CN115837011A - Injection for slowly releasing lactobionic acid and application of lactobionic acid microspheres in treatment of thin endometrium - Google Patents

Abstract

The invention relates to an injection containing slow-release lactobionic acid of lactobionic acid microballoons and application of lactobionic acid microballoons in treating thin endometrium, mainly utilize lactobionic acid microballoons to stimulate and substitute the traditional hysteroscope physical scratching and scrape and realize the effect of thickening endometrium, and because of small side effect of lactobionic acid, do not cause the injury to endometrium, very safe, in order to reach the effects of stimulating the endometrium continuously for a period of time at the same time, the invention uses collagen microballoons to wrap lactobionic acid and achieve the effects of slow-release lactobionic acid, the product of the invention can be injected into uterus directly after suspending with normal saline, it is convenient to apply clinically, can be used for replacing the endometrial stimulation therapy of the hysteroscope of the uterus.

Description

Injection for slowly releasing lactobionic acid and application of lactobionic acid microspheres in treatment of thin endometrium

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an injection for slowly releasing lactobionic acid and application of lactobionic acid microspheres in treatment of thin endometrium.

Background

Clinically, the thickness of the endometrium is less than or equal to 7mm and is defined as the thin endometrium, and the thickness of the endometrium is less than 5mm and is used as the diagnosis standard of the heavy thin endometrium.

Thin endometrium can cause the decrease of the endometrial receptivity and insufficient blood flow of women, influence the implantation and the development of embryos and finally possibly cause the occurrence of female infertility. The incidence of thin endometrium is about 2.4%.

Thin endometrium is usually caused by uterus injury, and endometrium injury can be caused by various factors such as intrauterine operation, endometrium infection, hormone medicine taking, radiotherapy, chemotherapy or other unknown reasons.

There are numerous clinical reports of thin endometrial therapy, mainly (1) hormonal drug therapy, including estrogen, gonadotropin-releasing hormone agonist, tamoxifen, growth hormone, chorionic gonadotropin; (2) Medicines for improving endometrial blood flow, including sildenafil vaginal gel, ketotheobromine, vitamin E, and low-dose aspirin; (3) granulocyte colony stimulating factor treatment; (4) Regenerative medical therapy, including stem cell therapy, platelet rich plasma therapy; (5) electrical stimulation therapy. These methods have respective disadvantages, such as high requirement on the integrity of the inner membrane itself by estrogen treatment, and the use of a large amount of estrogen in the follicular phase can inhibit the secretion of luteinizing hormone by the pituitary, which affects the development and quality of the follicle; the application of estrogen can cause high risk factors such as thrombus, endometrium lesion and the like. The treatment period for increasing blood flow is long and there is a great deal of risk of adverse effects of vasodilation.

However, the effectiveness of therapies such as granulocyte colony stimulating factor, stem cells, and platelet-rich plasma is not sufficiently proven, and further studies are required.

The thin endometrium has also one kind of operation therapy, hysteroscopic endometrium micro stimulating operation, and this operation has been proved by clinical practice to improve the endometrium thickness obviously, raise the pulse index of endometrium artery obviously, lower its resistance index and improve the tolerance of endometrium. The treatment mechanism is that mechanical stimulation is carried out on endometrium by scratching stimulation, endometrium trimming and other modes to cause pseudo-decidua reaction, thereby improving the success rate of embryo implantation. However, the scratching stimulation through the uterine cavity may still cause secondary damage to the endometrium, which leads to the risk of aggravation of the endometrium damage, meanwhile, the one-time scratching stimulation of the endometrium sometimes cannot achieve good effect of promoting the thickening of the endometrium, and the repeated hysteroscopy micro-stimulation operation for many times causes heavy burden to patients, and cannot be carried out clinically.

Disclosure of Invention

The invention aims to overcome a plurality of defects in the existing thin endometrium medical treatment technology, effectively avoid damage to endometrium and improve the treatment safety.

In order to achieve the purpose, the technical scheme provided by the invention comprises an injection for slowly releasing lactobionic acid, and is characterized in that the injection contains lactobionic acid microspheres.

Also comprises the application of the lactobionic acid microspheres in treating thin endometrium.

The preparation method of the lactobionic acid microspheres comprises the following steps:

the method comprises the following steps: weighing lactobionic acid powder, adding the lactobionic acid powder into purified water, and preparing a lactobionic acid water solution with the mass ratio of lactobionic acid being 5-20%;

step two: weighing non-hydrolyzed type I collagen, and fully dissolving the non-hydrolyzed type I collagen in a lactobionic acid aqueous solution to prepare a lactobionic acid-collagen aqueous solution with the mass ratio of collagen of 0.5-3%, wherein the lactobionic acid-collagen aqueous solution is used as a component A for later use;

step three: weighing isopropyl myristate, and adding water-in-oil emulsifier such as span 80, polyoxyethylene castor oil, lecithin and the like to ensure that the content of the water-in-oil emulsifier reaches the mass ratio of 10-30 percent, and the component B is the component B;

step four: heating the component B to 70-85 ℃, and then slowly adding the component A while stirring to obtain a mixed solution of the component A and the component B in a mass ratio of 1;

step five: shearing and homogenizing the mixed solution at a high speed by using a homogenizer until the mixed solution is clarified; then adding an absolute ethanol solution mixed with 5-20% of EDC by mass ratio at room temperature for 1/10-1/5 times dilution, and fully stirring; then standing for 6-72 hours at room temperature; then adding 2-5 times volume of ethanol to demulsify the whole system, centrifuging for 5-30 minutes by using 2000-10000G, and collecting precipitates; after collecting the precipitate, adding absolute ethyl alcohol for cleaning, centrifuging for 5-30 minutes at 2000-10000G, pouring out the supernatant, collecting the precipitate, and repeatedly cleaning for 1-5 times by the absolute ethyl alcohol; then, the precipitate was collected, and ethanol in the precipitate was removed by an evaporator, and the powder was collected as lactobionic acid microspheres.

The lactobionic acid microspheres and the sterile normal saline are mixed according to the mass ratio of 5-20% before application, and the mixed solution is injected into uterus after being fully and uniformly stirred, so that the lactobionic acid microspheres and the sterile normal saline are very convenient and safe to apply.

The lactobionic acid microspheres are used for stimulating to replace the traditional hysteroscope physical scratching to realize the effect of thickening the endometrium, the lactobionic acid has small side effect, does not damage the endometrium, is very safe, and can continuously stimulate the endometrium within a period of time.

The present invention will be described in further detail below with reference to the accompanying drawings.

Drawings

FIG. 1 shows microscopic pictures of lactobionic acid microspheres mixed with physiological saline under a 600-fold microscope.

FIG. 2 is a slow release profile of lactobionic acid microspheres.

Fig. 3 is a comparison chart of the thin endometrium treatment experiment of the animal before and after 1 month.

Fig. 4 is a comparison chart before and after 3 months of a thin endometrium treatment experiment of an animal.

Detailed Description

To further explain the technical means and effects of the present invention adopted to achieve the intended purpose, the following detailed description of the embodiments, structural features and effects of the present invention will be made with reference to the accompanying drawings and examples.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, it is to be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "aligned", "overlapping", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, are not to be construed as limiting the present invention.

The terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature; in the description of the present invention, "a plurality" means two or more unless otherwise specified.

Lactobionic acid is among the group of fruit acids, but it is less irritating to tissues than other types of fruit acid species, such as glycolic acid, lactic acid, citric acid, etc. In vitro research shows that lactobionic acid can stimulate endometrial cells to initiate related immune response, the expressions of HIF-1 alpha, VEGF, TNF alpha, integrin, osteopontin (OPN) and intercellular adhesion molecule (ICAM-1) are remarkably improved, the generation of blood vessels, stromal cells and glandular epithelium is promoted, the relaxation and the relaxation of micro blood vessels can be promoted, the blood perfusion of endometrium is improved, the blood flow resistance is reduced, the edema and the deciduation of stromal cells are promoted, the synergy on the molecular indexes related to the tolerance of the endometrium is obvious, and the implantation of embryos is facilitated.

The stimulation of the lactobionic acid on the endometrial cells belongs to weak stimulation, and in order to continuously give a certain micro-stimulation effect to the endometrium, the lactobionic acid is wrapped in the nano-microspheres based on collagen. The collagen is selected because of high biocompatibility and in vivo degradability, and meanwhile, the non-hydrolyzed collagen can be dissolved in a water phase under an acidic condition, so that the preparation of the sustained-release microspheres is facilitated. The water-in-oil nanoemulsion is used for preparing the microspheres, the collagen-lactobionic acid-water solution is uniformly dispersed in the oil phase, the particle size of the formed water phase is stable and uniform, and the stable crosslinking collagen microspheres containing the lactobionic acid can be formed after EDC crosslinking. The collagen nano-microspheres can release lactobionic acid along with slow degradation in the uterus, and can provide a period of continuous slight stimulation to the endometrium, promote the receptivity enhancement of the endometrium and improve the non-hormone-deficient thin endometrium. Therefore, the lactobionic acid microspheres are used for stimulating to replace the traditional hysteroscope physical scratching to realize thickening of the endometrium, so that the method is convenient and safer.

The specific preparation process of the lactobionic acid microspheres comprises the following steps:

1. weighing 10g of lactobionic acid powder, adding the lactobionic acid powder into 89g of purified water, and stirring to dissolve uniformly;

2. weighing 1g of non-hydrolyzed type I collagen, fully stirring and dissolving in 99g of lactobionic acid aqueous solution to obtain a component A;

3. weighing 250g of isopropyl myristate (IPM), adding a water-in-oil emulsifier such as 50g of span 80, and uniformly stirring to obtain a component B;

4. heating the A and B components to 80 ℃, and then slowly adding 100g of the A component into 300 g of the B component under the state of rapid stirring;

5. shearing and homogenizing the mixed solution at a high speed by using a high-speed homogenizer until the whole system is clear;

6. weighing 5g of EDC powder, dissolving in 45g of absolute ethyl alcohol, and then adding into the homogenized AB component mixed solution while rapidly stirring at room temperature;

7. standing the whole system for 18 hours at room temperature;

8. adding 1000G of ethanol to demulsify the whole system, centrifuging for 10 minutes by 5000G of centrifugal force, and collecting precipitates;

9. adding 200G of absolute ethyl alcohol into the precipitate, washing, centrifuging for 10 minutes by using a centrifugal force of 5000G, pouring out the supernatant, collecting the precipitate, and repeatedly washing for 3 times by using the absolute ethyl alcohol;

10. collecting the precipitate, evaporating to remove ethanol, and collecting the powder to obtain lactobionic acid microspheres (lactobionic acid-collagen microspheres).

Before application, the powder is mixed with sterile normal saline according to the mass ratio of 5-20% to prepare the injection of the slow-release lactobionic acid, which can be injected into the uterus and has convenient, efficient and safe operation.

Appearance of microspheres containing lactobionic acid

The lactobionic acid-collagen microspheres are purified and dried to be white powder, and are added into normal saline to ensure that the mass ratio reaches 5 percent, so that the lactobionic acid-collagen microspheres can be fully dispersed by the normal saline and suspended in the normal saline. Physiological saline mixed with lactobionic acid-collagen microspheres is smeared on a glass slide and observed under a microscope of 600 times, and the particle size of the microspheres is uniform, which is shown in figure 1.

Slow release assay of lactobionic acid microspheres as shown in figure 2:

adding 100mg lactobionic acid sustained-release microspheres into PBS buffer solution, simultaneously adding 10mg collagenase type I into phosphate buffer solution, then placing the system in a water bath at 37 ℃, taking a sample once a day, and detecting the content of lactobionic acid by using an HPLC method, wherein the detection result shows that the product releases clean 8.7mg lactobionic acid in 5 days and releases 10.9mg lactobionic acid in 10 days. The microspheres are proved to have the sustained-release effect of lactobionic acid.

Endometrium micro-stimulation animal experiment

20 SD rats, female SD rats in estrus, were intraperitoneally injected with 4% chloral hydrate (0.8 mL/100 g), and after corneal reflex and righting reflex disappeared, skin was prepared in the back region of the operation, and was sterilized with topical iodophor, and placed on a sterile operating table. A longitudinal incision of 1cm to 1.5cm is made at a position of 0.5 cm to 1cm on the ilium and beside the midline of the back, and the layer by layer separation is carried out to expose one uterus. Ligating the two ends of the ovary and the vagina by using a line with the number 0, soaking gauze by using isotonic saline, then filling the gauze in the position close to an incision, straightening the uterus by using toothless forceps, slowly injecting 95 percent ethanol by volume into the far end of the ovary by using a 1ml syringe, sucking the residual ethanol back after the needle is kept in the uterine cavity for 5 minutes according to the filling degree of the uterine cavity, and repeatedly washing the cavity of an official tube for 2-3 times by using the isotonic saline. After confirming no bleeding and liquid residue, the uterus is taken into the abdominal cavity, and then the layer by layer suture is carried out, and the iodophor sterilizes the skin suture part. Penicillin is continuously injected into muscles every day after operation to prevent infection.

After 3 months, 20 SD rats were divided into 10 test groups and 10 control groups. The abdominal cavity of the rat was opened again using the same protocol. Mixing 0.5g lactobionic acid microsphere powder with 4.5g sterile normal saline before treatment, clamping bilateral uterine proximal ends, uterine arteries and bilateral uterine distal ends and ovarian artery-uterine artery traffic branches by using a non-invasive blood vessel clamp after an abdominal cavity of a test group is opened, enabling a syringe needle to enter a uterine cavity at the lower end of the uterus and keep the uterine cavity parallel to a longitudinal axis of the uterus, slowly injecting 0.5mL collagen microsphere solution containing lactobionic acid to keep the uterine cavity in a filling state, and loosening the blood vessel clamp after 30min to perform uterine reperfusion. Meanwhile, normal saline is used for flushing the abdominal cavity and closing the abdomen. The rats were incubated prior to awakening. The same procedure was followed for 10 control rats, but saline was injected instead. Samples were taken at 1 month and 3 months and examined histologically for endometrial status.

The experimental results are shown in fig. 3 (the upper row in the figure is the picture of the experimental group, and the lower row is the picture of the control group), after 1 month of treatment, the glands of the uterine treatment group and the control group are enlarged, the number of blood vessels is obviously increased, and the experimental group with the thickness of the endometrium is slightly thicker than that of the control group. After 3 months of treatment, as shown in fig. 4 (the upper row in the figure is a picture of an experimental group, and the lower row is a picture of a control group), the distribution of the glands and blood vessels in the treated group is normal, the glands in the damaged group are slightly enlarged compared with the normal group, the number of the blood vessels is obviously more, and the recovery degree of the endometrium of the treated group is obviously better than that of the control group.

In conclusion, lactobionic acid microspheres are used for stimulation to replace traditional hysteroscopy physical scratching to achieve the effect of thickening endometrium, and because lactobionic acid is small in side effect, endometrium is not damaged, safety is high, and meanwhile, lactobionic acid is coated by collagen microspheres to achieve the effect of slowly releasing lactobionic acid, and the product can be directly injected into uterus after being suspended with normal saline, is convenient to clinically apply, and can be used for replacing a hysteroscope endometrium stimulation therapy.

Claims (3)

1. An injection for slowly releasing lactobionic acid contains lactobionic acid microspheres.

2. An injection formulation with extended release of lactobionic acid according to claim 1, characterised in that: the lactobionic acid microspheres are used for treating thin endometrium.

3. An injection formulation with extended release of lactobionic acid according to claim 1 or 2, characterised in that: the preparation method of the lactobionic acid microspheres comprises the following steps:

the method comprises the following steps: weighing lactobionic acid powder, adding the lactobionic acid powder into purified water, and preparing a lactobionic acid water solution with the mass ratio of lactobionic acid being 5-20%;

step two: weighing non-hydrolyzed type I collagen, and fully dissolving the non-hydrolyzed type I collagen in a lactobionic acid aqueous solution to prepare a lactobionic acid-collagen aqueous solution with the mass ratio of collagen of 0.5-3%, wherein the lactobionic acid-collagen aqueous solution is used as a component A for later use;

step three: weighing isopropyl myristate, and adding a water-in-oil emulsifier to enable the content of the water-in-oil emulsifier to reach a mass ratio of 10% -30%, wherein the component B is the component;

step four: heating the component B to 70-85 ℃, and then slowly adding the component A while stirring to obtain a mixed solution of the component A and the component B in a mass ratio of 1;

step five: shearing and homogenizing the mixed solution at a high speed by using a homogenizer until the mixed solution is clarified; then adding an absolute ethanol solution mixed with 5-20% of EDC by mass ratio at room temperature for 1/10-1/5 times dilution, and fully stirring; then standing for 6-72 hours at room temperature; then adding 2-5 times volume of ethanol to demulsify the whole system, centrifuging for 5-30 minutes by using 2000-10000G, and collecting precipitates; after collecting the precipitate, adding absolute ethyl alcohol for cleaning, centrifuging for 5-30 minutes at 2000-10000G, pouring out the supernatant, collecting the precipitate, and repeatedly cleaning for 1-5 times by the absolute ethyl alcohol; then, the precipitate was collected, and ethanol in the precipitate was removed by an evaporator, and the powder was collected as lactobionic acid microspheres.

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