CN102430112A - Atomization inhalant prepared from interferon alpha and salbutamol sulfate - Google Patents

Abstract

The invention belongs to the field of composites of antiviral medications, which relates to an atomization inhalant prepared from interferon alpha and salbutamol sulfate. The atomization inhalant comprises the interferon alpha with a curative effective dose, the salbutamol sulfate and a pharmaceutically acceptable adjuvant with sufficient quantity. Preferentially, the single dose comprises 2.5-30mug of the interferon alpha, 0.048-0.192mug of the salbutamol sulfate and the pharmaceutically acceptable adjuvant with the sufficient quantity, and more preferentially, the single dose comprises 10-20mug of the interferon alpha, 0.096-0.144mug of the salbutamol sulfate and the pharmaceutically acceptable adjuvant with the sufficient quantity. Compared with treating viral pneumonia by singly using the interferon alpha or the salbutamol sulfate, the curative effect can be obviously improved on treating the viral pneumonia by using the atomization inhalant prepared from the interferon alpha and the salbutamol sulfate.

Description

Nebulized Inhalation of Interferon α and Salbutamol Sulfate

technical field

The invention relates to a composition of antiviral drugs and its application in the preparation of drugs for treating viral pneumonia.

Background technique

Viral pneumonia is a common disease in infants and young children, which seriously endangers the life and health of infants and young children. According to incomplete statistics, more than 90% of infants and young children have been infected with viral pneumonia before the age of 2, of which more than 80% of the cases are within 1 year old, the peak age of onset is 2-6 months, and severe cases can be seen in 1-6 months . The death rate of infants and young children caused by infection of viral pneumonia is 0.5%-2.0% in developed countries, up to 7% in developing countries, and it is the leading cause of infant death in my country. In addition, viral pneumonia is very contagious and reinfectious. It has been reported that family members can be infected successively, and the incidence of reinfection in infected infants and young children is as high as 65% within 10 years.

The main viruses causing viral pneumonia are adenovirus (ADV) and respiratory syncytial virus (RSV), followed by influenza virus (IFV) and parainfluenza virus (PIV). Is cytomegalovirus (cytomegalovirus, CMV), herpes simplex virus (herpes simplex virus, HSV), enterovirus (enterovirus, EV), human metapneumovirus (hMPV) and so on. Because these viruses are very easy to mutate, and further bacterial infections are easily induced after viral infection, thus causing mixed infections and complications in children, it is difficult to achieve comprehensive and specific prevention and treatment of viral pneumonia in infants and young children, and there is a lack of effective clinical treatments. medical treatement.

Chemical drugs are currently the most important drugs for the treatment of viral pneumonia. The reason is that they have been used for the longest time in clinical practice and can quickly clear the infected virus. The body's immunity is against viruses, and drug resistance is prone to develop during long-term use. Currently, the main chemical drugs for the clinical treatment of viral pneumonia are ribavirin, ganciclovir, oseltamivir, vidarabine, and amantadine. For example, U.S. Patent No. 5,290,540 discloses a method for treating viral pneumonia with ribavirin.

In recent years, Chinese medicine has been widely used in the treatment of viral pneumonia, which is due to the mild and long-lasting properties of Chinese medicine, and also has immune regulation effect. However, the treatment of viral pneumonia by traditional Chinese medicine takes a long time, and traditional Chinese medicine is not suitable for infants and young children to a large extent. In addition, the long-term safety of traditional Chinese medicine treatment is also a problem that cannot be ignored. At present, it is reported that the traditional Chinese medicines used in clinical research for the treatment of viral pneumonia mainly include Daqingye, Banlangen, Yinhua, Forsythia, Shegan, Scutellaria, Coptis, Ephedra, Bitter Almond, etc. For example, Chinese patent CN 03131642.5 discloses a Qingfei oral liquid preparation for the treatment of children with viral pneumonia, which consists of ephedra, raw gypsum, bitter almond, Tinglizi, mulberry root, Peucedanum, silkworm, salvia miltiorrhiza, Polygonum cuspidatum, boxing ginseng Composition of Chinese herbal medicine. Another example is that Chinese patent CN 02138175.5 discloses a pharmaceutical composition for the treatment of children with viral pneumonia, which consists of ephedra, bitter almond, gypsum, licorice, Peucedanum, silkworm, salvia miltiorrhiza, Polygonum cuspidatum, boxing ginseng, Tinglizi, Morus alba The skin is composed of eleven traditional Chinese medicines.

With the rapid development of biotechnology drugs, more and more studies have focused on the treatment of viral pneumonia by biotechnology drugs in recent years. The biotechnology drugs used include interferon, interleukin, human gamma globulin, thymosin, Specific antiviral antibodies, etc., especially the most reported use of interferon. Biotechnology drugs, especially interferon, are used in the treatment of viral pneumonia. They have a long-lasting effect, can regulate and improve the immunity of children to fight against viruses, are less likely to develop drug resistance, and are safer. For example, Chinese patent CN03147580.9 discloses a recombinant human interferon α spray for the treatment of viral pneumonia, which is made of recombinant human interferon α stock solution with appropriate protective agent, mucosal absorption promoter, bacteriostatic agent, etc. The pH value is 5.0-8.0.

However, since the above-mentioned children are likely to further induce bacterial infection after being infected with viral pneumonia, thereby causing mixed infections and complications to the children, the clinical effect of using a single drug to treat infant viral pneumonia is not ideal, resulting in The drug combination or the pharmaceutical composition of two or more drug active ingredients is used for the treatment of viral pneumonia. For example, the article "Observation on the Curative Effect of Interferon Combined with Ribavirin in the Treatment of 30 Cases of Children with Viral Pneumonia" published in March 2009, Volume 4, Issue 9, Page 144-145 of "China Practical Medicine" reported that interferon combined with ribavirin The curative effect of treating children's viral pneumonia shows that the effective rate of the combined drug group is 96.7%, while the effective rate of the control group using ribavirin alone is only 76.6%, and there is a statistically significant difference between the two. However, the research on the use of combined drugs or combination drugs for viral pneumonia is not systematic enough, and the types of research are still small, so the research results are not suitable for large-scale clinical application.

In terms of drug delivery system for the treatment of viral pneumonia, nebulized inhalation is a new dosage form that has been clinically developed in recent years. The so-called nebulized inhalant refers to a dosage form in which the liquid medicine is atomized into a mist, and then inhaled to reach the target part of the respiratory system to play a therapeutic role. The administration of nebulized inhalation must rely on certain nebulized inhalation devices, including compression nebulizers, ultrasonic nebulizers, etc. Since nebulized inhalation can directly act on the lesions of the respiratory system without absorption after administration, it has significantly higher bioavailability and efficacy than other dosage forms. Therefore, nebulized inhalation has gradually become a clinical treatment for pediatric viruses The preferred dosage form for acute pneumonia.

In addition, salbutamol sulfate is a common drug for the clinical treatment of lung diseases. There have been a large number of literature reports that salbutamol sulfate is used for the treatment of infantile pneumonia, such as published on page 1108 of the 12th issue of volume 35 of "Shanxi Medicine Journal" in December 2006 The article "Observation on the Efficacy of Salbutamol Sulfate and Ketotifen Combined Treatment of Bronchopneumonia" reported the efficacy of salbutamol sulfate combined with ketotifen in the treatment of children with bronchopneumonia, and the effective rate can reach 65%.

Contents of the invention

The primary purpose of the present invention is to provide a nebulized inhalation of interferon alpha and salbutamol sulfate, to be used for the treatment of viral pneumonia in infants, and further improve the therapeutic effect.

In a basic embodiment, the nebulized inhalation of interferon-alpha and salbutamol sulfate of the present invention contains a therapeutically effective amount of interferon-alpha, salbutamol sulfate and a suitable amount of pharmaceutically acceptable adjuvants.

The preparation of nebulized inhalation can choose the method known to those skilled in the art, even the method similar to the injection solution. However, regardless of the composition of the nebulized inhaler or the preparation of the nebulized inhaler, the stability of interferon α and salbutamol sulfate should be fully considered, especially the thermal stability, pH stability and chemical stability of interferon α and albuterol sulfate. .

For example, the excipients should contain a buffer solution system that is conducive to the stability of interferon α and salbutamol sulfate. The pH of such a buffer solution system should be between 5.0-8.0. Such a buffer solution system includes but is not limited to a phosphate buffer solution system , citrate buffer solution system; should contain interferon α activity protection agent to prevent the interferon α activity from being reduced or lost during long-term storage, such protection agents include but not limited to various amino acids, albumin and multivariate Alcohols such as albumin, mannitol, trehalose, dextran. In addition, it is also preferable to contain an osmotic pressure regulator in the auxiliary material to adjust the osmotic pressure of the solution to be substantially isotonic with the body, such osmotic pressure regulator includes but not limited to NaCl, mannitol; preferably contains anti-interferon alpha, sulfuric acid Salbutamol and other excipient precipitation and adsorption solubilizers, such solubilizers include but not limited to Tween-80; preferably contain preservatives to prevent pharmaceutical preparations from being contaminated, such preservatives include but not limited to benzalkonium chloride, Parabens, Benzyl Alcohol, etc.

During the preparation of the nebulized inhaler, it is preferable to dissolve the poorly soluble excipients by a more suitable method and then mix them with the dissolved interferon-alpha, albuterol sulfate and other excipients. During the preparation process, it should be mixed quickly, and attention should be paid to avoiding sharp changes in pH, temperature, etc., which may cause bioinactivation and/or chemical degradation of the active ingredients of the drug interferon α and salbutamol sulfate.

After the active ingredient interferon α, salbutamol sulfate and auxiliary materials are mixed, there may be interactions between the active ingredients of the drug, between the auxiliary materials, and between each active ingredient of the drug and the auxiliary materials, causing biodegradation of the active ingredients of the drug. Live and/or chemical degradation, thereby reducing the clinical use effect of aerosol inhalation, and even bring safety problems to the clinical use of aerosol inhalation, so it is necessary to carry out necessary testing on the mixed aerosol inhalation to confirm Such biological inactivation and/or chemical degradation is absent or rarely present. Necessary detection methods for the nebulized inhalation include but are not limited to appearance detection (observing whether there is a color change, the appearance of gas and/or the generation of precipitation after interferon α, salbutamol sulfate and auxiliary materials are mixed), pH detection , interferon α biological activity detection, interferon α content detection, interferon α molecular weight detection, interferon α N-terminal sequence detection, interferon α C-terminal sequence detection, interferon α amino acid composition analysis and detection, salbutamol sulfate content detection, salbutamol sulfate purity detection etc. Preferably, the activity detection of interferon alpha, the molecular weight detection of interferon alpha, and the purity and content detection of salbutamol sulfate are carried out.

The detection of interferon-α activity preferably adopts the standard method stipulated in "Chinese Pharmacopoeia 2010 Edition (Part Three)".

Mass spectrometry rather than electrophoresis should be used to detect the molecular weight of interferon α, because mass spectrometry can detect small changes in the amino acid sequence of interferon α (degradation or substitution of several amino acids on the sequence of interferon α will lead to a change in the molecular weight of interferon α changes, which can be detected by mass spectrometry), while the sensitivity of electrophoresis cannot be achieved.

Salbutamol sulfate purity/content detection preferably adopts the standard reversed-phase HPLC method that " Chinese Pharmacopoeia 2010 edition (two) " stipulates, because such method can guarantee to detect the possible degradation products of salbutamol sulfate, also can make salbutamol sulfate and adjuvant in detection It can be fully separated in order to prevent the influence of excipients on the detection of salbutamol sulfate content. In order to determine whether the chromatographic peak at a certain retention time is a salbutamol sulfate degradation product peak or a soluble excipient component peak, each soluble excipient reference substance can be loaded under the same chromatographic conditions, and determined by retention time comparison.

According to the common knowledge of those skilled in the art, when adopting the HPLC method to carry out content detection to salbutamol sulfate, it is necessary to go up the salbutamol sulfate reference substance of the same volume and different concentrations or the salbutamol sulfate reference substance of the same concentration and different volumes on the HPLC packing column to draw the work curve.

In the above-mentioned various tests, if dissolution treatment is required, mild conditions should be used to prevent the bioinactivation and/or chemical degradation of interferon α and salbutamol sulfate during the dissolution process; in order to eliminate the influence of possible precipitates on the test, It is necessary to filter or centrifuge the precipitate; in order to eliminate the influence of large molecule interferon α on the purity/content detection of small molecule salbutamol sulfate, ultrafiltration can be used to remove interferon α in the solution; in order to eliminate the interference of small molecule soluble substances In order to avoid the influence of the detection of element α, ultrafiltration, dialysis and other methods can be used to remove small molecular substances in the solution.

In a preferred embodiment, the aerosolized inhalation of interferon-alpha and salbutamol sulfate of the present invention contains 2.5-30 μg of interferon-alpha and 0.048-0.192 mg of salbutamol sulfate in a single dose, and an appropriate amount of Pharmaceutical excipients.

In a preferred embodiment, the interferon-alpha of the present invention and salbutamol sulfate single administration dose contains 10-20 μg of interferon-alpha and 0.096-0.144 mg of salbutamol sulfate, and a suitable amount of Pharmaceutical excipients.

In a preferred embodiment, the interferon α in the nebulized inhalation of interferon α and albuterol sulfate of the present invention is one of interferon α 2a, interferon 2b and interferon α 1b or mixed in any proportion Several kinds.

In a preferred embodiment, the interferon α in the aerosol inhalation of interferon α and salbutamol sulfate of the present invention is interferon α 1b.

Another object of the present invention is to provide the application of the nebulized inhalation of interferon alpha and albuterol sulfate in the preparation of medicine for treating viral pneumonia. Utilizing the nebulized inhalation of the present invention to treat viral pneumonia can achieve significantly better therapeutic effect than using interferon alpha or albuterol sulfate alone to treat viral pneumonia.

Description of drawings

Fig. 1 is the chromatogram obtained after the albuterol sulfate reference substance is mounted on a DIKMA Platisil C18 reverse-phase high-performance liquid chromatography column (4.6 × 250mm).

Detailed ways

The implementation of the present invention will be further described through the following examples, but the implementation of the present invention is not limited to the following examples.

Embodiment 1: the preparation of the atomized inhalation of interferon alpha and albuterol sulfate

The nebulized inhalation of interferon alpha and albuterol sulfate was prepared according to the method in Table 1 below. Among them, "Anfulong" (trade name) is the recombinant human interferon α2b injection with a specification of 50 μg/ml/bottle produced by Tianjin Hualida Bioengineering Co., Ltd.; "Interfern" (trade name) is Shenyang Sansheng Pharmaceutical Co., Ltd. Recombinant human interferon α2a injection with a specification of 50 μg/ml/bottle produced by the limited liability company; Interferon α1b injection, "salbutamol sulfate injection" is produced by Jiangsu Yongda Pharmaceutical Co., Ltd., the specification is 0.48mg/2ml/bottle; "PBS" is 25mmol/L disodium hydrogen phosphate containing 0.15mol/L NaCl- Sodium dihydrogen phosphate buffer (pH7.0). Unless otherwise specified in the subsequent embodiments, the above trade names have the same meaning as in this embodiment.

The preparation of the nebulized inhalation of interferon α and albuterol sulfate of table 1

Example 2: Detection of the purity of albuterol sulfate after mixing interferon alpha and salbutamol sulfate

The aerosol inhalation of each interferon α and albuterol sulfate prepared by the method of Example 1 is carried out ultrafiltration with the Millipore ultrafiltration centrifuge tube with a molecular weight cut-off of 3000Da after placing the preset time, and the filtrate is treated with After diluting with deionized water to the same volume as before ultrafiltration and centrifugation, take 20 μl and put them on a DIKMAPlatisil C18 reversed-phase high-performance liquid chromatography column (5 μm filler particle size, column size 4.6×250mm), and perform chromatographic operation at a column temperature of 25°C. Phase 0.08mol/L sodium dihydrogen phosphate (phosphoric acid to adjust the pH value to 3.1±0.05)-methanol (85:15) elution, flow rate 1.0ml/min, detection wavelength 276nm. The measurement results are shown in Table 2 below.

Salbutamol sulfate purity detection result after table 2 interferon α and salbutamol sulfate mixed

serial number 0 hours after mixing 12 hours after mixing 24 hours after mixing 48 hours after mixing Nebulized inhalation 1 98.5% 98.4% 98.4% 98.5% Nebulized inhalation 2 98.4% 98.3% 98.2% 98.3% Nebulized inhaler 3 98.3% 98.3% 98.4% 98.4% Nebulized inhalation 4 98.5% 98.4% 98.3% 98.4% Nebulized inhalation 5 98.3% 98.5% 99.4% 99.3% Nebulized inhalation 6 98.4% 98.2% 98.3% 98.3% Nebulized inhaler 7 98.2% 98.3% 98.1% 98.2% Nebulized inhalation 8 98.2% 98.4% 98.4% 98.3% Nebulized inhalation 9 98.3% 98.2% 98.1% 98.1%

It can be seen that interferon-alpha mixed with salbutamol sulfate has no effect on the purity of salbutamol sulfate.

Embodiment 3: detection of salbutamol sulfate content after mixing interferon alpha and salbutamol sulfate

The salbutamol sulfate reference substance (prepared by China Institute for the Control of Pharmaceutical and Biological Products, batch number 100328-200502) is dissolved and diluted with water respectively to a concentration of 0.06, 0.12, 0.24, 0.48, 0.72mg/ml and then presses the high performance liquid chromatography conditions of Example 2 Samples were loaded to determine the peak area of the main peak. Use the peak area (y) to perform linear regression on the concentration (x) to obtain a working curve, the equation is y=6900000x+5158, and the regression coefficient is r=0.9999.

The aerosolized inhalation of each interferon α and albuterol sulfate prepared by the method of Example 1 is carried out ultrafiltration centrifugation according to the method of Example 2 respectively after placing the preset time, and the filtrate is deionized water respectively. After diluting to the same volume as before ultrafiltration and centrifugation, the sample was loaded according to the high-performance liquid chromatography conditions of Example 2 to determine the peak area of the main peak. The peak area obtained by each measurement is substituted into the working curve equation to obtain the concentration of salbutamol sulfate in each atomized inhalation. The measurement results are shown in Table 3 below.

Table 3 Interferon α and salbutamol sulfate content test results after mixing with salbutamol sulfate

It can be seen that interferon-alpha mixed with salbutamol sulfate has no effect on the content of salbutamol sulfate. Comprehensive Example 2 Interferon α and salbutamol sulfate purity detection results after mixing salbutamol sulfate, shows that interferon α and salbutamol sulfate mixed without chemical degradation of salbutamol sulfate.

Example 4: Interferon α molecular weight detection after interferon α is mixed with albuterol sulfate

Each interferon α prepared by the method of Example 1 and the aerosol inhalation of salbutamol sulfate were respectively dialyzed to 100 times the volume of deionized water (the molecular weight cut-off of the dialysis bag was 7000Da), during which the deionized water was changed once. MALDI-TOF mass spectrometry molecular weight detection was performed on the dialysate, and the detection results with a recorded molecular weight below 25000 Da are shown in Table 4. Only one molecular weight value was detected in each test result, indicating that interferon-alpha degradation products did not appear after mixing interferon-alpha and albuterol sulfate.

Interferon α molecular weight detection result after table 4 interferon α and albuterol sulfate mixing

Example 5: Detection of interferon-alpha activity after mixing interferon-alpha with albuterol sulfate

The aerosolized inhalation of each interferon α prepared by the method of Example 1 and salbutamol sulfate is measured respectively by the standard method of the interferon α assay activity specified in "Chinese Pharmacopoeia 2010 Edition (Three Parts)" Interferon α wherein activity. The measurement results are shown in Table 5 below.

Interferon α activity detection result after table 5 interferon α and albuterol sulfate mixing

The above test results show that interferon-alpha mixed with albuterol sulfate has no effect on the activity of interferon-alpha.

Based on the test results of Example 4 and Example 5, it shows that interferon α does not undergo biological inactivation and chemical degradation after mixing with salbutamol sulfate.

Embodiment 6: Animal experiment of interferon alpha and albuterol sulfate nebulized inhalation treatment viral pneumonia

1) RSV virus culture

The RSV standard strain RSV-Long (quoted from the Beijing Institute of Pediatrics) was inoculated on Hep-2 cells, harvested when the cytopathic rate reached more than 80%, and the virus liquid was frozen in liquid nitrogen and stored at 37°C Melt, centrifuge at 1000rpm for 10 minutes, and take the supernatant for later use.

2) Establishment of RSV mouse infection model

All 96 mice were anesthetized with ether, and 0.1ml of 10 ⁶ PFU/ml RSV virus solution was instilled through the nasal cavity. Positive reactions of rapid and abdominal muscle twitching indicated that the mouse model of RSV infection was established successfully.

3) Grouping and administration

The above 96 mice infected with RSV virus were randomly divided into 12 groups with 8 mice in each group. Groups 1 to 9 were ultrasonically inhaled 1ml each of the atomized inhalation 1 to atomized inhalation 9 described in the previous examples; group 10 was inhaled daily by ultrasonic atomization with the injection specification of 30 μg/ml/bottle " One bottle of "Yun Desu", group 11 daily ultrasonic atomization inhalation of 0.5 "albuterol sulfate injection" with a specification of 0.48mg/2ml/bottle; group 12 daily ultrasonic atomization inhalation of normal saline 1ml. All groups were only inhaled by ultrasonic nebulization once a day, for a total of 5 times (5 days). After 5 days, mouse blood was taken for BALF white blood cell count, and the results are shown in Table 6 below.

Table 6 BALF white blood cell count results

Claims (9)

1. the nebulized inhalation of interferon α and salbutamol sulfate, it is characterized in that described aerosol inhalation contains the interferon α of treatment effective dose, salbutamol sulfate and the pharmaceutically acceptable adjuvant of appropriate amount. 2. The atomized inhalation according to claim 1, characterized in that a single administration dose contains 2.5-30 μg of interferon alpha, 0.048-0.192 mg of salbutamol sulfate, and a suitable amount of pharmaceutically acceptable adjuvants. 3. The nebulized inhalation according to claim 2, characterized in that a single administration dose contains 10-20 μg of interferon alpha, 0.096-0.144 mg of salbutamol sulfate, and a suitable amount of pharmaceutically acceptable adjuvants. 4. The nebulized inhalation according to claim 1, characterized in that the pharmaceutically acceptable adjuvant comprises an interferon alpha active protective agent selected from one or more of albumin, mannitol, trehalose, and dextran The combination. 5. The nebulized inhaler according to claim 1, characterized in that the pharmaceutically acceptable excipients comprise an osmotic pressure regulator selected from one or a combination of NaCl and mannitol. 6. The nebulized inhalation according to claim 1, characterized in that the pharmaceutically acceptable adjuvant contains preservatives, selected from one or more of benzalkonium chloride, paraben, benzyl alcohol combination. 7. The nebulized inhaler according to claim 1, characterized in that said interferon α is one of interferon α2a, interferon 2b and interferon α1b or several mixed in any proportion. 8. The nebulized inhalation agent according to claim 7, characterized in that said interferon α is interferon α1b. 9. The application of the nebulized inhalation according to any one of claims 1-8 in the preparation of medicine for the treatment of viral pneumonia.