Use of proteasome inhibitors in the treatment of coronavirus infections

Abstract

An oral formulation comprising one or more proteasome inhibitors for use as a treatment for coronavirus infection in a subject. Use of such for the treatment or prevention of SARS-CoV-2 infection in humans is also disclosed.

Description

[Technical Field]

[0001] The present disclosure relates to the treatment of coronavirus diseases, including the use of oral formulations of proteasome inhibitors for the treatment of COVID-19 infection in humans. [Background technology]

[0002] The global emergence of a novel coronavirus, designated SARS-CoV-2, has affected virtually the entire world population. The virus has afflicted millions of individuals and caused the disease designated COVID-19. COVID-19 poses significant health risks, can be fatal, and places a high burden on healthcare resources and society at large.

[0003] SARS-CoV-2 is a single-stranded, positive-sense 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 access the nasal mucosa. Within the nasal mucosa, SARS-CoV-2 rapidly replicates and can be excreted in nasal secretions (sputum). The sputum can then be transmitted to other individuals via droplets, thus repeating the cycle of transmission. The SARS-CoV-2 virus can spread between individuals before the onset of symptoms, during symptoms, and even after recovery.

[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, its primary route of entry into the host; however, SARS-CoV-2 can also affect multiple organs in infected individuals. The severity of COVID-19 is usually associated with comorbidities, including, but not limited to, hypertension, diabetes, obesity, and / or advanced age, which may worsen the outcome of COVID-19.

[0005] Several vaccines against SARS-CoV-2 are now available and form an important part of health authorities' recommendations to mitigate the COVID-19 public health crisis and manage the stress that COVID-19 has placed on healthcare workers and hospitals. However, the SARS-CoV-2 virus has already demonstrated the ability to mutate into a variety of different variants, some of which are more transmissible than others and have proven successful in evading the protection offered by current vaccines.

[0006] There is a need for therapies that can mitigate the effects of the SARS-CoV-2 virus in a manner that delays, prevents, and / or treats the physiological effects on infected individuals. Summary of the Invention

[0007] Embodiments of the present disclosure provide one or more therapies 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.

[0008] Some embodiments of the present disclosure relate to the use of one or more proteasome inhibitors to ameliorate and / or inhibit some or substantially all of the risk, symptoms, and development of severe disease caused by coronavirus infection.

[0009] Some embodiments of the present disclosure relate to the use of an oral formulation comprising one or more proteasome inhibitors to prevent SARS-CoV-2 infection in a subject.

[0010] Some embodiments of the present disclosure relate to the use of an oral formulation comprising one or more proteasome inhibitors to treat a SARS-CoV-2 infection in a subject.

[0011] Some embodiments of the present disclosure relate to the use of an oral formulation comprising one or more proteasome inhibitors to prevent replication of the SARS-CoV-2 virus in a subject.

[0012] Some embodiments of the present disclosure relate to a method of treating an individual exposed to or infected with coronavirus, comprising providing a therapeutically effective amount of one or more proteasome inhibitors and administering the therapeutically effective amount of the one or more proteasome inhibitors to the individual to ameliorate and / or inhibit some or substantially all of the risk, symptoms, and development of severe disease in the subject exposed to or infected with coronavirus.

[0013] Some embodiments of the present disclosure relate to methods of making a drug / target cell complex, the method comprising administering a therapeutically effective amount of a drug to a subject, wherein the drug / target cell complex inhibits, delays, or prevents virion particles from entering, fusing with, and / or replicating within a target cell of which the complex is formed. In some embodiments of the present disclosure, the drug comprises one or more proteasome inhibitors.

[0014] Some embodiments of the present disclosure relate to a method of producing a drug / target virion complex, the method comprising administering a therapeutically effective amount of a drug to a subject, wherein the drug / target virion complex inhibits the drug / target cell complex from entering, fusing with, and / or replicating within the subject's cells. In some embodiments of the present disclosure, the drug comprises one or more proteasome inhibitors.

[0015] In an embodiment of the present disclosure, the proteasome inhibitor comprises bortezomib, ixazomib, carfilzomib, or a combination thereof. Ixazomib is currently available for oral administration and may have improved activity over bortezomib and / or carfilzomib. Both bortezomib and carfilzomib are typically administered by injection or intravenously. Bortezomib is a slow, reversible inhibitor (dissociation half-life = 110 minutes) of the β1 caspase-like subunit and β2 trypsin-like subunit, preferentially over the β5 chymotrypsin-like subunit of the 20S proteolytic site of the proteasome. Carfilzomib is an irreversible inhibitor with high specificity for the β5 chymotrypsin-like subunit of the proteasome. Ixazomib and bortezomib are mechanistically similar, and both have greater affinity for the β5 chymotrypsin-like subunit of the proteasome. However, ixazomib has a dissociation half-life of 18 minutes, which is thought to contribute to its excellent tissue penetration. Proteasomes are highly concentrated in blood cells, and bortezomib is known to maintain longer exposure in the circulation, where it exerts most of its inhibitory activity. At higher concentrations, ixazomib can also inhibit other proteolytic sites (e.g., β1, β2).

[0016] Initial in vitro studies described herein show promise in treating both kidney and lung cells with one or more proteasome inhibitors when these cells are exposed to the SARS-CoV-2 virus, with EC50 values ​​in the low nM range, demonstrating specificity and potency against SARS-CoV2.

[0017] Current formulations of bortezomib are limited to IV administration or subcutaneous injection. Advantages include 100% bioavailability and wide distribution to peripheral tissues. The time to peak plasma levels after IV administration is approximately 5 minutes. In vitro binding of IV-administered bortezomib to human plasma proteins averaged 83%.

[0018] Cyclin and CDK inhibitors regulate the activity of CDKs, which in turn regulate these proteins through the proteasome. Similarly, it has been shown that the combination of citreoviridin with the 26S proteasome inhibitor bortezomib can improve anticancer activity by enhancing ER stress, improving cyclin D3 compensation caused by citreoviridin, and contributing to CDK1 (cyclin-dependent kinase 1) inactivation and PCNA downregulation. Given this, the inventors hypothesized that bortezomib may be effective against SARS-CoV-2 because it inhibits CDK activity.

[0019] In vitro data show that SARS-CoV-2-infected Vero E6 cells treated with bortezomib demonstrated inhibition of virus-induced cytopathic effects at a concentration of 0.05 μM. However, cytotoxicity was observed at 0.002 μM. Cytotoxicity and inhibition of virus-induced cytopathic effects were observed at doses above 30 μM with shorter treatment schedules. Further data demonstrate efficacy in infected kidney and lung cells. However, oral dosage formulations of bortezomib may address these shortcomings.

[0020] In view of the above, the usefulness of bortezomib may be limited by its route of administration, narrow therapeutic index, the frequency and / or severity of clinical adverse effects (e.g., respiratory distress, cardiovascular problems) that may be observed after chronic exposure, including effects on the developing fetus and its potential to impair fertility.

[0021] Without being bound to any particular theory, the proteasome inhibitor bortezomib inhibits CDK activity and may therefore be useful in treating and / or preventing SARS-CoV-2 infection. Bortezomib is known to target proteasome subunit beta type 1 (0.5 nM), type 2 (N / A), type 5 (0.5 nM), type 7 (7 nM); type 8 (17 nM); 20S proteasome chymotrypsin-like (1.90 nM); proteasome subunit beta type 1 / beta type 5 (4 nM); proteasome subunit beta type 7 (7 nM); 26S proteasome (8.10 nM); proteasome subunit beta type 8 (17 nM); proteasome component C5 (30 nM); proteasome component C5 (130 nM); proteasome; macropein subunit (440 nM); and cathepsin G (520 nM). Bortezomib is known to target cathepsin A (9200 nM), cathepsin B (greater than 3000 nM), and cathepsin G (520 nM); chymase (mast cell protease 1) (1190 nM); multidrug resistance-associated protein 4 (ABCC4) (133 μM), canalicular multispecific organic anion transporter 1 (ABCC2) (133 μM), canalicular multispecific organic anion transporter 2 (ABCC3) (133 μM), and bile salt export pump (ABCB11) (133 μM).

[0022] Without being bound by any particular theory, the proteasome inhibitor ixazomib is known to target proteasome subunit beta types 1 (7.7 nM), 2 (N / A), and 5 (7.7 nM), and therefore, by its mechanism of action, may be useful in treating and / or preventing SARS-CoV-2 infection.

[0023] Without being bound by any particular theory, the proteasome inhibitor carfilzomib may be useful in treating and / or preventing SARS-CoV-2 infection due to its mechanism of action, as it is known to target proteasome subunit beta types 5 (9.6 nM), 7 (8.6 nM), and 8 (N / A); cathepsin A (greater than 30 μM), cathepsin B (11 μM), and cathepsin G (greater than 30 μM). [Brief explanation of the drawings]

[0024] Features of the present disclosure will become more apparent in the following detailed description, which refers to the accompanying drawings, which illustrate one or more embodiments of the present disclosure by way of example only and are not to be construed as limiting the scope of the present disclosure. [Figure 1] 1 is a graphical representation of the concentration-response curve for remdesivir from the second study series. [Figure 2] 1 is a graphical representation of the concentration-response curve for bortezomib from a second study series. [Figure 3] 1 is a graphical representation of the concentration-response curve for remdesivir in Calu3 cells. [Figure 4] 1 is a graphical representation of the concentration-response curve for carfilzomib in Calu3 cells. DETAILED DESCRIPTION OF THE INVENTION

[0025] Unless otherwise defined, all technical and scientific terms used herein have the meanings that would be commonly understood by those skilled in the art in the context of this description. Although any methods and materials similar or equivalent to those described herein can also 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.

[0026] 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.

[0027] 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%, and 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 referred to.

[0028] As used herein, the term "activity" is used interchangeably with the term "functionality," and both terms refer to the physiological action of a biomolecule.

[0029] As used herein, the terms "drug" and "therapeutic agent" refer to a substance that, when administered to a subject, elicits 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 immunological responses in the subject.

[0030] As used herein, the term "improve" refers to making better and / or making better and / or making more satisfactory.

[0031] 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. Administration of an agent to a cell includes in vivo, in vitro, and ex vivo administration and / or combinations thereof.

[0032] As used herein, the term "complex" refers to a direct or indirect association between one or more particles of a drug 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 virions. As used herein, the phrase "metabolic alteration" refers to an increase or decrease in the production of one or more protein targets and / or one or more of any post-translational modifications of one or more proteins. As used herein, the phrase "functional alteration" refers to a difference in the physiological function of one or more aspects of the target within a drug / target complex compared to a target that is not part of such a complex.

[0033] As used herein, the terms "dysregulation" and "dysregulated" refer to a situation or condition in which a homeostatic control system is disrupted and / or impaired, such that one or more metabolic, physiological and / or biochemical systems within a subject operate partially or completely without that homeostatic control system.

[0034] As used herein, the term "excipient" refers to any substance that is not itself a drug but that can be used in a composition to deliver one or more drugs or the like to a subject, or alternatively, can be used in combination with one or more carriers or the like (e.g., to create a pharmaceutical composition) to improve its handling or storage characteristics or to enable or facilitate the formation of a dosage unit of the composition (e.g., the formation of a topical hydrogel, which can then optionally be incorporated into a transdermal patch). Excipients include, by way of example and not limitation, binders, disintegrants, taste enhancers, solvents, thickeners or gelling agents (and optional neutralizing agents, if desired), penetration enhancers, solubilizers, humectants, antioxidants, lubricants, emollients, substances added to mask or neutralize unpleasant odors, fragrances, or tastes, substances added to improve the appearance or texture of a composition, and substances used to form a pharmaceutical composition. Any such excipient can be used in any dosage form according to the present disclosure. The foregoing classes of excipients are not intended to be exhaustive, but merely exemplary.

[0035] 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, a complete ablation 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.

[0036] As used herein, the term "pharmaceutical product" refers to a medicinal product and / or pharmaceutical composition, including a drug, that can promote recovery from a disease, disorder, or symptom thereof, and / or that can prevent a disease, disorder, or symptom thereof, and / or that can inhibit the progression of a disease, disorder, or symptom thereof.

[0037] As used herein, the term "pharmaceutical composition" refers to any composition containing, but not necessarily limited to, one or more pharmaceutical agents to be administered to a subject in need of therapy or treatment for a disease, disorder, or its symptoms. Pharmaceutical compositions may include additives such as pharmaceutically acceptable carriers, pharmaceutically acceptable salts, excipients, etc. Pharmaceutical compositions may also further include one or more additional active ingredients (e.g., antibacterial agents, anti-inflammatory agents, anesthetic agents, analgesics, etc.).

[0038] As used herein, the term "pharmaceutically acceptable carrier" refers to an essentially chemically inert and non-toxic ingredient in a pharmaceutical composition or drug that does not interfere with the efficacy and / or safety of one or more drugs. Some examples of pharmaceutically acceptable carriers and their formulations are described in Remington (1995, The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA), the disclosure of which is incorporated herein by reference. Typically, an appropriate amount of a pharmaceutically acceptable carrier is used in the formulation to make the formulation isotonic. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, saline solution, glycerol solution, ethanol, N-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), dioleolphosphotidylethanolamine (DOPE), and liposomes. Such pharmaceutical compositions contain a therapeutically effective amount of a drug together with a suitable amount of one or more pharmaceutically acceptable carriers and / or excipients to provide a suitable form for proper administration to a subject. The formulation is suitable for the administration route. For example, oral administration may require an enteric coating to protect the drug from degradation in the subject's gastrointestinal tract. In another example, an injectable administration route may be administered in a liposomal formulation to facilitate transport throughout the subject's vascular system and facilitate delivery across the cell membrane of the target intracellular site.

[0039] As used herein, the phrases "prevent," "prevention of," and "preventing" refer to averting the onset or progression of a disease, disorder, or symptom thereof.

[0040] As used herein, the term "subject" refers to any therapeutic target that receives a drug. A subject may be a vertebrate, e.g., 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.

[0041] As used herein, the term "target cell" refers to one or more cell types within a subject with which the virus can interact with a coronavirus by fusing with the outer membrane of one or more cell types, entering the cell, and / or replicating within it. Without being bound by any particular theory, target cells of a subject can include any cell within a subject that expresses receptors and / or cofactors necessary for viral interaction. Examples of these types of cells include, but are not limited to, epithelial cells of the upper and conducting airways (ciliated and non-ciliated), 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 of the gastrointestinal tract, enterocytes and glandular cells, cells of the blood, including immune effector cells, cardiovascular cells, and renal cells.

[0042] As used herein, the terms "target virion" and "virion" refer to one or more viral particles of a coronavirus that have the ability to cause a viral infection in a target cell. In some embodiments of the present disclosure, the viral particles are of one or more variants of SARS-CoV-2.

[0043] As used herein, the term "therapeutically effective amount" refers to the amount of a drug used that is sufficient to ameliorate, 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 can also affect the amount of drug that is therapeutically effective.

[0044] 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 that it completely or partially prevents the onset of a disease, disorder, or symptom thereof, and / or the effect may be therapeutic, in that it provides a partial or complete improvement or inhibition of the disease, disorder, or symptom thereof. Furthermore, the term "treatment" refers to any treatment of a disease, disorder, or symptom thereof in a subject, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., arresting its progression, and (c) ameliorating the disease.

[0045] As used herein, the terms "unit dosage form" and "unit dose" refer to a physically discrete unit suitable as a unitary dose for a patient. Each unit contains a predetermined amount of drug and, optionally, one or more suitable pharmaceutically 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.

[0046] In an embodiment of the present disclosure, the pharmaceutical compositions disclosed herein contain from about 0.1% to about 95% of one or more agents as described above, based on the total weight of the 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%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, or about 3.0%. %, approx. 2.9%, approx. 3%, approx. 3.1%, approx. 3.2%, approx. 3.3%, approx. 3.4%, approx. 3.5%, approx. 3.6%, approx. 3.7%, approx. 3.8%, approx. 3.9%, approx. 4%, approx. 4.1%, approx. 4.2%, approx. 4.3%, approx. 4.4%, approx. 4.5%, approx. 4.6%, approx. 4.7%, approx. 4.8%, approx. 4.9%, approx. 5%, approx. 5.1%, approx. 5.2%, approx. 5.3%, approx. 5.4%, approx. 5.5%, approx. 5.6%, approx. 5.7%, approx. 5.8%, approx. 5.9%, approx. 6% ,approx. 6.1%,approx. 6.2%,approx. 6.3%,approx. 6.4%,approx. 6.5%,approx. 6.6%,approx. 6.7%,approx. 6.8%,approx. 6.9%,approx. 7%,approx. 7.1%,approx. 7.2%,approx. 7.3%,approx. 7.4%,approx. 7.5%,approx. 7.6%,approx. 7.7%,approx. 7.8%,approx. 7.9%,approx. 8%,approx. 8.1%,approx. 8.2%,approx. 8.3%,approx. 8.4%,approx. 8.5%,approx. 8.6%,approx. 8.7%,approx. 8.8%,approx. 8.9%,approx. 9%,approx. 9.1%,approx. 9. It may be 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.

[0047] Where a range of values ​​is provided herein, it is understood that each intervening value between the upper and lower limit of that range, to one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0048]

[0003] Embodiments of the present disclosure relate to the use of one or more proteasome inhibitors to prevent, reduce, and / or treat infection by the SARS-CoV-2 virus in a subject. In embodiments of the present disclosure, the proteasome inhibitor comprises bortezomib, ixazomib, carfilzomib, or a combination thereof.

[0049] Bortezomib is indicated for the treatment of patients with multiple myeloma and mantle cell lymphoma. Bortezomib is administered as a combination therapy and can be administered subcutaneously (SC) or intravenously (IV). The recommended starting dose for injection is 1.3 mg / m² (based on an average body surface area of ​​1.8 m², corresponding to 2.3 mg / day if administered daily).

[0050] Bortezomib is currently used as a potent, selective, and reversible inhibitor of the 26S proteasome, a large protein complex that degrades ubiquitinated proteins in mammalian cells. The ubiquitin-proteasome pathway is important for regulating the intracellular concentrations of specific proteins to maintain intracellular homeostasis. Blocking this pathway in cancer cells can affect multiple cell signaling cascades that lead to NF-κB activation, inhibition of cell cycle progression, and the initiation of apoptosis. Proteasome inhibition can also lead to the accumulation of cyclin-dependent kinase (CDK) inhibitors, such as p27.

[0051] In vitro studies have shown that bortezomib inhibits virus-induced cytopathic effect (CPE) in SARS-CoV-2-infected Vero E6 cells at 0.05 μM. However, undesirable cytotoxicity occurred at 0.002 μM. At shorter drug treatment times, bortezomib was as effective as chloroquine (achieved at 15 μM) and failed to completely prevent CPE at doses above 30 μM.

[0052] Furthermore, bortezomib has been identified as a drug with potential activity against SARS-CoV-2 based on analytical and computational approaches. The open reading frame 10 (ORF10) viral protein has been identified as a key protein involved in the highly infectious nature of SARS-CoV-2 virus particles and has been reported to interact with an E3 ligase complex that plays a role in targeting cellular proteins for ubiquitination by the 26S proteasome. This suggests that ORF10 may bind to the proteasome complex and utilize it for the ubiquitination and degradation of restriction factors and other essential cellular proteins. Furthermore, bortezomib was classified as a cytotoxic drug in Vero E6 cells by an algorithmic prediction study using artificial intelligence, network diffusion, and network proximity to rank a large number of drugs for their predicted efficacy against SARS-CoV-2.

[0053] The SARS-CoV-2 virus has been shown to increase the phosphorylation and activation of CDKs, leading to an increased supply of essential nucleotides, DNA repair, and replication proteins, which are essential for viral replication. Without being bound by any particular theory, CDK inhibitors are a potential therapy for the treatment of SARS-CoV-2 virus infection, and inhibition of the proteasome leads to the accumulation of CDK inhibitors and downregulation of NF-κB-mediated inflammation. Therefore, the use of proteasome inhibitors may be advantageous in the context of alleviating SARS-CoV-2 infection and reducing the severity of COVID-19.

[0054] The unfolded protein response (UPR) is a signaling pathway activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER) of cells. Activation of this pathway leads to increased production of molecular chaperones, suppression of protein translation, and accelerated degradation of misfolded proteins. The SARS-CoV-2 virus utilizes the endogenous transcription machinery to generate viral proteins, and rapid viral replication often results in the accumulation of unfolded viral polypeptides in the ER. When the system is overloaded with viral proteins, endogenous protein production is suppressed, leading to cell death. Proteasome inhibitors initiate the UPR through the induction of protein kinase R-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4) to remove unfolded or aggregated proteins. Without being bound by any particular theory, one or more proteasome inhibitors may increase the ability of the UPR to maintain normal cellular integrity and function. Pharmacological chaperone therapy is being investigated to treat COVID-19 patients, however, prolonged UPR activation and severe ER stress may be associated with other disease states (e.g., Alzheimer's disease, pulmonary fibrosis).

[0055] Given the current state of the art, there is a need for therapeutic compounds that can reduce and / or ameliorate the physiological effects of the SARS-CoV-2 virus on infected individuals. The present disclosure fulfills this need by providing compositions and methods for the treatment of coronavirus infections, including SARS-CoV-2 infections, and related diseases and disorders.

[0056] In some embodiments of the present disclosure, the coronavirus is SARS-CoV-2, and the use of one or more proteasome inhibitors can inhibit replication of the SARS-CoV-2 virus. In some embodiments of the present disclosure, the subject may be infected with SARS-CoV-2, or may be at risk for infection by SARS-CoV-2, or may have been diagnosed with or is suspected of having COVID-19. In some embodiments of the present disclosure, the subject is a human. In some embodiments of the present disclosure, the proteasome inhibitor is provided orally or formulated for oral administration. In some embodiments of the present disclosure, the subject is provided with an oral formulation of bortezomib, including, but not limited to, the oral formulations set forth in any of Tables 4-8 herein below.

[0057] Protein-protein binding We performed a thorough evaluation of the potential binding of small therapeutics to COVID-19 virus particles. Three different mechanistic potential binding sites for small molecules were used to determine protein-protein binding potential. 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.

[0058] Antiviral peptides known to inhibit the SARS virus were used as targets, and fingerprints of these antiviral peptides (AVPs) were then generated to compare them with similarly generated fingerprints of individual drugs to identify the most closely related ones.

[0059] The AVPs used targeted three specific mechanisms: invasion, fusion, and replication. The most effective peptides were specifically filtered out and used to create three separate networks based on each peptide's known mechanism of action. This allowed for the identification of drugs with specific mechanism-based specificity.

[0060] The three mechanisms are related for the following reasons. Entry is crucial because inhibiting viral entry into cells reduces the amount of virus that can reach the cell. Similarly, inhibiting replication is important because it reduces the amount of viral load generated after a cell is infected and spread to other cells. Finally, fusion is noteworthy, though technically the least relevant, since not all viral entry occurs via the standard mechanism. Viruses can fuse directly with the cell membrane for infection. While this occurs at approximately one-tenth the speed of the standard entry mechanism, it is still a desirable mechanism to focus our efforts to inhibit.

[0061] These specific peptide fingerprints were generated using a large graph of the human proteome and all the proteins involved in the processes within it, and these fingerprints were then compared with drug fingerprints to identify drugs with similar (antiviral) effects on the human proteome as AVP.

[0062] First coupling mechanism Several therapeutic compounds were tested to determine their propensity to bind to SARS-CoV-2 viral particles according to the first binding mechanism. This interaction was further evaluated by assessing the therapeutic compounds' potential to affect COVID-19 entry into mammalian cells, fusion of SARS-CoV-2 viral particles with mammalian cells, and ultimately replication of COVID-19-infected cells. Table 1 summarizes the data obtained in this first round of modeling data analysis.

[0063] [Table 1]

[0064] Data collected in the first binding mechanism study showed that the majority of the analyzed compounds (those with a measurement score greater than 0.25) showed a tendency to bind to SARS-CoV-2 virus particles.

[0065] Second coupling mechanism The same therapeutic compounds were subsequently studied to determine their propensity to bind to SARS-CoV-2 viral particles according to a second binding mechanism. This interaction was further evaluated by assessing the potential of the therapeutic compounds to affect COVID-19 entry into mammalian cells, fusion of SARS-CoV-2 viral particles with mammalian cells, and ultimately replication of COVID-19-infected cells. Table 2 summarizes the data obtained in this second round of modeling data analysis.

[0066] [Table 2]

[0067] Data collected in the second binding mechanism study showed that all of the analyzed compounds (those with a measurement score greater than 0.5) showed a tendency to bind to SARS-CoV-2 virus particles.

[0068] Third coupling mechanism The same therapeutic compounds were then studied again to determine their propensity to bind to SARS-CoV-2 viral particles according to a third binding mechanism. This interaction was further evaluated by assessing the therapeutic compounds' potential to affect COVID-19 entry into mammalian cells, fusion of SARS-CoV-2 viral particles with mammalian cells, and ultimately replication of COVID-19-infected cells. Table 3 summarizes the data obtained in this third round of modeling data analysis.

[0069] [Table 3]

[0070] In some embodiments of the present disclosure, there is provided a method of inhibiting coronavirus replication in a mammal in need thereof, comprising providing to the mammal an effective amount of one or more proteasome inhibitors. In some embodiments of the present disclosure, the coronavirus is SARS-CoV-2. In some embodiments of the present disclosure, the subject in need of treatment with one or more proteasome inhibitors has been infected with SARS-CoV-2 or diagnosed with COVID-19.

[0071] In certain embodiments, the one or more proteasome inhibitors are (a) a therapeutically effective amount of one or more proteasome inhibitors; (b) one or more fatty acid glycerol esters; (c) one or more polyethylene oxide-containing phospholipids or one or more polyethylene oxide-containing fatty acid esters, and

[0072] In one embodiment, the one or more proteasome inhibitors are present in the formulation in an amount of about 0.5 to about 10 mg. In one embodiment, the one or more proteasome inhibitors are present in the formulation at about 3.5 mg. In certain embodiments, the formulation comprises (a) one or more proteasome inhibitors, (b) one or more fatty acid glycerol esters, (c) one or more polyethylene oxide-containing fatty acid esters, and, optionally, (d) tocopherol polyethylene glycol succinate.

[0073] In one embodiment, the fatty acid glycerol esters comprise from about 32 to about 52% by weight of fatty acid monoglycerides. In one embodiment, the fatty acid glycerol esters comprise from about 30 to about 50% by weight of fatty acid diglycerides. In one embodiment, the fatty acid glycerol esters comprise from about 5 to about 20% by weight of fatty acid triglycerides. In one embodiment, the fatty acid glycerol esters comprise greater than about 60% by weight of oleic acid monoglycerides, diglycerides, and triglycerides.

[0074] In one embodiment, the polyethylene oxide-containing phospholipid comprises a C8 to C22 saturated fatty acid ester of a phosphatidylethanolamine polyethylene glycol salt. In one embodiment, the polyethylene oxide-containing phospholipid comprises a distearoylphosphatidylethanolamine polyethylene glycol salt. In one embodiment, the distearoylphosphatidylethanolamine polyethylene glycol salt is selected from the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 350 salt, distearoylphosphatidylethanolamine polyethylene glycol 550 salt, distearoylphosphatidylethanolamine polyethylene glycol 750 salt, distearoylphosphatidylethanolamine polyethylene glycol 1000 salt, distearoylphosphatidylethanolamine polyethylene glycol 2000 salt, and mixtures thereof. In one embodiment, the distearoylphosphatidylethanolamine polyethylene glycol salt is present in the formulation in an amount of 1 mM to about 30 mM based on the volume of the formulation. In one embodiment, the distearoylphosphatidylethanolamine polyethylene glycol salt is an ammonium salt or a sodium salt.

[0075] In one embodiment, the polyethylene oxide-containing fatty acid ester comprises a polyethylene oxide ester of a C8 to C22 saturated fatty acid. In one embodiment, the polyethylene oxide-containing fatty acid ester comprises a polyethylene oxide ester of a C12 to C18 saturated fatty acid. In one embodiment, the polyethylene oxide-containing fatty acid ester is selected from the group consisting of laurate, palmitate, stearate, and mixtures thereof. In one embodiment, the polyethylene oxide-containing fatty acid ester comprises a polyethylene oxide having an average molecular weight of about 750 to about 2000. In one embodiment, the ratio of the fatty acid glycerol ester to the polyethylene oxide-containing fatty acid ester is about 20:80 to about 80:20 (v / v). In one embodiment, the ratio of the fatty acid glycerol ester to the polyethylene oxide-containing fatty acid ester is about 60:40 (v / v). In one embodiment, the formulation further comprises glycerol in an amount less than about 10% by weight. In one embodiment, the formulation is a self-emulsifying drug delivery system.

[0076] In certain embodiments, the formulation further comprises tocopherol polyethylene glycol succinate. In certain embodiments, the tocopherol polyethylene glycol succinate is present in the formulation at about 0.1 to about 10 volume percent based on the total volume of the formulation. Structurally, tocopherol polyethylene glycol succinate has polyethylene glycol (PEG) covalently attached to a tocopherol (e.g., α-tocopherol or vitamin E) via a succinate linker. Because PEG is a polymer, TPGS can be prepared using a variety of polymer molecular weights. In one embodiment, the TPGS is tocopherol polyethylene glycol succinate 1000, in which the average molecular weight of the PEG is 1000. One suitable tocopherol polyethylene glycol succinate is Vitamin E TPGS, commercially available from Eastman. In one embodiment, the tocopherol polyethylene glycol succinate is present in the formulation at about 5 volume percent based on the total volume of the formulation. In one embodiment, the formulation further comprises glycerol in an amount less than about 10% by weight. In one embodiment, the formulation is a self-emulsifying drug delivery system.

[0077] Certain bortezomib formulations disclosed herein include one or more fatty acid glycerol esters, typically a mixture of fatty acid glycerol esters. The fatty acid glycerol esters useful in the formulations can be provided by commercially available sources. A representative source of fatty acid glycerol esters is the mixture of monoesters, diesters, and triesters commercially available as PECEOL® (Gattefosse, Saint-Priest-Cedex, France), commonly referred to as "glyceryl oleate" or "glyceryl monooleate." When PECEOL® is used as the source of fatty acid glycerol esters in the formulation, the fatty acid glycerol esters comprise about 32 to about 52% by weight of fatty acid monoglycerides, about 30 to about 50% by weight of fatty acid diglycerides, and about 5 to about 20% by weight of fatty acid triglycerides. The fatty acid glycerol esters include greater than about 60% by weight of oleic acid (C18:1) monoglycerides, diglycerides, and triglycerides. Other fatty acid glycerol esters include esters of palmitic acid (C16) (less than about 12%), stearic acid (C18) (less than about 6%), linoleic acid (C18:2) (less than about 35%), linolenic acid (C18:3) (less than about 2%), arachidic acid (C20) (less than about 2%), and eicosenoic acid (C20:1) (less than about 2%). PECEOL® may also contain free glycerol (typically about 1%). In one embodiment, the fatty acid glycerol esters comprise about 44% by weight fatty acid monoglycerides, about 45% by weight fatty acid diglycerides, and about 9% by weight fatty acid triglycerides, and the fatty acid glycerol esters comprise about 78% by weight oleic acid (C18:1) monoglycerides, diglycerides, and triglycerides. Other fatty acid glycerol esters include esters of palmitic acid (C16) (about 4%), stearic acid (C18) (about 2%), linoleic acid (C18:2) (about 12%), linolenic acid (C18:3) (less than 1%), arachidic acid (C20) (less than 1%), and eicosenoic acid (C20:1) (less than 1%).

[0078] As used herein, the term "polyethylene oxide-containing fatty acid ester" refers to a fatty acid ester containing a polyethylene oxide group (i.e., a polyethylene glycol group) covalently attached to a fatty acid via an ester bond. Polyethylene oxide-containing fatty acid esters include mono- and di-fatty acid esters of polyethylene glycol. Suitable polyethylene oxide-containing fatty acid esters are derived from fatty acids containing saturated and unsaturated fatty acids having 8 to 22 carbon atoms (i.e., polyethylene oxide esters of C8 to C22 fatty acids). In certain embodiments, suitable polyethylene oxide-containing fatty acid esters are derived from fatty acids containing saturated and unsaturated fatty acids having 12 to 18 carbon atoms (i.e., polyethylene oxide esters of C12 to C18 fatty acids). Representative polyethylene oxide-containing fatty acid esters include saturated C8 to C22 fatty acid esters. In certain embodiments, suitable polyethylene oxide-containing fatty acid esters include saturated C12 to C18 fatty acids. The molecular weight of the polyethylene oxide group of the polyethylene oxide-containing fatty acid ester can be varied to optimize the solubility of the therapeutic agent (e.g., one or more proteasome inhibitors) in the formulation. A typical average molecular weight of the polyethylene oxide group can be about 350 to about 2000. In one embodiment, the average molecular weight of the polyethylene oxide group is about 1500. In this embodiment, the one or more proteasome inhibitor formulations include one or more polyethylene oxide-containing fatty acid esters, typically a mixture of polyethylene oxide-containing fatty acid esters (mono- and di-fatty acid esters of polyethylene glycol). The polyethylene oxide-containing fatty acid esters useful in the formulations can be provided by commercially available sources. An exemplary polyethylene oxide-containing fatty acid ester (a mixture of mono- and di-esters) is commercially available under the name GELUCIRE® (Gattefosse, Saint-Priest-Cedex, France).

[0079] Suitable polyethylene oxide-containing fatty acid esters can be provided by GELUCIRE® 44 / 14, GELUCIRE® 50 / 13, and GELUCIRE® 53 / 10. The numbers in these names refer to the melting point and hydrophilic / lipophilic balance (HLB) of these materials, respectively. GELUCIRE® 44 / 14, GELUCIRE® 50 / 13, and GELUCIRE® 53 / 10 are mixtures of (a) monoesters, diesters, and triesters of glycerol (glycerides) and (b) monoesters and diesters of polyethylene glycol (macrogols). GELUCIRE® may also contain free polyethylene glycol (e.g., PEG 1500). Lauric acid (C12) is the primary fatty acid component of the glycerides and polyethylene glycol esters in GELUCIRE® 44 / 14. GELUCIRE® 44 / 14 is a mixture of glyceryl dilaurate (a lauric acid diester with glycerol) and PEG dilaurate (a lauric acid diester with polyethylene glycol), commonly known as PEG-32 glyceryl laurate (Gattefosse), lauroyl macrogol-32 glyceride EP, or lauroyl polyoxylglyceride USP / NF. GELUCIRE® 44 / 14 is produced by the reaction of hydrogenated palm kernel oil with polyethylene glycol (average molecular weight 1500). GELUCIRE® 44 / 14 contains approximately 20% monoglycerides, diglycerides, and triglycerides, approximately 72% mono- and di-fatty acid esters of polyethylene glycol 1500, and approximately 8% polyethylene glycol 1500. GELUCIRE® 44 / 14 contains lauric acid (C12) esters (30-50%), myristic acid (C14) esters (5-25%), palmitic acid (C16) esters (4-25%), stearic acid (C18) esters (5-35%), caprylic acid (C8) esters (less than 15%), and capric acid (C10) esters (less than 12%). GELUCIRE® 44 / 14 may also contain free glycerol (typically less than about 1%).In a representative formulation, GELUCIRE® 44 / 14 contains lauric acid (C12) ester (about 47%), myristic acid (C14) ester (about 18%), palmitic acid (C16) ester (about 10%), stearic acid (C18) ester (about 11%), caprylic acid (C8) ester (about 8%), and capric acid (C10) ester (about 12%).

[0080] Palmitic acid (C16) (40-50%) and stearic acid (C18) (48-58%) are the major fatty acid components of the glycerides and polyethylene glycol esters in GELUCIRE® 50 / 13, also known as PEG-32 glyceryl palmitostearate (Gattefosse), stearoyl macrogolglycerides EP, or stearoyl polyoxylglycerides USP / NF). GELUCIRE® 50 / 13 contains palmitic acid (C16) esters (40-50%), stearic acid (C18) esters (48-58%) (greater than about 90% stearic and palmitic acid esters), lauric acid (C12) esters (less than 5%), myristic acid (C14) esters (less than 5%), caprylic acid (C8) esters (less than 3%), and capric acid (C10) esters (less than 3%). GELUCIRE® 50 / 13 may also contain free glycerol (typically less than about 1%). In a representative formulation, GELUCIRE® 50 / 13 contains palmitic acid (C16) ester (approximately 43%), stearic acid (C18) ester (approximately 54%) (stearic acid and palmitic acid esters approximately 97%), lauric acid (C12) ester (less than 1%), myristic acid (C14) ester (approximately 1%), caprylic acid (C8) ester (less than 1%), and capric acid (C10) ester (less than 1%). Stearic acid (C18) is the major fatty acid component of the glycerides and polyethylene glycol esters in GELUCIRE® 53 / 10. GELUCIRE® 53 / 10 is also known as PEG-32 glyceryl stearate (Gattefosse). In one embodiment, the polyethylene oxide-containing fatty acid ester is a laurate, palmitate, or stearate (i.e., mono- and dilaurates of polyethylene glycol, mono- and dipalmitates of polyethylene glycol, mono- and distearates of polyethylene glycol). Mixtures of these esters can also be used.

[0081] For embodiments including polyethylene oxide-containing fatty acid esters, the ratio of fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 20:80 to about 80:20 (v / v). In one embodiment, the ratio of fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 30:70 (v / v). In one embodiment, the ratio of fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 40:60 (v / v). In one embodiment, the ratio of fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 50:50 (v / v). In one embodiment, the ratio of fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 60:40 (v / v). In one embodiment, the ratio of fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 70:30 (v / v).

[0082] As used herein, the term "polyethylene oxide-containing phospholipid" refers to a phospholipid containing a polyethylene oxide group (i.e., a polyethylene glycol group) covalently attached to the phospholipid, typically via a carbamate or ester bond. Phospholipids are derived from glycerol and may contain a phosphate ester group and two fatty acid ester groups. Suitable fatty acids include saturated and unsaturated fatty acids having 8 to 22 carbon atoms (i.e., C8 to C22 fatty acids). In certain embodiments, suitable fatty acids include saturated C12 to C18 fatty acids. Representative polyethylene oxide-containing phospholipids include C8 to C22 saturated fatty acid esters of phosphatidylethanolamine polyethylene glycol salts. In certain embodiments, suitable fatty acids include saturated C12 to C18 fatty acids. The molecular weight of the polyethylene oxide group of the polyethylene oxide-containing phospholipid can be varied to optimize the solubility of the therapeutic agent (e.g., one or more proteasome inhibitors) in the formulation. Representative average molecular weights of the polyethylene oxide group can be about 200 to about 5000 (e.g., PEG200 to PEG5000).

[0083] In one embodiment, the polyethylene oxide-containing phospholipid is distearoylphosphatidylethanolamine polyethylene glycol salt.Representative distearoylphosphatidylethanolamine polyethylene glycol salts include distearoylphosphatidylethanolamine polyethylene glycol 350 (DSPE-PEG-350) salt, distearoylphosphatidylethanolamine polyethylene glycol 550 (DSPE-PEG-550) salt, distearoylphosphatidylethanolamine polyethylene glycol 750 (DSPE-PEG-750) salt, distearoylphosphatidylethanolamine polyethylene glycol 1000 (DSPE-PEG-1000) salt, distearoylphosphatidylethanolamine polyethylene glycol 1500 (DSPE-PEG-1500) salt, and distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG-2000) salt.Mixtures can also be used. For the distearoylphosphatidylethanolamine polyethylene glycol salts described above, the numbers (e.g., 350, 550, 750, 1000, and 2000) indicate the average molecular weight of the polyethylene oxide group. The abbreviations for these salts used herein are provided in parentheses above. Suitable distearoylphosphatidylethanolamine polyethylene glycol salts include ammonium and sodium salts.

[0084] The chemical structure of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG-2000) ammonium salt consists of a polyethylene oxide-containing phospholipid, containing a phosphate ester group and two fatty acid ester (stearate) groups, as well as a polyethylene oxide group covalently attached to the amino group of phosphatidylethanolamine via a carbamate bond.

[0085] The polyethylene oxide-containing phospholipid affects the formulation's ability to solubilize a therapeutic agent. Generally, the greater the amount of polyethylene oxide-containing phospholipid, the greater the formulation's ability to solubilize a poorly soluble therapeutic agent. The polyethylene oxide-containing phospholipid may be present in the formulation in an amount of about 1 mM to about 30 mM based on the volume of the formulation. In certain embodiments, the distearoylphosphatidylethanolamine polyethylene glycol salt is present in the formulation in an amount of 1 mM to about 30 mM based on the volume of the formulation. In one embodiment, the distearoylphosphatidylethanolamine polyethylene glycol salt is present in the formulation at about 15 mM based on the volume of the formulation.

[0086] In certain embodiments, the one or more proteasome inhibitors are provided to the subject in a solid dosage form (e.g., a solid or semi-solid dosage form) comprising bortezomib. In certain embodiments, the solid dosage form comprises an oral formulation disclosed herein. In some embodiments, the solid dosage form comprises bortezomib and at least one lipophilic component coated on a solid carrier. In other embodiments, the % (w / w) of bortezomib in the solid dosage form is greater than the % (w / w) of the at least one lipophilic component. In further embodiments, the % (w / w) of bortezomib ranges from about 20% to about 30% of the total weight of the solid dosage form. In some embodiments, bortezomib is present in the solid dosage form in an amount ranging from about 50 mg to about 200 mg. In other embodiments, bortezomib is present in an amount of about 100 mg. In yet other embodiments, bortezomib is present in an amount of about 150 mg. In certain embodiments, the formulation is present in a hard shell capsule. In certain embodiments, the bortezomib formulation is provided orally. In certain embodiments, the solid dosage form is any of those shown in Tables 4-10.

[0087] In some embodiments, the at least one lipophilic component is selected from the group consisting of polyethylene oxide-containing fatty acid esters, fatty acid glycerol esters, and combinations thereof. In some embodiments, the solid dose formulation comprises bortezomib, polyethylene oxide-containing fatty acid esters, and fatty acid glycerol esters.

[0088] The solid dosage forms of the present disclosure can be prepared by any suitable method, including granulating a therapeutic agent (e.g., one or more proteasome inhibitors) and excipients (e.g., fillers, glidants, lubricants, etc., known in the art and described herein), extruding a therapeutic agent and excipients, directly compressing a therapeutic agent and excipients to form a tablet, etc. In certain embodiments, the solid dosage forms of the present disclosure can be prepared by coating an active agent (e.g., bortezomib) onto a solid carrier. The solid carrier can be any material onto which a drug-containing composition can be coated and suitable for human consumption. Any conventional coating process can be used. For example, a therapeutic agent, such as bortezomib, along with an optional binder or one or more lipophilic components described herein, can be dissolved or suspended in a suitable solvent (e.g., ethanol) and deposited on the solid carrier by methods known in the art, such as fluidized bed coating or pan coating. The solvent can be removed, for example, by drying, or in situ during the coating process (eg, during fluidized bed coating), and / or by subsequent drying.

[0089] In some embodiments, the solid carrier may be inert beads or inert particles. In other embodiments, the solid carrier may be non-pareil seeds, acidic buffer crystals, alkaline buffer crystals, or encapsulated buffer crystals. In some embodiments, the solid carrier may be sugar spheres, cellulose spheres, lactose spheres, lactose-microcrystalline cellulose (MCC) spheres, mannitol-MCC spheres, or silicon dioxide spheres. In other embodiments, the solid carrier may be a sugar, a sugar alcohol, or a combination thereof. Suitable sugars include lactose, sucrose, maltose, and combinations thereof. Suitable sugar alcohols include mannitol, sorbitol, xylitol, maltitol, arabitol, ribitol, dulcitol, iditol, isomalt, lactitol, erythritol, and combinations thereof. In several embodiments, the solid carrier may be formed by combining any of the above with a filler. Examples of suitable fillers that can be used to form the solid carrier include lactose, microcrystalline cellulose, silicified microcrystalline cellulose, mannitol-microcrystalline cellulose, and silicon dioxide. In other embodiments, the dosage forms disclosed herein do not include a solid carrier. In other embodiments, the present disclosure provides a capsule comprising the solid dosage form described herein. Bortezomib oral administration formulations can be prepared using any of the formulations described in Tables 4-8.

[0090] [Table 4] Items a to h are internal phase components, and item i is an external phase component.

[0091] [Table 5] Items a to g are internal phase components, and item h is an external phase component.

[0092] [Table 6] Items a to g are internal phase components, and item h is an external phase component.

[0093] [Table 7] Items a to h are internal phase components, and item i is an external phase component.

[0094] [Table 8] Items a to g are internal phase components, and item h is an external phase component.

[0095] In vitro cell testing Several different compounds were tested for efficacy in an in vitro study using the virus strain: 2019 novel coronavirus, isolate USA-WA1 / 2020 (SARS-CoV-2) in a human non-small cell lung cancer cell line (Calu-3). The results are shown in Table 9 below.

[0096] Efficacy was tested in parallel in a human non-small cell lung cancer cell line (Calu-3 cells). Each test compound was tested individually. Technicians were blinded to the identity of the drug being tested. Each concentration was evaluated in triplicate for efficacy.

[0097] Calu-3 lung cells were cultured in 96-well plates the day before the assay. Cells were >90% confluent at the start of the study. Each test compound concentration was evaluated in triplicate.

[0098] The test substance concentrations were tested under two different conditions: 1) Pretreatment 24 ± 4 hours before virus inoculation followed by treatment immediately after removal of the virus inoculum, or 2) Treatment with test substance only immediately after removal of the virus inoculum. Remdesivir was added immediately after removal of the virus inoculum. For pretreatment and treatment, wells were covered with 0.2 mL of DMEM2 (Dulbecco's Modified Eagle Media (DMEM) containing 2% fetal bovine serum (FBS) containing the test substance).

[0099] After 24 ± 4 hours of pretreatment, cells were inoculated with SARS-CoV-2 at an MOI of 0.001 TCID50 / cell and incubated for 60–90 minutes. Immediately after the 60–90 minute incubation, the virus inoculum was removed, cells were washed, and appropriate wells were covered with 0.2 mL of DMEM2 (DMEM containing 2% FBS with test or control substances) and incubated in a humidified chamber at 37°C ± 2°C and 5 ± 2% CO2. 48 ± 6 hours after inoculation, cells were fixed and assessed for the presence of virus by immunostaining assay.

[0100] For immunostaining assays, the incubation time was changed to 48 hours, which included a 24±4 hour pretreatment of cells with selected test substances.

[0101] Immunostaining assay: After approximately 48 ± 6 hours, cells were fixed with paraformaldehyde and stained using an anti-SARS-2 nucleoprotein monoclonal antibody (Sino Biological) followed by peroxidase-conjugated goat anti-mouse IgG (SeraCare). The wells were developed using TMB substrate solution, and the reaction was stopped by acidification. The ELISA plate was read at 450 nm on a spectrophotometer using an ELISA plate reader.

[0102] For each well, viral inhibition was calculated as the percentage decrease in absorbance value relative to the virus control using the following formula: Percent inhibition = 100 - [(A450 of test substance dilution - A450 of cell control) / (A450 of virus control - A450 of cell control)] x 100. EC50 was defined as the reciprocal of the dilution that caused a 50% decrease in absorbance value of the virus control (50% A450 decrease).

[0103] Bortezomib showed a significant reduction in TCID50 titers, with a median effective concentration (EC50) of 7.8 nM.

[0104] [Table 9] * Significant viral activity

[0105] Second Test Series Efficacy was tested in parallel in African green monkey kidney (Vero E6) cells. Each test compound was tested individually. Technicians were blinded to the identity of the drug being tested. Each concentration was evaluated in triplicate for efficacy. Vero E6 cells were cultured in 96-well plates the day before the assay. Cells were greater than 90% confluent at the start of the test. Each test article concentration was evaluated in triplicate.

[0106] Test substance concentrations were tested under two different conditions: 1) pretreatment 24 ± 4 hours before virus inoculation, followed by treatment immediately after removal of the virus inoculum, or 2) treatment in which only the test substance was added immediately after removal of the virus inoculum. Remdesivir was added immediately after removal of the virus inoculum. For pretreatment and treatment, wells were covered with 0.2 mL of DMEM2 (Dulbecco's Modified Eagle Media (DMEM) containing 2% fetal bovine serum (FBS) containing various concentrations of the test compound. After 24 ± 4 hours of pretreatment, cells were inoculated with SARS-CoV-2 at an MOI of 0.001 TCID50 / cell and incubated for 60–90 minutes.

[0107] Immediately after the 60-90 minute incubation, the virus inoculum was removed, the cells were washed, and the appropriate wells were covered with 0.2 mL of DMEM2 (DMEM containing 2% FBS with test or control substances) and incubated in a humidified chamber at 37°C ± 2°C and 5 ± 2% CO2. Forty-eight ± 6 hours after inoculation, cells were fixed and evaluated for the presence of virus by immunostaining assay. The immunostaining assay utilized was modified to a 48 hour incubation period. For selected test substances, a 24 ± 4 hour pretreatment of the cells was included.

[0108] [Table 10] ** Activity is based on concentrations used to treat tapeworms (3.10-9 to 3.10-5 M) and systemic fungal infections (1000 to 2000 nM).

[0109] The above results in Table 10 confirm the results obtained in the first study series and confirm the versatility of bortezomib as a potent inhibitor of COVID-19, as different strains of COVID-19 were tested. The above results also show that in this study, bortezomib was clearly superior to remdesevir in terms of EC50, as also evidenced in Figures 1 and 2.

[0110] Figures 3 and 4 show IC50 and EC50 values ​​determined using the methods described above in Calu-3 cells exposed to SARS-CoV-2 virus and treated with remdesivir (Figure 3) or carfilzomib (Figure 4). Without being bound by any particular theory, the data presented in Figure 4 support the use of carfilzomib as a therapy for ameliorating and / or inhibiting some or substantially all of the risk, symptoms, and development of severe disease in subjects infected with the SARS-CoV-2 virus.

Claims

1. 1. Use of one or more proteasome inhibitors in the manufacture of a medicament for preventing SARS-CoV-2 infection, wherein at least one proteasome inhibitor is bortezomib, carfilzomib, or ixazomib.

2. 1. Use of one or more proteasome inhibitors in the manufacture of a medicament for treating SARS-CoV-2 infection, wherein at least one proteasome inhibitor is bortezomib, carfilzomib, or ixazomib.

3. 1. Use of one or more proteasome inhibitors in the manufacture of a medicament for preventing replication of the SARS-CoV-2 virus, wherein at least one proteasome inhibitor is bortezomib, carfilzomib, or ixazomib.

4. The use described in any one of claims 1 to 3, wherein the pharmaceutical is suitable for oral administration.

5. The pharmaceutical product, one or more fatty acid glycerol esters, and One or more polyethylene oxide-containing phospholipids or one or more polyethylene oxide-containing fatty acid esters The use of claim 4, further comprising:

6. The use according to any one of claims 1 to 5, wherein the proteasome inhibitor is bortezomib.

7. The use according to any one of claims 1 to 5, wherein the proteasome inhibitor is carfilzomib.

8. The use according to any one of claims 1 to 5, wherein the proteasome inhibitor is ixazomib.

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