The efﬁcacy of systemic enzyme therapy in the complex treatment of trophic ulcers of venous etiology

The current strategy for the treatment of venous trophic ulcers (VTU) suggests differentiated approach and a combination of conservative and surgical methods. This paper presents the results of the study of efficacy of systemic enzyme therapy (Phlogenzym by Mucos Pharma, Germany) in patients with varicose veins of lower extremities (CEAP class C6) and stage I, II and III VTU. The study included 38 patients aged 12 to 82 years. The patients were divided into the experimental (n = 20) and the control (n = 18) groups. The treatment lasted 1 month. Silcofix Professional wound dressings (Pharmaplast, Egypt) were used. All patients received Detralex (Les Laboratoires Servier, France) and wore class 2 and 3 knee-high compression socks. Patients of the experimental group also received Phlogenzym for 30 days (3 tablets 3 times a day). Total ulcer epithelization was observed in 8 (40 %) patients by week 3, and in 18 (90 %) patients by the end of treatment compared to 4 (22 %) and 9 (50 %) patients in the control group, respectively. In the control group, the regenerative process in the area of the ulcerous defect was less prominent compared to the experimental group. Immunoassays revealed a significant reduction in CD4 + CD25 Bright cells and increased levels of CD4 + CD45RO + T-lymphocytes in the experimental group, corresponding with the observed positive clinical response. The use of immunomodulatory drug Phlogenzym contributed to a more rapid regression of clinical symptoms of chronic venous insufficiency and faster healing of stage I–III venous trophic ulcers. Современная


статья ангиология
Varicose veins of lower extremities (VV) are one of the most common pathologic conditions of the peripheral vessels of lower extremities leading to the development of venous trophic ulcers (VTU) in some cases [1,2]. VTU of lower extremities are a serious problem in modern medicine: in 10 % of cases VTU are persistent and non-responsive to conservative therapy [3]. Relapse rates remain high and range from 4.8 to 31.6 % after surgery and from 15.0 to 100.0 % after complex conservative treatment [4]. It is clear that attempts to influence individual components involved in the pathogenesis of the disease are not effective.
Recent studies show that in the treatment of venous ulcers, elimination of venous hypertension and valvular incompetence seen as the main causes of chronic venous insufficiency (CVI) [5][6][7] does not provide a desired effect. It is necessary to treat microcirculatory disorders that lead to chronic inflammation in the ulcer, constant leukocyte infiltration and changes in the metabolism of endothelial cells [8]. A characteristic feature of chronic inflammation is an imbalance between the humoral and cell-mediated immunity; however, only limited data are currently available on the role of immune disorders in the pathogenesis of VV [9,10]. The few and rather contradictory results of the studies of the immune status of patients with VTU indicate that the severity of trophic disorders depends not only on the anatomical characteristics of the vein, but also on the inadequate response of immune cells, which is a chronic damaging factor [11][12][13][14].
One of the understudied methods of VTU treatment is systemic enzyme therapy (SET). Several studies have shown its efficacy, in particular when using Wobenzym (Mucos Pharma, Germany) for the treatment of CVI of various etiology (post-thrombotic syndrome disease and varicose vein disease) [15,16]. The aim of this study was to evaluate the efficacy of conservative therapy supported with systemic enzymes (Phlogenzym by Mucos Pharma).

METHODS
The study included 38 patients with VV (C6) who received treatment for 1 month. The inclusion criteria were stage I-III recurrent VTU with the area of the ulcerous defect up to 30 cm 2 , and patients' age from 18 to 82 years. The exclusion criteria were the presence of a concomitant pathology: type 1 and 2 diabetes, atherosclerosis of lower extremities, systemic vasculitis, rheumatoid arthritis, hormone and immune therapy.
The patients were divided into two groups. The control group received conservative treatment: wound dressings, compression and phlebotropic drug therapy. It included 18 patients (mean age of 60.1 ± 10.5 years) and consisted of 12 (66.7 %) women and 6 (33.3 %) men; 10 (55.6 %) patients were >60 years of age. The experimental group received conservative treatment and systemic enzyme therapy with Phlogenzym for 30 days, 3 tablets 3 times a day. It included 20 patients (mean age of 61.2±12.6 years) and consisted of 13 (65.0 %) women and 7 (35.0 %) men; 13 (65.0 %) patients were >60 years of age. Each group included 4 patients with stage III VTU and 3 patients with stage I VTU; the rest of the patients had stage II VTU.
In the course of treatment, wound dressings Silcofix Professional (Pharmaplast, Egypt) were used. In patients with stage I VTU, absorbent dressings (Fibrosorb, Fibroclean Ag) were used. Atraumatic povidone-iodine dressings (Silkofix POVI) were applied on the defect with persistent fibrin deposits after exudation has decreased. In patients with stage II-III VTU, hydrocolloid dressings with silver ions (Fibrocold Ag) were used. At the first sign of epithelialization, hypoadhesive mesh coatings with a lipidocolloid complex (Fibrotul, Fibrotul Ag) were used. All patients received Detralex (Les Laboratoires Servier, France) for 1 month and wore knee-high compression stockings (classes 2 and 3).
For the quantitative assessment of CVI symptoms ("heavy legs", pain, swelling, cramps) and the analysis of the wound healing dynamics (healing of the ulcer and the condition of the surrounding tissues), we used the Venous Clinical Severity Score (VCSS) and the ulcer and skin condition score. VCSS was used before and after treatment; ulcer assessment was performed before treatment and after 1 month of treatment.
To assess the discomfort in daily activities, the Visual Analogue Scale (VAS) was used.
Bacteriological analysis of ulcer discharge was performed at the initial visit and after 2 weeks of treatment. The cultures were assessed using semiautomatic analyzers Sceptor and Crystal (Becton Dickinson).
Statistical analysis was performed using Statistica 6.0 software (StatSoft, USA). Statistical significance was determined using Student's t-test. The differences between mean values were considered statistically significant at p <0.05. The data in the tables are presented as М ± m.
All patients provided written informed consent. The study protocol was approved by the Ethics Committee of Pirogov Russian National Research Medical University in 2013.

RESULTS
Complete healing of ulcers in the control group was observed in 4 (22.2 %) patients after 3 weeks of treatment and in 9 (50.0 %) patients after 5 weeks. In the experimental group regeneration was much faster: complete epithelialization of the ulcer was observed in 8 (40.0 %) patients after 3 weeks of treatment and in 18 (90.0 %) patients by the end of treatment (Table 1). Healing was confirmed by the evaluation of such parameters as wound pain, hyperpigmentation, maceration, hyperemia and eczematous dermatitis. In the experimental group, we observed faster ulcer surface clearing of purulent and necrotic tissues, reduction of induration and hyperemia, more active formation of granulation tissue, and marginal epithelialization ( Table 2). The patients of the experimental group also reported more significant pain relief and reduction of discomfort in the area of the ulcer in the course of treatment compared to the control group (Table 3).
Microbiological tests conducted before treatment identified the presence of Staphylococcus aureus in 40 % of patients and its microbial associations in 10 % of patients. After 2 weeks of treatment, only non-pathogenic and opportunistic microbes below the critical level of contamination were found in all patients.
In both groups, the analysis of cell subpopulations showed no significant changes in the absolute and relative counts of СТАТЬЯ КАРДИОЛОГИЯ   -cells), T-lymphocytes (CD3 +cells) and major subpopulations of T-lymphocytes (CD3 + CD4 +and CD3 + CD8 + -cells (Table 4). The analysis of Treg-lymphocytes showed a significant decrease in the absolute and relative counts of CD4 + CD25 Bright cells after treatment in patients who received Phlogenzym (p <0.05) ( Table 4). Conversely, in the control group increased levels CD4 + CD25 +Bright cells were detected. Also, a significant increase in the relative count of peripheral CD4 + CD45RА + T-lymphocytes was seen in the control group, while the experimental group demonstrated an increase in peripheral CD4 + CD45RO + T-lymphocyte count.
The analysis of CD45-T-lymphocytes revealed almost no changes in CD4 + CD45RA + /CD4 + CD45RO + and CD45RA + / CD45RO + ratios after treatment in the control group, while the experimental group demonstrated a significant decrease in these parameters (Table 4).

DISCUSSION
The results of the lymphocyte subpopulation analysis and lymphocyte functional activity in patients with VV (C6) showed beneficial clinical effects of systemic enzyme therapy with Note. The results are presented as M ± m. * -p <0.05 when comparing two values within a group. Phlogenzym, a statistically significant reduction in the number of regulatory CD4 + CD25 +Bright T-cells and an increase in CD4 + CD45RO + memory cells.
The described lymphocyte subpopulations are associated with the inhibition of the synthesis of proinflammatory cytokines and the antigen-presenting function of dendritic cells and macrophages, cell apoptosis induction, decreased generation of type 1 and 2 T-helpers (Th1, Th2) and reduced cytokine production by the latter. This leads to a less effective immune response and contributes to the development of chronic inflammation [17]. An important advantage of memory T-cells that constantly circulate in all organs and tissues of the body even in the absence of any inflammation over naive T-lymphocytes is their ability to detect antigens and eliminate them long before they reach secondary lymphoid structures [18].
After treatment completion, multidirectional dynamics of Treg and memory T-cells content in blood were revealed in the experimental group that corresponded with the observed positive clinical changes. This indicates a more effective immune response in patients who received Phlogenzym.

CONCLUSIONS
The present study demonstrated positive effects of systemic enzyme therapy on the regenerative processes in damaged tissues and on the function of T-cell mediated immunity in patients with VV (C6). The results of this study validate the inclusion of immunomodulatory drugs into the treatment protocol. Immunomodulatory drugs contribute to the regression of clinical symptoms of CVI and accelerate the healing process of stage I-III VTU.