Inhaled preparation of isoglycyrrhizic acid or salt thereof, and use in preparing drugs for treating respiratory system diseases

Abstract

The present invention belongs to the field of medicine, relates to an inhaled preparation of isoglycyrrhizic acid or a salt thereof, and in particular relates to an inhaled preparation of magnesium isoglycyrrhizinate and the use thereof in preparing drugs for treating respiratory system diseases.

Claims

1. A method for alleviating phlegm by administrating an inhaled preparation of magnesium isoglycyrrhizinate to a subject in need thereof, characterized in that the inhaled preparation delivers the magnesium isoglycyrrhizinate to lungs to exert a local or systemic effect, wherein the inhaled preparation is a liquid preparation for use in a nebulizer or powder fir inhalation, wherein the liquid preparation for use in a nebulizer comprising magnesium isoglycyrrhizinate, sodium chloride, ammonium hydroxide, and water for injection, with the pH of 6.5-7.0, the nebulizer being a continuous nebulizer or a quantitative nebulizer, the amount of the magnesium isoglycyrrhizinate being from 0.1 mg/ml to 5 mg/ml, and the amount of the sodium chloride being 9 mg/ml; wherein the powder for inhalation comprises magnesium isoglycyrrhizinate and one or more pharmaceutically acceptable carriers, the magnesium isoglycyrrhizinate being micronized and has a particle size of 0.5-10 μm, and the pharmaceutically acceptable carrier being ground lactose, sieved lactose or a mixture of sieved lactose and fine lactose, the particle size distribution of the ground lactose ranging from 1 to 350 μm, the particle size distribution of the sieved lactose ranging from 1 to 200 μm and the article size distribution of the time lactose ranges being from 1 to 60 μm, the ground lactose having a particle size distribution of X.sub.50 being 30-110 μm, the sieve lactose having a particle size distribution of X.sub.50 being 35-115 μm, and the fine lactose having a particle size distribution of X.sub.90<45 μm.

2. The method according to claim 1, characterized in that the amount of the magnesium isoglycyrrhizinate is from 0.1 mg/ml to 2.5 mg/ml.

3. The method according to claim 1, characterized in that the inhaled preparation is packaged in a single dose in a package size of 1 ml, 2 ml or 5 ml.

4. The method according to claim 1, characterized in that the magnesium isoglycyrrhizinate has a particle size of 0.5-5 μm.

5. The method according to claim 1, characterized in that the micronized magnesium isoglycyrrhizinate and the carrier are mixed and filled into a capsule or a blister.

6. The method according to claim 5, characterized in that each capsule or blister comprises 1-50 mg of micronized magnesium isoglycyrrhizinate and 0-50 mg of lactose.

7. The method according to claim 5, characterized in that each capsule or blister comprises 1-30 mg of micronized magnesium isoglycyrrhizinate and 1-40 mg of lactose.

8. The method according to claim 1, characterized in that the powder for inhalation further comprises: one or more pharmaceutically acceptable additives selected from the group consisting of surfactants, lubricants and flavoring agents.

9. The method according to claim 8, characterized in that the pharmaceutically acceptable additive is magnesium stearate and/or phospholipid.

10. The method according to claim 1, characterized in that the administration frequency of the inhaled preparation to a subject is selected from: up to three limes a day, up to twice a day, up to once a day and up to once every other day.

Description

DETAILED EMBODIMENTS OF THE INVENTION

(1) The present invention will be further illustrated below in conjunction with specific examples. It should be understood that these examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

(2) The assay methods which do not specify the specific conditions in the following examples may be carried out according to conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning known as those skilled in the art.

EXAMPLES

Example 1

Magnesium Isoglycyrrhizinate Powder for Inhalation

(3) The magnesium isoglycyrrhizinate was micronized to obtain samples having the following different particle size ranges.

(4) TABLE-US-00001 Particle size of the active ingredient X.sub.10/μm X.sub.50/μm X.sub.90/μm Large particle size 1.47 5.75 17.24 Medium particle size 1.38 5.08 12.64 Small particle size 0.58 2.03 5.69

Example 1a

(5) Prescription:

(6) TABLE-US-00002 Magnesium isoglycyrrhizinate (large particle size) 1 g Lactose A 2 g Amount of preparation 100 capsules

(7) Preparation Process:

(8) 1) The prescribed amount of magnesium isoglycyrrhizinate and the prescribed amount of lactose were taken;

(9) 2) Then sieved and mixed;

(10) 3) The capsules were filled according to 30 mg/capsule, and each capsule contained 10 mg of magnesium isoglycyrrhizinate;

(11) 4) The key quality indicators of powder for inhalation were detected according to requirements of general rule 0111 in the fourth part of the “Chinese Pharmacopoeia”.

Example 1b

(12) Prescription:

(13) TABLE-US-00003 Magnesium isoglycyrrhizinate (medium particle size) 1 g Lactose A 2 g Amount of preparation 100 capsules

(14) Preparation Process:

(15) 1) The prescribed amount of magnesium isoglycyrrhizinate and the prescribed amount of lactose were taken;

(16) 2) Then sieved and mixed;

(17) 3) The capsules were filled according to 30 mg/capsule, and each capsule contained 10 mg of magnesium isoglycyrrhizinate;

(18) 4) The key quality indicators of powder for inhalation were detected according to requirements of general rule 0111 in the fourth part of the “Chinese Pharmacopoeia”.

Example 1c

(19) Prescription:

(20) TABLE-US-00004 Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose A 2 g Amount of preparation 100 capsules

(21) Preparation Process:

(22) 1) The prescribed amount of magnesium isoglycyrrhizinate and the prescribed amount of lactose were taken;

(23) 2) Then sieved and mixed;

(24) 3) The capsules were filled according to 30 mg/capsule, and each capsule contained 10 mg of magnesium isoglycyrrhizinate;

(25) 4) The key quality indicators of powder for inhalation were detected according to requirements of general rule 0111 in the fourth part of the “Chinese Pharmacopoeia”.

(26) Comparison of key quality indicators:

(27) TABLE-US-00005 Quality indicator Example 1a Example 1b Example 1c Emptying rate 99% 99% 99% Fine particle fraction 13% 20% 32%

(28) The fine particle dose was an important parameter to evaluate the effectiveness of the inhaled preparation. The particle size of the magnesium isoglycyrrhizinate had a crucial influence on the fine particle fraction as the key quality indicator of the powder for inhalation. Therefore, the particle size of the magnesium isoglycyrrhizinate was controlled within the range of 0.5-10 μm. The fine particle fraction was >15%, in line with regulations of the pharmacopoeia.

Example 2

Magnesium Isoglycyrrhizinate Powder for Inhalation

(29) The effects of lactose with different types and different sizes on the key quality indicators of magnesium isoglycyrrhizinate powder for inhalation were compared.

(30) TABLE-US-00006 Lactose type Description particle size/μm Lactose A sieved lactose with a narrow X.sub.10: 30-60; X.sub.50: 70-110; X.sub.90: 110-150 Lactose B particle size distribution X.sub.10: 7-22; X.sub.50: 40-70; X.sub.90: 80-120 Lactose C ground lactose with the average X.sub.10: 5-15; X.sub.50: 50-100; X.sub.90: 120-160 particle size being strictly controlled Lactose D ground lactose with a wide 40%-60% < 45; 75%-100% < 100; particle size distribution 90%-100% < 150; 99.5%-100% < 315 Lactose E finely ground lactose with a 90%-100% < 45; 98%-100% < 63; narrow particle size distribution 100% < 150 Lactose F micronized fine lactose X.sub.50 < 5; X.sub.90 < 10 Note: a typical particle size range of lactose D is: X.sub.10: 1-10 μm; X.sub.50: 30-50 μm; X.sub.90: 70-150 μm.

Example 2a

(31) Prescription:

(32) TABLE-US-00007 Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose B 2 g Amount of preparation 100 capsules

(33) Preparation Process:

(34) 1) The prescribed amount of magnesium isoglycyrrhizinate and the prescribed amount of lactose were taken;

(35) 2) Then sieved and mixed;

(36) 3) The capsules were filled according to 30 mg/capsule, and each capsule contained 10 mg of magnesium isoglycyrrhizinate;

(37) 4) The key quality indicators of powder for inhalation were detected according to requirements of general rule 0111 in the fourth part of the “Chinese Pharmacopoeia”.

Example 2b

(38) Prescription:

(39) TABLE-US-00008 Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose C 2 g Amount of preparation 100 capsules

(40) Preparation Process:

(41) 1) The prescribed amount of magnesium isoglycyrrhizinate and the prescribed amount of lactose were taken;

(42) 2) Then sieved and mixed;

(43) 3) The capsules were filled according to 30 mg/capsule, and each capsule contained 10 mg of magnesium isoglycyrrhizinate;

(44) 4) The key quality indicators of powder for inhalation were detected according to requirements of general rule 0111 in the fourth part of the “Chinese Pharmacopoeia”.

Example 2c

(45) Prescription:

(46) TABLE-US-00009 Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose D 2 g Amount of preparation 100 capsules

(47) Preparation Process:

(48) 1) The prescribed amount of magnesium isoglycyrrhizinate and the prescribed amount of lactose were taken;

(49) 2) Then sieved and mixed;

(50) 3) The capsules were filled according to 30 mg/capsule, and each capsule contained 10 mg of magnesium isoglycyrrhizinate;

(51) 4) The key quality indicators of powder for inhalation were detected according to requirements of general rule 0111 in the fourth part of the “Chinese Pharmacopoeia”.

Example 2d

(52) Prescription:

(53) TABLE-US-00010 Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose E 2 g Amount of preparation 100 capsules

(54) Preparation Process:

(55) 1) The prescribed amount of magnesium isoglycyrrhizinate and the prescribed amount of lactose were taken;

(56) 2) Then sieved and mixed;

(57) 3) The capsules were filled according to 30 mg/capsule, and each capsule contained 10 mg of magnesium isoglycyrrhizinate;

(58) 4) The key quality indicators of powder for inhalation were detected according to requirements of general rule 0111 in the fourth part of the “Chinese Pharmacopoeia”.

Example 2e

(59) Prescription:

(60) TABLE-US-00011 Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose A 1.5 g Lactose F 0.5 g Amount of preparation 100 capsules

(61) Preparation Process:

(62) 1) The prescribed amount of magnesium isoglycyrrhizinate and the prescribed amount of lactose were taken;

(63) 2) Then sieved and mixed;

(64) 3) The capsules were filled according to 30 mg/capsule, and each capsule contained 10 mg of magnesium isoglycyrrhizinate;

(65) 4) The key quality indicators of powder for inhalation were detected according to requirements of general rule 0111 in the fourth part of the “Chinese Pharmacopoeia”.

Example 2f

(66) Prescription:

(67) TABLE-US-00012 Magnesium isoglycyrrhizinate (small particle size) 1 g Lactose B 1.5 g Lactose F 0.5 g Amount of preparation 100 capsules

(68) Preparation Process:

(69) 1) The prescribed amount of magnesium isoglycyrrhizinate and the prescribed amount of lactose were taken;

(70) 2) Then sieved and mixed;

(71) 3) The capsules were filled according to 30 mg/capsule, and each capsule contained 10 mg of magnesium isoglycyrrhizinate;

(72) 4) The key quality indicators of powder for inhalation were detected according to requirements of general rule 0111 in the fourth part of the “Chinese Pharmacopoeia”.

(73) Comparison of key quality indicators:

(74) TABLE-US-00013 Quality Indicators Example 2a Example 2b Example 2c Example 2d Example 2e Example 2f Emptying rate 99% 97% 97% 90% 97% 98% Fine particle 35% 40% 46% 48% 43% 45% fraction

(75) Different types and different particle sizes of lactose had crucial influence on the key quality indicators of powder for inhalation. The fine particle fraction in the sample prepared by sieved lactose alone with the active ingredient was lower, but the fine particle fraction could be significantly improved when adding a certain amount of fine lactose when preparing the sample. The fine particle fraction of the sample prepared by ground lactose and the active ingredient was higher.

Example 3

Magnesium Isoglycyrrhizinate Liquid Preparation for Use in Nebulizers Prescription

(76) TABLE-US-00014 Magnesium isoglycyrrhizinate 10 g Sodium chloride 18 g Ammonium hydroxide q.s. Water for injection to 2000 ml Amount of preparation 1000 preparations

(77) Preparation Process:

(78) The prescribed amount of magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizers containing 10 mg of magnesium isoglycyrrhizinate was obtained.

(79) Main Technical Evaluation Indicators:

(80) TABLE-US-00015 Acceleration Acceleration Acceleration Long term Time point 0 day for 1 month for 2 months for 3 months for 3 months Fne particle 38% 38% 39% 37% 38% fraction

Example 4

Magnesium Isoglycyrrhizinate Liquid Preparation for Use in Nebulizers Prescription

(81) TABLE-US-00016 Magnesium isoglycyrrhizinate 5 g Sodium chloride 18 g Ammonium hydroxide q.s. Water for injection to 2000 ml Amount of preparation 1000 preparations

(82) Preparation Process:

(83) The prescribed amount of magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizer containing 5 mg of magnesium isoglycyrrhizinate was obtained.

(84) Main Technical Evaluation Indicators:

(85) TABLE-US-00017 Time point Acceler- Acceler- Acceler- Long ation for ation for ation for term for 0 day 1 month 2 months 3 months 3 months Fine particle 39% 38% 36% 37% 40% fraction

Example 5

Magnesium Isoglycyrrhizinate Liquid Preparation for Use in Nebulizers

(86) Prescription:

(87) TABLE-US-00018 Magnesium isoglycyrrhizinate 1 g Sodium chloride 18 g Ammonium hydroxide q.s. Water for injection to 2000 ml Amount of preparation 1000 preparations

(88) Preparation Process:

(89) The prescribed amount of magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizer containing 1 mg of magnesium isoglycyrrhizinate was obtained.

(90) Main Technical Evaluation Indicators:

(91) TABLE-US-00019 Time point Acceler- Acceler- Acceler- Long ation for ation for ation for term for 0 day 1 month 2 months 3 months 3 months Fine particle 42% 38% 39% 39% 40% fraction

Example 6

Magnesium Isoglycyrrhizinate Liquid Preparation for Use in Nebulizers Prescription

(92) TABLE-US-00020 Magnesium isoglycyrrhizinate 0.4 g Sodium chloride 18 g Ammonium hydroxide q.s. Water for injection to 2000 ml Amount of preparation 1000 preparations

(93) Preparation Process:

(94) The prescribed amount of magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizer containing 0.4 mg of magnesium isoglycyrrhizinate was obtained.

(95) Main Technical Evaluation Indicators:

(96) TABLE-US-00021 Time point Acceler- Acceler- Acceler- Long ation for ation for ation for term for 0 day 1 month 2 months 3 months 3 months Fine particle 40% 39% 37% 41% 38% fraction

Example 7

Magnesium Isoglycyrrhizinate Liquid Preparation for Use in Nebulizers Prescription

(97) TABLE-US-00022 Magnesium isoglycyrrhizinate 0.2 g Sodium chloride 18 g Ammonium hydroxide q.s. Water for injection to 2000 ml Amount of preparation 1000 preparations

(98) Preparation Process:

(99) The prescribed amount of magnesium isoglycyrrhizinate and sodium chloride were taken and added into 1800 ml of water for injection, stirred until completely dissolved. Then ammonium hydroxide was added to adjust the pH value of the solution to 6.5-7.0, and the obtained solution was added with water for injection to 2000 ml, filtered and sterilized, filled according to 2 ml per package, before a liquid preparation for use in nebulizer containing 0.2 mg of magnesium isoglycyrrhizinate was obtained.

(100) Main Technical Evaluation Indicators:

(101) TABLE-US-00023 Time point Acceler- Acceler- Acceler- Long ation for ation for ation for term for 0 day 1 month 2 months 3 months 3 months Fine particle 40% 43% 42% 39% 40% fraction

Example 8

Pharmacokinetic Evaluation After Inhalation/Intragastric Administration to Rats

(102) Eight healthy male SD rats, weighing 223-252 g, were fed with standard formula granule feed of rats on time every day. The rats were fasted for 16 h before the experiment, and were refeeded at 4 h after the administration. Drinking water was free before, after and during the experiment. The rats were randomly divided into two groups, 4 rats in each group, and each rat was inhalation administrated with a single dose of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers (2.5 mg/mL) and intragastric administrated with magnesium isoglycyrrhizinate inhaled preparation (5.0 mg/mL, the amount of isoglycyrrhizic acid is 4.486 mg/mL) respectively. Each rat in the inhalation administration group was given 200 μL of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers (the actual dose given to the rat was 2.24-2.49 mg/kg). The dose in the intragastric administration group was 10.0 mg/kg. 0.2-0.3 mL of blood was taken from the fundus venous plexus before administration (0 h) and 0.0833 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h after administration. EDTA-K2 was used for anticoagulation, and the plasma was centrifuged. Then 50 μL was accurately measured, added with 10 μL internal standard solution to vortex and mixed, then added with 200 μL methanol, and mixed in high speed vortex mixer for 3 min, centrifuged for 10 min (4° C., 13000 rpm). The supernatant was collected, and 100 μL thereof was transferred to a 96-well plate. 50 μL of ultrapure water was added, vortexed, LC-MS/MS was used for detection, and the chromatogram was recorded.

(103) The pharmacokinetic results of inhalation administration of the magnesium isoglycyrrhizinate liquid preparation for use in nebulizers and the intragastric administration of isoglycyrrhizic acid injection were as follows:

(104) TABLE-US-00024 Group Inhalation Intragastric Sample size PK N = 4 N = 4 parameters Mean SD RSD % Mean SD RSD % Dose (mg/kg) 2.4 0.1 10 0 Cmax (μg/L) 3113 712 22.9% 27.3 17.2 63.0% AUC(0-t) (μg*h/L) 14567 6427 44.1% 89.8 105  118% AUC(0-∞) (μg*h/L) 14593 6427 44.0% 102 102  100% MRT(0-t) (h) 3.52 1.09 31.0% 2.35 1.58 67.1% t½ z(h) 2.13 0.69 32.3% 1.88 1.47 78.4% Tmax (h) 0.81 0.80 98.5% 1.75 2.84  162% CLz/F (L/h/kg) 0.195 0.103 53.0% 162 90.1 55.7% Cmax/Dose 1315 306 23.3% 2.73 1.72 63.0% AUC(0-t)/Dose 6108 2577 42.2% 8.98 10.5  118% relative F % 68052% 

(105) In addition, the pharmacokinetic parameters of inhalation administration were compared with the average pharmacokinetic parameters of intravenous administration reported in the literature, and the results were as follows:

(106) TABLE-US-00025 Group Inhalation Intravenous (N = 4) (N = 6) Dose (mg/kg ) 2.37 ± 0.13 30.0 Cmax (μg/L) 3113 ± 712  354500 AUC(0-t) (μg*h/L) 14567 ± 6427  209591 AUC(0-∞) (μg*h/L) 14593 ± 6427  212295 MRT(0-t) (h) 3.52 ± 1.09 1.71 t1/2 z(h) 2.13 ± 0.69 2.06 Tmax(h) 0.81 ± 0.8  0.0833 CLz/F(L/h/kg) 0.195 ± 0.103 141.3 Cmax/Dose 1315 ± 306  11817 AUC(0-t)/Dose 6108 ± 2577 6986 Relative F % 87%

(107) Compared with intragastric administration, the relative bioavailability of magnesium isoglycyrrhizinate by inhalation administration for rats was as high as 68052%. Compared with intravenous administration, the bioavailability of magnesium isoglycyrrhizinate by inhalation administration for rats was 87%. Therefore, inhalation administration can significantly improve the bioavailability of magnesium isoglycyrrhizinate by compared with gastrointestinal administration, and the bioavailability of magnesium isoglycyrrhizinate by inhalation administration was basically equal to that by gastrointestinal administration.

Example 9

Pharmacodynamic Experiment of Magnesium Isoglycyrrhizinate Inhaled Preparation for Rats with Chronic Obstructive Pulmonary Disease

(108) 9.1 Experimental Method:

(109) After modeling chronic obstructive pulmonary disease model induced by cigarette smoke inhalation, male SD rats were randomly divided into 6 groups according to the body weight: high dose tracheal instillation group (1.67 mg/ml of magnesium isoglycyrrhizinate, 100 μl/rat), low dose tracheal instillation group (1.67 mg/ml, 25 μl/rat), aerosolizing inhalation group, model group and blank control group, 10 rats in each group. During the intervention period, except that the control group and the model group were given normal saline, the other groups were successive administered every day, lasted for 15 days. During the administration period, the administration groups continued to be given with smoke stimulation after 30 minutes of administration, and the mental state, breathing, activity, hair luster, weight gain of the rats were recorded every day. 12 hour after the last administration, whole blood was taken from the rat eye for determination of white blood cell count and cell classified comparison. The rats were killed, the trachea and lungs were exposed by thoracotomy, and the morphology of the lungs and trachea was observed with the naked eye. The right lung was ligated at the right main branch, and the left lung was lavaged with normal saline 2 ml×3 times. The recovery rate was about 80%. The bronchoalveolar lavage fluid (BALF) was prepared for the determination of white blood cell count and classified comparison (white blood cells, neutrophils, lymphocytes and monocytes). Finally, the right lung of rat was fixed with 10% formalin and HE stained, lung injury was observed under microscope, and pathological lesions were scored. All scores were accumulated, and the average score of each animal in each group was calculated (mean±SD).

(110) Scoring indicators: (1) whether there was mucus and cell blockage in the small airway cavity; (2) whether the small airway epithelium had necrotic erosion; (3) small airway epithelial cells goblet cell metaplasia; (4) small airway epithelial cell squamous metaplasia; (5) small airway wall inflammation cell infiltration; (6) small airway wall fibrous connective tissue hyperplasia; (7) small airway wall smooth muscle hyperplasia; (8) small airway wall pigmentation; and (9) lung emphysema. Lesion score: according to the degree from light to heavy lesions, it was quantified as slight or very small amount for “0.5 point”, mild or small amount for “1 point”, moderate or more quantity amount for “2 points”, severe or plenty amount for “3 points”, very severe or large amount for “4 points”, and no obvious lesions for 0 point.

(111) 9.2 Experimental Results:

(112) The experimental result data was expressed in the form of mean±SD. One-way ANOVA combined with Post-Hoc (LSD method) was used to analyze the differences between groups. Statistical significance was expressed as a P value of less than 0.05. The model group was compared with the blank control group, #p<0.05, #p<0.01; each administration group of magnesium isoglycyrrhizinate was compared with the model group, *p<0.05, **p<0.01.

(113) 9.2.1 Effect of Magnesium Isoglycyrrhizinate Inhaled Preparation on Body Weight of COPD Rats

(114) The experimental results (Table 1) showed that after the successful modeling of COPD, the weight gain of the rats in each administration group was slow compared with that in the blank control group, and there existed a significant difference when compared with that in the blank control group (p<0.01). After 16 days of administration, compared with the model group, the weight of the rats in the high dose tracheal instillation group, low dose tracheal instillation group and the aerosolizing inhalation group of magnesium isoglycyrrhizinate increased slowly, wherein the weight gain of the aerosolizing inhalation group was more, and each group still differed from that in the blank control group (p<0.01).

(115) TABLE-US-00026 TABLE 1 effect of magnesium isoglycyrrhizinate on body weight of COPD rats Number of Body weight Group Dose animals Initial weight End weight Blank control group 0.9% NS 5 395.40 ± 16.50  460.60 ± 22.68  Model group 0.9% NS 10 316.30 ± 20.06.sup.##  343.00 ± 22.93.sup.##  High dose tracheal 1.67 mg/ml, 10  324.6 ± 12.22** 358.20 ± 16.17** instillation group 100 μl Low dose tracheal 1.67 mg/ml, 10 317.60 ± 24.23** 350.80 ± 27.79** instillation group 25 μl Aerosolizing 0.4 mg/ml, 9 319.22 ± 10.43** 360.11 ± 18.84** inhalation group 30 min

(116) 9.2.2 Effect of Magnesium Isoglycyrrhizinate Inhalation Preparation on General Symptoms of COPD Rats

(117) The experimental results showed that the rats in the blank control group had normal activities, sensitive reactions, body fat, and no symptoms such as cough, sneezing and dyspnea. After the model group was modeled, the rats tended to be prone, lack of spirit, stagnation, squint, bunching, unsteady walking, weight gain slowing, and grayish yellow fur, followed by symptoms such as cough, sneezing and dyspnea. After high dose and low dose tracheal infusion of magnesium isoglycyrrhizinate and nebulization, the symptoms of the rats in each group were relieved compared with the model group, wherein the magnesium isoglycyrrhizinate aerosolizing inhalation group was much more obvious than the other groups.

(118) 9.2.3 Effect of Magnesium Isoglycyrrhizinate Inhaled Preparation on the White Blood Cells Count and Cell Classified Comparison in COPD Rats

(119) The experimental results (Table 2) showed that the number of neutrophils and the percentage of neutrophils in the blood of rats in model group were increased to different degrees, and showed significant difference (p<0.05 or p<0.01). The above suggested that COPD rats had an inflammatory response mainly characterized by neutrophil infiltration after modeling. From the pharmacological point of view: high dose tracheal infusion group, low dose tracheal infusion group and aerosolizing inhalation group can significantly inhibit inflammatory factors mainly of neutrophils and percentage thereof in the blood of COPD rats, and the effect was obvious (p<0.05 or p<0.01). The effect of magnesium isoglycyrrhizinate in the aerosolizing inhalation group was the best (0.4 mg/ml, 30 min). The drug efficacy comparison was: magnesium isoglycyrrhizinate aerosolizing inhalation group>high dose tracheal instillation group>low dose tracheal instillation group.

(120) TABLE-US-00027 TABLE 2 Effect of magnesium isoglycyrrhizinate on the white blood cell count and cell classified comparison in blood of COPD rats (mean ± SD ) Number White blood Neutrophil Percentage of cell count count of neutrophils Group Dose animals (10/μl) (10/μl) (%) Blank control — 5 1399.00 ± 167.15 130.33 ± 14.01  9.40 ± 1.97 group Model group — 10 1277.50 ± 141.21 208.50 ± 44.83.sup.#  16.39 ± 3.63.sup.#  High dose 1.67 mg/ml, 10 1395.13 ± 270.32 153.25 ± 40.12* 11.31 ± 3.55* tracheal instillation 100 μl group Low dose tracheal 1.67 mg/ml, 10 1135.13 ± 155.73 155.13 ± 26.25* 13.80 ± 2.24* instillation group 25 μl Aerosolizing 0.4 mg/ml, 9 1123.33 ± 170.54  123.67 ± 23.24**  11.05 ± 1.61** inhalation group 30 min

(121) 9.2.4 Effect of Magnesium Isoglycyrrhizinate Inhaled Preparation on the White Blood Cell Count and Cell Classified Comparison in BALF of COPD Rats

(122) The experimental results (Table 3) showed that the numbers of white blood cells and neutrophils, as well as percentage of neutrophils in the BALF of rats in the model group were increased to different degrees (p<0.05 or p<0.01). The above suggested that the lungs of COPD rats had an inflammatory response mainly characterized by neutrophil infiltration. From the drug efficacy point of view: the magnesium isoglycyrrhizinate aerosolizing inhalation group can reduce the number of neutrophils and the percentage thereof.

(123) TABLE-US-00028 TABLE 3 Effect of magnesium isoglycyrrhizinate on the white blood cell count and cell classified comparison in BALF of COPD rats (mean ± SD ) White blood Neutrophil Percentage Number of cell count count of neutrophils Group Dose animals (10/μl) (10/μl) (%) Blank control — 5 55.67 ± 9.87 21.67 ± 5.69  38.57 ± 3.59  group Model group — 10 .sup. 155.38 ± 31.99.sup.## .sup. 62.38 ± 27.62.sup.# 39.18 ± 12.36 High dose 1.67 mg/ml, 10 184.38 ± 32.15  89.88 ± 21.57* 48.89 ± 9.69  tracheal 100 μl instillation group Low dose 1.67 mg/ml, 10 172.50 ± 26.52 66.38 ± 25.46 38.08 ± 12.86 tracheal 25 μl instillation group Aerosolizing 0.4 mg/ml, 9 170.33 ± 55.17 57.83 ± 20.78 38.83 ± 21.02 inhalation group 30 min

(124) 9.2.5 Effect of Magnesium Isoglycyrrhizinate Inhaled Preparation on Pathology of COPD Rats

(125) The experimental results (Table 4) showed that the lung tissue of rats in the blank control group consisted of alveolus, intrapulmonary bronchial branches and interstitial tissues with clear structure, no emphysema, minimal infiltration of inflammatory cells and goblet cell hyperplasia. The main lesions in the lung tissue of rats in the model group were interstitial pneumonia, edema around the perivascular tissue, with infiltration of inflammatory cells, increased goblet cells in the bronchial wall of the lung, degeneration and necrosis of bronchial wall cells, and a small amount of exudate in the bronchial lumen, wherein emphysema and inflammatory cell infiltration were particularly evident (p<0.01 or p<0.05). High dose tracheal infusion group, low dose tracheal infusion group and magnesium isoglycyrrhizinate aerosolizing inhalation group can significantly reduce lung inflammation, improve bronchial wall function, reduce bronchial mucus secretion and improve emphysema. Among them, magnesium isoglycyrrhizinate aerosolizing inhalation group (0.4 mg/ml, 30 min) was the most obvious to reduce inflammation and infiltration.

(126) TABLE-US-00029 TABLE 4 Effect of magnesium isoglycyrrhizinate on pathology of COPD rats Pathological score (mean ± SD) Number of Comprehensive Inflammatory cell Bronchial mucus Group Dose animals score Emphysema infiltration secretion Blank control group — 5 0.50 ± 0.60  0.000 ± 0.000 0.200 ± 0.274  0.100 ± 0.224 Model group — 10  2.20 ± 0.80.sup.## .sup. 0.450 ± 0.369.sup.# 1.100 ± 0.615** 0.550 ± 0.438 High dose tracheal 1.67 mg/ml, 10 0.90 ± 0.52* 0.300 ± 0.422 0.450 ± 0.550*   0.050 ± 0.158** instillation group 100 μl Low dose tracheal 1.67 mg/ml, 10 0.80 ± 0.50* 0.150 ± 0.337 0.400 ± 0.394** 0.200 ± 0.350 instillation group 25 μl Aerosolizing 0.4 mg/ml, 9 0.89 ± 0.65* 0.222 ± 0.441 0.389 ± 0.333** 0.278 ± 0.363 inhalation group 30 min

Example 10

Pharmacodynamic Experiment of Magnesium Isoglycyrrhizinate Inhaled Preparation for Mice with Chronic Obstructive Pulmonary Disease

(127) 10.1 Experimental Method:

(128) 90 ICR male mice, weighing 18-22 g, were divided into 9 groups, including blank control group, model group, low dose group (0.5 mg/ml, q.d.), medium dose group (1.5 mg/ml, q.d.), high dose group (5.0 mg/ml, q.d.), low dose group (0.5 mg/ml, b.i.d.), medium dose group (1.5 mg/ml, b.i.d.), high dose group (5.0 mg/ml, b.i.d.) of magnesium isoglycyrrhizinate inhaled preparation and positive control (Arformoterol) group. After the anesthesia of the mice, the trachea thereof was instilled with 30 μl the lipopolysaccharide (LPS) to model. After modeling for 30 minutes, 10 ml of the drug was administered by nebulizing inhalation device, and the nebulizing time was 30 min. 6 hours after LPS modeling, low dose group (0.5 mg/ml, b.i.d.), medium dose group (1.5 mg/ml, b.i.d.), high dose group (5.0 mg/ml, b.i.d.) and positive control group were continually administrated with 10 ml of drugs by nebulizing inhalation device. 24 hours after modeling, the mice were anesthetized and lung tissues thereof were lavaged. The bronchoalveolar lavage fluid (BALF) was taken to measure the number of inflammatory cells. Some lung tissues were taken for HE staining and the pathological section was used to detect the changes of inflammatory cells.

(129) TABLE-US-00030 TABLE 5-1 Effects of lipopolysaccharide on inflammatory cells (WBC) in the bronchial airways WBC Upward ratio Group (109/L ) ( % ) Blank control group 0.1350 ± 0.0778 — Model group 0.7971 ± 0.1931 490.48

(130) TABLE-US-00031 TABLE 5-2 Effect of magnesium isoglycyrrhizinate inhaled preparation on inflammatory cells (WBC) in bronchial airways WBC Downward ratio Group (109/L ) ( % ) Model group 0.7971 ± 0.1931 — Positive control group b.i.d. 0.5310 ± 0.1424 33.39 Low dose group b.i.d. 0.5644 ± 0.1599 29.19 Medium dose group b.i.d. 0.5350 ± 0.1277 32.89 High dose group b.i.d. 0.6710 ± 0.1723 15.82 Low dose group q.d. 0.6511 ± 0.2319 18.32 Medium dose group q.d. 0.6622 ± 0.1895 16.93 High dose group q.d. 0.6730 ± 0.1665 15.57

(131) 10.2 Experimental Results:

(132) The experimental results (Table 5-1) showed that a large number of inflammatory cells (white blood cells, WBC) were produced in the bronchial airway of the mice in the lipopolysaccharide-induced model group, which was up-regulated 490% compared with that in the blank control group, showing the successful induction of COPD. The histopathological examination of the lung tissue structure of the mice indicated that the model group showed obvious inflammatory reaction, the inflammatory cell infiltration was obvious, the exuded inflammatory cells were found in the alveolar and connective tissues of the alveolar septum. In particular, the neutrophils were arrested and aggregated obviously around the pulmonary blood vessels and all level of the bronchial tubes.

(133) The experimental results (Table 5-2) showed that in the model test of lipopolysaccharide-induced COPD mice, the number of white blood cells in each dose group of magnesium isoglycyrrhizinate inhaled preparation was significantly different from that of the model group. Among them, the therapeutic effects of the low dose group (down-regulated WBC of 29%) and the medium dose group (down-regulated WBC of 33%) of the magnesium isoglycyrrhizinate inhaled preparation administrating twice a day were comparable to that of the positive control group (down-regulated WBC of 33%), and the therapeutic effect of high dose group was slightly weaker than those of low dose group and medium dose group. The effect of magnesium isoglycyrrhizinate administrated twice a day was better than once a day at the same dose. In addition, histopathological examination showed that no obvious inflammatory reaction was observed in each dose group of magnesium isoglycyrrhizinate inhaled preparation.

Example 11

Effect of Magnesium Isoglycyrrhizinate Inhaled Preparation on Airway Secretion

(134) 11.1 Experimental Method

(135) 50 male ICR mice, weighing about 21 g, were randomly divided into 5 groups: control group 1 (normal saline, NS), control group 2 (normal saline, NS), positive control group (ammonium chloride, 1 g/kg), drug group 1 (0.2 mg/ml of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers, nebulizing for 30 min), drug group 2 (0.2 mg/ml of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers, nebulizing for 15 min). The mice in control group 1 and the positive control group were intragastrically administered with the corresponding drugs (0.1 ml/10 g), the mice in control group 2 and the drug group 1 were aerosolizing administered with a nebulizer for 30 min, while the mice in drug group 2 were 15 min. The administration frequency was once a day for 6 consecutive days. Before the 6th administration, the mice were starved for 16-18 hours, only allowed for drinking water. After 30 minutes of administration, i.p. 1% phenol red physiological in saline solution 0.2 ml/10 g was injected and 30 minutes later, the mice were killed by cervical dislocation. After waiting for a while until the blood in the mice coagulated, the neck skin thereof was cut without obvious bleeding (to avoid the phenol red in the blood mixed into the lavage), the trachea was separated, intubated (6 # syringe needle with a smooth tip was inserted into trachea for about 3 mm from laryngeal, fixed by silk thread ligation) and connected with the syringe, and then 0.6 ml of 5% NaHCO.sub.3 was slowly injected into the trachea, then gently sucked out, repeated 3 times, and 3 portions of lavage fluid were combined and centrifuged at 4000 rpm for 5 min, to obtain the supernatant.

(136) 11.2 Detection Indicators and Calculation Methods

(137) Production of Phenol Red Standard Curve:

(138) 1.95 mg of phenol red was weighted and dissolved by adding 5% NaHCO.sub.3 to 3.9 ml, and the obtained solution containing 0.5 mg/ml of phenol red was used as a stock solution. 0.1 ml of the stock solution was taken and added with 3.9 ml of 5% NaHCO.sub.3 to achieve the concentration of 12.5 μg/ml, which was sequentially diluted to 10 μg/ml, 7.5 μg/ml, 5 μg/ml, 2.5 μg/ml, 1.25 μg/ml, and 0.625 μg/ml. The phenol red standard curve was produced by colorimetric measuring the optical density (OD) value at the wavelength of 546 nm with an enzyme-labeling instrument.

(139) The sample OD value at a wavelength of 546 nm was measured by an enzyme-labeling instrument, and the phenol red amount in sample was calculated from the phenol red standard curve.

(140) 11.3 Experimental Results

(141) The experimental result data was expressed in the form of X±S, and the statistical significance was expressed with a P value of less than 0.05. Among them, each drug group was compared with the control group 2, .sup.ΔΔp<0.01. The experimental results were shown in Table 6. The phenol red excretion amount of drug group 2 (0.2 mg/ml of magnesium isoglycyrrhizinate liquid preparation for use in nebulizers, nebulizing for 15 min) was significantly increased compared with control group 2.

(142) TABLE-US-00032 TABLE 6 Effect of magnesium isoglycyrrhizinate inhaled preparation on tracheal secretion in mice X ± S) Number Amount of phenol Method of of red excretion Group dose administration animals (μg/ml) Control group 1 — i.g. 6 d 9 0.3962 ± 0.1715 (NS) Control group 2 — aerosolizing 10 0.3723 ± 0.1801 (NS) inhalation 30 min*6 d Positive control 1.0 g/kg/d*6 d i.g. 6 d 9 0.5924 ± 0.3781 group (ammonium chloride) Drug group 1 About aerosolizing 7 0.5381 ± 0.2307 6 mg/kg/d*6 d inhalation 30 min*6 d Drug group 2 About aerosolizing 10  .sup. 0.6926 ± 0.1792.sup.ΔΔ 6 mg/kg/d*6 d inhalation 15 min*6 d