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1 MINISTRY OF EDUCATION AND TRAININ THE THESIS WAS DONE IN108 INSTITUTE OF CLINICAL MINISTRY OF DEFENCE 108 INSTITUTE OF CLINICAL MEDICAL AND MEDICAL AND PHARMACEUTICAL SCIENCES PHARMACEUTICAL SCIENCES Supervisors: Dang Van Em, Associate professor, PhD LE THI HONG THANH Reviewers: RESEARCH ON SOME CYTOKINE CHANGES AND THE EFFICACY OF CYCLOSPORINE A IN PATIENTS WITH PSORIASIS VULGARIS The thesis defense was presentedtoThesis committee at: 108 Institute of Clinical Medical and Pharmaceutical Sciences Day Speciality: Dermatology Month Year 20 Code: 62720152 The thesis can be found at: SUMMARY OF PHD THESIS IN MEDICINE National Library of Vietnam Library of 108 Institude of Clinical Medical and Pharmaceutical Sciences Ha Noi – 2021 INTRODUCTION New contributions of the thesis - Quantification of serum levels of IL-2, IL-6, IL-8, IL-10, IL12, IL-17, TNF-α, IFN-γ before and after treatment with Cyclosporine A: + Before treatment: serum levels of IL-6, IL-8, IL-10, IL-12, IL-17, TNF-α, IFN-γ were higher than the control group There is a relationship between IL-6 and IL-8, IL-10, IL-12, TNF-α, between IL-8 and IL-12, TNF-α, between IL-10 and TNF-α, and between IL 12 with IFN-γ + After treatment: only the serum IFN- concentration decreased significantly, while the serum IL-2, IL-6, IL-8, IL-10, IL12, IL-17 levels did not decrease compared to that with pretreatment - Cyclosporine A is effective in the treatment of severe psoriasis: + PASI from 25,377±4,202 after 10 weeks of treatment reduced to 5.497±3,741 (reduced by 78.34%) In which, 74.28% good, 22.86% good and 2.86% poor results + Few unwanted effects (headache 5.71%, hypertension 2.86%, fatigue 11.43%) and liver function tests, kidney function tests, fat groups before and after treatment are within limits normal + However, when stopping treatment, the recurrence rate is quite high (after month 29,41%, months 58,82% and after months 82,35%) Chapter OVERVIEW 1.1 Psoriasis vulgaris 1.1.1 Epidemiology Psoriasis had been described since antiquity, in the Hippocratic literature, and the disease had many names Prevalence of Psoriasis: psoriasis occurred from children to the elderly, across the continents But depending on the author, depending on the location, the rate of the disease was different According to Egeberg A et al (2020), psoriasis accounted for 2-3% of the world population, in the Nordic countries from 8–11% of the population In Vietnam, according to documents from the Department of Dermatology, Hanoi Medical University, psoriasis accounted for 2.2% of the total number of patients were examined in 2010 at the National Hospital of Dermatology Sex ratio: Psoriasis prevalence is estimated to be equal in Psoriasis is a chronic inflammatory dermatosis that affects approximately 2- % of the world’s population Clinical symptoms of psoriasis are diverse, including red patches of skin covered with thick, silvery scales, clear boundaries The pathogenesis of the disease is still unclear, but up to now, the majority of authors have agreed that psoriasis is a genetic skin disease, a genetic skin disease, has an autoimmune mechanism, has epidermal cell overgrowth and is triggered by a number of factors (stress, infection, trauma, etc.) mechanical damage, physics, weather, the role of drugs, food, etc.) There are many cytokines involved in the pathogenesis of psoriasis, but the IL-23/Th17 axis plays a central role Treatment of psoriasis is still difficult even though there are many drugs and treatment methods In particular, Cyclosporin A has the effect of inhibiting the activation of TCD4+ cells, inhibiting the chemotaxis of neutrophils, thereby affecting the immune disorders in psoriasis, including the role of some cytokines such as IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, TNF-α, IFN-γ In Vietnam, there are no studies to determine the change of cytokines before and after treatment and the effectiveness of Cyclosporin A treatment for plaque psoriasis vulgaris Therefore, we conducted a study on the topic: “Research on some cytokine changes and the efficacy of cyclosporine a in patients with psoriasis vulgaris” with with the following objectives: To describe demographic and clinical characteristics of psoriasis patients at The clinical of outpatients with psoriasis National Hospital of Dermatology To investigate the change in the concentration of IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, TNF-α, IFN-𝜸 in the serum of patients with severe psoriasis vulgaris before and after treatment with Cyclosporin A To evaluate the efficacy of treatment by Cyclosporin A for severe psoriasis vulgaris 3 males and females Age: While it can begin at any age, psoriasis is high at age 5560 years Clinical types of psoriasis: Plaque-type psoriasis, occurring in 85%–90% of affected patients, is the most common form of psoriasis, following by special types (10-15%), including localized and generalized pustular psoriasis, erythrodermic psoriasis, and psoriatic arthritis 1.1.2 Clinical features The primary lesion is erythematous, thick, well-demarcated patches of scaly surface, often localized to the skin in areas of pressure, often symmetrical Psoriasis patients often itch more or less, depending on each person, each type, each stage of the disease There are several clinical types of psoriasis vulgaris, including plaque psoriasis, nummular psoriasis, guttate psoriasis According to Gudjonsson J et al (2007), common psoriasis is divided into types: Type 1: Age of early onset (before age 40), genetic factors, family history, carrier of HLA-Cw6 and DR7, age of onset less than 40 years Type 2: Late age of onset (after 40 years), no genetic factors, no family history, no HLA-Cw6 and DR7 carriers Histopathology:hyperkeratosis,parakeratosis,hyperpigmentatio n, Munro microabscess, papillary hyperplasia, inflammatory cell infiltration, dilated portal capillaries under the dermis 1.1.3 Pathogenesis The pathogenesis of the disease is still unclear, but up to now, the majority of authors have agreed that psoriasis is a genetic skin disease, a genetic skin disease, has an autoimmune mechanism, has epidermal cell overgrowth and is triggered by a number of factors (stress, infection, trauma, etc.) mechanical damage, physics, weather, the role of drugs, food, etc.) There are many cytokines involved in the pathogenesis of psoriasis, but the IL-23/Th17 axis plays a central role Genetic factors in psoriasis include family history; HLA plays a key role in the initiation, regulation and implementation of the immune response, which is passed on to the next generation; psoriasis-related genes (PSORS-1: 6p21.3, PSORS-2: 17q25, PSORS-3: 4q, ) Factors that trigger the disease, make it worse or worse: stress, localized infections, epidermal trauma, drugs, irritants, climate, weather, diet, etc Immune disorders associated with the pathogenesis of psoriasis are extremely complex Under the influence of many factors, first, Langerhans cells in the skin capture and process information about antigens (to date, the exact antigens have not been identified) and become sensitive antigen-presenting cells Langerhans cells penetrate the basement membrane, present to the T-lymphocytes in the lymph nodes, and the T-lymphocytes are activated The activated T lymphocytes are mainly CD4+, CD8+ When activated, T lymphocytes enter the circulatory system, interact with endothelial cells, migrate to inflammatory skin, gather around dermal blood vessels, and induce immune responses through cytokines, chemokines There are many cytokines secreted by T lymphocytes and other cells Under the action of cytokines, hyperplasia of epidermal cells, inflammation, angiogenesis In the pathogenesis of psoriasis, there are three important links: dendritic cells, keratinocytes and lymphocytes, whose main role is T lymphocytes (Th1 and Th17), which activate the production of corresponding cytokines such as IL-2, IL-4, IL-6, IL-8, IL-12, IL-17, IL-23, TNF-α, IFN-γ Figure 1.4 Pathogenesis of psoriasis (Source: J Am Acad Dermatol 2014., 71(1): 141 – 150) [69] 1.1.4 Treatment To date, there is no cure for psoriasis Therefore, the goal of treatment is to improve the patient's quality of life The treatment needs a specific and reasonable strategy, appropriate to each stage of the disease (attack and maintenance) and disease type In which the goal of the attack phase is to clean the lesion and minimize the unwanted effects of the treatment The goals of the maintenance phase are to prevent recurrence, eliminate and control the trigger factors and treat the lesions that reappear early Current psoriasis medications include as follows: topical drugs (topical, topical and bath) and systemic drugs and biologic drugs, phototherapy, and photochemotherapy 1.2 Pathophysiological role of cytokines in psoriasis Cytokines are biological, low-molecular-weight proteins, produced by immune cells, involved in cell differentiation, and in the regulation of immune responses and inflammation The cytokinemediated response is a complex, multi-component process that is essential for the body's natural defenses, but when overproduction of cytokines in the skin and serum are leads to proliferation and differentiation of keratinocytes as well as immune cells causing psoriasis lesions Cytokines are produced by many cells (dendritic cells, Langerhans, Th1, Th2, Th17 ) including IL-1, IL-2, IL-4, IL6, IL-8, IL-10, IL- 12, IL-15, IL-17, IL-18, IL-22, IL-23, TNF-α, IFN-γ These cytokines interact with each other to cause hyperkeratosis, inflammation of the epidermis and dermis, creating the clinical features of psoriasis 1.3 Treatment of psoriasis vulgaris with Cyclosporine A Cyclosporine A acts on many types of cells, but its strongest effect is on T-lymphocyte function (TCD4 +): Inhibits activation of T-lymphocytes (mainly TCD4 +), inhibits chemotaxis neutrophils, acting on keratinocytes, inhibiting KN expression of Langerhans cells in culture and in psoriasis patients CyA is indicated: Moderate, severe common psoriasis; Psoriasis treated with other methods failed such as PUVA, MTX Contraindications: Diseases causing increased serum creatinine (chronic glomerulonephritis, renal failure ), hypertension, history of cancer and immunodeficiency diseases, severe infections Initial dose: 2.5-3 mg/kg/day, divided into times morning and evening Side effects: Renal dysfunction, hypertension, infection, increased liver enzymes, isochromic anemia, hyperlipidemia, fatigue, weight loss, headache Chapter SUBJECTS AND METHODS 2.1 Research subjects 130 patients with confirmed psoriasis vulgaris were treated at the clinical of outpatients with psoriasis - National Hospital of Dermatology from October 2016 to October 2019 In which: - Objective 1: 130 patients with a definitive diagnosis of psoriasis vulgaris, to study some related factors and clinical features - Objective 2: 35 patients with severe psoriasis vulgaris (study group) and 44 normal people (control group) who are similar in age and gender with the research group to study changes in some cytokine levels before and after CyA treatment - Objective 3: 35 patients with psoriasis vulgaris were treated with CyA - Diagnostic criteria The diagnosis of psoriasis is primarily clinical: psoriasis vulgaris including plaque psoriasis, nummular psoriasis and guttate psoriasis Histopathology: keratosis, parakeratosis, granulomatous loss, squamous growth, infiltrates, Munro abscesses and capillary changes in the dermal papilla In our study, patients with atypical clinical symptoms were tested for histopathology - Inclusion criteria Objective 1: patients with definitive psoriasis vulgaris diagnosis Objective 2: + Patients with severe psoriasis vulgaris over 18 years old No contraindications to CyA, consent to study participation + Control group: Similar in age, gender and no metabolic disorders, cancer, autoimmune diseases, infections Obtained at 16A General Hospital, through periodic health examination Participants were given a clear explanation of the purpose of joining the control group, and agreed to participate Objective 3: patients aged at least 18 years suffering from severe psoriasis; had no contraindication to CyA and agreed to participate - Exclusion criteria Objective 1: Refused consent Objective 2,3: + Psoriasis patients with mild to moderate psoriasis; < 18 years old + There are contraindications to the use of CyA + Do not consent to participate in the study + The patient is using other systemic drugs: MTX, retinoids, probiotics within the past month + The patient is suffering from acute and chronic infections 2.2 Research materials - Neoral tablets 25 mg, 100 mg manufactured by Novartis - Cetaphil moisturizer produced by Gelderma, distributed by DSKH Vietnam - Kit and chemicals for testing cytokines (IL-2, IL-6, IL-8, IL10, IL-12, IL-17, TNF-α, IFN-γ) manufactured by Invitrogen (USA) export Luminex machine system 2.3 Research methods 2.3.1 Study some related factors and clinical features - Study design: Cross-sectional, prospective - Sample size: Convenient sampling: patients with psoriasis vulgaris were treated at the clinical of outpatients with psoriasis National Hospital of Dermatology from October 2016 to October 2019 In which: - Steps of the research process • Examination and selection of patients to participate in the study Data collection (according to the research case form) • Patient code, information on characteristics of research subjects: age, gender, occupation, geography, time of onset, duration of illness • Clinical features: Reason for admission, first lesion location, current lesion location, associated lesions • Clinical form: normal psoriasis • Factors associated with: family history, stress, medications, food, season of the year, comorbidities Summary of results 2.3.2 Investigate the change in the concentration of IL-2, IL-6, IL8, IL-10, IL-12, IL-17, TNF-α, IFN-𝜸 in the serum of patients with severe psoriasis vulgaris before and after treatment with Cyclosporin A - Study design: Cross-sectional, prospective, comparative control - Sample size: calculated according to the formula of the World Health Organization Calculation results of group sample size n1 = n2 ≥ 30 patients Thus, each group must be at least 30 patients - Steps of the research process + Study group of patients • Selection of qualified patients Collect data • Take blood for the first test: ml of venous blood for routine testing (CTM, AST, ALT, urea, creatinine, cholesterol, triglycerides); centrifuge, save serum for quantitative testing of IL-2, IL-6, IL-10, IL-12, IL-17, IFN-γ, TNF-α (freezer -80 degrees C continuously until during testing) and coding the patient number recorded on the sample and keeping a record • Carry out treatment according to the protocol • Take blood for the second test: after 10 weeks of treatment take 5ml of venous blood to the same test as the first time + Control group of patients • Similarity in age, gender with the study group • Take blood for one time for immunoassay: ml of venous blood, centrifuge and save serum to make quantitative tests for IL-2, IL-6, IL-10, IL-12, IL-17, IFN- γ, TNF-α Encode samples and save notes + Result evaluation: Levels of cytokines before and after treatment Compare the concentration of cytokines in the study group with the control group 2.3.3 Evaluate the efficacy of treatment by Cyclosporin A for severe psoriasis vulgaris - Study design: Self-assessment clinical trial before - after treatment - Sample size: As the sample size of the research group of target is ≥30 patients with normal psoriasis with severe severity - Steps of the research process • Select patients as objective group Treatment with CyA • Conduct treatment with dose: 2.5-3mg/kg/day Drink times morning and evening before meals 10 weeks treatment • Apply Cetaphil cream once a day in the morning • Evaluate the effectiveness of treatment after weeks, 10 weeks of treatment with PASI index (PASI decreases after treatment) • Evaluation of unwanted effects: clinical symptoms and liver, kidney function tests, blood lipids after weeks, 10 weeks of treatment Clinical: hypertension, fatigue, headache, weight loss • Changes in tests: AST, ALT, creatinine, cholesterol, Triglyceride • Evaluate recurrence after month, months, months after the end of treatment 10 weeks Patients were assessed for relapse when the PASI index was >20% compared with the PASI at the time of treatment discontinuation 9 2.3.4 Research tools and techniques - Assessment of disease severity based on PASI index: Mild (PASI=20) PASI= 0.1(E+I+D) Ah+0.2(E+I+D) Au+0.3(E+I+D) At+0.4(E+I+D) Al - Evaluation of treatment results: clinical effectiveness is calculated by percentage reduction of PASI according to the formula: (PASI before treatment - PASI after treatment)/ PASI before treatment ´100) Based on PASI reduction, it is divided into some levels: Very Good: PASI down 100% o Good: PASI down 75%-0.05 3.2.2 Cytokine serum levels before the treatment (X±SD) IL10 IL12 IL6 IL8 IL10 IL12 IL17 TNF-α IFN-γ r -0,23 0,24 -0,14 0,14 -0,04 0,12 -0,06 p r 0,19 0,17 0,57** 0,43 0,40* 0,43 0,37* 0,83 0,16 0,49 0,57** 0,75 0,08 0,000 0,02 0,23 0,19 0,03 0,58** 0,000 0,32 0,07 0,35 0,20 0,26 0,05 0,77 0,14 0,43 0,000 0,78** 0,000 0,36* 0,03 0,67** 0,000 0,66 0,16 0,37 0,13 0,45 0,45** 0,01 p r p r p r p 13 IL17 TNFα 14 r 0,17 0,28 p 0,32 0,10 r 0,26 p 0,13 Conclusion: There was no relationship between the concentration of cytokines and the PASI index (p>0.05) There was an association between IL-6 and IL-8 (r=0.57, p0.05 3.3.3 Side effects Table 3.26 Blood test results before and after treatment (n=35) Before After After 10 p (Sign (1) weeks (2) weeks (3) test) Creatinin 74,24 ± 75,78 ± 74,11 ± 17,75 p12 > 0,05 (𝜇𝑚𝑜𝑙/𝐿) 16,33 17,44 p13 > 0,05 Cholesteron 4,34 ± 4,66 ± 4,84 ± 0,98 p12 >0,05 (mmol/L) 0,91 0,94 p13 < 0,01 Triglycerid 1,33 ± 1,65 ± 1,48 ± 1,07 p12 > 0,05 (mmol/L) 0,67 1,31 p13> 0,05 GOT (U/L) 28,45 ± 25,18 ± 23,74 ± 7,35 p12 > 0,05 17 GPT (U/L) leukocytes (G/L) Neutrophil leukocytes (%) Erythrocyte (T/L) Hemoglobin (g/L) HCT (L/L) 11,65 29,32 ± 20,13 7,77 ± 1,67 64,30 ± 7,32 4,71 ± 0,56 140,09 ±13,29 0,42 ± 0,04 8,41 23,38 ± 14,66 8,27 ± 2,54 65,19 ± 9,61 4,76 ± 0,59 141,74 ± 12,47 0,42 ± 0,03 18 21,42 ± 10,08 8,10 ± 2,27 65,22 ± 9,46 4,83 ± 0,60 142,47 ± 13,68 0,43 ± 0,04 p13< 0,05 p12 > 0,05 p13< 0,05 p12 > 0,05 p13> 0,05 p12 > 0,05 p13> 0,05 p12 > 0,05 p13< 0,05 p12 > 0,05 p13 > 0,05 p12 > 0,05 p13> 0,05 Conclusion: Before and after weeks of treatment were all equivalent (p>0.05) Creatinine, Triglycerid, BC, %N, Hb, HCT before and after 10 weeks of treatment were all equivalent (p>0.05) Cholesterol, red blood cell count increased after 10 weeks of treatment, (p