Diphenhydramine and lactoferrin for the prevention and treatment of COVID-19

Abstract

Compositions and formulations containing diphenhydramine and lactoferrin are described for preventing or treating infection with SARS-CoV-related betacoronavirus. Methods of using the compositions and formulations are also described. The methods include administering the compositions or formulations containing diphenhydramine and lactoferrin to a patient at risk of infection with SARS-CoV-related betacoronavirus or suffering from a SARS-CoV-related betacoronavirus-associated disease.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 126,082, filed on 16 December 2020, which is incorporated herein by reference.

[0002] Sequence List The sequence listing described in the file T18371_SeqListing.txt is 1 kilobyte in size, was created on December 14, 2021, and is incorporated herein by reference.

[0003] Introduction COVID-19 is a global health crisis caused by the novel coronavirus SARS-CoV-2. In severe cases, SARS-CoV-2 infection can cause respiratory failure and death. SARS-CoV-2 enters airway cells by binding to angiotensin-converting enzyme 2 (ACE2).

[0004] The ACE2 gene encodes angiotensin-converting enzyme-2, which has been shown to be a receptor for both SARS-coronavirus (SARS-Cove) and human respiratory coronavirus NL63. Recent studies and analyses indicate that ACE2 may be a host receptor for the novel coronavirus 2019-nCoV / SARS-CoV-2.

[0005] There is an urgent need to identify therapies that can prevent SARS-CoV-2 infection and improve outcomes for COVID-19 patients. [Overview of the Initiative]

[0006] Combinations and formulations comprising diphenhydramine and lactoferrin are described for use in the prevention and / or treatment of COVID-19 and other SARS-CoV-related betacoronavirus diseases. The described combinations and formulations containing diphenhydramine and lactoferrin are useful as antiviral therapeutic agents in preventing and / or treating infections caused by SARS-CoV-related betacoronaviruses. In some embodiments, the SARS-CoV-related betacoronavirus is SARS-CoV or SARS-CoV-2. The infection caused by SARS-CoV-related betacoronavirus may be, but is not limited to, COVID-19. In some embodiments, the described combinations and formulations may be used to inhibit the replication and / or infection of SARS-CoV-related betacoronaviruses.

[0007] The described combinations and formulations may be used to prevent or treat SARS-CoV-2 infection in subjects. In some embodiments, the described combinations and formulations are administered to subjects at risk of SARS-CoV-2 infection. In some embodiments, the described combinations and formulations are administered to subjects who have tested positive for SARS-CoV-2. In some embodiments, the described combinations and formulations are administered to subjects exposed to SARS-CoV-2. In some embodiments, the described combinations and formulations are administered to subjects suspected of having been exposed to SARS-CoV-2. In some embodiments, the described combinations and formulations are administered to subjects at risk of SARS-CoV-2 exposure. In some embodiments, the described combinations and formulations are administered to subjects who have or have been diagnosed with COVID-19. In some embodiments, the described combinations and formulations are administered to subjects with a coronavirus infection such as COVID-19 to treat acute lung injury.

[0008] The described combinations and formulations may be used to prevent or treat SARS-CoV-associated beta-coronavirus infection in subjects. In some embodiments, the described combinations and formulations are administered to subjects at risk of infection with SARS-CoV-associated beta-coronavirus. In some embodiments, the described combinations and formulations are administered to subjects who have tested positive for SARS-CoV-associated beta-coronavirus. In some embodiments, the described combinations and formulations are administered to subjects exposed to SARS-CoV-associated beta-coronavirus. In some embodiments, the described combinations and formulations are administered to subjects suspected of being exposed to SARS-CoV-associated beta-coronavirus. In some embodiments, the described combinations and formulations are administered to subjects at risk of exposure to SARS-CoV-associated beta-coronavirus. In some embodiments, the described combinations and formulations are administered to subjects who have or have been diagnosed with SARS-CoV-associated beta-coronavirus disease.

[0009] In some embodiments, diphenhydramine is a diphenhydramine salt. The diphenhydramine salt may be, but is not limited to, diphenhydramine HCl or diphenhydramine citrate.

[0010] Lactoferrin may be, but is not limited to, unsaturated iron lactoferrin, hololactoferrin, recombinant lactoferrin, or lactoferrin fragments having antiviral activity. Recombinant lactoferrin can be produced from plants such as rice, from microorganisms such as yeast or bacteria, or from mammalian or insect cells grown in culture. Lactoferrin may be, but is not limited to, human lactoferrin or bovine lactoferrin. Lactoferrin may be derived from or obtained from milk or colostrum.

[0011] The combinations and formulations can be formulated for oral administration, parenteral administration, intravenous administration, injection, or inhalation (e.g., nasal delivery).

[0012] The combinations and formulations can be formulated or manufactured as solids, powders (e.g., lyophilized powders), tablets (e.g., pills), capsules, or liquids. In some embodiments, the combinations and formulations can be formulated for nasal delivery.

[0013] In some embodiments, the combination and formulation further contain an analgesic. In some embodiments, the combination and formulation further contain a nasal congestion decongestant. In some embodiments, the combination and formulation further contain an analgesic and a nasal congestion decongestant.

[0014] In some embodiments, pharmaceutical compositions for treating or preventing SARS-CoV-related beta-coronavirus diseases such as COVID-19 are described. The pharmaceutical composition comprises a therapeutically effective amount of an H1 receptor-blocking antihistamine and a therapeutically effective amount of lactoferrin. The H1 receptor-blocking antihistamine can be selected from the group consisting of diphenhydramine, hydroxyzine, cetirizine, azelastine, loratadine, levocetirizine, brompheniramine, fexofenadine, and chlorpheniramine. In some embodiments, the pharmaceutical composition further comprises one or more of an H2 receptor-blocking antihistamine, a non-steroidal anti-inflammatory drug (NSAID), a cough suppressant, a decongestant, and an antiemetic or antidiarrheal agent. In some embodiments, the pharmaceutical composition further comprises each of an H2 receptor-blocking antihistamine, a non-steroidal anti-inflammatory drug (NSAID), a cough suppressant, a decongestant, and an antiemetic or antidiarrheal agent. The H2 receptor blocking antihistamine may, but is not limited to, famotidine. The NSAID may, but is not limited to, acetaminophen. The cough suppressant may, but is not limited to, dextromethorphan. The decongestant may, but is not limited to, phenylephrine. The antiemetic or antidiarrheal agent may, but is not limited to, bismuth subsalicylate or loperamide.

[0015] In some embodiments, diphenhydramine and lactoferrin are formulated with one or more adjuvants, carriers, excipients, or combinations thereof. [Brief explanation of the drawing]

[0016] [Figure 1]Model of the antiviral mechanism mediated by specific antihistamines. Diphenhydramine and related antihistamines have the potential to inhibit SARS-CoV-2 entry (by binding to ACE2) and viral replication (by binding to the sigma-1 receptor). Diphenhydramine (asterisk) exhibits off-target inhibitory ACE2 activity by forming intermolecular interactions with the active site and inducing a conformational change from an open to a closed conformation. The conformational change shifts the position of ACE2 at the site contacting the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD), resulting in a decrease in intermolecular interactions at the ACE2 / RBD interface. The sigma-1 receptor is a membrane-bound chaperone hijacked by SARS-CoV-2 to link the replicase / transcriptase complex to the endoplasmic reticulum by directly binding to the non-structural protein NSP6. NSP6 forms a complex with NSP3 and NSP4. Diphenhydramine binds to the sigma-1 receptor and potentially interferes with the viral life cycle by blocking protein-protein interactions with NSP6. [Figure 2] Graph showing the efficacy of diphenhydramine (DPH), unsaturated iron human lactoferrin (hLNF milk), and recombinant human lactoferrin (rLFN) alone and in combination in reducing the cytotoxicity of the SARS-CoV-2 virus. Control samples (VEH) received no diphenhydramine or lactoferrin. Each data point was performed in triplicate. [Figure 3] Checkerboard plot showing inhibition of SARS-CoV-2-induced cytotoxicity in the presence of various concentrations of diphenhydramine and unsaturated iron lactoferrin (hLF). [Figure 4]A heatmap showing the inhibition of SARS-CoV-2-induced cytotoxicity against various concentrations of diphenhydramine and unsaturated iron lactoferrin alone or in combination. The inhibition of SARS-CoV-2-induced cytotoxicity was amplified when the two drugs were combined (100 μg / mL corresponds to 32 μM lactoferrin and 10 μg / mL corresponds to 35 μM diphenhydramine). [Figure 5] An effective concentration 50 (EC50) curve showing the dose-dependent inhibition of SARS-CoV-2-induced cytotoxicity by diphenhydramine. The LFN concentrations in the figure legend are in the order of the start of the curves on the y-axis (from highest to lowest). [Figure 6-1] A graph showing the anti-SARS-CoV-2 activity of diphenhydramine. Vero E6 cells were treated with various concentrations of diphenhydramine (DPH) without (black bars) or with (gray bars) SARS-CoV-2 at an MOI of 0.2, and cytotoxicity was measured by LDH release. [Figure 6-2] A graph showing the anti-SARS-CoV-2 activity of diphenhydramine. The EC50 (white circles) and CC50 (black circles) curves were determined by non-linear regression. The EC50 of diphenhydramine alone was 122 μg / ml. [Figure 6-3] A graph showing the anti-SARS-CoV-2 activity of diphenhydramine and lactoferrin. Vero E6 cells were treated with various concentrations of diphenhydramine and 400 μg / ml of lactoferrin (LFN) without (black bars) or with (gray bars) SARS-CoV-2 at an MOI of 0.2, and cytotoxicity was measured by LDH release. [Figure 6-4]This graph shows the anti-SARS-CoV-2 activity of diphenhydramine and lactoferrin. The EC50 (white circles) and CC50 (black circles) curves were determined by nonlinear regression. The EC50 of diphenhydramine with 400 μg / ml lactoferrin was 54.25 μg / ml. To compare the effect of LFN on DPH EC50, the EC50 curves for DPH (white circles), LFN (black diamonds), and DPH+LFN (black squares) are shown on the same graph. [Figure 6-5] This graph shows the anti-SARS-CoV-2 activity of diphenhydramine and lactoferrin. The combination of diphenhydramine and lactoferrin showed a synergistic effect against SARS-CoV-2. The EC50 curve shows that the inhibition of SARS-CoV-2-mediated cytotoxicity by diphenhydramine (DPH) is enhanced in the presence of lactoferrin (LFN). [Figure 6-6] This graph shows the anti-SARS-CoV-2 activity of diphenhydramine and lactoferrin. The combination of diphenhydramine and lactoferrin showed a synergistic effect against SARS-CoV-2. Measurement of viral genome equivalents by RT-qPCR of the SARS-CoV-2 N protein gene showed that DPH+LFN inhibited replication by approximately three logarithms. * indicates p≦0.05; ** indicates p≦0.01; *** indicates p≦0.001; **** indicates p≦0.0001; ns indicates not significant. [Figure 7-1] These are micrographs showing the susceptibility of ACE2-transfected human lung epithelial cells to SARS-CoV-2 infection. NCI-H23 (parental non-transduced cells), NCI-H23ACE2 pool (uncloned lentivirus-transformed cells), and NCI-H23ACE2 (clone A2) were infected with SARS-CoV-2, and the cytopathic effect was observed at 3 dpi. CPE is defined by cell rounding and detachment from the monolayer. The scale bar corresponds to 100 μm. [Figure 7-2]This graph shows the anti-SARS-CoV-2 activity of diphenhydramine and lactoferrin in human lung epithelial cells. The TCID50 experiment was performed three times by biological replication after infecting cells with a MOI of 0.01 for 72 hours. SARS-CoV-2 infection of human lung epithelial cell line H23 was dependent on heterologous expression of the human ACE2 receptor. [Figure 7-3] This graph shows the anti-SARS-CoV-2 activity of diphenhydramine and lactoferrin in human lung epithelial cells. Diphenhydramine, along with lactoferrin, significantly reduced the release of infectious SARS-CoV-2 particles from H23-hACE2 cells by approximately one logarithm compared to untreated H23-hACE2 cells. The data are from TCID50, which was performed in a technically triplicate manner. * indicates p ≤ 0.05; **** indicates p ≤ 0.0001; ns indicates not significant. [Modes for carrying out the invention]

[0017] Before describing this instruction in detail, it should be understood that this disclosure is not limited to any particular composition or process step and is therefore subject to change. Note that, as used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise explicitly indicated in the context. For example, a reference to "drugs" includes multiple drugs. The conjunction "or" should be interpreted in an inclusive sense, i.e., equivalent to "and / or" unless the inclusive meaning is unreasonable in the context.

[0018] In general, the term “approximately” refers to slight variations in the amount of a component of a composition that do not have any significant effect on the activity or stability of the composition. Where this specification discloses a specific value for a parameter, this specification should be understood as disclosing the parameter by that value “approximately.” All ranges should be interpreted as including the endpoint unless there is an explicit exclusion such as “not including the endpoint,” and therefore, for example, “10 to 15” or “10 to 15” includes the values ​​10 and 15. Furthermore, the use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” is not intended to be limiting. Both the general and detailed descriptions above are illustrative and descriptive only and should not be understood as limiting this teaching. Unless any material incorporated by reference is consistent with the express content of this disclosure, the express content shall prevail.

[0019] Unless otherwise specified, embodiments described herein as "including" various components are also intended to be described as "consisting of" or "essentially consisting of" the described components. Embodiments described herein as "essentially consisting of" various components are also intended to be described as "consisting of." "Essentially consisting of" means that additional components, compositions, or method steps that do not substantially alter the basic and novel features of the compositions and methods described herein may be included in those compositions or methods.

[0020] A "SARS-CoV-related beta-coronavirus" is a virus that is considered to be highly similar to or phylogenetically similar to 2003 SARS-CoV or 2019 SARS-CoV-2. SARS-CoV-related beta-coronaviruses may belong to lineage B, subgenus Sarbecovirus, or lineage D, subgenus Nobecovirus. Beta-coronaviruses of lineage A, subgenus Embecovirus (including common human coronaviruses OC43 and HKU1) and lineage C, subgenus Merbecovirus (including Middle East Respiratory Syndrome coronavirus) are not considered SARS-CoV-related beta-coronaviruses.

[0021] A “homologous” sequence (e.g., a nucleic acid sequence or amino acid sequence) is a sequence that is identical or substantially similar to a known reference sequence, and is identical to the known reference sequence by, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Sequence identity can be determined by aligning sequences using algorithms such as BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), either with default gap parameters or by scrutiny and best alignment (i.e., yielding the highest percentage of sequence similarity across the comparison window width). The sequence identity ratio is calculated by comparing two optimally sequenced sequences across the comparison window width, determining the number of positions where identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of matching and non-matching positions within the comparison window width (i.e., window width size), and multiplying the result by 100 to obtain the sequence identity ratio. Unless otherwise specified, the comparison window width between two sequences is determined by the total length of the shorter of the two sequences.

[0022] Peptide variants and derivatives are well understood by those skilled in the art and may include amino acid sequence modifications. Amino acid sequence modifications may be substitutions, insertions, or deletions. Insertions include amino and / or carboxyl termination additions, as well as intrasequential insertions of one or more amino acid residues. Deletions include the removal of one or more amino acid residues from a peptide sequence. Substitutions include the replacement of an amino acid residue at a given position in the amino acid sequence with a different amino acid. Insertions, deletions, and substitutions may occur at a single or multiple positions. Insertions, deletions, and substitutions may occur at adjacent and / or non-adjacent positions. In some embodiments, one or more of the substitutions are conserved amino acid substitutions. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final S protein polypeptide. For peptides that differ from the reference sequence by 0, 1, 2, or 3 amino acids, the peptide may have 0, 1, 2, or 3 amino acid substitutions, insertions, or deletions in any combination or order.

[0023] "Active ingredient" is any component of a drug product intended to provide pharmacological activity or other direct effects in the diagnosis, cure, alleviation, treatment, or prevention of a disease, or to affect the structure or any function of the body of a human or other animal. The active ingredient includes components of the product that may undergo chemical changes during the manufacture of the drug product and be present in the drug product in modified forms intended to provide a specific activity or effect. A dosage form for a pharmaceutical product contains the active pharmaceutical ingredient, which is the active pharmaceutical ingredient itself, and excipients, which are components of a tablet, or a liquid on which the active drug is suspended, or other pharmaceutically inert materials. During formulation development, excipients may be selected to allow the active ingredient to reach the target site in the body at a desired rate and degree.

[0024] "Pharmacologically effective dose," "therapeutic effective dose," or simply "effective dose" refers to the amount (dosage) of the active pharmaceutical ingredient or pharmaceutical composition described that produces the intended pharmacological, therapeutic, or prophylactic effect. "Effective dose" may also refer to the amount of, for example, an excipient in the pharmaceutical composition that is sufficient to achieve the desired properties of the composition. An effective dose may be administered in one or more doses, applications, or applications.

[0025] As used herein, “dose,” “unit dose,” or “administered amount” may refer to a physically distinct unit suitable for use in a subject, each unit containing a predetermined amount of an active pharmaceutical ingredient and / or its pharmaceutical composition calculated to produce a desired response or a combination of responses in connection with its administration.

[0026] The terms “to treat” and “treatment” refer to methods or steps taken to provide relief or mitigation of the number, severity, and / or frequency of one or more symptoms of a disease or condition in a subject. To treat generally refers to obtaining a desired pharmacological and / or physiological effect. An effect may be, but does not necessarily have to be, preventive in that it prevents or partially prevents the disease, its symptoms, or condition. An effect may be therapeutic in relation to the partial or complete cure of a disease, condition, symptoms, or adverse effects resulting from a disease, disorder, or condition. The term treatment may include: (a) preventing the development of a disease in a subject who is susceptible to the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., stopping its onset; and (c) reducing the disease, i.e., mitigating or improving the disease and / or its symptoms or condition. To treat may refer to therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Persons requiring treatment (subjects requiring treatment) may include those who already have a coronavirus infection or those for whom infection should be prevented. Treating a disease, disorder, or condition may include inhibiting its progression, for example, preventing its progression, and alleviating a disease, disorder, or condition, for example, causing regression of the disease, disorder, and / or condition. Treating a disease, disorder, or condition may include improving at least one symptom of a particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, for example, treating inflammation without preventing viral replication.

[0027] Diphenhydramine is a commercially available antihistamine with a long history of safety. It is readily available and has a favorable safety profile. In silico molecular docking suggests that diphenhydramine may interact with the sigma-1 receptor to inhibit or reduce SARS-CoV-related beta-coronavirus infection.

[0028] [ka]

[0029] Lactoferrin is an iron-binding protein found in colostrum, milk, exocrine secretions, and polymorphonuclear leukocytes. Lactoferrin is an approximately 80 kDa glycosylated protein consisting of about 700 amino acids (711 amino acids for human lactoferrin and 689 amino acids for bovine lactoferrin), exhibiting high interspecies homology (orthologism). Lactoferrin functions in non-immune defense. It has been shown to be involved in several physiological and protective functions, including the regulation of iron absorption, as well as antioxidant, anticancer, anti-inflammatory, and antibacterial activity. Orally administered lactoferrin has been shown to exhibit immunomodulatory activity, including antibacterial and anti-inflammatory activity. The antibacterial activity is thought to be due to iron deficiency and / or interaction with microbial cells. In addition to full-length lactoferrin, three lactoferrin-derived peptides with antibacterial (including antiviral) activity have been identified. These three peptides are Lf(1-11), lactoferricin (Lfcin), and lactoferrampin (Lfampin). Lf(1-11) contains the first 11 amino acid residues of lactoferrin and is inherently highly cationic. Lf(1-11) has been shown to interact with the membranes of several bacteria. Lfcin is an amphiphilic cationic peptide produced by pepsin-mediated digestion of lactoferrin (e.g., for bovine lactoferrin, amino acid residues 17-41, FKCRRWQWRMKKLGAPSITCVRRAF (SEQ ID NO: 1)). Lfampin contains residues 268-284 in the N1 domain of lactoferrin. Lactoferrin has been shown to exhibit antiviral activity against both enveloped viruses and unenveloped viruses, including cytomegalovirus (CMV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human hepatitis C virus (HCV), and human hepatitis B virus (HBV).

[0030] Each lactoferrin molecule contains two ferric (Fe) molecules. 3+ Lactoferrin can bind to iron ions, meaning that when saturated, each lactoferrin binds to two iron ions. Milk-derived lactoferrin typically contains 10-30% iron saturation. Unsaturated lactoferrin, also called reduced iron lactoferrin or apolactoferrin, is iron-depleted lactoferrin. In some embodiments, unsaturated lactoferrin has less than 10% iron saturation. In some embodiments, unsaturated lactoferrin has less than 9% iron saturation, less than 8% iron saturation, less than 7% iron saturation, less than 6% iron saturation, less than 5% iron saturation, less than 4% iron saturation, less than 3% iron saturation, less than 2% iron saturation, or less than 1% iron saturation. In some embodiments, unsaturated lactoferrin has less than 0.5% iron saturation, or less than 0.15% iron saturation.

[0031] Combinations and formulations comprising diphenhydramine and lactoferrin are described herein. The described combinations and formulations can be used in methods for the therapeutic treatment and / or prevention of symptoms and diseases associated with SARS-CoV-related beta-coronavirus infection. Such methods include the administration of the described combinations and formulations to a subject, e.g., a human or animal subject.

[0032] In some embodiments, the described combinations and formulations may be administered to a subject to reduce the disease burden of SARS-CoV-associated beta-coronavirus. In some embodiments, the described combinations and formulations may be administered to a subject to reduce the viral transmission of SARS-CoV-associated beta-coronavirus. In some embodiments, the described combinations and formulations may be administered to a subject to inhibit SARS-CoV-associated beta-coronavirus infection, reduce the likelihood of infection, reduce the severity of infection, and / or reduce the duration of infection. In some embodiments, the described combinations and formulations may be administered to a subject infected with SARS-CoV-associated beta-coronavirus to reduce the viral load in the subject. In some embodiments, the described combinations and formulations may be administered to a subject to inhibit the entry of SARS-CoV-associated beta-coronavirus into host cells. SARS-CoV-associated beta-coronavirus may be SARS-CoV-2. In some embodiments, the described combinations and formulations may be administered to a subject to reduce the likelihood that the subject will require hospitalization due to coronavirus infection.

[0033] Methods are described for reducing the viral load, reducing the disease load, reducing viral transmission, preventing infection, reducing the likelihood of infection, reducing the severity of infection, and / or reducing the duration of infection. These methods include administering one or more of the described combinations and formulations to subjects who are infected with, suspected of being infected with, or at risk of being infected with, SARS-CoV-associated beta-coronavirus.

[0034] In some embodiments, the described combinations and formulations may be used to inhibit the entry of SARS-CoV-related beta-coronavirus into ACE2-expressing host cells, for example, human airway epithelial cells, but not limited to. In some embodiments, the described combinations and formulations may be used to inhibit the entry of SARS-CoV-2 into ACE2-expressing host cells, for example, human airway epithelial cells, but not limited to.

[0035] Methods for interfering with the interaction of SARS-CoV-related beta-coronavirus with ACE2 in a subject are described. Interfering with the interaction of SARS-CoV-related beta-coronavirus with ACE2 inhibits viral entry and reduces viral transmission and disease burden. In some embodiments, the method involves administering one of the described combinations or formulations to a subject. In some embodiments, the method involves administering one of the described combinations or formulations to a subject to inhibit the entry of SARS-CoV-related beta-coronavirus into human airway cells.

[0036] Methods for interfering with the interaction of SARS-CoV-2 with ACE2 in a subject are described. Interfering with the interaction of SARS-CoV-2 with ACE2 inhibits viral entry and reduces viral transmission and disease burden. In some embodiments, the method involves administering one of the described combinations or formulations to a subject. In some embodiments, the method involves administering one of the described combinations or formulations to a subject to inhibit the entry of SARS-CoV-2 into human airway cells.

[0037] In some embodiments, diphenhydramine and lactoferrin are administered to the subject at dose levels recognized or recommended for treating other conditions.

[0038] In some embodiments, the diphenhydramine and lactoferrin described are administered according to their known routes of administration.

[0039] Diphenhydramine may be diphenhydramine citrate or diphenhydramine hydrochloride, but is not limited to these. Diphenhydramine may be provided as a liquid formulation, as a tablet, as a coated tablet, as a chewable tablet, as a powder, or as a capsule. Diphenhydramine may be administered orally, by inhalation (e.g., nasally), or parenterally. Parenteral administration may be, but is not limited to, intramuscular and intravenous administration. In some embodiments, diphenhydramine is administered orally. In some embodiments, diphenhydramine is administered by inhalation. In some embodiments, diphenhydramine is administered orally in water or phosphate-buffered saline at approximately pH 7.4. In some embodiments, diphenhydramine is administered parenterally.

[0040] In some embodiments, the effective dose of diphenhydramine is about 5–600 mg, about 38–468 mg, about 25–402 mg, or about 25–300 mg. In some embodiments, the effective dose of diphenhydramine is up to about 228 mg / day, up to about 300 mg / day, up to about 400 mg / day, or up to about 456 mg / day. In some embodiments, the effective dose of diphenhydramine is about 10–100 mg, about 38–78 mg, about 25–67 mg, or about 25–50 mg, administered orally every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is about 10–100 mg or about 10–50 mg, administered parenterally. In some embodiments, diphenhydramine is administered 1, 2, 3, 4, 5, or 6 times per day.

[0041] In some embodiments, the effective dose of diphenhydramine is about 10–50 mg or about 12.5–25 mg administered 3–4 times / day. In some embodiments, the effective dose of diphenhydramine is about 10 mg, about 12.5 mg, about 25 mg or about 50 mg administered 3–4 times / day. In some embodiments, the effective dose of diphenhydramine is about 5 mg / kg. In some embodiments, the effective dose of diphenhydramine is about 150 mg / m². 2 That is the case.

[0042] In some embodiments, the effective dose of diphenhydramine is approximately 19–38 mg every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is approximately 19 mg or approximately 38 mg every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is approximately 38–76 mg every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is approximately 38 mg or approximately 76 mg every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is approximately 6.25 mg every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is approximately 12.5–25 mg every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is approximately 12.5 mg or approximately 25 mg every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is approximately 25–50 mg every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is approximately 25 mg or approximately 50 mg every 4–6 hours. In some embodiments, the effective dose of diphenhydramine is approximately 1.25 mg / kg, administered up to four times per day. In some embodiments, the effective dose of diphenhydramine is approximately 37.5 mg / m2, administered up to four times per day.

[0043] Lactoferrin may be, but is not limited to, unsaturated iron lactoferrin (apolactoferrin), hololactoferrin, recombinant lactoferrin, or lactoferrin fragments having antiviral activity. Recombinant lactoferrin can be produced from plants such as rice, from microorganisms such as yeast or bacteria, or from mammalian or insect cells grown in culture. Lactoferrin may be, but is not limited to, human lactoferrin or bovine lactoferrin. Lactoferrin may be derived from or obtained from milk or colostrum. Lactoferrin may be provided as a liquid formulation, as a tablet, as a coated tablet, as a chewable tablet, as a powder, or as a capsule. Lactoferrin may be administered orally, by inhalation (e.g., nasally), or parenterally. Parenteral administration may be intramuscular or intravenous. In some embodiments, lactoferrin is administered orally. In some embodiments, lactoferrin is administered by inhalation. In some embodiments, diphenhydramine is administered parenterally.

[0044] In some embodiments, the effective dose of lactoferrin is approximately 100-5000 mg, approximately 250-4000 mg, approximately 500-3600 mg, approximately 1000-3600 mg, or approximately 1800-3600 mg. In some embodiments, the effective dose of lactoferrin is up to approximately 250 mg / day, up to approximately 500 mg / day, up to approximately 1000 mg / day, up to approximately 1800 mg / day, or up to approximately 3600 mg / day. In some embodiments, the effective dose of lactoferrin is approximately 250 mg / day, approximately 500 mg / day, approximately 1000 mg / day, approximately 1500 mg / day, approximately 1800 mg / day, approximately 1900 mg / day, approximately 2000 mg / day, approximately 2100 mg / day, approximately 2200 mg / day, approximately 2300 mg / day, approximately 2400 mg / day, approximately 2500 mg / day, approximately 2600 mg / day, approximately 2700 mg / day, approximately 2800 mg / day, approximately 2900 mg / day, approximately 3000 mg / day, approximately 3100 mg / day, approximately 3200 mg / day, approximately 3300 mg / day, approximately 3400 mg / day, approximately 3500 mg / day, or approximately 3600 mg / day. In some embodiments, lactoferrin is administered 1, 2, 3, 4, 5, or 6 times per day.

[0045] Diphenhydramine and lactoferrin are formulated for in vivo administration. Diphenhydramine and lactoferrin may be formulated together or separately. In some embodiments, diphenhydramine and lactoferrin are formulated together.

[0046] Diphenhydramine and lactoferrin may be provided together in liquid formulations, tablets, coated tablets, chewable tablets, powders, or capsules. Diphenhydramine and lactoferrin may be administered together orally, by inhalation (e.g., nasally), or parenterally. Parenteral administration may be, but is not limited to, intramuscular and intravenous administration. In some embodiments, diphenhydramine and lactoferrin are administered together orally. In some embodiments, diphenhydramine and lactoferrin are administered together by inhalation. In some embodiments, diphenhydramine and lactoferrin are administered together parenterally.

[0047] In some embodiments, diphenhydramine and / or lactoferrin are formulated with one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) to form a pharmaceutical composition or drug suitable for in vivo delivery to a subject such as a human. The term "pharmaceutically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other components of the formulation and / or the mammal to be treated with it.

[0048] A pharmaceutical composition or drug comprises a pharmacologically effective amount of diphenhydramine and / or lactoferrin, and optionally one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient is a substance other than the active pharmaceutical ingredient (API, therapeutic agent) that is intentionally included in the drug delivery system. Excipients do not exert, or are not intended to exert, a therapeutic effect at the intended dose. Excipients may act to a) assist in the processing of the drug delivery system during manufacturing, b) protect, support, or enhance the stability, bioavailability, or patient tolerance of the API, c) aid in product identification, and / or d) enhance any other attributes of the API's overall safety, efficacy, or delivery during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0049] Excipients include, but are not limited to, absorption enhancers, anti-adhesion agents, defoamers, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, fragrances, flow enhancers, humectants, lubricants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, tonic agents, vehicles, water repellents, and wetting agents.

[0050] A pharmaceutical composition may contain other additional components commonly found in pharmaceutical compositions. Such additional components may include, but are not limited to, antipruritic agents, astringents, topical anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.).

[0051] The carrier may be, but is not limited to, a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The carrier may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. The carrier may also contain isotonic agents, such as sugars, polyalcohols, and sodium chloride, in the composition.

[0052] Pharmacopoeia means properties and / or substances that are acceptable to a subject from a pharmacological / toxicological standpoint. The term pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and, when administered to a subject, typically do not cause allergic reactions or other adverse or toxic reactions. In some embodiments, pharmaceutically acceptable compounds are approved for use in animals, more specifically in humans, by federal or state regulatory authorities, or are listed in the United States Pharmacopeia or other generally recognized pharmacopoeias.

[0053] In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients. The additional active ingredients may be, but are not limited to, additional antiviral agents, analgesics, or decongestants. In some embodiments, the additional active ingredients include additional antiviral agents. In some embodiments, the additional active ingredients include an analgesic. The analgesic may be, but are not limited to, acetaminophen, an NSAID, ibuprofen, or naproxen. In some embodiments, the analgesic is acetaminophen. The amount of acetaminophen in the formulation may be about 325 to about 1000 mg. In some embodiments, the additional active ingredients include a decongestant. The decongestant may be, but are not limited to, phenylephrine and pseudoephedrine.

[0054] The pharmaceutical composition may be in the form of a liquid formulation, tablet, coated tablet, chewable tablet, powder (e.g., lyophilized powder), or capsule. The pharmaceutical composition may be administered orally, by inhalation (e.g., nasally), or parenterally. Parenteral administration may be, but is not limited to, intramuscular and intravenous administration. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered by inhalation. In some embodiments, the pharmaceutical composition is administered parenterally.

[0055] In some embodiments, the pharmaceutical compositions described are used to treat or manage clinical symptoms associated with SARS-CoV-associated beta-coronavirus. In some embodiments, a therapeutically effective or prophylactically effective dose of diphenhydramine and lactoferrin is administered to a subject in need of such treatment, prevention, or management. In some embodiments, the administration of diphenhydramine and lactoferrin may be used to reduce the number, severity, and / or frequency of disease symptoms in a subject.

[0056] The pharmaceutical compositions described may be used to treat at least one symptom associated with SARS-CoV-related beta-coronavirus in a subject. In some embodiments, the subject is administered a therapeutically effective dose of diphenhydramine and lactoferrin to treat the symptoms. In some embodiments, the subject is administered a prophylactically effective dose of one diphenhydramine and lactoferrin to prevent infection with SARS-CoV-related beta-coronavirus or to prevent the development of one or more symptoms associated with SARS-CoV-related beta-coronavirus infection, such as COVID-19.

[0057] Symptoms associated with SARS-CoV-associated beta-coronavirus infection may include, but are not limited to, inflammatory responses, cytokine storms, inflammasome-associated reactions, IL-1β-associated reactions, NLRP3-associated reactions, lung fibrosis, ground-glass opacities, acute respiratory distress syndrome (ARDS), pneumonia, and combinations thereof.

[0058] In some embodiments, the pharmaceutical compositions described may be administered to subjects infected with or suspected of being infected with SARS-related beta-coronavirus in order to improve mucociliary transport or to alleviate airway obstruction.

[0059] The description includes methods for shortening the duration of SARS-CoV-2 infection, reducing the severity of SARS-CoV-2 infection, reducing the likelihood of SARS-CoV-2 infection, reducing SARS-CoV-2, or reducing the likelihood of developing COVID-19 in a subject, wherein the method involves administering one of the described combinations or formulations comprising diphenhydramine and lactoferrin to the subject.

[0060] In some embodiments, the pharmaceutical compositions described are administered to subjects at risk of infection with SARS-CoV-2, subjects tested positive for SARS-CoV-2, subjects exposed to SARS-CoV-2, subjects suspected of being exposed to SARS-CoV-2, subjects at risk of being exposed to SARS-CoV-2, subjects who have or have been diagnosed with COVID-19, or subjects suffering from acute lung injury due to SARS-CoV-2 infection.

[0061] Methods are described for reducing the duration of SARS-CoV-associated betacoronavirus infection, reducing the severity of SARS-CoV-associated betacoronavirus infection, reducing the likelihood of SARS-CoV-associated betacoronavirus infection, reducing SARS-CoV-associated betacoronavirus replication, or reducing the likelihood of developing SARS-CoV-associated betacoronavirus-associated disease in a subject, wherein the method comprises administering one of the described combinations or formulations of diphenhydramine and lactoferrin (e.g., one of the described pharmaceutical compositions) to the subject. The SARS-CoV-associated betacoronavirus may be, but is not limited to, SARS-CoV-2. The method comprises administering a therapeutically effective amount of the described combination, formulation, or pharmaceutical composition to a subject in need of such treatment, e.g., a human or animal subject.

[0062] A combination or formulation of diphenhydramine and lactoferrin may be administered to individuals at risk of infection with SARS-CoV-related beta-coronavirus, individuals who have tested positive for SARS-CoV-related beta-coronavirus, individuals who have been exposed to SARS-CoV-related beta-coronavirus, individuals suspected of being exposed to SARS-CoV-related beta-coronavirus, individuals at risk of being exposed to SARS-CoV-related beta-coronavirus, or individuals who have or have been diagnosed with SARS-CoV-related beta-coronavirus disease. SARS-CoV-related beta-coronavirus may be, but is not limited to, SARS-CoV-2.

[0063] It should be understood that this disclosure should not be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Those skilled in the art will recognize many variations and adaptations of the embodiments described herein. These variations and adaptations are included in the teachings of this disclosure and are intended to be encompassed by the claims herein. [Examples]

[0064] Example 1. Hololactoferrin and unsaturated iron lactoferrin (apolactoferrin) have anti-SARS-CoV-2 activity. Hololactoferrin and unsaturated iron lactoferrin (apolactoferrin) were analyzed in vitro for their direct antiviral activity against SARS-CoV-2 isolates. Unsaturated iron lactoferrin more effectively inhibited SARS-CoV-2 infection directly compared to hololactoferrin. The results are shown in Figure 2.

[0065] Example 2. Diphenhydramine combined with lactoferrin. The combination of diphenhydramine and lactoferrin was tested in vitro to determine the antiviral effect of the combination against SARS-CoV-2. Hololactoferrin and unsaturated iron lactoferrin were used in combination with diphenhydramine. Both unsaturated iron lactoferrin and diphenhydramine inhibited SARS-CoV-2 infection when administered alone. When combined, diphenhydramine and unsaturated iron lactoferrin effectively inhibited viral infection. Furthermore, the combination of diphenhydramine and unsaturated iron lactoferrin had a synergistic effect on inhibiting SARS-CoV-2 infection (i.e., higher antiviral activity compared to the additive effect of the drugs administered individually).

[0066] A) Checkboard assay: Cells were incubated with various formulations of diphenhydramine, unsaturated iron lactoferrin, and recombinant iron-saturated lactoferrin and exposed to SARS-CoV-2 at an MOI of 0.3. Lactoferrin was used at 0, 50, 100, 200, 400, or 800 μg / mL. Diphenhydramine was used at 0, 5, 10, 20, 40, or 80 μg / mL. SARS-CoV-2 toxicity was then determined. The data are shown in Figures 2, 3, and 4. All treatments were effective in reducing virus-induced cytotoxicity. Unsaturated iron lactoferrin was more effective than recombinant lactoferrin. Both forms of lactoferrin showed a synergistic effect with diphenhydramine in reducing SARS-CoV-2 cytotoxicity (Figure 2). Figures 3 and 4 show the synergistic properties of DPH and LFN in reducing SARS-CoV-2-induced cytotoxicity.

[0067] As shown in Figure 4, 5 μg / mL of DPH was unable to reduce SARS-CoV-2-induced cytotoxicity. In vitro, EC 50 The diphenhydramine concentration was 17.4 μg / mL. However, when 50 μg / mL of unsaturated iron lactoferrin was administered simultaneously with 5 μg / mL of DPH, a significant reduction in SARS-CoV-2 induced cytotoxicity was observed, and when administered simultaneously with unsaturated iron lactoferrin, the EC of diphenhydramine was reduced. 50 This indicates unsaturation. The codependent dose-response effect can be seen as a darker color trend towards the upper right in Figure 4.

[0068] Hill coefficient and EC 50 The decision. The cytotoxicity rate of SARS-CoV-2 infected cells was determined for various concentrations of diphenhydramine and lactoferrin. The effective concentration of diphenhydramine showing dose-dependent inhibition of SARS-CoV-2-induced cytotoxicity was 50 EC2. 50)The curve is shown in Figure 5. The baseline cytotoxicity inhibition and inhibition rate of diphenhydramine against SARS-CoV-2-induced cytotoxicity were significantly increased in the presence of unsaturated human lactoferrin at various concentrations.

[0069] The data points were fitted, and the EC exemplified in Figure 5 50 The Hill equation was used to extrapolate the Hill coefficient related to the curve. The highly negative Hill gradient indicates an increase in the inhibition rate against SARS-CoV-2 near the EC 50 . Calculation of the Hill gradient: Y = 100 / (1 + 10 ((logIC50-X)*Hill slope) ) DPH + 0 μg / ml LFN: Y = 100 / (1 + 10 ((2.190-X)*-1.198) ) DPH + 50 μg / ml LFN: Y = 100 / (1 + 10 ((1.611-X)*-20.75) ) DPH + 100 μg / ml LFN: Y = 100 / (1 + 10 ((1.636-X)*-15.08) ) DPH + 200 μg / ml LFN: Y = 100 / (1 + 10 ((1.613-X)*-16.36) ) DPH + 400 μg / ml LFN: Y = 100 / (1 + 10 ((1.607-X)*-unstable) ) DPH + 800 μg / ml LFN: Y = 100 / (1 + 10 ((1.631-X)*-4.714) )

[0070] The EC extrapolated from Figure 5 50 value. The percentage of cytotoxicity was measured by the cytotoxicity after infecting Vero E6 cells with SARS-CoV-2 at an MOI of 0.3:1 for 72 hours. The EC of diphenhydramine in the absence of lactoferrin and in the presence of increasing lactoferrin concentrations 50 was demonstrated to decrease the EC of diphenhydramine by threefold as determined by the cytotoxicity assay, as the presence of LFN 50 .

[0071]

Table 1

[0072] Therefore, when low concentrations of lactoferrin are added in combination with diphenhydramine, it significantly reduces the negative hill gradient, while EC 50 It also reduces [the substance]. This demonstrates a synergistic effect in both efficacy and effectiveness against SARS-CoV-2-induced cytotoxicity when applied in combination.

[0073] Example 3. Combination therapy for the prevention and / or treatment of COVID-19 (SARS-CoV-2 infection). Based on the dosage of diphenhydramine and lactoferrin when administered for the treatment of other conditions, and based on the observed anti-SARS-CoV-2 activity, we propose a formulation of diphenhydramine HCl and unsaturated iron lactoferrin for the prevention and / or treatment of COVID-19 (SARS-CoV-2 infection).

[0074] In some embodiments, the formulation contains 12.5 to 50 mg of diphenhydramine HCl and 1.8 to 3.6 g of unsaturated iron lactoferrin.

[0075] In some embodiments, the formulation comprises 12.5, 15, 20, 25, 30, 35, 40, 45, or 50 mg of diphenhydramine HCl and 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.1, 3.3, 3.5, 3.5, or 3.6 g of unsaturated iron lactoferrin.

[0076] [Table 2]

[0077] In some embodiments, the formulation further comprises an analgesic. The analgesic may be, but is not limited to, acetaminophen, an NSAID, ibuprofen, or naproxen. In some embodiments, the analgesic is acetaminophen. Acetaminophen may be added to any of the formulations listed above. The amount of acetaminophen in the formulation may be about 325 to about 1000 mg.

[0078] In some embodiments, the formulation further comprises a nasal decongestant. The nasal decongestant may be, but is not limited to, phenylephrine and pseudoephedrine.

[0079] [Table 3-1]

[0080] [Table 3-2]

[0081] [Table 4]

[0082] Example 4. Synergistic antiviral activity by combining a sigma receptor ligand with lactoferrin. Diphenhydramine has recently been shown to inhibit SARS-CoV-2 infectivity, and the EC of SARS-CoV-2 was calculated by plaque reduction assays. 50 The EC2 level was 17.4 μg / ml (59.6 μM). This drug is safe, well-characterized, and widely available, making it highly relevant in the search for COVID treatments. Further studies have demonstrated the ability of diphenhydramine to inhibit SARS-CoV-2-induced cytotoxicity, with an EC2 level of 122.0 μg / ml. 50We found (418 μM, Figures 6A and 6B), which is approximately 7 times higher than that found in plaque reduction assays. The inventors have combined diphenhydramine with latoferrin and investigated its antiviral activity against SARS-CoV-2 in terms of EC 50 We tested whether it could be reduced.

[0083] The anti-SARS-CoV-2 activity of diphenhydramine combined with lactoferrin was tested. Host iron sequestration protein lactoferrin has been reported to exhibit direct antiviral activity against SARS-CoV-2. 28、29 It has broad antibacterial properties and host immune-stimulating characteristics. EC 50 To measure the effect on the reduction of EC, various combinations of lactoferrin and diphenhydramine were analyzed. Co-administration of 400 μg / ml lactoferrin and diphenhydramine further reduced SARS-CoV-2 induced cytotoxicity. 50 The concentration was reduced from 55.5% to 54.2 μg / ml (185.7 μM, Figures 6C and 6D). The antiviral enhancing effect of lactoferrin was more pronounced at lower concentrations of diphenhydramine (Figure 6E). Inhibition of viral replication was also investigated by qPCR (Figure 6F). Combining lactoferrin with diphenhydramine resulted in a synergistic effect on antiviral activity against SARS-CoV-2. Diphenhydramine alone at 40 μg / ml resulted in a 32.2% reduction in N protein RNA compared to DMSO alone as a control. Lactoferrin at 400 μg / ml was able to reduce N protein RNA copies by 28.0% 48 hours after infection. When combined, diphenhydramine and lactoferrin inhibited 99.97% of N protein RNA copies, and the 3-log reduction was highly significant. These data demonstrate that diphenhydramine and lactoferrin, both of which have well-characterized safety profiles, have a synergistic effect in inhibiting SARS-CoV-2.

[0084] Viral culture method. The SARS-CoV-2 strain used in this study was UF-1, isolated via nasal swab from a COVID-19 patient at UF Health Shands Hospital. Vero E6 cells were grown in DMEM + 2% FBS + PenStrep. SAEC, H23, and H23-hACE2 cells were grown in RPMI + 10% FBS + PenStrep with 4 μg / ml blastosidine to maintain ACE2 expression as needed. Cells were grown in a humidified incubator at 37°C and 5% CO2. Cells were visualized using an EVOS XL Core microscope.

[0085] Quantitative analysis of viral replication by qPCR. Using SARS-CoV-2, Vero E6 monolayers were biologically and technically triple-infected at a MOI of 0.01 in the presence of each treatment. Two days post-infection (dpi), the monolayers were scraped and recovered in viral lysis buffer (AVL buffer) from the QIAamp Viral RNA Kit (QIAGEN). AVL buffer is a CDC-approved method for viral inactivation. Samples were frozen at -80°C. RNA was purified according to the manufacturer's recommendations. Reverse transcription and cDNA synthesis were performed using the iTaq Universal SYBR Green One-Step Kit (BioRad) and primers targeting the SARS-CoV-2 nucleocapsid (N) gene (NproteinF-GCCTCTTCTCGTTCCTCATCAC SEQ ID NO: 2, NproteinR-AGCAGCATCACCGCCATTG SEQ ID NO: 3). qPCR was performed on a BioRad CFX96. The N protein copy level is calculated using the CT value from a standard curve created using a control plasmid containing the N protein gene, and is presented as genome equivalent (GE) (Integrated DNA Technologies).

[0086] Cytotoxicity Reduction Assay. Vero E6 cells were seeded into 96-well CellBind-treated plates (Corning) and allowed to adhere overnight. The drug was pre-aliquoted in DMEM + 2% FBS. The titrated SARS-CoV-2 aliquots were diluted to obtain a target MOI of 0.2 PFU / cell in solution at the final indicator drug concentration. Triple monolayers were infected by replacing the growth medium with 100 μl of drug / virus suspension. 72 hours post-infection, the supernatant was collected and lactate dehydrogenase (LDH) release was assayed using the Cytox96® non-radioactive cytotoxicity assay (Promega). The assay was performed as recommended by the manufacturer to produce formazan dye. Optical density at 450 nm was measured using a MultiSkan FC plate reader (ThermoFisher). Controls included total LDH release measured by lysis of all cells, spontaneous release from uninfected cells, and the culture medium alone. The toxicity of the treatment alone was also determined in parallel to identify the amount of SARS-CoV-2-induced cytotoxicity produced in the presence of the given treatment. After subtracting spontaneous occurrence and background, the OD450 values ​​were converted to a percentage (100%) of SARS-CoV-2-infected cells in the absence of any drug treatment to obtain the percentage of SARS-CoV-2-induced cytotoxicity. These experiments were performed twice.

[0087] Calculation of inhibitory and effective concentrations. CC 50 Value and EC 50 The values ​​were calculated using the GraphPad Prism9 software nonlinear regression module.

[0088] Example 5. Diphenhydramine and lactoferrin inhibit the production of infectious particles in human lung cells. A novel human lung epithelial cell line, H23-ACE2, susceptible to SARS-CoV-2 infection, was generated by lentiviral transduction to introduce the human ACE2 gene. Single-clonal isolation of the H23-ACE2 transduction cell pool yielded several healthy clones, including clone A2. Successful ACE2 expression was functionally demonstrated by increased cytopathic effects against SARS-CoV-2 infection in the H23-ACE2 cell pool and the isolated cell clone H23-ACE2 clone A2, but not in the parental H23 cell line (Figure 7A). ACE2 surface expression was confirmed by flow cytometry as a rightward peak shift on the X-axis for the H23-ACE2 cell pool and H23-ACE2 clone A2 compared to the untransduced parental H23 cell line and Vero E6 cells. H23-ACE2 clone A2 was used for further experiments. Human lung epithelial cell line H23 was infected with SARS-CoV-2 at an MOI of 0.01. This cell line cannot support SARS-CoV-2 infection without heterologous expression of the ACE-2 receptor. hACE2 expression was shown to be necessary for SARS-CoV-2 infection (Figures 7A and 7B). TCID50 was performed to measure infectious particles released during infection in the presence of diphenhydramine, lactoferrin, and diphenhydramine + lactoferrin. Cells were approximately 1.5 × 10⁶ 3 The cells were initially infected with an MOI of 0.01, equivalent to the virus. Figure 7C shows the ability of diphenhydramine alone, lactoferring alone, and a combination of diphenhydramine and lactoferring to reduce SARS-CoV-2 release during infection in this cell line.

[0089] Generation of ACE-2 lentiviral particles. Lentiviruses containing ACE2 were generated by co-transfecting an ACE expression vector also containing psPAX2, pMD2.G, and the blastosidine selection gene EX-U1285-Lv197 (GeneCopoeia). The plasmid was transfected into HEK293T cells using X-tremeGENE9 (Roche catalog number XTG9-RO) according to the manufacturer's instructions. Eighteen hours after transfection, the medium was replaced with DMEM containing 2% (w / v) bovine serum albumin (BSA), and then the lentiviruses were collected at 24 and 48 hours.

[0090] Transduction of NCI-H23 cells with ACE2 and selection of monoclonal cells. NCI-H23 (also known as H23) cells were obtained from ATCC (CRL-5800), and ACE2 lentivirus was filtered through a 0.45 μm filter and used to transduce H23 cells using reverse transduction. Briefly, the filtered virus particles were added to an H23 cell suspension containing RPMI1640 (Gibco catalog number 1185093) medium supplemented with 10% FBS and 8 μg / ml polyblen (Sigma catalog number TR-1003-G). 72 hours after transduction, the medium was replaced with RPMI1640 supplemented with 10% FBS and 4 μg / ml blastosidine S hydrochloride (Gibco catalog number R21001). Cells were grown in progressively larger cell culture plates, and ACE2 expression was confirmed by infection with SARS-CoV-2 (Canada / ON / VIDO-01 / 2020) and flow cytometry. Single clones were isolated from the H23-ACE2 cell pool using array dilution in 96-well plates. Single clones were collected 2-3 weeks after seeding and grown in progressively larger cell culture plates. After successful isolation, cells were maintained in complete medium containing 2 μg / ml blastosidine.

[0091] Analysis of cell surface ACE2 by flow cytometry. Healthy cells were separated from a monolayer using 0.5 mM EDTA in PBS and centrifuged at 1500 rpm for 3 minutes. The cell pellet was stained with primary ACE2 antibody (0.25 μg / 10⁶ cells, R&D Systems catalog number AF933) at 4°C for 1 hour. The cells were then washed twice with flow wash buffer (2% FBS in PBS), stained with secondary goat IgG APC conjugate antibody (10 μl / 10⁶ cells, R&D Systems catalog number F0108) and 1000× viability discrimination stain (Invitrogen catalog number L34958), and fixed with 2% PFA (diluted in flow wash buffer). The cells were analyzed using a Beckman CytoFLEX flow cytometer and CytoExpert software.

[0092] TCID50 assay in H23 cells. H23 or H23-hACE2 cells were seeded at 1.5 × 10⁵ cells in Corning CellBIND 24-well plates and allowed to adhere overnight. The following day, cells were infected with SARS-CoV-2 at a MOI of 0.01 in the presence of simulated treatment (PBS), diphenhydramine (40 μg / ml), human milk-derived lactoferrin (400 μg / ml), or a combination of diphenhydramine (40 μg / ml) and lactoferrin (400 μg / ml). TCID50 was performed in three independent experiments by diluting the supernatant from H23 infection across eight columns of Vero E6 cells after 48 hours. After 5 days, TCID plates were observed under a microscope for CPE. TCID50 / ml in the original H23-infected culture supernatant was calculated by the Spearman-Karber method. The TCID experiment was technically performed in three consecutive steps as described above, using individual TCID50 / ml values, their mean, and the standard deviation shown. The present invention provides, for example, the following items: (Item 1) A method for treating a subject who is infected with or susceptible to infection by SARS-CoV-related beta-coronavirus, comprising administering a therapeutically effective dose of diphenhydramine and lactoferrin to the subject. (Item 2) A method for treating a subject suffering from SARS-CoV-related beta-coronavirus-related illness, comprising administering a therapeutically effective amount of diphenhydramine and lactoferrin to the subject. (Item 3) A method for preventing infection by SARS-CoV-related beta-coronavirus, comprising administering a therapeutically effective dose of diphenhydramine and lactoferrin to the target. (Item 4) The method according to any one of items 1 to 3, wherein the lactoferrin is human lactoferrin or bovine lactoferrin. (Item 5) The method according to any one of items 1 to 4, further comprising administering one or more additional active ingredients. (Item 6) The method according to item 5, wherein the additional active ingredient is selected from the group consisting of additional antiviral agents, analgesics, and nasal congestion decongestants. (Item 7) The method according to item 6, wherein the analgesic comprises acetaminophen. (Item 8) The method according to any one of items 1 or 2 and 4-7, wherein the subject has been tested positive for SARS-CoV-related beta-coronavirus, has been exposed to SARS-CoV-related beta-coronavirus, is suspected of being exposed to SARS-CoV-related beta-coronavirus, is at risk of being exposed to SARS-CoV-related beta-coronavirus, has or has been diagnosed with SARS-CoV-related beta-coronavirus-related illness, or has acute lung injury due to SARS-CoV-related beta-coronavirus-related illness. (Item 9) The method according to any one of items 1 to 8, wherein the SARS-CoV-related beta-coronavirus is SARS-CoV-2. (Item 10) The method according to any one of items 2 and 4-9, wherein the SARS-CoV-related beta-coronavirus-related disease is COVID-19. (Item 11) A pharmaceutical composition comprising diphenhydramine and lactoferrin. (Item 12) The pharmaceutical composition according to item 11, further comprising pharmaceutically acceptable excipients. (Item 13) The pharmaceutical composition according to item 12, further comprising one or more additional active ingredients. (Item 14) The pharmaceutical composition according to item 13, wherein the one or more additional active ingredients are selected from the group consisting of additional antiviral agents, analgesics, and nasal congestion decongestants. (Item 15) The pharmaceutical composition according to any one of items 11 to 14, wherein the pharmaceutical composition is formulated as a liquid, tablet, coated tablet, chewable tablet, powder, capsule, or nasal preparation. (Item 16) A pharmaceutical composition according to any one of items 11 to 15, for use in the treatment of SARS-CoV-associated beta-coronavirus infection or SARS-CoV-associated beta-coronavirus-related disease. (Item 17) The pharmaceutical composition according to item 16, wherein the SARS-CoV-related beta-coronavirus is SARS-CoV-2. (Item 18) The pharmaceutical composition according to item 16, wherein the SARS-CoV-related beta-coronavirus-related disease is COVID-19. (Item 19) A method for treating a subject who is infected with or susceptible to infection by SARS-CoV-related beta-coronavirus, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an H1 receptor-blocking antihistamine and a therapeutically effective amount of lactoferrin. (Item 20) The method according to item 19, wherein the H1 receptor blocking antihistamine is selected from the group consisting of diphenhydramine, hydroxyzine, cetirizine, azelastine, loratadine, levocetirizine, brompheniramine, fexofenadine, and chlorpheniramine. (Item 21) The method according to item 19 or 20, wherein the pharmaceutical composition further comprises an H2 receptor blocking antihistamine. (Item 22) The method according to item 21, wherein the H2 receptor blocking antihistamine comprises famotidine. (Item 23) The method according to any one of items 19 to 22, wherein the pharmaceutical composition further comprises an NSAID. (Item 24) The method according to item 23, wherein the NSAID includes acetaminophen. (Item 25) The method according to any one of items 19 to 24, wherein the pharmaceutical composition further comprises a cough suppressant. (Item 26) The method according to item 25, wherein the cough suppressant comprises dextromethorphan. (Item 27) The method according to any one of items 19 to 26, wherein the pharmaceutical composition further comprises a decongestant. (Item 28) The method according to item 27, wherein the decongestant comprises phenylephrine. (Item 29) The method according to any one of items 19 to 28, wherein the pharmaceutical composition further comprises an antiemetic or antidiarrheal agent. (Item 30) The method according to item 29, wherein the antiemetic or antidiarrheal agent comprises bismuth subsalicylate or loperamide.

Claims

1. A combination for use in the treatment or prevention of SARS-CoV-associated betacoronavirus infection and / or SARS-CoV-associated betacoronavirus-associated disease, comprising diphenhydramine and lactoferrin.

2. The combination according to claim 1, wherein the lactoferrin is human lactoferrin or bovine lactoferrin.

3. The combination according to claim 1, wherein the combination further comprises one or more additional active ingredients.

4. The combination according to claim 3, wherein the additional active ingredient is selected from the group consisting of additional antiviral agents, analgesics, and nasal congestion decongestants.

5. The combination according to claim 4, wherein the analgesic comprises acetaminophen.

6. The combination according to claim 1, wherein the combination is formulated for administration to a subject, the subject has been tested positive for SARS-CoV-related beta-coronavirus, has been exposed to SARS-CoV-related beta-coronavirus, is suspected of being exposed to SARS-CoV-related beta-coronavirus, is at risk of being exposed to SARS-CoV-related beta-coronavirus, has or has been diagnosed with SARS-CoV-related beta-coronavirus-related disease, or has acute lung injury due to SARS-CoV-related beta-coronavirus-related disease.

7. The aforementioned SARS-CoV-related beta-coronavirus is SARS-CoV-2, and / or The aforementioned SARS-CoV-related beta-coronavirus-related disease is COVID-19. A combination according to any one of claims 1 to 6.

8. A pharmaceutical composition for use in the treatment or prevention of SARS-CoV-related beta-coronavirus infection and / or SARS-CoV-related beta-coronavirus-related disease, comprising diphenhydramine and lactoferrin.

9. The pharmaceutical composition according to claim 8, further comprising a pharmaceutically acceptable excipient.

10. The pharmaceutical composition according to claim 8, further comprising one or more additional active ingredients.

11. The pharmaceutical composition according to claim 10, wherein the one or more additional active ingredients are selected from the group consisting of additional antiviral therapeutic agents, analgesics, and nasal congestion decongestants.

12. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is formulated as a liquid, tablet, coated tablet, chewable tablet, powder, capsule, or nasal preparation.

13. The aforementioned SARS-CoV-related beta-coronavirus is SARS-CoV-2, and / or The aforementioned SARS-CoV-related beta-coronavirus-related disease is COVID-19. The pharmaceutical composition according to claim 8.

14. A combination for use in the treatment or prevention of SARS-CoV-associated betacoronavirus infection and / or SARS-CoV-associated betacoronavirus-associated disease, comprising a therapeutically effective amount of diphenhydramine and a therapeutically effective amount of lactoferrin.

15. The combination for use according to claim 14, wherein the combination further comprises an H2 receptor blocking antihistamine.

16. The combination for use according to claim 15, wherein the H2 receptor blocking antihistamine comprises famotidine.

17. The aforementioned combination is (a) NSAID, (b) Cough suppressants, (c) Decongestant, or (d) Antiemetics or antidiarrheal drugs A combination for use according to any one of claims 14 to 16, further comprising one or more of the above.

18. The aforementioned combination is (a) Acetaminophen, (b) Dextromethorphan, (c) Phenylephrine, and (d) Bismuth salicylate or loperamide A combination for use according to claim 17, further comprising one or more of the above.

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