CN115068453A - Application of salmeterol medicament for preventing and treating influenza - Google Patents

Abstract

The invention belongs to the technical field of medicines, and relates to a medicinal application of an antiasthmatic drug Salmeterol (Salmeterol) in preventing and treating influenza. The innovation of the invention is that the medicine is relocated: the research finds that the salmeterol has the function of resisting the influenza virus in vivo and in vitro; has the function of resisting amantadine and oseltamivir drug-resistant strains. Is expected to become a clinical medicine for preventing and treating influenza viruses and drug-resistant influenza virus infectious diseases. The invention provides a scientific experimental basis for expanding the clinical indications of salmeterol and provides a feasible and effective treatment scheme for clinically preventing and treating influenza.

Description

Application of salmeterol medicament for preventing and treating influenza

Technical Field

The invention belongs to the technical field of medicines, and relates to a medicinal application of salmeterol in preventing and treating influenza virus infectious diseases.

Background

Influenza virus is a common respiratory infectious agent and is particularly harmful to the elderly, infants and people with chronic diseases. Seasonal influenza and sudden new influenza outbreaks occurring in winter and spring every year threaten human life, health and safety. Because the influenza a virus is an RNA virus and consists of 8 gene segments, the virus is easy to mutate during replication, is the root cause of drug resistance of the influenza virus, and is also the root cause of seasonal influenza every year.

The current effective anti-influenza means mainly comprise vaccination and timely administration of chemotherapy drugs after infection. The novel H1N1 influenza comes, and the timely inoculation of the influenza vaccine plays an important role in protecting people. However, the effectiveness of influenza vaccines is based on the similarity between the virus strain of the vaccine and the influenza virus strain to be epidemic or present in the environment, and the high changeability of the influenza virus antigen greatly reduces the accuracy of people in predicting influenza outbreaks, so that the effectiveness of the conventional influenza vaccine in preventing influenza outbreaks and preventing influenza transmission is seriously affected, and the conventional influenza vaccine is not designed for unpredictable new influenza virus bundles. While the chemotherapy drugs clinically used against influenza virus are mainly of two types: one is an inhibitor of the viral M2 protein, such as amantadine (amantadine) and rimantadine (rimantadine). Such drugs are only effective against influenza a viruses, have developed relatively broad drug resistance, and are often associated with central nervous system side effects, which are not generally the first choice. Another class of drugs are Neuraminidase (NA) inhibitors, such as zanamivir (zanamivir), oseltamivir (oseltamivir). Although the drugs are effective for influenza A virus and influenza B virus, the raw material supply is short, the synthesis process is complex, and the large-scale production and application are greatly limited.

The existing anti-influenza chemotherapy drugs have complex preparation process, high production cost and high price, and the generation of drug-resistant strains becomes a difficult problem of clinical treatment along with increasingly wide application. According to statistics, drug-resistant strains account for 15-92% of strains which are interpersonal spread every year. In recent years, the drug resistance rate of the H3N 2A virus to amantadine is increasing and is over 90%, and the drug resistance is caused by mutation and replacement of a single amino acid on the M2 protein of the virus. The resistance to neuraminidase also tends to increase. Neuraminidase resistance has also recently emerged from the H5N1 strain. In order to inhibit the generation of drug resistance and the spread of drug-resistant strains, anti-influenza drugs are required to have a broad-spectrum anti-influenza effect, so that the search for finding anti-influenza drugs capable of avoiding the generation of drug resistance is particularly urgent.

Disclosure of Invention

The present invention relates to methods and results for the prevention and treatment of infection by influenza viruses and resistant strains thereof.

It was found that the antiasthmatic drug Salmeterol (Salmeterol), herein designated Compound I (LG0014), exhibited antiviral activity against influenza virus (A/PR/8/34(H1N1), A/Minfang/151/2000(H3N2) and the duffy and adamantane simultaneous resistant strain (A/Hebei Xinhua/SWL1106/2017H1N1 pdm).

The invention solves the technical problem of providing the application of salmeterol or pharmaceutically acceptable salt thereof in preparing medicaments for preventing and treating influenza virus infection.

Specifically, in order to solve the technical problem of the present invention, the following technical scheme is adopted:

the first aspect of the technical proposal of the invention provides the application of salmeterol shown as a structural formula I or pharmaceutically acceptable salt thereof in preparing the medicament for preventing or treating influenza virus,

the salmeterol pharmaceutically acceptable salt comprises pharmaceutically acceptable organic salt or inorganic salt, wherein the organic salt comprises sulfonate, carboxylate, amino acid salt and fatty acid salt, and the inorganic salt comprises hydrochloride, bromate, iodate, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate and nitrate.

The sulfonate comprises alkyl sulfonate containing 1-15 carbon atoms, benzene sulfonate, p-toluene sulfonate, o-toluene sulfonate and m-toluene sulfonate; carboxylates include tartrate, maleate, fumarate, citrate, malate, cinnamate, benzoate, malonate, succinate, glutarate, adipate, pamoate, and lactate; amino acid salts include glutamate, aspartate; the fatty acid salt comprises a long chain fatty acid salt having 2 to 18 carbon atoms.

Wherein the influenza virus infection comprises influenza induced by H1N1 and H3N 2.

The influenza virus infection comprises influenza induced by tamiflu and amantadine drug-resistant strains.

In a second aspect of the technical scheme of the invention, the invention provides an application of a pharmaceutical composition in preparing a medicament for preventing or treating influenza virus infection, wherein the pharmaceutical composition comprises salmeterol shown in structural formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

General description

The present invention is described above with the aid of terms in detail to facilitate a better understanding of the invention.

The term "antiviral drug" as used herein refers to a drug (compound, crude extract or biological agent) effective in inhibiting the formation or replication of a virus in a human body, including but not limited to drugs that interfere with host proteins or viral mechanisms necessary for the virus during the formation or replication of the human body.

"prevention" (prevention) refers to any treatment that prevents further progression of a disease or disorder. The term "preventing" also includes the use of a therapeutically effective amount of a compound or composition of the invention prior to exposure of an individual to a virus (i.e., pre-exposure prevention) to prevent the symptoms of disease from developing and/or to prevent the virus from reaching detectable levels in a sample of the patient's virus.

"treating" (or treating) means reversing, alleviating, or inhibiting one or more symptoms of the disease for which prevention is indicated. In certain embodiments, "treatment" refers to a compound or composition according to the present invention to reduce or eliminate symptoms of viral infection and/or reduce the viral load of a patient's viral sample.

The term "pharmaceutical composition" refers to a pharmaceutical composition prepared by mixing the compound of the present invention with adjuvants or other active ingredients in any ratio.

The compound names provided above are named according to literature, and one skilled in the art will appreciate that other recognized naming systems and symbols can be used to name or identify the compound structure. For example, a compound may be named or identified by a common name, a system or non-system name. Commonly accepted nomenclature systems and symbols in the chemical arts include, but are not limited to, the Chemical Abstracts Service (CAS) and the International Union of Pure and Applied Chemistry (IUPAC). Thus, the structure of compound formula I (LG0014) provided above is named 4- (1-hydroxy-2- ((6- (4-phenylbutoxy) hexyl) amino) ethyl) -2- (hydroxymethy) phenol using the ChemBioDraw Ultra name.

The beneficial technical effects are as follows: salmeterol is a selective beta 2 receptor agonist, has a bronchodilating effect and is a drug which is already on the market. Researches show that the salmeterol has the function of resisting influenza viruses, including influenza viruses H1N1 and H3N2 and duffy and amantadine double-drug-resistant influenza viruses, and has good clinical application prospects.

Drawings

FIG. 1 of formula I, LG0014 ¹ H NMR spectrum

FIG. 2 of formula I, LG0014 ¹³ C NMR spectrum

FIG. 3H-COSY-MOF map of formula I, LG0014

FIG. 4 Mass Spectroscopy of formula I, LG0014

FIG. 5 shows the body weight changes of the mice in each group

Detailed Description

Experimental example 1 evaluation of anti-influenza Virus Effect of salmeterol

The in vitro anti-influenza virus activity of the compounds was evaluated using a cytopathic effect (CPE) inhibition evaluation experiment. In this example, MDCK cell toxicity test of the drug and inhibition test of influenza virus cytopathic effect of the drug were carried out using MDCK cell (dog kidney cell). The virus is influenza A virus A/PR/8/34(H1N1) and A/Minfang/151/2000(H3N2), and the strains are from the Chinese center for disease prevention and control. Virus 10 ^-2 Infection, corresponding to 100 TCIDs ₅₀ 。

Cytotoxic assay: culturing the sample in the absence of serumDiluting the culture medium by multiple times, adding the diluted culture medium into MDCK monolayer cells, carrying out MTS (methyl thiazolyl tetrazolium) staining on the cells after 24 hours to examine the cell viability to obtain the maximum nontoxic concentration CC of each sample ₀ (μ g/mL) and half-toxic concentration CC ₅₀ (μg/mL)。

And (3) pharmacodynamic experiment: the experimental method adopts a cytopathic effect (CPE) method, 20-30 ten thousand MDCK cells per milliliter are inoculated to a 96-well cell culture plate, each well is 0.1mL, cultured for 24 hours at 37 ℃ and 5% CO2, and a culture solution is discarded. Sample treatment: the sample is prepared into 1000ug/ml mother liquor by culture solution, dissolved in cell maintenance solution before use to prepare 100ug/ml initial concentration, and diluted by culture solution 2 times and 6 dilutions. Four modes of administration: (1) first medicine and then toxin: after the drug is preincubated with MDCK cells for 2 hours, the cells are infected by the virus; (2) and (3) administration at the same time: simultaneously acting the virus and the medicine on MDCK cells for 2 hours, and then replacing maintenance liquid containing the medicine with different concentrations; (3) first toxic and then drug treatment: the virus is adsorbed for 2 hours, and then the administration is carried out (post treatment); (4) incubation of drug toxicity: after the virus and the drug are incubated for 2h at 37 ℃, MDCK cells are infected for 2h, virus liquid is removed, and maintenance liquid is added. And observing the CPE condition of the cells under a light microscope 24h after the influenza virus infection, carrying out crystal violet staining, and inspecting the cell viability. A cell control without drug, a virus control and a positive control drug are Oseltamivir (Oseltamivir). Calculating the half Effective Concentration (EC) of the drug ₅₀ ). Calculating selection index SI (TC) ₅₀ /EC ₅₀ )。

The administration mode of first administration, then toxicity and simultaneous administration is mainly used for investigating the preventive effect of the medicament; the administration mode of the first-toxic and the second-drug is mainly used for investigating the treatment effect of the drugs; the drug administration mode of drug toxicity incubation mainly inspects the influence of the drug on the virus infectivity.

The results are shown in tables 1 and 2. The results show that LG0014 has prevention and treatment effects on cytopathic effects induced by influenza virus A/PR/8/34(H1N1) and A/Minfang/151/2000(H3N2), and the protection effect of LG0014 on H1N1 and H3N2 infected cells is superior to that of the positive medicament oseltamivir.

TABLE 1 inhibition of the cytopathic effects induced by influenza virus A/PR/8/34(H1N1) by salmeterol on MDCK cells in vitro

TABLE 2 inhibition of influenza A/Minfang/151/2000(H3N2) induced cytopathic effects by salmeterol on MDCK cells in vitro

^a TC ₅₀ Average 50% cytotoxic compound concentration

^b EC ₅₀ Concentration of average 50% inhibition

^c SI selection index, TC ₅₀ /EC ₅₀ .

Experimental example 2 evaluation of Effect of Salmeterol against amantadine and Oseltamivir (Daphne) double drug-resistant strains

Cell: MDCK, frozen in passage in the laboratory, grown in DMEM high-sugar medium and containing 10% fetal bovine serum.

Influenza virus: the duel drug-resistant strain A/Hebei Xinhua/SWL1106/2017H1N1pdm of the tamiflu (Oseltamivir) and Amantadine (Amantadine) is a gift of the institute for preventing and controlling viral diseases in the Chinese disease prevention and control center, and is frozen at-80 ℃ after passage by a chick embryo method.

Virus strain: the duel-drug-resistant strain A/Hebei Xinhua/SWL1106/2017H1N1pdm of tamiflu and amantadine is cultured in a chick embryo allantoic cavity for passage and is preserved at-80 ℃. Sample treatment: the sample is prepared into 1000ug/ml mother liquor by culture solution, dissolved in cell maintenance solution before use to prepare 100ug/ml initial concentration, and diluted by culture solution 2 times and 6 dilutions. Positive control drug: tamiflu, amantadine and zanamivir. Four modes of administration: (1) first medicine and then toxin: after the drug is preincubated with MDCK cells for 2 hours, the cells are infected by the virus; (2) and (3) administration at the same time: simultaneously acting the virus and the medicine on MDCK cells for 2 hours, and then replacing maintenance liquid containing the medicine with different concentrations; (3) first toxic and then drug treatment: the virus was adsorbed for 2 hours and administered after discarding the virus (post treatment). Inoculating MDCK cells to 96-well culture plate, diluting the medicines by 2 times, and performing serial dilution on 6 medicinesDiluting, setting 2 holes for each concentration, culturing at 37 ℃ and 5% CO 2; (4) incubation of drug toxicity: after the virus and the drug are incubated for 2h at 37 ℃, MDCK cells are infected for 2h, virus liquid is removed, and maintenance liquid is added. After 24h of influenza virus infection, the cells were observed for CPE under an optical microscope, and examined for cell viability by crystal violet staining. No drug cell control and virus control were set. Observing the pathological change degree of each group when the pathological change degree (CPE) of the virus control group reaches 80% -100%, and calculating the half inhibitory concentration (EC) of anti-influenza virus of each sample ₅₀ ). The positive control drug is zanamivir.

The administration mode of first medicine and then toxin and simultaneous administration is mainly used for investigating the preventive effect of the medicine; the administration mode of the first-toxic and the second-drug is mainly used for investigating the treatment effect of the drugs; the drug administration mode of drug toxicity incubation mainly inspects the influence of the drug on the virus infectivity.

The experimental results are shown in table 3. the results show that in the administration modes of prevention and treatment, both tamiflu and amantadine do not show the effect of resisting drug-resistant strains, but both salmeterol (LG0014) and zanamivir have obvious effect of resisting drug-resistant strains, which indicates that LG0014 has the effects of prevention and treatment of influenza drug-resistant strains and inactivation of viruses, and has obvious advantages compared with tamiflu and amantadine.

TABLE 3 inhibition of the cytopathic effect induced by influenza A/Hebei Xinhua/SWL1106/2017H1N1pdm by salmeterol (LG0014) on MDCK cells in vitro

^a TC ₅₀ Average 50% cytotoxic compound concentration

^b EC ₅₀ Concentration of average 50% inhibition

^c SI selection index, TC ₅₀ /EC ₅₀ .

^d ND, no activity was detected.

Experimental example 3 evaluation of anti-influenza Activity of Salmeterol in mice

Animals: balb/c mice were purchased from Beijing Huafukang Biotech GmbH, all males, weighing 13-15 g. The mouse feed is a formula feed which is purchased from animals and is rich in various components, and the feeding environment is as follows: the temperature is 23 +/-2 ℃, and the relative humidity is 75 +/-10%.

Virus strain: the influenza A virus FM1 strain mouse lung adaptive strain, namely H1N1(A/PR/8/34), is provided by the research institute of virus diseases in the center of China disease prevention and control center, and the hemagglutination titer is 1: 1024, negative for bacterial culture, and low-temperature preservation at-80 ℃ in the laboratory.

Reagents and consumables: 2,2, 2-tribromoethanol, available from sigma; a pressure steam sterilizer, which is a product of Shanghai nan Hua medical instrument factory; an ultra-pure water device, which is a product of Millipore company; ultra low temperature refrigerator, product of Sanyo corporation, japan.

Experimental methods

1) Influenza virus mouse lung adapted strain LD ₅₀ Measurement of

Virus preparation: taking out the strain from a refrigerator at the temperature of-80 ℃, flushing the strain with running water, thawing the strain, and diluting the strain to 10 percent by using sterile normal saline ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 、10 ^-8 A total of 8 concentrations.

Grouping and infection of mice: 45 Balb/c mice, randomly divided into 9 groups of 5, 2,2, 2-tribromoethanol anaesthetised, were inoculated with 100. mu.l of virus dilutions of the above 8 concentrations, one in each nasal cavity, and one group at each dilution. The results were observed daily until 15 days after infection of the mice, and no statistics were given for non-specific results when the mice died within 24 h.

And (3) observation and recording: after 14 days of infection, the number of deaths per group was recorded, and the dead mice were dissected and observed for lung lesions to determine the number of deaths due to influenza. Finally calculating LD by Karber method ₅₀ 。

2) Observation of antiviral effects of salmeterol oral administration in mice

Virus preparation: according to LD ₅₀ The result of the determination, the virus seed is diluted to 2LD with sterile physiological saline ₅₀ And (4) concentration.

Grouping, infection and administration method of mice: 50 Balb/c mice were randomly divided into 7 salmeterol group No. 1 (oral salmeterol 30mg/kg), 7 salmeterol group No. 2 (intraperitoneal injection salmeterol 10mg/kg), 12 positive drug groups (oral Duffy 10mg/kg), 12 model groups and 12 blank control groups. Wherein salmeterol and duffy are prepared into a solution by 0.5 percent of CMC-Na and then are administrated, and the administration volume is 0.1mL/10 g. The model group and the blank control group were injected intraperitoneally with an equal volume of 0.5% CMC-Na solution. After mice were anesthetized with 2,2, 2-tribromoethanol, 100 μ l of each nasal cavity inoculated with virus diluent in the administration group, 100 μ l of sterile physiological saline was injected into the nasal cavity in the blank control group, and administration was started twice a day the following day for 5 days continuously.

And (3) observing and recording:

observing the change of the state of the mice each day, the survival number of each group and the change of the body weight of the mice

Results of the experiment

1) Influenza virus mouse lung adapted strain LD50 assay

LD of A/PR/8/34(H1N1) murine adapted strain ₅₀ The measurement results are shown in Table 4.

TABLE 4 determination of viral titer (LD) in influenza Virus-infected mice ₅₀ )

Calculating the formula:

distance ratio (percentage of death above 50% — 50)/(percentage of death above 50% — percentage of death below 50) — 10/60 ═ 0.16

LD50 log + distance ratio greater than 50% of the highest dilution of dead virus 4+ 0.16-4.16

The results show that the virus suspension is diluted 10 ^4.16 In time, 50% of mice inoculated with 100uL of virus solution through nasal cavity dieThe rate of death.

2LD was used in the subsequent experiments ₅₀ The virus amount of (a) is to infect mice, i.e. mice are infected by diluting the virus stock about 7000 times.

2) Observation of antiviral Effect of salmeterol in mice

After 21 days of continuous observation, survival and weight change of each group of mice were observed daily, and daily average body weight and survival rate were calculated, and the statistical results are shown in table 5 and fig. 5.

TABLE 5 daily survival rates of groups of mice infected with virus

# p <0.01VS normal group; p <0.01VS model group

As can be seen from the results, the survival rate of the mice in the model group after being infected with the virus is 58.3 percent, and the survival rates of the mice in the salmeterol oral administration group and the mice in the abdominal cavity injection group are both 100 percent. Compared with the model group, the weight of the mice of each administration group is higher than that of the model group and is close to that of the normal group, which shows that the salmeterol has obvious anti-influenza drug effect which is equivalent to that of the positive drug duffy group.

In conclusion, salmeterol has the effects of obviously resisting influenza virus H1N1 and H3N2 in vitro and resisting amantadine and oseltamivir (tamiflu) drug-resistant strains besides the effect of relieving asthma, has obvious drug effect of resisting influenza virus H1N1 in vivo and is expected to become an anti-influenza effective treatment drug. The invention lays a scientific experimental foundation for enlarging the clinical indications of salmeterol.

The invention belongs to the relocation of a medicine of a marketed antiasthmatic medicine Samette. The anti-influenza virus effect of the Samite provides a feasible and effective treatment scheme for clinically preventing and treating influenza.

Claims (6)

1. The application of the salmeterol shown in the formula I or the pharmaceutically acceptable salt thereof in preparing the medicaments for preventing and/or treating the influenza virus infection,

2. the use according to claim 1, wherein the pharmaceutically acceptable salt comprises a pharmaceutically acceptable organic salt or inorganic salt, wherein the organic salt comprises a sulfonate, a carboxylate, an amino acid salt, and a fatty acid salt, and the inorganic salt comprises a hydrochloride, a bromate, an iodate, a sulfate, a hydrogen sulfate, a phosphate, a hydrogen phosphate, a dihydrogen phosphate, and a nitrate.

3. Use according to claim 2, characterized in that said sulfonates comprise alkylsulfonates containing 1-15 carbon atoms, benzenesulfonates, p-toluenesulfonates, o-toluenesulfonates, m-toluenesulfonates; carboxylates include tartrate, maleate, fumarate, citrate, malate, cinnamate, benzoate, malonate, succinate, glutarate, adipate, pamoate, and lactate; amino acid salts include glutamate, aspartate; the fatty acid salt comprises a long chain fatty acid salt having 2 to 18 carbon atoms.

4. The use according to claim 1, wherein said influenza virus comprises H1N1, H3N 2.

5. The use according to claim 1, wherein the influenza virus infection comprises influenza induced by duffy and amantadine resistant strains.

6. The application of a pharmaceutical composition in preparing a medicament for preventing or treating influenza virus infection is characterized in that the pharmaceutical composition comprises salmeterol shown in a structural formula I or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient,

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