Application of interleukin-5 inhibitor in adjuvant treatment of vulvovaginal candidiasis with increased eosinophilic granulocyte

Abstract

The invention discloses an application of an interleukin-5 inhibitor in adjuvant treatment of vulvovaginal candidiasis with increased eosinophilic granulocyte. The invention belongs to the field of biomedicine, and particularly relates to application of an interleukin-5 inhibitor in adjuvant treatment of vulvovaginal candidiasis with increased eosinophilic granulocyte. The invention provides application of an interleukin-5 inhibitor in preparation of a medicine for treating or relieving or preventing vulvovaginal candidiasis, the medicine aims at subjects with increased eosinophilic granulocytes, and the subjects are animals with CARD9 gene defects. After the IL-5 antibody is used for treatment, CARD9-deficient mouse vulvovaginal candidiasis inflammation is obviously relieved, and PAS shows that hyphae are reduced.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to the application of an interleukin-5 inhibitor in the auxiliary treatment of vulvovaginal candidiasis with elevated eosinophils. Background Art

[0002] Vulvovaginal candidiasis (VVC), also known as candidal vulvovaginitis, is a common genital tract inflammation in clinical practice, causing discomfort such as vulvar itching and increased secretions. Candida albicans is the most common pathogen. The treatment of vulvovaginal candidiasis is mainly based on antifungal drugs. Clinical studies have found that Card9-deficient vulvovaginal candidiasis is characterized by severe itching, repeated attacks, and difficulty in treatment.

[0003] IL-5 (interleukin-5) was originally described as an eosinophil and B-cell growth factor and is a member of the hematopoietic family of Th2 cytokines. The IL-5 gene is located on chromosome 11 in mice and chromosome 5 in humans, in close proximity to genes encoding IL-3, IL-4, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are normally co-expressed in Th cells. IL-5 is also expressed by eosinophils and has been observed by immunohistochemistry in mast cells of asthmatic airways. IL-5 expression is regulated by multiple transcription factors, including GATA3. IL-5 has long been implicated in the etiology of a variety of allergic diseases, including allergic rhinitis and asthma, in which large increases in the number of circulating, airway tissue, and induced sputum eosinophils have been observed. Given the high degree of consistency in eosinophils, particularly in allergic asthma pathology, it has been widely speculated that eosinophils play an important role in the pathology of this disease. As of 2019, there are two FDA-approved monoclonal antibodies that inhibit IL-5, mepolizumab and reslizumab, both for the treatment of severe eosinophilic asthma and eosinophilic granulomatosis with polyangiitis (EGPA).

[0004] Caspase recruitment domain-containing protein 9 (CARD9 for short) is an adaptor protein of the CARD-CC protein family and is encoded by the CARD9 gene in humans. It mediates signals from pattern recognition receptors to activate pro- and anti-inflammatory cytokines and regulate inflammation. CARD9 is a member of the CARD protein family, which is defined by the presence of a characteristic caspase-associated recruitment domain (CARD). The protein was identified by its selective association with the CARD domain of BCL10, a positive regulator, and NF-κB activation. Card9 plays an important role in the innate immune response against yeast. Card9 mediates signals from the so-called pattern recognition receptors (Dectin-1) to downstream signaling pathways such as NF-κB, and thereby activates pro-inflammatory cytokines (TNF, IL-23, IL-6, IL-2) and anti-inflammatory cytokines (IL-10), followed by activation of appropriate innate and adaptive immune responses to clear the infection. Summary of the invention

[0005] The main problem to be solved by the present invention is how to treat or assist in the treatment of vulvovaginal candidiasis with elevated eosinophils.

[0006] In order to solve the above problems, the present invention provides the use of an interleukin-5 inhibitor in the preparation of a drug for treating, preventing or alleviating vulvovaginal candidiasis.

[0007] In the above application, the drug is for subjects with elevated eosinophils.

[0008] In the present invention, "subject" includes a person who is being treated for a disease or preventing a disease as a patient. The application described herein can be used to treat animal subjects belonging to any classification. Examples of such animals include mammals. Mammals include, but are not limited to, rodents (order Rodentia) mammals, such as mice and hamsters, and lagomorpha (order Logomorpha) mammals, such as rabbits. Mammals can be carnivores (order Carnivora), including felines (cats) and canines (dogs). Mammals can be even-toed ungulates (order Artiodactyla), including bovines (cows) and suids (pigs), or odd-toed ungulates (order Perssodactyla), including equines (horses). Mammals can be primates (order Primate), four-legged monkeys (order Ceboid) or monkeys (order Simoid) (monkeys) or anthropoids (order Anthropoid) (humans and apes). In some embodiments, mammals are humans.

[0009] The term "effective" as applied to dosage or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug or drugs employed, the mode of administration, and the like.

[0010] In the above application, the subject may be an animal with CARD9 gene defect.

[0011] The CARD9 gene defect may be Card9- / -.

[0012] After the CARD9 gene-deficient mice were infected with Candida albicans in the vulva and vagina, the eosinophils and IL-5 were significantly increased.

[0013] In the above application, the interleukin-5 inhibitor may be a substance that reduces the content of IL-5 or a substance that inhibits the expression of IL-5.

[0014] In the above application, the relief of vulvovaginal candidiasis can be reflected in the relief of the inflammatory response of vulvovaginal candidiasis and / or the increase of eosinophils, as well as the resulting vulvovaginal itching and other problems.

[0015] In the above application, the interleukin-5 inhibitor is an antibody against interleukin-5. In a specific embodiment, the antibody against interleukin-5 is IL-5 antibody (purchased from BioXcell, catalog number: BE0198)

[0016] In the above applications, the treatment may be adjuvant therapy.

[0017] In the above application, the dosage of the interleukin-5 antibody used for treatment is 1 mg / mouse.

[0018] The present invention also provides the use of the substance for reducing the content of IL-5 protein in the preparation of vulvovaginal Candida inhibitor or vulvovaginal Candida proliferation inhibitor.

[0019] The present invention also provides the use of a substance for reducing the content of IL-5 protein in the preparation of a drug for treating or / and preventing vulvovaginal Candida infection.

[0020] In the above application, the drug for treating vulvovaginal Candida infection or / and preventing vulvovaginal Candida infection is applied to subjects with CARD9 gene defect.

[0021] In the present invention, the vulvovaginal Candida is Candida albicans SC5314.

[0022] The study found that after CARD9-deficient mice were infected with Candida albicans, eosinophils and IL-5 increased significantly, which may be an important cause of its itching and recurrent attacks. After treatment with IL-5 antibodies, the inflammation of vulvovaginal candidiasis in CARD9-deficient mice was significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Card9 deficiency aggravates VVC. A shows the appearance of the mouse vulva and vagina; B shows the bacterial count of the vaginal lavage fluid of the mouse; C shows the desquamated epithelial cells and inflammatory cells in the vaginal lavage fluid; D shows the protein concentration of IL-5 in the vaginal lavage fluid of the mouse detected by ELISA.

[0024] Figure 2 The histological results show that Card9 deficiency aggravates VVC and causes an increase in eosinophils. A shows the vaginal mucosal tissue and fungal infection of mice by HE and PAS staining; B shows eosinophils by Sirius red staining; and C shows the flow cytometry detection and statistics of eosinophils.

[0025] Figure 3 The IL-5 antibody was used to reduce the levels of IL-5 and eosinophils in Card9-deficient mice. A is the protein level of IL-5 in vaginal lavage fluid of different groups; B is eosinophils shown by picrosirius red staining; C is flow cytometry detection of eosinophils and statistics. αIso represents the control antibody, and αIL-5 represents the IL-5 antibody.

[0026] Figure 4 The administration of IL-5 antibody reduces vaginal inflammation in Card9-deficient mice. A shows the appearance of the mouse vulva and vagina; B shows the bacterial count of the mouse vaginal lavage fluid. C shows the vaginal mucosal tissue and fungal infection of the mouse by HE and PAS staining. αIso represents the control antibody, and αIL-5 represents the IL-5 antibody. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0029] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0030] The Candida albicans SC5314 in the following examples was purchased from the ATCC website. The public can obtain the biological material from ATCC (www.atcc.org).

[0031] The Card9-deficient mice in the following examples were donated by Tsinghua University and have been recorded in: Jia XM, Tang B, Zhu LL, et al. CARD9 mediates Dectin-1-induced ERK activation by linking Ras-GRF1toH-Ras for antifungal immunity. J Exp Med. 2014; 211(11): 2307-2321, and were bred in Peking University First Hospital. The Card9-deficient VVC mice can also be purchased from companies such as Saiye Group, catalog number: S-KO-09574.

[0032] The following examples used GraphPad Prism 6.0 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation.

[0033] Example 1. Effect of anti-IL-5 antibodies in the treatment of vulvovaginal candidiasis

[0034] 1. Acquisition and cultivation of Candida albicans

[0035] The Candida albicans SC5314 strain was stored in glycerol at -80°C. A single colony of Candida albicans SC5314 was cultured in yeast peptone glucose medium at 30°C overnight with continuous stirring at 180 rpm. Yeast spores were washed in phosphate buffer, counted using an automated cell counter (LUNAII), and adjusted to a concentration of 5 × 10 in phosphate buffer. 8 / mL for subsequent experiments.

[0036] 2. Establishment of the mouse VVC model

[0037] All mice had a C57BL / 6J genetic background. Wild-type mice C57BL / 6J were obtained from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. 7-week-old wild-type female mice C57BL / 6J and Card9-deficient female mice (weight 18-22 g) were selected and housed together for at least 2 weeks before the experiment. The mice were randomly divided into 2 groups, 5 in each group.

[0038] 2.1 Establishment of VVC model using wild-type mice

[0039] Model group (abbreviated as WT): 72 h before infection, C57BL / 6 mice were subcutaneously injected with 0.1 mg of estrogen (β-17-estradiol valerate, Sigma) dissolved in 0.1 mL of sesame oil (Solarbio), and subcutaneously injected twice a week after infection. The mice were infected by intravaginal injection of 5×10 6 albicans spores (the spores were diluted with phosphate buffered saline (PBS) to a spore concentration of 5 × 10 8 / ml, 10μL was injected into the vagina) to obtain the VVC model of wild-type mice.

[0040] 2.2 Construction of VVC model using Card9- / - mice

[0041] Model group (referred to as Card9- / -): 72 h before infection, Card9-deficient mice were subcutaneously injected with 0.1 mg of estrogen (β-17-estradiol valerate, Sigma) dissolved in 0.1 mL of sesame oil (Solarbio), and subcutaneously injected twice a week after infection. The mice were infected by intravaginal injection of 5×10 6 albicans spores (the spores were diluted with phosphate buffered saline (PBS) to a spore concentration of 5 × 10 8 Card9-deficient VVC mice were obtained.

[0042] 3. Vaginal lavage, colony count and IL-5 detection in mice

[0043] The vagina of the mouse model obtained in step 2 was flushed with 100 μL of sterile phosphate-buffered saline to obtain vaginal lavage fluid. 10 μL of the lavage fluid sample was applied to yeast peptone glucose agar (produced by BD, 39 g powder dissolved in 1 L of water, high temperature and high pressure, poured into a 9 cm × 9 cm cell culture dish) supplemented with 50 μg / mL chloramphenicol, and cultured at 37 degrees for 24 hours for colony counting. In addition, 10 ul of the above lavage fluid was applied to a slide and stained with Wright-Giemsa to evaluate Candida albicans pseudohyphae and leukocyte recruitment.

[0044] A commercial ELISA kit (Solebio, SEKM-0006) was used to detect the concentration of cytokine IL-5 in vaginal lavage fluid, and the quantification was performed according to the instructions of the kit.

[0045] The results are as follows Figure 1 As shown: 14 days after infection, the vulva and vagina of Card9-deficient VVC mice were significantly swollen compared with wild-type VVC mice ( Figure 1 A), vaginal bacterial load increased significantly (such as Figure 1 Middle B); The results of Wright-Giemsa staining of vaginal lavage fluid showed that the shedding of vaginal epithelial cells in mice increased and the number of inflammatory cells increased significantly ( Figure 1 Middle C). The concentration of IL-5 in the vaginal lavage fluid of mice was significantly increased ( Figure 1 (middle D).

[0046] 4. Mouse vaginal tissue pathology, PAS staining, and Sirius red staining

[0047] The whole vaginal tissue of each group of mice obtained in step 2 was surgically removed and fixed in 4% formalin buffer for 24 hours. The samples were then embedded in paraffin, sectioned, and mounted on slides. Gross histological examination was performed using hematoxylin-eosin staining, and periodic acid-Schiff staining was used to detect pseudohyphae of Candida albicans. Immunohistochemistry used Sirius red staining to mark eosinophils.

[0048] The results are as follows Figure 2 As shown: Compared with wild-type VVC mice, Card9-deficient VVC mice showed a significant increase in vaginal mucosal inflammatory cells and significant epithelial damage by hematoxylin-eosin staining, and periodic acid-Schiff staining detected that Candida albicans pseudohyphae significantly invaded the vaginal mucosal epithelium ( Figure 2 Middle A); Sirius red staining showed that the number of eosinophils in the vaginal tissue of Card9-deficient VVC mice was significantly increased compared with that of wild-type VVC mice ( Figure 2 Middle B)

[0049] 5. Flow cytometry detection of eosinophils

[0050] The vaginal tissues of each group of mice obtained in step 2 were digested with collagenase IV (40 μg / mL) and DNaseI (1 mg / mL) in RPMI-1640 medium (Gibico, catalog number 27016021) at 37°C for 60 min. After passing through a 70 μm nylon cell strainer, the cells were stained with anti-mouse flow cytometry antibodies obtained from BioLegend and analyzed using a BD LSRF X-20 flow cytometer. The following antibodies were used for eosinophil analysis: BV510-Zombie (catalog number 423102), PEcy7-CD45 (catalog number: 157206), and BV421-Siglecf (catalog number: 155509) for labeling.

[0051] The results are as follows Figure 2 As shown in C: Compared with wild-type VVC mice, Card9-deficient VVC mice had significantly increased eosinophils.

[0052] 6. Anti-IL-5 antibody therapy

[0053] All mice had a C57BL / 6J genetic background. Wild-type mice C57BL / 6J were obtained from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. 7-week-old wild-type female mice C57BL / 6J and Card9-deficient female mice (weight 18-22 g) were selected and raised together for at least 2 weeks before the experiment. The mice were randomly divided into 4 groups, 5 mice in each group. The specific treatment methods were as follows:

[0054] 1) Experimental group: 72 h before infection, C57BL / 6 mice were subcutaneously injected with 0.1 mg of estrogen (β-17-estradiol valerate, Sigma) dissolved in 0.1 mL of sesame oil (Solarbio), and subcutaneously injected twice a week after infection. At the same time as the subcutaneous injection of estrogen, IL-5 antibody (purchased from BioXcell, catalog number: BE0198, dosage 1 mg / mouse) was intraperitoneally injected. The mice were infected by intravaginal injection of 5×10 6 albicans spores (the spores were diluted with phosphate buffered saline (PBS) to a spore concentration of 5 × 10 8 / ml, 10μL injected into the vagina) to obtain IL-5 antibody-treated wild-type VVC mice, referred to as αIL-5+WT in the text.

[0055] 2) Control group 1: 72 h before infection, C57BL / 6 mice were subcutaneously injected with 0.1 mg of estrogen (β-17-estradiol valerate, Sigma) dissolved in 0.1 mL of sesame oil (Solarbio), and subcutaneously injected twice a week after infection. At the same time as the subcutaneous injection of estrogen, rat IgG1 isotype control antibody (purchased from BioXcell, catalog number: BE0088, dosage 1 mg / mouse) was intraperitoneally injected. The mice were infected by intravaginal injection of 5×10 6 albicans spores (the spores were diluted with phosphate buffered saline (PBS) to a spore concentration of 5 × 10 8 / ml, 10μL injected into the vagina) to obtain the control antibody to treat wild-type VVC mice, referred to as αIso+WT in the text.

[0056] 3) Model group (αIL-5+Card9- / -): 72 h before infection, Card9-deficient mice were subcutaneously injected with 0.1 mg of estrogen (β-17-estradiol valerate, Sigma) dissolved in 0.1 mL of sesame oil (Solarbio), and subcutaneously injected twice a week after infection. At the same time as the subcutaneous injection of estrogen, IL-5 antibody (purchased from BioXcell, catalog number: BE0198, dosage 1 mg / mouse) was intraperitoneally injected. The mice were infected by intravaginal injection of 5×10 6 albicans spores (the spores were diluted with phosphate buffered saline (PBS) to a spore concentration of 5 × 10 8Card9-deficient VVC mice were obtained by treating them with IL-5 antibody, referred to as αIL-5+Card9- / - in this article.

[0057] 4) Control group 2 (abbreviated as αIso+Card9- / -): 72 h before infection, Card9-deficient mice were subcutaneously injected with 0.1 mg of estrogen (β-17-estradiol valerate, Sigma) dissolved in 0.1 mL of sesame oil (Solarbio), and subcutaneously injected twice a week after infection. At the same time as the subcutaneous injection of estrogen, rat IgG1 isotype control antibody (purchased from BioXcell, catalog number: BE0088, dosage 1 mg / mouse) was intraperitoneally injected. The mice were infected by intravaginal injection of 5×10 6 albicans spores (the spores were diluted with phosphate buffered saline (PBS) to a spore concentration of 5 × 10 8 / ml, 10μL injected into the vagina) to obtain control antibody-treated Card9-deficient mice, referred to as αIso+Card9- / - in the text.

[0058] The results are as follows Figure 3 and Figure 4 As shown: 1) Compared with the rat IgG1 isotype control antibody treatment, the concentration of IL-5 in the vaginal lavage fluid of Card9- / -deficient mice was significantly decreased after treatment with IL-5 antibody ( Figure 3 Middle A), Sirius red staining ( Figure 3 B) and flow cytometry ( Figure 3 C) shows a significant decrease in eosinophils; 2) Vulvovaginal inflammation in Card9- / - deficient mice was significantly reduced after IL-5 antibody treatment compared with rat IgG1 isotype control antibody treatment ( Figure 4 Middle A); The vaginal bacterial load of Card9- / - deficient mice was significantly decreased ( Figure 4 Middle B), hematoxylin-eosin staining showed that the inflammatory cells in the vaginal mucosa were significantly reduced, and the destruction of the epithelial mucosa was alleviated. Periodic acid-Schiff staining showed that the formation of pseudohyphae of Candida albicans was reduced, and the invasion of the vaginal epithelial mucosa was reduced ( Figure 4 Middle C).

[0059] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of interleukin-5 inhibitors in the preparation of drugs for treating, alleviating or preventing vulvovaginal candidiasis.

2. The use according to claim 1, characterized in that: The drug is intended for subjects with elevated eosinophils.

3. The use according to claim 1 or 2, characterized in that: The subject is an animal with CARD9 gene defect.

4. The use according to any one of claims 1 to 3, characterized in that: The interleukin-5 inhibitor is a substance that reduces the content of IL-5 or a substance that inhibits the expression of IL-5.

5. The use according to any one of claims 1 to 3, characterized in that: The interleukin-5 inhibitor is an antibody against interleukin-5.

6. The use according to any one of claims 1 to 3, characterized in that: The treatment may be adjuvant therapy.

7. Use of a substance that reduces IL-5 protein content in the preparation of a vulvovaginal Candida inhibitor or a vulvovaginal Candida proliferation inhibitor.

8. Use of a substance that reduces IL-5 protein content in the preparation of a drug for treating vulvovaginal Candida infection or / and alleviating vulvovaginal Candida infection.

9. The use according to any one of claim 8, characterized in that: The drug for treating vulvovaginal Candida infection and / or preventing vulvovaginal Candida infection is applied to subjects with CARD9 gene defect.

10. The use according to any one of claims 1 to 9, characterized in that: The vulvovaginal Candida is Candida albicans SC5314.

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