Lung (1990) 168:285-293

Effects of Erdosteine on Sputum Biochemical and Rheologic Properties: Pharmacokinetics in Chronic Obstructive Lung Disease C. F. M a r c h i o n i , j M. M o r e t t i , ~ M. M u r a t o r i , ~ M. C. C a s a d e i , ~ P. Guerzoni, 1

R. Scuri, ~-and G. B. Fregnan 3 qstituto di Tisiologia e Malattie dell'Apparato Respiratorio, University of Modena; -'Research Center, I.R.F.I., Ferentino, Frosinone; 3Research Center, Edmond Pharma, Milano, Italy

Erdosteine is a new thioderivative endowed with mucokinetic, mucolytic, and free-radical-scavenging properties. This study evaluated (in a double-blind design vs. placebo) its efficacy on biochemical and theologic properties of sputum and on some indices of respiratory function in chronic patients with chronic bronchitis (I0 per group), while receiving basic treatment with a controlled-release theophylline preparation. The pharmacokinetics oferdosteine and theophylline were also studied. We found that a 2 week treatment with erdosteine (300 mg 3 times daily) was able to reduce significantly (p < 0.05) the sputum apparent viscosity, fucose content, and macromolecular dry weight (MDW) with no statistically significant influence on sputum elasticity, DNA, albumin, total proteins, total IgA, lactoferrin, and lysozyme content, The treatment caused a significant increase in the following ratios: total IgA/albumin, lactoferrin/albumin,and lysozyme/albumin. The pharmacokinetics of erdosteine, its metabolites, and theophylline were the same after I or 14 days of treatment, evidence both o f absence of an enzymatic induction and of an accumulation process. Further confirmation that there was no interference between erdosteine and theophylline was obtained from the data available on the group of patients receiving only theophylline, since its plasma levels and related pharmacokinetic parameters were identical to those obtained in patients receiving both drugs. In conclusion, 2 weeks of therapy with erdosteine reduced the marker o f mucus glycoproteins (fucose) in patients with chronic bronchitis but did not interfere with the pharmacokinetics of xanthine derivatives. We also suggest that the significant increment in the IgA/albumin ratio might be related to a sum o f other local effects such as reduction of the inflammatory process and enhancement of the humoral defense mechanism. Abstract.

Key words: Bronchitis, chronic o b s t r u c t i v e - - M u c o l y t i c s - - F r e e radical scavengers--Bronchodilators. Address offprint requests to:

Milano, Italy

Prof. G. B. Fregnan, Edmond Pharma, via Gradi,sca 8, 20151

286

C.F. Marchioni et al.

Introduction Drugs that modify the physicochemical properties of bronchial secretions are generally used in the treatment of chronic obstructive bronchitis. In particular, mucolytic agents are widely administered in an attempt to reduce the viscosity of dense sputum and, therefore, to improve its expectoration [25]. A m o n g these drugs, erdosteine is a new c o m p o u n d [7] studied in animals and humans that modulates mucus production, viscosity, and ciliary transport [3, 7, 17, 20, 21], and also shows inhibitory activity against free radicals produced by cigarette smoke [1, 4, 23]. The c o m p o u n d contains 2 blocked SH groups that become free in vivo (De Bernardi, personal communication), which are responsible for its mucolytic and scavenging properties. In the present double-blind study, the activity of erdosteine was tested in patients with chronic bronchitis. Chemical and theologic properties of sputum and respiratory parameters were evaluated before and after 2 weeks of treatment. Markers of bronchial inflammation (albumin and DNA), of mucus glycoproteins (fucose and N-acetylneuraminic acid), and of humoral defence mechanisms (IgA), and the physical properties (apparent viscosity and elasticity) were tested in sputum. Lung function tests, such as forced expiratory volume in 1 sec (FEV 0 and forced vital capacity (FVC), were also assayed. The plasma levels o f erdosteine, its metabolites, and theophylline were evaluated contemporaneously after administration of single and multiple doses oferdosteine or placebo.

Materials and Methods Patient Selection Twenty male patients (4 smokers and t6 exsmokers, equally represented in the 2 groups) with chronic hypersecretive obstructive bronchitis [13] were selected for the study, after giving informed consent. All were clinically stable before and throughout the trial. They were receiving continuous bronchodilator therapy with xanthine derivatives ( I tablet every 12 hr containing300 mg of theophylline in a slow-release formulation). Mucolytics,/32-adrenergicagonists, antihistamines, steroids, antibiotics, or any other intermittent drug therapy that might interfere with the assessment of erdosteine was stopped at least 1 week (3 months in the case of steroids) before patients were admitted to the trial and use of these agents was forbidden throughout the study.

Patient Treatment Each patient entered the study on a double-blind basis and was randomly assigned to receive erdosteine or placebo treatment. One capsule containing 300 mg active principle or vehicle was administered orally every 8 hr (3 times daily) for 14 days.

Methods Sputum samples (2-3 ml) produced on awaking were collected from each patient, as described by Lopez-Vidriero [9], by placing dental cotton rolls on the outlets of the salivary glands for theologic

Pulmonary Pharmacodynamics and Pharmacokinetics of Erdosteine

287

and chemical analyses on the first 2 days preceding the treatment and on the 13th and 14th day of therapy. Apparent viscosity (n) and elasticity (G') were measured on each freshly produced sputum sample using an oscillatory viscometer (Contraves Low Shear 30 sinus) at a frequency of 0.352 Hz. Specimens were then stored at -30°C. Chemical, enzymatic, and immunoglobulin analyses were carried out on the specimens after being thawed [15] and homogenized by ultrasonication under cooling conditions [6, 14]. The following determinations were made: DNA by a photometric method [2]; albumin, lactoferrin, lgG, and total lgA by radial immunodiffusion technique [12]; lysozyme by photometric technique [18]; total proteins by folin phenol reagent [I I]; fucose by Gibbons's method [5]; N-acetylneuraminic acid (NANA) by thiobarbituric acid assay [24]; macromolecular dry weight (MDW) after lyophilization of the sputum. The rheologic and chemical measurements of sputum samples were expressed as the arithmetical mean of the two consecutive days at the beginning of the study (TO) and on the 13th and 14th day of observation (TI4). In each patient, lung function tests including FVC. FEV~, and FEVt/FVC %, were measured using a computerized spirometer on days 0 and 14.

Pharmacokinetic Study On the 15th day the same patients received 3 capsules all together of erdosteine (equal to 900 mg of active principle) or of placebo and I tablet containing 300 mg of theophylline in a slow-release formulation at 8:00 a.m. They fasted from the previous evening to I:00 p.m. of the experimental day. Blood samples were collected in the presence of EDTA at 0, 0.5, i, 2, 3, 4, 6, 8, 12, and 24 hr thereafter. One month after interruption of erdosteine and placebo treatment (while theophylline was continued) the same patients received an identical dose of the above substances for a further study of their plasma levels. Plasma specimens were stored at - 70°C until assayed for theophylline and erdosteine (or its metabolites). Homogeneous enzyme-multiplied immunoassay technique (EMIT, Syva Corp.) was used for measuring theophylline plasma concentrations and high-performance liquid chromatography (HPLC) for measurement of the levels of erdosteine and its metabolites [16]. Pharmacokinetic parameters (C max = peak concentration in plasma, t max = time to reach peak, t ~/~/3 = apparent half-life, AUC 0-24 hr = total area under the plasma concentration-time curve from 0 to 24 h, CI = total plasma clearance) were calculated according to the method of Sedman and Wagner [22], except for metabolite lIl, where C max and t max were obtained by visual inspection and AUC 0-24 hr was determined by the trapezoidal method.

Statistical Evaluations Analysis of variance was performed on the data to assess any statistical difference (p -< 0.05) either intratreatment or between the 2 treatments.

Results On admission the sputum biochemical and rheologic parameters and lung funct i o n t e s t s d i d n o t d i f f e r s t a t i s t i c a l l y b e t w e e n t h e 2 g r o u p s ( T a b l e 1). Two weeks after treatment with placebo most of the sputum chemical c o m p o n e n t s d i d n o t c h a n g e o r s h o w e d t e n d e n c y t o i n c r e a s e ; t h e f u c o s e inc r e a s e d s i g n i f i c a n t l y ( T a b l e 1). O n t h e o t h e r h a n d , in t h e g r o u p r e c e i v i n g e r d o steine the concentration of DNA, albumin, total proteins, MDW, and fucose generally decreased after 2 week treatment, the difference being significant for f u c o s e a n d M D W . I n t h e s a m e g r o u p w e o b s e r v e d a s l i g h t i n c r e a s e in t o t a l I g A , lactoferrin, and lysozyme in sputum. In order to understand better the role of

1103.50 79.05 129.46 38.82 94.37 59.29 142.11 126.42 6.94 42.40 1.05 0.61 1.40 0.05

Before

± 828.50 ± 46.66 ± 81.54 -+- 20.36 ± 44.32 + 32.34 ± 44.65 ± 44.10 -+ 6.30 ± 13.07 ± 0.46 -+ 0.36 ± 0.78 ± I).115

1244.40 89.63 175.81 47.89 92.89 58.06 151.31 147.47 6.78 44.59 0.68 0.54 1.23 0.05

After

Placebo

±- 783.48 ± 76.85 -± 129.04 ± 27.82 ± 65.95 -+ 36.66 ± 93.67 ± 61.54" ± 6.50 ± 18.03 -+ 11,43" -+ (I.48 -+ I).78 ± (I.I15

12.8 13.4 35.8 25.3 -1.6 -2.1 6.5 16.7 -2.3 5.2 -35.2 -11.5 -12.1 0

% 1139.117 80.75 108.45 28.07 911.18 58.95 142.57 119.80 6.54 44.05 I.I10 0.87 1.99 (I.(15

Before ± 472.07 ± 42.80 ± 64.70 ± 16.46 ± 37.43 ± 17.18 ± 26.39 _+ 411.II ± 3.09 ± 18.32 ± I).42 + 0.82 ± 1.37 - (I.(13

985.92 55.91 91.13 25.52 117.4{) 71.62 163.69 78.87 6.44 33.31 1.79 1.2(I 2.52 0.10

After ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Erdosteine

A na lys i s of variance p -< 0.05: *after vs. before wllues within each Ireatmenl, ~afler e rdos l e i ne vs. after placebo values %: Percentage chan g es in after vs. before values

Total proteins DNA A lb um i n lgG Total lgA Lacloferrin Lysozyme F u cos e NANA MDW Total lg A/a lbu m in Lactoferrin/albumin Lysozyme/albumin NANA/fucose

M ar ker s and ratios

446.78 38.72 59.94 17.15 42.36 25.67 52.84 30.23"t 5.7(I 10.69"1 1.26'~ 0.81"1 1.601 0.12"I

Table 1. S put um c o n c e n t r a t i o n s (/zg/dl) of some ma rke rs and relative ratios before and after treatment with erdosteine or placebo lbr 2 w e e k s (means +- SD)

- 13.5 -30.8 -16.0 -9.1 311.2 21.5 14.8 -34.2 - 1.5 -24.4 79.5 37.9 26.6 100.0

%

Pulmonary Pharmacodynamics and Pharmacokinetics of Erdosteine

289

inflammation in sputum, lgA/albumin, lactoferrin/aibumin, lysozyme/albumin, and NANA/fucose ratios were calculated (Table 1). In the group receiving erdosteine all the ratios significantly increased while in the placebo group, with the exception of lgA/albumin, they did not change. The sputum rheologic measurements before and after erdosteine showed a statistically significant reduction of viscosity while elasticity remained virtually unchanged .(Table 2). On the contrary, the sputum viscoelastic properties increased slightly during placebo treatment. The statistical comparison between groups verifies these significant differences at the end of the study. The respiratory function tests (FVC, FEV~, and FEVJFVC) did not appreciably change during treatment with either erdosteine or placebo (Table 2). The pharmacokinetic data reported in Table 3 indicate that the plasma levels of erdosteine and its metabolites remained the same after single and multiple treatments with the mucomodulator. In addition, the pharmacokinetic pattern of theophylline did not change with or without erdosteine treatment (Table 4).

Discussion

This double-blind study indicates that a 2 week treatment with erdosteine modifies sputum rheologic properites mainly by reducing viscosity in patients with chronic bronchitis as compared with placebo treatment. These results are in agreement with other findings on mucolytics and mucoregulators [25]. The changes in sputum physical properties induced by erdosteine are associated with some significant modifications in the chemical constituents of sputum. In particular, fucose (a marker of mucus glycoproteins) and MDW are highly reduced by erdosteine. The mucolytic drug also tends to reduce the sputum markers of bronchial inflammation (albumin, IgG, and DNA) and the absolute values of total proteins while increasing IgA, lactoferrin, and lysozyme. These latter findings are confirmed by significant increases in lgA/albumin, lactoferrin/ albumin, and lysozyme/albumin ratios (as verified by intra and intergroup comparison). The changes in mucus chemical components, mainly fucose and MDW reduction, may partially explain the significant decrease in sputum viscosity observed in patients with chronic bronchitis receiving erdosteine. In fact, the mucins are the principal macromolecular components of bronchial secretions [8] and their concentration greatly influences the mucus rheologic properties together with MDW, albumin and other sputum proteins [10, 19]. The NANA/ fucose ratio is significantly increased after administration of erdosteine, an indication that proportional increase of acid radicals in mucus glycoproteins might influence the intermolecular bridges. Furthermore, erdosteine shows an appreciable topical mucolytic activity by splitting the intra and intermolecular S-S bonds [7, 21], which contributes to the sputum theologic changes observed in our patients. Erdosteine did not induce any improvement in lung function tests. This lack of response is likely due to such factors as the short period of treatment, the

3792,7 -+ 2385.79 3736.80 -+ 2628.36 2065,50 -+ 323.11 1162.20 +- 295.52 55.20 -+ 8.56

Before 4889.90 4055.00 2070.20 1199.10 57.40

After

Placebo

_+ 2481.96" + 3189.37 -+ 407.43 +_ 315.21 -+ 9.92

28.9 8.5 0.2 3.2 3.9

4% 3679.20 2808.20 2009.60 1062.60 50.60

Before _+ 2797.00 -+ 2580.38 -+ 685.66 +_ 505,32 -+ 8.93

2342.70 2783.60 1944.00 1064.00 52.81)

After _+ 1749.77"t _+ 2534.07 + 641.81 +_ 494.51 _+ 10.58

Erdosteine

Analysis of variance p -< 0.05: *after vs. before values within each treatment, rafter erdosteine vs. after placebo values A%: Percentage changes in after vs. before values

Viscosity (mPa/sec) Elasticity (mPa) FVC (ml) FEV a (ml) F E V J F V C (%)

Parameters

-36.3 -0.9 -3.3 0.13 4.4

A%

Table 2. Rheologic properties of sputum and respiratory function before and after treatment with erdosteine or placebo for 2 weeks (means -+ SD)

_=.

O

P,

'-r]

t

*

[3.49 +_ 1.06]t 2855.96 -+ 1297.68

[12.08 -+ 4.00]'I842.72 -+ 299.07

-+ 1.16lt -+ 0.58 _+ 0.20]t + 0.65 -+ 0.59]t -+ 3.13

[3.46 1,66 [I.47 1.68 11.61 12.32

II.25 1.19 [I.18 1.29 [I.45 3.71

_+ 0.221"1 -+ 0.26 _+ 0.26]t _+ 0.47 - 0.60]t -+ 1.38

3.45-+ 1.25

1

1.34-+0.26

E

16.44 3.28 [3.09 2.63 [2.14 56.66

-+ 2.741t -+ 0.81 +_ 0.37]t _+ 2,05 + 0.39]t -+ 19.25

7.11-+2.65

11

[51.18 _+ 18.78]t 142.13 -+ 42.24

Single

2.62_+0.75 (2.04 -+ 0.82)* 12.02 -+ 1.081t 3.50 + 1.18 13.12 -+ 0.72]t --21.23 _+ 5.10 (6.79 -+ 3.60)* [(8.26 -+ 2.83]*t --

111

_+ 0.2lit -+ (I.23 -+ 0.29]t -+ 0.29 -+ (I.761¢ -+ 1.46

13.34 1.6(I 11.50 1.49 11.72 12.23

-+ 1.071t -4- (I.48 -+ 0.311t + 0.57 -+ 0.611t -+ 3.80

3.17-+0.84

I

[51,86 144.58

111 2.65_+0.65 (2.08 -+ 0.73)* -+ 2.981/ 12.02 -+ 1.071t -+ 1.04 3.70 -+ 1.06 -+ (1,32]t [3.50 -+ 11.52] _+ 2.08 --+ 0.38]t --+ 17.44 21.77 -+ 3.74 (8,(t4 + 4,66)* -+ 18.09]'i" 1(8.62 -+ 5.25]*1" -+ 54.26 --

II 7.29_+3.02 (7.02 3.59 [3.08 2.91 12.07 56.21

Multiple

[3.52 -+ 0,981"t 112,30 -+ 4.18]t 2841.65 -+ 901.31 832.59 -+ 286.46

11.20 1.35 [I.29 1.36 [I.58 3.55

1.15-+0.27

E

Erdosleine treatment

The basal value present at 0 hi was subtracted from all the values found at the others hours in order to calculate the specific parameters. These values were obtained fiom Prof. De Bernardi (personal communication) and concern healthy volunteers treated with oral erdosteine 900 rag.

Total CI (ml/min)

AUC 0-24 (/xg/ml/hr)

tl/2,/~ ( h r )

t max (hr)

Cmax (#g/ml)

Parameters

Table 3. Pharmacokinetic p a r a m e t e r s for erdosteine (E) and its metabolites (1, 11, and 1111 w h e n lheophylline w a s given in c o m b i n a t i o n with single and multiple d o s e s of erdosteine ( m e a n s --+ SD)

m

5

P~

e'~

3

@

F

G

292

C.F. Marchioni et al.

Table 4. Pharmacokinetic parameters for theophylline when the bronchodilator was given in combination with single and multiple doses of erdosteine or of placebo (means .+ SD) Parameters

c max t/zg/ml) t max (hr) t J/2/3(hr) AUC 0-24 (/.tg/ml/hr) Total CI (ml/min)

Erdosteine treatment

Placebo lrealment

Single

Multiple

Single

Multiple

15.60 --+ 2.32 2.40 -+ 0.54 30.32 + 5.36 290.79 --- 45.05

15.69 - 1.61) 2.60 -*- I).53 29.59 + 12.63 288.98-+ 34.28

15.1)3 -+ 1.93 2. t9 .+ 0.58 34.69 --- 4.62 288.62-+ 40.52

15.33 + 1.74 2.20 _+ 0.65 32.98 --_ 8.47 290.70-+ 38.80

7.19 .+ 1.74

7.57 .+ 1.97

6.54 -+ 1.43

6.97 .+ 2.17

clinically stable phase of the d i s e a s e d u r i n g the s t u d y period, a n d the a d v a n c e d c o m p r o m i s e of lung f u n c t i o n in p a t i e n t s a d m i t t e d to the study. T h e p r e s e n t findings also i n d i c a t e that single and multiple a d m i n i s t r a t i o n s o f e r d o s t e i n e are not c h a r a c t e r i z e d by c o n s i s t e n t c h a n g e s in blood levels o f the intact p r o d u c t and of its m e t a b o l i t e s , s u g g e s t i n g that n e i t h e r e n z y m a t i c a c t i v a tion n o r a c c u m u l a t i o n takes place d u r i n g t h e r a p y . In a d d i t i o n , by c o m p a r i n g the p h a r m a c o k i n e t i c p a r a m e t e r s in h e a l t h y v o l u n t e e r s for e r d o s t e i n e a n d its m e t a b o l i t e s I, It, a n d III (Table 3, d a t a in b r a c k e t s ) , it is clear that in p a t i e n t s with c h r o n i c b r o n c h i t i s the bioavai[ability o f the c o m p o u n d a n d its m e t a b o l i z a t i o n p r o c e s s are n o r m a l . M o r e o v e r , s i n c e the p a t i e n t s took a l o n g - a c t i n g t h e o p h y l l i n e p r e p a r a t i o n before, d u r i n g , a n d after e r d o s t e i n e or p l a c e b o t r e a t m e n t , e v i d e n c e is p r o v i d e d that the m u c o m o d u l a t o r a n d b r o n c h o d i l a t o r t h e r a p i e s do not influe n c e the p l a s m a levels of each other. In c o n c l u s i o n , a 2 w e e k t r e a t m e n t with e r d o s t e i n e f a v o r a b l y c h a n g e s the rheologic p r o p e r t i e s a n d m u c u s g l y c o p r o t e i n c o n t e n t in the s p u t u m of p a t i e n t s with c h r o n i c a n d clinically stable b r o n c h i t i s w i t h o u t i n d u c i n g its o w n m e t a b o l i c a c t i v a t i o n or a c c u m u l a t i o n . T h e s t u d y also s h o w s that e r d o s t e i n e does not interfere with the p h a r m a c o k i n e t i c s o f x a n t h i n e d e r i v a t i v e s , and vice v e r s a .

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Pulmonary Pharmacodynamics and Pharmacokine~.ics of Erdosteine

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