APPLICATION OF PROGESTIN IN PREPARATION OF DRUG INHIBITING CYTOKINE STORM

Abstract

The present application provides a use of hydroxyprogesterone caproate for treating multiple diseases that cause a 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, and acute pancreatitis. Moreover, the present application also provides a method for treating the diseases by using a corresponding pharmaceutical composition. Experimental results show that progestin hydroxyprogesterone caproate can effectively inhibit the cytokine storm and is expected to be an effective drug for treating diseases such as novel coronavirus pneumonia.

Claims

1. A method for inhibiting a cytokine storm syndrome, comprising administering protestogen.

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

3. The method according to claim 1, wherein the cytokine storm syndrome is induced by various virus infections, macromolecular antibody therapy, organ transplantation or chimeric antigen receptor T cell (CAR-T) therapy.

4. The method according to claim 3, wherein the cytokine storm syndrome is induced by coronavirus, influenza virus and other virus infections.

5. The method according to claim 4, wherein the cytokine storm syndrome is induced by novel coronavirus infection.

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

7. The pharmaceutical composition according to claim 6, wherein the progestogen is hydroxyprogesterone caproate.

8. The pharmaceutical composition according to claim 6, wherein the cytokine storm syndrome is induced by various virus infections, macromolecular antibody therapy, organ transplantation or chimeric antigen receptor T cell (CAR-T) therapy.

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

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

11. A method for treating a coronavirus-infected disease, comprising administering progestogen.

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

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

14. The method according to claim 12, wherein the coronavirus-infected disease is novel coronavirus pneumonia.

15. The method according to claim 12, comprising administering the hydroxyprotesterone caproate to inhibit a cytokine storm syndrome in the coronavirus-infected disease.

16. The method according to claim 12, wherein the hydroxyprotesterone caproate is administered by intraperitoneal injection at a dose of 5 mg/kg.

17. The method according to claim 15, wherein the cytokine comprises IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-γ and TNF-α.

Description

DETAILED DESCRIPTION

[0020] Test Animals and Materials:

[0021] Humanized mice bred under specific pathogen-free conditions were used in the test. All test procedures were approved by the Animal Ethics Committee. All antibodies used for flow cytometry were purchased from outside. The endotoxin levels of these antibodies were all less than 0.5 IU/mg.

[0022] Human peripheral blood mononuclear cells (PBMCs) were also used in this test. Isolated human PBMCs were washed with phosphate buffer. After lysing red blood cells, the nucleated cells were washed for three times with phosphate buffer.

[0023] Test Method and Procedures:

[0024] In general, the ratio of human CD45 cells to mouse CD45 cells in the spleen and blood of humanized severe combined immunodeficient (hu-SCID) mice was 3:1 and 0.4:1, respectively, after post-adaptive transfer of human peripheral blood mononuclear cells (PBMCs) for 10 days. This was equivalent to (1-2)×10.sup.7 human CD45 cells in the spleen and 1×10.sup.6 human

[0025] CD45 cells per milliliter of blood.

[0026] It was reported that muromonab-CD3 (OKT3) will produce severe adverse effects after infusion. After 10 days of adoptive transfer of human PBMCs, human cells isolated from the spleen of the hu-SCID mice were still able to bind to OKT3.

[0027] In the test, we chose both intravenous (IV) and intraperitoneal (IP) routes of administration because antibody absorption was slower after intraperitoneal injection.

[0028] Each humanized mouse for the test was first intravenously injected with 2×10.sup.7 PBMCs. After 10 days, each mouse was injected with OKT3 or control IgG (2 μg or 10 μg, IV or IP).

[0029] Blood was collected from the mice at 10 min, 20 min and 60 min after administration of the OKT3 and control IgG. A multiplex immunoassay system was then applied to the collected heparinized plasma to analyze cytokines. Cytokines measured included human IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IL-17, IFN-γand TNF-α. The mice were sacrificed after last blood collection (60 min). The activation markers thereof were then analyzed by flow cytometry. Human peripheral blood mononuclear cells in the mouse spleen were blocked with 2.4G2 and then stained with fluorescently labeled CD69, CD25, CD45 and CD3.

[0030] Body temperature measurement: the rectal temperature of mice was measured before treatment and the rectal temperature was measured again before each blood collection point. The temperature was measured by inserting a rectal thermocouple probe and waiting for the number to stabilize (about 10s) to obtain the reading. Mice with a body temperature of smaller than 32° C. were sacrificed.

[0031] Clinical score measurement: as recommended by the Canadian Council for Animal Care (www.ccac.ca/en /standards/guidelines), we conducted preliminary studies to determine the most appropriate clinical symptoms and scoring criteria. Mice were observed at each time point and given one clinical score. Score: 0=normal activity; 1=normal activity, hairs upright, and tiptoe gait; 2=arched back, decreased activity, but there are still activities; 3=too few activities, but there are still activities when prompted; 4=dying. Mice with a clinical score of 4 were sacrificed. Statistical analysis was then performed on inter-group differences in the test data.

[0032] Test Results:

[0033] Symptom observation after injection of antibody:

[0034] Animals Injected with OKT3:

[0035] IP injection group: in the administration of the IP route at a low dose of 2 μg, all hu-SCID mice were unaffected. Hu-SCID mice receiving IP administration of 10 μg OKT3 were clinically scored as moderate (arched back, too few activities) and one of 5 mice was dying and sacrificed at 2 h.

[0036] IV injection group: Hu-SCID mice showed moderate to severe response within lh when intravenously given 2 μg of OKT3. Two of four mice needed to be euthanized, while the remaining two mice showed moderate symptoms but slowly recovered within 5 hours. The response of mice to 10 μg of OKT3 intravenously injected was severe, so all mice were sacrificed at 1 h.

[0037] Animals Injected with Polyclonal Antibody ATG:

[0038] No clinical symptoms were found in the hu-SCID mice injected with control IgG by either IP or IV administration.

[0039] Hypothermia After Antibody Injection

[0040] Both hypothermia and hyperthermia occur in the cytokine storm. To this end, we made observations.

[0041] OKT3 group: the rectal temperature decreased significantly from 37° C. to 32° C. or below within 1 h, after IP or IV injection of 10 μg OKT3. At a lower dose of 2 μg, administration of the IV route resulted in transient hypothermia (about 32° C.) at lh with partial recovery after 5 h; the moderate changes that occurred after administration of the IP route were similar to those that occurred after injection of control human IgG.

[0042] IgG control group: no apparent change in body temperature.

[0043] Circulating cytokine changes after antibody injection:

[0044] After antibody injection, in order to determine if a cytokine storm was induced, we detected cytokines (human IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IL17, IFN-γ and TNF-α) in the plasma of the hu-SCID mice.

[0045] OKT3 group: OKT3-IP intraperitoneally injected at a high dose (10 μg) may induce production of multiple cytokines, including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10 and IFN-γ, and concentrations thereof gradually increased within 5 h of the test; in addition, production of TNF-α was also induced, but its peak appeared earlier (at 1 h). Hu-SCID mice that received IV injection of high-dose OKT3 were sacrificed after 1 h, so cytokines after this time point could not be detected. Induced cytokines were rarely detected at any time point, regardless of whether 2 μg of OKT3 was injected intraperitoneally or intravenously.

[0046] IgG control group: no cytokine changes were observed.

[0047] PR2005 treatment group:

[0048] On days 8 and 9 after intravenous injection of PBMCs in humanized mice (48 hours and 24 hours before antibody injection), a candidate drug PR2005 (5 mg/kg) was injected intraperitoneally to the animals. After the use of PR2005, the animals showed no significant changes in body temperature after receiving OKT3. In the animal group using PR2005 and OKT3 simultaneously, the concentration of cytokines in the blood was also substantially lower than that of animals using OKT3 alone. The results are detailed in Tables 1-4.

TABLE-US-00001 TABLE 1 Rectal temperature decline in hu-SCID mice induced by OKT3 injection. The data is the mean value of each group of animals. Compared with the control group at the same time point. IgG OKT3 Time 10 μg 2 μg 10 μg (hrs) 0 1 2 5 0 1 2 5 0 1 2 5 Temp 37.7 36.6 35.3 36.1 37.7 35.6 34.8 35.4 37.7 31.7 31.1 30.6 IP 37.7 35.7 34.5 35.0 37.7 31.7 33.8 34.9 37.7 31.9 IV

TABLE-US-00002 TABLE 2 Cytokines induced after OKT3 injection. Hu-SCID mice were intraperitoneally injected with 10 μg of OKT3 (black bars) or IgG control (grey bars). Blood samples were taken at 1 h, 2 h and 5 h respectively to determine the concentration of circulating cytokines. The sensitivity (limit of detection) of the analytical method was 1 ng .Math. ml.sup.−1. The data is expressed as the mean value of each group of animals. Compared with the control group at the same time point. Time (hrs) 1 2 5 IL-1β Concentration   30.0   44.0   30.0   77.5   30.0   97.4 (pg/ml) IgG OKT3 IgG OKT3 IgG OKT3 IL-4 Concentration 33 61 40 182 37 259 (pg/ml) IgG OKT3 IgG OKT3 IgG OKT3 IL-6 Concentration 35 96 35 160 35 252 (pg/ml) IgG OKT3 IgG OKT3 IgG OKT3 IFN-γ Concentration 1910  7015  2119  23574  1597  25783  (pg/ml) IgG OKT3 IgG OKT3 IgG OKT3 IL-2 Concentration   23.4   39.0   21.5   75.9   19.3   89.4 (pg/ml) IgG OKT3 IgG OKT3 IgG OKT3 IL-5 Concentration 57 72 57  98 57 158 (pg/ml) IgG OKT3 IgG OKT3 IgG OKT3 IL-10 Concentration 694  3319  694  11001  694  20052  (pg/ml) IgG OKT3 IgG OKT3 IgG OKT3 TNF-α Concentration 51 1409  51 1127  51 655 (pg/ml) IgG OKT3 IgG OKT3 IgG OKT3

TABLE-US-00003 TABLE 3 48 hours prior to antibody injection, a candidate drug PR2005 (5 mg/kg) was intraperitoneally injected to animals. Blood samples were taken at 1 h, 2 h and 5 h respectively to determine the concentration of circulating cytokines. The sensitivity (limit of detection) of the analytical method was 1 ng .Math. ml.sup.−1. The data is expressed as the mean value of each group of animals. Compared with the control group at the same time point. Time (hrs) 1 2 5 IL-1β Concentration   30.0   37.4   44.0   30.0   69.1   77.5   30.0   93.5   97.4 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-4 Concentration 33 49 61 40 151 182 37 221 259 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-6 Concentration 35 88 96 35 123 160 35 146 252 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IFN-γ Concentration 1910  5550  7015  2119  13231  23574  1597  18388  25783  (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-2 Concentration   23.4   32.7   39.0   21.5   65.7   75.9   19.3   70.4   89.4 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-5 Concentration 57 71 72 57  81  98 57 115 158 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-10 Concentration 694  3085  3319  694  6885  11001  694  15016  20052  (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 TNF-α Concentration 51 1383  1409  51 733 1127  51 442 655 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3

TABLE-US-00004 TABLE 4 24 hours prior to antibody injection, a candidate drug PR2005 (5 mg/kg) was intraperitoneally injected to animals. Blood samples were taken at 1 h, 2 h and 5 h respectively to determine the concentration of circulating cytokines. The sensitivity (limit of detection) of the analytical method was 1 ng .Math. ml.sup.−1. The data is expressed as the mean value of each group of animals. Compared with the control group at the same time point. Time (hrs) 1 2 5 IL-1β Concentration   30.0   35.4   44.0   30.0   44.1   77.5   30.0   39.8   97.4 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-4 Concentration 33 39 61 40 114  182 37 132 259 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-6 Concentration 35 51 96 35 70 160 35  69 252 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IFN-γ Concentration 1910  3960  7015  2119  4621  23574  1597  11359  25783  (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-2 Concentration   23.4   29.7   39.0   21.5   40.1   75.9   19.3   39.7   89.4 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-5 Concentration 57 64 72 57 76  98 57  81 158 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 IL-10 Concentration 694  882  3319  694  1961  11001  694  8614  20052  (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3 TNF-α Concentration 51 438  1409  51 412  1127  51 110 655 (pg/ml) IgG PR2005 + OKT3 IgG PR2005 + OKT3 IgG PR2005 + OKT3 OKT3 OKT3 OKT3

[0049] Conclusion: the test results show that PR2005 can inhibit the OKT3-induced immune storm in an animal model.