CN114557992B

CN114557992B - Application of compound pharmaceutical composition in preparation of medicine for treating interstitial pneumonia

Info

Publication number: CN114557992B
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: 2024-02-27
Anticipated expiration: 2042-03-11
Also published as: CN114557992A

Abstract

The invention discloses an application of a compound pharmaceutical composition taking Serratate 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) _2A R) Activity, in particular with 5-HT _2A The R antagonist sarpogrelate and 5-HT synthesis inhibitor carbidopa form a compound within a certain weight ratio range, and have obvious synergistic effect on treating interstitial pneumonia and 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 preparation of a medicament for treating interstitial pneumonia.

Background

Interstitial pneumonia (Interstitial Pneumonia, IP) is a lung 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 the IP can be very wide, and the causes of the IP are classified into rheumatic immune diseases, medicines or treatment-related diseases (antiarrhythmic medicines, chemotherapeutics, paraquat, sulfasalazine, radiotherapy and the like) with known causes, occupational and environment-related diseases (silicosis, asbestosis, pneumoconiosis and the like) and primary diseases (sarcoidosis, lymphangiomyoma, eosinophilic pneumonia and the like) or idiopathic diseases (idiopathic pulmonary fibrosis, nonspecific interstitial lung diseases and the like) with unknown causes in 1985 and in 1987. The pathological process of interstitial pneumonia is typically diffuse alveolitis and/or disturbance of alveolar structure, which progress slowly, eventually leading to destruction of alveolar structure, forming a completely fibrotic and vesicular alveolus lung in the alveolar space. The pulmonary fibrosis and the decline or even the loss of the pulmonary function caused by the IP and the further development thereof are still a great difficulty in the current medical and health industries in China and even the world, and the main reason is that the IP has not been treated effectively. The IP disease spectrum is up to more than 200, so far, the cause and pathogenesis of most interstitial pneumonia are still unknown, and rare diseases are not few.

Fibrosis is a chronic progressive disease characterized by tissue fibrosis, and the pathogenesis of pulmonary fibrosis includes the following: fibroblast activation; reduced autophagy; transforming growth factor-beta (Transforming Growth Factor-beta, TGF-beta) signaling pathway activation and immune disorders in the body. Macrophages are currently believed to play an important role in the pathogenesis of pulmonary fibrosis. Macrophages are mainly involved in the repair process of tissue injury in the body's immunomodulatory pathways, and are mainly derived from bone marrow mononuclear cells, which migrate into tissues to become tissue macrophages. Yang Lijie et al, yang Lijie, liu Gang. Role of macrophages in pulmonary fibrosis. J.respiratory and critical care, 11, 2020, volume 19, 6, P:626-629 states that: polarized M1 macrophages may promote pulmonary fibrosis by secreting pro-inflammatory factors; polarized M2 macrophages can promote pulmonary fibrosis by secreting TGF- β.

In summary, IP is typically characterized by: inflammatory factors such as tumor necrosis factor alpha (Tumor Necrosis Factor-alpha, TNF-a), interleukin 1 beta (Interleukin-1 beta, IL-1 beta) secretion increase, reactive oxygen species (Reactive Oxygen Species, ROS) increase, TGF-beta secretion increase, and pulmonary interstitial fibrosis.

5-hydroxytryptamine (5-HT), also known as serotonin, is a small molecular substance present in the periphery and in the center, and the physiological function of 5-HT is complex and has not been fully clarified so far.

The synthesis of 5-HT is divided into two steps, the first: tryptophan is converted to 5-hydroxytryptophan (Tph can be divided into two subtypes: tph1 and Tph2, present in the periphery and the center, respectively) under the catalysis of tryptophan hydroxylase (Tryptophan Hydroxylase, tph); and a second step of: 5-hydroxytryptophan is converted to 5-HT under the catalysis of aromatic amino acid decarboxylase (Aromatic Aminoacid Decarboxylase, AADC). Thus, inhibition of peripheral 5-HT synthesis can be achieved by inhibition of Tth 1 or AADC, respectively.

Compounds which directly inhibit Tth 1 activity have been reported, for example, p-phenylalanine (pCPA, alias: DL-4-phenylalanine, DL-p-phenylalanine, etc., molecular formula: C) ₉ H ₁₀ ClNO ₂ ) However, there is no report on clinical application because of the general disadvantage of low oral bioavailability.

Carbidopa (CDP), of formula: c (C) ₁₀ H ₁₄ N ₂ O ₄ The inhibitor is an AADC inhibitor, is clinically used for the adjuvant therapy of the 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, so that the content of the levodopa in the circulation is increased, and also can inhibit the conversion of 5-hydroxytryptophan into 5-HT, so that 5-HT produced by cells synthesizing the 5-HT is reduced. CDP has not been reported for direct use in the treatment of Interstitial Pneumonia (IP).

II.5-HT Receptor 5-HT Receptor (5-HT Receptor, 5-HTR) is present on the cell membrane, belonging to the membrane Receptor, the 5-HT Receptor subtype is complex, and it has been found that 7 receptors of the general class, 5-HT, are present _1-7 R，5-HT _1，4，5 R is mainly distributed in the center, 5-HT _{2，3，6，7} R is distributed mainly at the periphery. These 7 classes of receptors are further divided into several subtypes. 5-HT2 receptors (5-HT) ₂ R) can be divided into 5-HT _2A R、5-HT _2B R and 5-HT _2C R is defined as the formula. The distribution of both peripheral and central is 5-HT _2A R、5-HT _2B R，5-HT _2C R is mainly present in central nervous system, and 5-HT is distributed in kidney, liver, skeletal muscle, etc _2A R、5-HT _2B R,5-HT _2A R is believed to have major biological activity.

5-HT degradation 5-HT not only acts biologically on its receptor directly, but can also be degraded in the mitochondria of cells. The enzyme catalyzing 5-HT degradation is monoamine oxidase A (Monoamine Oxidase A, MAO-A), which can produce A large amount of ROS, mainly H, while catalyzing 5-HT degradation ₂ O ₂ . Excessive 5-HT will produce large amounts of ROS in the cell due to the catalytic degradation of MAO-A, thereby causing the cell to malfunction. ROS can reduce cellular mitochondrial ATP synthesis, mitochondrial injury, activation of inflammatory signaling pathway, increased secretion of inflammatory factors, activation of fibrotic pathway, and other cellular abnormalities.

Has the function of selectively blocking 5-HT _2A R has few drugs, sarpogrelate (Sar) is a specific 5-HT ₂ R antagonists, of the formula: c (C) ₂₄ H ₃₁ NO ₆ Mainly antagonizes 5-HT _2A R, pair 5-HT _2B R also has weak antagonistic action, next toIts hydrochloride salt is commonly used in bed applications. Sarpogrelate hydrochloride (Sarpogrelate Hydrochloride, SH) can inhibit platelet aggregation enhanced by 5-HT, inhibit vasoconstriction, etc., and is clinically used for improving ischemic symptoms such as ulcer, pain and cold feeling caused by chronic arterial occlusion.

At present, there is no 5-HT _2A The use of R antagonists and/or 5-HT synthesis inhibitors for the treatment of IP is reported.

Disclosure of Invention

The invention aims to: the invention aims to provide an application of a compound pharmaceutical composition in preparing medicines for treating interstitial pneumonia.

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

The sarpogrelate to the application, and the sarpogrelate to sarpogrelate or pharmaceutically acceptable salts of the sarpogrelate.

The use, the carbidopa is carbidopa or a pharmaceutically acceptable salt of carbidopa.

The compound pharmaceutical composition is applied in the 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 active component is the pharmaceutically acceptable salt of the sarpogrelate, converting the active component into sarpogrelate to weight ratio; and in the case of carbidopa pharmaceutically acceptable salt, converting into active component carbidopa for weight ratio.

We found in the study that:

I. in Bleomycin (BLM) -induced murine interstitial pneumonia (Interstitial Pneumonia, IP) model, 5-HT synthase-tryptophan hydroxylase 1 (Tph 1) and aromatic amino acid decarboxylase of lung tissueExpression of (AADC), 5-HT2A receptor (5-HT) _2A R) and monoamine oxidase A (MAO-A) were both significantly upregulated. Suggesting that BLM induces IP, 5-HT synthesis and 5-HT in kidney tissue _2A R is significantly activated and MAO-A catalyzed 5-HT degradation is also activated, possibly correlated with the occurrence of pneumoniA, pulmonary fibrosis at IP.

In BLM-induced mouse IP model, sarpogrelate Hydrochloride (SH) (5-HT inhibition) _2A R) and Carbidopa (CDP) (inhibiting 5-HT synthesis) form a compound, and SH+CDP forms different compounds according to different weight proportions of the two medicaments to treat the model. The results show that the pathological manifestations of IP are obviously reversed, and are expressed as follows: the alveolar collapse, atrophy and interstitial thickening of the model animal are obviously improved; the lung tissue inflammation of the model animal is obviously improved, the inflammatory cell infiltration of macrophages and the like is obviously reduced, the contents of lung tissue inflammatory factors TNF-alpha and IL-1 beta are obviously reduced, and the ROS content is obviously reduced; the pulmonary interstitial fibrosis of the model animal is obviously improved, and the TGF-beta content in the lung tissue is obviously reduced, so that the content of the pulmonary interstitial fibrosis tissue is obviously reduced. In addition, the SH+CDP compound medicine shows obvious synergistic effect on the treatment of IP according to a certain weight ratio of medicines, and the combined medicine can obviously improve the curative effect of the medicines.

The beneficial effects are that: compared with the prior art, the invention has the following advantages: the compound pharmaceutical composition of the invention can inhibit the synthesis of 5-HT and 5-HT of lung tissue cells (including macrophages) _2A R activity, in particular 5-HT _2A The R antagonist SH and the 5-HT synthesis inhibitor CDP form a compound within a certain weight ratio range, have obvious synergistic effect on treating interstitial pneumonia, and have obvious curative effect advantage when used for treating IP.

Drawings

FIG. 1 shows lung tissue Tpp 1, AADC, 5-HT after Bleomycin (BLM) induction of mouse IP _2A R, MAO-A expression assay (immunohistochemical staining to detect expression of lung tissue-associated protein) (. Times.400, typical macrophages indicated by the arrow in the BLM model group);

fig. 2 is the effect of sh+cdp combination drug treatment on BLM-induced IP mouse lung tissue 5-HT content: p < 0.05,: p < 0.01, n=8;

fig. 3 is the effect of sh+cdp combination drug treatment on BLM-induced ROS content in lung tissue of IP mice: p < 0.05,: p < 0.01, n=8;

fig. 4 is a graph of a BLM-induced IP mouse lung tissue section lignan-eosin (HE) staining results for sh+cdp compound drug treatment a. HE staining of typical lung tissue sites (x 400, photo topical, typical macrophages indicated by arrows in BLM model group) b. Whole photo macrophage count results: p < 0.05,: p < 0.01, n=8;

fig. 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,: p < 0.01, n=8;

fig. 6 is the effect of sh+cdp combination drug treatment on BLM-induced IL-1β content in lung tissue of IP mice: p < 0.05,: p < 0.01, n=8;

fig. 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,: p < 0.01, n=8;

fig. 8 is a graph of typical pulmonary interstitial fibrosis (x 400, model group visible pulmonary interstitial obvious fibrosis, pulmonary interstitial thickening, alveolar atrophy collapse) b. calculation of the percentage of pulmonary interstitial fibrosis area to total area in BLM induced IP mice lung tissue section Masson staining results a.masson staining photographs: p < 0.05,: p < 0.01, n=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

Male C57BL/6 mice (clean grade) for experiments, with weight of 20-24 g, are kept in common squirrel cages, keep room temperature of 24+ -2 ℃ and illumination control to ensure 12 hours of bright and dark conditions, and are fed with common water. After the animals were anesthetized, bleomycin (BLM, 3.5 mg/kg) was administered intratracheally by puncturing to establish an Interstitial Pneumonia (IP) animal model, and an equal volume of physiological saline was administered to the control group. Animals were sacrificed 21 days after molding, and lung tissue was formalin fixed, tissue sections, and immunohistochemical stainedDetection of lung tissue 5-HT2A receptor (5-HT _2A R), 5-HT synthase Tth 1 and AADC, 5-HT degrading enzyme monoamine oxidase A (MAO-A).

1.2 experimental results

The results are shown in FIG. 1. Immunohistochemical detection showed: 5-HT in control group lung tissue _2A R, MAO-A expression level is very low, while Tth 1 and AADC are obviously expressed and all cells are expressed; compared to control group, 5-HT in lung tissue of BLM model group _2A R, tph1, AADC and MAO-A expression levels are all obviously increased, and all cells Tth 1 and AADC expression levels are all increased, and the expression levels are most obvious by macrophages, but 5-HT _2A The most significant sites of increased R, MAO-A expression were macrophages and alveolar spaces.

Example 2

Therapeutic effect of drugs on BLM-induced mouse IP

The synergistic effect of SH in combination with CDP on IP treatment was verified by comparing the therapeutic effect of the administration of Sarpogrelate Hydrochloride (SH) in combination with Carbidopa (CDP) and each administration alone on BLM-induced Interstitial Pneumonia (IP) in mice.

2.1 Experimental methods

(1) Animal treatment

The experimental C57BL/6 mice (clean level) are kept in normal cages with weight of 20-24 g, the room temperature of 24+/-2 ℃ is kept, the illumination is controlled to ensure the bright and dark conditions for 12 hours, and the animals are fed with normal water.

Animals were randomly divided into 7 groups (8 per group): control group, BLM induced IP model group (BLM model group), IP model SH treatment group (BLM+SH group), IP model SH+CDP compound treatment group (SH: CDP) according to 3 compounds (BLM+compound-1 group, BLM+compound-2 group, BLM+compound-3 group respectively) of different weight ratios, IP model CDP treatment group (BLM+CDP group). In the experiment, three compounds of SH+CDP compounds according to different weight ratios are: compound-1-SH: cdp=5:1; compound-2-SH: cdp=2:1; compound-3-SH: cdp=1:4.

(2) Dosage for administration

Control group-oral administration (p.o.) 0.5% sodium carboxymethyl cellulose (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 group-p.o. each group of drugs, all administration groups were given the same dose: 60.0 mg/kg/time.

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

(3) BLM induced mouse IP method

Animals were kept acclimatized for 5 days. After the animals were anesthetized, bleomycin (BLM, 3.5 mg/kg) was administered intratracheally to the remaining animals in addition to normal saline administered intratracheally to the control group puncture method, and an Interstitial Pneumonia (IP) animal model was established. After 24h of molding, each drug treatment group was started to be administered with the drug, and the drug was administered by gavage 2 times a day for 21 days continuously, and the control group and the BLM group were given equal volumes of 0.5% cmc-Na solution (drug solvent). Animals were sacrificed 3 hours after the last dose had been anesthetized and lung tissue was harvested and formalin-fixed or cryopreserved at-80 ℃ for future use.

(4) Experimental index detection method

Taking lung tissue, and freezing and preserving for standby. ELISA method of the kit detects the contents of 5-HT, ROS, TNF-alpha, IL-1 beta and TGF-beta in lung tissues;

taking formalin-fixed lung tissue, performing conventional tissue section and HE staining, observing and photographing under a microscope, and counting the number of macrophages in the lung tissue photograph in 400 times of the photograph;

formalin-fixed lung tissue was taken, normal tissue sections, masson stained, observed under a microscope and photographed, and pulmonary interstitial fibrosis was observed in 400-fold photographs. A typical piece of lung tissue in the photograph was taken and Image J software was used to calculate the percentage of total area of fibrous tissue area blue stained with lung tissue: pulmonary interstitial fibrosis area (%) = pulmonary interstitial blue staining area +.total area x 100%.

2.2 experimental results

Statistical tests among multiple groups of data were performed using one-way anova and comparisons among groups were performed using LSD. P < 0.05 indicates a significant statistical difference and P < 0.01 indicates a very significant statistical difference. Data in the figures are shown as mean ± scaleDifference of accuracy And (3) representing.

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

1) Results of inhibition of increased 5-HT and ROS content in pulmonary tissue

The results are shown in FIG. 2 and FIG. 3. Compared with a control group, the lung tissue 5-HT and ROS content of the BLM model group are obviously increased, which indicates that the model animals have obvious increase of 5-HT synthesis and ROS production; compared with the model group, the three compounds SH, CDP and SH+CDP with the same administration dosage (120 mg/kg/day) can obviously reduce the contents of 5-HT and ROS in lung tissues. In addition, the effect of the BLM+compound-2 group is best, and the curative effects of the BLM+compound-1 group and the BLM+compound-2 group are better than those of the BLM+SH group and the BLM+CDP group. The obvious synergistic effect is suggested to be generated, and the compound-2 synergistic effect is the strongest. The result shows that SH and CDP form a compound with a certain proportion, and the compound has obvious synergistic effect on the inhibition of the increase of the synthesis of 5-HT and the increase of the generation of ROS of lung tissues in an IP model, and the SH is prepared according to the weight ratio: the CDP ratio ranges from: 5:1 to 1:4, SH is used in the experiment: cdp=2:1 (compound-2) was the best.

2) Treatment of lung injury and lung interstitial inflammation

The results are shown in FIG. 4. The results of HE staining of lung tissue sections indicated (fig. 4A): compared with the control group, the BLM model group mice have thickened lung interstitium, atrophic collapse of alveoli, obvious inflammatory cell infiltration, and massive macrophage infiltration can be observed in lung tissues. The pathological damage of lung tissues in the BLM+SH group and the BLM+CDP group is obviously improved; the improvement of the BLM+compound-1 group, the BLM+compound-2 group and the BLM+compound-3 group lung tissue pathological damage is more obvious; and the BLM+compound-2 group lung tissue pathological injury is almost completely inhibited, the lung interstitium is not obviously thickened, the alveoli are normal, and the occasional inflammatory cells infiltrate. The whole-picture macrophage count results indicated (fig. 4B): the control group has few and occasional macrophages in lung tissues, a large number of macrophages exist in the BLM model group, the macrophages in each administration group are obviously reduced, the number of the three compound groups is less than that of SH and CDP single groups, and the minimum number of BLM+compound-2 is used for prompting that the three compound groups all produce a synergistic effect.

The results of the inhibition of 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 lung tissue TNF-alpha and IL-1 beta content of the mice in the BLM model group are obviously increased; compared with the BLM model group, the contents of TNF-alpha and IL-1 beta in lung tissues of each administration group are obviously reduced, the BLM+compound-2 group is most obvious, and the contents of TNF-alpha and IL-1 beta in lung tissues of the BLM+compound-1 group and the BLM+compound-3 group are obviously lower than those of the BLM+SH group and the BLM+CDP group, so that the three compound groups are all indicated to generate a synergistic effect.

The result shows that SH and CDP form a compound with a certain proportion, and the compound has obvious synergistic effect on inhibiting lung tissue injury, inflammation and macrophage infiltration in an IP model, and SH is prepared according to the weight ratio: the CDP ratio ranges from: 5:1 to 1:4, the therapeutic effect in this experiment was best with SH:CDP=2:1 (Compound-2).

3) Therapeutic outcome for pulmonary interstitial fibrosis

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

The Masson staining results are shown in fig. 8A. The results show that: the control group has clear lung tissue structure, complete alveolus wall and little blue staining of fibrous tissue in lung interstitium; the BLM model group has obvious deposition of lung interstitial collagen fibers and thickening of the lung interstitial, and a large amount of blue dyeing and thickening of fibrous tissues are seen, and the alveolar structure is damaged; fibrous tissue deposition is improved in each administration group, and 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 two single groups, and the pulmonary interstitial fibrosis of the BLM+compound-2 group is almost completely inhibited. The calculation result of the pulmonary interstitial fibrosis area shows (fig. 8B): the percentage of the fibrous tissue among the alveoli of the control group is small, and the area percentage of the fibrous tissue among the 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, and the three compound groups are less than SH and CDP single groups, and the BLM+compound-2 group is the best, so that the three compound groups are all indicated to generate a synergistic effect.

The result shows that SH and CDP form a compound with a certain proportion, and the compound has obvious synergistic effect on the increase of TGF-beta secretion of lung tissues and the inhibition of pulmonary interstitial fibrosis in an IP model, and the proportion range of SH to CDP is as follows: 5:1 to 1:4, the therapeutic effect in this experiment was best with SH:CDP=2:1 (Compound-2). The molecular weight of the sarpogrelate (Sar) and the molecular weight of the Sarpogrelate (SH) are not greatly different, and the sarpogrelate (Sar) and the Sarpogrelate (SH) are converted into Sar: the CDP ratio ranges are also: 5:1 to 1:4, with Sar: CDP=2:1 (compound-2) the efficacy was best in this experiment.

Claims

1. An application of a compound pharmaceutical composition taking Serratia esters or pharmaceutically acceptable salts of Serratia esters and carbidopa or pharmaceutically acceptable salts of carbidopa as active ingredients in preparing medicines for treating interstitial pneumonia; the weight ratio of the sarpogrelate to the active ingredient carbidopa according to the pharmaceutically acceptable salt of the sarpogrelate to 5:1-1:4.

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