Efficacy and Safety of Letosteine in the Treatment of Sputum Thickening And

ORIGINAL ARTICLES

Department of Respiratory Medicine,1 the Third Xiangya Hospital of Central South University, Changsha; Department of Respiratory Medicine,2 Gansu Provincial People’s Hospital, Lanzhou; Department of Respiratory Medicine,3 Afﬁliated Hospital of Inner Mongolia Medical College, Hohhot; Department of Respiratory Medicine,4 Baotou Central Hospital, Baotou City; School of Public Health,5 Central South University, Changsha, China

Efﬁcacy and safety of letosteine in the treatment of sputum thickening and expectoration difﬁculty in patients with respiratory diseases: a multicenter, randomized, double-masked, double dummy, positive drug parallel controlled trial

Yan Zhou 1, Shenghua Sun 1, Hua Liu 2, Liying Cui 3, Xiaoyue Chang 4, Zhenqiu Sun 5

Received March 4, 2014, accepted April 25, 2014 Shenghua Sun, Department of Respiratory Medicine, the Third Xiangya Hospital of Central South Univer- sity, Changsha, Hunan, 410013, China [email protected] Pharmazie 69: 842–849 (2014) doi: 10.1691/ph.2014.4574

This study aimed to compare the efficacy and safety of letosteine and ambroxol hydrochloride for the treatment of sputum thickening and expectoration difficulty due to either acute or chronic respiratory diseases. Patients (n = 240) were randomized to receive either letosteine + placebo (50 mg thrice daily, Group A) or ambroxol hydrochloride + placebo (30 mg thrice daily, Group B) orally for 5-14 days. The primary outcomes comprised the total effectiveness rate and the total improvement rate. Secondary outcomes included: post- treatment IgA level changes and post-treatment therapeutic evaluation scoring of clinical symptoms. The full analysis set (FAS) comprised 113 patients in Group A and 116 in Group B. The total effectiveness rates were 95.58% for Group A and 95.69% for Group B. The total improvement rates were 99.12% and 99.14% for Group A and Group B, respectively. There were no significant differences between the two groups for any of the primary or secondary outcomes in either the FAS or the per protocol populations (PPS; all P-values > 0.05). Letosteine and ambroxol hydrochloride provided equivalent efficacy and safety in the treatment of sputum thickening and expectoration difficulty due to either acute or chronic respiratory diseases.

1. Introduction the viscosity of the sputum (Gachon et al. 1988). Letosteine is one of cyclic derivatives of cysteine, which have been widely As their name implies, mucolytic agents are used to ‘lyse’ or applied as mucokinetics in clinical practice (Rogers and Lethem decrease the viscosity of mucus (Rogers 2007). These drugs 1997; Wills and Cole 1996). Letosteine has potent apophleg- are beneficial adjuncts in the treatment of diseases such as matic ability and can reduce the viscosity of the sputum, dissolve asthma, chronic obstructive pulmonary disease (COPD), and mucus, increase the ciliary movement, and reduce the adhe- cystic fibrosis in which patients demonstrate hypersecretion of sion of sputum to the airway wall (Arroll 2005; Nespoli et al. airway mucus and excessive mucus in the airway lumen (Aikawa 1989; Olivieri et al. 1983; Search Medical Rx 2013). In addition, et al. 1989; Openshaw and Turner-Warwick 1989; Rogers 2004, letosteine has reactive oxygen scavenging ability, which is valu- 2007; Voynow and Rubin 2009). Mucolytic agents typically able for the treatment of several respiratory diseases (Gressier contain sulfhydryl groups that have the capacity to dissociate et al. 1995). To date, letosteine has been used in the treatment disulphide bonds in respiratory mucins, thereby reducing overall of bronchitis, asthma, emphysema, and chronic obstructive pul- mucus viscosity (Rogers 2007). Classic mucolytic agents, such monary disease (Gressier et al. 1995; Hansel and Barnes 2001; as N-acetylcysteine, contain either ‘free’ or ‘exposed’ sulfhydryl Macquet et al. 1979; Meroni et al. 1983; Nespoli et al. 1989; groups that break disulphide bonds. Other mucolytics, such as Search Medical Rx 2013). Letosteine is associated with few carbocysteine and letosteine, have ‘blocked’ sulfhydryl groups side effects and a favorable tolerability profile (Search Medical that are exposed through the body’s metabolic processes (Rogers Rx 2013). and Lethem 1997). Other examples of mucolytic compounds Letosteine has been available in Europe for many years, but is are cysteine, nacystelyn, ethylcysteine, nesosteine, erdosteine, yet to be approved for use in China. Letosteine, produced by the fudostein, and eltosteine (Rogers 2007). Other types of agents Huadong Medicine (Xi’ an) Bohua Pharmaceutical Co., Ltd, has with mucolytic properties, for example recombinant human been awarded with Investigational New Drug Approval by the deoxyribonuclease and proteolytic enzymes, break up mucus by State Food and Drug Administration (SFDA) (2007L01208). degrading DNA or actin. Some examples of peptide mucolytics Relatively little published information is available regarding the include: bromelain, streptokinase, and trypsin. clinical efficacy and safety of letosteine. The current study aimed Letosteine (2-[2’-(ethoxycarbonylmethyl-thio)-ethyl]-4-hydro to evaluate the efficacy and safety of letosteine and ambroxol xy-carbonyl thiazolidine) is a mucolytic agent used to regulate hydrochloride in the treatment of sputum thickening and 842 Pharmazie 69 (2014) ORIGINAL ARTICLES expectoration difficulty due to acute or chronic respiratory dis- two study groups. These results suggest that both drugs effec- eases. To the best of our knowledge, this is the first clinical trial tively improve sputum thickening and expectoration difficulty to be conducted in China to evaluate the safety and mucolytic due to acute or chronic respiratory diseases. efficacy of letosteine. A number of agents have demonstrated mucolytic activity in preclinical and clinical studies. Ambroxol, the positive control agent in the current study, is used clinically as a mucolytic and 2. Investigations and results expectorant to lower sputum viscosity (Gillissen and Nowak 2.1. Patient disposition 1998). This agent possesses other beneficial activities such as: secretolytic efficacy (promotes mucus clearance, facilitates A total of 240 patients enrolled and were randomized into group both expectoration and productive coughing), inhibition of lipid A (letosteine, n = 120) and group B (ambroxol, n = 120; Fig. 1). peroxidation induced by a variety of insults (ie, H2O2 and According to the definition of the FAS, PP, and safety popu- endotoxin), direct anti-oxidant actions, inhibition of human lations, group A had 113 patients in FAS, 109 in PP, and 114 phagocytes, and antiviral and antibacterial activity (Malerba in safety populations, respectively; group B had 116 patients in and Ragnoli 2008; Paleari et al. 2011). One study showed that FAS, 114 in PP, and 116 in safety populations, respectively. The fewer chronic bronchitis patients receiving ambroxol (67.2%) study design is shown in Table 1. experienced no exacerbations compared with those on placebo (50.5%) (Olivieri et al. 1987). N-Acetylcysteine has been shown 2.2. Demographics and baseline characteristics to cause a reduction in superoxide radical generation by alveolar macrophages in healthy smokers (Ziment 1986). Patients with Table 2 shows the patients demographics and baseline char- chronic bronchitis reportedly experienced an increased sputum acteristics for the FAS population. The average age of the volume, decreased sputum thickness, and a facilitation of expec- patients was 40.96 years (SD = 15.95) in group A and 41.91 toration (Aylward et al. 1980). Adverse effects associated with years (SD = 15.52) in group B, respectively. There were 49 males oral dosing of N-acetylcysteine include nausea, dyspepsia, and (43.4%) in Group A and 42 males (36.2%) in Group B. The diarrhea (Rogers 2007). comparisons show the demographic and baseline characteris- Letosteine has several actions that are beneficial in the treat- tics were all consistent between groups (All P-values > 0.05; ment of respiratory diseases. This mucolytic agent reduces the Table 2). viscosity of the sputum, dissolves mucus, increases the ciliary movement, and reduces the adhesion of sputum to the 2.3. Efficacy and safety analysis airway wall (Arroll 2005; Nespoli et al. 1989; Olivieri et al. 1987; Search Medical Rx 2013). Letosteine is readily absorbed Table 3 presents the efficacy analysis for primary outcomes. and several metabolites of the compound have been identified The primary outcomes included total effectiveness rate and total (Gachon et al. 1988). An animal model study showed splitting improvement rate after treatments. of the ester group of letosteine followed by cleavage of the thi- For the FAS population, patients in Group A experienced a total azolidinyl ring, which yielded cysteine, hypotaurine, taurine, effectiveness rate of 95.58% and a total improvement rate of and inorganic sulfate. Cysteine, in turn, is a substrate in the 99.12% Table 3). The Group B patients experienced a total effec- biosynthesis of glutathione, an important antioxidant (Tsan et al. tiveness rate of 95.69% and a total improvement rate 99.14%. 1985). Reactive oxygen species have been implicated in respira- There were no significant differences observed either in the total tory diseases such as asthma and respiratory distress syndrome effectiveness rates or in the total improvement rates between (Doelman and Bast 1990; Repine 1992). The beneficial clinical groups (FAS population; Both P-values > 0.05). effects of letosteine in the treatment of respiratory diseases are Regarding the PP populations, patients in Group A had total likely due in part to effects of the drug other than mucolysis. effectiveness rate of 96.33% and a total improvement rate of Relatively little information has been published to date on 99.08%. Group B patients had a total effectiveness rate of the clinical efficacy and safety of letosteine. To the best of 95.61% and total improvement rate of 99.12%. There were no our knowledge, this is the first clinical trial to be conducted significant differences observed in either the total effectiveness in China that compares the efficacy and safety of letosteine rates or in the total improvement rates between groups (PPS to another mucolytic agent. Nespoli et al. (1989) conducted population; Both P-vlaues > 0.05). a pediatric study that evaluated a 25 mg dose of letosteine The secondary outcomes included change of IgA from pre- to given three times daily. That study reported a statistically sig- post-treatments, improvement rates of single symptom score and nificant decrease in fever, a beneficial evolution of thoracic therapeutic efficacy for the FAS and PPS populations (Table 4). objectivity and an improvement in specific respiratory func- There were no significant differences observed in any of the sec- tion parameters (maximum expiratory flow at 75% [MEF 75], ondary outcomes between the two groups (All P-values > 0.05; peak expiratory flow [PEF]) for patients who received letosteine. Table 4). The authors theorized that the beneficial effects of the drug There were 38 patients, 23 in Group A and 15 in Group B, having were likely due to its actions on reducing mucus viscosity and at least once advise effect during the study period (Table 5). enhancing mucociliary clearance. A study on children suffering The total frequencies of AEs were 35 for the patients in Group from acute cough associated with upper respiratory infections A and 28 for the patients in Group B. In addition, 8 of the reported letosteine was superior to placebo in relieving cough- 35 AEs in Group A and 13 of the 28 AEs in Group B were ing symptoms (differences ranging from 0.1 to 0.3 points from probably or possibly related to study drug Moreover, no serious day 4 to day 10) (Schroeder and Fahey 2002). All of these AEs occurred during the course of this study. results are consistent with the findings of the current study in which patients suffering from a variety of respiratory diseases experienced significant improvements from baseline in spu- 3. Discussion tum characteristics, sputum viscosity, sputum volume, ease of The comprehensive analysis of primary parameters and sec- expectoration, and levels of coughing after receiving letosteine ondary parameters showed the therapeutic efficacy of letosteine treatment. (Group A) was not inferior to that for ambroxol hydrochloride A controlled double-masked clinical trial comparing letos- (Group B). The therapeutic efficacy was comparable between the teine to another mucolytic, S-carboxy-methyl-cysteine, was Pharmazie 69 (2014) 843 ORIGINAL ARTICLES

Fig. 1: Flowchart of patient enrollment. conducted on a total of 47 adult patients suffering from various between the two treatments for either changes in respiratory forms of chronic obstructive lung disease (Blaive et al. 1978). symptoms or sputum characteristics. Letosteine (50 mg t.i.d.) Daily observations, made by the patients themselves, and spu- was clinically equivalent to S-carboxy-methyl-cysteine (750 mg tum samples were obtained before, during, and after the course t.i.d.) for the treatment of chronic obstructive lung disease in of mucolytic therapy. No signiﬁcant differences were reported adults.

Table 1: Study design

Parameter Screening Day 1 Treatment Phase Days 5-14 First Follow Up

Informed consent X Demographics X Past history X Cough/expectoration symptoms evaluated X X Concomitant drugs recorded X X Physical exam1 X Laboratory examination2 XX Chest x-ray X X Electrocardiographic exam X X Human chorionic gonadotropin detection3 XX Inclusion/exclusion criteria evaluated X X Diary card provided X Sputum measured4 XX Randomization X Test drugs provided X Side effects recorded X Number of drugs recorded X Sign and symptoms recorded in diary X

844 Pharmazie 69 (2014) ORIGINAL ARTICLES

Table 2: Patient demographics and characteristics

Variables Group A (n = 113) Group B (n = 116) P-value

Demographics Agea, yrs 40.96 ± 15.95 41.91 ± 15.52 0.645 Sexc 0.269 Males 49 (43.4%) 42 (36.2%) Females 64 (56.6%) 74 (63.8%) Medical disease historyc 0.144 No 88 (77.9%) 99 (85.3%) Yes 25 (22.1%) 17 (14.7%) Surgical historyd 0.132 No 105 (92.9%) 113 (97.4%) Yes 8 (7.1%) 3 (2.65) History of drug allergyd 0.683 No 111 (98.2%) 112 (96.6%) Yes 2 (1.8%) 4 (3.4%) Drug use within 1 week of study inclusionc 0.383 No 81 (71.7%) 89 (76.7%) Yes 32 (28.3%) 27 (23.3%) BMIa, Kg/m2 22.90 ± 5.27 22.40 ± 3.15 0.386 Body temperaturea, oC 36.67 ± 0.44 36.7 ± 0.58 0.632 Heart ratea, beats/min. 75.43 ± 8.99 77.05 ± 10.5 0.212 SBPa, mmHg 119.32 ± 10.9 117.53 ± 11.58 0.229 DBPa, mmHg 73.6 ± 7.38 74.4 ± 7.68 0.426 Pre-treatment symptom observationb Sputum characteristics 1.76 ± 0.70 1.76 ± 0.73 0.922 Sputum viscosity 2.23 ± 0.42 2.29 ± 0.46 0.279 Ease of expectoration 1.77 ± 0.50 1.72 ± 0.50 0.498 Sputum volume 1.16 ± 0.58 1.26 ± 0.66 0.258 Cough level 1.51 ± 0.66 1.56 ± 0.70 0.544 Total scores 8.42 ± 1.73 8.59 ± 1.92 0.572 Routine blood testa Hemoglobin, g/L 142.61 ± 16.30 140.38 ± 19.00 0.345 RBC, 1012/L 4.75 ± 0.49 4.74 ± 0.67 0.858 WBC, 109/L 6.40 ± 2.16 6.44 ± 2.60 0.891 Neutrophils, % 59.76 ± 9.43 59.78 ± 8.44 0.984 Lymphocytes, % 31.15 ± 7.69 31.59 ± 7.86 0.666 Platelet, 109/L 204.48 ± 75.11 212.40 ± 67.46 0.405 Biochemical blood testb (M-W) ALT, U/L 22.32 ± 13.96 22.75 ± 15.69 0.770 AST, U/L 20.85 ± 8.27 22.72 ± 12.66 0.159 TBIL, mmol/L 14.37 ± 6.87 13.69 ± 5.83 0.649 BUN, mmol/L 4.72 ± 1.47 5.09 ± 1.93 0.208 Cr, umol/L 63.09 ± 15 62.93 ± 17.36 0.795 ALP, U/L 72.81 ± 25.48 77.58 ± 24.42 0.090 TP, g/L 75.05 ± 5.2 74.38 ± 7.86 0.745 ALB, g/L 45.66 ± 3.39 44.46 ± 4.22 0.092 GLU, mmol/L 5.26 ± 1.4 5.25 ± 1.49 0.593 IgA, g/L 2.04 ± 0.83 2.22 ± 2.01 0.947 Urinalysisb PH level 6.17 ± 0.81 6.17 ± 0.73 0.903 RBC, /HP 0.84 ± 3.32 1.42 ± 4.94 0.398 WBC, /HP 0.95 ± 2.75 1.63 ± 6.25 0.441

Data were presented as mean ± SD for continuous data and n(%) for categorical data by group. abcdDifference between groups were compared using: atwo-sample t-test for data followed normal distribution or bMann-Whitney U test for data didn’t follow normal distribution; cPearson Chi-square test or dFisher’s exact test were performed for categorical data. No significant difference between groups (P < 0.05) was observed from the comparison. Among the 63 side effects observed in the current study, 21 were tively. In addition, the incidences of first-degree atrioventricular related to the study drugs and mainly included dizziness, nausea, block (n = 1) and hiccups (n = 1) were 0.44% and 0.44%, respec- vomiting, dry mouth, elevated aminotransferases and stomach tively (both side effects occurred in Group A). Drowsiness in discomfort. First-degree atrioventricular block was noted in 1 Group B (n = 1) had an incidence of 0.44%. Four patients with patient, drowsiness in 1, leucopenia in 1, and hiccups in 1. The side effects were lost to follow up, and the remaining side effects overall (for both groups) incidences of dizziness, nausea, ele- recovered or improved without special treatment. vated aminotransferases, stomach discomfort, vomiting and dry Our findings confirm that letosteine and ambroxol hydrochloride mouth were low 1.74%, 1.3%, 1.3%, 1.3%, and 0.87%, respec- are both effective and safe for the treatment of sputum thickening

Pharmazie 69 (2014) 845 ORIGINAL ARTICLES

Table 3: Efﬁcacy analysis. Primary outcomes

Group A Group B Primary outcomes Response rate (95% CI) Response rate (95% CI) P-value

FAS population Total effectiveness rate 95.58% (108/113) (91.79%, 99.37%) 95.69% (111/116) (91.99%, 99.39%) 0.999 Total improvement rate 99.12% (112/113) (97.40%, 100%) 99.14% (115/116) (97.46%, 100%) 1.000 PPS population Total effectiveness rate 96.33% (105/109) (92.80%, 99.86%) 95.61% (109/114) (91.85%, 99.37%) 0.948 Total improvement rate 99.08% (108/109) (97.40%, 100%) 99.12% (113/114) (97.41%, 100%) 1.000

P-values were derived using Fisher’s exact test. FAS = full analysis set. PPS = per protocol set. No signiﬁcant difference between groups (P < 0.05) was observed.

Table 4: Efﬁcacy analysis. Secondary outcomes

Secondary outcomes Group A Group B P-value

FAS population (n = 113) (n = 116) Change of IgA from pre- to post-treatments, g/L 0.68 ± 7.15 0.07 ± 0.91 0.237 Improvement rate of single symptom score, % Sputum characteristics 92.04 ± 22.79 90.64 ± 23.47 0.651 Sputum viscosity 88.79 ± 20.71 86.06 ± 22.08 0.336 Ease of expectoration 96.61 ± 15.62 94.54 ± 18.09 0.356 Sputum volume 91.35 ± 29.92 84.74 ± 33.11 0.129 Cough level 64.45 ± 46.80 65.20 ± 42.34 0.899 Total scores 88.48 ± 15.61 85.88 ± 16.70 0.226 Therapeutic efﬁcacy 0.708 Clinically controlled 91 (80.53%) 85 (73.28%) Markedly improved 17 (15.04%) 26 (22.41%) Improved 4 (3.54%) 4 (3.45%) Ineffective 1 (0.88%) 1 (0.86%) PPS population (n = 109) (n = 114) Change of IgA from pre- to post-treatments, g/L -0.04 ± 0.91 0.06 ± 0.91 0.209 Improvement rate of single symptom score, % Sputum characteristics 92.04 ± 22.79 90.64 ± 23.47 0.347 Sputum viscosity 88.79 ± 20.71 86.06 ± 22.08 0.182 Ease of expectoration 96.62 ± 15.62 94.54 ± 18.09 0.375 Sputum volume 91.35 ± 29.92 84.74 ± 33.11 0.114 Cough level 64.45 ± 46.80 65.20 ± 42.34 0.936 Total scores 88.48 ± 15.61 85.88 ± 16.70 0.121 Therapeutic efﬁcacy 0.491 Clinically controlled 90 (82.57%) 83 (72.81%) Markedly improved 15 (13.87%) 26 (22.81%) Improved 3 (2.84%) 4 (3.51%) Ineffective 1 (0.92%) 1 (0.88%)

Data were presented as mean ± SD for continuous data and n(%) for categorical data by group. Difference between groups were compared using Mann-Whitney U test for data didn’t follow normal distribution; Fisher’s exact test were performed for categorical data. FAS = full analysis set. PPS = per protocol set. and expectoration difficulty due to acute or chronic respiratory were used for the study: clinical manifestations and laboratory findings diseases. showing acute tracheobronchitis, acute deterioration of chronic bronchitis, acute deterioration of chronic obstructive pulmonary disease, bronchiec- tasis, lung abscess, pneumonia or other respiratory diseases; concomitant ≥ 4. Experimental expectoration difficulty (score of expectoration: 1) and sputum thickening (score of sputum viscosity: ≥2); a conscious state and the ability to 4.1. Study characteristics autonomously expectorate; discontinuation of expectorants 2 days prior to the study. In addition, patients had to provide informed consent before the This was a multicenter, randomized, double-masked, double-dummy, posi- study commenced. tive drug (ambroxol hydrochloride) parallel controlled trial. The study was approved by the Ethics Committee of the Third Xiangya Hospital of Central South University. Informed consent was obtained from all these patients before study. The Third Xiangya Hospital (lead medical center, Gansu 4.2.2. Exclusion criteria Provincial People’s Hospital, Affiliated Hospital of Inner Mongolia Medical The following key exclusion criteria were used for this study: bronchial College, and Baotou Central Hospital participated in this study to evaluate asthma, severe airway obstruction, or severe respiratory failure; severe the clinical efficacy and safety of letosteine. cardiovascular and cerebrovascular diseases, blood diseases or immune dysfunction; liver or kidney diseases, liver or kidney failure; other cough 4.2. Patients suppressant or anti-sputum drugs taken concomitantly during the study; alcohol or drug abuse; known or suspected sensitivity to the investigational 4.2.1. Inclusion criteria drugs investigated or their components; participation in another study within Inpatients or outpatients 18-75 years of age (either gender) with favorable 3 months in which a drug was investigated; previous inclusion in a study compliance were allowed to participate. The following inclusion criteria involving letosteine. 846 Pharmazie 69 (2014) ORIGINAL ARTICLES

Table 5: Summary of adverse events (AEs)

Experience of AEs Group A (n = 114) Group B (n = 116) P-value

Number of patients having at least once AE 23 (20.18%) 15 (12.93%) 0.139 Have once times 16 (69.57%) 6 (40%) Have twice times 3 (13.04%) 5 (33.33%) Have three times 3 (13.04%) 4 (26.67%) Have four times AE 1 (4.34%) 0 (0%) Total frequency of AEs 35 28 NA Dry mouth, thirst, throat discomfort 4 3 ALT elevation 4 3 Dizziness 2 3 Abdominal pain, diarrhea 1 4 Neutrophil % increase 4 1 AST elevation 2 2 Nausea 2 1 Vomiting 0 3 Drowsiness 2 1 Abnormal WBC 2 1 Slightly elevated TBIL 2 0 ALP elevation 1 1 Blood urea nitrogen elevation 1 1 Positive urinary protein 1 1 Hiccups 1 0 Weakness 1 0 Hemoglobin elevation 0 1 Creatinine elevation 0 1 Erythrocyte elevation 0 1 Decreased blood sugar 1 0 Urinary RBC increase 1 0 Right lower back pain 1 0 Worsened vision 1 0 First degree AV block 1 0 Relationship of AEs to study drug Deﬁnitely related 0 0 NA Probably related 2 3 Possibly related 6 10 Probably not related 23 12 Not related 4 3

AEs, adverse events. P-values were derived using Pearson Chi-square test. NA, not assessed. No signiﬁcant difference between groups (P < 0.05) was observed from the comparison.

4.2.3. Criteria for study termination or patient withdrawal from the there were at least 200 cases for the final analysis, the number of cases was study increased by 20% (ie, 120 cases/group). Random numbers were generated using SAS according to a centre-stratified randomization scheme. Partici- Several criteria were used to determine the appropriate conditions for pants were randomized into the experimental groups and control groups at patients to withdraw from the study: 1. If the disease progressed during the a ratio of 1:1. Patients and investigators were masked as to the study design study, and therapeutic protocol was modified to the extent in which study and treatment. termination and appropriate treatment were required. 2. Patients had poor compliance and the physician or patients asked to terminate the treatment. 3. Side effects occurred, and treatment was terminated according to the advice 4.3. Drugs of the investigators. 4. Severe side effects occurred, and treatment needed to be terminated immediately. Investigational drug: Letosteine (50 mg; batch number: 0812001; period of validity: 2 years) was administered at a dose of 50 mg thrice daily. 4.2.4. Exclusion and expulsion Letosteine was provided by the Bohua Pharmaceutical Co., Ltd. (Xi’an, China). Letosteine was synthesized in a GMP certificated environment and Patients with misdiagnosis, meeting exclusion criteria, meeting inclusion has been qualified before use. criteria but not treated with letosteine, having no medical records during the Control drug: Ambroxol hydrochloride (30 mg; batch number: 08062451; treatment, or not compliant to the therapeutic protocol were excluded from period of validity: 5 years) was administered at a dosage of 30 mg thrice this study. daily. Ambroxol hydrochloride was purchased from Hengrui Medicine Co., Patients meeting the inclusion criteria but not completing the study, accord- Ltd (Jiangsu, China). ing to the predesigned protocol, were regarded as expulsion cases. Patients Placebo: The placebo had no active ingredient but was similar to letosteine were assigned as expulsions for the following reasons: intolerance to study and ambroxol hydrochloride in shape, odor, packaging, labeling, and other medications, poor compliance, unmasking of treatment regimens, progres- features. The placebo was produced by the Huadong Medicine (Xi’an) Bo sion of their disease, complications, severe side effects, and treatment Hua Pharmaceutical Co., Ltd in a GMP certified environment. It was given discontinuation. at the same dosage as that for letosteine and ambroxol hydrochloride. Patients who underwent expulsion received post-treatment follow up. The All drugs and placebos were encapsulated according to the requirements of expulsion rate was expected to be less than 20%. The therapeutic efficacy a double-masked, double-dummy trial. and safety were included for the intent to treat analysis.

4.2.5. Grouping 4.4. Therapeutic protocol According to the Registration and Management of Drugs, at least 200 par- Patients in the treatment group received a letosteine tablet (50 mg) thrice ticipants were required for ﬁnal analysis (n = 100/group). To assure that daily and a placebo thrice daily (after a meal) for 5-14 days. Patients in the Pharmazie 69 (2014) 847 ORIGINAL ARTICLES

Table 6: Clinical symptom index

Score Sputum characteristics Sputum viscosity Ease of expectoration Sputum volume Cough level

0 Foamy Normal No difficulty < 15 ml No cough 1 (mild) Mucous Thin Slightly difficult 15∼50ml Intermittent cough without affecting normal routine 2 (moderate) Mucopurulent Slightly viscous Significantly difficult 51∼100 ml Between mild and severe cough 3 (severe) Purulent Viscous Extremely difficult > 100 ml Frequent cough affecting normal routine and sleep

control group received an ambroxol hydrochloride tablet thrice daily and a 4.7. Statistical analysis placebo thrice daily (after a meal) for 5-14 days. 4.7.1. Demographics and baseline characteristics data analysis The schedule of study assessments and examinations is shown in Table 1. A diary card was provided to the patients. Participants filled in the diary Patient demographics and baseline characteristics were presented as before taking medication each day, and scoring was done according to the means ± standard deviations (SD) for continuous data if the data followed nature and viscosity of the sputum, the extent of expectoration difficulty, a normal distribution (or median with inter-quartiles (IQR: 1st and 3rd quar- and clinical symptoms of cough. The scores before treatment served as the tiles) if the data did not follow a normal distribution). The normal distribution baseline values, and scores after treatment were employed for evaluation was evaluated using Kolmogorov-Smirnov test considering a significance of therapeutic efficacy and to determine whether treatment termination was level of 0.20 (P > 0.20). Differences between groups were compared using required. a two-sample t-test or Mann-Whitney U test if data not follow a normal Treatment was administered for 5-14 days. Treatment was terminated if the distribution. For categorical data, data were presented as n(%) by group and symptoms and signs of participants were improved (acute bronchitis and the differences between groups were compared using a Pearson Chi-square pneumonia: score of sputum viscosity = 0; acute attack of chronic bronchitis: test or Fisher’s exact test if the cell number was less than five. score of sputum viscosity ≤ 1); or the investigators determined whether treatment should be terminated or whether other expectorants should be used if 4.7.2. Efficacy and safety analysis therapeutic efficacy was poor or intolerable side effects were observed. Once the symptoms and signs were not improved after treatment for 14 days (the The efficacy analyses were performed on the full analysis set (FAS) pop- criteria for treatment termination were not met), treatment was terminated ulation and per-protocol set (PPS) population. The safety analysis was and physicians determined whether other treatments were required. conducted on the safety set (SS) population. The definition of the populations were as follows: The full analysis set (FAS) referred to an ideal participant set meeting the 4.5. Observations requirements of the intent to treat analysis. This participant set was derived from the randomly assigned participants who were treated with predesigned Before inclusion, the general information, history of present illness, past drugs at least once. The FAS population was used for the evaluation of ther- medical history, history of medication, and history of sensitization of the apeutic efficacy. The last observation carried forward was used to impute patients were recorded. missing values. The per-protocol set (PPS) was a subset of the FAS pop- Clinical symptoms were recorded daily to evaluate therapeutic efficacy of the ulation meeting the inclusion criteria. Participants in PPS population had test drugs. The nature and viscosity of the sputum, the extent of expectoration favorable compliance, completed the entire study, had a complete CRF difficulty, sputum volume, and clinical symptoms of cough were scored chart, and complete information on the parameters used for the evaluation (Table 6). The sum of five scores before and after treatment was used for of therapeutic efficacy. the evaluation of therapeutic efficacy. The safety set (SS) referred to the participants who were treated with pre- The therapeutic efficacy was evaluated among this group of patients on the designed drugs at least once and recruited for the evaluation of therapeutic basis of the Criteria for Evaluation of Therapeutic Efficacy of Antitussives safety. and Antiasthmatics in the Guidelines for Clinical Studies on new Drugs. The following examinations were performed in participants at the first fol- 4.7.3. Efficacy analysis low up: routine blood test, routine urine test, detection of kidney and liver functions, detection of serum globulin A, electrocardiographic examination, The total effectiveness rate was calculated as follows: chest X ray, and detection of urine HCG in women of child-bearing age. With Total effectiveness rate (%) = [(patients with clinical control + patients with the exception of HCG, these tests were also performed at the third follow-up effectiveness) / total patients] × 100% visit. The total improvement rate was calculated as follows: Total improvement rate (%) = [(patients with clinical control + patients with effectiveness + patients with improvement) / total patients] × 100% 4.6. Evaluation of therapeutic efficacy The primary outcomes were represented as n(%) and corresponding 95% confidence intervals (95% CIs) of the response rate by group. Differences The primary outcomes of this study comprised the nature and viscosity of the between groups were compared using a Pearson Chi-square test or Fisher’s sputum, the extent of expectoration difficulty, sputum volume and clinical exact test if the cell number was less than five. symptoms of cough were scored, and these scores were summed up. Changes Post-treatment IgA level changes and post-treatment therapeutic evaluation in each symptom and serum globulin A after treatment were the secondary scoring of clinical symptoms comprised the secondary outcomes (Supple- outcomes. The changes in vital signs, laboratory findings, side effects, and mentary Table 1). Data were presented as mean ± SD. Differences between adverse effects related to drugs and reasons for withdrawing from this study groups were compared using a two-sample t-test or Mann-Whitney U test were used to evaluate therapeutic safety. if data did not follow a normal distribution. Comprehensive efficacy was calculated as follows: Comprehensive efficacy (%) = (total score before treatment – total score after treatment) / total score before treatment × 100% 4.7.4. Safety analysis Criteria for determination of comprehensive efficacy were calculated as The safety analysis included adverse event (AE) rate and serious adverse follows: event (SAE) rate. The rates of AEs and SAEs were summarized as n(%) and ≥ (1) Clinical control of disease: comprehensive efficacy of 80% compared between groups using Pearson Chi-square test or Fisher’s exact ≥ Effectiveness: comprehensive efficacy of 50% but < 80% test if the cell number was less than five. Improvement: comprehensive efficacy of ≥25% but < 50% Ineffectiveness: comprehensive efficacy of < 25% 4.7.5. Data management and analysis (2) Overall effectiveness rate: the sum of patients with clinical control and those with effectiveness. Data were created and double-entered using Epidata 3.0. A total of 240 Overall effectiveness rate (%) = [(patients with clinical control + patients participants were enrolled into this study. The sample size was determined with effectiveness) /total patients] × 100% based on the requirement of the national food and drug administration, hence (3) Overall improvement rate (%): clinical control, effectiveness and a sample size calculation was not performed. For the 240 patients, 120 were improvement were regarded as clinical improvement and used to evaluate randomized to receive Letosteine (group A) and 120 to receive Ambroxol the overall improvement rate. (group B), respectively. All statistical analyses were performed using SPSS Overall improvement rate (%) = [(patients with clinical control + patients 13.0 (SPSS Inc, Chicago, IL, USA). All statistical assessments were two- with effectiveness + patients with improvement)/total patients] × 100% tailed and considered significant if P < 0.05. 848 Pharmazie 69 (2014) ORIGINAL ARTICLES

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