WO2017092178A1 - Application of c-type natriuretic peptide in the preparation of topical contraceptive and in sperm function test - Google Patents

Abstract

The present invention provides application of C-type natriuretic peptide in the preparation of a topical contraceptive and a sperm function test reagent. It is found in studies that at the time of ovulation, C-type natriuretic peptide is expressed at the ampulla of oviduct, at a site of fertilization, and this can induce the chemotactic movement of sperms toward an ovum to accomplish the fertilization. If a certain amount of C-type natriuretic peptide is added into the genital tract of a female mammal, or the C-type natriuretic peptide is incubated with the sperms, such that the concentration gradient distribution of the C-type natriuretic peptide in the oviduct is changed, the sperms cannot chemotactically move toward the ovum along the concentration gradient, and thus the fertilization is blocked.

Description

Application of C-type natriuretic peptide in the preparation of topical contraceptives and sperm function test Technical field

The invention relates to the field of medicine and health, in particular to the application of a C-type sodium peptide in the preparation of a topical contraceptive and a sperm function test.

Background technique

The male infertility population has increased due to factors such as changes in the climate and the rapid pace of life and work. Some of the causes of male infertility have not yet been clarified. Conventional semen analysis can mainly understand the amount of semen, sperm density and the total amount of sperm in semen, sperm agglutination, sperm motility and viability, sperm morphology, semen liquefaction and other information. In fact, through the above conventional analysis, it is difficult to truly understand the function and fertilization potential of sperm, especially in artificial insemination and IVF, it is not possible to determine whether or not artificial insemination can be performed only by the information provided by routine analysis of semen. Therefore, it is necessary to explore new methods for detecting sperm function.

Family planning is China's basic national policy and is of great significance for improving the quality of our population, improving the level of reproductive health, and improving the overall strength of the nation. In this environment, the development of related birth control techniques and related contraceptives has developed rapidly, and various contraceptives have been introduced to the market. For example, common female physical barrier contraceptives have vaginal diaphragms and contraceptive cervical caps. These contraceptives Before use, it is necessary to undergo gynecological examination, select the appropriate type of vaginal diaphragm according to the size of the vagina, and then put it into the method of putting it in and out under the guidance of the doctor until it is fully used, but the vaginal diaphragm is needed after the woman gives birth. The model size may be different from the previous one, and often needs to be reconfigured, so the method of use is more cumbersome.

In addition, the existing female external contraceptives are cytotoxic spermicidal drugs, the mechanism of action is to damage or destroy sperm cells, but at the same time it is also toxic to other cells and has a certain stimulating effect on the vagina, so the clinical does not advocate long-term Continuous use. The most widely used representative drug is nonoxynol ether (N-9). If used frequently, N-9 can cause some gynecological inflammation, such as bacterial vaginitis, genital ulcer, vulvitis, toxic shock syndrome. (TSS), cervical vaginal epithelial shedding, etc.

Oral contraceptives include the following: short-acting oral contraceptives, long-acting oral contraceptives, Long-acting injectables and visiting contraceptives. Their mechanism of action mainly achieves contraception by inhibiting ovulation and preventing implantation of fertilized eggs. Long-term use of contraceptives can inhibit ovulation, which can easily make women gain weight and increase the risk of thrombosis and migraine. If you take it blindly and take it in large quantities, it will cause great harm to your health. Therefore, it has become an urgent need to further develop a safe, effective and long-term use of contraceptives.

Summary of the invention

The object of the present invention is to provide a C-type sodium peptide for use in the preparation of a topical contraceptive and sperm function detection based on the principle of sperm chemotaxis.

Another object of the present invention is to provide a novel, external contraceptive that is safe, effective, and suitable for long-term use by women of childbearing age.

It is still another object of the present invention to provide a sperm function detecting reagent and a detecting device.

In order to achieve the object of the present invention, the present invention provides the use of a C-type sodium peptide for the preparation of a topical contraceptive.

The present invention also provides a topical contraceptive, the active ingredient of which is a C-type sodium peptide.

The agent also includes a pharmaceutically acceptable excipient or ancillary ingredients. The excipients include plasticizers, fillers, wetting agents, binders, and the like.

The medicament is a liquid, suppository, gel, film, foam, tablet or soluble condom.

In the external contraceptive of the present invention, the single dose unit of the medicament is a C-type natriuretic peptide of 20 x 10 ^{-7 to} 200 x 10 ^-7 mg/kg body weight.

When in use, the contraceptive is placed in the reproductive tract of a female mammal (for example, after the vagina, or the contraceptive is delivered to the uterus of the female mammal by a topical contraceptive placer), and the contraceptive is dissolved and then mated to prevent contraception.

The invention also provides the use of a C-type sodium peptide in the detection of sperm function, wherein the tissue cell, liquid, gel or solid carrier containing the active ingredient C-type sodium peptide is used as a sperm chemotaxis source and placed in the capillary tube. One end, the other end is immersed in the semen droplets, and the number of sperm entering the capillary is observed under a microscope (10-fold objective), if the sperm enters the capillary The same amount as the control (the sperm chemotaxis source is not placed in the capillary) indicates that the sperm has no chemotactic ability, which may result in the inability to naturally fertilize.

The invention also provides a sperm function detecting reagent, which comprises a sperm chemotaxis source, which is a tissue cell, a liquid, a gel or a solid, and the active ingredient contained in the chemotactic source is a C-type sodium peptide. The concentration of the C-type natriuretic peptide in the sperm chemotaxis source is 0.01-10 nM (preferably 0.1 nM).

The present invention also provides a sperm function detecting kit containing the detecting reagent.

The present invention also provides a sperm function detecting reagent strip coated with the detecting reagent.

The present invention further provides a sperm function detecting device, wherein the detecting device is a capillary having a diameter of 1-10 mm and a length of 2-10 cm which is open at least at one end; and for a capillary closed at one end, the sperm is preset at a closed end of the capillary The function detecting reagent; for the capillary tube which is open at both ends, the sperm function detecting reagent is preset at one end of the capillary.

Studies have found that in the occurrence of ovulation, the expression of C-type natriuretic peptide in the ampulla of the tubal fertilization site can induce sperm chemotaxis to the egg and complete fertilization. If the ampulla at this time is taken out and placed in a capillary tube, the C-type sodium peptide slowly released from the ampulla can make the sperm move toward the capillary. Based on this, it can be judged whether the sperm has the ability to naturally fertilize. The ability of C-type natriuretic peptide to induce sperm chemotaxis can be used to detect the expression of NPR2 in sperm to detect sperm quality as a clinical criterion for judging sperm sterility.

The above-mentioned detection device or a capillary chemotaxis model can be established in vitro to detect the chemotactic ability of the sperm, and the cause of sperm natural infertility can be judged according to the ability of sperm chemotaxis, thereby realizing sperm function detection/identification. Specifically, the method for detecting sperm function provided by the present invention is to put a certain amount of C-type sodium peptide or tubal ampulla into one end of a capillary tube, and the other open end is connected with a sperm droplet, and observe under a microscope 10 times objective lens. The amount of sperm entering the capillary, if the number of sperm entering the capillary is the same as the control (the C-type natriuretic peptide or the fallopian tube ampulla is not placed in the capillary), it indicates that the sperm has no chemotactic ability, which may result in the inability to naturally fertilize. More specifically, the freshly collected semen is first incubated in a capacitation solution (e.g., T6) for 1.5 h, and then capacitated, and then added to 150 μl of the capacitor droplets, and the sperm density is about 10 ⁶ /ml. Capillaries with a length of 2.5 cm and a diameter of 3 mm were closed at one end. The ampulla of the mouse that had just ovulated was taken out, placed in the closed end of the capillary, and the open end of the capillary was connected to the semen droplet. The control capillaries did not contain the ampulla. The number of sperm in the field of view was continuously observed under a 10x objective of the microscope. At the same time, the number of sperm in the treated group and the control group was compared. If there was no significant difference, the reason why the semen could not be naturally fertilized may be the NPR2 gene. The mutation causes the chemotaxis to fail to the oocyte, causing fertilization to fail.

At the same time, using the above sperm chemotaxis principle, it interferes with sperm chemotaxis in the reproductive tract and can reduce the fertilization rate of female mammals. Therefore, the purpose of contraception can be achieved by interfering with sperm chemotaxis. For example, a certain amount of C-type natriuretic peptide is added to the reproductive tract of a female mammal, or the C-type natriuretic peptide is incubated with sperm to change the concentration gradient distribution of the C-type natriuretic peptide in the fallopian tube. In this way, the sperm cannot move toward the oocyte along the concentration gradient, and the sperm and the oocyte cannot meet, thereby preventing fertilization.

The natriuretic peptide type C (CNP, also referred to as NPPC) is a naturally occurring paracrine factor in follicular fluid. Studies have shown that CNP, as an oocyte maturation inhibitory factor, increases the levels of cyclic guanosine 3' (5'-monophosphate, cGMP) in cumulus cells and oocytes, and also increases oocytes. Cyclic adenosine 3', 5'-monophosphate (cAMP) levels. In addition, the expression characteristics of its receptor in sperm can be used as a criterion for judging the quality of sperm. Conventional semen analysis can mainly understand the amount of semen, sperm density and the total number of sperm in semen, sperm agglutination, sperm motility and viability, sperm morphology, semen liquefaction and other information. In fact, through the above conventional analysis, it is difficult to truly understand the function and fertilization potential of sperm, especially in artificial insemination and IVF, it is not possible to determine whether or not artificial insemination can be performed only by the information provided by semen routine. The C-type natriuretic peptide receptor (NPR2) has the activity of guanylate cyclase. When combined with CNP, it can stimulate the production of high levels of cGMP by NPR2, by increasing the intracellular calcium level in sperm cells. The sperm moves toward the oocyte to complete the fertilization. Therefore, it can express according to NPR2 Point to detect sperm quality as a clinical criterion for judging sperm infertility.

The novel external contraceptive pill provided by the invention has an active ingredient C-type natriuretic peptide which is an endogenous protein and is externally used, and therefore, its side effect is smaller than an oral contraceptive method. The medicament can be applied to the female genital tract, especially in the female vagina, and is suitable for contraception in most women of childbearing age, and the contraceptive has no toxic side effects and adverse reactions, and is convenient to use, and has broad application prospects. In addition, the use of C-type sodium peptide to identify sperm quality / function, the operation method is simple, the results are easy to observe, the amount of semen required is small, suitable for in vitro detection of male / male sperm function.

detailed description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the materials used are all commercially available.

The formulation of the energized liquid T6 (modified mTyrode's solution) in the following examples is as follows:

Table 1 Formulation of Capacitor T6

ingredient	Concentration (g/100ml)	Concentration (mmol/l)
NaCl	0.6660	114
KCl	0.0238	3.2
MgCl 2 ·6H 2 O	0.0100	0.5
CaCl 2 ·2H 2 O	0.0294	2.0
NaH 2 PO 4 ·2H 2 O	0.0062	0.4
NaHCO 3	0.2090	24.9
glucose	0.1000	5.6
Sodium pyrophosphate	0.0055	0.5
Sodium lactate solution	0.1121	10.0
4-hydroxyethylpiperazineethanesulfonic acid	0.2400	10.0
Phenol red	0.0010

Note: The sperm capacitation solution T6 also contains bovine serum albumin (BSA) at a final concentration of 10 mg/ml.

Example 1 Application of C-type natriuretic peptide in inducing sperm chemotaxis

Fresh sperm was taken from the epididymis tail of adult male mice and incubated for 1.5 h in the capacitative solution T6 for capacitation. The capacitated mouse sperm was added to 150 μl of T6 liquid droplets in the center of the culture dish, and the sperm density was about 5 × 10 ⁶ /ml. The capillary tube (length 2.5 cm, tube diameter 3 mm) was closed at one end, and a ampulla was placed at the closed end, and the ampulla was derived from the oviduct of the mouse that had just ovulated, as an experimental group. The ampulla was not placed in the control capillary (length 2.5 cm, tube diameter 3 mm). The open end of the capillary is connected to the semen droplet. The number of sperm in the capillary in the field of view was continuously observed under a 10x objective of the microscope. The experiment was repeated three times.

As a result, in the control group, at 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 minutes, the number of sperm in the visual field was 1, 1, 9, 20, 20, 17, 24, 37, respectively. 32, and the number of sperm in the experimental group was 2, 10, 28, 45, 68, 125, 280, 351, and 396, respectively. It can be seen that the ampulla containing the C-type sodium peptide can significantly increase the number of sperm in the capillary.

Further, by placing 0.1 nM of the C-type sodium peptide into the capillary, the number of sperm in the capillary was increased from 4.7 × 10 ⁵ /ml to 10.9 × 10 ⁵ /ml.

Example 2 Application of C-type natriuretic peptide in sperm function detection

The sperm that could not be naturally fertilized was subjected to a capacitation treatment in the capacitor T6, and then added to 150 μl of the T6 liquid droplet, and the sperm density was 10 ⁶ /ml. Capillaries with a length of 2.5 cm and a diameter of 3 mm were closed at one end. The ampulla of the mouse that had just ovulated was taken out, placed in the closed end of the capillary, and the open end of the capillary was connected to the semen droplet. The control capillary (length 2.5 cm, diameter 3 mm) did not contain the ampulla. The number of sperm in the field of view was continuously observed under a microscope 10 times objective lens. At the same time, the number of sperm in the capillary of the treated group and the control group was compared. If there was no significant difference, the reason why the semen could not be naturally fertilized may be NPR2. Genetic variation cannot chemotaxis to oocytes, leading to failure of fertilization.

Example 3 Application of C-type natriuretic peptide in reducing fertilization rate in mice

After ovulation induction treatment, adult female rats were injected with a certain concentration of C-type natriuretic peptide into the cervix, and then caged with male rats. The specific method is as follows: 30 adult female CD-1 females (6-8 weeks), each weighing about 35g, divided into five groups, six in each group, namely, groups A, B, C, D, E, each group The females are numbered 1, 2, 3, 4, 5, 6. Five groups of female rats were administered C-type sodium peptides of different concentrations, which were 0, 10×10 ^-10 , 5×10 ^-10 , 10 ^-9 , 10 ^-8 mol/l, and the injection volume was 20 μl. Adult female rats were induced with gonadotropin (PMSG 5IU/only, induced for 48 hours, then HCG 5IU/only, induced for 12 hours), and 20 μl of C-type sodium peptide was injected into the cervical cervix with a micro syringe. The cage was co-caged with adult CD-1, and the next morning, the second cell or the putative two cells in the oviduct of the female mouse were rushed out on the third day, and the fertilization rate was recorded at the two-cell rate. The experiment was repeated three times. The results showed that the fertilization rate was significantly lower than that of the control group A (no C-type sodium peptide) (the fertilization rate of the group A was 93.1%), and the fertilization rates of the treatment groups B, C, D, and E were significantly decreased, 54.9%, respectively. 45.0%, 24.8%, 38.7%, of which group D (injection of 10 ^-9 mol/l C-type sodium peptide) had the most significant effect on fertilization rate reduction.

Similarly, after the C-type sodium peptide was incubated with the sperm, artificial insemination was carried out, and the fertilization rate was also significantly lowered. The specific method is as follows: 12 adult female CD-1 females (6-8 weeks), each weighing about 35g, are divided into two groups, namely, groups A and B, each group of six, each group of females is numbered 1 , 2, 3, 4, 5, 6. The two groups of female rats were administered different concentrations of C-type sodium peptide, which were 0, ^{10 -9} mol/l. Adult female rats were induced by gonadotropin (PMSG 5 IU/only, induced for 48 hours, then HCG 5 IU/only, induced for 12 hours). The fresh sperm was treated with the capacitor T6 at 37 ° C in a 5% CO ₂ incubator, and then the capacitated sperm was incubated with C-type sodium peptide (0 or 10 ^-9 mol/l) for 30 min. Sperm (3 × 10 ⁶ -10 × 10 ⁶ , volume 50 μl) was injected into the cervix of the female mouse, and the male rats ligated with the vas deferens were immediately caged. The next morning, the female was checked, and the female rats were rushed out on the third day. Two cells or putative two cells in the fallopian tube are recorded as fertilization rate at the two cell rate. The experiment was repeated three times. The results showed that the fertilization rate was compared with that of the control group A (no C-type sodium peptide) (fertilization rate of group A was 74.3%), and fertilization of treatment group B ( ^10-9 mol/l C-type sodium peptide-incubated sperm) The rate dropped significantly and the fertilization rate was only 18.5%.

Example 4 Application of C-type sodium peptide in the preparation of topical contraceptives

The C-type natriuretic peptide is used in an amount of 20 × 10 ^{-7 to} 200 × 10 ^-7 mg / kg body weight, and a suppository or a soluble condom is added to the C-type sodium peptide to prepare a topical contraceptive. The base of the suppository can be further prepared into a spherical, oval or duckbill-shaped vaginal suppository using water-soluble glycerin gelatin. The C-type natriuretic peptide can be added to a suppository or a soluble condom according to the dose corresponding to the body weight to prepare a contraceptive.

When used, place the contraceptive pill in the reproductive tract of a female mammal (eg, after the vagina, Or the contraceptive is delivered to the female mammal's uterus through a topical contraceptive placer. After the drug is dissolved, it can be contraceptive.

Although the present invention has been described in detail with reference to the preferred embodiments of the present invention, it will be apparent to those skilled in the art Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.

Industrial applicability

The invention is based on the principle of sperm chemotaxis, and provides an external contraceptive pill with an endogenous protein-C-type sodium peptide as an active ingredient. The contraceptive has no toxic side effects and adverse reactions, and is suitable for contraception of most women of childbearing age, and is convenient to use. The application prospect is broad. In addition, the use of C-type sodium peptide to identify sperm quality / function, the operation is simple, the results are easy to observe, the amount of semen required is small, suitable for in vitro detection of sperm function.

Claims (10)

1. The use of C-type sodium peptide in the preparation of a topical contraceptive.
2. A topical contraceptive, characterized in that the active ingredient is a C-type natriuretic peptide.
3. The external contraceptive according to claim 2, wherein the agent further comprises a pharmaceutically acceptable adjuvant or an auxiliary ingredient; the adjuvant comprises a plasticizer, a filler, a wetting agent, a binder.
4. The external contraceptive according to claim 2 or 3, wherein the agent is a liquid, a suppository, a gel, a film, a foam, a tablet or a soluble condom.
5. The external contraceptive according to any one of claims 2 to 4, wherein the single dose unit of the medicament is a C-type natriuretic peptide of 20 x 10 ^{-7 to} 200 x 10 ^-7 mg/kg body weight.
6. The use of a C-type natriuretic peptide in the detection of sperm function is characterized in that the application is a tissue cell, a liquid, a gel or a solid carrier containing an active ingredient C-type sodium peptide as a sperm chemotaxis source, placed in a capillary tube One end and the other end were immersed in semen droplets, and the number of sperm entering the capillary was observed under a microscope.
7. A sperm function detecting reagent, characterized in that the detecting reagent contains a sperm chemotaxis source, which is a tissue cell, a liquid, a gel or a solid, and the active component contained in the chemotactic source is a C-type sodium peptide, wherein the C-type The concentration of the natriuretic peptide is from 0.01 to 10 nM, preferably 0.1 nM.
8. A sperm function detecting reagent strip coated with the detecting reagent according to claim 7.
9. A sperm function detecting kit comprising the detecting reagent according to claim 7 or the reagent strip according to claim 8.
10. The sperm function detecting device is characterized in that the detecting device is a capillary having a diameter of 1-10 mm and a length of 2-10 cm which is open at least at one end; and a capillary closed at one end, and the closed end of the capillary is preset with the method of claim 7. The sperm function detecting reagent; for the capillary tube having both ends open, the sperm function detecting reagent according to claim 6 or 7 is preset at one end of the capillary.