CN115814057A - Application of Caspofungi in preparation of pharmaceutical composition for treating cytokine storm caused by COVID19 - Google Patents

Abstract

The invention discloses application of Caspofungi in preparation of a pharmaceutical composition for treating cytokine storm caused by COVID 19. The Caspofungi of the invention can have protective action and anti-inflammatory action on SARS-CoV-2 infection in vivo.

Description

Application of Caspofungi in preparation of pharmaceutical composition for treating cytokine storm caused by COVID19

Technical Field

The invention belongs to the technical field of coronavirus treatment medicines, and particularly relates to application of Caspofungi in preparation of a pharmaceutical composition for treating cytokine storm caused by COVID 19.

Background

Human coronaviruses are a class of envelope-carrying positive-sense RNA viruses that cause highly pathogenic human diseases with clinical symptoms ranging from mild common cold to acute respiratory distress syndrome and death. Three highly pathogenic human coronaviruses that have exploded over the last two decades: severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS), and SARS-CoV-2, exhibit the pandemic potential of human coronavirus. SARS-CoV-2 can cause upper and lower respiratory tract infections often with fever, cough and loss of smell and taste, while some patients develop more severe symptoms including systemic inflammation, tissue damage, acute respiratory distress syndrome, thromboembolic complications, heart damage and/or cytokine storms, and even death.

Among these, cytokine storm is one of the key pathological features observed in SARS-CoV-infected patients, and is common in severe human coronavirus (CoV) (e.g., SARS and MERS) infections. CoVID-19 presents high levels of cytokines in critically ill patients. Among them, TNFa, IL6 and IL 1. Beta. Are mainly released by innate immune cells and may be one of the major drivers of cytokine release syndrome and severe systemic inflammatory response in patients with advanced SARS-CoV-2 infection. And the increase of chemokines and cytokines such as CCL2/3/5, CXCL8/9/10, IFN-gamma, TNF alpha, IL1 beta, IL1RA, IL6, IL7, IL8, IL12, IL33, granulocyte/granulocyte-macrophage colony stimulating factor (G-CSF and GM-CSF), vascular Endothelial Growth Factor A (VEGFA) and platelet-derived growth factor subunit B (PDGFB) further causes severe Acute Respiratory Distress Syndrome (ARDS) and tissue injury. Cytokine storms are mainly produced by the over-activation of a variety of immune cells, mainly macrophages, neutrophils, dendritic cells, NK cells, B cells and T cells. While SARS-CoV-2 specific T cells, activated by dendritic cells and macrophages through antigen presentation, may mediate antiviral responses early, the immune escape capacity of SARS-CoV-2 may make it difficult for T cells to mount an effective antiviral response, and importantly, T cell-mediated inflammation and sustained activation of innate immune cells may be factors leading to secondary complications that occur in lung pathology and in severe cases. Simultaneous studies have shown that patients with poor T or B cell activation are less sick, while CD4 ⁺ T cells and CD8 ⁺ Patients with over-activation of T cells are more severely affected and the role of T cells in mediating the inflammatory response in patients with COVID-19 is also suggested. These evidence suggests that COVID-19 may be an immune-related disease, the first strong evidence supporting severe acute infections with immunotherapy, and therefore treatment via immunomodulation would be important in reducing inflammatory responses, improving patient survival, and reducing mortality.

Facing the global pandemic situation of the new coronavirus, scientists have been looking for the development of vaccines and effective drugs for the prevention and treatment of SARS-CoV-2 infection, but no specific effective treatment methods exist at present. In addition to vaccine development, the main therapeutic drugs include antiviral drugs and immunomodulatory drugs. Antiviral drugs are directed against mediators of viral infection into cells, including the Receptor Binding Domain (RBD)/angiotensin converting enzyme 2 (ACE 2), the transmembrane protease serine 2 (TMPRSS 2), the 3C-like protease (M) ^pro ) RNA-dependent RNA polymerase (RdRp), etc., are generally used in early stages of disease. Immunomodulatory drugs are commonly used in the advanced stages of the disease to balance the immune response and relieve symptoms such as inflammatory responses, cytokine storms and secondary tissue damage and acute respiratory distress syndrome. The development of treatments aimed at reducing the severity of the disease is also the most important priority worldwide. Currently used immunomodulatory drugs mainly include three classes. The first group is the dexamethasone-based corticosteroids, and several studies have shown that dexamethasone can reduce patient mortality, and in particular has a significant beneficial effect in patients with increased disease during treatment. It is noteworthy, however, that corticosteroids may have deleterious effects on patients early in the treatment due to broad, non-specific immunosuppressive effects. The second class is kinase inhibitors, such as the JAK inhibitor baricitinib (baricitinib). Studies have shown that patients in the baracitinib-treated group recovered in shorter time compared to placebo and that baracitinib treatment was able to reduce patient mortality, with greater efficacy in subgroups requiring high flow of oxygen or non-invasive ventilation. The FDA recently approved baracitinib for use in the emergency treatment of COVID-19. In addition, imatinib (imatinib), a cytoplasmic polytyrosine kinase inhibitor, was also found to have a superior effect in a clinical trial in the netherlands on 400 codv-19 hospitalized patients, but these findings required follow-up trials to verify and determine which patients might benefit from imatinib treatment. Other kinase inhibitors in the research include Bruton's tyrosine kinase inhibitors (e.g., ibrutinib, acalburtinib, zanubrutinib), phosphatidylinositol-3 kinase (PI 3K)/rapamycin (mTOR) inhibitors (e.g., duvelisib, temsirolimus), and JAK inhibitors(e.g., ruxolitinib, tofacitinib). In addition to the beneficial effects exhibited by some tyrosine kinase inhibitors, previous studies have shown the pleiotropic properties of tyrosine kinases (which block cytokine signaling pathways and many immune effector pathways), as well as the well-known clinical safety profile of most tyrosine kinase inhibitors, suggesting that tyrosine kinase inhibitors are a potential COVID-19 treatment. The third class is cytokine-targeting drugs, which are currently dominated by anti-IL 1 and IL 6. Both IL1 and IL6 can cause local effects such as macrophage activation, endothelial leakage and fluid extravasation, as well as systemic effects. However, two studies have found that treatment with the IL1 inhibitor anakinra does not produce significant effects. Subsequent further studies have shown that anakinra is effective in patients with plasma high-soluble urokinase plasminogen receptor (suPAR), and therefore the application of anakinra is guided by the suPAR content. In addition, the IL1 β blocker canakinumab also produced no significant effect in the assay. In contrast, blocking therapy of IL6 appears to be more effective. Wherein both tocilizumab (tocilizumab) and salilumab show the effects of reducing mortality and improving patient survival in clinical trials. In addition to pro-inflammatory cytokines on the IL1-IL6 axis, other pro-inflammatory cytokines are involved in COVID-19 mediated inflammation. Clinical trials have shown that the anti-GM-CSF antibody, otilimab, has a significant effect in older than 70 years of age, but it is noted that this age dependence suggests possible side effects in younger patients. In addition, anti-TNF therapy is currently in clinical trials (NCT 04705844). Cytokine-targeted therapeutic strategies against COVID-19 appear to be an attractive approach, but given the complexity of the inflammatory response mediated by COVID-19, and the relatively single effect of cytokine-specific targeted therapies, random control trials to accurately detect biomarkers may be required to help further determine which patients are likely to benefit the most. In addition to the three classes of immunomodulatory drugs, other immune regulation strategies include anti-complement therapies (e.g., anti-C5 a antibody vilobelimab), interferons that stimulate antiviral defenses (e.g., IFN β, IFN γ), and the like.

Although the anti-inflammatory treatment of COVID-19 has been developed to a great extent, one major dilemma faced at present is that despite the use of immunotherapeutic drugs such as dexamethasone and IL6 blocking antibodies, some patients with COVID-19 have no improvement in treatment and still have a severe inflammatory response, and there are not enough randomized controlled trials to guide dosing. In addition, the host-pathogen response and the resulting immune environment are heterogeneous, dynamic, suggesting that not every patient will benefit from the same immunomodulatory treatment strategy. There is therefore a need to increase potential treatment options to address situations where patients fail to respond and to further alleviate symptoms in severe cases. It is also noted that some of the currently used antibody therapies or combination therapies face the problem of being expensive and difficult to ensure that all patients receive an equal chance of treatment. Therefore, in addition to increasing replacement therapy and improving efficacy, cheaper alternative drugs are found to better complete the therapeutic strategy of COVID-19.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of Caspofungi in preparing a pharmaceutical composition for treating cytokine storm caused by COVID 19.

Another object of the present invention is to provide the use of Caspofungi as a broad-spectrum inhibitor of inflammatory factors induced by COVID-19.

The technical scheme of the invention is as follows:

use of Caspofungi for the preparation of a pharmaceutical composition for the treatment of cytokine storm induced by COVID 19.

In a preferred embodiment of the invention, the inflammatory factor in the cytokine storm comprises at least CCL3, CCI4, CCL5, CKC12, IL1 β, IL2, IL4, IL5, IL6, IL8, IP10, IL13, IL18, GM-CSF, IFN β, IFN, TNF α, TNF β, CXCL12, LTA, IL22 and IL21.

Further preferably, the inflammatory factor in the cytokine storm consists of CCL3, CCL4, CCL5, CKC12, IL1 β, IL2, ILA, IL5, IL6, IL8, IP10, ILI0, IL13, IL18, GM-CSF, IFN β, IFN, TNF α, TNF β, CXCL12, LTA, IL22 and IL21.

Application of Caspofungi as a broad-spectrum inhibitor of inflammatory factors induced by COVID-19.

In a preferred embodiment of the invention, the inflammatory factor comprises at least CCL3, CCL4, CCL5, CKC12, IL1 β, IL2, IL4, IL5, IL6, IL8, IP10, IL13, IL18, GM-CSF, IFN β, IFN, TNF α, TNF β, CXCL12, LTA, IL22 and IL21.

Further preferably, the inflammatory factor consists of CCL3, CCL4, CCL5, CKC12, IL1 β, IL2, IL4, IL5, IL6, IL8, IP10, IL13, IL18, GM-CSF, IFN β, IFN, TNF α, TNF β, CXCL12, LTA, IL22 and IL21.

Drawings

FIG. 1 is a heat map showing the amounts of 34 cytokines and chemokines secreted in the cell supernatant measured by adding 4 screening drugs (# 1Felodipine, #2 Fascidull, #3Imatinib and #4 Caspofungi) to the cell supernatant under coculture of S-293T, SARS-CoV-2S CAR-T, THP-1 in example 1 of the present invention.

FIG. 2 is a schematic diagram of the experimental strategy for evaluating the in vivo effect of Felodipine, imatinib, caspofungi and Fasudil by SARS-CoV-2 virus challenge and drug therapy using golden hamster in example 2 of the present invention.

Fig. 3 is a graph showing the body weight change of golden mice in the control group, and the Felodipine, imatinib, caspofungi, and fadauil-treated groups in example 2 of the present invention, and a table showing the significance of the golden mice at each time point.

FIG. 4 is a graph showing the survival of golden hamsters in the control group and Felodipine, imatinib, caspofungi and Fasudil treatment groups in example 2.

Fig. 5 is a photograph of lung tissues of golden hamsters in the control group and Felodipine, imatinib, caspofungi, and Fasudil treatment groups in the end point (day 7) according to example 2 of the present invention.

FIG. 6 is a graph showing the results of HE staining of lung tissue sections of golden mice in the control group and Felodipine, imatinib, caspofungi, and Fasudil-treated groups in example 2 of the present invention (lung tissue of non-detoxified golden mice was used as a negative control).

FIG. 7 is a histogram of HE staining pathology scores of lung tissue sections of golden mice in the control group and Felodipine, imatinib, caspofungi, fasudil-treated groups of example 2 of the present invention (lung tissue of non-detoxified golden mice was used as a negative control).

FIG. 8 is a thermogram of inflammatory factor gene expression in lung tissue of golden hamster of control group and Felodipine, imatinib, caspofungi, fasudil treatment group detected by RT-qPCR in example 2 of the present invention (lung tissue of non-challenged golden hamster was used as negative control, and CXCL15 in golden hamster corresponds to IL 8).

Fig. 9 is a graph showing the expression of a representative inflammatory factor in lung tissues of golden hamsters in the control group and Felodipine, imatinib, caspofungi, and faudil treatment groups, which were detected by ELISA in example 2 of the present invention (lung tissues of non-challenged golden hamsters were used as a negative control).

FIG. 10 is a graph showing the results of experiments in which Caspofungi and antifungal similar drugs in example 2 of the present invention were simultaneously screened.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

Example 1

This example evaluates the anti-inflammatory effects of 4 drugs (Felodipine, imatinib, caspofungi, fasudil) screened in a cytokine storm model.

Cloning the S gene of the new corona strain into a pCAG-eGFP vector to construct an S gene expression vector pCAG-S-eGFP. 24h before transfection, 293T was applied at 1X 10 ⁶ Each well was laid in a six well plate. 5 μ L of transfection reagent Lipofectamine was used at the time of transfection ^TM 3000 (manufactured by Thermo Fisher Co., ltd.) was added to 125. Mu.L of basic medium RMPI 1640 to dilute it; in addition, 2.5. Mu.g of pCAG-S-eGFP plasmid and 5. Mu. L P3000 were added ^TM The reagent (manufactured by Thermo Fisher Co., ltd.) was added to 125. Mu.L of the basal medium RPMI 1640 to dilute, followed by dilution of the plasmid/P3000 ^TM Reagent addition to diluted Lipofectamine ^TM 3000, and adding the mixture into 293T cells after incubation for 10-15min at room temperature. 24h after transfection, cells were digested and subjected to subsequent experiments.

S-293T cells prepared as above were cultured at 2X 10 ⁴ The cells were inoculated into a 96-well plate,37℃、5％CO ₂ after adherent culture in the incubator, the ratio of 10:1, adding SARS-CoV-2S CAR-T (the CAR structure is Fc-SARS-CoV-2 CAR, the amino acid sequence is shown as SEQ ID NO.01, and the nucleotide sequence is shown as SEQ ID NO. 02) cells, adding THP-1 cells in a ratio of 10:1 relative to S-293T cells, simultaneously adding Felodipine, imatinib, caspofungi and Fasudil in a distributed manner to make the working concentration of the cells 10 muM, and co-culturing for 72 h; cell supernatants were collected and the amounts of 34 cytokines and chemokines secreted were measured by ProcartaPlex Hu Cytokine/Chemokine Panel (Invitrogen).

As shown in FIG. 1, the drugs Felodipine, imatinib, caspofungi and Fasudil screened in this example, in addition to inhibiting the secretion of IL8 and IFN γ, also produced a broad spectrum of inhibitory effects on other cytokines.

Example 2

This example demonstrates the effect of Felodipine, imatinib, caspofungi and Fasudil on SARS-CoV-2 infection and the resulting inflammatory response in vivo.

Using 8-14 weeks old golden hamster, narcotize with isoflurane, then inject 100 μ L PBS diluted 1X 10 ⁴ 5363 a dose of SARS-CoV-2 virus pFU; dividing golden mice into 5 groups of 6 mice per group, namely a control group (untreated), a Felodipine group, an Imatinib group, a Fasudil group and a Caspofungin group; treatment with 15mg/kg dose of drug on days 1, 2, 3 and 4 respectively, wherein Felodipine, fasudil and Caspofungin are administered intraperitoneally and Imatinib is administered by intragastric gavage; it was observed that the golden hamsters in the control group died (day 7), and the body weight of the golden hamsters was measured daily by electronic balance (experimental strategy shown in fig. 2). Killing golden hamster on the 7 th day, obtaining lung tissue to observe lung pathological changes and taking a picture, then obtaining lung tissue sections through methanol fixation, dehydration, paraffin embedding and section, detecting pathological conditions by HE staining, and carrying out comprehensive pathological scoring according to the conditions of alveolar septal thickening and consolidation, bleeding, exudation, pulmonary edema and mucus, and chemotaxis and infiltration of inflammatory cells on each lung lobe; in addition, the lung tissue was subjected to Extraction of Total RNA using Eastep Super Total RNA Extraction Kit (manufactured by Promega corporation), followed by RT-qPCR detectionA cytokine gene.

The results are shown in FIGS. 3 to 9. Figures 3 and 4 show that golden hamster continued to lose body weight on the 1 to 6 scale after infection, eventually by more than 20%, and died altogether within 7 days; while the weight loss was significantly reduced by treatment with Felodipine, imatinib, fasuil and Caspofungin, the average weight loss was 5.2%, 2.5%, 2.1% and 11% on day 7, respectively, and all golden mice of 4 treatment groups survived. Fig. 5 shows photographs of lung tissues of the groups on day 7, and severe lung lesions (including solid, multifocal and diffuse congestion) were observed in the control golden hamster, whereas no severe lung lesions were observed after Felodipine, imatinib and Fasudil treatment, and the lung lesions were also improved by Caspofungin treatment. Correspondingly, fig. 6 and 7 show the lung tissue HE staining results and the overall pathology score, and also demonstrate that Felodipine, imatinib, and faudin significantly reduced lung lesions with slightly less effect on Caspofungin. The RT-qPCR of fig. 8 and ELISA results of fig. 9 showed that the expression of inflammatory factor genes detected in lung tissues of Felodipine, imatinib, fasudil and Caspofungin treated groups was significantly down-regulated compared to the control group.

This example shows that Felodipine, imatinib, fasudil and Caspofungin have protective and anti-inflammatory effects on SARS-CoV-2 infection in vivo.

Further, caspofungi and similar antifungal drugs were screened simultaneously, the first round of IL8 faudil and Minoxidil performed well, but only faudil had strong inhibitory ability in IFN screening, and other similar drugs were eliminated, and the results are shown in fig. 10.

It will be appreciated by those of ordinary skill in the art that Caspofungin (Caspofungin) is an antifungal approved by the FDA for use in adults and children (3 months and older), for the treatment of putative fungal infections in febrile neutropenia, invasive Aspergillosis (IA) refractory or intolerant to azole antifungals, and for the treatment of severe candida infections. Liu et al found that Caspofungin could theoretically bind to the major protease Mpro of SARS-CoV-2 by visual docking, and thereby predicted that Caspofungin could be a potential antiviral drug. CN 111298098A discloses the application of Caspofungin in the preparation of products for inhibiting coronavirus, and Caspofungin can bind and inhibit nsp12 polymerase and shows an inhibition effect on SARS-CoV-2 virus replication in vitro experiments. In the research on anti-inflammatory aspect, kazuhiro et al found that Caspofungin could inhibit zymosan-induced cytokine and chemokine release in THP-1 cells by inhibiting the activation of spleen tyrosine kinase (syk), including TNFa, IL1 β, IL6, IL8, IP10, MCP-1, MIP1 α and MIP1 β. However, the anti-inflammatory action of Caspofungin has been studied mainly in relation to its antifungal activity, and its anti-inflammatory action in COVID-19 has not been reported.

SEQ ID NO.01：

SEQ ID NO.02：

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Claims (6)

1. Use of Caspofungi in the preparation of a pharmaceutical composition for the treatment of cytokine storm induced by COVID 19.
2. 2. The use of claim 1, wherein: the inflammatory factor in the cytokine storm comprises at least CCL3, CCL4, CCL5, CKC12, IL1 beta, IL2, IL4, IL5, IL6, IL8, IP10, IL13, IL18, GM-CSF, IFN beta, IFN, TNF alpha, TNF beta, CXCL12, LTA, IL22 and IL21.
3. 3. Use according to claim 2, characterized in that: the inflammatory factors in the cytokine storm consist of CCL3, CCL4, CCL5, CKC12, IL1 beta, IL2, IL4, IL5, IL6, IL8, IP10, IL13, IL18, GM-CSF, IFN beta, IFN, TNFa, TNF beta, CXCL12, LTA, IL22 and IL21.
4. Use of Fasudil as a broad-spectrum inhibitor of inflammatory factors induced by COVID-19.
5. 5. The use of claim 4, wherein: the inflammatory factor comprises at least CCL3, CCL4, CCL5, CKC12, II1 beta, IL2, IL4, IL5, IL6, IL8, IP10, IL13, IL18, GM-CSF, IFN beta, IFN, TNF alpha, TNF beta, CXCL12, LTA, IL22 and IL21.
6. 6. The use of claim 5, wherein: the inflammatory factor consists of CCL3, CCIA, CCL5, CKC12, IL1 beta, IL2, IL4, IL5, IL6, IL8, IP10, IL13, IL18, GM-CSF, IFN beta, IFN, TNFa, TNF beta, CXCL12, LTA, IL22 and IL21.

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