阅读说明：本技术 孕激素在制备抑制细胞因子风暴的药物中的应用 (Application of progestogen in preparing medicine for inhibiting cell factor storm ) 是由 杜涛 杜新 于 2020-04-28 设计创作，主要内容包括：本申请提供了己酸羟孕酮用于治疗多种引起细胞因子风暴的疾病,如新型冠状病毒肺炎,各种烈性病毒感染,单克隆抗体治疗后的副作用,CAR-T治疗后的副作用,炎症性肠病和进行胰腺炎等。同时本申请也提供了使用相应药物组合物来治疗上述疾患的方法。实验结果表明,孕激素己酸羟孕酮可以有效抑制细胞因子风暴,有望成为治疗新冠病毒肺炎等疾患的有效药物。(Hydroxyprogesterone caproate is used for treating various diseases causing cytokine storm, such as novel coronavirus pneumonia, various virulent virus infections, side effects after monoclonal antibody treatment, side effects after CAR-T treatment, inflammatory bowel disease, pancreatitis and the like. Methods of treating the above disorders using the corresponding pharmaceutical compositions are also provided. The experimental result shows that the progestational hormone hydroxyprogesterone caproate can effectively inhibit the cell factor storm and is expected to become an effective medicament for treating diseases such as new coronavirus pneumonia and the like.)

1. Use of a progestogen in the preparation of a medicament for inhibiting cytokine storm syndrome.

2. The use according to claim 1, wherein the progestogen is hydroxyprogesterone caproate.

3. Use according to claim 1 or 2, wherein the cytokine storm syndrome is caused by an infectious disease of a virus of all types, by a macroantibody treatment, by organ transplantation or by CAR-T treatment.

4. Use according to claim 3, wherein the cytokine storm syndrome is a cytokine storm syndrome caused by infection with coronavirus, influenza virus and other viruses.

5. Use according to claim 4, wherein the cytokine storm syndrome is a cytokine storm syndrome caused by a novel coronavirus infection.

6. A pharmaceutical composition for inhibiting cytokine storm syndrome comprising a progestin.

7. The pharmaceutical composition of claim 6, wherein the progestin is hydroxyprogesterone caproate.

8. The pharmaceutical composition according to claim 6 or 7, wherein the cytokine storm syndrome is a cytokine storm syndrome caused by an infectious disease of various viruses, a macroantibody treatment, an organ transplant or a CAR-T treatment.

9. The pharmaceutical composition according to claim 8, wherein the cytokine storm syndrome is a cytokine storm syndrome caused by coronavirus infection.

10. The pharmaceutical composition according to claim 9, wherein the cytokine storm syndrome is a cytokine storm syndrome caused by a novel coronavirus infection.

11. A method of treating a disease caused by a coronavirus infection, characterized by administering a progestogen.

12. The method according to claim 11, wherein the progestogen is hydroxyprogesterone caproate.

13. The method according to claim 11 or 12, wherein the coronavirus is a novel coronavirus.

14. The method according to any one of claims 11-13, wherein the disease of coronavirus infection is novel coronavirus pneumonia.

15. The method according to any one of claims 11-14, wherein the administration of a progestin inhibits the cytokine storm syndrome in said coronavirus infected disease.

Technical Field

The invention belongs to the field of treatment of immune diseases and infectious diseases, and particularly provides application of PR2005 in preparation of a medicine for inhibiting cytokine storm, particularly cytokine storm caused by diseases such as new coronavirus pneumonia.

Background

Immune cells communicate with each other through cytokines. The cell factor is a small molecule released by cells into blood, can enable immune cells to move infected parts, phagocytose damaged cells, and even penetrate through a blood vessel wall; at the same time, cytokines can also cause inflammation, causing swelling, fever and pain in the damaged body.

The phenomenon that the immune system is activated to a limited extent or loses control due to infection, CART treatment, imbalance of certain drugs or autoimmune regulation, etc., releases a large number of cytokines, and the level of a large number of pro-inflammatory cytokines is sharply increased is called cytokine storm syndrome (CSR). The cytokine storm syndrome is a systemic inflammatory reaction caused by hypersensitive activation of an immune system, and known specific symptoms comprise fever, headache, rash, arthralgia, myalgia, hypotension, vascular leakage, disseminated intravascular coagulation and multiple organ failure, and related indexes comprise lymphopenia, creatinine increase, coagulation parameter disorder, ferritin and C protein increase and the like besides cytokine levels. The clinical common cytokine storm inducing factors comprise CART treatment, H5N1, H1N1, SARS, MERS, COVID-19 and the like, and the related cytokines mainly comprise TNF-alpha, IL-1, IL-2, IL-6, IL-12, IFN-alpha, IFN-beta, IFN-gamma, MCP-1, IL-8, G-CSF, MCP-1 and the like.

The specific pathophysiological mechanism of the cytokine storm syndrome is not clear (is presumed to be related to cytolytic immune response), no special corresponding therapeutic drugs exist, most of the clinical treatment experiences of the cytokine storm syndrome are from CRS symptoms caused by immunotherapy, especially adoptive cell therapy, no clear therapeutic guidelines are provided in influenza virus and coronavirus infection, and drugs which are tried to treat the cytokine storm syndrome comprise peroxisome proliferator activated receptor agonists, sphingosine-1-phosphate receptor agonists, cyclooxygenase inhibitors, antioxidants, antitumor necrosis factor therapy, intravenous immunoglobulin and other therapies.

The new coronavirus pneumonia (COVID-19) caused by the new coronavirus is a great threat to human health and socioeconomic development. A significant proportion of cases of death due to the corona virus pneumonia are associated with cytokine storms (Mehta P et al, CODVI-19: conditioner cytokine stocks syndrome and immunity, Lancet, Vol.395, 16 p.2020; Fu B et al, nutritional T cells and antibiotics in tissue stocks in section COVID-19 titles, J Transl Med, 4.2004, Vol.18, No. 1). The inhibition of the cytokine storm syndrome is one of the main ways of treating the new coronavirus pneumonia and reducing the death rate of the new coronavirus pneumonia like the inhibition of virus invasion/replication.

Disclosure of Invention

In order to inhibit cytokine storm and treat a series of diseases related to the cytokine storm, a large number of candidate drugs are screened, wherein hydroxyprogesterone caproate in the development stage is a candidate drug with high curative effect and low side effect potential. The test result proves that the hydroxyprogesterone caproate can inhibit the cytokine storm occurring on an animal model. The test results prove that the PR2005 is possible to become a clinical medicine for treating the new coronavirus pneumonia.

In one aspect, the application claims the use of a progestogen in the preparation of a medicament for suppressing a cytokine storm syndrome.

In another aspect, the present application claims a pharmaceutical composition for inhibiting cytokine storm syndrome, characterized by comprising a progestogen.

In yet another aspect, the application claims a method for treating a coronavirus infection condition, characterized in that a progestogen is administered.

The progestogen can be selected from hydroxyprogesterone caproate, medroxyprogesterone acetate, progesterone, etc., preferably hydroxyprogesterone caproate.

The cytokine storm syndrome can be caused by various virus infections, macromolecular antibody therapy, organ transplantation or CAR-T therapy.

The infection may be a coronavirus, influenza virus and other viral infection.

The coronavirus infectious diseases can be new type coronavirus pneumonia, middle east respiratory syndrome and severe acute respiratory syndrome, preferably new type coronavirus pneumonia, and can be treated by inhibiting cytokine storm in these diseases by administering progestogen

There are a number of nomenclature for the novel coronaviruses described in this application, including but not limited to the novel coronaviruses, 2019-nCov, SARS-CoV-2 (official nomenclature of the international committee for classification of viruses); the diseases caused by the virus infection can be called novel coronavirus pneumonia, NCP, COVID-19 (official name of International health organization), and the like.

Hydroxyprogesterone caproate is also referred to herein as 17- α hydroxyprogesterone caproate, 17-HPC, 17-hydroxyprogesterone caproate, 17-hydroxyprogestin, CAS number 630-56-8, and the code for PR2005 was used in the experimental section.

The medicament in the application can be in any clinically acceptable dosage form, and the medicament in the treatment of the application can be applied in any clinically acceptable dosage form. Specific dosage forms include, but are not limited to: tablet, capsule, oral liquid, injection, powder for injection, etc.

The medicament prepared by the invention can also contain other known and unknown medicaments for treating related diseases, and other known and unknown medicaments for treating related diseases can also be used in the treatment method of the invention, and the medicaments comprise but are not limited to: drugs for the treatment of autoimmune diseases, such as immunosuppressants, peroxisome proliferator-activated receptor agonists, sphingosine-1-phosphate receptor agonists, cyclooxygenase inhibitors, antioxidants, anti-tumor necrosis factor therapy, intravenous immunoglobulin, and other therapies. Drugs for treating infections such as antibiotics, antifungals, inhibitors of viral replication, inhibitors of viral entry, and known and unknown drugs for treating symptoms of fever, vomiting, skin problems, etc. in related diseases.

According to the preparation/administration dosage form, various pharmaceutically acceptable adjuvants can be selected from the medicine, including but not limited to coating materials, solvents, solubilizers, binders, stabilizers, antioxidants, pH regulators and flavoring agents, and the ingredients of the adjuvants can be selected by those skilled in the art according to the common knowledge in pharmacy.

Detailed Description

Test animals and materials:

humanized mice grown in the absence of a particular pathogen were used in the experiment. All experimental procedures were approved by the animal ethics committee. All antibodies used for flow cytometry analysis were from outsourced sources. The endotoxin levels of these antibodies were less than 0.5IU/mg on average.

Human Peripheral Blood Mononuclear Cells (PBMCs) were also used in this experiment. Isolated PBMCs were washed first with phosphate buffer. After the erythrocytes were dissolved out, the nucleated cells were washed three more times with phosphate buffer.

The test method and the process are as follows:

generally, the ratio of human CD45 cells to mouse CD45 cells in the spleen and blood of humanized severe combined immunodeficiency (hu-SCID) mice was 3:1 and 0.4:1, respectively, 10 days after adoptive transplantation of human Peripheral Blood Mononuclear Cells (PBMCs) (post-adaptive transfer). This corresponds to 1-2X 10 in the spleen⁷Personal CD45 cells, 1X 10 per ml of blood⁶Human CD45 cells.

Moluzumab-CD 3(OKT3) was reported to produce severe adverse reactions after infusion. Human cells isolated from the spleen of hu-SCID mice were still able to bind OKT3 after 10 days of adoptive transplantation of human PBMCs.

In the experiment, we chose both Intravenous (IV) and Intraperitoneal (IP) routes of administration, as the absorption of antibody after intraperitoneal injection was slower.

Humanized mice for each trial were first injected intravenously with 2X 10⁷And (5) PBMCs. After 10 days, each mouse was injected with OKT3 or control IgG (2 μ g or 10 μ g, IV or IP).

Mice were bled at time points of 10min, 20min and 60min after administration of OKT3 and control IgG. The collected heparinized plasma was then analyzed for cytokines using a multiplex immunoassay system. Cytokines measured include human IL-1 β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, IL-17, IFN- γ, and TNF- α. After the last blood draw (60min), the mice were sacrificed. The activation markers were then analyzed by flow cytometry. Mouse spleen human peripheral blood mononuclear cells were blocked with 2.4G2 and then stained with fluorescently labeled CD69, CD25, CD45, and CD 3.

Body temperature measurement: rectal temperature of the mice was measured before treatment and again before each blood collection point. The temperature was measured by inserting a rectal thermocouple probe and waiting for the number to stabilize and taking a reading (10 s). Mice with a body temperature <32 ℃ were sacrificed.

Measurement of clinical score: according to the Canadian animal protection Association (www.ccac.ca/en _/standards/guidelines) recommendations, we conducted preliminary studies to determine the most appropriate clinical symptoms and scoring criteria. Mice were observed at each time point and given a clinical score. And (4) score: 0-normal activity; 1, standing hair and standing tiptoe gait, namely normal activity; 2, the movement is reduced but the movement is still available; if the activity is too little, the activity is still available when the prompt is given; 4-dying. Mice with a clinical score of 4 were sacrificed. Statistical analysis was then performed on the differences between groups of experimental data.

And (3) test results:

observation of symptoms after antibody injection:

OKT3 injected animals:

IP injection group: all hu-SCID mice were unaffected by the low dose 2 μ g IP route of administration. The Hu-SCID mice receiving 10 μ g OKT3 IP dose were clinically scored as moderate (arch back, less activity), with 1 out of 5 mice showing moribund and sacrificed at 2 h.

IV injection group: Hu-SCID mice developed moderate to severe reactions within 1h when given 2. mu.g IV of OKT 3. Only 2 of 4 mice were euthanized, while the remaining 2 mice showed moderate symptoms but slowly recovered within 5 hours. Mice responded heavily to 10 μ g OKT3 IV, so all mice were sacrificed at the 1h time point.

Polyclonal antibody ATG injected animals:

neither the IP nor IV control IgG injected hu-SCID mice developed clinical symptoms.

Hypothermia after antibody injection

Both hypothermia and hyperthermia occur in cytokine storms. For this reason, we have made observations.

OKT3 group: rectal temperature dropped severely from 37 ℃ to below 32 ℃ within 1h after injection of 10 μ g OKT3, either IP or IV. At a lower dose of 2 μ g, IV route administration resulted in transient hypothermia (-32 ℃) at 1h, with partial recovery after 5 h; the modest changes that occurred after administration by the IP route were similar to those seen with the injection of control human IgG.

IgG control group: body temperature did not change significantly.

Circulating cytokine changes after antibody injection:

to determine whether cytokine storm was induced after antibody injection, we tested the cytokines (human IL-1. beta., IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, IL17, IFN-. gamma.and TNF-. alpha.) in the plasma of hu-SCID mice.

OKT3 group: high doses (10 μ g) of IP-injected OKT3-IP induced the production of a variety of cytokines including IL-1 β, IL-2, IL-4, IL-5, IL-6, IL-10 and IFN- γ at increasing concentrations over the 5h test period; TNF-. alpha.production was also induced, but its peak appeared earlier (at 1 h). IV injected Hu-SCID mice receiving high doses of OKT3 were sacrificed after 1h, so cytokines after this time point could not be detected. Little induced cytokine was detected at any time point whether 2 μ g OKT3 was injected IP or IV.

IgG control group: no cytokine changes were seen.

PR2005 treatment group:

on days 8 and 9 (48 hours and 24 hours before antibody injection) after the intravenous injection of PBMCs in the humanized mouse, the (5mg/kg) candidate PR2005 was injected into the animal in the form of IP. After PR2005, the animals received OKT3 without significant changes in body temperature. In the group of animals with both PR2005 and OKT3, the concentration of cytokines in the blood was also significantly lower than in animals with OKT3 alone. The results are detailed in tables 1 to 4.

TABLE 1 rectal temperature drop in hu-SCID mice induced after OKT3 injection. Data are mean values for each group of animals. Compared to a control group at the same time point.

TABLE 2 cytokines induced after OKT3 injection. Hu-SCID mice were injected with 10 μ g of OKT3 IP (black bars) or IgG control IP (gray bars). Blood is collected for 1h, 2h and 5h respectively, and the circulating cytokine concentration is determined. The sensitivity (detection limit) of the assay method was 1ng ml^-1. Data are presented as mean values for each group of animals. Compared to a control group at the same time point.

TABLE 3 animals were injected IP with (5mg/kg) candidate PR2005 48 hours prior to antibody injection. Blood is collected for 1h, 2h and 5h respectively, and the circulating cytokine concentration is determined. The sensitivity (detection limit) of the assay method was 1ng ml^-1. Data are presented as mean values for each group of animals. Compared to a control group at the same time point.

TABLE 4. 24 hours before antibody injection, (5mg/kg) candidate PR2005 was injected IP-wise into animals. Blood is collected for 1h, 2h and 5h respectively, and the circulating cytokine concentration is determined. The sensitivity (detection limit) of the assay method was 1ng ml^-1. Data are presented as mean values for each group of animals. Compared to a control group at the same time point.

And (4) conclusion: the test results show that PR2005 can inhibit immune storm caused by OKT3 on an animal model.