Abstract of medical phd thesis: research on immunological changes and the efficacy of combined methotrexate and narrowband UVB phototherapy in the treatment of psoriasis vulgaris

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENCE 108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES ---PHAM DIEM THUY RESEARCH ON IMMUNOLOGICAL CHANGES AND THE EFFICA

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENCE

108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES

-PHAM DIEM THUY

RESEARCH ON IMMUNOLOGICAL CHANGES AND THE EFFICACY OF COMBINED METHOTREXATE AND NARROWBAND UVB PHOTOTHERAPY IN THE TREATMENT OF PSORIASIS VULGARIS

Speciality: Dermatology Code: 62720152

ABSTRACT OF MEDICAL PhD THESIS

Hanoi – 2020

THE THESIS WAS CONDUCTED AT 108 INSTITUTE OF CLINICAL

MEDICAL AND PHARMACEUTICAL SCIENCES

Supervisors:

1 Ass Prof Dang Van Em, MD, PhD

2 Ass Prof Ly Tuan Khai, MD, PhD

Reviewers:

1

2

3

This thesis will be defended to the Thesis Committee at 108 Institute

of Clinical Medical and Pharmaceutical Sciences

Day Month Year

The thesis can be found at:

1 National Library of Vietnam

2 Library of 108 Institute of Clinical Medical and Pharmaceutical Sciences

3 Central Institute for Medical Science Infomation and

Technology

INTRODUCTION

Psoriasis is a chronic inflammatory disease that affects men and women of all ages, regardless of ethnic origin, in all countries The pathogenesis of psoriasis is still not fully understood

Like other autoimmune diseases, psoriasis has been shown to have a strong genetic component with external and internal triggers, including stress, skin trauma, and infections The serum and tissue levels of cytokines were reported to be elevated in psoriasis patients compared with normals, especially Th1‑ and Th17‑associated cytokines

The treatment strategy consists of induction and maintenance phases with topical and systemic medications Methotrexate (MTX) remains the gold standard for the treatment of moderate to severe psoriasis NBUVB is indicated

to have quick treatment response, low rates of adverse effect, long relapse-free period, and reduction in levels of the pro-inflammatory cytokines

To our knowledge, no study has been conducted on changes in cytokine profile during combination therapy of low-dose MTX and NBUVB in patients with psoriasis vulgaris Therefore, we conducted the study “Research on

immunological changes and the efficacy of combined methotrexate and narrowband UVB phototherapy in the treatment of psoriasis vulgaris” with the following objectives:

1 To describe demographic and clinical characteristics of psoriasis patients at the Department of Dermatology,

Venerology, and Allergology of 108 Military Central Hospital between August 2015 and May 2018

2 To investigate immunological changes in peripheral blood

(CD4, CD8, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α, and INF-γ) of patients with moderate and severe psoriasis vulgaris before and after the combined treatment of methotrexate and narrowband UVB phototherapy

3 To evaluate the efficacy of combined methotrexate and

narrowband UVB phototherapy for moderate and severe psoriasis

Chapter 1 OVERVIEW 1.1 Psoriasis vulgaris

1.1.1 Epidemiology

Incidence and prevalence: Psoriasis occurs in 2–3% of

the population worldwide The prevalence of psoriasis was 1,5

% of the total population in Vietnam

Sex ratio: Psoriasis prevalence is estimated to be equal in

males and females

Age: While it can begin at any age, psoriasis has 2 peaks

of onset, the first at age 30 to 39 years and the second at age 60 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 a symmetric, well-demarcated erythematous plaque with a silvery scale The prevalence of pruritus and nail involvement in psoriasis is 20-30% and 30-50% respectively There are several clinical types of psoriasis vulgaris, including plaque psoriasis, nummular psoriasis, guttate psoriasis, and mixed type

Type 1 begins on or before age 40 years; Type II begins after the age of 40 years Patients with early-onset, or type I psoriasis, tended to have more relatives affected and more severe disease than patients who have a later onset of disease or type II psoriasis In addition, strong associations have been reported with human leukocyte antigen (HLA)-Cw6 in patients with early onset, compared with later onset of psoriasis

Psoriasis severity is defined using PASI score: mild (PASI<10), moderate (PASI=10-20), severe (PASI >20)

Histopathology: Parakeratosis without hyperkeratosis, acanthosis with a downward elongation of rete ridges, thin / no granular cell layer, suprapapillary thinning, Munro microabscesses, prominent dermal capillaries, mixed dermal infiltrate of lymphocytes, macrophages, and neutrophils

1.1.3 Pathogenesis

There are three main factors:

- Genetic factors: Psoriasis is associated with certain human leukocyte antigen (HLA) alleles, such as HLA-Cw6,

HLA-DR7 The psoriasis susceptibility loci include PSORS1: 6p21.3, PSORS2: 17q, PSORS3: 4q, PSORS4: 1q21, PSORS5: 3q21, PSORS6: 19p, PSORS7: 1p, PSORS8:16q, PSORS9: 4q31, PSORS10: 18p11, PSORS11: 5q31-q33, and PSORS12: 20q13 4 - 91% of patients have a positive family history of psoriasis

- Immune disorders (role of cytokines, especially a key role of the IL-17 / IL-23 axis)

- Environmental a factors a may a exacerbate a its manifestations, or even trigger the disease, such as mental stress, infection, trauma drugs, drinking, smoking, diet, climate, and weather

1.1.4 Treatment

- Goal of treatment: to achieve skin clearance and maintain control of the lesions as well as improve patient’s quality of life

- Treatment strategy: induction and maintenance phase

- Strategy for medication administration: single-agent, combination, rotational, and sequential approaches

as TNF-α inhibitors (infliximab, adalimumab), IL-17A inhibitor (secukinumab)

1.2 Pathophysiological role of cytokines in psoriasis

Cytokines are small polypeptides (8-80 kD) produced in response to antigens, microorganisms, or other non-infectious stimuli They are capable of regulating immune and inflammatory reactions and interacting with the endocrine and nervous systems

Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells There is a close relationship between cytokine and adipokine Adipokine’s cytokine-mediated response is a complex process involving many components and stages This is a natural protective response of the body However, overproduction of cytokines, adipokines will cause a variety of diseases, including psoriasis

1.3 NBUVB phototherapy and methotrexate in the treatment of psoriasis vulgaris

1.3.1 NBUVB phototherapy

Narrowband ultraviolet B (NBUVB) emitting light with a peak around 311 nm The exact mechanism of its therapeutic action remains poorly understood Several genetic and molecular factors are induced by NBUVB, such as inhibition of proliferation of keratinocytes, increased keratinocyte apoptosis, and the effect on other skin cells like epidermal T lymphocytes, melanocytes, and Langerhans cells NBUVB can be safely used

in children and pregnancy UVB can be combined with other topical, systemic, or biologic agents to enhance efficacy

1.3.2 Methotrexate in psoriasis

Methotrexate (MTX) was the first systemic agent used in the treatment of psoriasis (1955) and remains the gold standard for the treatment of moderate to severe psoriasis The US FDA approved the use of MTX for the treatment of psoriasis in 1972 Methotrexate produces significant mitotic depression (S phase) and has potent anti-inflammatory The usual adult dose of methotrexate is 7.5 to 25 mg weekly

Chapter 2 SUBJECTS AND METHODS 2.1 Research subjects

- 260 inpatients were diagnosed with psoriasis at the Department of Dermatology, Venerology, and Allergology of

108 Military Central Hospital between August 2015 and May

2018

2.1.1 Diagnostic criteria

The diagnosis of psoriasis is primarily clinical: psoriasis vulgaris (plaque psoriasis, nummular psoriasis, guttate psoriasis), special types (pustular psoriasis, erythrodermic psoriasis, and psoriatic arthritis)

Histopathology: Parakeratosis without hyperkeratosis, acanthosis with a downward elongation of rete ridges, thin / no granular cell layer, suprapapillary thinning, Munro microabscesses, prominent dermal capillaries, mixed dermal

infiltrate of lymphocytes, macrophages, and neutrophils Skin biopsy is usually reserved for the evaluation of atypical cases

+ Control group: 35 volunteers, matched on age, gender, had no autoimmune or infectious diseases

- Objective 3: patients aged at least 16 years suffering from moderate-to-severe plaque psoriasis and had no contraindication to methotrexate and NBUVB phototherapy and

no history of systemic treatment at least 1 month prior to enrollment

Narrowband UVB unit manufactured by Daavlin of Bryan, USA

Objective 2: Prospective cross-sectional study with a control group to investigate immunological changes in peripheral blood of patients after treatment

Objective 3: Randomized prospective clinical trial to evaluate the efficacy of combined methotrexate and narrowband UVB phototherapy for psoriasis vulgaris

2.3.2 Sample size

Objective 1: Convenience sampling 260 inpatients

Objective 2: The sample size is calculated using the WHO’s formula Experimental group (35 patients) and control group (35 volunteers)

Objective 3: Estimating the sample size for a clinical trial with equal-sized groups The experimental group (35 patients) and control group (35 patients) were matched for age, sex, and severity

+ First testing (before treatment): CD4, CD8, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α, IFN-γ, AST, ALT, urea, creatinine, and CBC

+ Inpatient treatment: NBUVB phototherapy + methotrexate 7.5 mg PO weekly for 4 weeks

+ Second testing (after treatment): CD4, CD8, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α, IFN-γ, AST, ALT, urea, creatinine, and CBC

- Control group: 35 volunteers, matched on age, gender with the experimental group Cytokine testing (IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α, and IFN-γ)

2.3.3.3 To evaluate the efficacy of combined methotrexate and narrowband UVB phototherapy

- 70 patients with moderate-to-severe plaque psoriasis who satisfy the inclusion criteria were divided into 2 equal groups matched for age, sex, and severity

- First testing (before treatment):

+ Experimental group: CD4, CD8, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α, IFN-γ, AST, ALT, urea, creatinine, and CBC

+ Control group: AST, ALT, urea, creatinine, and CBC

- Inpatient treatment for 4 weeks

- Second testing (after treatment): similar to the first testing for 2 groups

Patient treatment process:

- Experimental group: Methotrexate + NBUVB phototherapy + Physiogel for 4 weeks

+ Methotrexate 7.5 mg PO weekly

+ NBUVB phototherapy: Patients were started at an initial dose of 800 mJ/cm2 and increased by 100 mJ/cm² in subsequent visits until reaching a dose of 2500 mJ/cm2 in the last treatment Treatment once daily, five days per week

+ Physiogel once daily

- Control group: Methotrexate + Physiogel for 4 weeks

Evaluating treatment effectiveness:

- Clinical outcome measures using PASI after 2 weeks, 3 weeks, and 4 weeks

- Side effects: Clinical findings (erythema and pruritus) and laboratory test results (AST, ALT, urea, creatinine, and CBC)

- Recurrence rate after 1, 2, and 3 months of the treatment

2.3.4 Laboratory techniques and procedures in research

2.3.4.1 Cytokine measurements

3 ml of blood were drawn from each patient Tubes were centrifuged at 4000xg for 30 minutes and divided into two equal parts in Eppendorf tubes 1.5 mL Serum samples were stored at

−80°C until analyzed

Cytokine detection could be accomplished by sandwich immunoassay on microspheres surface This was carried out by Bio-Plex System and Bio-Plex Manager Software The test was conducted at Military Medical University

2.3.4.2 CD4 and CD8 T cell counting

2ml of venous blood samples collected in containing tubes should not be kept for more than 1 hour before counting CD4 and CD8 T cells by flow cytometry method on FACSCalibur system The test was conducted at 108 Military Central Hospital

EDTA-2.3.5 Outcome assessment

2.3.5.1 Severity of psoriasis

Psoriasis severity is defined using PASI score: mild (PASI<10), moderate (PASI=10-20), severe (PASI >20)

2.3.5.2 Evaluating treatment effectiveness

- We calculated PASI score reduction, classifying it into one of four groups: poor (< 25%), fair (25% or more to less than 50%), moderate (50% or more to less than 75%), and good (≥ 75%)

- Adverse effects:

+ Side effects of NBUVB: itchy, redness, blister formation, etc

+ Side effects of Methotrexate: Systemic side-effects (headache, fever, chills, dizziness); skin (erythema, blisters, urticaria, alopecia); abnormal laboratory test result (AST, ALT, urea, creatinine, and CBC)

2.3.5.3 Recurrence rate

Relapse was defined as a loss of at least 25% of PASI improvement over baseline

Chapter 3 RESULTS 3.1 Demographic and clinical characteristics of psoriasis patients

Trigger factors Frequency %

Infection (sinuses, nose, throat) 17 6,5

Drugs (antibiotics, pain killer) 22 8,5

Conclusion: The most common triggers cited in this study were stress (44.2 %), followed by food (30.4 %) overall

3.1.2 Clinical characteristics of psoriasis

Table 3.5 Clinical subtypes of psoriasis (n = 260)

Table 3.7 Frequencies of CD4 and CD8 in the blood of patients

with psoriasis vulgaris (n = 35)

Before treatment (X±SD) After treatment (X±SD) (sign test) p

CD4 (cell/µl) 682,3 ± 266,2 511,4 ± 196,7 <0,001

CD8 (cell/µl) 611,9 ± 365,6 419,7 ± 191,0 <0,001 Conclusion: After treatment, the frequencies of both CD4 and CD8 decreased significantly (both p<0.001)

3.2.2 Cytokine serum levels in patients with psoriasis vulgaris

3.2.2.2 Cytokine serum levels before the treatment

Table 3.13 Cytokine serum levels in the study group and

control group before the treatment

Cytokine Study group (n=35)

(X±SD)

Control group (n=35)

IL-2 (pg/ml) 86,259 ± 70,50 5,000 ± 0,000 <0,001 IL-4 (pg/ml) 8,041 ± 9,646 3,829 ± 5,541 <0,01 IL-6 (pg/ml) 16,687 ± 79,244 3,084 ± 6,413 >0,05 IL-8 (pg/ml) 53,024 ± 228,239 25,264 ± 66,821 <0,05 IL-10 (pg/ml) 1,984 ± 2,113 1,000 ± 0,000 <0,05 IL-17 (pg/ml) 1,798 ± 2,062 2,000 ± 0,000 <0,001 TNF-α (pg/ml) 3,438 ± 11,090 1,715 ± 1,371 >0,05 INF-γ (pg/ml) 8,311 ± 3,214 9,099 ± 2,894 >0,05 Conclusion: Compared with the control group, psoriatic patients had significantly different serum concentrations of several cytokines (IL-2, IL-4, IL-8, IL-10)

Table 3.16 Cytokine serum levels in psoriatic patients of

different severity

Cytokine Severe (n=12)

X±SD

Moderate (n=23)

IL-2 (pg/ml) 102,509 ± 66,116 77,780 ± 72,645 >0,05 IL-4 (pg/ml) 14,022 ± 13,416 4,920 ± 4,908 <0,05