WO2016095778A1

WO2016095778A1 - Method for treatment and prevention of air pollution-related diseases

Info

Publication number: WO2016095778A1
Application number: PCT/CN2015/097277
Authority: WO
Inventors: 张卫国; 贺庆; 陈迥; 埃格斯多费尔·曼弗雷德·L
Original assignee: 帝斯曼知识产权资产管理有限公司
Priority date: 2014-12-15
Filing date: 2015-12-14
Publication date: 2016-06-23
Also published as: BR112017012810A2; CN107106535A

Abstract

A method for treatment and prevention of air pollution-related diseases, especially diseases related to atmospheric PM2.5 in subjects. The method comprises administering an effective amount of a polyunsaturated fatty acid and vitamin E to subjects in need thereof.

Description

Method of treating or preventing diseases associated with air pollution

Technical field

The present invention relates to a method of treating or preventing a disease associated with air pollution in an individual. In particular, the invention relates to a method of treating or preventing a disease associated with PM2.5 in the air.

Background technique

Air pollution, especially PM2.5, seriously affects human health. PM2.5 is a generic term for solid particles or droplets having an aerodynamic diameter of less than or equal to 2.5 microns, also known as fine particles or into particulate matter. Due to its small size and light weight, PM2.5 can stay in the air for a long time. Moreover, PM2.5 has a large specific surface area and can absorb various bacteria, viruses and various pollutants harmful to human health. PM2.5 in the air can enter the alveoli through the respiratory tract, accumulate in the alveoli, interfere with the gas exchange in the lungs, and cause various diseases. Therefore, PM2.5 is particularly harmful to health.

Studies have shown that atmospheric particulate matter such as PM2.5 that enters the respiratory tract can cause damage to the respiratory mucosa, irritating and corroding the alveolar wall, leading to impaired lung function and inflammation of the lungs, and respiratory symptoms such as coughing, coughing, and wheezing. The incidence of chronic bronchitis, bronchial asthma and emphysema increased. (Calderon-Garciduenas L, Am. J. Respir. Cell Mol. Biol., 2001, Vol. 24, pp. 132–138; Grig J, Proc Am Thorac Soc, 2009, Vol 6, pp. 564–569) Evidence It is shown that PM2.5 damage to the respiratory system is related to oxidative stress and inflammatory response. (Romieu I et al., Eur Respir J, 2008, Vol. 31, pp. 179-196)

The inflammatory mediators and oxygen free radicals produced in the lungs due to PM2.5 are not confined to the lungs, and these factors can enter the blood circulation, thereby damaging the cardiovascular system. Experiments have shown that inflammatory factors such as IL-6, IL-1, TNF, interferon and IL-8 in the blood will increase after exposure to particulate air pollution, while IL-6 and TNF play a key role in the inflammatory response. It plays a central role in the process of damaging the cardiovascular system. Studies have shown that PM2.5 is associated with an increased incidence of acute myocardial infarction, stroke, arrhythmia, heart failure, thrombosis, hypertension, and atherosclerosis, with cerebrovascular disease, peripheral vascular disease, ischemic heart disease, Arrhythmias are associated with increased hospitalization rates for heart failure. (Wan Zheng et al., Chinese Journal of Evidence-based Cardiovascular Medicine, December 2011, Vol. 3, No. 5, pp. 332-335)

PM2.5 can also enter the central nervous system through the blood-brain barrier, olfactory nerves, etc., causing an inflammatory response in the central nervous system. In addition, various inflammatory factors that enter the circulatory system generated by PM2.5 enter the brain through diffusion and active transport, activate immune cells in the brain, produce neuroinflammation, and cause damage to cells in the brain to produce neurodegenerative diseases. (Wu Yuanshou et al., Life Sciences, August 2011, Vol. 23, No. 8, pp. 784-789) have reported that PM2.5 and ischemic cerebrovascular disease, cognitive impairment, It is associated with central nervous system diseases and/or damage such as Alzheimer's disease. (Calderon-Garciduenas L et al, Toxicol Pathol, 2008, Vol. 36, pp. 289-310)

Exposure to PM2.5 is also associated with an increased risk of disease characterized by inflammation, such as diabetes and insulin resistance. (Carol Petera, Environment and Health Outlook, August 2014, Vol 122 (4C), p. 30) In addition, PM2.5 can cause toxic effects on different levels of genetic material such as chromosomes and DNA, including changes in chromosome structure, DNA. Injury and genetic mutations, etc., thereby inducing cancer. Recent studies have also found that PM2.5 can cause genetic DNA damage, leading to birth defects in newborns. (Guo Xinyu et al., Science Bulletin, 2013, Vol. 58, pp. 1171–1177)

Due to the above-mentioned hazards of air pollution, especially PM2.5, there is an urgent need for a method of preventing or treating these hazards. The present inventors have surprisingly found that the combination of polyunsaturated fatty acids and vitamin E has a synergistic effect on the treatment or prevention of diseases associated with air pollution, particularly PM2.5 in the air, with unexpected effects.

Summary of the invention

Accordingly, the present invention provides a method of treating or preventing a disease associated with air pollution, particularly PM2.5 in the air, comprising administering to an individual in need thereof an effective amount of a polyunsaturated fatty acid and a vitamin E. .

In the present invention, the term "treating" means alleviating a disease or condition, ie preventing or reducing the progression of the disease or at least one of its clinical symptoms, such as ameliorating at least one physical parameter, or inhibiting a disease or condition, or delaying the disease. Or the onset of a condition. The term "prevention" refers to reducing the risk of acquiring a disease or condition.

In the present invention, the term "effective amount" is an amount that prevents an individual's deficiency, treats its disease or medical condition, or more generally, reduces the symptoms of the disease, controls the progression of the disease, or provides a nutritional, physiological or medical benefit to the individual.

In the present invention, the term "individual" generally refers to a person, but is not limited thereto, and includes any animal, such as a mammal, which can benefit from the treatment or prevention of the present invention.

In the present invention, the polyunsaturated fatty acid is preferably a linear fatty acid having two or more double bonds and having a carbon chain length of 18 to 22 carbon atoms, including but not limited to an omega-3 unsaturated fatty acid such as ten. Hexatrienoic acid (HTA), alpha-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), twenty carbon Pentaenoic acid (EPA), docosapentaenoic acid (HPA), docosahexaenoic acid, docosapentaenoic acid (DPA), docosahexaenoic acid (DHA) and twentieth Tetrapentaenoic acid, etc., omega-6 unsaturated fatty acids such as linoleic acid (LA), conjugated linoleic acid (CLA), γ-linolenic acid (GLA), eicosatrienoic acid (DGLA), peanut four Acetate, adrenal acid, and eicosatetraenoic acid (ETA), etc., are mixed therewith. The polyunsaturated fatty acids may be of various origins such as, but not limited to, linseed, walnut, fish oil, scale shrimp, algae oil and various vegetable oils such as safflower oil, corn oil, soybean Oil and sunflower oil, etc. Preferably, the polyunsaturated fatty acid is DHA, EPA or a mixture thereof. More preferably, the unsaturated fatty acid is fish oil, algal oil or a mixture thereof, such as commercially available fish oil and algal oil.

In the present invention, the vitamin E may be d-α-tocopherol, dl-α-tocopherol, d-α-tocopherol acetate, dl-α-tocopherol acetate, mixed tocopherol concentrate, d-α - Tocopheryl succinate and / or dl-α-succinic acid tocopherol. It can be synthesized or obtained commercially.

In the present invention, the disease refers to a disease associated with air pollution, particularly oxidative stress or inflammatory reaction caused by PM2.5 in the air, and specific examples include, but are not limited to, respiratory diseases such as respiratory infections. , cough, cough, wheezing, asthma, allergic rhinitis, bronchitis, bronchitis, pneumonia, chronic obstructive pneumonia, respiratory failure, emphysema and lung cancer; cardiovascular diseases such as vascular inflammation, arrhythmia, and arteries Atherosclerosis, ischemic heart disease, myocardial infarction, heart failure, thrombosis, hypertension, stroke, pulmonary heart disease and myocarditis; neurological diseases such as neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Cognitive dysfunction, etc.; and depression, impotence, liver disease, diabetes, insulin resistance, cancer, and birth defects in newborns.

According to the method of the present invention, administration of an effective amount of polyunsaturated fatty acids and vitamin E to a person in need thereof has a synergistic effect in the treatment and prevention of diseases associated with air pollution, particularly PM2.5 in the air. The “synergistic effect” refers to the fact that two or more substances are related to each other, cooperate with each other, and coordinate with each other, and the effect is greater than the sum of the effects of each substance acting alone, that is, the phenomenon of 1+1>2, or refers to A phenomenon in which a substance is enhanced by the presence of another substance, and the latter phenomenon may also be referred to as a "mutual promotion effect." The synergy of the present invention can be verified by cytological or animal tests well known in the art.

The amount of polyunsaturated fatty acid and vitamin E administered depends on the specific condition of the individual being administered. Specifically, the weight ratio of the polyunsaturated fatty acid to be administered and vitamin E (calculated as d-α-tocopherol) is from 0.1 to 30:1, preferably from 0.3 to 20:1, more preferably from 0.8 to 10:1, Most preferably it is 5-8:1.

In a specific embodiment, the weight ratio of polyunsaturated fatty acid and vitamin E (calculated as d-alpha-tocopherol) applied is 0.2:1, 0.4:1, 0.6:1, 1:1, 2:1 , 3:1, 4:1, 5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 25:1 or 30 :1.

In another specific embodiment, for an individual having a body weight of about 60 kg, the polyunsaturated fatty acid is 50 mg to 2 g per day, such as 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/day, 100 mg. /day, 110 mg / day, 120 mg / day, 130 mg / day, 140 mg / day, 150 mg / day, 160 mg / day, 170 mg / day, 180 mg / day, 190 mg / day, 200 mg / day, 250 mg / day, 300 mg / day 350mg/day, 400mg/day, 450mg/day, 500mg/day, 550mg/day, 600mg/day, 650mg/day, 700mg/day, 750mg/day, 800mg/day, 850mg/day, 900mg/day, 950mg / day, 1 g / day, 1.2 g / day, 1.5 g / day, 1.8 g / day or 2 g / day, and the vitamin E is 10 mg - 2 g / day, such as 10 mg / day, 20 mg / day, 25mg/day, 30mg/day, 35mg/day, 40mg/day, 45mg/day, 50mg/day, 55mg/day, 60mg/day, 65mg/day, 70mg/day, 75mg/day, 80mg/day, 85mg/ Day, 90 mg/day, 95 mg/day, 100 mg/day, 110 mg/day, 120 mg/day, 130 mg/day, 140 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 300 mg/day, 350 mg/day, 400mg/ Day, 450 mg/day, 500 mg/day, 550 mg/day, 600 mg/day, 700 mg/day, 800 mg/day, 900 mg/day, 1 g/day, 1.2 g/day, 1.5 g/day or 2 g/day ( Administration is based on d-alpha-tocopherol). Based on the above application rates, one of ordinary skill in the art can readily convert the amount of administration to individuals of other body weights. In a preferred embodiment, the polyunsaturated fatty acids and vitamin E are used in the above ratios in accordance with the recommended amount of the Chinese Nutrition Association.

The administration may be in the form of one or more dosage units, such as capsules, tablets or lozenges, for example, it may be administered once a day in a daily dosage unit, or in two or more doses in a multi-dose unit. Times. In addition, the polyunsaturated fatty acid and vitamin E may be administered simultaneously, or may be administered separately, for example, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours or 4 hours apart from each other. Apply.

Additionally, the polyunsaturated fatty acids and vitamin E can be administered to the individual in the form of a composition. Accordingly, in one embodiment of the present invention, the present invention provides a composition comprising a polyunsaturated fatty acid and vitamin E for the preparation of a disease for treating or preventing an individual associated with air pollution, particularly PM2.5 in the air. Use in medicines and / or nutrients.

Preferably, in the composition, the weight ratio of the polyunsaturated fatty acid and vitamin E (calculated as d-α-tocopherol) is from 0.1 to 30:1, preferably from 0.3 to 20:1, more preferably 0.8-10:1, most preferably 5-8:1.

In a specific embodiment, the weight ratio of polyunsaturated fatty acids and vitamin E (calculated as d-alpha-tocopherol) in the composition is 0.2:1, 0.4:1, 0.6:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 25: 1 or 30:1.

In another specific embodiment, the composition comprises 50 mg to 2 g, such as 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1g, 1.2g, 1.5g, 1.8g and 2g of polyunsaturated fatty acids; and 10mg -2g, for example, 10mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 110mg, 120mg, 130mg, 140mg, 150mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 1.2 g, 1.5 g and 2 g of vitamin E (calculated as d-α-tocopherol).

The following non-limiting examples are intended to further illustrate the invention.

Example

Example 1: Effect of fish oil and vitamin E combination on cell viability

I. Materials and pretreatment

1. Reagents and instruments

RPMI1640 cell culture medium (GIBICO, American Thermo Electron Corporation), trypsin (GIBICO, American Thermo Electron Corporation), cell cryopreservation with dimethyl sulfoxide (DMSO), and other reagents are analytically pure reagents.

Thermo Anderson G-2.5 (American Thermo Electronics), CO ₂ cell incubator (NAPCO, France), inverted microscope (Leica, Germany), Infinite-M200 microplate reader (TECAN), 6-well, 24-well and 96-well culture Board (Nunc, Denmark).

2. Sampling and processing of fine particles in the atmosphere

In the roof of a building in downtown Shanghai (about 10 meters high, there is no obvious pollution source around), the Thermo Anderson G-2.5 large flow sampler uses a glass fiber filter to collect fine particles of the atmosphere. The filter membrane with fine particles was cut into a size of 1 cm×3 cm, immersed in deionized water, ultrasonically shaken for 30 min×3 times, and fine particles were eluted. The oscillating liquid was filtered with six layers of gauze, and the filtrate was vacuum-dried and stored in a low-temperature refrigerator.

3. Preparation of fine particle exposure solution

The collected fine particulate matter was weighed and prepared into a fine particle suspension stock solution having a concentration of 1000 μg/ml in phosphate buffered saline (PBS), thoroughly mixed, and sealed in a refrigerator at 4 ° C for use. When the cells were infected, the stock solution was ultrasonically shaken and added to the cell culture solution at an exposure concentration of 50 μg/ml.

4. Preparation of nutrients

Using RPMI1640 cell culture medium as a solvent, the nutrient vitamin E (obtained from DSM, the Netherlands) was configured to be 1000 μmol/L, and the fish oil (obtained from DSM, the Netherlands) was configured to be 600 mg/L, and thoroughly mixed, respectively. Store at 4 ° C for later use. The mixture was sterilized by ultrasonic shaking before use, and then configured with the RPMI1640 cell culture medium to the desired concentration for the test.

The fish oil contained 180 mg/g EPA and 110 mg/g DHA, and the total omega-3 unsaturated fatty acid content was 360 mg/g, both in the form of triglyceride.

5. Test cell preparation

Human umbilical vein endothelial cells HUVEC (Nanjing Kaiji, China) were purchased and cultured in RPMI1640 cell culture medium. The newly purchased cells were cultured by changing the solution and placed in a 37 ° C incubator. After 24 hours, the cells were changed again and the culture was continued. The cell density was adjusted to 1 × 10 ⁵ /ml, inoculated in a culture flask, and placed in a 5% CO ₂ and cultured in a 37 ° C incubator. When the cells grew to form a monolayer with 80% to 90% confluence, the culture solution was aspirated and washed 3 times with PBS. Digestion was carried out by adding 0.25% trypsin, cell counting, and then subcultured at a ratio of 1:3.

2. Experimental methods

The intervention effect of nutrients on the survival rate of HUVEC exposed to fine particles was investigated by MTT assay.

Adjust the cell concentration to 1×10 ⁵ /ml, inoculate 100 μl of cell suspension per well on a 96-well flat-bottomed plate, incubate at 37 ° C, 5% CO ₂ for 24 h, change the culture solution, and then add the fines prepared as above. The particulate matter exposure solution was applied to the concentration of the drug and the nutrient suspension prepared as above to the test concentration, and three parallel holes were set in each group, and three control wells were set. The control wells were only added with the same concentration of fine particle suspension without inoculation of cells, mainly to eliminate the influence of the toxic solution on the absorbance value. After continuing to culture for 24 hours, 50 μl (concentration 5 mg/ml) of MTT solution was added to each well, and incubated at 37 ° C for 4 h. The medium was removed and 150 μl of DMSO was added. After shaking for 10 min, the absorbance value (OD value) was measured by zeroing the control well on a microplate reader, and the measurement wavelength was 550 nm. Cell viability is calculated according to the following formula:

Cell viability = (experimental group OD value - control well OD value) / control well OD value x 100%.

III. Test results

The test results are averaged as shown in Table 1:

Table 1

As can be seen from the results in Table 1, the combination of vitamin E and fish oil has a synergistic effect on increasing the survival rate of human umbilical vein endothelial cells in PM2.5.

Example 2: Effect of combination of fish oil and vitamin E on cellular antioxidant activity

I. Materials and pretreatment

Same as Example 1.

2. Experimental methods

Superoxide dismutase (SOD) is an antioxidant enzyme present in cells. Determination of SOD in cells can reflect the antioxidant capacity of cells. l×10 ⁵ /ml test cells were seeded in 24-well culture plates, cultured to 80%-90% confluence, the cell culture medium was discarded, the cells were washed 3 times with PBS, and the concentration of fine particles and the test were added. Concentration of nutrients, 3 parallel holes per group, and 3 control wells. After 24 h, it was washed 3 times with PBS solution, then treated with 0.1% Triton X-100 (Nanjing Kaiji, China) for 30 min at room temperature, and the cell lysate was assayed for SOD activity by xanthine oxidase method.

III. Test results

The test results are averaged as shown in Table 2:

Table 2

As can be seen from the results in Table 2, the combination of vitamin E and fish oil has a synergistic effect on increasing the antioxidant activity of human umbilical vein endothelial cells in PM2.5.

Example 3: Intervention of cell oil and vitamin E combination on anti-inflammatory injury of cells

I. Materials and pretreatment

Same as Example 1.

2. Experimental methods

Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) play an important role in regulating inflammation, measuring IL-6 and The expression of TNF-[alpha] can reflect the extent of the inflammatory response of the cells. l×10 ⁵ /ml test cells were seeded in 24-well culture plates, grown to 80%-90% confluence, the cell culture medium was discarded, the cells were washed 3 times with PBS, and the concentration of fine particles and test concentration were added. For the nutrients, set up 3 parallel holes in each group and set 3 control holes. After 24 h, the cell supernatant was collected. The expression levels of inflammatory factors IL-6 and TNF-α in HUVEC cells of each treatment group were determined by ELISA.

III. Test results

The test results are averaged as shown in Table 3:

table 3

As can be seen from the results in Table 3, the combination of vitamin E and fish oil has a synergistic effect on increasing the anti-inflammatory damage of human umbilical vein endothelial cells in PM2.5.

Claims

A method of treating or preventing a disease associated with air pollution, particularly PM2.5 in the air, comprising administering to an individual in need thereof an effective amount of a polyunsaturated fatty acid and vitamin E.
The method of claim 1 wherein said polyunsaturated fatty acid is selected from the group consisting of omega-3 unsaturated fatty acids such as hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA), stearidonic acid ( SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), docosapentaenoic acid (HPA), docosahexaenoene Acid, docosapentaenoic acid (DPA), docosahexaenoic acid (DHA) and docosapentaenoic acid, omega-6 unsaturated fatty acids such as linoleic acid (LA), conjugated linoleum Acid (CLA), γ-linolenic acid (GLA), eicosatrienoic acid (DGLA), arachidonic acid, adrenal acid, and arachidonic acid (ETA), and mixtures thereof.
The method according to claim 1 or 2, wherein the polyunsaturated fatty acid has various sources such as, but not limited to, linseed, walnut, fish oil, scale shrimp, algae oil and various vegetable oils such as safflower oil, Corn oil, soybean oil and sunflower oil.
The method according to any one of claims 1 to 3, wherein the disease is a disease associated with oxidative stress or an inflammatory reaction caused by air pollution, particularly PM2.5 in the air.
The method according to claim 4, wherein the disease is selected from the group consisting of respiratory diseases such as respiratory infections, cough, cough, wheezing, asthma, allergic rhinitis, bronchitis, bronchitis, pneumonia, chronic obstructive pneumonia, and breathing. Failure, emphysema and lung cancer; cardiovascular diseases such as vascular inflammation, arrhythmia, and atherosclerosis, ischemic heart disease, myocardial infarction, heart failure, thrombosis, hypertension, stroke, pulmonary heart disease and myocarditis; Nervous system diseases such as neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, cognitive dysfunction; or depression, impotence, liver disease, diabetes, insulin resistance, cancer or neonatal birth defects.
The method according to any one of claims 1 to 5, wherein the weight ratio of the polyunsaturated fatty acid to be administered and vitamin E (calculated as d-α-tocopherol) is from 0.1 to 30:1, preferably from 0.3 to 20 :1, more preferably from 0.8 to 10:1, most preferably from 5 to 8:1.
The method according to any one of claims 1 to 6, wherein the polyunsaturated fatty acid is administered in an amount of from 50 mg to 2 g per day, and the vitamin E is in an amount of from 10 mg to 2 g per day (d-α- Tocopherol calculations) administration.
The method of any of claims 1-7, wherein the administering is administered one or more times a day in one or more dosage units.
The method according to any one of claims 1 to 8, wherein the polyunsaturated fatty acid and vitamin E are administered simultaneously or separately.
Use of a composition comprising a polyunsaturated fatty acid and vitamin E in the manufacture of a medicament and/or nutrient for the treatment and prevention of a disease associated with air pollution, in particular PM2.5 in the air.
The use according to claim 10, wherein the weight ratio of said polyunsaturated fatty acid to vitamin E is from 0.1 to 30:1, preferably from 0.3 to 20:1, more preferably from 0.8 to 10:1, most preferably 5 -8:1.
The use according to claim 11 or 12, wherein the composition comprises 50 mg to 2 g of fish oil; and 10 mg to 2 g of vitamin E (calculated as d-α-tocopherol).