European Review for Medical and Pharmacological Sciences 2004; 8: 259-264 Early protective effects of in patients with airways hyperres p o n s i v e n e s s

C. TERZANO, A. PETROIANNI, A. RICCI, L. D’ANTONI, L. ALLEGRA*

Department of Cardiovascular and Respiratory Sciences, Respiratory Diseases Unit, Fondazione E. Lorillard Spencer Cenci, “La Sapienza” University - Rome (Italy) *Institute of Respiratory Diseases, University of Milan, Ospedale Maggiore IRCCS – Milan (Italy)

Abstract. – Tiotropium is an anticholiner- Introduction gic drug for Ch ronic Obstructive Pulmonary Di sease (COPD) patients, with a peak b ron- Ti o t rop ium is a qu atern a ry amm o niu m chodilator effect observed after 1.5 to 2 hours and a long duration of action. The aim of our co ngener of . It has been developed study was to quantify the early protection of a as a lon g-acting an ticho linergic bro n c h o d i l a- single dose of inhaled tiotropium against metha- tor for inhalation by patients with chronic ob- -induced in asthmat- s t ructive pu lmon ary d isease (COPD). Dru g ic patients with airway hyperresponsiveness. p ro p e rties o f ant icho lin ergic agent s have Ten subjects (7M, 3F), with history of been well-kno wn to man y cultures for many and a baseline FEV (Forced Expiratory Volume 1 1 1 c e n t u r i e s . In the 1920s the d iscovery o f the sec) >80% of pred icted, were enrolled in the s t u d y. Each subject perfo rmed three meth a- adrenergic agonists displaced these agents as choline challenge tests, with a time of 72 hours th e first-line treatmen t for obstru ctive dis- between each challenge: Test A ( eases. Nevertheless in last decades there has challenge test), an d successively, at random, been a renewed interest in the use of anti- Test B (methacholine 30 minutes after inhaled drugs, given the increase in preva- Tiotropium) and Test C (methacholine 30 minutes lence an d m orb idity of asth ma and COPD, after inhaled Placebo). PD20 (Provocative Dose and the better understanding of the choliner- causing a 20% decrease in basal FEV1 value) was reached to assess airways responsiveness. gic mechanisms that control airway calibre in All the subjects showed in Test A and Test C obstructive diseases. The newer anticholiner- a mild-moderate airway hyperresponsiveness. gic agents are water-soluble, quatern a ry am- In Test B no PD20 was reached at the inhaled mo n iu m co m p o u n d s th at are p o o rly ab - maximu m do se o f meth ach ol ine (1600 µg), sorbed, and via inhalatio n cause fewer sys- F E V before tiotropium was 88.6% ± 4.4, begin- 2,3 1 temic side effects . ning test FEV1 92.6% ± 4.3, end test FEV1 8 5 . 7 % ± 4.6. In patients with acute asthma the m ech a- Inhaled tiotropium bromide 18µg has shown nism s for broncho constriction and dilatatio n a pro tective effect ag ain st methacholin e-in- a re mediated by the autono mic nervo us sys- du ced bro nchoconstriction in asth matic p a- tem . The majo rity of au ton om ic nerves in tients, with mild-moderate airways h yp err e- hu man airways are b ran ch es of th e vagu s sponsiveness, already 30 minutes after its ad- n e rve; it s eff e ren t in nervat io n is d erived m i n i s t r a t i o n . f ro m th e p ostgan glionic fibres that en d in smoo th muscle of the airways, in submu cosal Key Words: glan d s an d in vascu lar st ru c t u re s 4. Met h ach o lin e, Airwa ys h yp erre s p o n s i v e n e s s , Ph ysio logically th e release of Tio t r op iu m, Muscarin ic rec e p t o r s . stimulates muscarinic receptors at these sites and resu lts in smo o th mu scle co ntract io n Abbreviations: an d th e release of secretions from su bm u- CO PD = C hronic Obstructive Pulmonary D isease; cosal gland s.

MCh = Methacholine; ACh = Acetylcholine; F EV1 In humans there are 5 muscarinic receptor = Forced Expiratory Volume in 1 second; PD20 = subtypes (M1-M5) all encoded by individual Pr ovocative Dose causing a 20% decrease in basal genes and exhibit distinct pharmacology and 5,6 FE V 1 va l u e . tissue distribution .

259 C. Terzano, A. Petroianni, A. Ricci, L. D’antoni, L. Allegra

The M1 receptors are lo cated in parasym- Th is m ay weak en th e effect o f th e p at hetic g ang lia and are resp o nsib le fo r postjunctional M3 muscarinic receptor block- c h o l i n e rgic neurotransm ission . They may fa- ade on airway smooth muscle and submucos- cilitate nicotinic transmission or be responsi- al glands. Otherwise antagonists that bind se- b le fo r m ain tain in g ch o lin ergic to n e. lectively t o M3 an d M1 recep to rs m ay be Inhibition would reduce cholinergic tone and m o re effective in inh ibitin g cholin ergic ef- thu s wou ld red uce bro nchoco nstriction. M1 fects in the airways. recep tors are also fo un d on alveo lar walls, Ti o t ropium brom id e is a recen tly devel- where their function is unknown, on the air- oped , lo ng-actin g, selective antimuscarin ic way ep ith elium , an d in vario u s exo crin e drug. It is structurally similar to ipratropium glands7. with the exception of the presence of th io- Prejunctional M2 receptors are located on phen e rin gs o n tiotropium , which resu lt in po stganglio nic nerves in the smoo th muscle g reater lipoph ilicity and faster systemic m e- fibres and submucosal glands and inhibit the tabolism12,13. release o f acetylch olin e. Released acetyl- Tiotropium is a competitive muscarinic re- cho line is hydro lysed by the , cepto r an tago nist th at disp lays a 6-20-fold an d th e cho line is tak en u p. M2 re c e p t o r s higher affinity for m uscarinic recepto rs than wo rk as auto recep tors, provid in g negative i p r a t ro p i u m8. Ti o t ro pium bind s with sim ilar feed b ack on th e release o f acetylcho lin e. a ffinity to all subtypes of m uscarinic re c e p- They are activated by th e release of acetyl- tors, but it dissociates rapidly from M2 recep- cho line, lim iting the degree o f b ro n c h o c o n- tors and very slowly from M1 and M3 recep- striction. Agents that block M2 receptors in- tors, resulting in a prolonged duration of ac- hib it the negative feedback an d result in in- tion in physiologic experiments6,14. c reased acetylcholine release and there f o re , In hu man b ron chi, tiotropium has an in- incr eased tone in the bronchial smooth mus- hibitory reported effect with a slow onset of c l e8. M2 receptors predom inate in the heart action, a peak effect observed and mediate the bradycardic effects of Ach 7. after 1.5 to 2 h, and a prolonged duration. M3 recep tors are located on th e airw a y Methach olin e (MC h) ch allen ge test is a smooth muscle. M3 receptors typically medi- method of assessing airways responsiveness. It ate the direct con tract ile effects o f ACh . may vary over time, often increasing during ex- Their activation leads to the con traction o f acerbations and d ecreasing d uring tre a t m e n t airway smooth muscle. M3 receptors also are with antin flamm atory medications1 5. Airw a y located on submucosal glands, where they are h y p e rrespo nsiveness can be d emon strated in likely to be involved in secretion9,10. almost all patients with current sym pto matic Although M4 and M5 receptors have been asthma an d even subjects with normal h ista- identified, the physiological roles of receptors mine or MCh airway responsiveness can devel- remain unknown. op symptoms of asthma if exposed to specific It should be n oted that in hibiting M1 and stimuli to which they are sensitized, such as an M3 r eceptors results in a beneficial reduction inhaled or toluene diisocyanate. in b ro nch oconstriction , wh ereas inh ib itin g M e a s u rements of airways re s p o n s i v e n e s s M2 receptors p ro duces th e opp osite eff e c t . to in h aled bro n ch o co n stricto r m ed iato rs T h e re f o re the id eal drug for (such as MCh) can be useful in making a di- airways bronchoconstriction would preferen- agn osis o f asth ma, particu larly in p atien ts tially antagonize M1 and M3 receptors or the with symptoms of asthma with no evidence of M 3 receptor alone, with little affinity for the airflow obstruction. MCh inhalation tests can M 2 receptor. be performed safely and reproducibly by ex- Non-selective anticholinergic agents such as perien ced t ech n ician s , o n ce th e fact or s at r opine, , and oxitrop i - known to influence the tests are appropriate- u m b rom id e b lo ck bo th the p re j u n c t i o n a l ly controlled for. (M2) an d po stju nction al (M3) re c e p t o r s . The ob servation th at airway re s p o n s i v e- Th us, due to th e blockade of the prej u n c t i o n a l ness to agents such as MCh progressively in- M2 recepto rs, non -selective an tagon ists may c reases after the inh alatio n challenge, h as i n c rease the am ount of acetylcholine th at is provided a useful model for observing the ef- released by cholinergic nerve stimulation11 . fect of drugs.

260 Early protective effects of tiotropium bromide in patients with airways hyperresponsiveness

Our stu dy d escribes the evaluation of the Table I. Ae r osol characteristics of MCh delivered by the early pro tection and safety o f tiotropium at ME F AR MB3 dosimeter and standard MB2 ampoule. t h e d o se o f 18 µ g again st MCh -in du ced b ro nch oco nstrictio n in asth matics p atients MMAD (mm) GSD with m ild an d mo derate airways h yperre- Diluent 2.15 1.45 s p o n s i v e n e s s . MCh 0.125% 1.31 1.50 MCh 0.250% 1.38 1.49 MCh 1% 1.76 1.49 Materials and methods Subjects Ten su bjects (7M, 3F, age 26.6 ± 8) with h i s t o ry of asth ma and d ocu mented airw a y s Th e aeroso l characteristics o f MCh d eliv- h y p e rresp o nsiven ess were enrolled in th e e red by th e MEFAR MB3 d osimeter, was study. Baseline FEV1 was >80% of predicted; m e a s u red with a high-resolution instru m e n t no sym ptom s and treatments were re p o rt e d . ( A e ro sizer Mach2; API, Am herst, MA) in Written informed consent was obtained from our aerosol research laboratory (Table I). each su b ject , an d Eth ics Co m m it tee ap - The measurement of FEV1 was made using pr oved the research study. a co mp uterized spirograp h (C osm ed PFT1, Pavona, Italy). Study design and drugs Ti o t rop ium dose 18 µ g was adm inistere d Each sub ject p erf o rm ed th ree MCh chal- through a dry powder (HandiHaler). lenge tests, each at distance of 72 hours. Test Placebo was administered via a lactose-based A was performed only as MCh challenge test dry-powder inhaler device. with PD20 measure, and successively, at ran- do m, Test B as MCh challen ge test 30 min- Statistics utes after inh aled tio tropium and Test C as The primary efficacy variable was MCh re- MC h ch allen ge t est 30 m in after in haled sponsiveness, measured as PD20. FEV1 p r e - placebo. t reatmen t, at begin ning test, and test, an d S p i rom etric p aram eters were measu re d PD20 were compared using mean ± standard b e f o re in h aled treatm en t (t io t ro p ium o r deviation. p laceb o ), at th e b egin nin g o f MC h ch al- lenge tests and at the cum ulative dose steps (12.5, 25, 50, 100, 200, 400, 800, 1600 µg). PD20 was reached to assess airway re s p o n- Results s i v e n e s s . The p atien ts u nderwent an initial forc e d All participants perf o rm ed the three type exp iration test to assess their suitability for of Tests. They showed in Test A (MCh) and the test (FEV1 >80% of the predicted) in ac- Test C (placebo + MCh) a mild-moderate air- c o r dance with the p rotocol of the American way h yp erresp o n siv en ess. In t est A was Thoracic Society 16. achieved a PD20 value between 583 µg an d MCh aerosolization, preceded by an initial 1446 µg as well as in test C a value between adm inistration of diluent so lutio n, was car- 624 µg and 1552 µ g. In test B (tiotrop ium + ried out with a MEFAR MB3 dosimeter and MC h) n o PD20 valu e was reach ed b y any s t a n d a rd MB2 amp ou le with the fo llowing subject at the inhaled MCh dose 1600 µg. f e a t u res: (1) comp ressed air at 1.75 atmos- In Test A PD20 and spirometric parameters p h e res; (2) air flow 9 l/min; (3) nebulization we r e (mean±SD): PD20 878.95 µg ± 284.3, be- µ time: 1 second; (4) output: 10 ± 0.2 l; (5) vol- ginn in g test FEV1 90.71% ± 4.54, end test ume of MB2 ampoule: 3 ml; (6) particle size F E V1 66% ± 2.9 . In t est B (tio trop ium + µ of 0.5-5 m, with mass m edian aero d y n a m i c MCh) the FEV1 di d n ’ t fall by at least 20% af- diameter (MMAD): 1.70 and geometric stan- ter the highest MCh concentration (1600 µg) d a rd deviation (GSD): 1.37 (aero sol charac- in all the subjects, FEV1 be f o r e pre- t r ea t m e n t teristics obtained by using NaCl 0.9% in the was 8 8.64 % ± 4.46 , b egin n in g t est F EV 1 quality control tests). 92.58% ± 4.27, end test FEV1 85.75% ± 4.6.

261 C. Terzano, A. Petroianni, A. Ricci, L. D’antoni, L. Allegra

Table II. F E V1 values and PD 20 for each subject .

Test A (MCh) Test B (tiotropium + MCh) Test C (placebo + MCh)

Subject BT PD20 ET PT BT ET PT BT PD20 ET

FEV1 (µg) FEV1 FEV1 FEV1 FEV1 FEV1 FEV1 (µg) FEV1 (%) (%) (%) (%) (%) (%) (%) (%)

1 88.7 762.3 64.2 88.2 91.6 84.4 87.2 88.4 859.2 65.6 2 89.6 621.2 65.8 87.4 92.5 85.8 86.9 87.8 824.5 65.3 3 94.2 962.4 68.1 90.3 94.8 87.2 91.4 92.8 894.2 68.6 4 96.3 1225.3 69.7 95.8 98.1 91.4 96.1 96.9 1480.3 72.6 5 85.3 583.2 63 83.4 86.8 81.1 84.5 85.1 624 64.2 6 92.3 1006.9 66.4 88.9 93.2 86 90 91.5 982.6 66.8 7 84.2 681.3 62.2 84.1 88.1 80.6 83.8 84.5 724.4 63.7 8 87.9 644.8 64.4 85.7 88.7 81.3 85.2 86.8 683.8 65.7 9 90.5 855.6 64.9 86.2 91.8 84.7 91.3 91.2 1022.4 67.5 10 98.1 1446.5 71.6 96.4 100.2 95 95.2 96.3 1552.8 72.4 Mean 90.7±4.5 878.9±284 66±2.9* 88.6±4.4 92.5±4.3 85.7±4.6* 89.1±4.3 90.1±4.3 964.8±316 67.24±3.1* ± SD

BT = beginning test; PT = pre treatment; ET = end test. *p <0.0001.

In test C (placebo+MCh) PD20 was 964.82 tions. Similar changes can be seen after ozone 1 6 µ g ± 31 6.9, F EV 1 b e f o re p re - t re a t m e n t e x p o s u re o r an tigen ch allenge . Wh en the 89.16% ± 4.34, beginning test FEV1 90.13% ± M2 receptors are dysfunctional, the resulting 4.35, end test FEV1 67.2% ± 3.1. (Table II). excessive con centratio ns o f acetylcholin e at Statistical analysis h as shown a significant th e neuro e ffecto r ju nction can promo te sig- difference for PD20 between test A and B (p nificant bronchoconstriction9,14. This might be <0.0001) an d between test B an d test C (p c o n t r i b u t o ry to exaggerated cho lin ergic re- <0.0001), and a significant difference for end flex bronchoconstriction in asthma17. 1 3 t est F EV1 b etween test A an d t est B (p In exp eriments re p o rted by Disse et al , <0.0001) an d between test B an d test C (p bo th t io trop iu m an d ip rat ro pium b lo ck ed <0.0001). No significant statistical differences c h o l i n e rgic nerve stim ulation with MCh. In h ave been demo nstrated for beginning test this study, concentration effect curves indicat- FEV1 and pre-treatment FEV1 in all the tests. ed reversible antagonism, with the tiotropium In Figure 1 were reported the average val- samples showing a much slower recurrence of ues for all MCh steps. muscarinic effects, further suggesting a slow- No ad verse effects of in haled tio tro p i u m er dissociation from muscarinic receptors 13. were reported after MCh administration. In pharmacologic studies tiotropium has a prolonged duration of blockade in guinea pig trachea in vitro and after inhalation in dogs in vivo13. Pharmacokinetic studies of tiotropium Discussion have also shown that disso ciation from M1 and M3 receptors is virtually 100 times slower Th e ro le o f cho lin ergic m ech anism s in than for ipratropium8,18,19. b ron chial hyperresp on siveness is m uch de- A comp arative stud y2 0 has evaluated the bated. Feedback inhibitory muscarinic recep- e fficacy an d safety o f t io t ro p iu m 18µ g tors (M2) are thought to be dysfunctional al- once/day with ipratropium 40 µg 4 times/day so in asthma, resulting in exaggerated cholin- and demonstrated tiotropium was significant- ergic reflexes. The loss of M2 receptor func- ly more effective than ipratropium in improv- tion has been demonstrated after viral infec- ing trough and peak lung function over the 13

262 Early protective effects of tiotropium bromide in patients with airways hyperresponsiveness

In our stu dy we d emonstrate that tiotrop i - um b ro mid e 18 µg, com pared with p laceb o and administered 30 minutes in ad vance, have an early significant protection again st a MCh challenge in subjects with mild -moderate air- ways hyperrespon siveness, b efo re re a c h i n g the peak b ronchodilator effect. In conclusion, today anticholinergic drugs are established as first lin e b ro n ch o d ilato rs in C OPD. Tio t r opiu m has been d eveloped as a new gen- eration antim uscarinic following ipratrop i u m . The high potency and slow receptor dissocia- tion fo und its clinical correlate in significant and long lasting bronchod ilatation an d bron - Figure 1. Average values for MCh tests, fall of FEV1 (%) and inhaled MCh (µg). ch o p r otection in patients with obstructive air- way diseases. In our stu dy Ti o t ropiu m pro- tects asthm atic patients with m ild-mo derate ai r ways hyperresponsiveness, against MCh-in- week treatment period. The safety profile of duced bronchoconstriction, already 30 min af- Tiotropium was similar to ipratropium. ter its adm in istration , suggestin g h ow the In clinical trials, tiotropium appears to be a d ru g, b efo re achievin g th e m axim um bro n - safe drug during long term treatment and it is ch o d i l a t o r y effect (about 120 min), carries out generally well tolerated, with th e most co m- a protective effect on bron cho constriction. mo n side effect being dry mo uth, which oc- In future, it would be interesting to explore curs in 6-15% of patients20,21,22. the mechanisms involved in the early cholin- The role for anticholinergic medications in ergic effect and the potential role of tiotropi- acute asthma is not well-defined. The use of um in th e bro n c h o p rotection against oth er therapy with an ticholinergics and b eta2a g o - stimuli such as cold air, exercise and ultrason- nists, eith er simultan eo usly o r in seq uence, ically nebulized distilled water. has produced positive as well as not consider- able results in trials 23. The effect of tiotropium bromide on MCh- References induced bronchoconstriction in asthmatic sub- jects has been in vestigated by O’Co nno r et 1) G A N D E V I A B . Historic al review of the use of al 10 after three doses of tiotropium , res p e c t i v e - parasympatholytic agents in the treatment of res- ly 10, 40 and 80 µg. In th is double-blind, four- pirat ory dis orders . Post grad Med J 1975; period crossover stu dy th at in cluded 12 pa- 51(Suppl): 13-20. t ien ts with mild asth m a, th e efficacy o f 2) WEBER R. Role of in asthma. Ann t i o t rop iu m was d eterm in ed in com parison Allergy 1990; 65: 348-350. with placebo. Each treatment period was sep- 3) B EAKES DE. The use of anticholinergics in asthma. arated by 8-24 days. After administration of J Asthma 1997; 34: 357-368. the study d rug, p atients underwent re p e a t e d 4) RICHARDSON JB. Nerve supply to the lungs. Am Rev MCh Test 2, 12, 24, 36, and 48 hours later. In Respir Dis 1979; 119: 785-802. th is stu dy clinically significant pro t e c t i o n 5) B AR N E S PJ. Muscarinic receptor subtypes in air- against a MCh challenge was sustained in nine ways. Life Sci 1993; 52: 521-527. patients 48 hours after receiving tiotro pium 10 6) B ARNES PJ. Novel approaches and targets for treat- µ g. Th e results o f th is st ud y su ggest th at ment of chronic obstructive pulmonary disease. ti o t r opium has bro nch odilator prop e r ties and Am J Respir Crit Care Med 1999;160:S72-79. p r otection against a MCh challenge that be- 7) L E F K O W I T Z RJ , HO F F M A N B B, TA Y L O R P. gins with in 2 hours after ad ministration and Neurohumoral transmission: the autonomic and persists fo r at least 24 hours10 . somatic motor nervous systems. In: Gilman AG, Rall TW, Nies AS, Taylor P, eds. Goodman and The maximu m bro n c h o d i l a t o ry effects o f Gilman's the pharmacological basis of therapeu- t i o t ropium are reached 90-120 m in after in- tics, 8th ed. Elmsford, NY: Pergamon Press, Inc., halation24,25. 1990: 84-121.

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