Methods and compounds for treating coronavirus diseases

Abstract

Topotecan for use in treating COVID-19 and / or its associated symptoms.Also disclosed is a method of treating an individual infected with a coronavirus, comprising providing topotecan and administering said topotecan to said individual in a dosage sufficient to prevent / stabilize / reduce risk and / or symptoms associated with coronavirus infection.

Description

[Technical field]

[0001] Field

[0001] The present disclosure relates to compounds and compositions for use in the treatment of coronavirus diseases. More specifically, embodiments of the present disclosure relate to compounds and compositions that can target the mode of interaction between coronaviruses and cells of interest. [Background technology]

[0002] background

[0002] The emergence of a new coronavirus, designated SARS-CoV-2, onto the global stage has affected virtually all populations worldwide. The virus has afflicted millions of individuals and has resulted in a disease designated COVID-19. COVID-19 can develop into a significant health risk and result in death, which places a strain on healthcare resources and society at large.

[0003]

[0003] SARS-CoV-2 is a single-stranded positive-stranded ribonucleic acid (RNA) virus with a receptor-binding domain structure similar to that of SARS-CoV and MERS-CoV. SARS-CoV-2 is transmitted between individuals via droplets that reach the nasal mucosa. Within the nasal mucosa, SARS-CoV-2 can rapidly reproduce and be released into nasal secretions (sputum). The sputum can then be transmitted to other individuals via airborne droplets, thus repeating the cycle of transmission. The SARS-CoV-2 virus can be transmitted between individuals before the onset of symptoms, during the symptomatic period, and even after recovery.

[0004]

[0004] The clinical spectrum of infection is broad, ranging from mild symptoms of upper respiratory tract infection to severe pneumonia, multiple organ failure and death. At onset, SARS-CoV-2 primarily attacks the respiratory system, as this is the main entry point into the host, but SARS-CoV-2 can also affect multiple organs in infected individuals. The severity of COVID-19 is typically associated with comorbidities such as, but not limited to, hypertension, diabetes, obesity, cardiovascular disease and / or advanced age, which may exacerbate the outcome of COVID-19. Summary of the Invention [Problem to be solved by the invention]

[0005]

[0005] There is a need for treatments that can mitigate the effects of COVID-19 in a manner that blunts the physiological impact on infected individuals. [Means for solving the problem]

[0006] overview

[0006] Embodiments of the present disclosure provide one or more treatments for ameliorating and / or inhibiting some or substantially all of the risk, symptoms, and development of severe disease in subjects infected with a coronavirus. In some embodiments of the present disclosure, the coronavirus is SARS-CoV-2. Given the rapidity and scale of the SARS-CoV-2 pandemic, it may be advantageous to identify compounds and compositions for treatment that are already approved to treat other indications.

[0007]

[0007] Some embodiments of the present disclosure relate to the use of topotecan to ameliorate and / or inhibit some or substantially all of the risk, symptoms and onset of severe disease resulting from coronavirus infection.

[0008]

[0008] Some embodiments of the present disclosure relate to a method of treating an individual exposed to or infected with coronavirus, the method comprising the steps of providing a therapeutically effective amount of topotecan and administering the therapeutically effective amount of topotecan to the individual to ameliorate and / or inhibit some or substantially all of the risk, symptoms and development of severe disease in a subject infected with coronavirus.

[0009]

[0009] Some embodiments of the present disclosure relate to a method for producing a topotecan / target cell complex, the method comprising administering a therapeutically effective amount of topotecan to a subject, wherein the topotecan / target cell complex inhibits coronavirus from entering the topotecan / target cell complex, fusing with the topotecan / target cell complex, and / or replicating within the topotecan / target cell complex.

[0010] Some embodiments of the present disclosure relate to pharmaceutical compositions comprising topotecan and at least one excipient.

[0011]

[0011] Without being bound to any particular theory, it is hypothesized that topotecan may target the SARS-CoV-2 virus by inhibiting viral entry or fusion into the subject's cells and / or inhibiting viral replication once inside the subject's cells. While topotecan is a known anti-cancer drug, surprisingly, it may also be useful in the treatment of COVID-19.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] These and other features of the present disclosure will become more apparent from the following detailed description, which refers to the accompanying drawings. [Brief description of the drawings]

[0013] [Figure 1]

[0013] FIG. 2 is a line graph depicting in vitro data from samples obtained according to an embodiment of the present disclosure regarding the viral inhibition efficacy and toxicity of topotecan used according to an embodiment of the present disclosure. [Diagram 2]

[0014] 1 shows a line graph of in vitro data from samples obtained according to embodiments of the present disclosure regarding cell viability for various concentrations of topotecan. [Diagram 3]

[0015] 1 shows a line graph of in vitro data from samples obtained according to embodiments of the present disclosure regarding viral inhibition for various concentrations of topotecan. [Figure 4A]

[0016] 1 shows a line graph of in vitro data from samples taken as a control according to embodiments of the present disclosure regarding cell viability for various concentrations of remdesivir. [Figure 4B]

[0017] 1 shows a line graph of in vitro data from samples taken as controls according to embodiments of the present disclosure regarding viral inhibition for various concentrations of remdesivir. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description

[0018] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art in the context of this disclosure. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described here. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0015]

[0019] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, reference to "an agent" includes one or more agents, and reference to "a subject" or "the subject" includes one or more subjects.

[0016]

[0020] As used herein, the term "about" or "approximately" refers to within about 25%, preferably within about 20%, preferably within about 15%, preferably within about 10%, preferably within about 5% of a given value or range. It is understood that such a variation is always included in any given value provided herein, whether or not it is specifically mentioned.

[0017]

[0021] As used herein, the term "activity" is used interchangeably with the term "functionality" and both terms refer to the physiological action of a biomolecule.

[0018]

[0022] As used herein, the terms "drug" and "therapeutic agent" refer to a substance that, when administered to a subject, produces one or more chemical responses, and / or one or more physical responses, and / or one or more physiological responses, and / or one or more pharmacological responses, and / or one or more immune responses in the subject.

[0019]

[0023] As used herein, the term "improve" refers to restore and / or make better and / or make more satisfactory.

[0020]

[0024] As used herein, the term "cell" refers to a single cell as well as multiple cells or populations of the same or different cell types. Administering an agent to a cell includes in vivo, in vitro and ex vivo administration and / or combinations thereof.

[0021]

[0025] As used herein, the term "complex" refers to a direct or indirect association between one or more particles of an agent and one or more target cells or target virions. This association results in a change in the metabolism or functionality of the target cells or target virions. As used herein, the phrase "metabolic change" refers to an increase or decrease in the production of one or more proteins and / or the post-translational modification of one or more proteins of one or more targets. As used herein, the phrase "functional change" refers to a difference in the physiological function of one or more aspects of the target / target complex within the agent compared to a target that is not part of such a complex.

[0022]

[0026] As used herein, the terms "dysregulation" and "dysregulated" refer to a state or condition in which a homeostatic control system is disturbed and / or impaired, such that one or more metabolic, physiological and / or biochemical systems in a subject function partially or completely without said homeostatic control system.

[0023]

[0027] As used herein, the term "excipient" refers to any substance, not itself a drug, that may be used in a composition for delivery of one or more drugs, etc. to a subject, or alternatively combined with one or more carriers, etc. (e.g., to make a pharmaceutical composition) to improve its handling or storage characteristics, or to allow or facilitate the formation of a dosage unit of the composition (e.g., the formation of a topical hydrogel, which may then be optionally incorporated into a transdermal patch). Excipients include, by way of example and without limitation, binders, disintegrants, taste enhancers, solvents, thickeners or gelling agents (and optional neutralizing agents, if necessary), penetration enhancers, solubilizers, humectants, antioxidants, lubricants, emollients, substances added to mask or counter unpleasant odors, scents or tastes, substances added to improve the appearance or texture of the composition, and substances used to form a pharmaceutical composition. Any such excipient may be used in any dosage form according to the present disclosure. The above classes of excipients are not meant to be exhaustive, but merely exemplary.

[0024]

[0028] As used herein, the terms "inhibit", "inhibiting" and "inhibition" refer to a decrease in the activity, response, or other biological parameter of a biological process, disease, disorder, or symptom thereof. This can include, but is not limited to, the complete disappearance of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any amount between the specifically recited percentages compared to native or control levels.

[0025]

[0029] As used herein, the term "medicament" refers to medicines and / or pharmaceutical compositions, including pharmaceutical agents, that may promote recovery from a disease, disorder, or a symptom thereof, and / or that may prevent a disease, disorder, or a symptom thereof, and / or that may inhibit the progression of a disease, disorder, or a symptom thereof.

[0026]

[0030] As used herein, the term "pharmaceutical composition" refers to any composition containing one or more agents that is administered to a subject in need of treatment or therapy for a disease, disorder, or a symptom thereof, but is not necessarily limited thereto. A pharmaceutical composition may include additives such as a pharmaceutical acceptable carrier, a pharmaceutical acceptable salt, an excipient, etc. A pharmaceutical composition may also further include one or more additional active ingredients, such as an antimicrobial agent, an anti-inflammatory agent, an anesthetic agent, an analgesic agent, etc.

[0027]

[0031] As used herein, the term "pharmaceutical acceptable carrier" refers to an essentially chemically inert and non-toxic ingredient in a pharmaceutical composition or medicament that does not interfere with the efficacy and / or safety of one or more agents. Some examples of pharmaceutical acceptable carriers and their formulations are described in Remington (1995, The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA), the disclosure of which is incorporated herein by reference. Typically, an appropriate amount of a pharmaceutical acceptable carrier is used in a formulation to make the formulation isotonic. Examples of suitable pharmaceutical acceptable carriers include, but are not limited to, saline, glycerol solution, ethanol, N-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylethanolamine (DOPE) and liposomes. Such pharmaceutical compositions contain a therapeutically effective amount of the drug together with an appropriate amount of one or more pharma- ceutically acceptable carriers and / or excipients to provide a suitable form for proper administration to a subject.The formulation is adapted to the route of administration.For example, oral administration may require an enteric coating to protect the drug from being decomposed in a part of the subject's gastrointestinal tract.In another example, the route of administration by injection may be administered in a liposomal formulation to facilitate transport throughout the subject's vascular system and to facilitate delivery across the cell membrane of the targeted intracellular site.

[0028]

[0032] As used herein, the phrases "prevent", "prevention" and "preventing" refer to averting the onset or progression of a disease, disorder, or a symptom thereof.

[0029]

[0033] As used herein, the term "subject" refers to any therapeutic target that receives an agent. A subject may be a vertebrate, such as a mammal, including a human. The term "subject" does not denote a particular age or sex. The term "subject" also refers to one or more cells of an organism, in vitro cultures of one or more tissue types, in vitro cultures of one or more cell types, ex vivo preparations, and / or samples of biological material, such as tissues and / or biological fluids.

[0030]

[0034] As used herein, the term "target cell" refers to one or more cell types in a subject where the virus may interact with a coronavirus by fusing with the outer membrane of one or more cell types, entering the cell, and / or replicating therein. Without being bound to any particular theory, a target cell of a subject may include any cell in a subject that expresses a receptor and / or cofactor required for viral interaction. Examples of these types of cells include, but are not limited to, epithelial cells (ciliated and non-ciliated) of the upper and conducting airways; alveolar epithelial cells (both type 1 and type 2); epithelial cells and neurons of the olfactory system; neurons of the central or peripheral nervous system; epithelial cells, enterocytes, and glandular cells of the gastrointestinal tract; cells of the blood, including immune effector cells; cardiovascular cells, and renal cells.

[0031]

[0035] As used herein, the term "target virion" refers to one or more virus particles of a coronavirus capable of causing viral infection in a target cell. In some embodiments of the present disclosure, the virus particles are of one or more variants of SARS-CoV-2.

[0032]

[0036] As used herein, the term "therapeutically effective amount" refers to the amount of drug used that is sufficient to improve, prevent, treat and / or inhibit a disease, disorder or one or more of its symptoms. A "therapeutically effective amount" varies depending on the drug used, the route of administration of the drug, and the severity of the disease, disorder or its symptoms. The age, weight, and genetic makeup of the subject may also affect the amount of drug that is a therapeutically effective amount.

[0033]

[0037] As used herein, the terms "treat", "treatment" and "treating" refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing a disease, disorder or a symptom thereof from occurring, and / or the effect may be therapeutic in achieving a partial or complete amelioration or inhibition of a disease, disorder or a symptom thereof. Furthermore, the term "treatment" refers to any treatment of a disease, disorder or a symptom thereof in a subject, including (a) preventing a disease from occurring in a subject susceptible to, but not yet diagnosed as having, the disease, (b) inhibiting a disease, i.e., arresting its occurrence, and (c) ameliorating a disease.

[0034]

[0038] As used herein, the terms "unit dosage form" and "unit dose" refer to a physically discrete unit suitable as a single dose for a patient. Each unit contains a predetermined amount of drug and optionally one or more suitable pharma- ceutically acceptable carriers, one or more excipients, one or more additional active ingredients, or a combination thereof. The amount of drug in each unit is a therapeutically effective amount.

[0035]

[0039] In an embodiment of the present disclosure, the pharmaceutical compositions disclosed herein include one or more of such agents in an amount of about 0.1% to about 95% by weight of the total composition. For example, the amount of agent by weight of the pharmaceutical composition may be about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, approximately 2.4%, approximately 2.5%, approximately 2.6%, approximately 2.7%, approximately 2.8%, approximately 2.9%, approximately 3%, approximately 3.1%, approximately 3.2%, approximately 3.3%, approximately 3.4%, approximately 3.5%, approximately 3.6%, approximately 3.7%, approximately 3.8%, approximately 3.9%, approximately 4%, approximately 4.1%, approximately 4.2%, approximately 4.3%, approximately 4.4%, approximately 4.5%, approximately 4.6%, approximately 4.7%, approximately 4.8%. 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6% , about 8.7%, about 8.8%, about 8.9%, about 9%, about 9.1%, about 9.2%, about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% or more.

[0036]

[0040] Where a range of values ​​is provided herein, it is understood that, to the tenth of the unit of the lower limit, each intervening value between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure, unless the context clearly dictates otherwise. The upper and lower limits of these narrower ranges may be independently included in the narrower ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit values ​​in the stated ranges. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0037]

[0041] The emergence of COVID-19 onto the world stage has affected all populations around the world, with millions of individuals infected and the numbers continuing to rise.

[0038]

[0042] Topotecan has a mechanism of action related to binding to the topoisomerase I / DNA complex and is currently indicated for the treatment of metastatic carcinoma of the ovary and susceptible small cell lung cancer. Topotecan is a semisynthetic structural analogue of the naturally occurring alkaloid camptothecin, which inhibits topoisomerase I, an enzyme that functions to facilitate DNA replication during cell division. Topotecan binds covalently to the enzyme and single-stranded DNA, ultimately resulting in both single- and double-stranded DNA breaks, which cause cell death. To date, no data have been published in clinical trials or in vitro studies that show topotecan to be an effective treatment for coronavirus-related disease in humans.

[0039]

[0043] After oral administration, topotecan is rapidly absorbed, with peak plasma concentrations occurring within 1-2 hours, and is widely distributed to tissues after IV administration. It undergoes pH-dependent hydrolysis to the inactive ring-open hydroxyl acid form, which is prevalent in plasma. Topotecan is excreted both via bile and urine. The plasma elimination half-life is 2-3 hours.

[0040]

[0044] The dose-limiting toxicity associated with conventional topotecan therapy is myelosuppression. Other commonly reported effects include nausea and / or vomiting, headache, alopecia (hair loss), diarrhea or constipation, abdominal pain, fatigue / weakness, body / back / joint pain, rash, and respiratory distress (dyspnea). All effects are reversible with cessation of treatment. The most severe adverse effects reported include interstitial lung disease and severe cases of extravasation and tissue injury.

[0041]

[0045] Based on the mechanism of action and the effects noted in repeated dose and developmental toxicity studies, the possibility that topotecan may adversely affect rapidly dividing cells, including sperm and the developing fetus, cannot be excluded in the absence of conclusive data. Furthermore, based on the mechanism of action and the positive genotoxicity findings, topotecan should be considered a possible carcinogen.

[0042]

[0046] In view of the prior art, potential applications of the use of topotecan to achieve several therapeutic benefits with respect to infections by coronaviruses, such as SARS-CoV-2, have been identified.

[0043]

[0047] According to embodiments of the present disclosure, topotecan may be used for the treatment of COVID-19 and / or symptoms thereof, and / or prophylaxis to avoid or mitigate infection following exposure to the SARS-CoV-2 virus.

[0044]

[0048] Some embodiments of the present disclosure relate to a method of treating a subject exposed to the SARS-CoV-2 virus. The steps of the method include providing a therapeutically effective amount of topotecan and administering the therapeutically effective amount to a subject. The therapeutically effective amount of topotecan may ameliorate or inhibit some or substantially all of the symptoms of a coronavirus-associated disease. While SARS-CoV-2 virus and COVID-19 associated diseases are provided as examples of coronaviruses and associated diseases, one of skill in the art will understand that the present disclosure is not limited to only SARS-CoV-2 and COVID-19 - other coronaviruses and associated diseases are contemplated herein.

[0045]

[0049] Some embodiments of the present disclosure relate to a method of forming a complex between topotecan and a target cell. The method includes administering a therapeutically effective amount of topotecan to a target cell and forming a target cell / topotecan complex. The complex may inhibit fusion of coronavirus with the target cell. The complex may inhibit entry of coronavirus particles into the target cell. The complex may inhibit replication of coronavirus within the target cell. Some embodiments of the present disclosure relate to administering a therapeutically effective dose of topotecan to form a target cell / topotecan complex.

[0046]

[0050] Some embodiments of the present disclosure relate to the use of topotecan to treat a subject exposed to or infected with coronavirus. Some embodiments of the present disclosure relate to a pharmaceutical composition useful for treating a subject exposed to or infected with coronavirus. The pharmaceutical composition comprises a therapeutically effective amount of topotecan and one or more excipients.

[0047]

[0051] In some embodiments of the present disclosure, topotecan may be used as a therapeutic agent. By way of non-limiting example, topotecan may be provided in one or more pharmaceuticals that deliver a dose of 100 ng to 100 mg of topotecan.

[0048]

[0052] In some embodiments of the present disclosure, topotecan may be administered intravenously (IV). When administered by IV, the therapeutically effective amount of topotecan is about 1.5 mg / m per day for five consecutive days. 2 This can occur once, twice or three times over the first three week cycle. This dose can be about 0.75-2 mg / m for each administration during the first cycle. 2 The second cycle may be given 1, 2, 3 or 4 times over a period of 3 to 4 weeks, which may be in the range of about 1.25 mg / m2 for 24 hours on 5 consecutive days. 2The third cycle may be administered once a week for about 4-6 weeks, which may include about 1-2 mg / m2 per day for 5 consecutive days. Those skilled in the art will recognize that subjects experiencing myelosuppression may be treated with reduced or delayed dosing of topotecan.

[0049]

[0053] In some embodiments of the present disclosure, target cells may be exposed to a therapeutically effective amount when treated with topotecan at a concentration of about 50 nM to about 2200 nM, about 75 nM to about 2100 nM, about 100 nM to about 2000 nM, about 150 nM to about 1800 nM, about 200 nM to about 1600 nM, about 300 nM to about 1400 nM, about 400 nM to about 1200 nM, about 500 nM to about 1000 nM, about 600 nM to about 800 nM, and all concentration ranges therebetween.

[0050]

[0054] In some embodiments of the present disclosure, the pharmaceutical composition further comprises one or more carriers and / or one or more excipients. In a non-limiting example of the present disclosure, the pharmaceutical composition comprises topotecan, at least one monoglyceride, and / or at least one diglyceride and / or tocopherol polyethylene glycol succinate. In a further non-limiting example of the present disclosure, the pharmaceutical composition may further comprise a lipid-based carrier, such as an emulsion of micro-sized particles and / or nanoparticles. At least one example of a suitable nanoparticle comprises poly(lactide-co-glycolide). EXAMPLES

[0051] Working Example

[0055] Using the DeepDrug™ computational artificial intelligence (AI) system, we identified that topotecan is likely to be effective against SARS-CoV-2 based on its similarity to antiviral peptides (AVPs) known to target SARS-CoV-1 and other viruses. AVPs are fragments of human proteins that respond to viral infection by targeting key steps in the viral replication cycle, including (1) virus binding to the cell surface and internalization into the endosomal compartment (entry), (2) virus release from the endosomal compartment into the cytosol (fusion), and (3) viral protein processing and replication of the viral genome (replication) protein-protein binding.

[0052]

[0056] AI techniques were used to generate "fingerprints" for topotecan and AVP in a mathematical representation of all protein interactions in the cell (as described by Grover, A. & Leskovec, J. (2016) node2vec: Scalable Feature Learning for Networks. Kdd, 2016, 855-864). This mathematical representation was generated based on the following datasets: AVPdb, a dataset of 2,683 AVPs, including 98 from SARS-CoV-1; HPIDB, a dataset of 981 HIV AVPs; hu.map, a dataset of 17.5 million protein-protein interactions; Corum, a dataset of 4,274 mammalian protein complexes; STRING, a dataset of 4,584,628 proteins from 5,090 organisms; DrugBank, a dataset of 13,491 drugs; and BindingDB, a dataset of 846,857 drugs and 7,605 proteins.

[0053]

[0057] An AI technique called Siamese Network (SNet) was used to compare the fingerprints of topotecan with those of AVP. SNet predictions were based on a small number of SARS-CoV-1 AVPs that showed the strongest antiviral effects. In addition, SNet provided separate predictions for the three mechanisms of viral infection (e.g., entry, fusion, and replication), which led to a higher degree of specificity in drug screening. SNet projects the fingerprints into a multidimensional space and calculates the distance between them; the closer the prediction is to 0, the more similar the fingerprint pair is and the more similar the drug is to AVP. Predictions below the optimal threshold of 0.63 indicate significant similarity between the fingerprints of the drug and AVP (i.e., the drug with antiviral effects).

[0054]

[0058] Topotecan achieved significance (i.e., SNet distances less than 0.63) for all three of the viral mechanisms (e.g., entry, fusion, and replication). Table A below summarizes the SNet prediction data for topotecan.

[0055] [Table 1]

[0056]

[0059] An evaluation of the potential of topotecan to bind coronavirus particles was performed. Three different mechanistic potential binding sites for the small molecule were used to determine protein-protein binding potential using an AI system. A template of the crystal structure of the essential SARS-CoV-2 protease was used to identify the functional center of the protease inhibitor binding pocket.

[0057]

[0060] Antiviral peptides known to inhibit the SARS virus were used as targets by generating fingerprints of the AVPs, which were then compared to similarly generated fingerprints of topotecan (embedded) to identify those with the closest relatives.

[0058]

[0061] The AVPs used targeted three specific mechanisms: invasion, fusion and replication. The most effective peptides were specifically selected and used to generate three separate networks based on the known mechanism of action of each peptide. This allowed the identification of drugs with specific specificity based on mechanism.

[0059]

[0062] The three mechanisms are relevant for the following reasons: Entry is extremely important because inhibiting viral entry into the cell reduces the amount of virus that acts on the cell. Similarly, inhibiting replication is important because it reduces the amount of viral load that occurs after the cell is infected and spreads to other cells. Finally, fusion is the least technically relevant, but is notable because not all viral entry occurs via the standard mechanism. Viruses can directly fuse with the cell's membrane for infection. Although this occurs at about one-tenth the rate of the standard entry mechanism, it is still a desirable mechanism to use as a focus to try to inhibit.

[0060]

[0063] These specific peptide fingerprints were generated by using a large graph of the human proteome and the proteins involved in all processes within it, and these fingerprints were then compared with drug fingerprints to identify drugs that have similar (antiviral) effects on the human proteome as AVP.

[0061]

[0064] First binding mechanism

[0065] Topotecan was studied to determine its propensity to bind to coronavirus particles by the first binding mechanism. The interaction was further evaluated by assessing the potential for topotecan to affect coronavirus entry into mammalian cells; coronavirus particle fusion with mammalian cells; and ultimately coronavirus-infected cell replication. Table 1 summarizes the data obtained from this first round of modeling data analysis.

[0062] [Table 2]

[0063]

[0066] Without being bound by any particular theory, the data collected in the first binding mechanism study for topotecan showed theoretical trends that may affect SARS-CoV-2 viral replication within host cells.

[0064]

[0067] In vitro testing

[0068] The in vitro efficacy of topotecan against SARS-CoV-2 (USA-WA1 / 2020 isolate) was evaluated in human lung cancer (Calu-3) cells. A stock solution of 4 μM topotecan was prepared in DMSO and tested at eight concentrations: 5000 nM, 1670 nM, 555.6 nM, 185.2 nM, 61.7 nM, 20.6 nM, 6.9 nM, and 2.3 nM. Calu-3 cells were cultured in 96-well plates and tested in triplicate. A pretreatment / treatment regimen was utilized, where cells were incubated with topotecan for 24 ± 4 hours and then 0.005 TCID per cell. 50 Multiplicity of infection (MOI) (median tissue culture infectious dose) (200TCID 50 SARS-CoV-2 was inoculated into wells (0.2 mL / well) and incubated for 60–90 min. Immediately after incubation, the virus inoculum was removed, cells were washed, and wells were covered with 0.2 mL of Eagle's modified essential medium (EMEM) with 2% fetal bovine serum (FBS) containing topotecan or control and incubated at 37 °C ± 2 °C in 5 ± 2% CO2 in a humidified chamber. Cells were fixed 48 ± 6 h after virus inoculation and evaluated for the presence of virus by immunostaining assays (for cytopathic effect (CPE) by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay).

[0065]

[0069] For each well on the assay plate, inhibition of SARS-CoV-2 was measured using the positive control (A 450 Virus control; no drug and 0% inhibition) and negative control (cell control A 450Absorbance values ​​(A of topotecan dilutions) relative to the average absorbance values ​​from wells designated as (no virus and 100% inhibition). 450 ) was calculated as a percentage of reduction in

[0001]

number

[0066]

[0070] Effective concentration (IC 50 ) was defined as the concentration of topotecan that resulted in a 50% reduction in the mean absorbance value of the virus control (0% inhibition) relative to the cell control (100% inhibition). The cytotoxicity of topotecan at eight concentrations was also assessed as determined by the percent inhibition of cell viability.

[0067]

[0071] IC of 35 nM 50 Complete or near-complete inhibition of SARS-CoV-2 with cytotoxicity was determined for topotecan (see Figure 1). 50 ) was 169 nM, indicating a relatively wide margin of safety between the effective and cytotoxic concentrations reported in these human lung cells.

[0068]

[0072] Further experimental testing

[0073] Figure 2 shows in vitro cell viability data for topotecan according to the above protocol, and Figure 3 shows in vitro viral replication inhibition data. Figure 4A shows in vitro cell viability data, and Figure 4B shows in vitro viral inhibition data according to the above protocol for remdesivir as a control.

[0069]

[0074] Table 2 shows the IC obtained from the in vitro inhibition data shown in Figures 3 and 4B. 50 Summarize the values.

[0070] [Table 3]

[0071]

[0075] Without wishing to be bound by any particular theory, IC 50 Data collected from the study showed that topotecan had a significantly lower IC compared with remdesivir. 50 It was shown that

Claims

1. 1. A method of treating a subject exposed to a coronavirus, comprising: (a) providing a therapeutically effective amount of topotecan; (b) administering said therapeutically effective amount to said subject; wherein said treating comprises ameliorating and / or inhibiting some or substantially all of the symptoms of coronavirus-related disease in said subject.

2. 2. The method of claim 1, wherein the coronavirus is SARS-CoV-2 or a variant thereof.

3. 1. A method for forming a target cell / topotecan complex, comprising: (a) administering a therapeutically effective amount of topotecan to said target cells; (b) forming said target cell / topotecan complex; wherein the target cell / complex inhibits fusion of the coronavirus with the target cell, inhibits entry of the coronavirus into the target cell, and / or inhibits replication of the coronavirus within the target cell.

4. 1. A method for forming a target virion / topotecan complex, comprising: (a) administering a therapeutically effective amount of topotecan to the target virions; (b) forming the target virion / topotecan complex; wherein the target virion / complex inhibits fusion of the target virion with a target cell, inhibits entry of the target virion into the target cell, and / or inhibits replication of the target virion within the target cell.

5. The method of claim 3 or 4, wherein the therapeutically effective amount is from about 75 nM to about 2100 nM.

6. The method of claim 3 or 4, wherein the therapeutically effective amount is about 100 nM to 2000 nM.

7. 4. The method of claim 3, wherein the coronavirus is SARS-CoV-2 or a variant thereof.

8. 5. The method of claim 4, wherein the target virion is SARS-CoV-2 or a variant thereof.

9. 4. The method of claim 3, wherein the target cells are epithelial cells (ciliated and non-ciliated) of the upper respiratory tract and conducting airways; alveolar epithelial cells (either type 1 or type 2); epithelial cells of the olfactory system, neurons of the olfactory system; neurons of the central nervous system, neurons of the peripheral nervous system; epithelial cells of the gastrointestinal tract, enterocytes of the gastrointestinal tract, glandular cells of the gastrointestinal tract; white blood cells, red blood cells, platelets, immune effector cells; cardiovascular cells and renal cells.

10. Use of topotecan to treat a subject exposed to or infected with coronavirus.

11. The use according to claim 10, wherein the coronavirus is SARS-CoV-2 or a variant thereof.

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