CN113244368A

CN113244368A - Application of polypeptide in preparing medicine for treating endometriosis

Info

Publication number: CN113244368A
Application number: CN202110662689.4A
Authority: CN
Inventors: 朱小琳; 韩亚光; 韩延华; 贾丽研; 韩亚鹏
Original assignee: Heilongjiang University of Chinese Medicine
Current assignee: Heilongjiang University of Chinese Medicine
Priority date: 2021-06-15
Filing date: 2021-06-15
Publication date: 2021-08-13
Anticipated expiration: 2041-06-15
Also published as: CN113244368B

Abstract

The invention relates to application of a polypeptide in preparing a medicament for treating endometriosis, wherein the polypeptide has an amino acid sequence shown as SEQ ID NO.1 or an amino acid sequence which is lack of, replaces any one of amino acids or is added with one of amino acids compared with the sequence of SEQ ID NO. 1. The polypeptide of the present invention can improve endometriosis, and is particularly suitable for alleviating abdominal pain and/or abnormal menstruation caused by endometriosis.

Description

Application of polypeptide in preparing medicine for treating endometriosis

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of polypeptide in preparation of a medicine for treating endometriosis.

Background

Endometriosis (endometrisis) refers to a common gynecological disease in women, in which activated endometrial cells are seeded at a site other than the endometrium. The endometrial cells should grow in the uterine cavity, but because the uterine cavity is communicated with the pelvic cavity through the fallopian tube, the endometrial cells can enter the pelvic cavity through the fallopian tube to grow ectopically. There are a number of indications as to the mechanism of the onset of this disease, among which the theory of endometrial implantation is generally accepted. The disease mostly occurs in women of childbearing age, the disease does not occur before puberty, and the ectopic focus after menopause can gradually shrink and degenerate. The main pathological changes of endometriosis are ectopic intimal periodic hemorrhage and fibrosis of surrounding tissues, formation of ectopic nodules, and main symptoms of dysmenorrhea, chronic pelvic pain, abnormal menstruation and infertility. Lesions can spread to all pelvic tissues and organs, are most common in parts such as ovary, uterine rectum pouch, uterosacral ligament and the like, and can also occur in abdominal cavity, thoracic cavity, limbs and the like. Clinical practice shows that patients often show progressive aggravated pelvic fibrosis, adhesion, pain and infertility (about 30-50% of EMS patients have secondary infertility), so that physical and mental health and life quality of wide females are seriously affected, and harmony of the whole family and the society is even caused.

Despite the last half century of research, there are still too many unknowns on the pathogenesis of EMS. Drug and surgical therapy remain the current primary treatment, but relapse remains a problem that is confusing to the industry. Therefore, deep analysis of the pathogenesis of the internal abnormality lays a foundation for better understanding of the disease and finding more effective treatment measures in the industry, and has important strategic significance for improving the health quality of women and the health harmony of the whole family and the society.

The theory of endometrial reflux is the most well-recognized theory, however, reflux occurs in the menstrual cycle in most women, and only 10% to 15% of women suffer from EMS; there are studies showing that intrinsic abnormalities in Endometrial Stromal Cells (ESCs) are associated with EMS, including aberrant gene expression, response of the endometrium to hormones, increased nerve density and oxidative stress; under normal conditions, the endometrium which flows back to the pelvic cavity is identified as being cleared by the immune system of an organism as a 'foreign body', while the local immune microenvironment is likely to be changed in the pelvic cavity of an EMS patient, and the abnormal function of immune cells can not effectively clear ectopic endometrial cells, even help the ectopic endometrial cells to be planted and grow in the ectopic position; therefore, in recent years, there is increasing evidence that local immune microenvironment abnormalities in the pelvic cavity of EMS patients play an essential role in the development and progression of EMS, especially the various immune cells and cytokines produced in ectopic foci.

Current pharmacotherapy for treating endometriosis is primarily the use of drugs to combat or inhibit the cyclic endocrine stimulation of the ovary. Testosterone androgen is used initially, and has been abandoned gradually because of large side effect and insufficient efficacy. Later, the method gradually develops into pseudopregnancy therapy and pseudomenopause therapy.

(1) The progestogen medicine for the pseudopregnancy therapy is taken uninterruptedly for a long time with larger dosage, so that the menstruation stops and the endometrium and the ectopic endometrium generate a reaction similar to pregnancy under the action of the medicine, and the pseudopregnancy therapy is also called as the pseudopregnancy therapy. The drugs used for this therapy are numerous and are still in development, mainly medroxyprogesterone, prevela, endometin, etc. taken orally, and progesterone caproate, injected intramuscularly. This treatment lasts at least six months before the ectopic intima becomes inactive and eventually atrophy occurs, resulting in a therapeutic effect.

(2) Danazol, a derivative of androgen, is used for pseudo-menopausal therapy, and has better effect but larger side effect. At present, a gonadotropin releasing hormone agonist (GnRHA), commonly called goserelin, is widely used, and mainly can strongly inhibit the function of ovaries and almost completely lose the function of the ovaries so as to achieve the treatment purpose. These drugs can cause the endometrium to produce a phenomenon similar to endometrial atrophy in menopausal women, and are called pseudo-menopausal therapy.

In view of the above known drug treatment methods, which mainly use androgens or progestogens, some patients have a significant resistance to hormonal drugs. In the previous work of the applicant, the polypeptide Met-Trp-Trp-His-His-Met is found to have a good curative effect on the treatment of polycystic ovarian syndrome, and the research result is applied to Chinese patent (application number: 202010605265X). As a continuation of the above work, the applicants further found that the polypeptide not only has a superior therapeutic effect on polycystic ovarian syndrome, but surprisingly that the polypeptide Met-Trp-His-Met also has a superior therapeutic effect on endometriosis. The present invention has been completed based on the above-mentioned unexpected findings of the present applicant.

Disclosure of Invention

In order to treat endometriosis, the invention provides the use of a polypeptide in the manufacture of a medicament for the treatment of endometriosis. Specifically, the invention adopts the following technical scheme:

the invention relates to the application of polypeptide in preparing medicine for treating endometriosis, wherein the polypeptide has an amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence which is lack of, replaces any one of or adds one amino acid compared with the sequence of SEQ ID NO. 1.

In a preferred embodiment of the invention, the medicament is for alleviating abdominal pain and/or abnormal menstruation caused by endometriosis.

In a preferred embodiment of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

In a preferred embodiment of the present invention, the drug is in an injectable formulation, such as a liquid injectable formulation or a lyophilized powder injection.

In another aspect, the present invention relates to a medicament for treating endometriosis, which comprises as an active ingredient a polypeptide having an amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence which is one of the amino acids lacking, substituted or added as compared with the sequence of SEQ ID NO. 1.

Yet another aspect of the present invention relates to an injection for treating endometriosis, which comprises a polypeptide having an amino acid sequence shown in SEQ ID No.1 or an amino acid sequence lacking, replacing or adding one amino acid thereto as compared with the sequence of SEQ ID No.1, as an active ingredient; preferably, the polypeptide is the only active ingredient.

Advantageous effects

The polypeptide Met-Trp-Trp-His-His-Met can improve endometriosis, and is particularly suitable for relieving abdominal pain and/or abnormal menstruation caused by endometriosis.

Detailed Description

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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 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.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1

Establishing an endometriosis animal model

In the study of endometriosis, clinical patients have limited access to endometriosis tissues, and therefore it is very necessary to establish an endometriosis animal model. The inventor selects 45 female mature SPF SD rats to establish an endometriosis model, and identifies that 30 SD rats are successfully modeled, and the success rate is 67.7%. The specific animal experimental procedure (refer to CN102988401A) is as follows:

(1) obtaining 45 sexually mature SPF grade healthy unmated female SD rats from the animal center of the first hospital affiliated to the university of traditional Chinese medicine of heilongjiang;

(2) 1% sodium pentobarbital (40mg/kg) was anesthetized by intraperitoneal injection;

(3) after conventional disinfection, a longitudinal incision of about 3cm is made on 1cm of the middle lower abdomen and pubic symphysis of the rat;

(4) finding the uterus on the dorsal side of the bladder after entering the abdominal cavity;

(5) dissociating the right uterus, ligating the uterine horn at the proximal end about 1cm, and ligating the uterine horn at the distal end about 1cm away from the ovary;

(6) cutting off uterus tissue in the middle of the ligature, rapidly placing in a culture dish containing sterile normal saline, and shearing three pieces of endometrium tissue (containing muscle layer) with the size of 5mm multiplied by 5 mm;

taking a piece of endometrial tissue, pasting the endometrial surface of a No. 40 absorbable thread on the adipose tissue around the left ovary, and sewing four corners;

taking an inner membrane tissue, pasting the inner membrane surface on the right abdominal wall by using a No. 40 absorbable thread, and sewing four corners;

(7) suturing the incision of the rectus abdominis and dripping 40 million U/injection penicillin sodium into the abdominal cavity;

(8) a tunnel is formed between subcutaneous fascia layers of abdominal muscles on the right side of the abdominal wall incision, so that another endometrial tissue can be implanted. Implanting the last piece of endometrial tissue into the bottom of the right tunnel in a smooth manner, so that the intima surface is tightly attached to the abdominal muscles;

(9) placing the rest uterus tissue in formalin solution;

(10) conventional abdominal closing;

(11) after operation, 40 million U of penicillin sodium for injection is injected into the abdominal cavity of each rat to prevent infection for 3 days continuously;

(12) starting on day 5 after surgery, each SD rat was intramuscularly injected with estradiol benzoate injection (0.1mg/kg) once every 5 days for 3 consecutive times;

(13) the breeding is carried out in a clean environment, and the growth condition of the transplanted intima tissue is observed after 4 weeks of laparotomy.

(14) The transplanted ectopic endometrium is observed to grow well, the volume is obviously increased, the ectopic endometrium is in a small sac shape with a transparent bulge, the inside is full of effusion, and the surface blood vessel is clearly visible. Morphological examination revealed endometrial epithelial, glandular and interstitial cell growth, secretory activity and old bleeding. According to the standard, 30 of 45 rats are identified to be successfully modeled, and the success rate of modeling is 67.7%.

Example 2

Testing the therapeutic Effect of Polypeptides on endometriosis

The drugs used were:

gestrinone: the gestrinone capsule produced by Beijing Shibata pharmaceutical company is dissolved in normal saline. A polypeptide having the amino acid sequence of SEQ ID No. 1: synthesized and sequenced from Shanghai, the polypeptide was diluted to 10. mu.g/kg with physiological saline.

The experimental steps are as follows:

the 32 SD rats successfully molded were randomly divided into 3 groups, each group containing 10 rats, i.e., 10 rats in the negative control group (saline-infused group), 10 rats in the positive control group (gestrinone-infused group, 0.5mg/kg daily) and 10 rats in the polypeptide injection group (10 ml/kg daily). The physiological saline dosage of the control group was the same as the volume of the drug used in the polypeptide injection group for 4 consecutive weeks.

After 4 weeks, the abdominal wall is again dissected, and the ectopic intima tissue of the corresponding site is taken, placed in formalin solution, embedded in paraffin, and HE-stained after sectioning. The morphology of the endometrium was observed under a microscope.

Pathological result interpretation standard

The + and + shows that the growth state of the ectopic growth endometrium is good, the stroma is more, and a plurality of glands are visible;

+ indicates that the growth state of the endometrium of the ectopic growth is not good enough, the stroma is obviously reduced, and 12 expanded glandular cavities can be seen;

+ indicates that the ectopically growing endometrium is atrophic, with few interstitium (about 1/2 + + only), and only 1 distended glandular cavity;

indicating no visible intimal tissue and disappearance of the glands.

Results

The effects of the saline group (group 1), the gestrinone group (group 2) and the ATP instillation group (group 3) were +++, ++, and were respectively scored as 3, 2, 1, and 0. The results of the mean scores in each group were then recorded as shown in the following table:

as can be seen from the above table, the morphological changes of the ectopic endometrial tissues of the negative control group (normal saline infusion group) and the polypeptide injection group are obvious, and the differences have significant statistical significance. The glands and the interstitium of the ectopic endometrium of the polypeptide injection group are obviously reduced compared with the negative control group. The experiments show that the therapeutic effect of the polypeptide injection group in the treatment of endometriosis is even better than that of the current clinically common therapeutic drug, namely gestrinone.

EXAMPLE 3 clinical trials

1 data of

1.1 general data

40 patients of black dragon river Chinese medicine university affiliated to the first hospital in 2019, 10 months to 2019, 12 months are selected, the patients are divided into two groups by a random digital table method, the two groups are respectively a treatment group and a control group, each group comprises 20 patients, before treatment, the two groups of patients have no significant difference in the aspects of sex, age, disease course and the like, and the balance among the groups is good and comparable.

1.2 diagnostic criteria

According to the specified standard of the third academic conference of the special Committee of obstetrics and gynecology of the Chinese medical and western medicine integration society and the "clinical research guiding principle of novel Chinese medicine treatment EM", the following diagnosis standards are drawn up:

step one, gradual dysmenorrhea;

the lower abdomen and lumbosacral part during the menstrual period bulge gradually;

③ periodical drop pain of anus;

fourthly, the posterior fornix, the uterosacral ligament or the uterine isthmus are contacted with painful nodules;

ovarian chocolate cyst;

sixthly, there is an internal different focus in the pelvic cavity.

1 of the two items are clinical diagnosis when the two items coexist. And conforms to the traditional Chinese medicine dampness-heat stasis syndrome: a pain in the lower abdomen, an increased abdominal pain during menstruation, burning sensation, a premature or dribbling menstruation, low fever, a downward swelling or pain in the waist, a yellow color of the leucorrhea, a petechia on the tongue edge, and a slippery pulse.

1.3 inclusion criteria

(1) Meets the EM diagnosis standard;

(2) the subject signs an informed consent.

1.4 rejection criteria:

cases that were misincorporated without meeting inclusion criteria; cases that were not treated with the study protocol after inclusion, although meeting inclusion criteria; the abscission cases stopped in the middle of the test due to non-curative effect reasons and adverse reactions; adding other medicines; the data is not complete and cannot be counted.

1.5 termination and withdrawal of clinical trial criteria: other diseases appear in the test, affecting the test performers; other situations are not foreseeable.

2 method

2.1 methods of treatment

Treatment groups were given the following drugs: the patients were treated daily at the following doses: the injection is administered 1 time per day, 5ml each time (polypeptide is diluted to 10 μ g/kg with physiological saline), 2 months is a treatment course, the administration is stopped during menstrual period, 20 patients are sampled before and after treatment with the same fasting time in the 1 st day of menstrual cycle, and the treatment effect is recorded.

The control group was administered the following drugs: the patients in the control group are administered danazol 200mg each time 2 times a day, the total amount is not more than 800mg for 1 day, and 3 months are 1 course of treatment. 20 patients were sampled on empty stomach at the same time as the 1 st day of menstrual cycle before and after treatment and the treatment effect was recorded

2.2 Scoring criteria

(1) Abdominal pain: the abdominal pain disappeared, score 0; occasionally abdominal pain, 2 points; abdominal pain often occurs, 4 points; the abdominal pain persists and affects the work by 6 points.

(2) Menstruation abnormalities: abnormal menstruation disappeared, 0 point; occasionally, abnormal menstruation occurred, 2 points; menstrual abnormalities often appear, 4 points; menstrual abnormalities persisted for 6 points.

All patients fill in a unified symptom form before and after treatment, and psychological disorders (melancholy, schizophrenia, etc.) are excluded. The curative effect on dysmenorrhea is judged by comparing the components before and after the score-keeping method.

2.3 therapeutic efficacy criteria

The efficacy index (pre-treatment score-post-treatment score)/pre-treatment score was calculated from the pre-and post-treatment scores.

And (3) healing: after treatment, the integral value is reduced by more than 95 percent, and symptoms such as abdominal pain and the like disappear;

the effect is shown: after treatment, the integral value is reduced by 70-95%, and symptoms such as abdominal pain and the like are obviously improved;

the method has the following advantages: after treatment, the integral value is reduced by 30-70%, and symptoms such as abdominal pain and the like are relieved;

and (4) invalidation: the integral value after treatment is reduced by less than 30 percent, and symptoms such as abdominal pain and the like are not obviously improved.

3 results

3.1 Total therapeutic Effect

The treatment results are that in 20 patients in the treatment group, 5 patients are cured, 8 patients are obviously effective, 5 patients are effective, 2 patients are ineffective, and the total effective rate is 90%; in 20 patients in the control group, 3 patients are cured, 6 patients are effective, 4 patients are effective, 7 patients are ineffective, and the total effective rate is 65%.

4 conclusion

The experimental results show that the polypeptide injection has obvious therapeutic effect on endometriosis. The medicine has the advantages of quick action and good curative effect.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims

1. Use of a polypeptide having an amino acid sequence as shown in SEQ ID No.1 or an amino acid sequence which is missing, substituted or added with an amino acid compared to the sequence of SEQ ID No.1 for the preparation of a medicament for the treatment of endometriosis.

2. Use according to claim 1, for the relief of abdominal pain and/or abnormal menstruation caused by endometriosis.

3. The use according to claim 1 or 2, the medicament comprising a pharmaceutically acceptable carrier.

4. The use according to any one of claims 1 to 3, wherein the medicament is an injectable formulation.

5. The use according to any one of claims 1 to 3, wherein the medicament is in the form of a liquid injection or a lyophilized powder for injection.

6. A medicament for treating endometriosis, which comprises as an active ingredient a polypeptide having an amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence lacking, replacing or adding one amino acid as compared with the sequence of SEQ ID NO. 1.

7. An injection for treating endometriosis, which comprises a polypeptide as an active ingredient, the polypeptide having an amino acid sequence shown in SEQ ID No.1 or an amino acid sequence which is one of lacking, substituted or added compared with the sequence of SEQ ID No. 1; preferably, the polypeptide is the only active ingredient.