Pharmaceutical composition containing Nafamostat and K777 and application thereof

Abstract

The invention provides a pharmaceutical composition containing Nafamostat and K777, and an application of the pharmaceutical composition containing Nafamostat and K777. The pharmaceutical composition comprises the following active components: Nafamostat and K777, wherein the active components are nafamostat and K777. The pharmaceutical composition disclosed by the invention can show a remarkable synergistic effect in related research of anti-coronavirus, and shows improvement of efficacy by more than 10 times in an anti-new coronavirus test.

Description

A drug combination comprising Nafamostat and K777 and its application

[0001] This application claims priority to Chinese patent application No. PCT / CN2022 / 125676, filed on October 17, 2022. This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to the field of biopharmaceutical technology, and specifically to a drug combination comprising Nafamostat and K777 and its use in preparing a drug for inhibiting the novel coronavirus or treating / preventing diseases caused by the novel coronavirus. Background Art

[0003] Coronavirus (CoV) is a type of single-stranded positive-sense RNA enveloped virus that can infect a variety of organisms including humans, mammals, birds, and rodents. It can also cause cross-species transmission between animals and humans, causing a variety of chronic or acute infectious diseases.

[0004] The entire coronavirus life cycle is divided into multiple steps, including viral invasion of cells, viral transcription and replication, and the assembly and release of virions. Proteins that play key roles in these vital processes, such as the viral spike protein (Spike), main protease (Mpro), papain (PLpro), host angiotensin-converting enzyme 2 (ACE2), serine protease TMPRSS2, and cathepsin L, are ideal targets for developing anti-COVID-19 therapeutics. Spike, ACE2, TMPRSS2, and Cathepsin L all play a crucial role in viral invasion. For example, SARS-CoV-2 requires recognition and binding of its spike protein to angiotensin-converting enzyme 2 (ACE2) on the host cell surface. After binding to the receptor, the Spike needs to be cleaved by the host's protease to complete the conformational transition, thereby releasing its own fusion peptide to mediate the fusion of the viral envelope and cell membrane (Glowacka, Ilona et al. Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response. Journal of virology, (2011)).

[0005] Main protease (Mpro) is a cysteine ​​protease that is essential for viral replication and assembly. It contains 11 cleavage sites and plays a crucial role in the replication and transcription of the coronavirus life cycle. Papain (PLpro) is an important regulatory protein molecule in the formation of the replicase complex (RC) and is also crucial for the transcription and replication of the viral genome.

[0006] Angiotensin-converting enzyme 2 (ACE2) is a zinc metalloproteinase present in normal lung tissue, such as type I and type II alveolar epithelial cells. Coronaviruses invade the lungs by binding to ACE2 via the S protein. Following coronavirus infection, ACE2 levels decrease, ultimately leading to increased capillary permeability in the lungs, resulting in pulmonary edema, acute severe lung injury, and acute lung failure. This suggests that ACE2 not only facilitates viral invasion but also worsens the condition of infected individuals.

[0007] TMPRSS2 is a type II transmembrane serine protease located on the cell membrane surface. It is regulated by androgens and its main recognition cleavage sequence is a single arginine or lysine (AFAR DE, VIVANCO I, HUBERT RS, et al. Catalytic cleavage of the androgen-regulated TMPRSS2 protease results in its secretion by prostate and prostate cancer epithelia. Cancer Res, (2001)). TMPRSS2 not only plays an important role in the invasion of coronaviruses, but is also highly correlated with a variety of diseases, such as influenza virus infection and prostate cancer (WANG Z, WANG Y, ZHANG J, et al. Significance of the TMPRSS2: ERG gene fusion in prostate cancer. Mol Med Rep, (2017); ZMORA P, MOLAU-BLAZEJEWSKA P, BERTRAM S, et al. Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin. PLoS One, (2017)). Therefore, the development of drugs based on TMPRSS2 has a certain research basis. At the same time, because it is located on the cell membrane surface, small drug molecules do not need to enter the cell, which makes drug development more advantageous. Cathepsin L belongs to the cysteine ​​protease in the papain family. Its substrate binding site and catalytic site have a key amino acid, namely cysteine, which plays a key role in functioning.

[0008] Currently, there are no drugs or vaccines targeting SARS-CoV and MERS-CoV, and treatment options are scarce. In contrast, the successful development of vaccines and antibodies against the novel coronavirus provides an effective means of preventing and treating the novel coronavirus. However, the continuous emergence of SARS-CoV-2 variants poses a more severe challenge to the effectiveness of existing vaccines and antibodies (E. Cameroni et al. Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift. Nature, (2021); L. Liu et al. Striking antibody evasion manifested by the Omicron variant of SARS-CoV-2. Nature, (2021); Y. Cao et al. Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature, (2021); S. Cele et al. Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization. Nature, (2021)). Similarly, although a series of small molecule drugs, such as Pfizer's Paxlovid, have been approved for marketing, the highly variable nature of the novel coronavirus as an RNA virus means that antiviral drugs developed targeting the virus itself face the risk of drug resistance during subsequent use. Therefore, the development of new antiviral therapies is urgent.

[0009] Summary of the Invention

[0010] In view of the lack of host-targeted antiviral therapy for the new coronavirus and the lack of effective broad-spectrum drug treatment options in the existing technology, the present application provides a drug combination including Nafamostat and K777 and its application.

[0011] Nafamostat (structural formula see Formula 1) is an oral drug with broad-spectrum serine protease inhibitory activity. It has been approved for marketing in Japan. Its main indication is acute pancreatitis. It also plays an anticoagulant role during hemodialysis, coronary artery bypass grafting, and liver resection.

[0012] K777 (structural formula shown in Formula 2) is an orally available, irreversible cysteine ​​protease inhibitor. K777 antagonizes infections by inhibiting cathepsins B and L, including SARS-CoV and Ebola. K777 is also an antagonist of the chemokine receptor CCR4.

[0013] Studies have shown that mice with Cathepsin L and TMPRSS2 knockout genes, respectively, exhibit no significant phenotypic changes compared to wild-type mice, effectively validating the safety of these host-derived targets (Kim, TS et al. Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease. Molecular and Cellular Biology, (2006)). Therefore, the technical solution of this application presents no safety risks.

[0014] Since SARS-CoV-2 and its variants can simultaneously complete the cleavage of the Spike through the proteases Cathepsin L and TMPRSS2 during the process of invading host cells, and the degree of dependence of these two entry methods is variable and will be affected by multiple factors such as host cell type and viral variants, it is difficult to effectively eliminate viral infection by inhibiting TMPRSS2 or Cathepsin L alone.

[0015] The present invention found that the combination of Nafamostat and K777 significantly improved the efficacy of the drug compared with the use of a single drug through in vitro enzyme activity experiments and in vitro cell virus experiments. In the in vitro cell experiment against the Delta strain of the new coronavirus, the EC 50 The increase in value can be used to treat related diseases caused by the new coronavirus.

[0016] The first aspect of the present invention provides a pharmaceutical combination comprising active ingredients Nafamostat and K777.

[0017] In some embodiments, the molar ratio of Nafamostat to K777 is 1:0.5 to 1:2.

[0018] In some preferred embodiments, the molar ratio of Nafamostat to K777 is 1:1.

[0019] In some preferred embodiments, the pharmaceutical combination further comprises a pharmaceutically acceptable carrier or excipient.

[0020] In some embodiments, the dosage form of the pharmaceutical composition is selected from one of granules, tablets, pills, capsules, and injections.

[0021] The second aspect of the present invention provides a use of the drug combination described in the first aspect of the present invention in the preparation of a drug for inhibiting coronavirus.

[0022] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, HCoV-229E.

[0023] In some preferred embodiments, the coronavirus is SARS-CoV-2.

[0024] The third aspect of the present invention provides a use of the drug combination described in the first aspect of the present invention in the preparation of a drug for treating / preventing diseases caused by coronavirus.

[0025] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, HCoV-229E.

[0026] In some preferred embodiments, the coronavirus is SARS-CoV-2.

[0027] The fourth aspect of the present invention provides a method for treating / preventing diseases caused by coronavirus, wherein the method comprises administering the drug combination as described in the first aspect of the present invention, or a drug containing the drug combination, to a subject in need.

[0028] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, HCoV-229E.

[0029] In some preferred embodiments, the coronavirus is SARS-CoV-2.

[0030] A fifth aspect of the present invention provides a medicine kit, wherein the medicine kit comprises a medicine kit A and a medicine kit B, the main active ingredient of the medicine kit A is Nafamostat, and the main active ingredient of the medicine kit B is K777.

[0031] The sixth aspect of the present invention provides a drug combination as described in the first aspect of the present invention, which is used to inhibit coronavirus.

[0032] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, HCoV-229E.

[0033] In some preferred embodiments, the coronavirus is SARS-CoV-2.

[0034] The seventh aspect of the present invention provides a drug combination as described in the first aspect of the present invention, which is used to treat / prevent diseases caused by coronavirus.

[0035] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, HCoV-229E.

[0036] In some preferred embodiments, the coronavirus is SARS-CoV-2.

[0037] To provide an effective and broad-spectrum treatment for diseases caused by various coronavirus infections, the present invention employs a combined strategy of TMPRSS2 and Cathepsin L inhibitors, demonstrating a synergistic effect that significantly improves efficacy compared to single-drug use. This synergistic effect completely blocks the viral invasion pathway, providing an effective and broad-spectrum drug treatment for SARS-CoV-2 and its variants, and also offering an effective and broad-spectrum drug use strategy for treating major infectious diseases caused by the novel coronavirus.

[0038] The advantages of the present invention are:

[0039] (1) The combination of Nafamostat and K777 has shown significant synergistic effects in anti-coronavirus studies, and has demonstrated a more than 10-fold increase in efficacy in anti-COVID-19 trials.

[0040] (2) Nafamostat and K777 have reliable drug safety, and clinical trials of the dual-drug combination can be rapidly advanced, providing effective and broad-spectrum treatment options for combating the COVID-19 pandemic and its emerging variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1A is a schematic diagram showing the inhibition of the host protease TMPRSS2 by the small molecule Nafamostat; FIG1B is a schematic diagram showing the inhibition of the host protease Cathepsin L by the small molecule K777.

[0042] FIG2A is a schematic diagram showing that the small molecule Nafamostat has no inhibitory activity against Cathepsin L; FIG2B is a schematic diagram showing that the small molecule K777 has no inhibitory activity against TMPRSS2.

[0043] Figure 3 is a schematic diagram of an in vitro cell virus experiment in which Nafamostat and K777 inhibit the infection of SARS-CoV-2 Delta into cells. DETAILED DESCRIPTION

[0044] Example 1 In vitro enzyme activity inhibition experiment

[0045] A 384-well plate (PerkinElmer, Catalog No. 6007270) was filled with 40 μL of TMPRSS2 (Gill Biochemical, Catalog No. 117075) protein solution per well, distributed across 4 rows x 20 columns. 5 μL of 14 concentration gradients of nafamostat (initial concentration: 400 nM, 2.5-fold serial dilutions; Selleck, Catalog No. S1386) were added to the first 14 columns of protein solution, starting at 400 nM, as the experimental control. An equal volume of DMSO was added to columns 15-20, serving as the positive control, and an equal volume of buffer was added to columns 18-20, serving as the negative control. Finally, 5 μL of substrate (Boc-Gln-Ala-Arg-AMC (Gill Biochemical, Catalog No. 117075)) was added to each well. The 384-well plate was placed in a microplate reader for assay. The slope of the fluorescence intensity increase during the first 2 minutes of the enzymatic reaction was used as the protein activity for subsequent calculations.

[0046] The substrate was replaced with Cathepsin L-specific catalytic substrate, namely Cbz-Phe-Arg-AMC (brand: Jier Biochemical, product number: 974233), and the drug was replaced with K777 (brand: MCE, product number: HY-119293), and the same operation as above was performed.

[0047] The above-mentioned in vitro biochemical enzyme activity inhibition experiment proved that Nafamostat has a very high inhibitory effect on the serine protease TMPRSS2. 50 K777 showed significant inhibitory activity against cathepsin L, with IC 50 reached 4.55 nM ( FIG1B ).

[0048] Example 2 In vitro enzyme activity inhibition experiment cross validation

[0049] The operation is basically the same as in Example 1, except that the drug and substrate are cross-exchanged.

[0050] Through cross-validation of in vitro enzyme inhibition experiments, it was found that Nafamostat had no inhibitory activity against Cathepsin L and K777 had no inhibitory activity against TMPRSS2 (Figure 2A and Figure 2B), thus confirming that both drugs are specific for their targets.

[0051] Example 3 In vitro cell virus experiment

[0052] The half-maximal effective concentration (EC) of the test substances Nafamostat and K777 was determined by cytopathic effect assay. 50 ).

[0053] (1) The day before, Calu-3 cells (brand: ATCC, catalog number: HTB-55) were cultured at a rate of 2×10 5 / well inoculated into 24-well plates (brand: Thermo Fisher, catalog number: 142475);

[0054] (2) Nafamostat was diluted 5-fold at a maximum concentration of 20 μM, K777 was diluted 5-fold at a maximum concentration of 40 μM, and Nafamostat combined with K777 was diluted 5-fold at a maximum concentration of 20 μM, for a total of 6 or 8 dilutions. Three replicates were set for each dilution. Cells were pretreated for 1 h, and an equal volume of DMSO was added to the control group.

[0055] (3) Enter the BSL-3 laboratory and add 500 μL of SARS-CoV-2 Delta virus suspension (Accession No.: Delta-IM2175251-P3-YQ, Guangzhou Customs Technology Center) with a multiplicity of infection (MOI) of 1 to each well and incubate at 37°C for 1 hour;

[0056] (4) Aspirate and discard the cell culture supernatant, add 500 μL PBS (brand: Gibco, product number: 10099) to each well to rinse the cells three times, then add the corresponding concentration of drug and place in a 37°C, 5% CO2 constant temperature and humidity incubator for 24 hours;

[0057] (5) After 24 hours of culture, the cell growth was observed and recorded under a microscope. The cell supernatant was collected and the titer was determined using Vero E6 cells (brand: ATCC, catalog number: CRL-1586);

[0058] (6) After 72 hours, the cytopathic rate was read using a full-field cell analyzer (Celigo Image Cytometer, Celigo), and the viral titer was calculated using the Reed-Muench method;

[0059] (7) Calculation of EC 50 : 100×(1-mean titer of drug group / mean titer of DMSO group).

[0060] Through in vitro cell virus experiments, it was found that both Nafamostat and K777 could significantly inhibit the infection of SARS-CoV-2 Delta to cells. 50 When the two drugs were used in combination at a 1:1 (molar ratio), it was found that their antiviral ability was significantly improved compared with the use of a single drug. 50 reached 0.002 μM ( FIG3 ).

[0061] The present invention confirms that the combined use of Nafamostat and K777, inhibitors of host proteases TMPRSS2 and Cathepsin L, can show an efficacy improvement of more than 10 times in anti-new coronavirus tests, reflecting a significant synergistic effect between the two.

[0062] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A drug combination, characterized in that It contains the active ingredients Nafamostat and K777.

2. The pharmaceutical combination according to claim 1, wherein The molar ratio of Nafamostat to K777 is 1:0.5 to 1:

2.

3. The pharmaceutical combination according to claim 2, wherein The molar ratio is preferably 1:

1.

4. The pharmaceutical combination according to claim 2, wherein The pharmaceutical combination further includes a pharmaceutically acceptable carrier or excipient.

5. The pharmaceutical combination according to claim 1, wherein The dosage form of the pharmaceutical composition is selected from granules, tablets, pills, capsules and injections.

6. Use of the drug combination according to any one of claims 1 to 5 in the preparation of a drug for inhibiting coronavirus.

7. The use according to claim 6, characterized in that The coronaviruses are SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, and HCoV-229E.

8. The use according to claim 7, characterized in that The coronavirus is SARS-CoV-2.

9. Use of the drug combination according to any one of claims 1 to 5 in the preparation of a drug for treating / preventing diseases caused by coronavirus.

10. The use according to claim 9, characterized in that The coronaviruses are SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, and HCoV-229E.

11. The use according to claim 10, characterized in that The coronavirus is SARS-CoV-2.

12. A method for treating / preventing a disease caused by coronavirus, characterized in that: The method comprises administering the pharmaceutical combination according to any one of claims 1 to 5, or a medicament comprising the pharmaceutical combination, to a subject in need thereof.

13. The method according to claim 12, wherein: The coronaviruses are SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, and HCoV-229E.

14. The method according to claim 13, wherein The coronavirus is SARS-CoV-2.

15. A medicine kit, characterized in that: The medicine kit comprises medicine kit A and medicine kit B. The main active ingredient of medicine kit A is Nafamostat, and the main active ingredient of medicine kit B is K777.

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