Use of latanoprost nitrate in the preparation of a drug for treating diseases related to coronavirus disease 2019

Abstract

The application discloses application of latanoprost nitrate in preparation of a therapeutic drug for a disease related to new coronavirus infection. According to the research of the application, the latanoprost nitrate has a strong effect of inhibiting RNA-dependent RNA polymerase of the new coronavirus, has significant antiviral activity on the new coronavirus, and the latanoprost nitrate is an existing clinical drug, and therefore has important application value in development of an anti-new coronavirus infection drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine. More particularly, it relates to the application of latanoprostene bunod in the preparation of a drug for treating diseases related to coronavirus disease 2019 (COVID-19). BACKGROUND

[0002] Latanoprostene bunod (CAS: 860005-21-6, also known as Latanoprostene bunod) is a nitric oxide (NO) donor prostaglandin F2a analogue, which is clinically used for local ophthalmic research to reduce intraocular pressure in patients with open-angle glaucoma or high intraocular pressure. It has completed phase IV clinical trials.

[0003] The structure of latanoprostene bunod is as follows:

[0004] In addition, COVID-19 is an acute respiratory disease caused by SARS-CoV-2, which is mainly characterized by respiratory distress, fever, cough, fatigue, and muscle pain. Severe cases can develop into acute respiratory distress syndrome, septic shock, and multiple organ failure. The evolution of SARS-CoV-2 virus is very fast, with a replacement every 10-12 days, so it is increasingly important to make drugs effective against new variants of SARS-CoV-2. Current studies have shown that SARS-CoV-2 has many mutations in the spike protein, while the amino acid sequence of nsp12 (with RdRp activity) remains highly conserved in early and new variants, so nsp12 is an excellent target for SARS-CoV-2 drugs.

[0005] According to data from the World Health Organization (WHO), as of early 2023, the cumulative number of confirmed COVID-19 cases worldwide reached 753 million, and the cumulative number of deaths reached 6.8 million. In the face of the continuous variation and long-term trend of SARS-CoV-2 virus, we urgently need to develop a new type of highly effective and low-toxicity anti-COVID-19 drug. SUMMARY

[0006] The present application aims to overcome the shortcomings of existing anti-COVID-19 drugs and provide the application of latanoprostene bunod in the preparation of a drug for inhibiting COVID-19.

[0007] The first object of the present application is to provide the application of latanoprostene bunod in the preparation of a drug for inhibiting COVID-19.

[0008] The second object of the present application is to provide the application of latanoprostene bunod in the preparation of a drug for treating, ameliorating, or preventing diseases related to COVID-19 infection.

[0009] The third object of the present application is to provide an application of latanoprost nitrate in preparing a drug for treating, improving or preventing a symptom caused by a novel coronavirus infection.

[0010] The fourth object of the present application is to provide an application of latanoprost nitrate in preparing a novel coronavirus RNA-dependent RNA polymerase inhibitor.

[0011] The fifth object of the present application is to provide an anti-novel coronavirus drug.

[0012] The sixth object of the present application is to provide a RNA-dependent RNA polymerase inhibitor.

[0013] The above objects of the present application are achieved by the following technical solutions.

[0014] The present application provides an application of latanoprost nitrate in preparing an inhibitor of a novel coronavirus.

[0015] The present application provides an application of latanoprost nitrate in preparing a drug for treating, improving or preventing a disease caused by a novel coronavirus infection.

[0016] The present application provides an application of latanoprost nitrate in preparing a drug for treating, improving or preventing a symptom caused by a novel coronavirus infection.

[0017] Optionally, the drug in the above application comprises a pharmaceutically acceptable salt or a carrier.

[0018] Optionally, the carrier can be a dendrimer, a liposome, a micelle, a microsphere or a microcapsule, etc.

[0019] The present application provides an application of latanoprost nitrate in preparing a novel coronavirus RNA-dependent RNA polymerase inhibitor.

[0020] Specifically, the RNA-dependent RNA polymerase comprises one or more of nsp12 protein, nsp7 protein or nsp8 protein.

[0021] The present application provides an anti-novel coronavirus drug, which comprises latanoprost nitrate and a pharmaceutically acceptable salt or a carrier.

[0022] The present application provides a RNA-dependent RNA polymerase inhibitor, wherein the inhibitor comprises latanoprost nitrate and a pharmaceutically acceptable salt or a carrier.

[0023] Optionally, the carrier can be a dendrimer, a liposome, a micelle, a microsphere or a microcapsule, etc.

[0024] In addition, the above-mentioned drugs can also be used in combination with other antiviral drugs (especially against the new coronavirus), such as Remdesivir and the like.

[0025] The present application has the following beneficial effects:

[0026] The present application provides a new application of latanoprost nitrate in inhibiting the new coronavirus. Latanoprost nitrate is originally a nitric oxide (NO) donor prostaglandin F2a analogue, which is clinically used for local ophthalmic research to reduce intraocular pressure in patients with open-angle glaucoma or high eye pressure. The present application provides a new choice and vitality for the old drug with new use.

[0027] The present application proves that the therapeutic window of latanoprost nitrate is large by comparing the CC 50 (>40μM) with the half maximal effective dose EC 50 (1.7μM) on the hACE2-293T cell model level to inhibit the new coronavirus (Omicron BA.2), which proves that latanoprost nitrate has a large therapeutic window and strong inhibitory activity on the RNA-dependent RNA polymerase of the new coronavirus, and has a good application prospect as a new anti-new coronavirus infection drug. DETAILED DESCRIPTION

[0028] The present application will be further described below in combination with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0029] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] The following virus experiments were completed in the Biosafety Level 3 (BSL-3) laboratory of the Key Laboratory of Tropical Disease Prevention and Treatment Research, Ministry of Education, Sun Yat-sen University.

[0031] Test virus strain: new coronavirus strain SARS-CoV-2 (BA.2GDPCC 2.00299) from the Guangdong Center for Disease Prevention and Control.

[0032] Cell line: hACE2-293T (high expression of human angiotensin converting enzyme 2 293T cell line), self-built, which has been disclosed in the article "New Sorbicillinoids from the Mangrove Endophytic Fungus Trichodermareesei SCNU-F0042, Jialin Li et.al., Marine Drugs, 2023.8".

[0033] Example 1

[0034] (I) Test method

[0035] 1. Drug

[0036] Latanoprostene bunod, Ribavirin, Remdesivir.

[0037] 2. Cell

[0038] Test virus strain: Novel coronavirus strain SARS-CoV-2 (BA.2GDPCC 2.00299)

[0039] Cell line: hACE2-293T (293T cell line with high expression of human angiotensin converting enzyme 2)

[0040] 3. Anti-novel coronavirus EC of each drug 50 Test

[0041] The activity of each drug in inhibiting the novel coronavirus at the cell level was detected by RT-qPCR method (antiviral 50% effective concentration, 50% Effective Concentration, EC 50 ):

[0042] Latanoprostene bunod was set at a concentration gradient of 0.03125, 0.125, 0.5, 2, and 8 μM;

[0043] Ribavirin was set at a concentration gradient of 5, 10, 20, and 40 μM;

[0044] Remdesivir was set at a concentration gradient of 5, 10, 20, and 40 μM.

[0045] The above concentration gradient doses of each drug were added to the hACE2-293T cell supernatant 1 h in advance, then the culture medium containing the new coronavirus (MOI = 0.2) was added for 1 h, and then the virus-free culture medium containing the corresponding concentration gradient drug was added for 48 h. The cell supernatant was collected, and the viral RNA was extracted using the QIAGEN viral nucleic acid extraction kit (QIAGEN, #74104); then the new coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) (Guangzhou Daan Gene Co., Ltd., DA0932) was used for qPCR detection of viral infection after compound at different doses and solvent group (DMSO) Ct value, and the following formula was used to calculate the inhibition rate of new coronavirus RNA level. Corresponding drug inhibition rate (%) = 2^ (solvent control group Ct value-given group Ct value) x 100%, calculated by EXCEL 2013 Forecast formula (select 3 data points closest to 50% inhibition rate for calculation), when the inhibition rate is equal to 50%, the concentration of the corresponding drug is taken as EC 50 , and the average value of three repeated experiments is taken.

[0046] 4. Cytotoxicity test of latanoprostene bunod

[0047] MTT method was used to test the 50% cytotoxic concentration (CC 50 ) of each drug:

[0048] The concentration gradient of latanoprostene bunod was set to 0.6125, 2.5, 10, and 40 μM;

[0049] The concentration gradient of Ribavirin was set to 5, 10, 20, and 40 μM;

[0050] The concentration gradient of Remdesivir was set to 5, 10, 20, and 40 μM.

[0051] Latanoprostene bunod, Ribavirin, and Remdesivir were added to the hACE2-293T cell supernatant at different concentration gradient doses, and incubated with MTT for 4 h after 48 h of maintenance. The culture medium was aspirated, DMSO was added to detect the 490 nm absorbance value, and compared with the DMSO solvent control group. The inhibition rate (%) = (1-dose group 490 nm absorbance value / solvent control group 490 nm absorbance value) 100%, calculated by EXCEL 2013 Forecast formula (select 3 data points closest to 50% inhibition rate for calculation), when the inhibition rate is equal to 50%, the concentration of the corresponding drug is taken as CC 50, and the average value was taken for triplicate experiments.

[0052] (ii) Test results

[0053] EC of each drug in inhibiting SARS-CoV2 BA.2 strain in hACE2-293T cells 50 and CC 50 The results are shown in Table 1 and Table 2, respectively. The results show that latanoprostene bunod can significantly inhibit the replication and infection of the novel coronavirus in the cell model, has good antiviral activity, and has no obvious cytotoxicity at the therapeutic dose.

[0054] Table 1

[0055]

[0056] Table 2

[0057]

[0058]

[0059] Example 2

[0060] (i) Test method

[0061] Test of inhibition of latanoprostene bunod on the RNA-dependent RNA polymerase activity of the novel coronavirus (50% Inhibitory Concentration, IC 50 ):

[0062] Test virus strain: Novel coronavirus strain SARS-CoV-2 (BA.2 GDPCC 2.00299)

[0063] Cell line: hACE2-293T (293T cell line with high expression of human angiotensin converting enzyme 2)

[0064] Drugs: Latanoprostene bunod, Remdesivir, Ribavirin;

[0065] Latanoprostene bunod was set at a concentration gradient of 0, 0.033, 0.11, 0.33, 1, 3 μM;

[0066] Ribavirin was set at a concentration gradient of 0, 0.33, 1, 3, 9 μM;

[0067] Remdesivir was set at a concentration gradient of 0, 1, 3, 9, 27 μM.

[0068] Plasmid: The nsp12, nsp7, nsp8 genes of the novel coronavirus were constructed into the vector pcDNA3 respectively to obtain three recombinant plasmids pcDNA3-nsp12, pcDNA3-nsp7, pcDNA3-nsp8. The 5'UTR and 3'UTR sequences of SARS-CoV-2 were added upstream and downstream of the Luciferase gene in the pcDNA3 plasmid to obtain the pcDNA3-SARS-CoV-2-Luciferase reporter plasmid.

[0069] In the 293T cell system, the pcDNA3-nsp12: pcDNA3-nsp7: pcDNA3-nsp8 (mass ratio 1:3:3, 6-hole plate transfection total 1 μg / hole, 10 5 μg pcDNA3-SARS-CoV-2-Luciferase reporter plasmid were co-transfected, and after 6 hours, the culture medium containing different drug concentration gradient doses was replaced, and the expression was continued for 24 hours. Finally, the cell precipitate was collected to detect the luciferase fluorescence intensity (A Ctrl The fluorescence intensity of the 0 μM concentration drug culture medium treatment was A C1 , A C2 , A C3 , A C4 , A C5 The fluorescence intensity detected by the above corresponding concentration drug culture medium was A mock .

[0070] The corresponding drug group inhibition rate (%) = (A Cx -A mock ) / (A Ctrl -A mock ) × 100%, calculated by the Forecast formula of EXCEL 2013 (select the 3 data points closest to 50% inhibition rate for calculation), when the inhibition rate is equal to 50%, the concentration of the corresponding drug is the IC 50 , and the average value of three repeated experiments was taken.

[0071] (B) Test results

[0072] The IC 50The results are shown in Table 3. The results show that latanoprostene bunod has an inhibitory effect on the activity of the RNA-dependent RNA polymerase of the novel coronavirus.

[0073] Table 3

[0074]

[0075] Example 3

[0076] The present embodiment provides an anti-novel coronavirus drug containing latanoprostene bunod.

[0077] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. Use of latanoprost nitrate in the preparation of a drug for inhibiting a novel coronavirus.

2. Use according to claim 1, characterized in that, The drug comprises a pharmaceutically acceptable carrier.

3. Use according to claim 1, characterized in that, The carrier is a liposome, a micelle, a dendrimer, a microsphere or a microcapsule.

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