Effects of Inhalable Microparticles of  on Chronic Obstructive Pulmonary Disease in a Mouse Model

Eung-Seok Lee; Jong-min Han; Min-Hee Kim; Uk Namgung; Yoon Yeo; Yang-Chun Park; Lee, Eung-Seok; Han, Jong-min; Kim, Min-Hee; Namgung, Uk; Yeo, Yoon; Park, Yang-Chun

doi:10.13048/jkm.13012

JKM > Volume 34(3); 2013 > Article

Lee, Han, Kim, Namgung, Yeo, and Park: Effects of Inhalable Microparticles of Socheongryong-tang on Chronic Obstructive Pulmonary Disease in a Mouse Model

Original Article

Journal of Korean Medicine 2013; 34(3): 54-68.

Published online: September 30, 2013

DOI: https://doi.org/10.13048/jkm.13012

Effects of Inhalable Microparticles of Socheongryong-tang on Chronic Obstructive Pulmonary Disease in a Mouse Model

Eung-Seok Lee¹, Jong-min Han¹, Min-Hee Kim², Uk Namgung², Yoon Yeo⁴, Yang-Chun Park¹

¹Division of Respiratory System, Dept. of Internal Medicine, College of Oriental Medicine, Daejeon University

²Dept. of Neurophysiology, College of Oriental Medicine, Daejeon University

³Institute of Traditional Medicine and Bioscience, Daejeon University

⁴College of Pharmacy, Purdue University

Correspondence to: 박양춘 (Yang-Chun Park), 대전광역시 중구 대흥동 22-5 대전대학교대전한방병원 한방내과, Tel : +82-42-229-6919, Fax : +82-42-254-3403, E-mail : omdpyc@dju.kr

Received April 23, 2013 Revised May 21, 2013 Accepted May 21, 2013

Abstract

Objectives:

This study aimed to evaluate the effects of microparticles of Socheongryong-tang (SCRT) on chronic obstructive pulmonary disease (COPD) in a mouse model.

Methods:

The inhalable microparticles containing SCRT were produced by spray-drying with leucine as an excipient, and evaluated with respect to the aerodynamic properties of the powder by Andersen cascade impactor (ACI). Its equivalence to SCRT extract was evaluated using lipopolysaccharide (LPS) and a cigarette-smoking (CS)-induced murine COPD model.

Results:

SCRT microparticles provided desirable aerodynamic properties (fine particle fraction of 49.6±5.5% and mass median aerodynamic diameter of 4.8±0.3 μm). SCRT microparticles did not show mortality or clinical signs over 14 days. Also there were no significant differences in body weight, organ weights or serum chemical parameters between SCRT microparticle-treated and non-treated groups. After 14 days the platelet count significantly increased compared with the non-treated group, but the values were within the normal range. Inhalation of SCRT microparticles decreased the rate of neutrophils in blood, granulocytes in peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid (BALF) and level of TNF-α and IL-6 in BALF on COPD mouse model induced by LPS plus CS. This effect was verified by histological findings including immunofluorescence staining of elastin, collagen, and caspase 3 protein in lung tissue.

Conclusions:

These data demonstrate that SCRT microparticles are equivalent to SCRT extract in pharmaceutical properties for COPD. This study suggests that SCRT microparticles would be a potential agent of inhalation therapy for the treatment of COPD.

Keywords: Socheongryong-tang, inhalable microparticles, chronic obstructive pulmonary disease, spray drying, cigarette smoking

Fig. 1.

Experimental plan of repeated LPS+CS exposure.

Fig. 2.

Effect of SCRT-MP on neutrophil % of blood in COPD mice. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle (50, 100 mg/kg) for 21 days (n=4).

*: p<0.05 †: p<0.01 compared to COPD-CT by T-test.

‡: p<0.05 compared to Intact by T-test.

Fig. 3.

Effect of SCRT-MP on CD11b/Gr-1 cell rate of PBMC (A) and BALF (B) in COPD mice. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle (50, 100 mg/kg) for 21 days.

Fig. 4.

Effects of SCRT-MP on TNF-α and IL-6 production in COPD mice. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle(50, 100 mg/kg) for 21 days (n=4). (A) TNF-α, (B) IL-6.

*: p<0.05 †: p<0.01 ‡: p<0.001 compared to COPD_CT by T-test.

∮ : p<0.001 compared to Intact by T-test.

Fig. 5.

Histological analysis(A) of lung tissues. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle (50, 100 mg/kg) for 21 days. Lung tissues were examined under microscope (200 × magnification) after H&E staining. Asterisks denote alveoli. Immunofluorescence analysis(B) for caspase 3, elastin, collagen in lung tissue. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle(50, 100 mg/kg) for 21 days. Lung tissues were examined under microscope (200 × magnification) after immunofluorescence staining against caspase 3, elastin, collagen. Asterisks denote alveoli.

Table 1.

The Composition of Socheongryong-tang (SCRT)

Herbal name	Pharmacognostic name	Lot No.	Country of origin	Amount (g)
Mahwang	Ephedrae Herba	M011005	China	6.0
Baekjakyak	Paeoniae Radix	J031008	Gyeongbuk, Korea	6.0
Omija	Schizandrae Fructus	A151008	Gyeongbuk, Korea	6.0
Banha	Pinelliae Tuber	B031003	China	6.0
Sesin	Asiasari Radix et Rhizoma	S381004	China	4.0
Geongang	Zingiberis Rhizoma	K021008	Chungnam, Korea	4.0
Gyeji	Cinnamomi Cortex	K031006	Vietnam	4.0
Gamcho	Glycyrrhizae Radix et Rhizoma	K370910	China	4.0
Total amount				40.0

Table 2.

Aerodynamic Properties of Microparticle of SCRT

ED^* (%)	FPF^† of CD^‡ (%)	FPF of ED (%)	FPF of ND^∮ (%)	MMAD‖ (μm)
89.0±18.0	49.6±5.5	27.8±6.5	25.4±9.7	4.8±0.3

* ED: Emitted dose,

† FPF: fine particle fraction,

‡ CD: collection dose,

∮ ND: nominal dose (11.1±1.7 mg),

‖ MMAD: mass median aerodynamic diameter.

Table 3.

Body Weight Changes of Male Mice on 14 Day Single Dose Toxicity Study of SCRT Microparticle

	Body weight (g)
	Intact^*	Mock^†	SCRT-MP^‡
0 day	22.54±0.27	21.69±0.28	22.14±0.20
5 day	23.29±0.46	22.00±0.31	22.57±0.30
10 day	24.43±0.37	23.36±0.39	23.64±0.34
14 day	24.58±0.49	23.64±0.53	24.00±0.61

Values are expressed as mean±S.D. (n=7).

* Intact: negative, not inhaled;

† Mock: inhaled air;

‡ SCRT-MP: inhaled SCRT microparticle (240 mg/kg).

Table 4.

Organ Weight of Male Mice Inhaled SCRT Microparticle on 14 Day Single Dose Toxicity Study

	Intact^*	Mock^†	SCRT-MP^‡
Liver (g)	1.35±0.06	1.26±0.11	1.12±0.06
Kidney (g)	0.35±0.02	0.32±0.01	0.31±0.01

Values are expressed as mean±S.D. (n=7). Intact: negative, not inhaled;

† Mock: inhaled air;

‡ SCRT-MP: inhaled SCRT microparticle (240 mg/kg).

Table 5.

Hematological Values of Male Mice Inhaled SCRT Microparticle on 1 and 14 Day in Single Dose Toxicity Study

	Intact^*		Mock^†		SCRT-MP^‡
	1 Day	14 Day	1 Day	14 Day	1 Day	14 Day
WBC (10³/mm³)	3.93±2.23	5.99±2.14	4.06±2.33	4.55±1.55	6.02±1.03	6.49±1.84
RBC (10⁶/mm³)	7.96±1.52	9.02±0.84	8.75±0.06	8.03±2.46	7.19±4.20	9.02±1.30
HGB (g/dl)	9.83±2.11	10.80±0.93	10.47±0.86	9.66±3.11	8.40±5.29	10.28±2.46
HCT (%)	41.50±7.62	45.49±4.11	44.23±0.68	40.52±12.39	35.37±21.10	44.28±7.44
PLT (10³/mm³)	467.00±139.86	598.14±83.23	524.00±128.64	604.20±204.95	411.67±285.63	1,015.20±142.25^*

Values are expressed as mean±S.D. (n=7).

* Intact: negative, not inhaled;

† Mock: inhaled air;

‡ SCRT-MP: inhaled SCRT microparticle (240 mg/kg).

*: p<0.01 compared to Intact by T-test.

Table 6.

Clinical Chemistry Values of Male Mice Inhaled SCRT Microparticle on 1 Day Single Dose Toxicity Study

	Intact^*		Mock^†		SCRT-MP^‡
	1 Day	14 Day	1 Day	14 Day	1 Day	14 Day
GOT (IU/L)	10.00±3.43	19.29±6.66	7.57±1.78	14.86±2.48	11.29±2.78	10.00±5.08
GPT (IU/L)	0.33±0.56	8.86±3.93	0.00±0.00	2.00±0.38	0.14±0.14	1.17±0.48
ALP (IU/L)	9.17±1.05	7.29±1.29	10.29±1.04	6.71±0.89	8.57±1.15	6.17±0.60
Creatinine (IU/L)	0.05±0.02	0.00±0.00	0.04±0.02	0.00±0.00	0.04±0.02	0.00±0.00

Values are expressed as mean±S.D. (n=7).

* Intact: negative, not inhaled;

† Mock: inhaled air;

‡ SCRT-MP: inhaled SCRT microparticle (240 mg/kg).

References

1.. Statistics Korea. Cause of death statistics 2011. 2012. [2screens]. Available at: URL:http://kosis.kr/ups/ups_01List01.jsp?pubcode=YD. Accessed Sep 20, 2012.

2.. Mannino DM, Kiriz VA. Changing the burden of COPD mortality. Int J Chron Obstruct Pulmon Dis. 2006; 1:3. 219–33.

3.. Mannino DM. COPD: epidemiology, prevalence, morbidity and mortality, and disease heterogeneity. Chest. 2002; 121:121S–26S.

4.. Celli BR, MacNee W. Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J. 2004; 23:6. 932–46.

5.. Barnes PJ, Hansel TT. Prospects for new drugs for chronic obstructive pulmonary disease. Lancet. 2004; 364:9438. 985–96.

6.. Jung SK, Jung HJ, Kim JD, Choi HY, Park MY, Park YC, et al. Pye-gye-nae-gwa-hak. Seoul: Nado;2011. p. 510–1.

7.. Lee H, Kim Y, Kim HJ, Park S, Jang YP, Jung S, et al. Herbal Formula, PM014, Attenuates Lung Inflammation in a Murine Model of Chronic Obstructive Pulmonary Disease. Evid Based Complement Alternat Med. 2012; 2012:769830Epub 2012 Jun 12.

8.. Lee JG, Yang SY, Kim MH, Namgung U, Park YC. Protective effects of Socheongryong-tang on Elastase-Induced Lung Injury. J Korean Oriental Med. 2011; 32:4. 83–99.

9.. Yoon JM, Park YC. Protective effects of Seonpyejeongcheon-tang on Elastase-induced Lung Injury. J Korean Oriental Med. 2010; 31:1. 84–101.

10.. Choi HJ, Bang NY, Song BW, Kim NJ, Rhyu BH. Survey on the preference for the dosage forms of oriental herbal medicine. Kyunghee Medicine. 2004; 20:1. 46–57.

11.. Derendorf H, Nave R, Drollmann A, Cerasoli F, Wurst W. Relevance of pharmacokinetics and pharmacodynamics of inhaled corticosteroids to asthma. Eur Respir J. 2006; 28:5. 1042–50.

12.. Courrier HM, Butz N, Vandamme TF. Pulmonary drug delivery systems: recent developments and prospects. Crit Rev Ther Drug Carrier Syst. 2002; 19:4–5. 425–98.

13.. Zhang J. Shang-han-za-bing-lun. Shijiazhuang. Hebei Kezue Jishu Chubanshe. 1994; 27

14.. Jung S, Cho SJ, Moon KI, Kim HW, Kim BY, Cho SI. Effects of Socheongryong-Tang on Immunoglobulin Production in Asthmatic Mice. Kor. J. Herbology. 2008; 23:1. 23–8.

15.. Kim KY, Lee JH, Kim YJ, Choi SY, Kim TH, Lyu YS, et al. Anti-allergic effects of Socheongyoung-tang on RBL-2H3 mast cell and mice-mediated allergy model. Korean J. Oriental Physiology & Pathology. 2007; 21:5. 1260–70.

16.. Hwang WS, Lee JS, Choi JY, Jung HJ, Rhee HK, Jung SK. Two Cases of Chronic Sinusitis with Asthma Improved by Socheongryong-tang. J Korean Oriental Med. 2003; 24:1. 207–12.

17.. Jung SK, Heo TS, Hwang WS, Ju CY, Kim YW, Jung HJ. The Effects of Sochongryong-tang on Serum IL-4, IL-5, and IFN-ɣ in asthmatic Patients. J Korean Oriental Med. 2002; 23:2. 70–7.

18.. Ibrahim BM, Jun SW, Lee MY, Kang SH, Yeo Y. Development of inhalable dry powder formulation of basic fibroblast growth factor. Int J Pharm. 2010; 385:1–2. 66–72.

19.. Thiel CG. Cascade impactor data and the log-normal distribution: nonlinear regression for a better fit. J Aerosol Med. 2002; 15:4. 369–78.

20.. Nemzek JA, Bolgos GL, Williams BA, Remick DG. Differences in normal values for murine white blood cell counts and other hematological parameters based on sampling site. Inflamm Res. 2001; 50:10. 523–7.

21.. Hillery AM, Lloyd AW, Swarbrick J. Drug delivery and targeting; for pharmacists and pharmaceutical scientists. London: Taylor and Francis;2001. p. 2

22.. Tayab ZR, Hochhaus G. Pharmacokinetic/pharmacodynamic evaluation of inhalation drugs: application to targeted pulmonary delivery systems. Expert Opin Drug Deliv. 2005; 2:3. 519–32.

23.. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P, et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med. 2007; 176:6. 532–55.

24.. Geller DE. Comparing clinical features of the nebulizer, metered-dose inhaler, and dry powder inhaler. Respir Care. 2005; 50:10. 1313–21.

25.. Borgstrom L. On the use of dry powder inhalers in situations perceived as constrained. J Aerosol Med. 2001; 14:3. 281–7.

26.. Yang Y, Tsifansky MD, Shin S, Lin Q, Yeo Y. Mannitol-guided delivery of Ciprofloxacin in artificial cystic fibrosis mucus model. Biotechnol Bioeng. 2011; 108:6. 1441–9.

27.. Yang Y, Tsifansky MD, Wu CJ, Yang HI, Schmidt G, Yeo Y. Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis. Pharm Res. 2010; 27:1. 151–60.

28.. Lipworth BJ. Targets for inhaled treatment. Respir Med. 2000; 94:S13–6.

29.. Azarmi S, Lobenberg R, Roa WH, Tai S, Finlay WH. Formulation and in vivo evaluation of effervescent inhalable carrier particles for pulmonary delivery of nanoparticles. Drug Dev Ind Pharm. 2008; 34:9. 943–7.

30.. Molfino NA, Jeffery PK. Chronic obstructive pulmonary disease: Histopathology, inflammation and potential therapies. Pulm Pharmacol Ther. 2007; 20:5. 462–72.

31.. Yoo CG. Pathogenesis and pathophysiology of COPD. The Korean Journal of Medicine. 2009; 77:383–400.

32.. Niewoehner DE, Kleinerman J, Rice DB. Pathologic changes in the peripheral airways of young cigarette smokers. N Engl J Med. 1974; 291:15. 755–8.

33.. Schaberg T, Haller H, Rau M, Kaiser D, Fassbender M, Lode H. Superoxide anion release induced by platelet-activating factor is increased in human alveolar macrophages from smokers. Eur Respir J. 1992; 5:4. 387–93.

34.. Wright JL, Cosio M, Churg A. Animal models of chronic obstructive pulmonary disease. Am J Physiol Lung Cell Mol Physiol. 2008; 295:1. L1–15.

35.. Wright JL, Sun JP. Effect of smoking cessation on pulmonary and cardiovascular function and structure: analysis of guinea pig model. J Appl Physiol. 1994; 76:5. 2163–8.

36.. Yang IA, Clarke MS, Sim EH, Fong KM. Inhaled corticosteroids for stable chronic obstructive pulmonary disease. Cochrane Database Syst Rev. 2012; 7:CD002991

37.. Tanino M, Betsuyaku T, Takeyabu K, Tanino Y, Yamaguchi E, Miyamoto K, et al. Increased levels of interleukin-8 in BAL fluid from smokers susceptible to pulmonary emphysema. Thorax. 2002; 57:5. 405–11.

38.. Keatings VM, Collins PD, Scott DM, Barnes PJ. Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med. 1996; 153:2. 530–4.

39.. Demedts IK, Demoor T, Bracke KR, Joos GF, Brusselle GG. Role of apoptosis in the pathogenesis of COPD and pulmonary emphysema. Respir Res. 2006; 7:53

40.. Foronjy R, D’Armiento J. The role of collagenase in emphysema. Respir Res. 2001; 2:6. 348–52.