Резюме
Бутко Я.О. Вивчення впливу препаратів з глюкокортикостероїдами та керамідами на гематологічні показники в щурів на моделі алергічного контактного дерматиту.
Проведено вивчення впливу препаратів з глюкокортикостероїдами та керамідами на гематологічні показники за умов алергічного контактного дерматиту в щурів. Аналіз співвідношення клітинних елементів у лейкоцитарній формулі крові показав, що розвиток експерименталь­ного дерматиту супроводжується запаленням, алергійною реакцією сповільненого типу та порушенням імунного захисту. В ході лікування тварин кремом «Мометазон з керамідами» та маззю «Метилпреднізолон з керамідами» спостерігали нормалізацію гематологіч­них показників: ШОЕ, рівня лейкоцитів – лейкоформули та інтегральних показників, що свідчить про зменшення запальних процесів та відновлення імунного захисту за умов патології. За лікувальної дією розроблені препарати не поступалися референс-препаратам.
Ключові слова: глюкокортикостероїди, кераміди, алергічний дерматит, гематологічні показники.
Резюме
Бутко Я.А. Изучение влияния препаратов с глюкокортикостероидами и керамидами на гематологические показатели у крыс на модели аллергического контактного дерматита.
Проведено изучение влияния препаратов с глюкокортикостероидами и керамидами на гематологические показатели в условиях аллергического контактного дерматита у крыс. Анализ соотношения клеточных элементов в лейкоцитарной формуле крови показал, что развитие экспериментального дерматита сопровождается воспалением, аллергической реакцией замедленного типа и нарушением иммунной защиты. В ходе лечения животных кремом «Мометазон с керамидами» и мазью «Метилпреднизолон с керамидами» наблюдали нормализацию гематологических показателей: СОЭ, уровня лейкоцитов – лейкоформулы и интегральных показателей, что свидетельствует об уменьшении воспалительных процессов и восстановления иммунной защиты в условиях патологии. По лечебному действию разработанные препараты не уступали референс-препаратам.
Ключевые слова: глюкокортикостероиды, керамиды, аллергический дерматит, гематологические показатели.
Summary
Butko Y.O. The studying of medications with glucocorticosteroids and ceramides influence on rats hematological parameters on model of allergic contact dermatitis.
Studying of influence of medications with glucocorticosteroids and ceramides on rats hematological parameters in condition of allergic contact dermatitis was carried. Analysis of the ratio of cellular elements in leukocytic formula showed, that the development of the experimental dermatitis was accompanied with inflammation, delayed hypersensitivity and impairment of the immune protection. During animals treatment period with cream “Mometasone with Ceramides” and ointment “Methylprednisolone with Ceramides” the normalization of hematologic parameters was observed: ESR, the level of leukocytes (leukocytic formula) and integral indicators, that testifies reduction of inflammatory processes and restoration of immune protection in conditions of pathology. Developed medications didn’t concede to reference preparations on therapeutic effect.
Key words: glucocorticosteroids, ceramides, allergic dermatitis, hematological parameters.
Рецензент: д.мед.н., проф. А.І. Березнякова

УДК 615.015:615.454.122

Національний фармацевтичний університет (Харків)

Национальный фармацевтический университет (Харьков)

61002, г. Харьков, ул. Пушкинская, 53

National University of Pharmacy (Kharkov) Ukraine,

61002, Kharkov, 53, str. Pushkinska

yaroslavabutko79@mail.ru

Allergic contact dermatitis (ACD) is the most common skin disease. It is a delayed-type hypersensitivity reaction (cellular type), mediated by sensitized lymphocytes [4, 9]. Antigen-presenting cells are very important in ACD pathogenesis. They are macrophages, dendritic cells and Langerhans cells, which accept antigenic information and transmit it to lymphocytes [9]. Activated lymphocytes and Langerhans cells amplify immune response and inflammation reaction. The triggering role in the dynamics of allergic inflammation mediators release belongs to histamine and neuro-lipidomediators. They cause pronounced vasodilation, and induce expression of intercellular interaction molecules on the leukocytes and endothelial cells’ surface. As the result, stimulate their migration and production of cytokines and macrophages, affect the immunogenesis and modulate lymphocyte proliferation [4, 10]. It should be noted, that there is a sequence in the migration of blood cells to the inflammatory lesion: at first – polymorphonuclear leukocytes (neutrophils, eosinophils and basophils), then monocytes and in the end lymphocytes. When the inflammatory process ends, these blood cells disappear from the lesion [4, 9].

Glucocorticosteroids (GCS) are drugs of choice for treating immune-dependent inflammatory skin diseases, because they have influence on different links in the pathogenesis of the disease [2, 4]. But it should be noted, that they have immunosuppressive effect due to the disturbance of the leukocyte migration, inhibition of lymphocyte proliferation, and as a consequence reducing of the synthesis of immunoglobulins, cytokines, interleukins and interferon occurs [2]. Therefore, it is important to search and develop drugs with GCS, which would act locally in the area of inflammation and would not show suppression of the immune and circulatory systems to avoid serious side effects.

The main aim of this study was investigation of the influence of medications with GCS and ceramides on haematological indicators in rats in allergic contact dermatitis caused by dynitrochlorbenzol (DNCB).

Materials and methods. Objects of the study were cream “Mometasone with Ceramides” and ointment “Methylprednisolone with Ceramides”. These medications were developed under the direction of prof. Lyapunov M. O. Comparison medications were cream “Elocom” (containing mometasone furoate, manufacturer “Schering Plough”, Belgium) and ointment “Advantan” (containing methylprednisolone atseponat, manufacturer "Schering AG", Germany).

In experiment 36 female rats weighting 220-250 g were used divided in 6 groups (6 rats each): the first group – intact animals, the second – control pathology, the third – animals treated with cream “Mometasone with ceramides”, the fourth – with cream “Elocom”, the fifth – with ointment “Methylprednisolone with ceramides”, the sixth – with ointment “Advantan”. To induce pathology in rats we depilated areas of skin on both sidepieces, initial sensitization was performed by coating the depilated skin areas on the left side with 0.1 ml of 5% alcoholic solution of DNCB [3]. We began treatment in 24 hours after the second application of allergen on the right animal sidepiece (the 3^rd day of the experiment). To evaluate the severity of systemic manifestations of allergic inflammation we measured blood parameters in the dynamics of the experiment: the total leukocyte count, ESR, leukocyte formula.

According to leukocyte formula we counted integral hematological indices: leukocyte and VES ratio (LVESR), leukocyte index (LI), leukocytic and granulocytic index (LGI), neurophil-monocyte ratio (NMR), lymphocyte-monocyte ratio (LMR), lymphocyte-eosinophil ratio (LER), leukocyte intoxication index (LII), leukocyte shift index (LSI), neurophil-lymphocyte ratio (NLR) [1, 6]. Integrated assessment of hematological indices allows estimating development, severity and progress of the pathological process, the state of non-specific and specific parts of the immune system, and evaluating the effectiveness of therapy.

Statistical analysis of results was performed by methods of variational statistics using the Mann-Whitney test, discrepancy within р<0,05 was considered reliable [7].

Results and discussions. During the experiment after re-sensitization with DNCB in animals we observed the development of dermatitis, which manifested in hyperemia, diffuse edema and erythema. Development of inflammation is characterized by impaired blood circulation, changes in blood rheology and involvement of different types of cells, including cells of bone marrow origin: polymorphonuclear leukocytes (neutrophils and eosinophils), lymphocytes and monocytes [4, 8].

The dynamics of hematological parameters gives reliable evidences of ACD pathology development (peak of pathology): ESR level increase in 5-6 times and leukocytes increase in 1.7-1.9 times over the initial data (tab. 1). Increase in total leukocyte count indicates the immune stress of the organism associated with activation of phagocytosis and increase in the number of cells that produce inflammatory mediators, humoral and cellular immunity factors. On the 7^th day of observation in CP group a significant 2.3 times decrease in ESR level occurred, and in groups treated with creams “Elocom” and “Mometasone with ceramides” – 3.1, 3.9, 3.6, and 3.7 times decrease respectively, below the peak of pathology. It should be noted, that normalization of ESR in the groups treated with medications containing GCS and ceramides was slightly higher than in groups which received applications of reference medications that indicates the effectiveness of the local therapy.

Also we observed the normalization of the level of leukocytes in all groups. In the CP group, the level of leukocytes had 1.2 times decrease, and in groups treated with creams “Elocom” and “Mometasone with ceramides” - 1.3 times decrease, with ointment “Methylprednisolone with ceramides” - 1.4 times and ointment “Advantan” - 1.5 times decrease respectively, below the peak of pathology. It should be mentioned, that in groups treated with medications, the level of leukocytes was significantly lower than in the CP group, but significantly higher than the output data, that indicates a gradual decrease in the inflammatory process.

Table 1

The influence of studied medications on the hematological parameters of blood in rats with ACD (n=6)

Notes: 

1. ¹ – significant deviations from intact animals (p<0.05, Mann-Whitney criterion);

2. ² – significant deviations from peak of pathology (р<0.05);

3. ³ – significant deviations from control pathology (р<0.05);

4. SN – segmented neutrophils, SNN – stabnuclear neutrophils;

5. MF – mometasone furoate, MPA – methylprednisolone atseponat, C – ceramides;

6. n – number of animals in the group.

The predominance of various forms of leukocytes in the pathological process is determined by the specific and etiology of disease. In the course of ACD development the increase in general leukocyte level occurs in parallel with the increase in the level of eosinophils, which is the diagnostic sign of allergic inflammation according to the literature. Increasing number of monocytes in the leukocyte formula is associated with the activation of phagocytosis of damaged cells in the inflammatory lesion.

Analysis of the leukocyte formula (tab. 1) showed that in the peak of ACD a significant increase in eosinophils in 2.8 times on average occurred, in segmented forms of leukocytes in 1.5 times, in monocytes – in 2.0 times, and 1.2 times decrease in lymphocytes, that indicates inflammation and increasing cellular link of immunity. Significant changes in the number of stabnuclear forms of leukocytes in the leukocyte formula in animal studies were not observed, that confirms the absence of the infectious component.

At the end of the experiment in all groups we observed normalization of the leukogram: level of SN and lymphocytes normalized, but high level of eosinophils remained. So, on the 7^th day in groups of animals treated with creams “Elocom” and “Mometasone with ceramides, ointments “Methylprednisolone with ceramides” and “Advantan” a significant increase in the percentage of lymphocytes (in 1.1-1.2 times) and a significant reduction in SN (in 1.2-1.3 times), in eosinophils (in 1.4-1.6 times) and in monocytes comparing with the peak of pathology occurred. Significantly lower percentage of lymphocytes was in groups treated with ointment “Advantan” and cream “Elocom” compared to CP. Dynamics of other leukocyte fractions was not essential.

Analysis of leukocyte indices in animals with ACD showed that this pathology is accompanied by inflammation and immune responsiveness violation. Thus, the development of ACD (peak of pathology) in rats in all groups was accompanied by a significant 11.3 times increase of LVESR compared to the output data, resulting from leukocytosis and increase in ESR that indicates the intensity of the inflammatory response in simulation dermatitis (fig. 1). This probable LSI increase (in 1.8 times) and LER index reduction (in 3.8 times) indicates active inflammation and the development of delayed-type hypersensitivity reactions in accordance to the literature available [4, 9]. Significant reduction of LI (in 1.7 times) and NLR increase (in 1.8 times), that indicates suppression of humoral and cell-level prevalence of immune protection. Reduction of LMR (in 2.5 times) indicates the predominance of effective level of immune protection over affective and as a result reduce in LGI (in 1.9 times). Reduce in NMR (in 1.2 times) indicates increased inhibition of macrophage and microphagocyte level in animals’ protection. It should be noted, that due to eosinophilia and monocytosis LII tended to decrease in 1.3 times on average, which indicates the absence of significant intoxication in rats with DNCB dermatitis.

At the end of the experiment in all groups the normalization of haematological indices in the blood of rats with ACD compared to the peak of pathology was observed, that indicates reduce of inflammation and normalization of immunity in animals.                          

Fig. 1. The level of haematological indices in rats with ACD in treatment with medications

Thus, in animals that were not treated, the level of LVESR significantly decreased in 2.7 times compared to the peak of pathology, but was significantly higher compared to output data, that indicates the presence of expressed inflammation. In animals receiving treatment the highest level of LVESR normalization was observed in application of ointment “Methylprednisolone with ceramides” (7.5times decrease in index) and cream “Mometasone with ceramides” (5.2times decrease) compared to the peak of pathology, that indicates the effectiveness of local therapy. According to the influence on LVESR level the efficiency of ointment “Methylprednisolone with ceramides” was 1.5 times higher compared to ointment “Advantan”, and of cream “Mometasone with ceramides” 1.3 times higher compared to cream “Elocom”.

As the result of suppression of inflammation and reactivation of immune responsiveness, in all groups 1.4 times reduction in LSI on average and 1.6 times increase in LER were observed, compared to the peak of pathology. The normalization of LI, NLR, LMR, NMR and LGI indices occurred, that indicates rebalance in the immune protection. Thus, the CP group and groups treated with the study medications had significantly increased LI, LGI indices and decreased NLR index in 1.4 times on average, compared to the peak of pathology.

Influence of the study medications on NMR and LMR indices was somehow ambiguous. In animals that were not treated the level of NMR practically didn’t change. In groups treated with ointment “Methylprednisolone with ceramides” and cream “Mometasone with ceramides” 1.2 times increase in this parameter occurred and in group treated with ointment “Advantan” and cream“Elocom” 1.1 times reduction in NMR was observed, compared to the peak of pathology. Also, the level of LMR increased the most in CP group and groups treated with ointment “Methylprednisolone with ceramides” and cream “Mometasone with ceramides” 1.5 times increase on average compared to the peak of pathology, caused by normalization of lymphocytes. In groups treated with ointment “Advantan” and cream “Elocom” only 1.2 times increase in LMR level occurred compared to the peak of pathology, which may indicate the systemic exposure of the reference preparations and the manifestation of immunosuppressive action of GCS.

Thus, the studied ointment “Methylprednisolone with ceramides” and cream “Mometasone with ceramides” in ACD model in rats, according to the haematological parameters and evaluation of leukocyte indices, inhibit the inflammation, reduce delayed-type allergic reaction and have positive effect on recovery of the immune protection. Analysis of haematological studies showed that the influence of cream “Mometasone with ceramides” and ointment “Methylprednisolone with ceramides” on the indices that reflect the effectiveness of the anti-inflammatory therapy (ESR, total leukocyte count, and LVESR, LSI) did not inferior to therapeutic action and tend to exceed it of cream “Elocom” and ointment “Advantan”. This fact indicates that adding ceramides to GCS does not decrease its anti-inflammatory action and there is no antagonism between them. Also, the highest normalization of immune protection (LI, NLR, NMR and LMR) was observed in treatment with cream “Mometasone with ceramides” and ointment “Methylprednisolone with ceramides” that indicates a reduced suppressor action of GCS on the immune system. This fact can be explained by the adding of ceramides, which act in the superficial layers of the skin and can concentrate the active substance in the epidermis, GCS flow to the blood have been reduced and the risk of SE have been decreased.

Results:

1. Hematological parameters were analyzed in the model of allergic contact dermatitis in rats. It was found that development of experimental dermatitis is accompanied by inflammation, allergic reactions and delayed-type immune protection violation.

2. During the treatment of animals with cream “Mometasone with ceramides” and ointment “Methylprednisolone with ceramides” we observed the normalization of hematological parameters: ESR, level of leukocytes – leukocyte formula and integral parameters (LVESR, NLR, LII LMR, NMR, LER and LGI), indicating the reduction of inflammatory processes and improvement of immune protection in conditions of pathology. Study medications were not inferior to cream “Elocom” and ointment “Advantan” in therapeutical effectiveness.

3. Further pharmacological study of drugs with GCS and ceramides is perspective in order to improve the effectiveness and safety of the inflammatory skin diseases treatment.

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