CN114557992A

CN114557992A - Application of compound pharmaceutical composition in preparation of medicine for treating interstitial pneumonia

Info

Publication number: CN114557992A
Application number: CN202210244761.6A
Authority: CN
Inventors: 傅继华
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2022-05-31
Anticipated expiration: 2042-03-11
Also published as: CN114557992B

Abstract

The invention discloses application of a compound pharmaceutical composition taking sarpogrelate and carbidopa as active ingredients in preparing a medicament for treating interstitial pneumonia, wherein the compound pharmaceutical composition can inhibit 5-hydroxytryptamine (5-HT) synthetase and 5-HT2A receptor (5-HT) of lung tissue cells (including macrophages)_2AR) Activity, in particular with 5-HT_2AThe R antagonist sarpogrelate and the 5-HT synthesis inhibitor carbidopa form a compound within a certain weight ratio range, have obvious synergistic effect on treating interstitial pneumonia, and have obvious treatment effect.

Description

Application of compound pharmaceutical composition in preparation of medicine for treating interstitial pneumonia

Technical Field

The invention relates to application of a compound, in particular to application of a compound pharmaceutical composition in preparing a medicament for treating interstitial pneumonia.

Background

Interstitial Pneumonia (IP) is a pulmonary inflammation and fibrotic lesion caused by various factors such as radiation, chemicals (Bleomycin, BLM), silica, asbestos, etc.), infection (respiratory viruses, bacteria, etc.), etc. The causes of IP are very extensive, and in 1985, Hill's Interscience and 1987, Hashima's Interscience classify the IP into rheumatism immune diseases, drugs or treatment-related diseases (antiarrhythmic drugs, chemotherapeutic drugs, paraquat, sulfasalazine, radiotherapy and the like), occupational and environmental-related diseases (silicosis, asbestosis, smut and the like), and primary diseases (sarcoidosis, lymphangiosarcoidosis, eosinophilic pneumonia and the like) or idiopathic diseases (idiopathic pulmonary fibrosis, nonspecific interstitial lung disease and the like) with unknown causes according to the causes. The pathological process of interstitial pneumonia is generally diffuse alveolitis with slow progress and/or alveolar structure disorder, and finally the alveolar structure is destroyed to form complete fibrosis and vesicular honeycomb lung in the alveolar cavity. The pulmonary fibrosis and the decline and even loss of the lung function caused by the further development of IP are still a big problem in the medical and health industry in China and even the world at present, and the main reason is that IP has not been treated with effective pertinence. The IP disease spectrum is more than 200, so far, the etiology and pathogenesis of most interstitial pneumonia are still unclear, and rare diseases are rare.

Fibrosis is a chronic progressive disease characterized by tissue fibrosis, and the pathogenesis of pulmonary fibrosis includes the following: fibroblast activation; decreased autophagy ability of the cell; transforming Growth Factor-beta (TGF-beta) signaling pathway activation and immune dysregulation of the body. Macrophages are currently thought to play an important role in the pathogenesis of pulmonary fibrosis. Macrophages are mainly involved in the process of repairing tissue injury in the immunoregulation pathway of the body, and mainly come from bone marrow mononuclear cells, and the bone marrow mononuclear cells become tissue macrophages after migrating into tissues. The role and research progress of Yanglijie et al in Yanglijie, Liugang, macrophage in pulmonary fibrosis, China journal of respiratory and Critical Care, Vol.19, No. 6, 11/2020, P: 626 + 629 > states that: polarized M1 macrophages may promote pulmonary fibrosis by secreting proinflammatory factors; polarized M2 macrophages can promote pulmonary fibrosis by secreting TGF- β.

In summary, the typical characteristics of IP are: inflammation of lung tissue, increased secretion of inflammatory factors such as Tumor Necrosis Factor alpha (TNF-a), Interleukin 1 beta (IL-1 beta), increased secretion of Reactive Oxygen Species (ROS), increased secretion of TGF-beta, and pulmonary interstitial fibrosis.

5-hydroxytryptamine (5-HT), also called serotonin, is a small molecule substance existing in the periphery and the center, and the physiological function of 5-HT is complex and is not completely clarified so far.

The synthesis of 5-HT is carried out in two steps, the first step: tryptophan is converted into 5-hydroxytryptophan under the catalysis of Tryptophan Hydroxylase (Tph) (Tph can be divided into two subtypes, i.e., Tph1 and Tph2, which are respectively present at the periphery and the center); the second step is that: 5-hydroxytryptophan is converted to 5-HT under the catalysis of Aromatic Amino Acid Decarboxylase (AADC). Thus, inhibition of peripheral 5-HT synthesis can be achieved by inhibiting Tph1 or AADC, respectively.

Although compounds that directly inhibit the activity of Tph1 have been reported, such as p-chlorophenylalanine (pCPA, alternative name: DL-4-chlorophenylalanine, DL-p-chlorophenylalanine, etc., molecular formula: C₉H₁₀ClNO₂) However, the disadvantage of low oral bioavailability is common, so no clinical application report exists.

Carbidopa (Cabidoba, CDP), molecular formula: c₁₀H₁₄N₂O₄The inhibitor is an AADC inhibitor, is clinically used for adjuvant therapy of Parkinson's disease, and has the action characteristics that the peripheral AADC inhibitor is not easy to enter the center, only inhibits the conversion of peripheral levodopa into dopamine, increases the levodopa content in circulation, and also inhibits the conversion of 5-hydroxytryptophan into 5-HT, and reduces the generation of 5-HT by cells synthesizing the 5-HT. CDP has not been reported in studies directed to the treatment of Interstitial Pneumonia (IP).

5-HT receptors (5-HT receptors, 5-HTR) are present on the cell membrane, belong to the membrane receptors, and the 5-HT Receptor subtype is complex, and it has been found that 7 major classes of receptors exist, namely 5-HT receptors_1-7R，5-HT_1，4，5R is mainly distributed in the center, 5-HT_{2，3，6，7}R is mainly distributed on the periphery. These 7 classes of receptors are further divided into several subtypes. 5-HT2 receptor (5-HT)₂R) can be classified as 5-HT_2AR、5-HT_2BR and 5-HT_2CAnd R is shown in the specification. Peripherally and centrally distributed is 5-HT_2AR、5-HT_2BR，5-HT_2CR is mainly present in the central nervous system, kidney, liver, skeletal muscle, etc. with 5-HT distributed therein_2AR、5-HT_2BR, wherein 5-HT_2AR is believed to have a major biological activity.

III.5-HT degradation5-HT is not only biologically active by acting directly on its receptor, but is also degraded within the mitochondria of the cell. The enzyme catalyzing 5-HT degradation is Monoamine Oxidase A (MAO-A), and the MAO-A can generate A large amount of ROS (reactive oxygen species), mainly H, while catalyzing 5-HT degradation₂O₂. Too much 5-HT will be degraded in cells by MAO-A catalysis, generating A large amount of ROS, thus causing the cells to malfunction. ROS can reduce cell mitochondrial ATP synthesis, mitochondrial damage, inflammatory signal pathway activation, increased inflammatory factor secretion, fibrotic pathway activation and other cell abnormalities.

Has the function of selectively blocking 5-HT_2AR is rare, Sarpogrelate (Sar) is a specific 5-HT₂An R antagonist of the formula: c₂₄H₃₁NO₆Antagonizing mainly 5-HT_2AR, p-5-HT_2BR also has weak antagonistic action, and hydrochloride thereof is commonly used in clinical application. Sarpogrelate Hydrochloride (SH) can inhibit platelet aggregation enhanced by 5-HT and inhibit vasoconstriction, and is clinically used for improving ischemic symptoms such as ulcer, pain and cold caused by chronic arterial occlusion.

At present, there is no 5-HT_2AThe use of R antagonists and/or 5-HT synthesis inhibitors for the treatment of IP is reported.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide application of a compound pharmaceutical composition in preparing a medicament for treating interstitial pneumonia.

The technical scheme is as follows: the application of a compound pharmaceutical composition taking sarpogrelate and carbidopa as active ingredients in preparing a medicament for treating interstitial pneumonia.

In the application, the sarpogrelate ester is sarpogrelate ester or a pharmaceutically acceptable salt of sarpogrelate ester.

In the application, the carbidopa is carbidopa or a pharmaceutically acceptable salt of carbidopa.

The compound pharmaceutical composition is in the dosage form of tablets, capsules, granules, powder, syrup, oral liquid or injection.

In the application, the weight ratio of the sarpogrelate to the carbidopa is 10: 1-1: 8.

In the application, the weight ratio of the sarpogrelate to the carbidopa is 5: 1-1: 4.

In the application, the weight ratio of the sarpogrelate to the carbidopa is 2: 1.

If the salt is pharmaceutically acceptable salt of sarpogrelate, converting the salt into the active ingredient sarpogrelate for weight ratio; if the salt is pharmaceutically acceptable carbidopa, the weight ratio is converted into the active ingredient carbidopa.

We found in the study:

I. expression of 5-HT synthetase system-tryptophan hydroxylase 1(Tph1) and aromatic Amino Acid Decarboxylase (AADC), 5-HT2A receptor (5-HT 2) in Bleomycin (BLM) -induced mouse Interstitial Pneumonia (IP) model_2AR) and monoamine oxidase A (MAO-A) were both significantly up-regulated. Suggesting that BLM induces IP, 5-HT synthesis and 5-HT in renal tissue_2ABoth R are activated significantly and 5-HT degradation catalyzed by MAO-A is also activated, possibly in association with the occurrence of pneumoniA, pulmonary fibrosis at IP.

Sarpogrelate Hydrochloride (SH) (inhibition of 5-HT) in BLM-induced mouse IP model_2AR) and Carbidopa (CDP) (inhibiting 5-HT synthesis) to form a compound, and SH + CDP is mixed according to different weight ratios of the two medicines to form different compounds to treat the model. The results indicate that the pathological manifestations of IP are significantly reversed, appearing as: the model animal shows obviously improved alveolar collapse, atrophy and interstitial thickening; the lung tissue inflammation expressed by the model animal is obviously improved, inflammatory cell infiltration such as macrophage and the like is obviously reduced, the contents of lung tissue inflammatory factors TNF-alpha and IL-1 beta are obviously reduced, and the content of ROS is obviously reduced; the model animal shows obviously improved pulmonary interstitial fibrosis, and shows that the content of TGF-beta in lung tissue is obviously reduced, and the content of pulmonary interstitial fibrosis tissue is obviously reduced. In addition, the SH + CDP compound has a remarkable synergistic effect on IP treatment according to a certain weight proportion of the medicine, and the curative effect of the medicine can be remarkably improved by combined use.

Has the advantages that: compared with the prior art, the invention has the following advantages: the compound pharmaceutical composition can inhibit 5-HT synthesis and 5-HT of lung tissue cells (including macrophages)_2AR Activity, in particular with 5-HT_2AThe R antagonist SH and the 5-HT synthesis inhibitor CDP form a compound in a certain weight ratio range, have obvious synergistic effect on treating interstitial pneumonia, and have obvious curative effect advantage when being used for treating IP.

Drawings

FIG. 1 shows the lung tissue Tph1, AADC, 5-HT after Bleomycin (BLM) induces mouse IP_2AR, MAO-A expression assay (immunohistochemical staining for expression of lung tissue-associated protein) (. times.400, typical macrophages are indicated by the arrows in the BLM model group);

figure 2 is the effect of SH + CDP combination drug treatment on BLM induced 5-HT levels in IP mouse lung tissue: p < 0.05, x: p is less than 0.01, and N is 8;

figure 3 is the effect of SH + CDP combination drug treatment on BLM induced ROS content in IP mouse lung tissue: p < 0.05, x: p is less than 0.01, and N is 8;

fig. 4 shows SH + CDP combination drug treatment on BLM-induced IP mouse lung tissue microtomine-eosin (HE) staining results a.he stained photographs of typical lung tissue sites (x 400, local to photograph, arrow in BLM model group indicates typical macrophages) b. P < 0.05, x: p is less than 0.01, and N is 8;

figure 5 is the effect of SH + CDP combination drug treatment on BLM induced TNF- α content in lung tissue of IP mice: p < 0.05, x: p is less than 0.01, and N is 8;

figure 6 is the effect of SH + CDP combination drug treatment on BLM induced IL-1 β levels in lung tissue of IP mice: p < 0.05, x: p is less than 0.01, and N is 8;

figure 7 is the effect of SH + CDP combination drug treatment on BLM induced TGF- β content in lung tissue of IP mice: p < 0.05, x: p is less than 0.01, and N is 8;

fig. 8 is mason staining results of BLM-induced IP mouse lung tissue sections on SH + CDP combination drug treatment a. mason staining photographs are representative of pulmonary interstitial fibrosis (x 400, model group visible significant pulmonary interstitial fibrosis, pulmonary interstitial thickening, alveolar atrophy collapse) b. percentage calculation of pulmonary interstitial fibrosis area to total area: p < 0.05, x: p is less than 0.01, and N is 8.

Detailed Description

Example 1

BLM induces alterations in lung tissue 5-HT synthase, 5-HT2A receptor, and MAO-A expression in mouse IP

1.1 Experimental methods

For experiments, male C57BL/6 mice (clean grade) are used, the weight of the mice is 20-24 g, the animals are raised in cages in common mouse cages, the room temperature is kept at 24 +/-2 ℃, the illumination control is carried out to ensure the light and dark conditions for 12 hours, and the mice are fed with common water. After animals were anesthetized, bleomycin (BLM, 3.5mg/kg) was administered intratracheally by puncture to establish an Interstitial Pneumonia (IP) animal model, and an equal volume of physiological saline was administered to the control group. The animals were sacrificed 21 days after molding, and lung tissue was formalin-fixed, tissue sectioned, immunohistochemically stained, and examined for lung tissue 5-HT2A receptor (5-HT 2A)_2AR), 5-HT synthetase Tph1 and AADC, 5-HT degradation enzyme monoamine oxidase A (MAO-A).

1.2 results of the experiment

The results are shown in FIG. 1. Immunohistochemical detection shows that: 5-HT in Lung tissue of control group_2AR, MAO-A expression level is very low, and Tph1 and AADC expression are obvious and all cells express; 5-HT in lung tissue of BLM model group compared to control group_2AR, Tph1, AADC and MAO-A expression level are obviously increased, and all cells Tph1 and AADC expression level are increased, and macrophage is most obvious, but 5-HT_2AThe most significant sites of increased R, MAO-A expression are macrophages and alveolar spaces.

Example 2

Therapeutic effect of drug on BLM-induced mouse IP

The therapeutic effects of the combined administration of Sarpogrelate Hydrochloride (SH) and Carbidopa (CDP), and of each administration alone on BLM-induced Interstitial Pneumonia (IP) in mice, were compared, thus verifying the synergistic effect of the combined administration of SH and CDP on IP treatment.

2.1 Experimental methods

(1) Animal treatment

For experiments, C57BL/6 mice (clean grade) are used, the weight of the mice is 20-24 g, the animals are raised in cages in common mouse cages, the room temperature is kept at 24 +/-2 ℃, the illumination control is carried out to ensure the light and dark conditions for 12 hours, and the mice are fed with common water.

Animals were randomized into 7 groups (8 per group): the control group comprises a BLM induction IP model group (BLM model group), an IP model SH treatment group (BLM + SH group), an IP model SH + CDP compound treatment group (SH: CDP) and 3 compounds (BLM + compound-1 group, BLM + compound-2 group and BLM + compound-3 group) and an IP model CDP treatment group (BLM + CDP group) according to different weight ratios. In the experiment, three SH + CDP compounds in different weight proportions are as follows: compound-1-SH: CDP is 5: 1; compound-2-SH: CDP is 2: 1; compound formula-3-SH: CDP is 1: 4.

(2) Dosage to be administered

Control group-oral administration (p.o.) 0.5% sodium carboxymethylcellulose (CMC-Na) solution 0.20ml/10g body weight/time;

model group- -p.o.0.5% CMC-Na 0.20ml/10g body weight/time;

drug treatment groups-p.o. groups of drugs, all groups administered the same dose: 60.0 mg/kg/time.

Each drug was suspended with 0.5% CMC-Na at the same concentration: 2.0mg/ml, and the p.o. administration volume of each group of the medicines is 0.20ml/10g body weight/time; the medicine is taken twice a day, once every morning and afternoon, so the daily dosage is as follows: 120 mg/kg.

(3) BLM induction mouse IP method

Animals were acclimatized for 5 days. After animals are anesthetized, bleomycin (BLM, 3.5mg/kg) is administered intratracheally to the puncture methods of other animals except for the puncture method of the control group to establish an Interstitial Pneumonia (IP) animal model. After 24h of molding, each drug treatment group starts administration treatment and gavage administration 2 times a day for 21 consecutive days, and the control group and the BLM group are administered with an equal volume of 0.5% CMC-Na solution (drug solvent). After 3 hours of the last administration, the animals were sacrificed after anesthesia, and then formalin was fixed after collection of lung tissues or frozen at-80 ℃ for storage.

(4) Test index detection method

And (5) taking lung tissues for freezing and storing for later use. Detecting the contents of 5-HT, ROS, TNF-alpha, IL-1 beta and TGF-beta in lung tissues by using a kit ELISA method;

taking a lung tissue fixed by formalin, carrying out conventional tissue section and HE staining, observing and photographing under a microscope, and counting the number of macrophages in the lung tissue picture in a 400-time picture;

formalin-fixed lung tissue was taken, sectioned by routine tissue, stained by Masson, observed under a microscope and photographed, and pulmonary interstitial fibrosis was observed in a 400-fold photograph. Taking a typical piece of lung interstitium in the picture, calculating the percentage of the area of the lung interstitial blue-stained fibrous tissue to the total area by using Image J software: area (%) of pulmonary interstitial fibrosis ═ area of pulmonary interstitial blue staining ÷ total area × 100%.

2.2 results of the experiment

Statistical tests among the groups of data were performed using one-way anova, and comparisons among the groups were performed using LSD. P < 0.05 indicates a significant statistical difference, and P < 0.01 indicates a very significant statistical difference. Mean. + -. standard deviation of data in the plot

And (4) showing.

(1) Therapeutic effect of SH + CDP compound on BLM induced mouse IP

1) Inhibition of elevated 5-HT and ROS levels in lung tissue

The results are shown in FIGS. 2 and 3. Compared with the control group, the lung tissue of the BLM model group has obviously increased 5-HT and ROS content, which indicates that the model animal has obviously increased 5-HT synthesis and increased ROS generation; compared with the model group, the three compound preparations of SH, CDP and SH + CDP with the same administration dose (120 mg/kg/day) can obviously reduce the 5-HT and ROS content of the lung tissue. Moreover, the action effect of the BLM + FUFANG-2 group is the best, and the curative effect of the BLM + FUFANG-1 group and the BLM + FUFANG-2 group is better than that of the BLM + SH group and the BLM + CDP group. The suggestion shows that the obvious synergistic effect is generated, and the compound-2 synergistic effect is strongest. The result shows that the SH and the CDP form a compound with a certain proportion, and the compound has obvious synergistic effect on the inhibition effect of increasing the synthesis of 5-HT and the generation of ROS of lung tissues in an IP model, and the SH: the CDP ratio ranges are: 5: 1-1: 4, in the experiment, SH: CDP is 2: 1 (Fufang-2) with the best curative effect.

2) Therapeutic results on Lung injury and Lung interstitial inflammation

The results are shown in FIG. 4. Lung tissue section HE staining results indicated (fig. 4A): compared with a control group, the lung interstitium of the mice of the BLM model group is thickened, the alveoli are atrophied and collapsed, obvious inflammatory cell infiltration is realized, and a large amount of macrophage infiltration can be observed in lung tissues. The pathological damage of the lung tissues of the BLM + SH group and the BLM + CDP group is obviously improved; the improvement of the pathological injury of the lung tissues of the BLM + compound-1 group, the BLM + compound-2 group and the BLM + compound-3 group is more obvious; and the pathological damage of the lung tissues of the BLM + compound-2 group is almost completely inhibited, and the lung interstitium is not obviously thickened, the alveoli are normal and inflammatory cell infiltration is occasionally seen. The full photograph macrophage count results showed (fig. 4B): the control group has few and rare macrophages in lung tissues, the BLM model group has a large number of macrophages in lung tissues, the number of macrophages in each administration group is obviously reduced, the number of macrophages in the three compound groups is less than that of SH and CDP single groups, and the number of BLM + compound-2 is minimum, which indicates that the three compound groups all generate synergistic effect.

The results of the inhibition effect on the secretion of inflammatory factors TNF-alpha and IL-1 beta are shown in FIG. 5 and FIG. 6. The results show that: compared with a control group, the content of TNF-alpha and IL-1 beta in lung tissues of mice in the BLM model group is obviously increased; compared with the BLM model group, the lung tissues of each administration group have obviously reduced contents of TNF-alpha and IL-1 beta, the BLM + compound-2 group is most obvious, and the lung tissues of the BLM + compound-1 group and the BLM + compound-3 group have obviously lower contents of TNF-alpha and IL-1 beta than the BLM + SH group and the BLM + CDP group, which indicates that the three compound groups generate synergistic effect.

The result shows that the SH and the CDP form a compound with a certain proportion, and the compound has obvious synergistic effect on the inhibition effect of lung tissue injury, inflammation and macrophage infiltration in an IP model, and the SH: the CDP ratio ranges are: 5: 1-1: 4, the best curative effect is achieved by taking SH to CDP as 2: 1 (compound-2) in the experiment.

3) Therapeutic outcome to pulmonary interstitial fibrosis

The results of inhibition of TGF-beta secretion, a fibrosis-promoting factor, are shown in FIG. 7. The results show that: compared with a control group, the content of TGF-beta in lung tissues of mice in the BLM model group is obviously increased; compared with the BLM model group, the TGF-beta content of the lung tissues in each administration group is obviously reduced, the BLM + compound-2 group is most obvious, and the TGF-beta content of the lung tissues in the BLM + compound-1 group and the BLM + compound-3 group is also obviously lower than that of the BLM + SH group and the BLM + CDP group, so that the three compound groups are prompted to generate synergistic effect.

The Masson staining results are shown in FIG. 8A. The results show that: the control group has clear lung tissue structure, complete alveolar wall and a very small amount of fibrous tissue blue staining in the pulmonary stroma; the collagen fiber of the pulmonary interstitium in the BLM model group is obviously deposited and the pulmonary interstitium is thickened, so that a great amount of fibrous tissues are blue-stained and thickened, and the structure of the pulmonary alveoli is damaged; the fibrous tissue deposition of each administration group is improved, while the improvement effect of the BLM + compound-1 group, the BLM + compound-2 group and the BLM + compound-3 group is better than that of the two single formula groups, and the pulmonary interstitial fibrosis of the BLM + compound-2 group is almost completely inhibited. The calculation of the area of pulmonary interstitial fibrosis showed that (fig. 8B): the percentage of fibrous tissues between alveoli of the control group is very small, and the percentage of the fibrous tissues between alveoli of the BLM model group is greatly increased; compared with the BLM model group, the fibrosis area of each administration group is obviously reduced, the three compound groups are less than SH and CDP single group, and the BLM + compound-2 group is the best, which indicates that the three compound groups all generate synergistic effect.

The result shows that the SH and the CDP form a compound with a certain proportion, and the compound has obvious synergistic effect on the inhibition effect of increasing the secretion of TGF-beta of lung tissues and pulmonary interstitial fibrosis in an IP model, and the proportion range of the SH to the CDP is as follows according to the weight ratio: 5: 1-1: 4, the best curative effect is achieved by taking SH to CDP as 2: 1 (compound-2) in the experiment. The molecular weights of the sarpogrelate (Sar) and Sarpogrelate Hydrochloride (SH) are not greatly different, and are converted into Sar: the range of CDP ratios is also: 5: 1-1: 4, the best curative effect is achieved by using Sar to CDP as 2: 1 (compound-2).

Claims

1. An application of a compound pharmaceutical composition taking sarpogrelate and carbidopa as active ingredients in preparing a medicament for treating interstitial pneumonia.

2. The use according to claim 1, wherein the sarpogrelate ester is sarpogrelate ester or a pharmaceutically acceptable salt of sarpogrelate ester.

3. The use of claim 1, wherein said carbidopa is carbidopa or a pharmaceutically acceptable salt of carbidopa.

4. The use of claim 1, wherein the compound pharmaceutical composition is in the form of tablet, capsule, granule, powder, syrup, oral liquid or injection.

5. The use according to claim 1, wherein the weight ratio of sarpogrelate to carbidopa is from 10: 1 to 1: 8.

6. The use according to claim 5, wherein the weight ratio of sarpogrelate to carbidopa is from 5: 1 to 1: 4.