CN116236579A - Application of IL-34 antibody in preparation of medicament for treating psoriasis - Google Patents

Application of IL-34 antibody in preparation of medicament for treating psoriasis Download PDF

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CN116236579A
CN116236579A CN202310281116.6A CN202310281116A CN116236579A CN 116236579 A CN116236579 A CN 116236579A CN 202310281116 A CN202310281116 A CN 202310281116A CN 116236579 A CN116236579 A CN 116236579A
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psoriasis
skin
antibody
mice
medicament
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周利君
谈智
林震嘉
李莹
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Sun Yat Sen University
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Sun Yat Sen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses an application of an IL-34 antibody in preparing a medicament for treating psoriasis. The invention discovers that the psoriasis-like symptoms and pruritus of a psoriasis mouse model can be relieved by inhibiting an IL-34 signal pathway, the characterization and pathological changes of the skin damage area and severity index (PASI) of the psoriasis are obviously improved, the increase of the thickness of the epidermis and the proliferation of lesion skin cells are inhibited, and the inflammatory reaction is inhibited; that is, psoriasis can be treated by inhibiting IL-34 signaling pathway, so that agents inhibiting IL-34 signaling pathway, such as IL-34 antibodies, etc., can be used for the preparation of psoriasis therapeutic drugs. The invention not only widens the application range of the IL-34 antibody, but also enriches the medicines for treating psoriasis, and is beneficial to the treatment of psoriasis patients and the development of novel safe, efficient and economic psoriasis treatment medicines.

Description

Application of IL-34 antibody in preparation of medicament for treating psoriasis
Technical Field
The invention belongs to the technical field of psoriasis treatment. More particularly, the application of IL-34 antibody in preparing medicine for treating psoriasis.
Background
Psoriasis is a common chronic, recurrent, inflammatory autoimmune skin disease, with typical skin appearing as well-defined scales and plaques, with recurrent episodes, and nearly half of patients with discomfort such as itching. Psoriasis can be classified into four major categories according to its clinical manifestations, psoriasis vulgaris, arthrosis type psoriasis, erythrodermic type psoriasis and pustular type psoriasis, with psoriasis vulgaris being one of the most common types of psoriasis.
At present, the treatment of psoriasis is mainly divided into two types of traditional treatment and biological agent targeting treatment. Although biological agents or small molecule drugs targeting key cytokines in psoriasis treatment mechanisms achieve satisfactory curative effects, the biological agents or small molecule drugs cannot thoroughly radically treat psoriasis and effectively prevent psoriasis recurrence, and meanwhile, the biological agents or small molecule drugs have the conditions of poor curative effects of partial patients and the like. Therefore, the development of a novel psoriasis therapeutic drug which is safe, efficient and economical has important clinical significance.
IL-34 (Interleukin 34) is a novel interleukin large family cytokine identified in 2008 by high throughput screening of proteins secreted by humans (Lin, H., et al, discovery of a cytokine and its receptor by functional screening of the extracellular proteome. Science,2008.320 (5877): p.807-11.). Through more than ten years of research, IL-34 has been found to have specific physiological functions and key pathological roles in various diseases, but the role in psoriasis is not clear, and no report on the treatment of psoriasis aiming at IL-34 targets exists at present.
Disclosure of Invention
The invention provides an application of an IL-34 antibody in preparation of psoriasis therapeutic drugs, which overcomes the defect of poor curative effect of the existing psoriasis therapeutic drugs.
It is a first object of the present invention to provide the use of an agent that inhibits the IL-34 signaling pathway in the manufacture of a medicament for the treatment of psoriasis.
It is a second object of the present invention to provide the use of an agent that inhibits the IL-34 signaling pathway in the manufacture of a medicament for alleviating the progression of psoriasis lesions.
A third object of the present invention is to provide a medicament for the treatment of psoriasis.
The above object of the present invention is achieved by the following technical scheme:
the invention discovers that IL-34 mediated activation of signal paths participates in the pathogenesis of psoriasis and immune inflammatory disorder by analyzing the expression conditions of IL-34 genes and three receptor genes (Csf 1r, sdc1, ptprz 1) thereof in the lesion skin, the parapathic skin and the normal human skin of clinical psoriasis patients and constructing a psoriasis mouse model and detecting the expression conditions of IL-34 and receptors thereof in the skin of the psoriasis mouse model. Meanwhile, the invention suppresses inflammatory cytoma and keratinocyte proliferation and relieves itching symptoms by injecting IL-34 antibody into the psoriasis mouse model subcutaneously and suppressing IL-34 signal path, thereby remarkably relieving the progress of skin damage of the psoriasis mouse model. Accordingly, the present application protects the following applications:
the invention claims the use of a formulation that inhibits the IL-34 signaling pathway in the preparation of a medicament for the treatment of psoriasis.
In particular, the psoriasis is psoriasis vulgaris, arthropathy type psoriasis, erythroderma type psoriasis or pustular type psoriasis.
The invention also claims the use of a formulation that inhibits the IL-34 signaling pathway in the preparation of a psoriasis lesion progression remission medicament.
In particular, the relief of psoriatic lesion progression is achieved by inhibiting inflammatory cytosis, inhibiting keratinocyte proliferation and alleviating itching symptoms.
In particular, the psoriasis is psoriasis vulgaris, arthropathy type psoriasis, erythroderma type psoriasis or pustular type psoriasis.
Specifically, according to the application, the medicine contains pharmaceutically acceptable auxiliary materials besides the preparation for inhibiting the IL-34 signal path.
As an alternative embodiment, the dosage form of the drug may be an injection or a microneedle.
Specifically, the agent that inhibits the IL-34 signaling pathway is an agent that targets inhibition of IL-34 function.
In particular, the agent that inhibits the IL-34 signaling pathway is an IL-34 antibody.
In particular, the IL-34 antibody is an anti-IL-34 antibody having neutralizing biological activity, i.e., an IL-34 neutralizing antibody.
Specifically, the IL-34 antibody is used in an amount of at least 5. Mu.g/kg.
The invention also provides a medicament for treating psoriasis, which contains a preparation for inhibiting the IL-34 signal path and pharmaceutically acceptable auxiliary materials thereof.
In particular, the agent that inhibits the IL-34 signaling pathway is an IL-34 antibody.
The invention has the following beneficial effects:
the invention discovers that the psoriasis-like symptoms and itch can be relieved by inhibiting the IL-34 signaling pathway, the characterization and pathological changes of the skin damage area and severity index (PASI) of the psoriasis are obviously improved, the alleviation and relief of the erythema, the scaling and the itch symptoms at the psoriasis patient are particularly embodied, the increase of the epidermis thickness and the proliferation of lesion skin cells are simultaneously inhibited, and the inhibition of inflammatory reaction is realized, so that the psoriasis can be improved or treated by inhibiting the IL-34 signaling pathway, and preparations inhibiting the IL-34 signaling pathway, such as IL-34 antibodies and the like, can be used for preparing medicaments for treating the psoriasis. The invention not only widens the application range of the IL-34 antibody, but also enriches the medicines for treating psoriasis, and is beneficial to the treatment of psoriasis patients and the development of novel safe, efficient and economic medicines for treating psoriasis.
Drawings
FIG. 1 shows the expression of IL-34 gene and its 3 receptor genes (Csf 1r, sdc1, ptprz 1) in clinical psoriatic lesions (PP), parapathogenic (PN) and normal human (NN) skin using the GEO database (GSE 13355); NN is 64 normal skin samples, PN is 58 parapsoriatic skin samples, and PP is 58 psoriatic lesion skin samples; comparison of data between groups with One-Way ANOVA, tukey's post hoc test,/P<0.01，***P<0.001 is PP vs NN group; #### P<0.0001 is PP versus PN group.
FIG. 2 shows IL-34 and its receptor expression in skin of imiquimod-induced psoriasis-like mice (WT IMQ) and petrolatum control mice (WT CON); panel A shows the results of bulk RNA-seq data of the dorsal skin of IL-34 and its receptor mRNA at day 2.5 after molding in different groups of mice; panel B shows the expression of IL-34 protein in the dorsal skin of the 2.5 and 4.5 days after molding in different groups of mice as determined by enzyme-linked immunosorbent assay (ELISA); c and D in the figures are the results of CSF1R immunohistochemical staining of skin tissue from different groups of mice and their statistical plots; FIG. E shows the immunofluorescence double staining results of CSF1R in the skin sample of the skin lesion at day 4.5 after model construction; comparison was performed using two independent samples, mann-Whitney test, P <0.05, P <0.01.
FIG. 3 shows the results of treatment of mice with a model psoriasis with IL-34 antibody by subcutaneous injection; figure a is a back skin image of day 4 post-molding psoriasis mice; panel B is a time chart of Psoriasis Area and Severity Index (PASI) score statistics (left), skin fold thickness variation (middle), and touch induced itch behavioral testing (right) for different groups of mice; in the figure, vehi is a solvent control group, namely a mouse group with the back skin externally coated with equal amounts of Vaseline and subcutaneous injection of physiological saline, and Anti-IL-34 is a control group with the back skin externally coated with equal amounts of Vaseline and subcutaneous injection of Anti-IL-34 antibody; vehi+IMQ is a model mouse group which is injected subcutaneously with normal saline 30min in advance and then is applied with Imiquimod (IMQ) on the back skin, and Anti-IL-34+IMQ is a treatment mouse group which is injected subcutaneously with Anti-IL-34 30min in advance and then is applied with IMQ on the back skin; tukey's post hoc test using Two-Way ANOVA analysis<0.0001 is vehi+imq vs. Vehi group; ### P<0.001， #### P<0.0001 is Anti-IL-34+IMQ compared to the Vehi+IMQ group.
FIG. 4 is a graph showing the effect of IL-34 antibody treatment on skin inflammatory cytosis, epidermal keratinocyte proliferation and sensory neuron excitability in psoriatic mice; FIG. A is an immunofluorescent staining pattern of macrophages (marker F4/80), CSF1R, cell proliferation (marker Ki 67) and neutrophils (marker NIMP-R14) in the skin tissue of mice in the Vehi+IMQ group and Anti-IL-34+IMQ group; b in the graph is a statistical plot of the results shown in A, post hoc inspection of Tukey's using One-Way ANOVA analysis, & ltP & gt<0.01，****P<0.0001 Vehi+IMQ group was compared to Vehi group; ## P<0.01， ### P<0.001， #### P<0.0001 is an Anti-IL-34+IMQ group versus the Vehi+IMQ group; c in the figure is the change in neuronal excitability (marker p-ERK) in the Dorsal Root Ganglion (DRG) and spinal cord dorsal horn (SDH) shallow layer; d in the graph is a statistical graph of the results shown in C; a separate sample is used for the t-test, #### P<0.0001 is an Anti-IL-34+IMQ group versus the Vehi+IMQ group.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 expression changes of IL-34 Gene and its receptor
The invention detects the expression change of IL-34 gene and its receptor of skin injury skin, paradisease skin and normal human skin of clinical psoriasis patient. Specifically, the present invention utilizes GEO databases to find a biopsy skin total RNA data (GSE 13355) covering 58 cases of psoriasis (including diseased skin and paradiseased skin) and 64 normal healthy controls. The expression of the IL-34 gene and its three receptor genes (Csf 1r, sdc1, ptprz 1) in normal healthy control samples (NN), parapsoriatic skin samples (PN) and diseased skin samples (PP) was analyzed by platform annotation (GPL 570).
The expression of the IL-34 gene and its 3 receptor genes (Csf 1r, pdc 1, ptprz 1) in the diseased skin, the parapathogenic skin and the normal human skin of clinical psoriasis patients, analyzed by the GEO database (GSE 13355) is shown in FIG. 1; wherein NN is 64 normal skin samples, PN is 58 parapsoriatic skin samples, and PP is 58 psoriatic lesion skin samples. As can be seen from FIG. 1, although there was no statistical difference between the expression of IL-34 mRNA in the psoriatic diseased skin group (PP) and the normal healthy control group (NN), the expression of IL-34 mRNA in the parapsoriatic tissue group (PN) was significantly higher than in the NN and PP groups. Furthermore, CSF1R (CSF 1R) mRNA was significantly reduced in both psoriatic lesions and parapathic skin in the receptor for IL-34, but SDC1 (SDC 1) and PTP- ζ (Ptprz 1) mRNA was expressed significantly higher in psoriatic lesions and skin groups than in healthy human skin groups; the results suggest that IL-34 may be involved in the development and progression of psoriasis.
EXAMPLE 2 imiquimod-induced expression of IL-34 and its receptor in murine psoriasis-like skin
The invention utilizes Imiquimod (IMQ) to induce a mouse psoriasis-like model, and detects the expression condition of IL-34 and a receptor thereof in the induced mouse psoriasis-like skin.
1. Construction of mice psoriasis-like model and acquisition of skin tissue sample
The experimental mice used in this example were female C57BL/6J mice purchased from the university of Zhongshan animal experiment center, and were fed under SPF-grade environment until 8-10 weeks of age, and then animal experiments were started. Animal experiments in the present invention have been approved by the laboratory animal ethics committee of the university of Zhongshan.
Mice were numbered and randomized into IMQ-induced psoriasis-like model groups and petrolatum control groups (n=16-18/group). The back of the mice was skinned with an electric razor 3 days before the start of the experiment, forming 2X 3cm 2 An exposed area of a size. A5% imiquimod cream (available from Inova Pharmaceuticals company in an amount of 10 mg/cm) was applied to the back skin of mice in the psoriasis model group 2 1 time per day) for 5 times. The skin of the mice can be observed to appear light red spots and scales successively on the 1 st day of modeling, and the model is successfully constructed from the 4.5 th day to the most serious day, which indicates that the psoriasis-like mouse model is successfully constructed; vaseline control mice were given an equal amount of petrolatum (CON) to the back skin. Two groups of mouse back skin tissues were collected on day 2.5 of modeling (IMQ 2.5 d) for subsequent Bulk RNA-seq sequencing analysis, and two groups of mouse back skin tissues were collected on day 4.5 of modeling (IMQ 4.5 d) for ELISA, immunohistochemistry and immunofluorescent staining treatments.
2. Bulk RNA-seq sequencing and analysis
Sending back skin tissues of psoriasis-like model group mice (IMQ group) and vaseline control group mice (CON group) molded for 2.5 days to Baimeike biotechnology limited company for RNA transcriptome analysis (n=4/group) of eukaryotes with reference genome, wherein the reference genome is GRCm38_release95, differential expression analysis software is edge_DESeq2, and the screening condition is FDR < 0.05; fold Change (FC) is more than or equal to 1.5.
The results of bulk RNA-seq data of the back skin of IL-34 and its receptor mRNA at day 2.5 after the molding of an IMQ psoriasis-model mouse (WT IMQ) and a Vaseline control mouse (WT CON) are shown as A in FIG. 2, and as can be seen from A in FIG. 2, the mRNA expression level of IL-34 and its receptor CSF1R is significantly decreased in the skin of the psoriasis-model mouse, but the expression levels of the other two receptors PTP- ζ (Ptprz 1) and Syndecan-1 (Sdc 1) mRNA of IL-34 are significantly increased; n=4/group.
3. ELISA detection of protein expression level of IL-34 in skin lesion skin and healthy control skin of psoriasis model mice
The back skin of the mice was thoroughly lysed with RIPA lysate (containing 1% protease inhibitor), centrifuged at 4℃and 10000g for 10 min, and the supernatant was taken. Protein standards were prepared and samples were diluted according to ELISA kit (Elabscience, catalog #: E-EL-M0720C-96T) instructions; 2-3 multiple wells are arranged on each standard substance and each sample, and the standard substances and the samples are added into a 96-well plate of an ELISA kit by utilizing a multi-channel pipette. And incubating the standard substance and the sample according to the conditions recommended by the kit instruction, and sequentially adding the primary antibody, the HRP, the substrate working solution and the stop solution to complete the ELISA operation step. Detecting the absorbance of each well of the 96-well plate by using a full-automatic enzyme-linked immunoassay (Bio-Rad company); the concentration of IL-34 in each skin sample was calculated from the concentration-fitted linear working curve of the standard.
The results of measuring the IL-34 content in the skin lesion skin tissue of the psoriasis model mice homogenized by the skin sample and ELISA and the Vaseline control skin tissue are shown as B in FIG. 2, and as can be seen from B in FIG. 2, the IL-34 content in the skin lesion skin of the psoriasis model mice is not significantly different from that in the Vaseline control group, and n=4 to 6 mice/group.
4. Immunohistochemical detection of protein expression level of CSF1R in skin lesion and Vaseline control skin of psoriasis model mice
Collecting the back skin of the mice, placing the mice in 10% formaldehyde for fixation, embedding paraffin, and slicing; after dewaxing, hydration, antigen retrieval and endogenous peroxidase removal of the flakes, primary antibodies (Anti-CSF 1R Anti, santa Cruz, catalog#: SC-692) and secondary antibodies (enhanced goat Anti-mouse/rabbit IgG polymer, china fir gold bridge, catalog#: PV-9000) were added dropwise and developed with DAB kit (Vector Laboratories, catalog#: sk-4100); slides were scanned using a pathology section scanning imager and the gray scale value of CSF1R in each set of skin samples was measured by ImageJ software.
The results of CSF1R immunohistochemical staining of skin tissues of different groups of mice and their statistical plots are shown as C and D in fig. 2, respectively, showing relative fluorescence gray scale values (RelOD) and cell changes of CSF1R positive signals between groups, n=6/group.
5. Immunofluorescence double-staining detection of CSF1R expression cell types
Collecting skin tissues of each back after 4.5 days of the model, placing the skin tissues in 4% paraformaldehyde for fixation, and placing 30% sucrose solution for dehydration for 2 days after 4 hours until the skin tissues are dehydrated and sunk in the sucrose solution; embedding the dehydrated specimen with OTC embedding liquid for 5min, cutting the tissue into slices of 16-18 mu m by using a frozen microtome, adhering the slices to an adhesive glass slide, and airing the slices at room temperature overnight; selecting and placing the dried glass slide into a washing tank, and slowly shaking and washing the glass slide for three times with PBS (phosphate buffer solution) for 5min each time; slowly shaking and sealing for 1 hour at room temperature by using 5% donkey serum sealing liquid; the blocking solution was aspirated, primary antibody was added and the mixture was slowly shaken overnight at 4℃for 18 hours. One antibody included an Anti-CSF1R antibody (Santa Cruz, catalog#: SC-692), an Anti-F4/80 antibody (Biolegend, catalog#: 123102), an Anti-Iba1 antibody (Abcam, catalog#: ab 5076), an Anti-CD3 antibody (Biolegend, catalog#: 100214), an NIMP-R14 antibody (Santa Cruz, catalog#: SC-59338), an Anti-PECAM1 antibody (R & D, catalog#: AF 3628); the primary antibody was blotted and washed three times with PBS for 15 minutes each in a wash tank. Then incubating the corresponding secondary fluorescent antibody (diluted by TBST), and slowly shaking the secondary fluorescent antibody at room temperature in dark place for 1 hour. After the secondary antibody is sucked off, the secondary antibody is washed three times by PBS for 15 minutes each time; finally, dropping anti-fluorescence quenching sealing tablet (with DAPI), slowly covering the cover glass to prevent bubble generation, and dropping a small amount of nail polish to adhere to the cover glass. The fluorescent pictures are taken by a common fluorescent microscope (EVOS FL Imaging System), and the shooting parameters such as exposure time, gain and the like of the same batch of experiments are fixed so as to ensure comparability between each sample and reduce experimental errors.
Analysis of results: fluorescence intensity and cell count were analyzed using ImageJ software. The fluorescence intensity was quantified with relative gray values (relative integrated density, intDen), the locations to be measured were circled with ImageJ software and the average gray value was automatically calculated.
Immunofluorescence double staining results of CSF1R in skin samples of skin lesions at day 4.5 after model assembly are shown in FIG. 2 as E, which shows that CSF1R is predominantly expressed in macrophages (F4/80, iba 1) but not in T cells (CD 3), endothelial cells (PECAM 1) and neutrophils (NIMP-R14).
From the above results, it was found that the expression level of IL-34 protein in skin of mice with lesions in Vaseline control and psoriasis-like models was not significantly changed, but the receptor expression was significantly changed: CSF1-R, PTP- ζ and Syndecan-1 are abnormally high expressed in psoriatic skin lesions. In combination with the high expression of IL-34 in clinical parapsoriasis tissues in the results of FIG. 1, it is suggested that IL-34-mediated activation of signaling pathways may be involved in the pathogenesis of psoriasis, affecting its phenotype and immunoinflammatory disorders.
Example 3 Effect of neutralizing antibodies to IL-34 on psoriasis
To determine whether activation of the IL-34 signaling pathway in the psoriatic lesions is involved in the course of psoriasis, the present invention employs subcutaneous injections of neutralizing antibodies to IL-34 in Imiquimod (IMQ) -induced psoriasis-like mouse models to see if inhibition of the IL-34 signaling pathway is effective in treating psoriasis.
1. Grouping mice
24 female C57BL/6J mice were purchased at the university of Zhongshan animal experiment center and the animal experiment was started after 8-10 weeks of age of feeding in SPF-grade environment. Mice were numbered and randomly divided into 4 groups: solvent control group (Vehi), anti-IL-34 antibody alone group (Anti-IL-34), silverPsoriasis group (vehi+imq) and Anti-IL-34 antibody plus psoriasis treatment group (Anti-IL-34+imq), n=6/group. The back of the mice was skinned 3 days before the start of the experiment using an electric razor to form 2X 3cm 2 An exposed area of a size. Animal experiments in the present invention have been approved by the laboratory animal ethics committee of the university of Zhongshan.
2. Model construction and therapeutic intervention for psoriasis of mice
The modeling procedure for psoriatic mice was the same as in example 2.
The anti-IL-34 antibody is selected from anti-IL-34 antibody (R & D company, catalog #: AF 5195) with neutralizing bioactivity for treating psoriasis and anti-IL-34 antibody control group mice. The specific procedure was back subcutaneous injection of 100. Mu.L of anti-IL-34 antibody (5. Mu.g/kg, 1 time/day, 5 times total) at four points 30 minutes prior to each imiquimod cream or petrolatum application. The solvent control group and the psoriasis group mice were subcutaneously injected with 100 μl of physiological saline (1 time per day, 5 times total) at the back 30 minutes before the daily cream application. Two groups of mice were individually collected by camera on day 4.5 of molding for gross phenotypes of skin lesions on the back (fig. 3A), psoriasis areas were measured and severity index (PASI score) was calculated (left in fig. 3B), skin fold thickness changes were measured with vernier calipers (in fig. 3B), and then touch-induced itching behavioural tests were examined (Alloknesis, right in fig. 3B).
The results of treatment of mice with subcutaneously injected IL-34 antibody on psoriasis model are shown in FIG. 3; a in fig. 3 is a back skin image on day 4 after molding of psoriatic mice; b in fig. 3 is a time chart of psoriasis area versus severity index (PASI) score statistics (left), skin fold thickness variation (middle), and touch induced itch behavioral testing (right) for different groups of mice; in the figure, vehi is a solvent control group, namely a mouse group with the back skin externally coated with equal amounts of Vaseline and subcutaneous injection of physiological saline, and Anti-IL-34 is a control group with the back skin externally coated with equal amounts of Vaseline and subcutaneous injection of Anti-IL-34 antibody; vehi+imq is a model group for applying imiquimod to back skin after 30min of subcutaneous injection of normal saline, and Anti-IL-34+imq is a treatment group for applying IMQ to local skin after 30min of subcutaneous injection of Anti-IL-34.
As can be seen from fig. 3, the psoriasis model group (vehi+imq, n=6/group) developed psoriasis-like dermatitis symptoms including oedema, erythema and scaling (fig. 3A), skin loss PASI scores (fig. 3B left), pre-model varying skin fold thickness (fig. 3B), and touch-induced itching behavior (Alloknesis, fig. 3B right) were all significantly increased compared to the solvent control group (Vehi). Psoriasis group (Anti-IL-34+imq) psoriasis-like skin symptoms were significantly improved following subcutaneous injection of Anti-IL-34 neutralizing antibodies: if erythema and scaling were visually reduced (right in fig. 3A), PASI scores were significantly reduced (left in fig. 3B) and skin fold thickness was also reduced (in fig. 3B); in addition, there was also a significant improvement in touch-induced itching behavior (Alloknesis, right in fig. 3B). The results show that subcutaneous injection of IL-34 antibody is significantly effective in alleviating the progression of skin lesions in psoriatic mice.
Example 4
To further observe the effect of anti-IL-34 antibody treatment on skin inflammation, skin thickness, and pruritic behaviours in psoriatic mice, the present invention examined the effect of anti-IL-34 antibody treatment on inflammatory cytosis, epidermal keratinocyte proliferation, and sensory neuron excitability using immunofluorescence.
Samples of each set of dorsal skin tissue, dorsal Root Ganglion (DRG) and dorsal horn of spinal cord (SDH) were collected after molding for 4.5d and fixed in 4% paraformaldehyde, followed by immunofluorescence experiments performed in the same manner as in example 2. Among the newly added primary antibodies are: anti-Ki67 antibody (Abcam, catalog#: ab 16667), p-ERK antibody (Santa Cruz, catalog#: SC-59338).
The results of the effect of IL-34 antibody treatment on the excitability of dermal macrophage cells (F4/80 label), neutrophils (NIMP-R14 label) inflammatory cells, epidermal keratinocyte proliferation (Ki 67 label), dorsal Root Ganglion (DRG) sensory neurons and spinal cord dorsal horn (SDH) superficial projection neurons of psoriatic mice are shown in FIG. 4. A in FIG. 4 is an immunofluorescent staining pattern of macrophages (marker F4/80), CSF1R, cell proliferation (marker Ki 67), neutrophils (marker NIMP-R14) in the skin tissue of mice in the Vehi+IMQ group and Anti-IL-34+IMQ group; b in fig. 4 is a statistical chart of the results shown in a; c in fig. 4 is the change in neuronal excitability (marker p-ERK) in DRG and SDH shallow layers; d in FIG. 4 is a statistical graph of the results shown as C.
As can be seen from fig. 4, the proportion of macrophages (F4/80, CSF1R markers), cell proliferation (Ki 67 marker), neutrophils (NIMP-R14 marker) which are dominant in diseased skin after treatment by subcutaneous injection of anti-IL-34 antibody was significantly reduced to the recovery control level, and neuronal excitability (marker p-ERK) in DRG and SDH shallow layers was also significantly reduced. The cell molecular level proves that 5 mug/kg of the anti-IL-34 antibody can effectively play roles in relieving the development of skin lesions of psoriasis mice in terms of inhibiting inflammatory cytoma, inhibiting proliferation of epidermal keratinocytes, relieving itching symptoms and the like.
It is also contemplated that the present invention employs topical injection of the skin lesion to reduce the systemic effects of systemic drug delivery. As shown in FIG. 4, the anti-IL-34 antibody+IMQ group and the anti-IL-34 antibody control group are similar to the solvent control group mice, and the skin thickness and inflammatory cells and proliferative cells indirectly demonstrate the safety of the anti-IL-34 antibody administration. The above results further demonstrate the potential of subcutaneous injection of anti-IL-34 antibodies for the treatment of psoriasis, and can be used for the preparation of psoriasis therapeutic drugs.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. Use of an agent that inhibits the IL-34 signaling pathway in the manufacture of a medicament for the treatment of psoriasis.
2. Use of an agent that inhibits the IL-34 signaling pathway in the manufacture of a medicament for alleviating the progression of psoriasis lesions.
3. The use according to claim 2, wherein said alleviation of psoriatic lesion progression is achieved by inhibition of inflammatory cytosis, inhibition of keratinocyte proliferation and alleviation of pruritic symptoms.
4. The use according to any one of claims 1 to 3, wherein the medicament comprises a pharmaceutically acceptable adjuvant in addition to the agent which inhibits the IL-34 signalling pathway.
5. The use according to claim 4, wherein the pharmaceutical dosage form comprises an injection, a microneedle.
6. The use of claim 4, wherein the agent that inhibits the IL-34 signaling pathway is an agent that targets inhibition of IL-34 function.
7. The use of claim 6, wherein the agent that inhibits the IL-34 signaling pathway is an IL-34 antibody.
8. The use according to claim 7, wherein the IL-34 antibody is used in an amount of at least 5 μg/kg.
9. A medicament for treating psoriasis, which is characterized by comprising a preparation for inhibiting an IL-34 signal pathway and pharmaceutically acceptable auxiliary materials thereof.
10. The medicament of claim 9, wherein the agent that inhibits the IL-34 signaling pathway is an IL-34 antibody.
CN202310281116.6A 2023-03-20 2023-03-20 Application of IL-34 antibody in preparation of medicament for treating psoriasis Pending CN116236579A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130302322A1 (en) * 2012-05-11 2013-11-14 Five Prime Therapeutics, Inc. Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (csf1r)
KR20150104275A (en) * 2014-03-05 2015-09-15 울산대학교 산학협력단 Use of IL-34 for the diagnosis and treatment of obesity
US20170342148A1 (en) * 2014-12-19 2017-11-30 Universite De Nantes Anti il-34 antibodies
CN113855807A (en) * 2021-10-25 2021-12-31 孙良丹 Application of reagent in preparation of medicine for treating/inhibiting psoriasis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130302322A1 (en) * 2012-05-11 2013-11-14 Five Prime Therapeutics, Inc. Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (csf1r)
KR20150104275A (en) * 2014-03-05 2015-09-15 울산대학교 산학협력단 Use of IL-34 for the diagnosis and treatment of obesity
US20170342148A1 (en) * 2014-12-19 2017-11-30 Universite De Nantes Anti il-34 antibodies
CN113855807A (en) * 2021-10-25 2021-12-31 孙良丹 Application of reagent in preparation of medicine for treating/inhibiting psoriasis

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