Резюме
Бутко Я.О.¹, Дроговоз С.М.¹, Ляпунов М.О.² Вплив крему «Декспантенол з керамідами» на процес загоєння шкірних ран на тлі імуносупресії.
Проведено вивчення впливу крему «Декспантенол з керамідами» на загоєння шкірних ран на тлі імуносупресії в щурів. Аналіз планіметричних та біохімічних показників показав, що на фоні введення гідрокортизону, тваринам з ранами, спостерігали зниження швидкості загоєння ран, а також порушення показників білкового обміну та імунного захисту. В ході лікування тварин кремами з декспантенолом або з керамідами, або їх комбінації, спостерігали прискорення загоєння ран та нормалізацію біохімічних показників на фоні введення гідрокортизону. Найбільш швидке загоєння ран (до рівня природної регенерації), а також нормалізацію біохімічних показників спостерігали при лікуванні кремом «Декспантенол з керамідами». Слід зазначити, що додаван­ня керамідів у крем, що містить декспантенол, робить позитивний вплив на його ранозагоювальну дію, що свідчить про раціональність даної комбінації з метою підвищення ефективності загоєння ускладненого ранового процесу.
Ключові слова: декспантенол, кераміди, швидкість загоєння ран, імуносупресія.

Резюме
Бутко Я.А., Дроговоз С.М., Ляпунов Н.А. Влияние крема «Декспантенол с керамидами» на процесс заживление кожных ран на фоне иммуносупрессии.
Проведено изучение влияния крема «Декспантенол с керамидами» на заживление кожных ран на фоне иммуносупрессии у крыс. Анализ планиметрических и биохимических показателей показал, что на фоне введения гидрокортизона, животным с ранами, наблюдали снижение скорости заживления ран, а так же нарушение показателей белкового обмена и иммунной защиты. В ходе лечения животных кремами с декспантенолом или  керамидами, или их комбинации, наблюдали увеличение скорости заживления ран и нормализацию биохимических показателей на фоне введения гидрокортизона. Наибольшее ускорение заживление ран (до уровня естественной регенерации), а также нормализацию биохимических показателей, наблюдали при лечении кремом «Декспантенол с керамидами». Следует отметить, что добавление керамидов в крем, содержащий декспантенол, оказывает положительное влияние на его ранозаживляющее действие, что свидетельствует о рациональности данной комбинации с целью повышения эффективности заживления осложненного раневого процесса.
Ключевые слова: декспантенол, керамиды, скорость заживления ран, иммуносупрессия.

Summary
Butko Y.A., Drogovoz S.M., Lyapunov N.А. The study of effect cream «Dexpanthenol with ceramides» on the healing process of skin wounds with immunosuppression.
The influence of the cream «Dexpanthenol with ceramides» on the healing of skin wounds on rats with immunosuppression was study. Analysis of planimetric and biochemical parameters were show that the introduction of hydrocortisone, the animals with injuries, observed a decrease in the rate of wound healing, as well as a violation of the indicators of protein metabolism and immune defense. During the treatment of the animals were the creams with dexpanthenol or ceramides, or a combination thereof, were observed acceleration of wound healing and normalization of biochemical parameters with administration of hydrocortisone. The most accelerating wound healing (the level of the natural regeneration) as well as the normalization of biochemical parameters were observed of during treatment cream «Dexpanthenol with ceramides.» It should be noted that the addition of a ceramides in the cream containing  dexpanthenol, it has a positive influence on its wound healing effect, indicating that this combination is the rational for increase the efficiency of complicated the wound healing process.
Key words: dexpanthenol, ceramides, the rate of wound healing, immunosuppression.

Рецензент: д.мед.н., проф. Л.М. Деримедвідь

УДК 615.015:615.454.122

¹Національний фармацевтичний університет (Харків)

Национальный фармацевтический университет (Харьков)

61002, г. Харьков, ул. Пушкинская, 53

National University of Pharmacy (Kharkov)

Ukraine, 61002, Kharkov, 53, str. Pushkinska

²Державний науково-дослідний центр лікарських засобів (Харків)

«Государственный научный центр лекарственных средств и медицинской продукции» (Харьков)

"State Scientific Center of Drug and Medical Products" (Kharkiv)

yaroslavabutko79@mail.ru

Skin repair is a complex biological process involving interaction between different types of cells and growth factors. Thus, during the skin wound regeneration the cascade of different immunological factors is activated promoting leukocytes migration into the wound, activating neutrophils in the lesion, stimulating growth of conjunctive tissue (causing proliferation of fibroblasts) as well as improving epithelization by regulation of ceratinocyte’s proliferation [1, 5, 8]. Deviation in one or a number of these interactions may significantly effect regeneration of the wound. Thus, low immunity brings into wound-healing process additional difficulties, which should be investigated in order to provide optimal results of the treatment [2, 4].

Skin injuries are followed by the damage of epidermal barrier, which consists of horny layer of the epidermis with the protein-lipid complex [5, 9, 10]. Resulting from the epidermal barrier damage the skin protective function is decreased (increasing the risk of contamination with infection, toxins, allergens and etc.). Expended loss of fluids through epidermis occurs, migration of epithelial cells decreases and the wound healing is delayed. Therefore, recently much attention is dedicated to the state of barrier function and its restoration in the skin injury [10].

Current findings about wound-healing process allow optimizing the treatment of wound and its complications as well as targeted creation of novel or improve already known wound-healing preparations in order to increase efficacy of wound treatment. In this regard, dexapanthenol and ceramides containing cream was developed under the supervision of prof. Lyapunov N.А. Dexapanthenol is a widely known medical substance that stimulates regeneration processes, has anti-inflammatory and dermatoprotective action. Ceramides are important cell membrane lipid components that stimulate cell proliferation, increase skin barrier properties and normalize skin water balance. They are widely used in cosmetology to prevent skin aging and also to improve its elasticity and smoothness[10].

         This study objective was to investigate the efficacy of “Dexapanthenol with ceramides” cream on the skin wound-healing dynamics in immunosuppressed rats.

Materials and methods. Wound regeneration in laboratory immunosuppressed rats was investigated. Immunosuppression was induced by hydrocortisone injections, because this model allows reducing of the wound process speed and assessing the influence of the preparation on skin regeneration in complicated wound process. Glucocorticoids exhibit anti-proliferative properties and can delay normal healing processes in laboratory animals. Hydrocortisone administration leads to reduced number of cell elements in granulation tissue and also to its delayed formation and maturation [2, 4].

          In experiment 30 rats weighting 190-230 g were used. Skin wounds were reproduced on depilated skin area of anesthetized with thiopental animals. The skin area 2х2 cm² (400mm²) was rejected in this case [1]. Solution of hydrocortisone (Pharmak, Ukraine) in dose 25 mg/kg was injected intramuscular: first – a day before wound application and then everyday during the experiment (14 days) [4]. Animals with wounds that received hydrocortisone injections and those that did not, were used as control groups. The treatment was initiated a day after wound reproduction and till the end of the experiment. Animals were divided into 5 groups: group 1 - control pathology (untreated animals with pathology that didn’t receive injections of hydrocortisone); group 2 - control pathology with immunosuppression background (untreated animals with pathology that received hydrocortisone injections); groups 3-5 – immunosupressed animals with pathology that received creams “Ceramides”, “Dexapanthenol with ceramides” and “Bepanthen” (preparation of comparison containing dexapanthenol produced by the company «bayer», Germany) treatment, respectively. Regeneration process of skin wounds was assessed with the help of planimetric rates: wound area (S_stud, mm²) measured a day before the end of the study; healing area (S_heal, mm²) calculated by the formula S_gen=S_outcome–S_exp; wound-healing coefficient (k) calculated by the formula k= S_gen/S_outcome [1].

Protein turnover indices – levels of general protein (GP) and urea estimated with the help of “Filisit-Diagnostic” reagent kits (Ukraine) – have been evaluated at the end of the study in order to assess the influence of preparations on the regeneration process at biochemical level [3]. The immune system stage evaluated with the help of medium sized circulating immune complexes (CIC) (“Granum” kit, Ukraine) [6].

All the interventions and euthanasia of animals were carried out according to standards of the treatment of laboratory animals (Strasburg, 1986). and the І-st National Congress on Bioethics (Kiev, 2001). The results obtained were treated with the program Statistica 6.0 at a significance level of p£0.05 [7].

         Results and discussion. Treatment outcomes of creams study samples in the model of wounds with immunosuppreson background provided in Tabl. 1.

Outcomes analysis (Tabl. 1) have shown that the wound area reduces slowly while natural healing of wound in untreated rats that didn’t receive hydrocortisone injections: on the 5^th day - the wound-healing area was 40.3 mm² and wound-healing coefficient was 0.10; on the 7^th day – 83.0 mm² and 0.21; on the 9^th day – 128.2 mm² and 0.32; at the end of the study – 227.2 mm² and 0.57, respectively. Planimetric rates examination have shown that in untreated rats receiving hydrocortisone injections on the 7^th day immunosuppression contributed to statistically significant suppression of the wound healing – the wound area increased to 449.0 mm2 and violation of the skin barrier function: suppuration was observed in the wound that confirms development of immunosuppression. Subsequently the wounds in rats cleaned slowly from suppuration and  healed: at the end of the study the healing area was 26.2 mm² and healing coefficient0.06, which is 9.1 times lower compared to natural regeneration.

Tabl. 1

Dynamics of the planimetric rates on the model of skin wounds in immunosuppressed rats (n=6)

Примітки: 

1. ^$ – significant deviations from outcome data (р<0.05, t-criteria);

2. * – significant deviations from control pathology (р<0.05);

3. ^# – significant deviations from control pathology with immunosuppression background (р<0.05);

4. n – number of animals in the group.

         Planimetric analysis of wound process with immunosuppression background examination have shown in all groups of treated animals increased wound healing and absence of their suppuration compared to the control pathology with immunosupression. Thus, in animals treated with cream “Ceramides” wound healing starts from the 9^th day of treatment: healing area was 18.0 mm² and healing coefficient 0.04; at the end of the treatment – 99.2 mm² and 0.25, respectively. These findings indicate that application of cream “Ceramides”contributes to the healing process in average by 3.3 times faster than in the control group of immunosuppressed animals, though it doesn’t gain the rate of the natural regeneration. Treatment of complicated wound process with cream “Dexapanthenol with ceramides” have shown that from the first days cream contributes to wound healing and from the 11^th day of treatment the healing area (141.2 mm²) and healing coefficient (0.34) were almost similar to the natural regeneration rates. Treatment of complicated wound process with cream “Bepanthen” is also efficient from the first days but at the end of the study is somewhat inferior to the natural regeneration speed. Thus, on the 11^th day of treatment with cream “Bepanthen” healing area was 108.7 mm² and healing coefficient (0.27); on the 13^th day – 160.0 mm² and 0.40, respectively and at the end of treatment it is on average in 6.4 times faster compared the control pathology with immunosuppression background but in 1.4 times slower than the natural regeneration.

Overall, planimetric study analysis have shown that all examined creames containing dexapanthenol, ceramides or their combination improve regeneration of wounds with immunossupresson background. By the wound healing speed they can be placed in the following way: cream “Dexapanthenol with ceramides” > cream “Bepanthen” > cream “Ceramides”.

Biochemical rates examination (Tabl. 2) have shown the most significant changes in protein turnover – general protein decreased in 1.5 times and urea level increased in 1.8 times – in the blood of immunosuppressed rats with wounds and at the end of the treatment. These changes indicate significant tissue destruction and prevalence of catabolic processes during wound healing. Changes in the immune reaction also suggest the pathological process development, particularly immune complexes formation. The level of CIC of medium sized molecules significantly increased in 2.6 times compared to the intact measures and in 1.2 times compared to the rats with natural regeneration background. Such a significant increase of CIC level in immunosuppression may be possible due to hydrocortisone injections ability to suppress monocyte-macrophage system and increase antigen load, that supports pathogen immuno-complexex formation [4].

Tabl. 2

Biochemical rates investigation in rats with complicated wound process (n=6)

Notes:

1. *– significant deviations from intact animals (р<0.05, t-criteria);

2. # – significant deviations from control pathology (р<0.05);

3. ^$– significant deviations from control pathology with immunosuppression background (р<0.05);

4. n – number of animals in the group.

Biochemical rates normalization appeared in rats with naturally healing wounds: GP level was higher in 1.3 times, urea and CIC levels were lower in 1.3 and 1.2 times, respectively compared to control pathology with immunosuppression.

While treating animals with complicated wounds all preparations contributed to restoration of protein metabolic deviations and immune reaction. The most significant normalization in studied rates was observed in treatment with cream “Dexapanthenol with ceramides” and “Bepanthen” – GP level increased in 1.4 times, urea level decreased in 2.1 and 2.5 times, CIC concentration decreased in 1.5 and 1.6 times, respectively compared to the control pathology with immunosuppression. While treating with cream “Ceramides” moderate restoration of biochemical rates was observed: protein level was higher in 1.2 times, urea level was lower in 1.4 times, CIC level was lower in 1.4 times, respectively compared to the control pathology with immunosupression.

Thereof, analysis of animal serum biochemical investigation results indicates significant deviations in protein metabolism and immune reaction that follow wound healing with immunosuppression. Treatment with study preparations improved normalization of biochemical rates and by the efficacy that can be placed in the following order: cream “Bepanthen” = cream “Dexapanthenol with ceramides” > cream “Ceramides”.

Thus, according to the planimetric and biochemical results in immunosuppressive model of wound the wound healing speed decrease and protein metabolism rates deviations (increased catabolism) and immune reaction were observed. Wound healing increase and biochemical rates normalization along with hydrocortisone injections were observed during local application of creams with dexapanthenol or ceramides, or their combination. The fastest wound healing (to the level of natural regeneration) and also normalization of biochemical rates were observed in treatment with cream “Dexapanthenol with ceramides”. It should be noted, that adding of ceramides into the cream containing dexapanthenol has a possitive effect on its wound-healing action that supports rationality of this combination in order to increase wound-healing process efficiency.

         Conclusions

1. On the model of skin wounds with immunosuppressive background in has been found that utilization of creams containing dexapanthenol or ceramides and their combination improves wound healing in rats.
2. Cream “Dexapanthenol with ceramides” improves wound healing of wounds with complicated wound-healing process to the level of natural regeneration and also supports biochemical rates normalization (GP, urea and medium sized CIC). According to the study results efficacy of cream “Dexapanthenol with ceramides” is higher than the efficacy of reference-preparation.
3. Cream “Dexapanthenol with ceramides” creates favorable conditions for successful healing of the wound with complicated healing process and is a perspective preparation for further pharmacological study in order to have a clinical practice impact.

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