CN114432303A - Application of panobinostat in preparation of medicine for preventing and treating coronavirus - Google Patents

Abstract

The invention relates to application of panobinostat in preparation of a medicament for preventing and treating coronavirus. In particular discloses application of panobinostat or pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs thereof in preparing medicaments for preventing and/or treating diseases caused by coronavirus. The coronavirus is novel coronavirus SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV. The disease caused by coronavirus is pneumonia or its complication caused by any one of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 or MERS-CoV. The invention proves the inhibition effect of panobinostat on the novel coronavirus of new coronary pneumonia for the first time, and the panobinostat has low half effective concentration and great potential.

Description

Application of panobinostat in the preparation of drugs for preventing and treating coronavirus

technical field

The invention belongs to the field of medicines, and specifically relates to the application of panobinostat in the preparation of medicines for preventing and treating coronavirus.

Background technique

The new coronavirus pneumonia (Corona Virus Disease 2019) is an infectious disease caused by the infection of the human body by the new coronavirus (SARS-Cov-2). , 2020, 395(10223): 507-513; The Lancet, 2020, 395(10223): 497-506]. Corona virus belongs to the genus Corona virus in the family Coronaviridae. The virus of the genus Coronavirus is a positive-stranded single-stranded RNA virus with an envelope, with a diameter of about 80-120 nm. Its genetic material is the largest among all RNA viruses, and generally only infects humans, mice, pigs, cats, and dogs. , avian vertebrates. The coronavirus was first isolated from chickens in 1937. The shape of coronavirus particles is irregular, with a diameter of about 60-220nm. The virus has an envelope structure with three proteins on it: spike glycoprotein (S, Spike Protein), small envelope glycoprotein (E, Envelope Protein) and membrane glycoprotein (M, Membrane Protein), and a few species also have hemagglutinin Glycoprotein (HE protein, Haemaglutinin-esterase) [Nederlands Tijdschrift Voor Geneeskunde, 2014, 158(158):A8119-A8119].

The diameter of SARS-Cov-2 virus particles is between 60 and 140 nm, and there are spikes of 9 to 12 nm outside the envelope, which are shaped like a corolla. Genome sequencing revealed that SARS-Cov-2 is a single-stranded RNA coronavirus. By comparing the gene sequences of other virus samples, it was found that SARS-Cov-2 was similar to SARS-CoV (79.5%) [Nature, 2020] and bat coronavirus (96%) [bioRxiv, 2020, 2020.01.22.914952], and speculated that The virus may have originated from bats [bioRxiv, 2020, 2020.01.24.915157; Nature, 2020]. The 2019-nCoV virus belongs to βCoV and is the seventh member of the HCoV family that is different from SARS-CoV and MERS-CoV [New England Journal of Medicine, 2020]. The remaining six members include HCoV 229E, NL63, OC43, HKU1, SARS -CoV and MERS-CoV.

The novel coronavirus pneumonia is caused by a new type of coronavirus. It is the same coronavirus as the well-known SARS-CoV that causes atypical pneumonia, but the type is different. Although its fatality rate is lower than that of SARS-CoV, it is far more infectious. to SARS-CoV.

Panobinostat is a histone deacetylase (HDAC) inhibitor that catalyzes the removal of acetyl groups from lysine residues in histones and some non-histone proteins. Inhibition of HDAC activity leads to increased acetylation of histones, resulting in epigenetic changes in chromatin relaxation, leading to transcriptional activation, which in turn induces cell cycle arrest and induces tumor cell apoptosis. Clinically, panobinostat is approved as a drug for the treatment of multiple myeloma, and it is combined with bortezomib and dexamethasone as a third-line drug to treat multiple myeloma after receiving 2 immunosuppressive treatments ( multiplemyeloma, MM) patients.

So far, there is still no effective drug that can cure the new coronavirus pneumonia. Most of the current treatment methods are symptomatic treatment, especially for some severe patients with poor efficacy. Therefore, the development of effective drugs for the treatment of coronavirus pneumonia has become an urgent issue that needs to be solved.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of panobinostat in the preparation of a drug for preventing or treating the novel coronavirus of novel coronavirus pneumonia.

Specifically, in order to solve the technical problem of the present invention, the following technical solutions are adopted:

The present invention provides panobinostat or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs in the preparation of preventing and/or treating coronary Use in medicines for diseases caused by viruses.

Another aspect of the present invention provides panobinostat or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs in the preparation of preventing coronavirus The application of drugs that enter cells.

In the technical solution of the present invention, the coronaviruses are novel coronaviruses SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV.

In the technical solution of the present invention, the disease caused by the coronavirus is pneumonia or its complications caused by any virus of SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 or MERS-CoV.

In the technical solution of the present invention, panobinostat is represented by structural formula (1)

In the technical solution of the present invention, panobinostat or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs are used as the only active ingredient Application in the preparation of medicaments for preventing and/or treating diseases caused by coronavirus.

In the technical solution of the present invention, panobinostat or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs and other antiviral The application of the composition prepared by the medicine as an active ingredient in the preparation of a medicine for preventing and/or treating diseases caused by coronavirus.

In the technical scheme of the present invention, other antiviral drugs are selected from ganciclovir, acyclovir, amantadine, rimantadine, oseltamivir, abacavir, acemannan, acyclovir Sodium, Adefovir, Alovudine, Avesuto, Tricyclodecamine Hydrochloride, Alanodine, Aridone, Ateviridine Mesylate, Afrilidine, Cidofovir, Silicate panthefylline, emtricitabine, cytarabine hydrochloride, delavirdine mesylate, desilovir, didanosine, dioxalisine, eduridine, emivirine, eltra Sitapine, Enviraden, Enviroxime, Heptin, Famciclovir, Chlorphenhydramine Hydrochloride, Noncitabine, Nonauridine, Phosphorate, Foscarnet Sodium, Phosphonoacetate Sodium, Ganciclovir Sodium, iodine, indinavir, ethoxybutyraldehyde, lamivudine, lobcavir, lordadenosine, lopinavir, memodine hydrochloride, thiocarbamide, nelfinavir , nevirapine, penciclovir, pirodavir, ribavirin, saquinavir mesylate, ritonavir, somantamide hydrochloride, solivudine, penicillin, stavudine , tenofovir, tidrolone hydrochloride, trifluridine, valacyclovir hydrochloride, vidarabine, vidarabine phosphate, vidarabine sodium phosphate, tipranavir, viroxime, Citabine, Zidovudine, Jingwei oxime.

Another aspect of the present invention provides a pharmaceutical composition for treating or preventing diseases caused by coronaviruses of the family Coronaviridae, which comprises panobinostat or its pharmaceutically acceptable salts, isotopes, stereoisomers, stereoisomers Mixtures, tautomers, esters, amides or prodrugs.

In the technical solution of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

In the technical solution of the present invention, the dosage form of the pharmaceutical composition is an oral preparation, a pulmonary inhalation preparation, a mucosal administration preparation, an ophthalmic preparation or an injection.

In the technical solution of the present invention, the oral preparation is selected from granules, powders, pills, tablets, capsules or oral liquids.

Another aspect of the present invention provides the use of panobinostat as an anti-coronaviridae virus disinfectant.

Another aspect of the present invention provides a method for treating diseases caused by coronaviruses, comprising adding a therapeutically effective amount of panobinostat or a pharmaceutically acceptable salt, isotope, stereoisomer, stereoisomer Mixtures of isomers, tautomers, esters or prodrugs are administered to the subject.

Another aspect of the present invention provides a method for preventing a subject from being infected with a coronavirus of the family Coronaviridae, comprising adding a therapeutically effective amount of panobinostat or a pharmaceutically acceptable salt, isotope, stereoisomer, Stereoisomer mixture, tautomer, ester or prodrug administration is administered to the subject prior to infection.

beneficial effect

The present invention proves for the first time that panobinostat has an inhibitory effect on the novel coronavirus of new coronary pneumonia. Panobinostat is used as an effective drug in the treatment of novel coronavirus infection.

Description of drawings

Figure 1 shows the production and validation of the SARS-2-S pseudotype particle.

Among them, (A) SARS-2-S protein can be successfully expressed in mammalian cells. (B) SARS-2-S pseudovirion has S protein modification. (C) The SARS-2-S pseudovirion can successfully infect host cells and integrate the reporter gene. (D) SARS-2-S pseudovirions enter susceptible cells by recognizing ACE2 receptors. (E) SARS-2-S pseudovirions were able to infect human 293T cells expressing ACE2-GFP, but not into 293T cells without ACE2 expression. ACE2-GFP, green; Flag tag, red, showing the S protein; (F) SARS-2-S pseudovirion capable of binding to the ACE2 receptor with colocalized signals at different locations within the cell (membrane and cytoplasm). (G) Time-dependent entry of SARS-2-S pseudovirions into ACE2-GFP/293T cells.

Figure 2 shows that panobinostat effectively inhibits pseudovirion infection. Immunofluorescence staining was used to determine the inhibitory effect of the screened clinical drugs on the infection of host cells by SARS-2-S pseudovirus particles. After the pseudovirus particles infect ACE2-GFP expressing 293T cells, the intracellular localization of pseudovirus particles was observed by fixed staining. Blue, DAPI; red, Flag antibody labeled S protein; green, ACE2-GFP signal.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below, but should not be construed as limiting the scope of the present invention.

In the present invention, the term treatment as used herein refers to reversing, alleviating, inhibiting the progression of, or preventing the diseases or conditions to which the term applies, or one or more symptoms of these diseases or conditions, or the One or more symptoms of a disorder.

The term "therapeutically effective amount" as used herein is the amount of Compound 1, or a pharmaceutically acceptable salt thereof, present in the compositions described herein that, when such compositions are administered by the chosen route of administration, at The desired level of drug is provided in the secretions and tissues of the airways and lungs, or alternatively in the bloodstream of the subject to be treated, to produce the desired physiological response or desired biological effect. The precise amount will depend on many factors, such as the specific activity of the composition, the delivery device used, the physical properties of the composition, its intended use, and animal considerations such as the severity of the disease state, etc., and can be determined by one of skill in the art based on The information provided herein makes it easy to determine the exact amount.

With regard to the route of administration, the active ingredients of the present invention are administered by any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and the like. It will be appreciated that the preferred route may vary depending, for example, on the condition of the recipient. An advantage of the compounds of the present invention is that they are orally bioavailable and can be administered orally.

The effective dose of the active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against an active viral infection, delivery method and pharmaceutical formulation, and will be determined by the clinician using routine dose escalation studies.

In the technical solution of the present invention, panobinostat or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs and other antiviral The composition of the pharmaceutical preparation is used as an active ingredient in the preparation of a medicine for preventing and/or treating a disease caused by a coronavirus, and for the above-mentioned two or more active ingredients in a unit dosage form combination is administered to a patient simultaneously or sequentially. Combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

In the technical solution of the present invention, the cell is any cell, such as eukaryotic or prokaryotic cells, especially refers to a host cell that can be used as a coronavirus, especially a cell containing an ACE2 receptor.

Example 1 Construction, Production and Verification of Pseudovirus Particles Modified by New Coronavirus S Protein

In order to simulate the natural process of the new coronavirus pneumonia virus (SARS-CoV-2) using the S protein to recognize the ACE2 receptor for host cell infection, a pseudovirus with replication-defective lentivirus as the core and modified with the SARS-2-S protein was constructed. particles. The construction method adopts the article Xiuyuan Ou et al. (published on March 27, 2020). Characterization of spikeglycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nature communications, 11(1), 1-12 . Published method.

First, the DNA sequence of SARS-2-S protein was modified without changing its amino acid sequence by codon optimization method, which facilitated the abundant expression of SARS-2-S in 293T cells (Fig. 1A). Subsequently, using the replication-defective HIV lentivirus commonly used in the laboratory as the core of the pseudovirion, the three-plasmid expression system was transfected in 293T cells, and SARS-2-S was modified on the outer membrane of the pseudovirion to form SARS-2 -S pseudovirion. It can be judged by immunoblotting that this pseudoviral particle removed the original VSV-G envelope glycoprotein and replaced it with SARS-2-S, the envelope glycoprotein of the new coronavirus (Figure 1B). In order to verify the infection efficiency of the SARS-2-S pseudovirion, the pseudovirion carrying the Luciferase reporter gene was used to infect host cells 293T or ACE2/293T cells. The Luciferase cell activity assay showed that the SARS-2-S pseudovirus particles had high infectivity to ACE2/293T cells, and the infection efficiency was about 100 times higher than that of 293T cells (Figure 1C). In addition, pseudovirus particles were also used to infect monkey kidney epithelial Vero-E6 cells with endogenous ACE2 expression, and immunofluorescence staining showed that SARS-2-S pseudovirus particles could well enter Vero-E6 cells; Knockdown of the endogenous ACE2 receptor greatly reduced the infection efficiency of pseudovirions (Fig. 1D). Similarly, ACE2-GFP overexpressed 293T was also used as a host cell to observe the entry process of pseudovirions. The results of immunofluorescence staining showed that SARS-2-S could efficiently enter ACE-GFP/293T cells, but not into 293T cells without ACE2 expression (Fig. 1E). Therefore, the above results prove that the constructed SARS-2-S pseudovirus system can simulate the natural process of S protein recognizing ACE2 and has infectious activity.

The infection process of SARS-2-S pseudovirus particles was further observed under a high magnification microscope by laser confocal microscopy, and it was found that pseudovirus particles were not only localized on the cell membrane, but also could enter cells with ACE2 receptors and transported to the perinuclear cells of cells perinuclear area (FIG. 1F). In addition, the entry of SARS-2-S pseudovirions into host cells was also time-dependent, reaching a saturation phase after approximately 2 h (Fig. 1G).

Example 2 Screening of clinical drugs by cell biology platform to inhibit the infection of SARS-2-S pseudovirus particles

Using the SARS-2-S pseudovirus particle in vitro infection model (Luciferase reporter system and immunofluorescence staining localization system) constructed in Example 1, the in vitro cell biological verification of panobinostat was carried out. 293T cells were stably inoculated with ACE2-GFP in a 96-well plate, and the cells were pretreated with different concentrations of panobinostat for 2 hours, then SARS-2-S pseudovirus particles were added to infect for 3 hours, and then the supernatant was removed and replaced with fresh complete culture medium. After 40 hours of infection, the cells were lysed by Luciferase Assay System (Promega) and the reaction substrate was added, and Glomax 96 was used to measure the luminescence intensity of luciferase, which was proportional to the infection efficiency of virus particles.

The results of Luciferase activity assay showed that panobinostat had an obvious concentration-dependent effect and could effectively inhibit the infection efficiency of SARS-2-S pseudovirus particles. The EC50 concentration of panobinostat is approximately 2.8 ± 1.0 μM.

The results of immunofluorescence staining also showed that pretreatment of host cells with panobinostat at a concentration of 50 μM for 2 h could significantly inhibit the entry of SARS-2-S pseudovirus particles into ACE2-GFP/293T (Figure 2). From the results in Figure 2, in the control group, a large number of pseudovirus particles entered the cells and were located in the area around the nucleus, forming a red aggregation area; most of the virus particles in the administration group were located in and around the cell membrane and membrane, indicating that panobinostat is effective The invasion process of pseudovirus particles is inhibited, and the pseudovirus particles are blocked on the cell membrane.

It is worth mentioning that since immunofluorescence staining requires imaging of virus particles, the amount of pseudovirus particles used for infection is much higher than that of Luciferase reporter assay, which is closer to the amount of virus infecting human cells under physiological conditions. . Therefore, the Luciferase reporter system can better reflect the antiviral effect of clinical drugs under physiological conditions. This shows that panobinostat has a great potential for anti-coronavirus infection. The action pathway and target of this clinical drug are clear steps in inhibiting the infection of host cells by the new coronavirus pneumonia virus, which has great research value and drug screening potential.

Claims (11)

1. Panobinostat or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs in the preparation of prevention and/or treatment of coronavirus Use of disease-causing drugs. 2. Panobinostat or its pharmaceutically acceptable salts, isotopes, stereoisomers, mixtures of stereoisomers, tautomers, esters, amides or prodrugs in the preparation of drugs for preventing coronavirus from entering cells Applications. 3. The application according to claim 1 or 2, wherein the coronaviruses are novel coronaviruses SARS-Cov-2, SARS-CoV, HCoV 229E, NL63, OC43, HKU1 and MERS-CoV. 4. The application according to claim 1 or 2, wherein the disease caused by coronavirus is pneumonia or its complication caused by any virus of SARS-Cov-2, SARS-CoV, HCoV229E, NL63, OC43, HKU1 or MERS-CoV . 5. The use according to claim 1 or 2, panobinostat or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or pro medicine as the only active ingredient. 6. The use according to claim 1 or 2, panobinostat or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomer, ester, amide or pro and other antiviral drugs as active ingredients. 7. application according to claim 6, other antiviral drugs are selected from ganciclovir, acyclovir, amantadine, rimantadine, oseltamivir, abacavir, acemannan, Ciclovir Sodium, Adefovir, Alovudine, Avesuto, Tricyclodecamine Hydrochloride, Alanodine, Aridone, Ateviridine Mesylate, Afrilidine, Cidofo Wei, sipanphylline, emtricitabine, cytarabine hydrochloride, delavirdine mesylate, deciclovir, didanosine, dioxin, eduridine, emivirine , Etricitapine, Enviraden, Enviroxime, Heptin, Famciclovir, Chlorphenhydramine Hydrochloride, Fecitabine, Feauridine, Phosphorate, Sodium Foscarnet, Sodium Phosphonate, Glycine Cilovir sodium, iodine, indinavir, ethoxybutanone aldehyde, lamivudine, lobcavir, lordadenosine, lopinavir, memotine hydrochloride, methyl red thiourea, that Finavir, nevirapine, penciclovir, pirodavir, ribavirin, saquinavir mesylate, ritonavir, somantamide hydrochloride, solivudine, penicillin, division Tavudine, tenofovir, tidrolone hydrochloride, trifluridine, valacyclovir hydrochloride, vidarabine, vidarabine phosphate, vidarabine sodium phosphate, tipranavir, virol Oxime, Zhacitabine, Zidovudine, Jingwei oxime. 8. a pharmaceutical composition for the treatment or prevention of diseases caused by coronaviruses, comprising panobinostat or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers, tautomerism Conforms, esters, amides or prodrugs as active ingredients; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients; Preferably, the dosage form of the pharmaceutical composition is an oral formulation, a pulmonary inhalation formulation, a mucosal administration formulation, an ophthalmic formulation or an injection. 9. The pharmaceutical composition according to claim 8, further comprising other antiviral drugs in the pharmaceutical composition; Preferably, the other antiviral drugs are selected from ganciclovir, acyclovir, amantadine, rimantadine, oseltamivir, abacavir, acemannan, acyclovir sodium, adefo Wei, alovudine, avesuto, tricyclodecylamine hydrochloride, alanodine, aridone, ateviridine mesylate, alfridine, cidofovir, sipanphylline, en Tricitabine, cytarabine hydrochloride, delavirdine mesylate, desilovir, didanosine, dioxalid, eduridine, emivirine, etricitapine, en Veladen, Enviroxime, Heptin, Famciclovir, Chlorphene Hydrochloride, Fecitabine, Non-Auridine, Phosphate, Foscarnet, Foscarnet, Ganciclovir, Iodine, Indinavir, ethoxybutanone aldehyde, lamivudine, lobcavir, lordadenosine, lopinavir, memodine hydrochloride, methylred thiourea, nelfinavir, nevirapine, penxi Lovir, pirodavir, ribavirin, saquinavir mesylate, ritonavir, somantamide hydrochloride, solivudine, penicillin, stavudine, tenofovir , Trifluridine Hydrochloride, Trifluridine, Valacyclovir Hydrochloride, Vidarabine, Vidarabine Phosphate, Vidarabine Sodium Phosphate, Tipranavir, Veroxime, Zalcitabine, Qi Dovudine, net Wei oxime. 10. A method for treating diseases caused by coronaviruses, comprising adding a therapeutically effective amount of panobinostat or a pharmaceutically acceptable salt, isotope, stereoisomer, mixture of stereoisomers thereof , tautomers, esters or prodrugs are administered to a subject. 11. A method for preventing a subject from being infected with a coronavirus of the family Coronaviridae, comprising adding a therapeutically effective amount of panobinostat or a pharmaceutically acceptable salt, isotope, stereoisomer, stereoisomer thereof. The mixture, tautomer, ester or prodrug is administered to subjects prior to infection with the coronavirus.

Images