Nitric oxide gel for treating endometrial injury

Abstract

The nitric oxide gel for treating endometrial injury is composed of a gel matrix and a nitric oxide gas solution, the nitric oxide gas solution is uniformly dispersed in the gel matrix, and the volume ratio of the gel matrix to the nitric oxide gas solution is 3: 1-1: 3. The gel matrix is composed of one or more of poloxamer and derivatives thereof, povidone, carbomer and cellulose derivatives, and the nitric oxide gas solution is formed by dispersing nitric oxide gas in absolute ethyl alcohol or water-free and oxygen-free polyhydric alcohol which is in a liquid state at the temperature of 25 DEG C. According to the nitric oxide gel for treating the endometrial injury, the limitation bottleneck of the nitric oxide gel for treating the endometrial injury is overcome, meanwhile, the safe, effective, convenient and economical requirements of clinical treatment are met, and the effect of nitric oxide on treating the endometrial injury is fully exerted.

Description

Technical Field

[0001] The invention relates to a pharmaceutical preparation for treating endometrial damage, in particular to a nitric oxide gel for treating endometrial damage. Background Art

[0002] Endometrial damage is the most common gynecological disease, such as endometrial damage and endometritis. It can occur in people of all ages and has great harm, such as affecting the quality of life and causing female infertility. After suffering from endometrial damage, women will experience symptoms such as increased vaginal discharge and vulvar itching, which seriously affect their normal life. If a woman suffers from endometrial damage during pregnancy, it will have a great impact on the development of the fetus, especially during childbirth, and the disease may be transmitted to the fetus, affecting the health of the fetus.

[0003] Studies have found that nitric oxide has a good therapeutic effect on inflammation. Through animal experiments on inflammation models, it was found that in the early stages of inflammation, nitric oxide mainly plays an anti-inflammatory role, including: maintaining microcirculatory integrity, relaxing vascular smooth muscle, increasing intestinal mucosal blood flow and inhibiting platelet and leukocyte adhesion and accumulation on the surface of endothelial cells, preventing thrombosis, inhibiting myeloperoxidase activity, protecting epithelial barriers and promoting epithelial tissue repair. However, in the late stages of inflammation, accompanied by the production of superoxide anion oxygen, nitric oxide can synthesize with anion oxygen to produce peroxynitrite, and its biological oxidizing property can cause damage to normal tissues.

[0004] Nitric oxide plays a dual role in the progression of endometrial damage, not only protecting the reproductive system mucosa, but also killing sperm and causing infertility. In the hypoxic reproductive cavity environment, nitric oxide molecules mainly play an anti-inflammatory and protective role. Most of the methods reported for supplementing nitric oxide in the body are to use nitric oxide donor compounds. Nitric oxide donor compounds are divided into two categories: non-enzymatic and enzymatic. Most non-enzymatic nitric oxide donor compounds come from nitro compounds, including nitroprusside, organic or inorganic nitrites and nitrates, nitrosamines, nitrogen mustard, hydrazine, etc., with small doses and large toxic side effects. Enzymatic nitric oxide donor compounds (such as arginine) need to be decomposed by biological enzymes in the body to produce nitric oxide molecules. The nitric oxide molecules produced by the above nitric oxide donor compounds cannot be concentrated in the reproductive cavity to take effect, so the effect of using nitric oxide donor compounds to treat endometrial damage is very poor.

[0005] The nitric oxide molecule is in gaseous form. Nitric oxide gas (NO) is slightly soluble in water, with a solubility of only 5.6×10-3 g / L (equivalent to 0.186 μmol / L) in water at 20°C. It cannot be made into an ordinary dosage form to play a therapeutic role in endometrial damage.

[0006] In situ gel is a type of preparation that can undergo a phase transition immediately at the site of administration after being administered in a solution state, transforming from a liquid state to form a non-chemically cross-linked semi-solid gel. Gels have good tissue compatibility and a long retention time at the site of administration; they can also store drugs and protect them from environmental influences. According to the different formation mechanisms, in situ gels can be divided into temperature-sensitive, pH-sensitive, and ion-sensitive types. As a new type of drug dosage form, in situ gels are widely used in new drug delivery systems such as sustained release, controlled release, and pulsed release. In situ gels can be used for multiple routes of administration such as skin, eyes, nasal cavity, oral cavity, vagina, and rectum, and have become a research hotspot in the fields of pharmacy and biotechnology.

[0007] Although in situ gel has many advantages in local administration of uterine drugs, nitric oxide is a gas, and there is no research report on the efficient delivery of nitric oxide gas in in situ gel. Therefore, the preparation of in situ gel that efficiently carries nitric oxide gas is a limiting bottleneck for the use of nitric oxide to treat endometrial damage. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art - the lack of a safe, stable and efficient gel for carrying nitric oxide (NO) gas, to provide a safe, stable NO highly dispersed nitric oxide gel, to break through the restrictive bottleneck of NO in the treatment of endometrial damage, and at the same time meet the safety, effectiveness, convenience and economy requirements of clinical treatment.

[0009] The inventors found that NO molecules have poor stability and are easily oxidized. When they encounter oxygen, they easily generate brown-red NO2, which generates nitrous acid after dissolving in water, which not only loses the pharmacological effects of NO, but also produces serious toxic side effects. To this end, the inventors have prepared a highly dispersed nitric oxide gel with stable NO gas after a large number of experiments, achieving the purpose of safety, effectiveness, convenience and economy, breaking through the restrictive bottleneck of NO in the treatment of body diseases (especially the treatment of skin and mucosal inflammation), and giving full play to the NO therapeutic effect of body diseases, especially the NO therapeutic effect of skin and mucosal inflammation.

[0010] The nitric oxide gel for treating endometrial damage of the present invention is composed of a gel matrix and a nitric oxide gas solution, wherein the nitric oxide gas solution is uniformly dispersed in the gel matrix;

[0011] The volume ratio of the gel matrix to the nitric oxide gas solution is 3:1 to 1:3;

[0012] The gel matrix is ​​composed of one or a combination of poloxamer and its derivatives, povidone, carbomer, and cellulose derivatives;

[0013] The nitric oxide gas solution is formed by dispersing nitric oxide gas in anhydrous ethanol or anhydrous and oxygen-free polyol that is liquid at 25°C;

[0014] The above-mentioned nitric oxide gel is used to achieve the therapeutic effect of nitric oxide on endometrial damage;

[0015] The nitric oxide gas solution is formed by saturating nitric oxide gas in an anhydrous ethanol solution;

[0016] The volume ratio of the gel matrix to the nitric oxide gas solution is 2:1 to 1:2;

[0017] The volume ratio of the gel matrix to the nitric oxide gas solution is 1:1.

[0018] The cellulose derivative is one or a combination of hydroxypropyl methylcellulose, ethyl cellulose, cellulose acetate, carboxymethyl cellulose and its sodium salt, and methyl cellulose;

[0019] The above-mentioned polyol which is liquid at 25°C is one or a combination of polyethylene glycol 200-800, glycerol, and propylene glycol;

[0020] A method for preparing the above-mentioned nitric oxide gel for treating endometrial damage is characterized by comprising the following steps:

[0021] a. Take 100 ml of polyol that is liquid at 25°C, heat it to 100°C to remove oxygen, transfer it into a stoppered glass bottle, saturate the inner space of the glass bottle with nitrogen, cool it to 25°C, add nitric oxide gas to replace the nitrogen, and form a nitric oxide gas solution;

[0022] b. The gel matrix is ​​dispersed in 2 times the mass of a polyol that is liquid at 25°C, mixed to form a solution, and the NO gas solution prepared in step a is added according to the specified volume ratio, mixed to form a NO highly dispersed nitric oxide gel, and stored in a cool and sealed place.

[0023] Another method for preparing the above-mentioned nitric oxide gel for treating endometrial damage is characterized by comprising the following steps:

[0024] a. Take 100 ml of anhydrous ethanol and heat it to 77°C to remove oxygen, transfer it into a stoppered glass bottle, saturate the inner space of the glass bottle with nitrogen, cool it to 25°C, add nitric oxide gas to replace the nitrogen, and form a nitric oxide gas solution;

[0025] b. Disperse the gel matrix in 2 times the mass of anhydrous ethanol, mix well to form a solution, add the NO gas solution prepared in step a according to the specified volume ratio, mix well to form NO highly dispersed nitric oxide gel, and store in a cool and sealed place.

[0026] One or a combination of antioxidants, emulsifiers, antibacterial agents and pH regulators commonly used in medicine are added to the above-mentioned nitric oxide gel for treating endometrial damage.

[0027] The NO highly dispersible nitric oxide gel of the present invention has the following advantages: 1. converting gas molecules into semisolid preparations not only enhances the stability of NO but also facilitates application in local pharmaceutical treatment; 2. highly dispersing NO in the nitric oxide gel matrix, which has good compatibility with tissues after entering the uterine cavity; 3. not using any NO donor compound, thus avoiding the adverse reactions and toxic side effects of NO donor compounds on body tissues; 4. the nitric oxide gel is simple to prepare and easy to use, which is conducive to achieving the purpose of rapid and long-term treatment of endometrial damage. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention will be described in detail below. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be considered isolated, and they can be combined with each other to achieve better technical effects.

[0029] Example 1 Preparation of nitric oxide gel for the treatment of endometrial damage

[0030] According to the design in Table 1, gel samples were prepared using the following two methods:

[0031] Method 1:

[0032] a. Take 100 ml of polyol that is liquid at 25°C, heat it to 100°C to remove oxygen, transfer it into a stoppered glass bottle, saturate the inner space of the glass bottle with nitrogen, cool it to 25°C, add nitric oxide gas to replace the nitrogen, and form a nitric oxide gas solution;

[0033] b. The gel matrix is ​​dispersed in 2 times the mass of a polyol that is liquid at 25°C, mixed to form a solution, and the NO gas solution prepared in step a is added according to the specified volume ratio, mixed to form a NO highly dispersed nitric oxide gel, and stored in a cool and sealed place.

[0034] Method 2:

[0035] a. Take 100 ml of anhydrous ethanol and heat it to 77°C to remove oxygen, transfer it into a stoppered glass bottle, saturate the inner space of the glass bottle with nitrogen, cool it to 25°C, add nitric oxide gas to replace the nitrogen, and form a nitric oxide gas solution;

[0036] b. Disperse the gel matrix in 2 times the mass of anhydrous ethanol, mix well to form a solution, add the NO gas solution prepared in step a according to the specified volume ratio, mix well to form NO highly dispersed nitric oxide gel, and store in a cool and sealed place.

[0037] The gel of the control group was prepared with reference to the experimental group according to the component ratios in Table 1. Each experimental group was configured according to the components and ratios within the protection scope of the claims of this application, while each control group had a component replaced by another component or a component value beyond the protection scope of the claims of this application.

[0038] Table 1 Sample preparation of experimental group and control group

[0039]

[0040]

[0041] Note: NO gas is nitric oxide gas; NO saturation state 100% means that the NO gas in the NO gas solution is in a saturated state at 25°C, that is, the volume proportion of NO is 100%; NO saturation state 60%~90% means that the NO gas and nitrogen in the NO gas solution are in a mixed state at 25°C, and the volume proportion of NO is 60%~90%; * represents that the item has obvious differences in components or values ​​from the experimental group.

[0042] Example 2 Effect of Nitric Oxide Gel on the Treatment of Endometrial Damage

[0043] (1) Establishment of endometrial injury model animals

[0044] A mechanical endometrial injury model was established in rats by mechanical curettage. The method was as follows: in a sterile operating room, SPF female SD rats weighing 250-300g were taken, and the vaginal smears of the rats were observed daily. Female rats in estrus were used for model construction. The experimental rats were anesthetized with 10% chloral hydrate, the lower abdomen was disinfected with 75% alcohol, the skin was cut 1 cm above the urethra to enter the abdominal cavity, the uterus was exposed, and an incision was made at 1 / 3 of the right Y-shaped uterus. The endometrium was scraped with a homemade scraper to simulate clinical human endometrial curettage, causing pathological endometrial damage, and the surgical incision was sutured after surgery. After the rats woke up from anesthesia, they were placed in the animal room for feeding. The feeding environment was changed to clean cages for single cage feeding, the bedding was changed frequently and kept dry, free drinking water and food were given, and nutrition was strengthened. Within three days after modeling, 200,000U / kg of penicillin sodium was routinely injected into the thigh muscle every day to prevent wound infection.

[0045] (2) In vivo effects of each group of gels

[0046] Using the rat endometrial injury model established above, each group of hydrogels was injected in situ into the damaged endometrium, and the repair effect of each group of hydrogels on endometrial damage was compared and analyzed, and the animal's fur color and diet were observed. At the corresponding experimental time point in each group, the rats were anesthetized with 10% chloral hydrate, and quickly rinsed with 250ml normal saline through cardiac perfusion. After the right atrial appendage outflow liquid became clear, it was replaced with 4% paraformaldehyde for rapid perfusion, and the tissues of various parts were fixed in the body. After the rat's limbs became stiff, it was slowly dripped. The incision used in the preparation of the abdominal model of the perfused rat was cut open to expose the Y-shaped uterine tissue, and the uterus of 0.25cm above and below the right side of the injury was carefully removed, and placed in 4% paraformaldehyde for fixation for 24h. The uterine tissue stored in 4% paraformaldehyde was taken out, wrapped with gauze and rinsed overnight under a thin stream of water. Embedded with hard wax, tissue sections were made with a thickness of 5μm.

[0047] The pathological sections of the above rat uterine tissues were used for determination of the repair effect of the rat endometrium by HE staining, TUNEL cell apoptosis and immunohistochemistry. Transmission electron microscopy (TEM) was used to observe the electron microscopic specimens of the endometrium in each experimental group, and the tissue morphological changes of the endometrial damaged sites after the application of different groups of hydrogels were compared.

[0048] Based on the above results, the double-blind method was used to evaluate the application effect of the hydrogel in each group, and a total score (out of 100 points) was given. The higher the score, the better the overall effect.

[0049] Table 2 Effects of gel in the experimental and control groups on the treatment of endometrial damage

[0050]

[0051]

[0052] As can be seen from the experimental results in Table 2, the nitric oxide gel for the treatment of endometrial damage in the experimental group has a good therapeutic effect on endometrial damage, especially in experimental groups 4, 5 and 6, all indicators are good, which verifies that the experimental group has a good repair effect on rat endometrial damage. Compared with the experimental group, the comprehensive score of the control group is significantly poorer, especially in control groups 1, 3 and 5, all evaluation indicators are very poor. As can be seen from the above, each experimental group has a good repair effect on rat endometrial damage and can be used as a gel for repairing endometrial damage. Therefore, the nitric oxide gel for the treatment of endometrial damage of the present invention plays a therapeutic role of nitric oxide for endometrial damage and has a good application prospect.

[0053] The above detailed description is a specific description of a feasible embodiment of the invention, and the embodiment is not intended to limit the scope of the invention. Any equivalent implementation or change that does not deviate from the invention should be included in the scope of the invention. In addition, those skilled in the art can also make various modifications, additions and substitutions in other forms and details within the scope and spirit disclosed in the claims of the invention. Of course, these various modifications, additions and substitutions made according to the spirit of the invention should be included in the scope of the invention.

Claims

1. A nitric oxide gel for treating endometrial damage, wherein the main features are as follows: the nitric oxide gel is composed of a gel matrix and a nitric oxide gas solution, and the nitric oxide gas solution is uniformly dispersed in the gel matrix; the volume ratio of the gel matrix to the nitric oxide gas solution is 3:1 to 1:3; the gel matrix is ​​composed of one or more combinations of poloxamer and its derivatives, povidone, carbomer, and cellulose derivatives; the nitric oxide gas solution is formed by dispersing nitric oxide gas in anhydrous ethanol or anhydrous and oxygen-free polyol that is liquid at 25°C; the nitric oxide gel is used to achieve the therapeutic effect of nitric oxide on endometrial damage.

2. The nitric oxide gel for treating endometrial damage according to claim 1, characterized in that: The nitric oxide gas solution is formed by saturated dispersion of nitric oxide gas in anhydrous ethanol solution.

3. The nitric oxide gel for treating endometrial damage according to claim 1, characterized in that: The volume ratio of the gel matrix to the nitric oxide gas solution is 2:1 to 1:

2.

4. The nitric oxide gel for treating endometrial damage according to claim 1, characterized in that: The volume ratio of the gel matrix to the nitric oxide gas solution is 1:

1.

5. The nitric oxide gel for treating endometrial damage according to claim 1, characterized in that: The cellulose derivative is one or a combination of hydroxypropyl methylcellulose, ethyl cellulose and cellulose acetate.

6. The nitric oxide gel for treating endometrial damage according to claim 1, characterized in that: The polyol which is liquid at 25° C. is one or a combination of polyethylene glycol 200-800, glycerol and propylene glycol.

7. A method for preparing the nitric oxide gel for treating endometrial damage according to claim 1, characterized in that: The steps include: a. Take 100 ml of polyol that is liquid at 25°C, heat it to 100°C to remove oxygen, transfer it into a stoppered glass bottle, saturate the inner space of the glass bottle with nitrogen, cool it to 25°C, add nitric oxide gas to replace the nitrogen, and form a nitric oxide gas solution; b. The gel matrix is ​​dispersed in 2 times the mass of a polyol that is liquid at 25°C, mixed to form a solution, and the NO gas solution prepared in step a is added according to the specified volume ratio, mixed to form a NO highly dispersed nitric oxide gel, and stored in a cool and sealed place.

8. A method for preparing the nitric oxide gel for treating endometrial damage according to claim 1, characterized in that: The steps include: a. Take 100 ml of anhydrous ethanol and heat it to 77°C to remove oxygen, transfer it into a stoppered glass bottle, saturate the inner space of the glass bottle with nitrogen, cool it to 25°C, add nitric oxide gas to replace the nitrogen, and form a nitric oxide gas solution; b. Disperse the gel matrix in 2 times the mass of anhydrous ethanol, mix well to form a solution, add the NO gas solution prepared in step a according to the specified volume ratio, mix well to form NO highly dispersed nitric oxide gel, and store in a cool and sealed place.

9. A nitric oxide gel for treating endometrial damage according to claims 7 to 8, characterized in that: One or a combination of antioxidants, emulsifiers, antibacterial agents and pH regulators commonly used in pharmacy are added to the nitric oxide gel for treating endometrial damage.

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