WO2022161381A1 - Use of endostatin in treating and preventing coronavirus-related diseases - Google Patents

Abstract

A method for preventing or treating coronavirus-related diseases (especially coronavirus disease 2019) in a subject, the method comprising administering an effective amount of endostatin to a subject.

Description

Application of endostatin in the treatment and prevention of coronavirus-related diseases technical field

The present invention relates to the treatment and prevention of coronavirus-related diseases, in particular, to the application of endostatin and its functional variants in the treatment and prevention of coronavirus-related diseases.

Background technique

Novel coronavirus pneumonia (referred to as "new coronary pneumonia") is an acute infectious pneumonia for which there is currently no specific medicine. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen that caused the outbreak of new coronary pneumonia in 2019-2020 ^[1-4] . At present, the virus has spread widely around the world. As of June 28, 2020, there have been more than 10.04 million confirmed cases worldwide and 500,000 deaths ^[5] . There is no specific drug for the treatment of new coronary pneumonia in the world so far, and the development of specific drugs for the treatment of new coronary pneumonia is crucial for controlling the epidemic, restoring normal social order and economic development.

In the process of new coronary pneumonia drug development, many single-component chemical drugs with high expectations are not satisfactory, while traditional Chinese medicines with complex components have shown unexpected efficacy ^[6] . It can be seen that the treatment of new coronary pneumonia is likely to require the synergy of multiple targets. Nucleolin (NCL) on the cell membrane is a receptor for Endostatin (ES) and is associated with the infection of a variety of RNA viruses, including respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), Influenza A virus (Influenza A), polio virus (Polro virus), human parainfluenza virus (HPIV), etc. ^[7-10] . For example, NCL is the receptor of RSV fusion protein ^[10] , and in cell culture and animal models, interfering with the interaction of NCL-RSV fusion protein can effectively treat RSV infection ^[7-10] . NCL is closely related to the infection and replication process of viruses ^[11] . During the infection of various viruses, NCL will first undergo membrane localization, and the NCL after membrane localization will directly contact the virus to help the virus enter the cell, and then carry out Virus replication and subsequent infection. It has been reported that the presence of non-structural protein 1 (nsp1) of SARS-COV will increase the content of NCL in the cytoplasm ^[11] . The similarity of non-structural protein 1 in SARS-COV and SARS-CoV-2, the pathogen of new coronary pneumonia virus, is as high as 91.1%. As an RNA virus associated with severe acute respiratory disease, SARS-CoV-2, its infection and replication process May also be closely related to NCL.

SUMMARY OF THE INVENTION

The present invention provides the use of endostatin in the preparation of a medicament for preventing or treating coronavirus-related diseases in a subject.

The present invention also provides a method of preventing or treating a coronavirus-related disease in a subject, comprising administering to the subject an effective amount of endostatin.

The present invention also provides a pharmaceutical composition for preventing or treating coronavirus-related diseases in a subject, comprising: endostatin; and a pharmaceutically acceptable carrier.

The present invention also provides a kit for preventing or treating a coronavirus-related disease in a subject, comprising the pharmaceutical composition described above.

In some embodiments, the coronavirus is selected from the group consisting of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2.

In some embodiments, the coronavirus-related disease is selected from SARS, MERS, and COVID-19.

In some embodiments, the subject is a mammal or avian, preferably a human.

In some embodiments, the dosage form of the medicament is selected from: intravenous injection, oral spray, nasal spray, eye drops.

In some embodiments, the endostatin is human endostatin.

In some embodiments, the endostatin is YH-16 or M2ES.

In some embodiments, the endostatin is pegylated human endostatin or a functional variant thereof.

Detailed ways

Unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art.

Endostatin (ES) is an approved endogenous protein drug that can effectively inhibit angiogenesis. The inventor first reported that the receptor of ES is NCL ^[12] . M2ES is a long-acting recombinant endostatin whose receptor is also NCL. 273 patients have been enrolled in the phase II clinical trial of this drug for lung cancer indications, and its safety has been verified. We speculate that M2ES may affect the infection and replication process of SARS-CoV-2 by binding to NCL. Therefore, exploring the effects of M2ES on SARS-CoV-2 infection and replication is of great significance for the development of new antiviral drugs.

"Coronavirus" refers to a class of enveloped viruses that can cause disease in vertebrates, especially mammals or birds, having a single-stranded positive-stranded RNA genome. They belong to the order Nidovirales, the family Coronaviridae. Animal or human diseases caused by coronaviruses are referred to herein as "coronavirus-related diseases" or "coronavirus infections". Seven coronaviruses have been found to infect humans so far, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Some of the diseases they cause are mild, like the common flu, while others can be deadly, such as SARS, MERS, and COVID-19. The coronavirus that causes COVID-19 is SARS-CoV-2. The most common symptoms of COVID-19 include fever, dry cough and weakness. Patients with mild symptoms may experience only a runny nose or sore throat; in severe cases, breathing difficulties, cytokine storms, and even eventual organ failure may occur.

The present invention provides a method of preventing or treating a coronavirus-related disease in a subject, comprising administering to the subject an effective amount of endostatin.

The term "subject" refers to an individual who has or is suspected of having a disease (eg, COVID-19), or, in predicting risk of disease or for prophylactic applications, may also include healthy individuals . The term is often used interchangeably with "patient," "subject," "subject," and the like. In an embodiment of the present invention, the subject is a vertebrate, preferably a mammal and a bird, more preferably a human.

"Prevention" refers to avoiding, reducing or delaying the occurrence of a particular disease or disease-related symptom in a subject that has not occurred prior to administration of the relevant drug. "Prevention" does not necessarily require complete prevention of the occurrence of a disease or disease-related symptoms, for example, to reduce the risk of a particular disease or disease-related symptoms in a subject after administration of the relevant drug, or to reduce the severity of subsequent related symptoms, All can be considered to "prevent" the appearance or development of the disease. "Treatment" refers to alleviating, alleviating, ameliorating, or inhibiting (eg, arresting the development) of a disease that a subject has exhibited or has experienced. With respect to a specific disease, "treating" may include "curing" the disease, but in most cases it is not necessary to completely eliminate all its symptoms, for example, administration of the relevant drug results in the reduction or elimination of at least one symptom in the subject, The subject can then be considered to have been treated.

A "therapeutically effective amount" refers to an amount sufficient to induce a biological or medical response expected by a clinician in a subject, which can usually be determined by those skilled in the art according to the route of administration, the subject's weight, age, condition and other factors . A "therapeutically effective amount" can also be considered a "prophylactically effective amount" when used for prophylactic purposes. For example, a typical daily dose may range from 0.01 mg to 100 mg of the active pharmaceutical ingredient per kg body weight of the subject.

Depending on the medical application, the term "effective amount" can include "therapeutically effective amount" and "prophylactically effective amount".

In some embodiments, a pharmaceutically active ingredient, such as endostatin (including functional variants or derivatives thereof), can be used alone or formulated with a pharmaceutically acceptable carrier as a pharmaceutical composition.

The term "pharmaceutically acceptable carrier" refers to substances such as solid or liquid diluents, fillers, antioxidants, stabilizers, etc., which can be safely administered to animals or humans, and that are suitable for human and/or Animals were administered without excessive adverse side effects. Depending on the route of administration, a variety of carriers well known in the art can be employed. Common routes of administration include, for example, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, oral spray, nasal spray or inhalation, transdermal, and the like. Commonly used carriers include, but are not limited to, carbohydrates, starch, cellulose and derivatives thereof, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and/or pyrogen-free water, etc.

The pharmaceutical composition can be prepared in any clinically acceptable dosage form, such as granules, powders, injections (eg, intravenous injections), eye drops or sprays, and the like.

The present invention also provides drug combinations including endostatin and other drugs such as remdesivir.

Herein "drug combination" refers to the combination of two or more pharmaceutically active ingredients. In the case of a pharmaceutical combination comprising two active pharmaceutical ingredients, it not only includes the coexistence of the two active pharmaceutical ingredients in the same pharmaceutical preparation (eg, a pharmaceutical composition), but also means that the two active pharmaceutical ingredients can be used as separate pharmaceutical preparations Present in the same pharmaceutical kit, or even the two pharmaceutically active ingredients may be presented as separate pharmaceutical formulations in different pharmaceutical kits. In effect, such a mode of administration employs such a "drug combination" as long as the administration is such that the two pharmaceutically active ingredients are simultaneously present in the subject at a certain time. These pharmaceutically active ingredients can optionally be formulated with a pharmaceutically acceptable carrier.

In other words, a pharmaceutical combination includes not only the pharmaceutical composition form, but also other forms in which different pharmaceutical active ingredients may coexist in a subject. In some embodiments, the drug combination is in the form of a pharmaceutical composition; in other embodiments, the drug combination is in the form of a non-pharmaceutical composition, eg, endostatin and remdesivir are present in separate pharmaceutical formulations.

The term "co-administration" or "combination", eg, for endostatin and remdesivir, includes separate administration of the two drugs, eg, endostatin before or after remdesivir administration. Drugs, also include the simultaneous administration of two drugs in the same pharmaceutical formulation or in separate pharmaceutical formulations.

The term "endostatin" or "endostatin" (Endostatin, ES for short) should be understood in a broad sense, including natural endostatin, preferably human endostatin, but not limited thereto, for example, it can also be Native endostatins from other mammals such as mice, rats, pigs, dogs, rabbits, sheep, goats, cats, etc. "Endostatin" also includes functional variants of endostatin, such as engineered functional variants, which have one or more amino acid substitutions, deletions, or additions compared to native endostatin, and It has basically the same biological functions, such as the activity of inhibiting vascular endothelial cell proliferation, migration and angiogenesis in vivo. "Endostatin" also includes derivatives or modified products of native endostatin or functional variants thereof, such as polyethylene glycol modified products.

The term "functional variant" as used herein includes one or several (eg 1-5, 1-10 or 1-15, specifically, for example, may be 1, 2, 3, 4) in the amino acid sequence , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or even more) amino acid substitutions, deletions or additions of endostatin mutants, and the mutants have vascular Endostatin has similar biological activities of inhibiting vascular endothelial cell proliferation, migration and angiogenesis in vivo. The biological activity of a "functional variant" of endostatin may be, for example, native endostatin, eg, 30% or more, 50% or more, 60% or more, of native human endostatin. 70% or higher, 80% or higher or 90% or higher. The "functional variant" may be a naturally occurring mutant or an artificial mutant, such as a mutant obtained by site-directed mutagenesis, or a mutant generated by genetic recombination methods.

The biological activity of the "functional variant" can be detected by methods well known in the art for detecting endostatin activity. For example, HMEC cells can be selected, the migration (Tranwell Assay) method can be used to analyze the inhibition rate of functional variants on HMEC cell migration, and the number of cells can reflect the protein activity (refer to Luo yongzhang et al., Endostatin inhibits tumorlymphangiogenesis and lymphatic metastasis via cell surface nucleolin on lymphangiogenic endothelial cells (J Pathol 2010;222:249–260).

Functional variants of endostatin can be obtained by engineering native endostatin (eg, native human endostatin or native mammalian endostatin).

Specific examples of endostatins (including functional variants, derivatives or modified products thereof) that can be used in the present invention can be found in the following patents: Chinese Patent Application Nos. CN201610997233.2, 201510079486.7, 201280078261.4, 200610011247.9.

It is known in the art that when human endostatin is recombinantly expressed in E. coli, its first amino acid M may be randomly deleted. When ES is expressed recombinantly in yeast, ES variants with 4 amino acids missing from the N-terminus may be generated due to random splicing of the N-terminus.

Preferred ESs include YH-16 (also known as Endo), an ES variant obtained by adding 9 additional amino acids (MGGSHHHHH) to the N-terminus of ES for increased soluble expression and ease of purification (Fu Y et al. al. IUBMB Life 2009; 61: 613-626; Wang J et al. Zhongguo fei ai za zhi 2005; 8: 283-290; Han B et al. J Thorac Oncol 2011; 6(6): 1104-1109, via is incorporated herein by reference in its entirety).

Preferred ESs also include those ES variants disclosed in PCT International Application PCT/CN2012/081210, such as ES006, ES008, ES011, S02, S09, Z006, Z008, ZN1, etc. (the entire contents of which are incorporated herein by reference) ).

Preferred ES also include those endostatin variants disclosed in PCT International Publication No. WO2016/070798, such as 003, 007, Z101, 009, S03, 36, 249, 381, 57, 114, 124, 125, 160, 163, 119 (incorporated by reference in their entirety).

Preferred ESs also include M2ES (see eg 200610011247.9). "M2ES" refers to polyethylene glycol recombinant human endostatin. M2ES is a long-acting recombinant endostatin, which has entered Phase II clinical trials for lung cancer indications.

Preferred ESs also include PEG double-modified ESs.

The term "corresponding to the position of native endostatin" as used herein means that the endostatin functional variant is aligned with the amino acid sequence of native endostatin using software or algorithms well known in the art. Positions on the functional variant of inhibin that correspond to corresponding amino acid residues in the amino acid sequence of native endostatin. The software or algorithms include, but are not limited to, BLAST, FASTA.

Unless otherwise indicated, the methods and techniques used in this specification are generally performed according to well-known and conventional methods in the art and in the manner described in the various references set forth in this specification or cited.

references:

[1] Zhu, N.; Zhang, D.; Wang, W.; Li, X.; Yang, B.; Song, J.; Zhao, X.; Huang, B.; Shi, W.; Lu, R.et al.A Novel Coronavirus from Patients with Pneumonia in China,2019.N Engl J Med.2020,382,727.

[2] Adegboye, O.A.; Adekunle, A.I.; Gayawan, E.Early Transmission Dynamics of Novel Coronavirus(COVID-19) in Nigeria.Int J Environ Res Public Health.2020,17.

[3] Zhou, P.; Yang, X.L.; Wang, X.G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.R.; Zhu, Y.; Li, B.; Huang, C.L. et al .A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature.2020,579,270.

[4] Wu, F.; Zhao, S.; Yu, B.; Chen, Y.M.; Wang, W.; Song, Z.G.; Hu, Y.; Tao, Z.W.; Tian, J.H.; Pei, Y.Y. et al. A new coronavirus associated with human respiratory disease in China.Nature.2020,579,265.

[5]COVID-19Coronavirus Pandemic. https://www.worldometers.info/coronavirus/, 2020-6-28.

[6] Runfeng, L.; Yunlong, H.; Jicheng, H.; Weiqi, P.; Qinhai, M.; Yongxia, S.; Chufang, L.; Jin, Z.; Zhenhua, J.; Haiming, J.et al.Lianhuaqingwen exerts anti-viral and anti-inflammatory activity again novel coronavirus(SARS-CoV-2).Pharmacol Res.2020,156,104761.

[7] Tayyari, F.; Marchant, D.; Moraes, T.J.; Duan, W.; Mastrangelo, P.; Hegele, R.G.Identification of nucleolin as a cellular receptor for human respiratory syncytial virus.Nat Med.2011,17, 1132.

[8] Mastrangelo, P.; Hegele, R.G. The RSV fusion receptor: not what everyone expected it to be. Microbes Infect. 2012, 14, 1205.

[9] Mastrangelo, P.; Hegele, R.G. RSV fusion: time for a new model. Viruses. 2013, 5, 873.

[10] Chaudhry, U.; Malik, D.A.; Saleem, N.; Malik, M.T. Nucleolin: Role in Bacterial and Viral Infections. EC Microbiol. 2018, 9, 631.

[11] Shakeri, A.; Mastrangelo, P.; Griffin, J.K.; Moraes, T.J.; Hegele, R.G.

[12] Shi, H.; Huang, Y.; Zhou, H.; Song, X.; Yuan, S.; Fu, Y.; Luo, Y. Nucleolin is a receptor that mediates antiangiogenic and antitumor activity of endostatin. Blood.2007,110,2899.

Claims (37)

1. Use of endostatin in the preparation of a medicament for preventing or treating coronavirus-related diseases in a subject.
2. The use of claim 1, wherein the coronavirus is selected from the group consisting of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2.
3. The use of claim 1, wherein the coronavirus is SARS-CoV-2.
4. The use of any one of claims 1-3, wherein the coronavirus-related disease is selected from the group consisting of SARS, MERS and COVID-19.
5. The use of any one of claims 1-4, wherein the coronavirus-related disease is COVID-19.
6. The use of any one of claims 1-5, wherein the subject is a mammal or a bird.
7. The use of any one of claims 1-6, wherein the subject is a human.
8. The use according to any one of claims 1-7, wherein the dosage form of the medicament is selected from: intravenous injection, oral spray, nasal spray, and eye drops.
9. The use of any one of claims 1-8, wherein the endostatin is human endostatin.
10. The use of any one of claims 1-8, wherein the endostatin is YH-16.
11. The use of any one of claims 1-8, wherein the endostatin is M2ES.
12. The use of any one of claims 1-8, wherein the endostatin is pegylated human endostatin or a functional variant thereof.
13. A method of preventing or treating a coronavirus-related disease in a subject, comprising administering to the subject an effective amount of endostatin.
14. The method of claim 13, wherein the coronavirus is selected from the group consisting of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2.
15. The method of claim 13, wherein the coronavirus is SARS-CoV-2.
16. The method of any one of claims 13-15, wherein the coronavirus-related disease is selected from the group consisting of SARS, MERS, and COVID-19.
17. The method of any one of claims 13-15, wherein the coronavirus-related disease is COVID-19.
18. The method of any one of claims 13-17, wherein the subject is a mammal or a bird.
19. The method of any one of claims 13-17, wherein the subject is a human.
20. The method of any one of claims 13-19, wherein the drug is administered by intravenous injection, oral spray, nasal spray, or eye drops.
21. The method of any one of claims 13-20, wherein the endostatin is human endostatin.
22. The method of any one of claims 13-20, wherein the endostatin is YH-16.
23. The method of any one of claims 13-20, wherein the endostatin is M2ES.
24. The method of any one of claims 13-20, wherein the endostatin is pegylated human endostatin or a functional variant thereof.
25. A pharmaceutical composition for preventing or treating a coronavirus-related disease in a subject, comprising: endostatin; and a pharmaceutically acceptable carrier.
26. The pharmaceutical composition of claim 25, wherein the coronavirus is selected from the group consisting of HCoV-229E,

    HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2.
27. The pharmaceutical composition of claim 25, wherein the coronavirus is SARS-CoV-2.
28. The pharmaceutical composition of any one of claims 25-27, wherein the coronavirus-related disease is selected from the group consisting of SARS, MERS, and COVID-19.
29. The pharmaceutical composition of any one of claims 25-27, wherein the coronavirus-related disease is COVID-19.
30. The pharmaceutical composition of any one of claims 25-29, wherein the subject is a mammal or a bird.
31. The pharmaceutical composition of any one of claims 25-29, wherein the subject is a human.
32. The pharmaceutical composition according to any one of claims 25-31, wherein the dosage form of the pharmaceutical composition is selected from: intravenous injection, oral spray, nasal spray, and eye drops.
33. The pharmaceutical composition of any one of claims 25-32, wherein the endostatin is human endostatin.
34. The pharmaceutical composition of any one of claims 25-32, wherein the endostatin is YH-16.
35. The pharmaceutical composition of any one of claims 25-32, wherein the endostatin is

    M2ES.
36. The pharmaceutical composition of any one of claims 25-32, wherein the endostatin is pegylated human endostatin or a functional variant thereof.
37. A kit for preventing or treating a coronavirus-related disease in a subject, comprising the pharmaceutical composition of any one of claims 25-36.