A composition for treating psoriasis comprising a beta-glucan

Abstract

The present invention is to provide a composition for treating psoriasis comprising the beta-glucan, and a method of use for manufacturing a composition for treating psoriasis comprising the beta-glucan.

Description

A COMPOSITION FOR TREATING PSORIASIS COMPRISING A BETA-GLUCAN

[0001] Disclosed herein is safety and efficacy of Neu REFIX 1,3-1,6 beta-glucans in improving the clinical parameters, amelioration of immune-inflammatory biomarkers and skin lymphocyte infiltration in psoriasis such as Psoriasis vulgaris with results of a randomized clinical trial.

[0002] Introduction: Psoriasis is a chronic, immune-mediated inflammatory skin condition that leads to erythema, thickening, and scaling of the skin. The primary histological change associated with psoriasis is increased keratinocyte cell proliferation [1]. Although early theories about the etiology of psoriasis centred on keratinocyte proliferation and differentiation, subsequent research has revealed that immune system dysregulation plays a significant role in the development of psoriasis. The interactions of dendritic cells, T cells, keratinocytes, neutrophils, and the cytokines generated by immune cells are the central mechanism of psoriasis development. Psoriasis pathology is assumed to be influenced by genetic, environmental, and behavioural factors. Adult psoriasis prevalence is estimated to range from 0.91% to 8.5% globally [2]. Psoriasis vulgaris (PV) is the most prevalent subtype of psoriasis, affecting over 90% of patients. Topical medications, UV light, systemic medicines, and biologicals are the most often used therapies for psoriasis. Psoriasis Vulgaris is a chronic, inflammatory, debilitating, relapsing disease which is associated with systemic manifestations such as arthritis and cardiovascular disease.　Current treatment approaches include methotrexate, ciclosporin, biological agents including anti-tumour necrosis factor (TNF)-α), Vitamin D3 analogues, and combination therapies but the desired outcome of complete remission has not been achieved yet and there are associated serious adverse effects of taking medications long term including an increased incidence of bacterial and fungal infections and cancer. First-line therapy for PV includes topical medicines such as corticosteroids, retinoid analogues, and vitamin D analogues [3]. Systemic medications are presently used to treat severe psoriasis, whereas ultraviolet radiation and biologics are utilized when appropriate and necessary. The main drawbacks of these therapies are that present approaches to treatment of psoriasis don ft induce a complete remission and require continuous long term therapy which is associated with serious side effects including susceptibility to bacterial and fungal infections and cancer [4]. The recent classes of biologicals for treatment of psoriasis include the interleukin (IL)-16 inhibitor (clazakizumab), IL- 2 / 23 inhibitor (ustekinumab), the tumor necrosis factor (TNF)-inhibitors (secukinumab, ixekizumab, and brodalumab), the tumor necrosis factor (TNF)-inhibitors (etanercept, infliximab, adalimumab, and certolizumab pegol) [5]. Though these biologicals have raised the standard and efficacy of psoriasis treatments, most medicines lose effectiveness with time and are associated with serious side effects [6]. Therefore additional more effective therapies are required.

[0003] Natural food substances have been considered as promising candidates because they are safer than novel synthetic drugs even after prolonged administration and with high patient compliance. One of the potential natural compounds, is a novel 1,3-1,6 beta-glucan produced by a novel N-163 strain of Aureobasidium pullulans (also referred to as Neu REFIX herein) which has shown significant anti-inflammatory, immune-modulatory and anti-fibrotic effects in clinical, pre-clinical studies of genetic conditions such as Duchenne muscular dystrophy (DMD) [7,8] and in clinical trials for autoimmune conditions like multiple sclerosis (MS) [9]. Additionally, it has shown metabolic and immune regulatory effects in pre-clinical studies [10,11].

[0004] In an animal study of obese diabetic mice model of KK-Ay mice, Neu REFIX beta-glucan was able to regulate the levels of non-esterified fatty acids (NEFA) in 28 days

[0010] . NEFA is associated with metabolic syndrome induced inflammation

[0010] . In an animal study of non-alcoholic steatohepatitis (NASH)

[0011] , Neu REFIX beta-glucan was effective in reducing IL-6 expression as well as other fibrotic and inflammatory markers. In COVID-19 clinical trials, a combination of Neu REFIX beta-glucan and another strain, AFO-202 of A. pullulans derived beta-glucan, demonstrated a reduction in inflammatory markers associated with cytokine storms in trials lasting 15 days

[0012] and 30 days

[0013] . Furthermore, this combination safely yielded an efficient control over biomarkers of dysregulated metabolism, inflammation, and immune dysfunction in healthy volunteers

[0014] .

[0005] In a clinical trial involving patients with DMD, orally consumed Neu REFIX beta-glucan decreased inflammatory markers such as IL-6 and TGF-β, increased dystrophin levels, and improved muscle strength within 45 days [7]. In the study with MS patients, oral consumption of Neu REFIX beta-glucan resulted in decreased IL-6, improved cell counts (CD4+ve, CD19+ve, CD3+ve, and CD8+ve), increased Lymphocyte to C-reactive protein ratio (LCR), Leukocyte to CRP ratio (LeCR), and decreased Neutrophil to Lymphocyte ratio (NLR). Additionally, there was an improvement in the Expanded Disability Status Scale (EDSS) [8]. Effective gut microbiome reconstitution, with a decrease in harmful bacteria and an increase in beneficial bacteria, was observed in both the pre-clinical study in NASH mice and clinical studies in DMD and MS [15,16].

[0006] IL-23, a heterodimeric cytokine of the IL-12 family, comprises a unique p19 subunit and the shared p40 subunit of IL-12. Primarily produced by activated macrophages and DCs in peripheral tissues, IL-23 forms a disulfide-linked complex with the p40 subunit.　Emerging evidence suggests a critical role for IL-23 in autoimmune disorders like colitis, gastritis, psoriasis, and arthritis.　While structurally similar to IL-12 and sharing the ability to enhance IFN-γ production and memory T cell proliferation, IL-23 possesses a distinct function due to its capacity to induce IL-17, a key player in autoimmune inflammation

[0028] .　Within the psoriatic dermis, dendritic cells (DCs) secrete IL-23, which promotes the production of IL-17 by CD4+ T cells.　This IL-23 / IL-17 signaling pathway is established as the critical driver of cellular and molecular alterations in psoriasis. Moreover, it is linked to the aberrant proliferation of epidermal keratinocytes, ultimately manifesting as psoriatic plaques with characteristic scaling and a persistent inflammatory milieu.　Extensive research has demonstrated significantly elevated and overexpressed IL-23 levels in both the serum and psoriatic skin, further solidifying its role in the pathogenesis of both psoriasis and psoriatic arthritis (PsA)

[0029] .　Based on these findings, selective IL-23 inhibitors such as guselkumab, tildrakizumab, and risankizumab have emerged of which Guselkumab has been already approved by the US Food and Drug Administration (FDA), as well as the European Medicines Agency (EMA) for the treatment of adult patients with moderate-to-severe plaque psoriasis

[0030] .　The most common side effects for guselkumab reported are upper respiratory tract infections, headache, injection site reactions, joint pain, diarrhea, gastroenteritis, fungal skin infections and herpes simplex infections

[0031] .SUMMARY OF THE INVENTION

[0007] The present invention relates to the following: (1) A composition for treating psoriasis, comprising a beta-glucan. (2) A composition for decreasing lymphocyte infiltration in a subject who suffers from psoriasis or has a risk of suffering from psoriasis, comprising a beta-glucan. (3) A composition for decreasing an expression of IL-23 in a subject who suffers from psoriasis or has a risk of suffering from psoriasis, comprising beta-glucan. (4) The composition of any one of (1)-(3), wherein the beta-glucan comprises a 1,3-1,6-beta-glucan produced by Aureobasidium pullulans N-163 (NITE BP-03377). (5) The composition of any one of (1)-(4), wherein psoriasis is psoriasis vulgaris (PVS).

[0008] [Rectified under Rule 91, 19.03.2026]Figure 1: A. Decrease in PASI score significant in Neu REFIX group compared to control group between pre-and post-intervention; B Percentage change in PASI in control and Neu REFIX groups. C. Reduction of PASI 75 not achieved in any patient in Control group with PASI worsening in one patient. Highly significant reduction of PASI 75 - benchmark achieved in five patients in Neu REFIX group while seven showed improvement.

[0009] Figure 2: A. Significant decrease in IGA score was observed in the Neu REFIX group compared to the control group between pre- and post-intervention; B. A greater reduction in the change in IGA score was noted in the Neu REFIX group compared to the control group. P-value significance < 0.05

[0010] Figure 3: Clinically evident decrease in erythema, scaling, and thickening in psoriatic plaques in the treatment arm (A-C), while there was no evident decrease in the control arm (D and E). A: Neu REFIX - Upper back showing a significant decrease in thickening and scaling; B. Neu REFIX - Left forearm showing a significant decrease in thickening and scaling, post-intervention; C: Neu REFIX - Right forearm showing a decrease in scaling, thickening, and erythema of the skin significantly after 28 days.

[0011] Figure 4: Histopathological evaluation by H&E staining revealed a significant reduction of infiltrating lymphocytes in (n=8) specimens, represented by A & B, and a moderate reduction in (n=3) specimens, represented by C & in the Neu REFIX group of patients, represented by D. In the control group (n=8) specimens showed an increase or no change in the infiltration, represented by E & F (magnification x40)

[0012] Figure 5:Greater reduction in the thickness of the epidermis and significant decrease in lymphocyte infiltration score was noted in the Neu REFIX group compared to the control group.Figure 6: Correlation of different parameters with BMI wherein the improvement in score was greater in subjects with BMI > 30 than the improvement in all the subjects put together; A. PASI-BMI correlation; B. PLR- BMI correlation; C. CAR-BMI correlation and D. A:G ratio-BMI correlation

[0013] Figure 7: A. PLR ratio and B. CAR ratio increased in the control arm, while it decreased in the Neu REFIX arm post-intervention. C. NLR ratio increased in both the control arm and treatment arm post-intervention; D. LCR and E. LeCR decreased in both the control arm and treatment arm post-intervention.

[0014] Figure 8: A, B. hsCRP decreased in the Neu REFIX treatment arm, while it significantly increased from baseline in the control arm post-intervention; C,D. IL-6 decreased in the Neu REFIX treatment arm, while it increased from baseline in the control arm post-intervention

[0015] Figure 9: IL-2, IL-12, IL-17 and TNF-Alpha increased in both the control arm and treatment arm post-intervention

[0016] Figure 10: A: IL-23 expression pre-Neu REFIX, B. IL-23 expression decreased post Neu REFIX; C. Graph showing the difference in expression of IL-23 pre-and post-Neu REFIX.DETAILED DESCRIPTION OF INVENTION

[0017] In an aspect, the present invention relates to a composition for treating psoriasis, comprising a beta-glucan.　The beta-glucan used in the present invention can be a beta-glucan derived from Aureobasidium pullulans strain APNN-M163 (Also referred to herein as "strain M163", or "strain N-163"), and preferably 1,3-1,6 beta-glucan derived from N-163 (Also referred to herein simply as "N-163 glucan" or "N-163 beta-glucan").　"Aureobasidium pullulans strain APNN-M163" has been deposited at the Patent Microorganisms Depositary Center, National Institute of Technology and Evaluation (Room. 122, 2-5-8, Kazusa Kamatari, Kisarazu City, Chiba, 292-0818 Japan), under the deposit number NITE P-03377, on February 9, 2021.

[0018] While the domestic deposition was made on February 9, 2021, Aureobasidium pullulans strain APNN-M163 has then been transferred to international deposition at the International Patent Organism Depositary, National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depository (Room. 122, 2-5-8, Kazusa Kamatari, Kisarazu-shi, Chiba, 292-0818 Japan) on September 14, 2021 with the accession number: NITE BP-03377.

[0019] The beta-glucan produced by N-163 strain was estimated to have the following chemical structure (Japanese Patent Application No. 2021-087255).Chem 1

[0020] The composition of the present invention exerts its function when ingested by mammals including humans. The term gingestion h as used herein is not limited to any administration route as long as it can enter the human body, and is realized by all known administration methods such as oral administration, tube administration, and enteral administration. Typically, oral ingestion and enteral ingestion via the digestive tract are preferable.

[0021] The dose of the present invention can be appropriately set in consideration of various factors such as administration route, age, body weight, and symptoms. The dose of the composition of the present invention is not particularly limited, but the amount of glucan is preferably 0.05 mg / kg / day or more, more preferably 0.5 mg / kg / day or more, particularly preferably 1.0 mg / kg / day. However, when ingested over a long period of time, the amount may be smaller than the preferable amount described above. In addition, the beta-glucan used in the present invention has a sufficient dietary experience, and there is no problem in terms of safety. Therefore, an amount far exceeding the above amount (for example, 10 mg / kg / day or more).

[0022] The composition of the present invention can be used as a food or drink.　The composition of the present invention, as a special-purpose food such as a food for specified health use and a nutritionally functional food, by administering to animals such as humans, treatment or prevention can be achieved against various diseases related to fibrosis.

[0023] When the composition of the present invention is used as food or drink, the type of food or drink is not particularly limited. Further, the shape of the food or drink is not particularly limited, and may be any shape of food or drink that is usually used. For example, it may be in any form such as solid form (including powder and granule form), paste form, liquid form and suspension form, and is not limited to these forms.

[0024] When used as a pharmaceutical, a dosage form that can be orally administered is preferable because the composition of the present invention reaches the intestine. Examples of preferable dosage forms of the drug according to the present invention include tablets, coated tablets, capsules, granules, powders, solutions, syrups, troches, and the like. These various preparations are prepared according to a conventional method by using beta-glucan, which is the active ingredient, an excipient, a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent, a solubilizing agent, a suspending agent, a coating agent, etc. It can be formulated by admixing the auxiliaries usually used in the technical field of pharmaceutical formulation.

[0025] In some embodiments, the present invention can be used in combination with other food, drink, drugs and any other substances in order to enhance the efficacy of the present invention.

[0026] In one embodiment, the beta-glucan, the composition, or the pharmaceutical of the present invention can treat psoriasis.　As used herein, psoriasis includes, but is not limited to, psoriasis vulgaris, psoriatic arthritis, dropsy psoriasis, psoriatic erythroderma, and pustular psoriasis.　Preferably, the beta-glucan, the composition, or the pharmaceutical of the present invention can treat psoriasis vulgaris.

[0027] In one embodiment, the beta-glucan, the composition, or the pharmaceutical of the present invention can decrease lymphocyte infiltration in a subject who suffers from psoriasis or has a risk of suffering from psoriasis.　In one embodiment, the beta-glucan, the composition, or the pharmaceutical of the present invention can decrease IL-23 in a subject who suffers from psoriasis or has a risk of suffering from psoriasis.

[0028] In another aspect, the present invention relates to a method for treating psoriasis, such as psoriasis vulgaris, comprising administering a beta-glucan, a composition or pharmaceutical comprising a beta-glucan to a subject in need thereof.　The subject may suffer from psoriasis or have a risk of suffering from psoriasis.

[0029] Example 1: Given the safety and immune-modulating potential of a 1,3-1,6 beta-glucan produced by a novel N-163 strain of Aureobasidium pullulans (Neu REFIX), a randomized clinical trial was conducted to evaluate the safety and efficacy in terms of clinical outcome and immune parameters in patients with moderate to severe psoriasis vulgaris.　In the present study, we evaluated the effects of Neu REFIX beta-glucan in patients with moderate to severe psoriasis.

[0030] Methods: Thirty patients with plaque psoriasis vulgaris were enrolled in this randomized, 28-day, single-centre study to investigate the effects of Neu REFIX beta-glucan. Informed consent was obtained, and eligible patients were randomized at a 1:2 ratio, being assigned to either the control group or the Neu REFIX group. The study was registered in Clinical trials registry of India, CTRI / 2022 / 05 / 042497 and approved by the institutional ethics committee of Government Thoothukudi Medical College, Thoothukudi, India (IEC Ref No: 02 / 2023-09).

[0031] The interventions were as follows: Group I-Control (n=10): Standard treatment: Topical corticosteroids and liquid paraffin with oral multivitamin tablets Group II-Neu REFIX (n=20): Standard treatment + N-163 beta-glucan (Neu REFIX) (16g in gel form per day in 2 sachets of each 8g) (n =20)

[0032] Inclusion Criteria: ・Males and females at least 18 years of age. ・Plaque psoriasis with or without arthritis in subjects who were otherwise is in generally good health. ・Subject exhibits acutely active at least moderate to severe plaque-type psoriasis (> 5% body surface area (BSA) involvement). ・Signed, written informed consent. Willing and able to comply with study visits according to protocol for the full study period.

[0033] Exclusion Criteria: ・Patients with forms of psoriasis other than chronic plaque-type psoriasis or drug-induced psoriasis. Use of any investigational drug within 90 days prior to inclusion. ・Patients suffering from significant cardiac, renal or hepatic failure or any other disease that may risk the patient or interfere with the ability to interpret the results. ・Patient with any active or chronic infection. ・Pregnancy or risk of pregnancy. ・Patients who are seropositive for HIV1, HIV2, Hepatitis B Surface Antigen, and Hepatitis C. ・Patients unable to give written informed consent

[0034] Dietary Requirements: Patients were asked to refrain from excessive alcohol intake during treatment.

[0035] Clinical Examinations: Clinical examinations occurred at screening, baseline, and after 28 days. Standardized scoring tools for psoriasis, including the Psoriasis Area and Severity Index (PASI) score and the Investigator's Global Assessment (IGA) Scale, were used to assess psoriasis severity. The PASI is a widely utilized instrument in psoriasis trials that grades the severity of psoriatic lesions and the patient's response to treatment. It produces a numeric score ranging from 0 to 72. Generally, a PASI score of 5 to 10 is considered moderate disease, while a score over 10 is deemed severe. A 75% reduction in the PASI score (PASI 75) serves as the benchmark for most clinical trials in psoriasis and the criterion for the efficacy of new treatments. When calculating the PASI, severity is determined by dividing the body into four regions: head (h), upper extremities (u), trunk (t), and lower extremities (l), accounting for 10%, 20%, 30%, and 40% of the total body surface area (BSA), respectively. Each area is assessed separately for erythema, induration, and scaling on a scale from 0 (none) to 4 (very severe). The IGA scale is based on a point-in-time assessment, with possible scores on the IGA modified (mod) 2011 scale as follows: 0 = clear, 1 = almost clear, 2 = mild, 3 = moderate, 4 = severe.

[0036] Histological Analysis: Skin punch biopsy samples were obtained from a psoriatic lesion and assessed for histopathology at baseline and after 28 days. Histopathological analysis included the degree of epidermal thickness, dermal inflammation, and lymphocytic infiltration. For estimation of lymphocyte infiltration, the absolute number of cells was counted in three high-power fields with the highest cellularity. The field with the highest average cell count was then used for analysis.

[0037] Laboratory Analyses: Blood samples were analysed to ascertain hemogram and biochemical parameters such as immune-inflammatory parameters using the same controls and parameters as those routinely used. Serum levels of biomarkers: Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-17 (IL-17) and Interleukin-12 (IL-12) were measured using ELISA kits obtained from Wuhan Fine Biotech Limited, China (IL-17 Cat. No: EH3267; IL-12 Cat. No: EH0176; IL-2 Cat. No: EH0189).

[0038] Adverse Events: Before and after 28 days of treatment, medical histories were recorded for each patient. Standard laboratory examinations included assessment of hematological parameters, urinalysis, serum electrolytes, indices of renal function (creatinine and urea), and hepatic function (alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and g-glutamyl-transpeptidase). All adverse events (AEs) were collected and graded for severity and potential relation to treatment. The safety evaluations include the incidence of treatment-induced or serious adverse events, dropout due to adverse events and laboratory parameter changes.

[0039] Statistical Analysis: All data were analysed using GraphPad Prism 10 software. Results were expressed as means ± SD. T-tests and non-parametric tests such as Mann-Whitney test were performed for statistical analysis as indicated. P values of < 0.05 were considered to be significant.

[0040] Results: Patient Outcomes: Table 1 presents the demographic information and relevant safety parameters of the study subjects, with a focus on biochemical characteristics. There were 8 males and 2 females in the control arm while there were 7 males and 8 females in the treatment arm. The mean age was 43.8±14.56 years in the control arm and 48.10±12.51 years in the treatment arm. The Body Mass Index (BMI) was 24.19±2.930 in the control arm and 24.16±6.11 in the treatment arm. There were no treatment-emergent adverse events (TEAEs) or serious AEs in any of the patients in the study. Two subjects in the control arm and five subjects in the Neu-REFIX treatment arm discontinued the study (dropout rate: 20% control, 25% Neu-REFIX). Although the dropout rate was higher in the Neu-REFIX group, this difference was proportional to the initial randomization ratio (1:2, control:Neu REFIX). The primary reasons for discontinuation were logistical challenges associated with travel from distant rural locations to the study site.

[0041] Hematology and biochemistry: Serum creatinine values increased from 0.850 ± 0.371 mg / dl at baseline to 0.930 ± 0.33 mg / dl post intervention in the control arm (p-value=0.17) while it decreased from 0.76 ± 0.11 mg / dl at baseline to 0.74 ± 0.06 mg / dl in the treatment arm, post-intervention(p-value =0.92). ESR values decreased from 26.99 ± 24.78 mm / hr at baseline to 21.50 ± 7.93 mm / hr post intervention in the control arm (p-value=0.92) while it increased from 17.28 ± 10.49 mm / hr at baseline to 19.99 ± 10.08 mm / hr in the treatment arm, post-intervention (p-value =0.26). Random blood sugar decreased from 142.3 ± 78.58 mg / dl at baseline to 119.9 ± 45.17 mg / dl post intervention in the control arm (p-value=0.92) while it increased from 113.2 ± 57.04 mg / dl at baseline to 114.6 ± 59.24 mg / dl in the treatment arm, post-intervention (p-value =0.26). The albumin to globulin ratio decreased from 0.93 ± 0.05 to 0.88 ± 0.08 in the control group from baseline while it increased from 0.89 ± 0.08 to 0.91 ± 0.08 from the baseline in the treatment arm. Additionally, there were no clinically significant laboratory abnormalities or changes from baseline in vital signs in the study. There was no change in the BMI from baseline at the end of the study.

[0042] Table 1: Demographics and Changes in Clinical and Safety-Associated Biochemical Parameters from Baseline to Post-Intervention

[0043] Table 2: Baseline Values of Clinically Significant Parameters in the Control and Neu REFIX Groups

[0044] Clinical Correlates of Histological Changes in Psoriasis: Clinically meaningful reduction of PASI score to 50% (PASI 50) from baseline was achieved in six patients in the treatment arm (40%) with five of them achieving the clinical trials f benchmark score of PASI 75 (75 % reduction in PASI score) while only one patient in the control arm achieved PASI 50 in the control arm and none achieved PASI 75 in the control arm. The PASI score improved in 12 subjects in the treatment arm and remained unchanged in 3 subjects while in the control arm, it worsened in one subject, slightly improved in five subjects and remained unchanged in four subjects (Figure 1). The mean PASI score was 18.46 ± 8.51 at baseline in the control arm which reduced to 15.21 ±7.142 at the end of 28 days but the difference was not significant (p-value=0.46). The mean PASI score in the Neu REFIX treatment arm which was 22.41 ±7.386 at baseline significantly decreased to 12.15 ±7.325 at the end of 28 days (p-value =0.0007) (Figure 1). See also Figure 1.

[0045] The IGA score improved in 67% of the patients in the treatment arm, while it worsened in 60% of patients in the control arm. In the treatment arm, the IGA score decreased from 3.4± 0.63 at baseline to 2.4± 0.91 at the end of 28 days, and the difference was significant (p-value = 0.002). The IGA score decreased from 3.4± 0.51 at baseline to 2.9± 0.87 at the end of 28 days in the control arm (p-value = 0.22), but the difference was not significant. (Figure 2). See also Figures 2 and 3.

[0046] Histological Analysis: Histological analysis revealed that the mean thickness score of the epidermis decreased from a baseline of 0.64± 0.35 to post-intervention 0.50± 0.22 in the control arm (p-value=0.4), while it decreased from 0.59± 0.22 to 0.47± 0.14 in the treatment arm (p-value=0.16). In terms of lymphocyte infiltration, there was a significant decrease in the lymphocyte infiltration score in the treatment arm from a baseline value of 193.5± 76.83 to 181.1± 67.97 post-treatment (p-value=0.02), while it decreased from a baseline of 183.1± 55.64 to 144.3± 35.95 post-treatment (p-value=0.97) in the control arm, which was not significant. See also Figures 4 and 5.

[0047] BMI based correlations: We measured the BMI of patients enrolled in the study since severity of psoriasis has been reported to correlate with an increase in BMI

[0018] .　Interestingly, reduction in PASI score (improvement in clinical outcome) was higher in those Neu REFIX treated subjects with BMI >30 (60.41 ± 31.99 %) compared to all subjects　(40.16 ± 33.64 %) (p-value =0.39). A similar pattern was observed in PLR. Subjects with BMI > 30 had a higher reduction in PLR, CAR ( PLR: 0.004 ± 0.003) compared to all study subjects (0.007 ± 0.007) (p-value =0,48); CAR (0.201 ± 0.174 in subjects with BMI>30 compared to all study subjects (0.259 ± 0.290) (p-value=0.93) Finally, the increase in A:G ratio was　also higher in subjects with BMI>30　(0.105 ± 0.169) compared to all the subjects (0.020 ± 0.127) (p-value=0.34) The significance of these findings warrants further studies. See also Figure 6.

[0048] Blood immune-inflammatory parameters: Platelet to Lymphocyte ratio (PLR) increased from 0.080 ± 0.0316 at baseline to 0.0935 ± 0.0373 post-intervention in the control group (p-value = 0.39) and it decreased from 0.08053 ± 0.0401 at baseline to 0.078 ±0.043 post-intervention (p-value=0.76). Neutrophil to Lymphocyte ratio (NLR) values increased from 2.139 ± 1.207 at baseline to 2.784 ± 2.114 post-intervention in the control group (p-value=0.52) while it increased from 1.947 ± 0.87 at baseline to 2.116 ± 0.90 post-intervention (p-value=0.76). Lymphocyte to CRP ratio (LCR) decreased from 66.36 ± 40.91 at baseline to 47.58 ± 85.29 post-intervention in the control group (p-value=0.04) and from 93.72 ± 86.23 at baseline to 63.69 ± 53.62 post-intervention (p-value=0.54). Leukocyte to CRP ratio (LeCR) decreased from 12152 ±　4698　at baseline to 8369 ± 11676 post-intervention in the control group (p-value = 0.04) and from 26032 ± 29011 at baseline to 14016 ± 9621 post-intervention (p-value =0.57). The C-reactive protein to albumin ratio (CAR) increased from 0.17 ± 0.11 to 0.73 ± 0.92 in the control group (p-value=0.02) while it decreased from 0.34 ± 0.41 to 0.32 ± 0.35 in the treatment group (p-value = 0.51). See also Figure 7.

[0049] Inflammatory mediators: High-sensitivity C-reactive protein (hsCRP) values significantly increased from 0.67± 0.43 mg / L at baseline to 2.93± 3.828 mg / L post-intervention in the control group (p-value = 0.01), while they decreased from 1.218± 1.43 mg / L at baseline to 1.098± 1.127 mg / L post-intervention (p-value = 0.50) in the Neu REFIX treatment group. IL-6 values decreased from a baseline of 3.575± 4.454 pg / ml to 3.401± 2.21 pg / ml (p-value=0.19) post-treatment in the treatment group, against an increase from a baseline of 3.401± 2.556 pg / ml to 8.401± 7.585 pg / ml post-intervention (p-value=0.06) in the control group. See also Figure 8.

[0050] IL-2 values increased from a baseline of 15.01± 10.88 pg / ml to post-intervention 16.03± 16.08 pg / ml (p-value=0.83), while in the treatment arm, they increased from a baseline of 14.16± 12.42 pg / ml to 16.83± 12.90 pg / ml post-intervention (p-value = 0.63). IL-12 increased from 7.78± 5.963 pg / ml at baseline to 9.968± 6.769 pg / ml post-intervention (p-value=0.39) in the control group, while it increased from a baseline value of 10.79± 11.41 pg / ml to 10.86± 8.24 pg / ml post-intervention in the treatment group (p-value=0.75). IL-17 values increased from 20.95± 19.44 pg / ml at baseline to 25.06± 19.47 pg / ml post-intervention in the control group (p-value=0.40), while they increased from a baseline of 18.49± 14.28 pg / ml to 21.31± 14.72 pg / ml post-intervention in the treatment group (p-value=0.49). Tumor Necrosis Factor-alpha (TNF-Alpha) values increased from 7.549± 7.137 pg / ml at baseline to 18.61± 12.84 pg / ml post-intervention in the control group (p-value=0.01), while they increased from 6.47± 4.59 pg / ml at baseline to 11.55± 7.21 pg / ml post-intervention (p-value=0.008). See also Figure 9.

[0051] Discussion: Psoriasis vulgaris is an inflammatory skin disease that involves infiltration of leukocytes, activation of skin-resident cells and increased production of numerous inflammatory cytokines, and chemokines. Activated dendritic cells (DCs) and T cells are also key players in the pathogenesis of psoriasis causing 'inflammation' via a pathway of sequential interleukin-23 (IL-23) synthesis, interferon-γ(IFN-γ) production, activation of STAT1 (signal transducer and activator of transcription 1) activation, and subsequent transcription of a wide range of IFN- and STAT-1-regulated genes

[0018] . In situ expression of macrophage inflammatory protein-3 (MIP-3; CCL19), secondary lymphoid tissue chemokine (SLC; CCL21), and other chemokines generally seen in formal lymphoid tissues may aid to sustain DC accumulation and overall inflammatory pathway activation

[0018] .

[0052] Though different components of the immune system is involved, current mainstay approaches to treatment include the use of topical or systemic steroids and biologicals that reduce immune mediated inflammation.　More recently systemic immunosuppressive therapies for psoriasis have been employed including the use of biologic agents such as adalimumab, infliximab, etanercept (tumor necrosis factor (TNF)α blockers), ustekinumab (interleukin 12 / 23 monoclonal antibody), and alefacept (T-cell inhibitor), nonbiologic immunosuppressive agents that include cyclosporine and methotrexate and the non-immunosuppressive agent, acitretin. The main issue of these immune-suppression agents is that although they suppress the clinical and histologic manifestations of psoriasis, their use is associated with major side effects

[0019] . For instance, Adalimumab and infliximab have been demonstrated to increase tuberculosis risk, histoplasmosis, and other granulomatous diseases as well as herpes zoster and malignancy

[0019] . Ustekinumab has been implicated to increase cardiovascular risk

[0019] . Even the use of steroids along with associated side effects, when used in elderly, in whom there is higher incidence of psoriasis, immunosenescence i.e an immunosuppressed state of the elderly due to natural aging which is characterized by decrease in T-cell function with aging as well as other age-related changes in innate immunity further exacerbates the side effects and co-morbidities

[0019] . Therefore, there is still a need for agents that can regulate the immune system without associated serious side effects.　 In the present study, we have shown that Neu REFIX beta-glucan markedly improves the clinical and histologic severity of psoriasis with a significant reduction in lymphocyte infiltration, thickness of epidermis and decrease in levels of IL-6 and　PLR ratio. Of interest,　the clinical improvement observed was greater in patients with high BMI (>30). High BMI has been reported to be an associated risk factor for incidence and severity of psoriasis

[0017] although the mechanism for this is not known. As NEU REFIX is known to alter the gut microbiome [16,20], the beneficial effect may be thought this mechanism although further studies are required to firmly make this conclusion

[0021] .

[0053] Although the cause of psoriasis is unknown, it is thought to be multifaceted, with multiple critical components such as genetic vulnerability, environmental triggers, skin barrier disturbance, and immunological dysfunction

[0022] . Two basic grading systems are used to determine the severity of plaques: the psoriasis area and severity index (PASI) and the investigator global assessment (IGA). The PASI scale quantifies disease severity by measuring area and intensity (erythema, induration, desquamation, and body surface area). The IGA was established as an alternate assessment metric due to the complexity of the PASI calculation. The IGA determines if the lesion severity is static or changing. The normal appearing "uninvolved" skin directly adjacent to the affected area is an interesting aspect of chronic plaque psoriasis

[0022] . Several recent investigations

[0023] have shown that uninvolved tissue contains a distinct set of genes that, when triggered, may account for the transformation of uninvolved skin to involved plaque. However, considering the numerous cell types known to participate in this conversion, it is doubtful that a single triggering factor can be isolated. Therefore, while the search for the triggers continue, Neu REFIX beta-glucans can be considered as a safe-biologic agent which has produced clinically significant reduction in PASI score (PASI 50 achieved in 40% of the treatment arm patients) and the patients in the treatment arm have demonstrated improvement both clinically and in biological markers of immune-inflammation dysfunction. Immunological-inflammatory parameters showed significant reduction in the treatment arm such as IL-6, lymphocyte infiltration and thickness of epidermis. Further, the reduction in PLR in the current study is important because earlier literature has pointed out that platelets-to-lymphocytes (PLR) is a simple and inexpensive marker of systemic inflammatory response and a significant positive correlation found in terms of PASI score and PL ratio

[0024] .　Derangement of renal function is more prevalent in psoriatic patients, especially with those with psoriatic arthritis

[0025] and one of its indicators is elevated serum creatinine levels which has been observed in patients with psoriasis and with certain treatments for psoriasis. Therefore, its levels are monitored in clinical trials as a safety parameter and in the present study, the decrease in creatinine levels post-Neu REFIX intervention is an indicator of the safety and efficacy profile of Neu REFIX apart from its potential applications for psoriatic co-morbidities such as kidney disease and arthritis.

[0054] There are important criteria / mechanisms that need further exploration, for example, in some patients of treatment arm, in whom there was significant reduction in PASI score, there was no evident change in the lymphocyte infiltration score. This mismatch between the clinical manifestation and underlying immune-mediated pathology needs further studies. Additionally, the levels of IL-2, IL-17, IL-21, NLR and ESR whose values have shown increase in the treatment group as well needs further long term follow up studies.

[0055] Example 2: We wanted to study the effects of the safe beta-glucan food supplement Neu REFIX on IL-23 expression in the psoriasis patients.

[0056] Methods: Paraffin-embedded tissue sections from patients with psoriasis before and after Neu REFIX intervention, were deparaffinized and rehydrated using standard protocols. Antigen retrieval was performed using heat-induced method.　Tissues were fixed with 20% high-concentration formalin instead of 10% neutral buffered formalin.

[0057] Following antigen retrieval, sections were blocked with a species-appropriate serum to prevent non-specific antibody binding. The primary antibody, IL-23 abcam clone ab45420, was diluted in blocking solution and incubated on the sections for 30-60 minutes at room temperature. After washing with Tris-buffered saline with Tween 20 (TBS-T), a secondary antibody was applied and incubated for a similar duration. Sections were then washed again with TBS-T.

[0058] Diaminobenzidine (DAB) chromogenic substrate was used for visualization of the primary antibody target. Sections were incubated with DAB solution (prepared by mixing 250 L of 1% DAB and 250 L of 0.3% hydrogen peroxide with 5 mL of PBS) for 1-3 minutes at room temperature. Following washes with distilled water, sections were counterstained with Giemsa stain to differentiate pigment-bearing cells. Finally, sections were dehydrated and mounted using standard procedures.

[0059] An automated staining system, Leica Bond III, was used to perform the immunohistochemistry staining process. Lymphocyte infiltration sites were identified in the tissue sections. The number of positively stained cells expressing the target antigen was manually counted at 400x magnification.

[0060] The IL-23 positive stained cells were reduced by 40% post-Neu REFIX intervention. See also Figure 10.

[0061] Conclusion: Here we demonstrate the safety and effectiveness of Neu REFIX beta-glucans as a treatment in patients with moderate to severe psoriasis with substantial reductions in PASI, IGA, lymphocyte infiltration, and epidermal thickness, along with improvements in blood immune-inflammatory parameters. In this trial, Neu REFIX beta-glucans are shown to be safe in psoriasis and have yielded a significant reduction in PASI, IGA, lymphocyte infiltration, epidermal thickness apart from improvement in blood immune-inflammatory parameters in this short 28-day clinical trial. Larger longer clinical trials for validation in both cutaneous and systemic psoriasis, associated co-morbidities will help to prove the potentials of this oral Neu REFIX beta-glucan as an adjuvant in the treatment of auto-immune skin and systemic diseases.

[0062] References: 1.　Deng J, Yao D, Lu C, Wen Z, Yan Y, He Z, Wu H, Deng H. Oral Chinese herbal medicine for psoriasis vulgaris: protocol for a randomised, double-blind, double-dummy, multicentre clinical trial. BMJ Open. 2017 Nov 3;7(11):e014475. 2.　Griffiths CE, Barker JN. Pathogenesis and clinical features of psoriasis. Lancet 2007;370:263-71. 10.1016 / S0140-6736(07)61128-3 3.　Nast A, Boehncke WH, Mrowietz U, et al.. German S3-guidelines on the treatment of psoriasis vulgaris (short version). Arch Dermatol Res2012;304:87-113 4.　Balak DMW, Gerdes S, Parodi A, Salgado-Boquete L. Long-term Safety of Oral Systemic Therapies for Psoriasis: A Comprehensive Review of the Literature. Dermatol Ther (Heidelb). 2020 Aug;10(4):589-613. 5.　Armstrong A.W., Read C. Pathophysiology, Clinical Presentation, and Treatment of Psoriasis: A Review. JAMA. 2020;323:1945-1960. doi: 10.1001 / jama.2020.4006. 6.　Jo HG, Kim H, Baek E, Lee D, Hwang JH. Efficacy and Key Materials of East Asian Herbal Medicine Combined with Conventional Medicine on Inflammatory Skin Lesion in Patients with Psoriasis Vulgaris: A Meta-Analysis, Integrated Data Mining, and Network Pharmacology. Pharmaceuticals (Basel). 2023 Aug 15;16(8):1160. 7.　Raghavan K, Dedeepiya VD, Srinivasan S, Pushkala S, Subramanian S, Ikewaki N, Iwasaki M, Senthilkumar R, Preethy S, Abraham S. Beneficial immune-modulatory effects of the N-163 strain of Aureobasidium pullulans-produced 1,3-1,6 Beta glucans in Duchenne muscular dystrophy: Results of an open-label, prospective, exploratory case-control clinical study. IBRO Neuroscience reports 2023; 15: 90-99. doi: 10.1016 / j.ibneur.2023.06.007. 8.　Preethy S, Aoki Y, Minegishi K, Iwasaki M, Senthilkumar R, Abraham SJK. Resolution of fibrosis in mdx dystrophic mouse after oral consumption of N-163 strain of Aureobasidium pullulans produced β-glucan. Sci Rep. 2023 Oct 9;13(1):17008. 9.　Dedeepiya V, Vetrievel C, Ikewaki N, Ichiyama K, Yamamoto N, Kawashima H,　Bharatidasan SS,　Srinivasan SS, Senthilkumar R, Preethy S, Abraham S. Improvement in Expanded Disability Status Scale (EDSS) and anti-inflammatory parameters in patients with multiple sclerosis following oral consumption of N-163 strain of Aureobasidium pullulans produced beta glucan in a pilot clinical study. medRxiv 2023.05.14.23289953v2; doi: 10.1101 / 2023.05.14.23289953 10.　Ikewaki N, Ikeue Y, Nagataki M, Kurosawa G, Dedeepiya VD, Rajmohan M, Vaddi S, Senthilkumar R, Preethy S, Abraham SJK. Beneficial effects of 1,3-1,6 β-glucans produced by Aureobasidium pullulans on non-esterified fatty acid levels in diabetic KKAy mice and their potential implications in metabolic dysregulation. J Diabetes MetabDisord. 2022 Dec 11;22(1):487-494. 11.　Ikewaki N, Levy GA, Kurosawa G, Iwasaki M, Dedeepiya VD, Vaddi S, Senthilkumar R, Preethy S, Abraham SJK. Hepatoprotective Effects of Aureobasidium pullulans Derived β1,3-1,6 Glucans in a Murine Model of Non-alcoholic Steatohepatitis. J Clin Exp Hepatol. 2022 Nov-Dec;12(6):1428-1437. 12.　Raghavan K, Dedeepiya VD, Suryaprakash V, Rao KS, Ikewaki N, Sonoda T, Levy GA, Iwasaki M, Senthilkumar R, Preethy S, Abraham SJ. Beneficial effects of novel aureobasidium pullulans strains produced beta-1,3-1,6 glucans on interleukin-6 and D-dimer levels in COVID-19 patients; results of a randomized multiple-arm pilot clinical study. Biomed Pharmacother. 2022 Jan;145:112243. 13.　Pushkala S, Seshayyan S, Theranirajan E, Sudhakar D, Raghavan K, Dedeepiya VD, IkewakiN,Iwasaki M, Preethy S, Abraham S. Efficient control of IL-6, CRP and Ferritin in Covid-19 patients with two variants of Beta-1,3-1,6 glucans in combination, within 15 days in an open-label prospective clinical trial.medRxiv 2021.12.14.21267778; doi: 10.1101 / 2021.12.14.21267778 14.　Ikewaki N, Sonoda T, Kurosawa G, Iwasaki M, Dedeepiya VD, Senthilkumar R, Preethy S, Abraham S . Beta 1,3-1,6 glucans produced by two novel strains of Aureobasidium pullulans exert immune and metabolic beneficial effects in healthy middle-aged Japanese men: Results of an exploratory randomized control study. J Aging Res & Lifestyle 2023;12:61-71. doi: 10.14283 / jarlife.2023.11. 15.　Raghavan K, Dedeepiya VD, Yamamoto N, Ikewaki N, Iwasaki M, Dinassing A, Senthilkumar R, Preethy S, Abraham SJK. Randomised trial of Aureobasidium pullulans-produced beta 1,3-1,6-glucans in patients with Duchenne muscular dystrophy: Favourable changes in gut microbiota and clinical outcomes indicating their potential in epigenetic manipulation. medRxiv 2022.12.09.22283273; doi: 10.1101 / 2022.12.09.22283273 16.　Vidyasagar DevaprasadDedeepiya, ChockanathanVetrievel, NobunaoIkewaki, Naoki Yamamoto, Hiroto Kawashima, Koji Ichiyama, Rajappa Senthilkumar, SenthilkumarPreethy, Samuel JK Abraham. Beneficial changes in the gut microbiome of patients with multiple sclerosis after consumption of Neu REFIX B-glucan in a clinical triaL. medRxiv 2023.09.07.23295172; doi: https: / / doi.org / 10.1101 / 2023.09.07.23295172 17.　Norden A, Rekhtman S, Strunk A, Garg A. Risk of psoriasis according to body mass index: A retrospective cohort analysis. J Am Acad Dermatol. 2022 May;86(5):1020-1026. d 18.　Ben Abdallah H, Johansen C, Iversen L. Key Signaling Pathways in Psoriasis: Recent Insights from Antipsoriatic Therapeutics. Psoriasis (Auckl). 2021 Jun 29;11:83-97. doi: 10.2147 / PTT.S294173. 19.　Wong JW, Koo JY. The safety of systemic treatments that can be used for geriatric psoriasis patients: a review. Dermatol Res Pract. 2012;2012:367475. doi: 10.1155 / 2012 / 367475. 20.　Preethy S, Ikewaki N, Levy GA, Raghavan K, Dedeepiya VD, Yamamoto N, Srinivasan S, Ranganathan N, Iwasaki M, Senthilkumar R, Abraham SJK. Two unique biological response-modifier glucans beneficially regulating gut microbiota and faecal metabolome in a non-alcoholic steatohepatitis animal model, with potential for applications in human health and disease. BMJ Open Gastroenterology 2022;9:e000985. doi: 10.1136 / bmjgast-2022-000985. 21.　Ely PH. Is psoriasis a bowel disease? Successful treatment with bile acids and bioflavonoids suggests it is. Clin Dermatol. 2018 May-Jun;36(3):376-389. 22.　Rendon A, Schakel K. Psoriasis Pathogenesis and Treatment. Int J Mol Sci. 2019 Mar 23;20(6):1475. doi: 10.3390 / ijms20061475. 23.　Ayala-Fontanez N, Soler DC, McCormick TS. Current knowledge on psoriasis and autoimmune diseases. Psoriasis (Auckl). 2016 Feb 22;6:7-32. doi: 10.2147 / PTT.S64950. 24.　Mondal S, Guha S, Saha A, Ghoshal L, Bandyopadhyay D. Evaluation of Neutrophil-to-Lymphocyte Ratio and Platelet-to-Lymphocyte Ratio in Chronic Plaque Psoriasis. Indian J Dermatol. 2022 Jul-Aug;67(4):477. 25.　Khan A, Haider I, Ayub M, Humayun M. Psoriatic Arthritis Is an Indicator of Significant Renal Damage in Patients with Psoriasis: An Observational and Epidemiological Study. Int J Inflam. 2017;2017:5217687. 26.　Kannan AK, Su Z, Gauvin DM, Paulsboe SE, Duggan R, Lasko LM, Honore P, Kort ME, McGaraughty SP, Scott VE, Gauld SB. IL-23 induces regulatory T cell plasticity with implications for inflammatory skin diseases. Sci Rep. 2019 Nov 27;9(1):17675. 27.　Fragoulis GE, Siebert S. The role of IL-23 and the use of IL-23 inhibitors in psoriatic arthritis. Musculoskeletal Care. 2022 Nov;20 Suppl 1(Suppl 1):S12-S21. 28.　Tang C, Chen S, Qian H, Huang W. Interleukin-23: as a drug target for autoimmune inflammatory diseases. Immunology. 2012 Feb;135(2):112-24. 29.　Sally El-Leithy, Nahed Sherif, Nadia H. El-Arousy, Rana El-Hilaly, Marwa M. Shakweer. Cutaneous immunohistochemical expression of interleukin-23 receptor (IL-23R) in psoriasis and psoriatic arthritis patients: Relation to musculoskeletal ultrasound findings. The Egyptian Rheumatologist 2020; 42(4): 313-318 30.　Nogueira M, Torres T. Guselkumab for the treatment of psoriasis - evidence to date. Drugs Context. 2019 Jul 9;8:212594. 31.　Kim PJ, Lansang RP, Vender R. A Systematic Review and Meta-Analysis of Injection Site Reactions in Randomized-Controlled Trials of Biologic Injections. J Cutan Med Surg. 2023 Jul-Aug;27(4):358-367. d

Claims

1. A composition for treating psoriasis, comprising a beta-glucan.

2. A composition for decreasing lymphocyte infiltration in a subject who suffers from psoriasis or has a risk of suffering from psoriasis, comprising a beta-glucan.

3. A composition for decreasing an expression of IL-23 in a subject who suffers from psoriasis or has a risk of suffering from psoriasis, comprising a beta-glucan.

4. The composition of any one of claims 1-3, wherein the beta-glucan comprises a 1,3-1,6-beta-glucan produced by Aureobasidium pullulans N-163 (NITE BP-03377).

5. The composition of any one of claims 1-4, wherein psoriasis is psoriasis vulgaris (PVS).

Images