmaceutically acceptable salt thereof;

。

14. the coating of claim 13, wherein the implement comprises a container, vessel, instrument.

15. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a device for contacting sperm or sperm;

。

16. the use of claim 15, wherein the compound of formula i or a pharmaceutically acceptable salt thereof is used to form a coating for said device.

17. Use according to claim 15 or 16, wherein the appliance comprises a container, vessel, instrument.

18. A culture system comprising a basal culture system for sperm culture and a compound of formula i or a pharmaceutically acceptable salt thereof;

。

19. the culture system of claim 18, wherein the culture system comprises a culture medium and a broth.

20. A kit for use in assisted reproduction or for the treatment of asthenospermia, which comprises a compound of formula i or a pharmaceutically acceptable salt thereof and optionally a culture system for sperm culture; the auxiliary reproduction is in vitro improvement of sperm-egg combination and fertilization capability; the treatment of asthenospermia is to promote sperm motility, prolong sperm motility time and/or prolong the survival time of thawed sperm;

。

21. the kit of claim 20, wherein the kit comprises the culture system of claim 18 or 19.