CN111135166A - Pharmaceutical composition consisting of GC376 and GS-441524 and application thereof in inhibiting new coronavirus - Google Patents

Pharmaceutical composition consisting of GC376 and GS-441524 and application thereof in inhibiting new coronavirus Download PDF

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CN111135166A
CN111135166A CN202010145969.3A CN202010145969A CN111135166A CN 111135166 A CN111135166 A CN 111135166A CN 202010145969 A CN202010145969 A CN 202010145969A CN 111135166 A CN111135166 A CN 111135166A
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composition
cell
cov
pharmaceutical composition
virus
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彭贵青
刘紫微
陈西
田大勇
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Wuhan Kangmat Biotechnology Co ltd
Huazhong Agricultural University
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Wuhan Kangmat Biotechnology Co ltd
Huazhong Agricultural University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Abstract

The invention discloses a pharmaceutical composition, which consists of GC376 and GS-441524. The results of in vitro cell experiments show that: the composition can inhibit the proliferation of novel coronavirus SARS-CoV-2 in cells, has the drug effect superior to that of single drug application, has obvious synergistic effect, can be used as a novel coronavirus SARS-CoV-2 inhibitor for treating COVID-19 pneumonia, and has the advantages of good curative effect, high safety, reduction of virus resistance, reduction of drug toxic and side effects and the like.

Description

Pharmaceutical composition consisting of GC376 and GS-441524 and application thereof in inhibiting new coronavirus
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to a pharmaceutical composition for inhibiting novel coronavirus SARS-CoV-2 and application thereof.
Background
The new type coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is called new type coronavirus pneumonia for short, and is a contact infectious Disease mainly characterized by fever, dry cough, hypodynamia and certain lethality rate caused by the new type coronavirus SARS-CoV-2.
Since 12 months of new coronary pneumonia outbreak in 2019, the cases of confirmed infection exceed the SARS, the number of death cases is increasing, serious social hazard and economic loss are caused, and the world health organization declares that the epidemic situation of new coronary pneumonia constitutes an international concerned emergent public health event. Since outbreak of epidemic diseases, the research on the infectious diseases mainly focuses on epidemic investigation, sequence comparison and analysis of clinical separated strains, virus detection methods and other aspects, the research on the invasion, replication and immune regulation and control mechanism of novel coronavirus is still in the primary exploration stage, and no vaccine can provide sufficient protection, so the research on the antiviral drugs of SARS-CoV-2 is of great significance.
GC376 is a broad-spectrum polypeptide analog inhibitor targeted to inhibit the virally encoded 3C-like protease, binding to the active site of the 3C-like protease. Upon reaction, GC376 loses the sulfite group structure exposing the aldehyde group, which reacts with the nucleophilic cysteine residue of 3CLpro to form a covalent bond. This prevents the catalytic action of the 3C-like protease, which in turn inhibits viral replication. The research shows that GC376 has good inhibitory activity on various viruses such as Norovirus (NV), transmissible gastroenteritis virus (TGEV), transmissible peritonitis virus of cat (FIPV), hepatitis virus of Mouse (MHV) and the like.
GC376 has the chemical name 3-pyrrolidineppancuronic acid, α -hydroxy- β - [ [ (2S) -4-methyl-1-oxo-2- [ [ (phenylmethyloxy) carbonyl]amino]pentyl]amino]-2-oxo-, sodium salt (1:1), (β S) -, formula C21H30N3NaO8S, molecular weight is 507.53, and the structural formula is as follows:
Figure BDA0002400732510000021
GS-441524 is a nucleoside analog that primarily targets viral RNA polymerase, interfering with the viral RNA transcription/replication process. GS-441524 is phosphorylated to active triphosphate metabolites (NTP structural analogs) in cells through cell kinases, the active NTP structural analogs serve as competitors of natural nucleoside triphosphates to compete with natural Nucleosides (NTP) for participating in RNA transcription/replication in viral RNA synthesis, and when GS-441524 molecules are inserted into transcription/replication products, the transcription/replication is terminated early, so that the transcription/replication process of viral RNA is inhibited. GS-441524 has been found to exhibit certain inhibitory activity against Ebola, SARS, Huning virus, respiratory syncytial virus, and the like.
GS-441524 is chemically named D-Altrinitronitrile, 2-C- (4-aminopyrrolo [2, 1-f)][1,2,4]triazin-7-yl) -2, 5-anhydro. Molecular formula C12H13N5O4Molecular weight of 291.267, and structural formula as follows:
Figure BDA0002400732510000022
the two medicines are mainly used for treating Feline Infectious Peritonitis (FIP) clinically at present, and the applicant previously filed a patent of 'a feline coronavirus inhibitor composition consisting of GC376 and GS-441524', publication No. CN110215456A, and researches show that the composition can inhibit the proliferation of feline coronavirus in cells and has a remarkable synergistic effect, but no relevant report on the aspect of inhibiting the human susceptible coronavirus and the COVID-19 pneumonia caused by the coronavirus is found at present.
Disclosure of Invention
The invention aims to provide a pharmaceutical composition consisting of GC376 and GS-441524 and a new application thereof in inhibiting novel coronavirus SARS-CoV-2 and treating related diseases.
The results of in vitro cell experiments show that: the composition has little toxic effect on the normal cells of Vero cells, has obvious inhibition effect on novel coronavirus SARS-CoV-2, and can obviously reduce the toxic value of the virus in the cells; the result of the virus valence measurement in the cell shows that the composition can inhibit the proliferation of the novel coronavirus SARS-CoV-2 in the cell, has obviously higher effect than that of single medicine application, and has obvious synergistic effect.
Therefore, the composition of GC376 and GS-441524 can be used as a novel coronavirus SARS-CoV-2 inhibitor for treating COVID-19 pneumonia, and has the advantages of good curative effect, high safety, reduction of virus resistance, reduction of drug toxic and side effects and the like.
In the composition, the molar ratio of GC376 to GS-441524 is 1: 1.
drawings
FIG. 1 shows the results of toxicity tests of the composition against Vero-E6 cells.
FIG. 2 is a cell micrograph of the composition inhibiting the novel coronavirus SARS-CoV-2.
FIG. 3 is EC of composition for inhibiting novel coronavirus SARS-CoV-250The curve was measured.
FIG. 4 is a TCID of the composition for inhibiting the novel coronavirus SARS-CoV-250The measured results show that ＊＊ P is less than 0.01, ＊＊＊ P is less than 0.001。
FIG. 5 is a composition and TCID administered alone50Comparative results, ＊ P < 0.05, ＊＊P＜0.01。
Detailed Description
The present invention will be described in detail with reference to examples. It should be noted that the embodiments of the present invention are only illustrative and not restrictive. The experimental procedures and other experimental procedures described in the examples are conventional in the art and are not specifically described herein, and those skilled in the art can refer to various conventional tool books, technical literatures or related specifications, manuals, etc. before the filing date of the present application.
Test materials: Vero-E6 is preserved in the laboratory of Huazhong university of agriculture; the SARS-CoV-2 strain is 19# strain separated from CDC in Zhejiang; the cell growth solution is DMEM containing 10% FBS; the cell maintenance solution was DMEM containing 2% FBS.
EXAMPLE 1 toxicity test of pharmaceutical composition on cells
The test method comprises the following steps:
1) taking Vero-E6 cell (African green monkey kidney cell) with good growth state for digestion passage, adjusting cell density to 1 × 10 with cell growth liquid5Inoculate a 96-well plate at 100. mu.L/well in 37 ℃ 5% CO2Culturing in an incubator for 16 h;
2) after 16h, the medium in the wells was discarded, washed three times with 1 × PBS, spun-dried, and added with the cell maintenance solution, along with the composition (molar ratio 1:1) the final concentration of the composition in the wells was set to 10000. mu.M, 1000. mu.M, 500. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, and cell controls were placed at 37 ℃ in 5% CO2Culturing in an incubator;
3) after 72h, use
Figure BDA0002400732510000041
And (5) carrying out cell viability detection by using the reagent.
And (3) test results:
the results are shown in FIG. 1. The determination of cell viability in response to the toxic effect of the drug on Vero-E6 cells shows that the composition has less toxic effect on Vero-E6 cells and half the Cytotoxic Concentration (CC)50) Greater than 500. mu.M.
EXAMPLE 2 inhibitory Effect of pharmaceutical compositions on viruses at cellular level
The test method comprises the following steps:
1) Vero-E6 cells with good growth state are taken for digestion and passage, and cell density is adjusted to 1 × 10 by cell maintenance liquid5The cells were seeded at 100. mu.L/well in 96-well plates and placed at 37 ℃ in 5% CO2Culturing in an incubator for 16 h;
2) after 16h, the wells were discarded with medium, washed three times with 1 XPBS, spun-dried, 50. mu.L of 100. mu.M GC376 and GS-441524 were added to each well, and 100. mu. L0.01MOI SARS-CoV-2 virus was added to each well to give a final concentration of 50. mu.M. Setting cell control and virus control at the same time, placing at 37 deg.C and 5% CO2Culturing in an incubator;
3) after 48h of virus inoculation, the cell state change of the compound and the virus after combined action is observed by a microscope and photographed for storage.
And (3) test results:
the results are shown in FIG. 2. The normal Vero-E6 cell can be observed to have good growth state, complete cell shape and clear cell boundary by microscope pictures in the picture; the wiredrawing phenomenon of virus infected cells does not have complete cell morphology, and the pathological changes are obvious; after the composition is added to treat cells, the growth state of the cells is good, and no obvious lesion exists. Therefore, the composition has certain inhibition effect on the novel coronavirus SARS-CoV-2 at the cellular level.
EXAMPLE 3 antiviral Activity of pharmaceutical compositions (EC)50) Test of
The test method comprises the following steps:
1) taking Vero-E6 cells with good growth state for digestion and passage, and adjusting the cell density to 1 × 10 with cell growth liquid5The cells were seeded at 100. mu.L/well in 96-well plates and placed at 37 ℃ in 5% CO2Culturing in an incubator for 16 h;
2) after 16h, the composition of GC376 and GS-441524 was diluted with a cell maintenance solution at a molar ratio of 1:1 to give a composition concentration of 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M;
3) labeling 96-well plate cover, discarding culture medium in well, washing with 1 × PBS for three times, spin-drying, adding 100 μ L diluted composition into each well according to labeling order, adding 100 μ L0.01MOI virus solution into each well to make final concentration of composition 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.125 μ M, and 1.5625 μ M, setting cell control group and virus control group, placing at 37 deg.C and 5% CO2Culturing in an incubator;
4) after 72h, use
Figure BDA0002400732510000051
And (5) carrying out cell viability detection by using the reagent.
And (3) test results:
the results are shown in FIG. 3. As can be seen from the figure, the inhibitory effect of the composition on the novel coronavirus SARS-CoV-2 is dose-dependent. Half maximal Effect Concentration (EC) of composition50) Comprises the following steps: 1.045 μ M, significantly lower than GC376 (3.1) alone33 μ M) and GS-441524(1.424 μ M).
EXAMPLE 4 antiviral Activity of pharmaceutical compositions (TCID)50) Measurement of (2)
The test method comprises the following steps:
1) the Vero-E6 cells are subjected to digestion and passage, and the cell density is adjusted to 1 x 10 by using a cell growth solution5Inoculate a 96-well plate at 100. mu.L/well in 37 ℃ 5% CO2Culturing in an incubator for 16 h;
2) after 16h, the GC 376-441524 composition was diluted with cell maintenance medium at a molar ratio of 1:1 to make the composition concentrations 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.5625. mu.M, 0.78125. mu.M, respectively;
3) after dilution, the 96-well plate was removed, the medium in the well of the 96-well plate was discarded, washed three times with 1 XPBS, and after spin-drying, 100. mu.L of the diluted composition was added to each well, and each well was inoculated with 100. mu. L0.01MOI of virus to give a final concentration of 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.5625. mu.M, 0.78125. mu.M, 0.390625. mu.M, at 37 ℃ with 5% CO2Culturing in an incubator;
4) after about 72h, supernatants from each well were collected for titer.
And (3) test results:
the results are shown in FIG. 4. As the concentration of the composition increases, the viral titer decreases and is dose-dependent. The inhibitory effect is most pronounced at composition concentrations greater than 1.5625. mu.M. TCID at the concentration of 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.5625. mu.M, 0.78125. mu.M, 0.390625. mu.M, 0. mu.M of the composition50Are respectively 0, 10-2.44/mL、10-3.685/mL、10-4.585/mL、10-6.5mL, no virus was detected at 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, and the proliferation of the virus was completely inhibited, and about 4.06 titers were reduced at 1.5625. mu.M, about 2.815 titers were reduced at 0.78125. mu.M, and about 1.915 titers were reduced at 0.390625. mu.M.
The results of the composition compared to the individual doses are shown in FIG. 5. TCID of 1.5625. mu.M GC376 or GS-441524 administered alone50Are respectively 10-4.61/mL、10-3.14Per mL, composition1.5625 μ M TCID50Is 10-2.44The composition reduced 2.17 titers compared to GC376 alone and 0.7 titer compared to GS-441524 alone/mL. TCID for single drug use of 0.78125 mu MGC376 or GS-44152450Are respectively 10-5.345/mL、10-4.41Perml, composition 0.78125. mu.M TCID50Is 10-3.685The composition decreased 1.66 titers compared to GC376 alone and 0.725 titers compared to GS-441524 alone/mL. Separately used 0.390625 μ M TCID of GC376 or GS-44152450Are respectively 10-5.59、10-5.47Perml, composition 0.390625. mu.M TCID50Is 10-4.585The composition decreased 1.005 titers compared to GC376 alone and 0.885 titers compared to GS-441524 alone/mL. The above results show that the composition can better inhibit the virus proliferation, and the two have synergistic effect.

Claims (4)

1. A pharmaceutical composition is characterized by consisting of GC376 and GS-441524.
2. The pharmaceutical composition of claim 1, wherein: the mole ratio of GC376 to GS-441524 was 1: 1.
3. use of the pharmaceutical composition of claim 1 or 2 for the preparation of a novel inhibitor of coronavirus SARS-CoV-2.
4. Use of a pharmaceutical composition according to claim 1 or 2 in the manufacture of a medicament for the treatment of COVID-19 pneumonia.
CN202010145969.3A 2020-03-05 2020-03-05 Pharmaceutical composition consisting of GC376 and GS-441524 and application thereof in inhibiting new coronavirus Pending CN111135166A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022047441A1 (en) * 2020-08-28 2022-03-03 Emphascience, Inc. Formulations of anti-viral compounds
WO2024027844A1 (en) * 2022-08-05 2024-02-08 临港国家实验室 Pharmaceutical composition and use thereof

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Publication number Priority date Publication date Assignee Title
US20180296584A1 (en) * 2017-03-14 2018-10-18 Gilead Sciences, Inc. Methods of treating feline coronavirus infections
CN110215456A (en) * 2019-06-25 2019-09-10 华中农业大学 A kind of cat coronavirus inhibitor combination being made of GC376 and GS-441524

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180296584A1 (en) * 2017-03-14 2018-10-18 Gilead Sciences, Inc. Methods of treating feline coronavirus infections
CN110215456A (en) * 2019-06-25 2019-09-10 华中农业大学 A kind of cat coronavirus inhibitor combination being made of GC376 and GS-441524

Non-Patent Citations (3)

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Title
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022047441A1 (en) * 2020-08-28 2022-03-03 Emphascience, Inc. Formulations of anti-viral compounds
GB2613516A (en) * 2020-08-28 2023-06-07 Sayvaa Pharmaceuticals Inc Formulations of anti-viral compounds
WO2024027844A1 (en) * 2022-08-05 2024-02-08 临港国家实验室 Pharmaceutical composition and use thereof

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