Pharmaceutical combination containing nafamostat and K777 and use thereof

Abstract

The present invention provides a pharmaceutical combination comprising nafamostat and K777, and uses thereof. The pharmaceutical combination comprises the active ingredients nafamostat and K777. The pharmaceutical combination of the present invention has demonstrated significant synergistic effects in anti-coronavirus studies, demonstrating a 10-fold or greater improvement in efficacy in anti-coronavirus tests.

Description

Detailed Description of the Invention

[0001] The present invention claims priority from Chinese patent application PCT / CN2022 / 125676, filed on October 17, 2022. This application cites the full text of the above Chinese patent application.

[0002] [Technical field] The present invention relates to the field of biomedical technology, and in particular to a pharmaceutical combination comprising nafamostat and K777, and its use in the preparation of a medicament for inhibiting or treating / preventing a novel coronavirus disease (COVID-19)-caused disease.

[0003] [Background technology] Coronaviruses (CoVs) are a type of single-stranded, positive-sense RNA enveloped virus that can infect a variety of organisms, including humans, mammals, birds, and rodents, cause interspecies transmission between animals and humans, and can cause a number of chronic or acute infections.

[0004] The entire coronavirus life cycle is divided into several steps, including viral cell invasion, viral transcription and replication, and viral particle assembly and release. Proteins that play key roles in these vital vital processes, such as the viral spike protein (Spike), main protease (Mpro), papain-like protease (PLpro), and host angiotensin-converting enzyme 2 (ACE2), serine protease TMPRSS2, and cathepsin L, are ideal targets for the development of anti-coronavirus therapeutics. Among these, Spike, ACE2, TMPRSS2, and cathepsin L all play important roles in the process of viral cell invasion. For example, SARS-CoV-2 must first recognize and bind to angiotensin-converting enzyme 2 (ACE2) on the surface of host cells to invade the host. After binding to the receptor, the spike protein must be cleaved by host proteases to complete the conformational change, thereby releasing its own fusion peptide and mediating fusion of the viral envelope with the 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] The main protease (Mpro) is a cysteine ​​protease essential for viral replication and assembly, containing 11 enzyme cleavage sites and playing a key role in the replication and transcription processes of the coronavirus life cycle. The papain-like protease (PLpro) is a key regulatory protein molecule in the formation of the replicase complex (RC) and is also essential for the transcription and replication of the viral genome.

[0006] Angiotensin-converting enzyme 2 (ACE2) is a zinc metalloprotease present in normal lung tissues, such as type I and type II alveolar epithelial cells. Coronaviruses enter the lungs by binding to ACE2 via the S protein. After coronavirus infection, ACE2 levels are downregulated, ultimately leading to increased pulmonary capillary permeability, resulting in pulmonary edema, acute severe lung injury, and acute lung failure. This suggests that ACE2 not only aids virus cell entry but may also worsen the condition of infected individuals.

[0007] TMPRSS2 is a type II transmembrane serine protease located on the cell membrane surface that is regulated by androgen, and its primary 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 coronavirus invasion, but is also closely linked to various diseases, including 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 influenza virus hemagglutinin. PLoS One, (2017)). Therefore, there is a solid research foundation for the development of TMPRSS2-based drugs. Furthermore, its location on the surface of the cell membrane offers an additional advantage in drug development, as it eliminates the need for small molecule drugs to enter the cell. Cathepsin L belongs to the papain-like protease family of cysteine ​​proteases. Its substrate-binding and catalytic domains contain cysteines, a key amino acid that plays a crucial role in its function.

[0008] Currently, there are no therapeutic drugs or vaccines for SARS-CoV and MERS-CoV, and treatments are scarce. In contrast, the successful development of vaccines and antibodies against SARS-CoV-2 provides effective means of preventing and treating the virus. However, the continued emergence of SARS-CoV-2 mutant strains poses even more serious challenges to the preventive and therapeutic efficacy 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, a series of small molecule drugs, such as Pfizer's Paquilobid, have been approved for sale, but because the novel coronavirus itself is an RNA virus and highly mutable, antiviral drugs developed to target the virus itself will face the risk of drug resistance in subsequent use. Therefore, the development of novel antiviral therapies is urgently needed.

[0009] [Summary of the Invention] In response to the lack of host-targeted antiviral therapies for novel coronavirus in the prior art and the lack of effective broad-spectrum drug treatment options, the present application provides a drug combination comprising nafamostat and K777 and uses thereof.

[0010] Nafamostat (see Formula 1 for the structural formula) is an oral drug with broad-spectrum serine protease inhibitory activity. It has been approved for sale in Japan and its primary indication is acute pancreatitis. It also functions as an anticoagulant during hemodialysis, coronary artery bypass grafting, and liver resection surgery.

[0011] [ka]

[0012] K777 (structural formula: see Formula 2) is an oral, irreversible cysteine ​​protease inhibitor. K777 antagonizes various viral infections, including SARS-CoV and Ebola hemorrhagic fever, by inhibiting cathepsins B and L. K777 is also an antagonist of the chemokine receptor CCR4.

[0013] [ka]

[0014] Research has shown that no significant phenotypic changes were observed in mice with cathepsin L and TMPRSS2 genes knocked out and wild-type mice, effectively verifying the safety of the above-mentioned 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 does not pose any safety risks.

[0015] During the process of invading host cells, SARS-CoV-2 and its mutant strains can simultaneously complete spike protein cleavage via the proteases cathepsin L and TMPRSS2. The degree of dependence on these two entry methods varies and is affected by multiple factors, such as the type of host cell and the viral mutant strain, making it difficult to effectively eliminate viral infection by inhibiting TMPRSS2 or cathepsin L alone.

[0016] Through in vitro enzyme activity experiments and in vitro cell virus experiments, the present inventors have found that the combination of nafamostat and K777 significantly improves efficacy compared to the use of a single drug. 50 The value increased by more than 10 times, making it possible to treat related diseases caused by the new coronavirus.

[0017] A first aspect of the present invention provides a pharmaceutical combination comprising the active ingredients nafamostat and K777. 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. In some preferred embodiments, the pharmaceutical combination further comprises pharmaceutically acceptable carriers and excipients.

[0019] In some embodiments, the dosage form of the pharmaceutical combination is selected from one of granules, tablets, pills, capsules, and injections. A second aspect of the present invention provides the use of a pharmaceutical combination according to the first aspect of the present invention in the preparation of a medicament for inhibiting coronavirus.

[0020] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, or HCoV-229E.

[0021] In some preferred embodiments, the coronavirus is SARS-CoV-2. A third aspect of the present invention provides the use of a pharmaceutical combination according to the first aspect of the present invention in the preparation of a medicament for treating / preventing a disease caused by a coronavirus.

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

[0023] In some preferred embodiments, the coronavirus is SARS-CoV-2. A fourth aspect of the present invention provides a method for treating / preventing a disease caused by coronavirus, comprising administering to a subject in need thereof a pharmaceutical combination according to the first aspect of the present invention, or a medicament containing said pharmaceutical combination.

[0024] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, or HCoV-229E.

[0025] In some preferred embodiments, the coronavirus is SARS-CoV-2. A fifth aspect of the present invention provides a medicine box set comprising medicine box A and medicine box B, wherein the main active ingredient of medicine box A is nafamostat and the main active ingredient of medicine box B is K777.

[0026] A sixth aspect of the present invention provides a pharmaceutical combination according to the first aspect of the present invention for use in inhibiting coronavirus. In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, or HCoV-229E.

[0027] In some preferred embodiments, the coronavirus is SARS-CoV-2. A seventh aspect of the present invention provides a pharmaceutical combination according to the first aspect of the present invention for use in treating / preventing a disease caused by a coronavirus.

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

[0029] In some preferred embodiments, the coronavirus is SARS-CoV-2. To provide an effective and broad-spectrum treatment protocol for diseases caused by various coronavirus infections, the present invention employs a combination strategy of TMPRSS2 and cathepsin L inhibitors, demonstrating synergistic effects and significantly improving therapeutic efficacy compared to administration of a single drug. The synergistic effect of the two drugs completely blocks the viral entry pathway, providing an effective and broad-spectrum drug treatment protocol for SARS-CoV-2 and its mutant strains, as well as an effective and broad-spectrum drug strategy for the treatment of serious infections caused by novel coronaviruses.

[0030] Advantages of this invention: (1) The combination of nafamostat and K777 has shown significant synergistic effects in anti-coronavirus related studies, demonstrating a more than 10-fold improvement in efficacy in anti-coronavirus tests.

[0031] (2) Nafamostat and K777 have high drug safety, allowing clinical trials of the two drugs in combination to proceed quickly, providing an effective and widespread treatment to combat the novel coronavirus pandemic and its new variants. [Brief explanation of the drawings]

[0032] [Figure 1A] FIG. 1 is a schematic diagram of the inhibitory results of the small molecule nafamostat against the host protease TMPRSS2. [Figure 1B]FIG. 1 is a schematic diagram of the inhibitory results of the small molecule K777 on the host protease cathepsin L. [Figure 2A] FIG. 1 is a schematic diagram showing that the small molecule nafamostat has no inhibitory activity against cathepsin L. [Figure 2B] FIG. 1 is a schematic diagram showing that the small molecule K777 has no inhibitory activity against TMPRSS2. [Figure 3] Schematic diagram of in vitro cell-virus experiments in which nafamostat and K777 inhibit infection of cells by SARS-CoV-2 Delta. DETAILED DESCRIPTION OF THE INVENTION

[0033] Example 1 In vitro enzyme activity inhibition experiment TMPRSS2 (GLS, 117075) protein solution was dispensed into a 384-well plate (PerkinElmer, Catalog Number: 6007270) in 4 rows and 20 columns, at 40 µL per well. 5 µL of 14 gradient Nafamostat (starting concentration: 400 nM, 2.5-fold gradient dilution, Selleck, Catalog Number: S1386) was added to the protein solution in column 14. The starting concentration served as the experimental group. Columns 15-20 were filled with the same volume of DMSO as the positive control group. Columns 18-20 were filled with the same volume of buffer as the negative control group. Finally, 5 μL of substrate, i.e., Boc-Gln-Ala-Arg-AMC (Brand: GLS, Catalog No.: 117075), was added to each well, and the 384-well plate was placed in a microplate reader for detection. The slope of the increase in fluorescence intensity during the first 2 minutes of the enzyme reaction was used for subsequent calculations as the protein activity.

[0034] The same procedure as above was performed, replacing the substrate with a cathepsin L-specific catalytic substrate, i.e., Cbz-Phe-Arg-AMC (Brand: GLS, Catalog No.: 974233), and the drug with K777 (Brand: MCE, Catalog No.: HY-119293).

[0035] The above in vitro biochemical enzyme activity inhibition experiments demonstrated that nafamostat has an extremely high inhibitory effect on serine protease TMPRSS2, and IC 50 The IC value reached 0.62 nM (Fig. 1A), indicating that K777 had significant inhibitory activity against cathepsin L. 50 was shown to reach 4.55 nM (Figure 1B).

[0036] Example 2 Cross-validation of in vitro enzyme activity inhibition experiments The procedure was basically the same as in Example 1, except that the drug and substrate were interchanged.

[0037] Cross-validation of in vitro enzyme activity inhibition experiments revealed that nafamostat did not exhibit inhibitory activity against cathepsin L, and K777 did not exhibit inhibitory activity against TMPRSS2 (Figures 2A and 2B), confirming the specificity of both drugs for their respective targets.

[0038] Example 3 In vitro cell virus experiments Using the cytopathic effect method, the half-maximal effective concentrations (EC) of the test substances nafamostat and K777 were measured. 50 ) was measured.

[0039] (1) On the previous day, Calu-3 cells (ATCC, Catalog No. HTB-55) were plated at 2 × 10 in a 24-well plate (Thermo Fisher, Catalog No. 142475). 5 / well.

[0040] (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 the combination of Nafamostat and K777 was diluted 5-fold at a maximum concentration of 20 μM. A total of 6 or 8 dilutions were performed, with three replicate wells for each dilution. The cells were pretreated for 1 hour, and the same volume of DMSO was added to the control group.

[0041] (3) In the BSL-3 laboratory, a SARS-CoV-2 Delta virus suspension (accession number: Delta-IM2175251-P3-YQ-500 μL, Guangzhou Customs Technical Center) at a multiplicity of infection (MOI) of 1 was added to each well and co-cultured at 37 °C for 1 hour.

[0042] (4) The cell culture supernatant was aspirated and discarded, and 500 μL of PBS (Brand: Gibco, Catalog No.: 10099) was added to each well to rinse the cells three times. Then, the corresponding concentrations of drugs were added and the cells were cultured in a 37°C, 5% CO2, temperature- and humidity-controlled incubator for 24 hours.

[0043] (5) After 24 hours of culture, cell growth was observed and recorded under a microscope, and the cell supernatant was collected and titered on Vero E6 cells (Brand: ATCC, Catalog No.: CRL-1586).

[0044] (6) After 72 hours, the cytopathic rate was read using a high-speed cell imaging system (Celigo Image Cytometer, Celigo), and the viral titer was calculated using the Reed-Muench method.

[0045] (7)EC 50 Calculation: 100 x (1 - mean titer of drug group / mean titer of DMSO group). In vitro cell virus experiments showed that both nafamostat and K777 significantly inhibited SARS-CoV-2 Delta infection in EC 50 The EC values ​​were 0.02 μM and 0.66 μM, respectively. When the two drugs were used in a 1:1 molar ratio, the antiviral effect was significantly improved compared to when a single drug was used, and the EC values ​​were 0.02 μM and 0.66 μM. 50 It was found that the concentration reached 0.002 μM (Figure 3).

[0046] The present invention demonstrated that the combination of nafamostat, an inhibitor of host protease TMPRSS2 and cathepsin L, and K777 showed a more than 10-fold improvement in efficacy in anti-coronavirus tests, demonstrating significant synergistic effects between the two.

[0047] Although specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are merely examples and that various changes or modifications can 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 limited by the appended claims.

Claims

1. A pharmaceutical combination comprising 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, characterized in that the molar ratio is preferably 1:

1.

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

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

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

7. The pharmaceutical combination according to claim 6, wherein the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, or HCoV-229E.

8. The pharmaceutical combination according to claim 7, wherein the coronavirus is SARS-CoV-2.

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

10. The pharmaceutical combination according to claim 9, wherein the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, or HCoV-229E.

11. The pharmaceutical combination according to claim 10, wherein the coronavirus is SARS-CoV-2.

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

13. The pharmaceutical combination according to claim 12, wherein the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, or HCoV-229E.

14. The pharmaceutical combination according to claim 13, wherein the coronavirus is SARS-CoV-2.

15. A medicine box set comprising medicine box A and medicine box B, wherein the main active ingredient of medicine box A is nafamostat and the main active ingredient of medicine box B is K777.

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