CN110833549B

CN110833549B - Use of pyrazolopyrimidine derivative for the treatment of chronic pelvic inflammatory disease

Info

Publication number: CN110833549B
Application number: CN201810927528.1A
Authority: CN
Inventors: 钟杰敏; 赖树生; 巫鑫; 聂雪玫; 陈丹; 覃秋琳
Original assignee: Guangxi Wuzhou Pharmaceutical Group Co Ltd
Current assignee: Guangxi Wuzhou Pharmaceutical Group Co Ltd
Priority date: 2018-08-15
Filing date: 2018-08-15
Publication date: 2023-05-02
Anticipated expiration: 2038-08-15
Also published as: CN110833549A

Abstract

The invention relates to the technical field of medicines, in particular to application of pyrazolopyrimidine derivative in treating chronic pelvic inflammatory disease. The chronic pelvic inflammatory disease is associated with increased blood viscosity in inflammatory tissues and enhanced expression of ICAM-1 and FGF-2. The pyrazolopyrimidine derivative can inhibit combined action mechanisms such as ICAM-1, FGF-expression and the like in tissues by reducing blood viscosity, and has good treatment effect on chronic pelvic inflammatory disease.

Description

Use of pyrazolopyrimidine derivative for the treatment of chronic pelvic inflammatory disease

Technical Field

The invention relates to the technical field of medicines, in particular to application of pyrazolopyrimidine derivative in treating chronic pelvic inflammatory disease.

Background

Chronic Pelvic Inflammatory Disease (CPID) refers to a series of sequelae that occur when pelvic inflammatory disease is not treated correctly in time, and is mainly manifested by recurrent pelvic pain, lumbosacral soreness, dysmenorrhea, and easy to cause infertility and ectopic pregnancy. The main pathological changes of CPID are mainly inflammatory exudation, adhesion and hyperplasia of pelvic cavity, bacterial infection is different from PID, and the antibacterial treatment has better effect on acute phase, but has insignificant effect on CPID.

Pyrazolopyrimidine derivative (formula I) is a small molecule inhibitor targeting FLT3 kinase, is a novel compound independently developed by the applicant, and has been granted in China, the United states, japan and other areas.

At present, only pyrazolopyrimidine derivative is reported in the aspect of treating acute myelogenous leukemia and psoriasis, but the pyrazolopyrimidine derivative has no application in the aspect of treating chronic pelvic inflammatory disease.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides the application of pyrazolopyrimidine derivative in treating chronic pelvic inflammatory disease.

The aim of the invention can be achieved by the following technical scheme:

use of pyrazolopyrimidine derivative shown as formula I or pharmaceutically acceptable salt and hydrate thereof in preparation of medicines for treating chronic pelvic inflammatory disease

Preferably, the chronic pelvic inflammatory disease is associated with increased blood viscosity in inflammatory tissue, and increased expression of ICAM-1, FGF-2.

Preferably, the pharmaceutically acceptable salts include hydrochloride, sulfate, mesylate, phosphate.

The invention also provides application of the pharmaceutical composition in preparing medicines for treating chronic pelvic inflammatory disease, and the pharmaceutical composition is characterized by comprising pyrazolopyrimidine derivative shown as a formula I or pharmaceutically acceptable salt and hydrate thereof as active ingredients and pharmaceutically acceptable excipients.

Preferably, the pharmaceutical composition is an injection preparation, an oral preparation, an external preparation, a slow release preparation or a controlled release preparation.

Preferably, the oral preparation comprises a tablet, a granule and a capsule.

Preferably, the pharmaceutical composition is a controlled release preparation, a sustained release preparation or an immediate release preparation.

ICAM-1 is one of important adhesion molecules, and is usually expressed at a certain level on the cell surface, and certain stimulus factors (such as hypoxia and the like) can induce the expression to be increased, participate in the activation and migration of inflammatory cells, promote chronic inflammatory reaction and increase of collagen secretion, so that tissue interstitial proliferation and local fibrosis occur; down-regulating ICAM-1 expression or inhibiting cytokine induction of ICAM-1 expression can effectively inhibit inflammatory reaction.

FGF-2 is a cell growth regulating factor with strong proliferation and differentiation promoting effect, and in vivo FGF-2 moderate expression is normally effective in improving local microcirculation and tissue nutrition condition, but in stress state, such as ischemia, hypoxia, infection, etc., FGF-2 expression is increased, vascular endothelial cells, tissue epithelial cells and fibroblasts can be induced to proliferate and differentiate rapidly, so that the FGF-2 is a promoting factor for tissue adhesion and proliferation. Therefore, inhibition of FGF-2 overexpression and reduction of capillary vessel neogenesis are one of important mechanisms for preventing tissue adhesion.

Blood viscosity is one of the most important indices in blood rheology examination, and is a comprehensive index reflecting blood rheology. When the proinflammatory cytokines stimulate the body, the local hemodynamics and the rheology can be changed, and various indexes (high cut, medium cut, low cut, plasma viscosity and the like) of the haemorheology are higher than normal values, so that the body blood circulation disorder is caused, and the blood is in a sticky, thick, coagulated and aggregated state.

The pyrazolopyrimidine derivative is a FLT3 inhibitor, and researches show that the pyrazolopyrimidine derivative can inhibit combined action mechanisms such as ICAM-1 and FGF-2 expression in tissues by reducing blood viscosity, and has good treatment effect on chronic pelvic inflammatory disease.

Compared with the prior art, the method has the following advantages:

the pyrazolopyrimidine derivative provided by the invention is a novel medicament for treating chronic pelvic inflammatory disease, not only expands the selectivity of the existing medicament for treating chronic pelvic inflammatory disease and provides more medicament choices for effective treatment of the disease, but also further expands the application range of the pyrazolopyrimidine derivative as an FLT3 inhibitor.

Drawings

FIG. 1 is a pathological observation of tissue morphology of the uterus of a control group of rats stained with HE.

FIG. 2 is a pathological observation of tissue morphology of the uterus of sham operated rats by HE staining.

FIG. 3 is a histomorphometric pathological observation of model group rat uterus by HE staining.

Fig. 4 is a histomorphology pathology of the HE-stained uterus of pyrazolopyrimidine derivative high-dose group rats.

Fig. 5 is a histomorphology pathology of the HE stained uterus of rats in dose groups of pyrazolopyrimidine derivative.

Fig. 6 is a histomorphology pathology of the HE-stained uterus of pyrazolopyrimidine derivative low-dose group rats.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

EXAMPLE 1 study of the Effect of rats with chronic pelvic inflammatory disease model

1. Experimental animals: SPF-class female SD rats 90, mass (200+ -20 g), provided by the university of Sichuan laboratory animal center.

2. Experimental materials: pyrazolopyrimidine derivative is synthesized by the inventors. Castor oil with ethanol 1:1, passing through a sterile filter membrane with the diameter of 0.22 mu m to obtain an ELE solution, weighing a certain mass of pyrazolopyrimidine derivative powder, dissolving the powder by the ELE solution, adding a certain volume of sterilized water after the powder is completely dissolved, and uniformly mixing for later use. The volume ratio of the ELE solution to the sterile water is 1:3. coli, staphylococcus aureus, by the university of chinese medicine, university of guangxi, microbiology teaching and research laboratory. According to the escherichia coli: staphylococcus aureus = 1:1 ratio, formulated to a density of 3X 10 ⁹ Bacterial suspension/mL. 3. And (3) establishing a model: 80 female SD rats are taken, and the female SD rats are placed in an indoor adaptation environment for 7 days before the experiment, and are fed with standard feed at the room temperature of 18-20 ℃ and are free to drink water. Fasted 24 hours before molding, normally drinking water, injecting 10% chloral hydrate into the abdominal cavity of an anesthetized rat the next day, taking the dosage of 0.3mL/100g, shearing off the middle hair of the lower abdomen after the anesthesia of the rat, exposing the skin, sterilizing with iodophor and medical alcohol, cutting a small opening at the middle of the lower abdomen for about 0.8-1 cm, exposing and fixing the uterus after opening the abdomen,the needle is respectively inserted into the uterus of the left and right sides of the rat at the uterine bifurcation by a 4-gauge needle (the tip of the needle is ground off), and is pulled back and forth in the uterine cavity for 2 times, and the injection density is 3 multiplied by 10 ⁹ After 0.lmL of bacterial mixed suspension, the rat abdomen was sutured in layers and the surgical field was sterilized. The rats which are only opened and closed and are pulled back and injected with bacterial liquid in the future according to the method are a sham operation control group. The blank group was not subjected to any treatment. Drinking water is recovered after the operation, normal clean feeding is performed, and the operation area 7d is disinfected by iodophor.

4. Grouping and administration of animals: on day 15 after modeling, model rats were weighed one by one, except for the blank control group and the sham operation group, and randomly divided into 6 groups: model group, pyrazolopyrimidine derivative high dose group (hereinafter referred to as high dose group), pyrazolopyrimidine derivative medium dose group (hereinafter referred to as medium dose group), pyrazolopyrimidine derivative low dose group (hereinafter referred to as low dose group), each group is 15. High dose group lavage administration of pyrazolopyrimidine derivative of the invention 15mg/kg/d (1.5 mg/mL of the drug solution), medium dose group lavage administration of pyrazolopyrimidine derivative of the invention 10mg/kg/d (1.0 mg/mL of the drug solution), low dose group lavage administration of pyrazolopyrimidine derivative of the invention 5mg/kg/d (0.5 mg/mL of the drug solution); the model group, the sham operation group and the blank control group are given with a mixed solution of the ELE solution and distilled water in a volume ratio of 1:3, and the daily gastric lavage amount is calculated as 10 mL/kg. 1 time per day for 14 days.

5. Detection index

(1) After the last administration for 24 hours, the anesthetized rats were intraperitoneally injected with 10% chloral hydrate at a dose of 0.3mL/100g, the rats were fixed on a plate, the median hair of the lower abdomen was cut off, sterilized with iodophor and medical alcohol, a small opening about 1cm long was cut in the center of the lower abdomen to expose the abdominal aorta, blood was taken from the abdominal aorta with a blood taking needle, 4mL was placed in an EDTA-K2 anticoagulation tube, 1mL was placed in a heparin-treated blood taking tube, blood rheology analysis was performed with a full-automatic blood flow-changing rapid analyzer, and the whole blood viscosity, plasma viscosity and hematocrit were detected, blood routine detection analysis was performed with a full-automatic five-class hemocyte analyzer, and the total number of white blood cells and the percentage of neutral granulocytes were recorded.

(2) Histopathological examination: after blood collection, the uterus of the two sides of the rat is picked, adipose tissues are removed, the rat is fixed by a 10% paraformaldehyde solution after weighing, gradient ethanol is used for dehydration, xylene is transparent, paraffin is embedded, the rat is cut into tissue slices with the thickness of 2-3 mu m, HE (human-animal-derived) staining is performed, a neutral resin sealing piece is performed, and pathological morphological changes of the uterus are observed under a light microscope.

(3) The level of ICAM-1 and FGF-2 in rat uterine tissues is detected by adopting an immunohistochemical S-P method, and the specific operation method is according to a kit instruction provided by Nanjing building. Quantitative analysis of ICAM-1 and FGF-2 expression in uterine tissue, namely, selecting a region with concentrated positive cells under an optical microscope and 20 times of an objective lens, collecting 3 optical lens visual fields, and inputting the visual fields into an HPIAS-1000 pathological image-text analysis system for computer complete quantitative analysis. According to the analysis result of the system, the percentage content of positive cells in each slice to the total cells is calculated, and the calculation formula is [ total positive target area/total statistical field area (surface density) ]/[ total negative target area/total statistical field area (surface density) +total positive target area/total statistical field area (surface density) ].

6. Statistical methods: SPSS17.0 is adopted to process data, metering data is expressed by (mean ± standard deviation), multi-group comparison is performed by single factor analysis of variance, the inter-group comparison is performed by LSD and SNK test, and P <0.05 is the difference and has statistical significance.

7. Experimental results

(1) Influence on changes in the haemorheological index of rats with chronic pelvic inflammatory disease model

Table 1 change in the hemorheology index of rats in each group (n=15)

P <0.05 compared to the normal group; compared with the model group, deltaP < 0.05.

As shown in table 1, compared with the blank control group, the whole blood viscosity of the rats of the model group was significantly increased (P < 0.05) by low cut, medium cut, high cut, plasma viscosity and erythrocyte aggregation index; compared with the model group, the whole blood viscosity of the rats in the high dose group, the medium dose group and the low dose group is obviously reduced in the low cut, the medium cut and the high cut (P < 0.05); the plasma viscosity was significantly reduced (P < 0.05) in both the high and medium dose groups, and the low dose group showed only a reduced trend.

(2) Effects on expression of ICAM-1 and FGF-2 in uterine tissue of rats with chronic pelvic inflammatory disease

Table 2 expression of ICAM-1, FGF-2 by uterine tissue of rats of each group (n=15)

Group of	ICAM-1/％	FGF-2/％
			Blank control group	23.65±3.25	24.62±3.56
False operation group	24.62±3.42	25.37±3.84
			Model group	46.27±3.75*	58.44±4.25*
High dose group	32.75±3.19△	36.46±3.74△
			Medium dose group	36.48±3.27△	42.75±4.12△
Low dose group	41.85±3.66	54.92±3.88

As shown in Table 2, ICAM-1 and FGF-2 expression in uterine tissue of rats in the model group was significantly enhanced (P < 0.05) compared to the blank group; compared with the model group, ICAM-1 and FGF-2 expression in uterine tissues of the high-dose group and the medium-dose group of rats are significantly reduced (P < 0.05), and the low-dose group only shows a reduction trend.

(3) Influence on pathological morphology of rat uterine tissue

As can be seen from fig. 1 and 2, no obvious abnormalities were seen in the uterine tissue of rats in the blank control group and the sham operation group. As can be seen from fig. 3, the main lesions of the model group are lesions such as disordered structures and unclear layering of endometrium and mucous membrane layers of rats, most of the lesions are visible as intrauterine adhesion or expansion, slight congestion and edema of endometrium, degeneration, necrosis and shedding of epithelial cells, slight hyperplasia of epithelial cells, infiltration of a large number of inflammatory cells of submucosa and the like, and the lesions basically accord with clinical CPID lesion characteristics. As can be seen from figures 4-6, the pyrazolopyrimidine derivative of the invention has the major pathological changes of mild congestion and edema of endometrium in the high, medium and low dosage groups, and is in dose-effect relationship.

Conclusion(s)

The pyrazolopyrimidine derivative has a good treatment effect on chronic pelvic inflammatory disease, and the main mechanism of the pyrazolopyrimidine derivative is related to reducing blood viscosity and inhibiting ICAM-1 and FGF-2 expression in tissues.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. Use of pyrazolopyrimidine derivative shown as formula I or pharmaceutically acceptable salt thereof in preparation of medicine for treating chronic pelvic inflammatory disease

Formula I.

2. The use according to claim 1, wherein the chronic pelvic inflammatory disease is associated with increased blood viscosity in inflammatory tissue, increased ICAM-1, FGF-2 expression.

3. The use according to claim 1, wherein the pharmaceutically acceptable salts comprise hydrochloride, sulfate, mesylate, phosphate.

4. Use of a pharmaceutical composition for the preparation of a medicament for the treatment of chronic pelvic inflammatory disease, characterized in that the pharmaceutical composition comprises a pyrazolopyrimidine derivative of formula i as defined in claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable excipient.

5. The use according to claim 4, wherein the pharmaceutical composition is an oral formulation.

6. The use according to claim 5, wherein the oral formulation comprises a tablet, a granule, a capsule.

7. The use according to claim 4, wherein the pharmaceutical composition is a controlled release formulation, a sustained release formulation, an immediate release formulation.