Abstract
Introduction
Erdosteine, a mucoactive drug, is known to be an antioxidant agent also. 1 Erdosteine is a commonly used agent as a mucolytic for chronic obstructive bronchitis and acute exacerbation of chronic bronchitis in our life practices and pediatric policlinics. When prescribing erdosteine syrup for children, its use is generally recommended for 3–5 days. In a number of studies, erdosteine has been successfully used as protective agent against various toxic agents.2,3 Upon the entry of erdosteine into the body, it rapidly enables formation of three active thiol group metabolites. The first group forms N-thioglycolyl-homocysteine (metabolite I), while the second and third form N-acetyl-homocysteine (metabolite II) and homocysteine (metabolite III), respectively. The substance features mucolytic and antioxidant characteristics through N-thioglycolyl-homocysteine, one of the common metabolites of the thiol derivative erdosteine and N-acetylcysteine; it is used for children because of these properties.4,5
We previously evaluated the effect of erdosteine (10 mg/kg, 7 days) on methotrexate-induced testicular toxicity in C57BL/6 mice (8 weeks, 20–30 g). 2 We noticed that erdosteine qualitatively accelerated clotting in mice. 2 The prospectus details of ERDOSTIN® and literature have been reviewed, but we could not find any information available therein on erdosteine’s effects on hemostasis and coagulation. In man, data resulting in hemostasis and coagulation abnormalities in relation to the unstable disulfide bond with N-acetylcysteine protein exists in the literature. 6 Similarly, increased homocysteine, one of the thiol derivative erdosteine metabolites with a disulfide bond, may enhance platelet aggregation through adenosine diphosphate induction. 7 For the above-mentioned reasons, we have planned to examine the effects of erdosteine on platelet functions and coagulation parameters.
Materials and methods
All the animal experiment protocols were approved by the Animal Ethical Committee of Mustafa Kemal University. Experiments were performed on 29 mixed strain young albino Wistar rats (3 weeks old) which were obtained from the experimental research center of Gaziantep University. Rats were kept in a room maintained at ambient temperature and humidity (25 ± 5°C, 55% ± 5%) under a day/night regime (day 7:00–19:00 and night 19:00–7:00) and allowed a commercial standard rat diet and water ad libitum. Rats were weighed before the experiment and those weighing 40–50 g were divided randomly into four groups.
A total of six young rats were involved in the first group, which is also the control group, and were treated with a placebo. A total of seven young rats were gathered as the second group, receiving 3 mg/kg erdosteine orally (gavage) once every 24 hours for 3 days (Group 1); the third group of seven young rats, received 10 mg/kg erdosteine orally once every 24 hours for 3 days (Group 2); and the fourth group, which included nine young rats, received 30 mg/kg erdosteine orally once every 24 hours for 3 days (Group 3). One ERDOSTIN® capsule was prepared at an appropriate concentration by diluting with sodium bicarbonate. At the end of the experiment, 24 hours after the last dose was given at the end of day 3, general anesthesia with ketamine (50 mg/kg) and xylazine (10 mg/kg) was administered subcutaneously, and the rats' blood was drawn by opening them at the midline and dissecting the portal vein. The blood samples were put into a tube containing sodium citrate 3.8% (4.5 mL blood sample and 0.5 mL citrate mixture) for coagulation analysis. A thin smear between lamina and lamella was performed for peripheral blood smears and they were allowed to dry out. Plasma samples were taken by centrifuge at 3000 g for 10 minutes. Prothrombin time (PT), activated partial thromboplastin time (aPTT) and international normalized ratio (INR) were measured from a tube containing sodium citrate 3.8% by an automatic coagulation analyzer (automatic coagulation analyzer system, Biomeireux, Inc., USA). Normal reference range was PT: 10–15 sec, aPTT: 23–38 sec, INR: 0.8–1.2 sec. By inspecting all areas with light microscopy, platelet counts, sizes, and clumping trends of peripheral blood were assessed and examined for any statistical difference between groups.
Data were analyzed by using a commercially available statistics software package (SPSS for Windows v. 15.0, Chicago, IL, USA). Distribution of the groups was analyzed with a one-sample Kolmogrov-Smirnov test. All groups showed normal distribution, so that parametric statistical methods were used to analyze the data. One-way ANOVA test was performed and post hoc multiple comparisons were made using least-squares differences. Results are presented as mean ± SEM; p < 0.05 was regarded as statistically significant.
Results
PT and INR values in Group 1 were prolonged compared to the control group, which is presented in Table 1. We could not measure PT, INR and aPTT values in Group 2 because the blood in the citrated tubes of Group 2 quickly became clots. There was no difference in values among groups. There were no differences in any parameters in Group 3 compared to control rats. In peripheral blood samples from control, Group 1, and Group 3, platelets were in sufficient amount and their sizes were normal; their clumping tendencies were also of sufficient count. Although all areas were scanned with light microscopy, the peripheral blood from Group 2 presented with insufficient and low platelet counts and no clump formation (Figure 1 ). While drawing blood from Group 2 rats into the tube containing 3.8% sodium citrate, the blood samples in Group 2’s tubes became rapidly clotted compared with control (71% vs. 0%, p < 0.001; Figure 2 ). The clotting occurred in two tubes of both Group 1 and Group 3 but was not statistically significant.
Hemostatic parameters in all groups
ND: Not determined. Results were represented mean ± SEM.
a The clotting occurred in two tubes of both Group 1 and Group 3.
b p < 0.05 versus control group.
Platelets count in peripheral blood of all groups. A. Control group (n = 6), B. Group 1 (n = 7): Rats were given 3 mg/kg erdosteine, C. Group 2 (n = 7): Rats were given mg/kg erdosteinee 10 mg/kg erdosteinee, D. Group 3 (n = 9): Rats were given 30 mg/kg erdosteinee. ⇒ The aggregated platelets.
The coagulated samples of blood tubes in all groups. a, Control group (n = 6), b. Group 1 (n = 7): Rats were given 3 mg/kg erdosteine. c, Group2 (n = 7): Rats were given mg/kg erdosteinee 10 mg/kg erdosteinee, d. Group 3 (n = 9): Rats were given 30 mg/kg erdosteine. All tubes are not seen in the pictures.
Discussion
In humans, data resulting in hemostasis and coagulation abnormalities in relation to the unstable disulfide bond with N-acetylcysteine protein are present in the literature. 6 Similarly, increased homocysteine, one of the thiol derivative erdosteine metabolites with a disulfide bond, may enhance platelet aggregation through an adenosine diphosphate induction. 7 Surprisingly, however, various unknown coagulation mechanisms of erdosteine were observed in this study to be enabled, and hemostasis to be affected, in a dose-dependent manner. We realized that, in the group receiving 3 mg/kg erdosteine, unlike other groups, PT and INR values increased. No platelet counts and clumping occurred in the peripheral blood of Group 2, while the group’s blood coagulated upon being drawn into a tube containing 3.8% sodium citrate. At the same time, we found no significant difference in coagulation tests and peripheral blood of Group 3, which received 30 mg/kg erdosteine when compared to the controls. We discussed N-acetyl cysteine, as our study is the first in the literature to indicate erdosteine’s effects on coagulation defects and hemostasis; since they are common in the thiol group and the disulfide group and have similar antioxidant characteristics, they have similar uses as mucolytics in clinics and policlinic practices. Knudsen et al. gave N-acetyl cysteine IV at therapeutic doses to 10 healthy volunteers in a study in Denmark in 2005. For patients in this group, they observed lower Factor 2, Factor 7, Factor 9 and Factor 10 levels and therefore PT prolongation in relation to the defect in the extrinsic pathway of the coagulation mechanism. 6 We observed a significant increase in Group 1, to which we had given 3 mg/kg erdosteine, in the PT and INR values of erdosteine with similar characteristics to N-acetylcysteine in the study of Knudsen et al., and with common thiol and disulfide bonds. The study Knudsen et al. had conducted is an important one defining the relationship between N-acetylcysteine and coagulation and hemostasis; they have demonstrated the extrinsic pathway to be corrupted and PT to be prolonged. We did not measure the factor levels, but it has been shown that PT prolongation stating the extrinsic pathway to be corrupted is affected in a dose-dependent manner. In two different studies by Jepsen et al. and Burns et al., N-acetylcysteine was shown to have no effect on aPTT.8,9 N-acetylcysteine has been reported to be effective on Factor 2, Factor 5, Factor 7 and Factor 9 but mainly on Factor 1010–12 in the literature. Finding erdosteine molecules to have similar metabolites to N-acetylcysteine supports our study, in which they have affected PT levels by corrupting the extrinsic pathway. Interestingly, it has been shown that erdosteine molecules cause coagulation defects by unknown dose-related mechanisms, particularly affecting the extrinsic pathway and PT levels, and at certain doses, it makes platelet counts insufficient and corrupts clumping tendencies.
Erdosteine given orally is known to be of protective effect with antioxidant activity.13–16 However, no scientific research or prospectus information is present on coagulation and hemostasis. We used 3-week-old young rats in this study to mimic the age of children admitted to a pediatrics unit. Newborns' coagulation systems are underdeveloped, but the hemostatic system of older children is no different than that of adults. The rate of platelet aggregation and platelet microviscosity increased with age in rats in a previous study. 17 In the study referenced, the age differences in the rats were too great: young rats were aged 8 weeks; adult, 35 weeks; elderly, 100 weeks. In fact, hemostasis is not impaired in young rats, while there is a tendency toward thrombosis in old rats. In this experimental study, when using erdosteine frequently in our policlinic practice because of its mucolytic and antioxidant characteristics, it should be taken into account that hemostasis and coagulation defects may be dose-related and the drug, therefore, should be used at an appropriate dose range. Generally, we give 350 mg/day dose of erdosteine for children 15–19 kg, 525 mg/day for children 20–30 kg, and 750 mg/day for children over 30 kg in clinical practice. 18 Doses are calculated per kilogram; the result is 15–25 mg/kg/day. Doses of 3–10 mg/kg result in deterioration of hemostasis, according to our study. Hemostasis was not a problem in higher doses (30 mg/kg). Even so, this is the first and only study revealing erdosteine effects on hemostasis and coagulation mechanisms in a dose-related manner. We suggest that erdosteine should not be used at doses of 10 mg/kg and below. We also suggest that different metabolites of erdosteine may have different influences on hemostasis.
Footnotes
This research received no specific grant from any funding agency in the public, commercial, or not for-profit sectors.