COPD M 3 M 2 ( 2 COPD 45 3 M 3 COPD ( 1 M 2 M 1 M 3 ( M 3 M 3 2 in vitro in vivo ( 3 COPD 1.1 ( (EFS In vivo ( ( 1 1. ( (( ( MCh pa (in vitro 2 =9.51

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1 . 1. M 1 M ( M 1 M 2 M 3 3 M 4 M 5 2 M 1 M 2 M 3 2 ( 1 M 1 M 2 M 3 1 GOLD(Global initiative for chronic Obstructive Lung Disease (Chronic Obstructive Pulmonary DiseaseCOPD 1 3 ( 1 Malcolm P Caulfield, Nigel J. M. Birdsall, International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors, Pharmacol Rev, 5 (2, , Peter J Barnes, Airway muscarinic receptors: Anticholinergic agents in the upper and lower airways, Lung Biology in Health and Disease edited by SL Spector, Mercel Dekker Inc. New York, Basel, Global Initiative for Chronic Obstructive Lung Disease (GOLD guidelines, NHLI/WHO workshop report, Executive summary, 21 1

2 COPD M 3 M 2 ( 2 COPD 45 3 M 3 COPD ( 1 M 2 M 1 M 3 ( M 3 M 3 2 in vitro in vivo ( 3 COPD 1.1 ( (EFS In vivo ( ( 1 1. ( (( ( MCh pa (in vitro 2 =9.51 (62 pa 2 =9.37 (58 pa 2 =9.17 (67 1 EFS IC 5 =.17 nm (5 (in vitro IC 5 =.58 nm (5 IC 5 =.74 nm (5 IC 5 =.24 nm (5 (in vitro IC 5 =5.5 nm (5 1 ACh.313 g/kg 13 g/kg ( (67 ( mg/ml 13, 1 mg/ml ( (51 (1 2.1,.3 1 mg/ml (.3,.9,.1,.3 1, 1 mg/ml (.3 g/kg (.3,.9,.3, 3. 1 (6 g/kg (6 MCh ACh 4 Mahler DA, Donohue JF, Barbee RA, Goldman MD, Gross NJ, Wisniewski ME, Yancey SW, Zakes BA, Rickard KA, Anderson WH, Efficacy of salmeterol xinafoate in the treatment of COPD, Chest, 115(4:957-65, Jones PW, Bosh TK, Quality of life changes in COPD patients treated with salmeterol, Am J Respir Crit Care Med, 155(4:1283-9,

3 1.2 in vivo ( 2 2. ( EFS t 1/2 (onset=34.8 min (5 t 1/2 (offset=54 min (5 (in vitro (( t 1/2 (onset=7.6 min (5 t 1/2 (offset=81.2 min (5 t 1/2 (onset=3.8 min(5 1 t 1/2 (offset=31.6 min(5 t 1/2 (onset=43.5 min (5 t t 1/2 (offset>3 min (5 1/2 (onset=6.8 min(5 1 (in vitro t 1/2 (offset=64. min(5 t 1/2 (onset=74.4 min (5 t 1/2 (onset=24. min (6 t 1/2 (offset>15 hr (5 t 1/2 (offset=83.3 min (6 (in vitro 2 ACh.31 mg/ml (.9,.3 1 mg/ml (3. g/kg g/kg 6 3 ( (6 ( mg/ml mg/ml 12 ( (51 ( mg/ml (2 g/kg 1 mg/ml (2. g/kg ( 24 (1 (1 t 1/2 (onsetefs ( 5 t 1/2 (offset EFS ( 5 ACh ( (( ACh ( (4 4 ( (4 ACh (115 (6 5 (1114 (6 3

4 ACh 2.5 g (.9 g/kg 2 g (7.2 g/kg 6 ( ( (5 ( 2 3 g (.12 g/kg ( 2 mg/kgi.v. 1 7 ( mg(.4 mg/kg (5 ( 2.5g (.1 g/kg 8 ( +1 mg/kg, i.v. (5 His His 1g/kg His 63g/kg, i.v. (6 9 1g/kg His ( 3g/kg, i.v. (6 ACh His 1.5 ( M 3 1 in vivo M 2 M 1 M 3 ( M 3 M 3 ( 2 ( 1 M 1 M 2 M 3 M 1 M 2 M 3 M 1 M 2 M 3 4

5 5. ( (( Hm1.41 (4 Hm1.183 (3 Hm11.5 (3 (CHO in vitrohm2.21 (3 Hm2.195 (3 Hm21. (3 1 K D (nm Hm3.14 (5 Hm3.24 (3 Hm31.1 (3 11 ( Hm4.5 (1 Hm4.89 (3 Hm41.3 (3 K i Hm5.12 (2 Hm5.815 (3 Hm53.2 (3 Hm114.6 (5 Hm1.11 (3 (CHO in vitro t 1/2 (hr Hm23.6 (4 Hm334.7 (4 Hm43.7 (3 Hm579 (2 Hm2.35 (4 Hm3.258 (3 Hm4.98 (3 Hm5.23 (3 1 (CHO in vitro t 1/2 (hr Hm334.8 (3 Hm3.21 (3 Hm3.7 (3 12 K D.39 nm (4 [ 3 H]- B max 48 fmol/mg (4 t 1/2 212 min (3 3 K i.26 nm [ 3 H]-N- (36 K i.51 nm (36 ACh 1-9 M (EFS (in vitro ACh ( 2 ( M ACh 1 ( 2 (68 H 1 (K i =81 nm 13 ACh ACh ACh MAO MAO MAO His 3 g/kg (His 131, g/kg 14 (6 (BK BK (7 (LTC 4 LTC 4 (6 14 (LTD 4 LTD 4 (6 Hm: CHO, ACh His:, BK:, LT: (Ba 338 BR BIIH 27 SE (BIIH 27 NA (SCH 731 BRBIIH 28 XX 1, 15 5

6 (BINEB, MDI RBG1 BINEB(Boehringer Ingelheim Neblizar MDI(Metered Dose Inhalar ( (, (, RBG1 in vitro in vivo S.E. S.D. pa 2 K i K D IC 5 ID 5 EC 5 ED 5 p.o. i.v. s.c. CHO HEK EFS BINEB MDI APD HERG IC 5 Scatchard 5% 5% 5% 5% K1 (Electrical Field Stimulation Boehringer Ingelheim Neblizer Metered Dose Inhaler Action Potential Duration human ether-a-go-go related gene 6

7 b c 1.1 ( a Krebs-Henseleit 2 g pa ( 3 6 pa ( 6 4 (11 (6 3 ( (8 ( log[ ] 3. S.E ( DR: Drug Ratio ( ED 5 / ED 5 ( log (DR-1 * pa 2 Slope n 9.51 ( ( ( ( ( ( Schild plot ( 95% n *: Schild plot slope 1 apparent pa 2 7

8 1.1.2 (EFS 1 a Krebs-Henseleit 1 g( 2 g( EFS( 4 Hz,.5 msec duration4 V15 sec, 8 Hz,.5 msec duration4 V15 sec EFS ( 1-5 M 3 ( (- 1-6 M ( 1-5 M b EFS EFS 5% (IC.17 nm EFS IC 5.74 nm.58 nm ( 7 EFS IC 5.24 nm IC nm ( 7 c EFS ( 7 EFS IC 5 (nm IC N a (2 g/kg (.313 (1, 3 g/kg b.31 3 g/kg g/kg ( 4 c (7 1 (6 (6 75 (6 ( S.E.( ( 8

9 a 1% ( (.1.31 mg/ml (.3131 mg/ml1 b.1 mg/ml.3 1 mg/ml ( 5.3 mg/ml 1 mg/ml ( 5 c **: p.1 (unpaired t-test, S.E., n51( a Konzett-Röβler 2 ( mg/ml (.1,.3,.9,.3 g/kg ( mg/ml (.3,.9,.3, 3. g/kg b.1.3, 1 mg/ml (.3,.9,.3 g/kg 52, mg/ml (.3,.9,.3, 3. g/kg 28, 47, 58 1 ( 6 c 9

10 S.E.n=6( 1

11 a Krebs-Henseleit 1 g( 2 g( EFS( 4 Hz,.5 msec duration4 V15 sec, 8 Hz,.5 msec duration4 V15 sec EFS ( 1-5 M3 (- 1-6 M ( 1-5 M b (1-9 M (1-8 M (1-7 M ( 8 (1-9 M (1-7 M ( 8 7 c ( 8. EFS t 1/2 (onset(min t 1/2 (offset(min > t 1/2 (onsetefs ( 5% t 1/2 (offset EFS ( 5% --- S.E.n=5 A. B hr hr 7. EFS 1-9 M( 1-7 M( S.E.n=5 11

12 a Krebs-Henseleit 1 g EFS(4 Hz.5 msec duration3 V15 sec EFS ( 1-5 M3 (- 1-6 M ( 1-5 M N - -L- (NO 1-4 M b EFS t 1/2 onset( ( t 1/2 onset.4.2 (24.1. ( 8 15 EFS 8 t 1/2 offset( 5 15 (9 t 1/2 offset ( ( 8 c A. B. % hr hr 8. EFS 1-9 M( n=5 3 x 1-9 M( n=6, S.E. 12

13 a Konzett-Röβler 2 (.3,.1.31 mg/ml (.1,.3,.9,.3 g/kg ( mg/ml (.3,.9,.3, 3. g/kg1 b.3 mg/ml (.1 g/kg (.1 mg/ml (.3 g/kg 4.31 mg/ml (.9,.3 g/kg 6 ( 9 1 mg/ml (3. g/kg 3 ( 9 c mg/ml (hr 1 mg/ml S.E.n a 1 ( (.1.31 mg/ml ( mg/ml1 b.3 1 mg/ml 24 ( 1.1 mg/ml 1 mg/ml 9 c

14 mg/ml 2.1 mg/ml 16.3 mg/ml 12.3 mg/ml 1. mg/ml 8 1. mg/ml ( ( 1 mg/ml 3 n5, mg/ml 5 n=6 S.E.*: p<.5, **: p<.1 (unpaired t-test a 1 ( ( (1 mg/ml (2. g/kg (1 mg/ml (2 g/kg1 b 1 mg/ml (2. g/kg 24 ( 11 1 mg/ml (2 g/kg c ** ** ** ** ** ** ** 11. ( 1 mg/ml(2. g/kg ( 1 mg/ml(2 g/kg ( S.E.n1**: p.1 (unpaired t-test 14

15 a 1 BINEB 2.3 g/ (.2 g/kg rotating brush generation device(rbg (28 (12 g/kg Konzett-Röβler b % 24 ( ( 9 c ( S.D., n a ( mg/ml ( ( 3 b ( 1 c 1. EC 5 (mg/ml 5.34 (51.45 ( (6 EC 5 ( 5 EC 5 ( ( EC 5 (EC

16 a (BINEB 2.5 g/ (.9 g/kg (MDI 2 g/ (7.2 g/kg b (2.5 g/ (.9 g/kg (2 g/ (7.2 g/kg ( 12A ( (2.5 g/ (.9 g/kg ( 12C (2.5 g/ (.9 g/kg (2 g/ (7.2 g/kg ( (2 g/ (7.2 g/kg ( 12B 12C ( (2 g/ (7.2 g/kg ( 12C c A. TP Res C dyn C. 3 B (min (min HR -2 HR slvp dp/dt max BPs BPd 12. B C S.E.n5 TP: transpulmonary pressureres: C dyn : HR: slvp:, dp/dt max : BPs: BPd:, *: p.5**: p.1(unpaired t-test, ( 16

17 a (BINEB 3 g/ (.12 g/kg ( ( mg/kg + ( 1 MDI 1 mg/ (.4 mg/kg b (3 g/ (.12 g/kg (3 g/ (.12 g/kg ( 13A ( 13B ( ( 13C ( 13B (3 g/ (.12 g/kg c ( A. C. TP Res C dyn B. 1-1 HR slvp dp/dt max BPs BPd 13. B C S.E.n5(C n=3 TP: transpulmonary pressureres: C dyn : HR: slvp: dp/dt max : BPs: BPd: ( 17

18 a (BINEB 2.5 g/ (.1 g/kg (1 mg/kg i.v. (1 b (2.5 g/ (.1 g/kg ( 14A (2.5 g/ (.1 g/kg (1 mg/kgi.v. ( 14A14C (1 mg/kgi.v. (2.5 g/ (.1 g/kg ( 14B c A. B TP Res C TP Res dyn dvn C. HR slvp dp/dt max BPs BPd 14. B C S.E.n5 TP: transpulmonary pressureres: C dyn : HR: slvp: dp/dt max : BPs: BPd:. ( 18

19 in vitro a Konzett-Röβler ( ( g/kgi.v. 1 (63 g/kg (3 g/kg ( , 17, (3 g/kgi.v. 1 g/kgi.v.( 1 b ml/1g ( 15 c ml/1g A B A. B. S.D.n6 ( 19

20 a (Hm1Hm2Hm3Hm4Hm5 CHO [ 3 H]- [ 3 H]- (K D.2 nm [ 3 H]-N- (K i b ( 1112 c 11. (K D K D (nm Hm1 Hm2 Hm3 Hm4 Hm5.12 ( ( ( (5.5 (1 [ ] ( ( ( ( (3 S.D. ( [ ] 12. (K i K i (nm Hm1 Hm2 Hm3 Hm4 Hm ( ( ( ( (3 S.D.( 2

21 a (Hm1Hm2Hm3Hm4Hm5 CHO 2 1 [ 3 H]- (5 1-1 M2 [ 3 H]- (1-9 M1 (1-5 M [ 3 H]-N- 3 b Hm2 Hm1 Hm3 ( 13Hm3 1 ( 14 c M 2 M 1 M 3 ( M 3 M ( hr Hm1 Hm2 Hm3 Hm4 Hm5 [ 3 H] ( ( ( (3 79 [8573] (2 [ 3 H] ( ( ( ( (3 S.D.( [ ] 14. (Hm3 (hr S.D.n3 21

22 a [ 3 H]-.63 nm [ 3 H]- 2 1 M [ 3 H]-N- b [ 3 H]- 3 ( 16A 7.5 ( K D.39 nm( 15 1 M [ 3 H]- (.63 nm ( 15 16B[ 3 H]-N- ( 16 c A. B [ 3 H]- A ( triplicate B ( n=3 S.E. 15. [ 3 H]- K D (nm.39.1 (4 Bmax (fmol/mg 487 (4 (min (3 S.E.( 16. K i (nm S.E.n36 22

23 M 2 M 3 M 1 M 2 EFS a [ 3 H]- Krebs-Henseleit [ 3 H]- 1 ml/min EFS(.5 msec duration4 V4 Hz1 b (1-9 M (1-8 M EFS M 2 2 ( 17 c M 3 (in vitro M 3 M 2 3 A. B EFS 2 S.E.n68 (1-8 M (1-9 *: p.5 (Wilcoxon s rank-order test, 23

24 a ( ( ( ( ( (MAO-A-B ([ 3 H] (MAO-A[ 14 C] (MAO-B b H 1 (K i =81 nm ( 17 1, nm ( 18 (EC 5 =36, nm (1-8 M MAO-A MAO-B ( 18 c 17. IC 5 (nm 1 [ 3 H]- 25,7 (3 >1, (2 35,9 (4 [ 3 H]- >1, (2 >1, (1 >1, (1 1 [ 3 H]-DH- >1, (3 >1, (2 >1, (2 [ 3 H]-DH- >1, (3 >1, (2 >1, (2-1 [ 3 H]-SCH2339 >1, (2 >1, (2 >1, (2-2 [ 3 H]->1, (3 >1, (2 >1, (2-1A [ 3 H]-8-OH-DPAT >1, (2 >1, (2 >1, (2-2 [ 3 H]->1, (2 >1, (2 >1, (2 -H 1 [ 3 H]- 81 (4 >1, (3 >1, (2 -H 2 [ 3 H]- >1, (2 >1, (2 >1, (2 [ 3 H]- >1, (1 12,287 (2 >1, (1 [ 3 H]- >1, (3 >1, (3 >1, (3-1 [ 3 H]-DPCPX >1, (1 >1, (1 >1, (1 NMDA [ 3 H]-MK-81 >1, (1 >1, (1 >1, (1 M 1 [ 3 H]-.8 (4.65 (4.71 (5 M [ 3 H]-N- -.2 (3.42 (4.81 (4 M [ 3 H]-N- -.1 (3.5 (4 1.4 (5 Ki (nm( 8-OH-DPAT(-o- -2-(-n- DPCPX1, NMDAN- -D- 18. MAO IC 5 (nm >1, (2 >1, (2 >1, (2 >1, (2 >1, (2 >1, (2 >1, (2 >1, (2 >1, (2 >1, (2 >1, (2 >1, (2 MAO-A >1, (2 >1, (2 >1, (2 MAO-B >1, (2 >1, (3 >1, (3 ( 24

25 1.5.6 C 4 D 4 14 In vitro (ED 5.3 g/kgi.v. 1 1 in vivo a Konzett-Röβler ( ( g/kgi.v. 1 (3131, g/kg 1 ( (17g/kgi.v. (3 g/kg i.v.3 C 4 (3 g/kg i.v. D 4 (3 g/kg i.v. (1 g/kg i.v. b 3 g/kg 1 g/kg 13 1, g/kg ( 18in vivo (ED 5.3 g/kg, i.v g/kg C 4 D 4 ( 19 c 1 in vivo ml/1 g (ml/1 g n=4 n=6 (3 µg/kg, i.v ( 1. n=5 n= (3 µg/kg, i.v..25. ( (1 µg/kg, i.v. 1 1 (1 µg/kg, i.v S.D. ( 3g/kg 17g/kg n2 ( 25

26 (ml/1 g A. B. C. (ml/1 g min 3 6 min 19. C 4 D 4 A. C 4 ( (n=6 (n=7b. D 4 (n=6c. (n=6 S.D a (Hm1Hm2Hm3Hm4Hm5CHO.2 nm [ 3 H]-N- (Ba 338 BR BIIH 27 SE (BIIH 27 NA (SCH 731 BRBIIH 28 XX IC 5 b 1, ( 19 c 19. (CHO IC 5 (nm Hm1 Hm2 Hm3 Hm4 Hm5.23 (2.69 (2.36 (2.2 ( (2 Ba 338 BR >1, (2 >1, (2 42,56 (2 >1, (2 >1, (2 BIIH 27 NA >1, (2 >1, (2 43,851 (2 >1, (2 >1, (2 SCH 731 BR >1, (2 >1, (2 >1, (2 >1, (2 >1, (2 BIIH 28 XX >1, (2 >1, (2 33,228 (2 >1, (2 >1, (2 ( (ml/1 g min (3 µg/kg, i.v. 26

27 (COPD COPD Tc ( 3 ( -24 ( ( ( (irritant receptor (A-fiber ( 3 COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD guidelines, NHLI/WHO workshop report, Executive summary, 21 2 Pavia D. et al., Effect of Four-Week Treatment with Oxitropium Bromide on Lung Mucociliary Clearance in patients with Chronic Bronchitis or Asthma Respiration 55: : Phipps RJ et al., A physiologicalbiochemical and histological study of goose tracheal mucin and its secretion Phios Trans Royal Soc Lond (Biol. 9: Newman TM et al., Microanatomy of secretory granule release from guinea pig tracheal goblet cells Am J Respir Cell Mol Biol 15: Baker B et al., Nervous control of mucin secretion into human bronchi J Physiol 365: Ramnarine SI et al., On muscarinic control of neurogenic mucus secretion in ferret trachea J Physiol 15;494 ( Pt 2: Rogers DFMotor control of airway goblet cells and glands. 21 Respir Physiol 125: :

28 COPD 28

29 ( mg/kgs.c. foot hold 1 mg/kg s.c. 1 mg/kg s.c. 1 mg/kgs.c..1 mg/kgi.v. 1, mg/kgi.v. HERG mg/kgp.o. 1 mg/kgs.c. (ED 5 =.32 mg/kgs.c nm 11, nm 11, nm 31 5 Na K Cl mg/kgp.o (1 mg/kgs.c..1 mg/kgi.v. (ED 1.3 mg/kgi.v. 1 ( ( 2 (n (mg/kg ( mg/kg foot hold 1 16 mg/kg ( ( ( mg/kg 18 29

30 ( ( (n (mg/kg 1 mg/kg 33 (1 3 mg/kg (.1 2 ( ( ( mg/kg ( ( mg/kg ( mg/kg ( (4.1.3 ( (4.1.3 ( (LVPdp/dt max (4.1.3 (6 1g/15L (.36 g/kg 25 (6 1% 26 (4.1.3 (6 1 g/15l (.36 g/kg (6 1% 26 ( % ( (6 ( g/kg ( (6 (6 ( 1g/15L (.36 g/kg 25 (6 1% 26 ( (.92.9 g/kg ( (4.1.3 ( 1 24 PQQRSQT 3

31 ( (n (mg/kg In vitro APD 1, 3, 9 (5 11 nm HEK HERG 293 In vitro (3 11 nm 3 (6.11 ( mg/kg 27 ED 5 =72.7 mg/kg ( ED 5 =.32 mg/kg ( 11 nm in vitro ( (8 ( 11, nm 3 Na + Cl - ( K + ( (ED 5 = mg/kg ( ( (5.1.1 (61.13 ED 5 =.71 mg/kg.1.1 (45 ED 5 =.5 mg/kg ID (34 5 =.15 mg/kg 35 (.3 mg/kg 3 ( (5 ( mg/kg 37 31

32 ( (n (mg/kg ( (1 11 (1.33 (1.1.1 (1.33 ( (34 (4.3.1 mg/kg 38 3 mg/kg (ED 2.69 mg/kg (ED 2.17 mg/kg 39 (ED mg/kg (ED 5.34 mg/kg.3 mg/kg 35 (324 (1 (1 (4.5 mg/site 4 (48 ( (mg/ml/day (mg/kg/day.5.5 (mg/kg/day.3.6 (mg/kg/day.11 (mg/kg/day 14 (1 mg/ml (ED 5 = mg/kg 12 (ED 5 = mg/kg (ED 5 =.3 mg/kg

33 (mg/ml (L/min (min (kg Evans blue

/ / A/ B 16/17 COPD 18mcg COPD COPD COPD 1

/ / A/ B 16/17 COPD 18mcg COPD COPD COPD 1 11 1 -------------------- 19 205.117/205.128 14/15 205.126A/205.126B 16/17 COPD 18mcg COPD 11.1 11.1.1 COPD 100 19 205.227 COPD 1 FEV1.0 %FEV1.0 70% 1 FEV1.0 / FVC 70% 2 1 200 3 40 COPD 1 10mg ACE 1 1

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