WO2022010802A1 - Methods for detecting and treating infection - Google Patents

Abstract

The present disclosure provides methods for diagnosing and treating infection in a subject, as well as disorders that arise from infection and/or risk of severe infection. Also provided are systems to diagnose, prognose, monitor, profile, classify, and/or otherwise determine presence of a disease or disorder, such as pathogenic infection, as well as severity of such infections.

Description

METHODS FOR DETECTING AND TREATING INFECTION

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/705,555, filed on July 4, 2020, and U.S. Provisional Patent Application No. 62/705,566, filed on July 5, 2020, which are herein incorporated by reference in their entireties.

FIELD

[0002] The present disclosure relates generally to diagnostics and treatment of disease, and more specifically to methods for detecting and treating infection in a subject.

BACKGROUND

[0003] The emergence of treatment-resistance infections has been on the rise around the world. Recent pandemics such as severe acute respiratory syndrome (SARS), H1N1 flu and most recently, COVID-19 resulted in unprecedented mortality and life-long societal impact. Hospitalized patients are at risk for contracting secondary antibiotic resistant bacterial infections, which account for as much as 50% of the mortality and morbidity associated with viral infections. Therapies such as antibiotics and vaccines require a targeted approach that is only available for some diseases; thus, it is imperative that treatment strategies for severe infectious diseases focus on common underlying mechanisms.

[0004] Complications from serious infections, such as COVID-19 and antibiotic resistant bacterial infections, occur by unresolved systemic hyperinflammation from the body's effort in clearing the infection. This phenomenon is referred to as life-threatening “cytokine storm syndrome” that is a result of activated immune cells releasing excessive pro-inflammatory cytokines including TNFα, IL-6, IL-lp, IL-1, IL-8, G-CSF, MCP-1, IP-10, and MIP-1 in an often failed attempt at eliminating bacterial and viral infections, such as COVID-19. Treatment strategies often focus on targeting a single cytokine (e.g., tocilizumab or anakinra) to reduce systemic inflammation: however, due to the complex interaction and involvement of multiple cytokines, this single cytokine approach often has failed to prevent the cytokine storm syndrome and to result in the increase of mortality. Therefore, decreasing these inflammatory cytokines is important in preventing morbidity and mortality. In addition, conventional anti-inflammatories, such as corticosteroids or NSAIDs (e.g., ibuprofen), while effective in reducing inflammatory cytokines, also prevent the resolution of inflammation and actually increase viral shedding which may aggravate serious infectious diseases, but are not effective for treating severe infections, such as COVID-19. Additionally, the cytokine storm response correlates more directly with death than virus titers, suggesting that regulating the host-immune response is more effective than anti-viral therapies in COVID-19 disease. Thus, focusing on resolving inflammation broadly and preventing both the eicosanoid storm and cytokine storm syndrome is an important treatment strategy that can be implemented across both viral and antibiotic resistant bacterial infections.

[0005] Prior to or in parallel with the induction of the cytokine storm, infections trigger the release of inflammatory eicosanoids, or “eicosanoid storm”, which has not been well- studied in severe infectious diseases. These eicosanoids, produced from the metabolism of regulatory lipids, induce pathological ER-stress that leads to cell senescence and production of inflammatory cytokines and eventually contributing to the cytokine storm syndrome. They also show a positive feedback system where the activated ER stress pathway increases the production of inflammatory eicosanoids. In contrast, cytochrome P450-mediated epoxy-fatty acids (EpFA) derived from the corresponding polyunsaturated fatty acid (PUFA) turn down production of inflammatory eicosanoids and cytokines acting as pro-resolving lipid mediators and moving the ER stress pathway back toward maintaining homeostasis, cell health and resolution. However, EpFA are rapidly metabolized in the body by an enzyme named soluble epoxide hydrolase (sEH) to their corresponding inactive or even inflammatory vicinal diols. Inhibitors of sEH (sEHIs) can stabilize EpFA and increase these pro-resolving mediators. Thus, sEHIs cause a shift in the metabolism of polyunsaturated fatty acids from a pattern of initiation of inflammation to resolution. Moreover, sEHIs are effective in blocking pathological fibrosis by preventing ventilation-perfusion mismatch through regulation of the pulmonary hypoxic vasoconstriction phenomenon, with a dramatic reduction in IL-1 and IL- 6, as well as preventing the induction of ER stress. These findings become particularly important in improving outcomes in pulmonary infections and diseases that require mechanical ventilation.

[0006] Recent data demonstrate that hospitalized COVID-19 patients have increased eicosanoids, specifically the sEH formed metabolites of linoleic acid epoxides, 9,10 and 11,12 DiHOME. These data correlate with inflammatory cytokines and are increased in patients requiring longer hospitalized stay and intubation. The pattern of eicosanoids in COVID-19 patients can be used to predict severity of disease. Additionally, increases in these pathways will identify patients that can benefit from steroid treatments or other therapies that target the lipid signaling pathways, such as sEH inhibitors, NSAIDs or LOX inhibitors. SUMMARY

[0007] In one aspect, disclosed herein is a method of identifying infection in a subject.

The method includes: a) obtaining a sample from the subject; b) identifying lipids present in the sample; c) generating a lipid profile from the identified lipids; and c) analyzing the lipid profile and classifying the lipid profile as being indicative of infection and degree of infection, or not indicative of infection, thereby identifying infection in the subject.

[0008] In another aspect, disclosed herein is a method of treating a subject having, or at risk of having, an infection. The method includes: a) performing the method of the disclosure to determine a status of infection; and b) administering to the subject a therapeutic agent or regime to inhibit or alleviate the infection, thereby treating the subject.

[0009] In another aspect, disclosed herein is a method of treating a subject having, or at risk of having, an infection. The method includes administering to the subject a therapeutic agent to inhibit or alleviate the infection, thereby treating the subject.

[0010] In another aspect, the disclosure provides a non-transitory computer readable storage medium encoded with a computer program. The computer program includes instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method of the disclosure.

[0011] In still another embodiment, the disclosure provides a computing system. The system includes a memory, and one or more processors coupled to the memory, with the one or more processors being configured to perform operations that implement a method of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGURE 1 shows structure of linoleic acid (18:2n6, LA), epoxy octadecenoic acids (EpOMEs), and dihydroxyoctadecenoic acids (DiHOMEs).

[0013] FIGURE 2A is a graphical plot depicting data associated with the methodology of the disclosure as discussed herein.

[0014] FIGURE 2B is a graphical plot depicting date depicting data associated with the methodology of the disclosure as discussed herein. DESCRIPTION

[0015] Disclosed herein are methods for diagnosing and treating infection in a subject, as well as disorders that arise from infection and/or risk of severe infection, e.g., inflammation, sepsis and the like.

[0016] Conventional methodologies tentatively associate use of sEHIs to prevent cytokine storm, reduce ER stress, and reduce complications of sepsis and pulmonary disease. Regardless, the methodology described herein, addresses the pivotal role of lipids as an indicator of disease and/or disease status. Additionally, conventional methodologies do not address the realization that increasing EpFA through sEH inhibition blocks the production of lipids that are ultimately responsible for inducing cytokine storm syndrome and pulmonary damage associated with infection and mechanical ventilation which occurs during many types of bacterial and viral infections. Further, the present disclosure provides that monitoring a lipid profile of a subject, e.g., eicosanoid profiles and the like, can be utilized in identifying treatments for infection by various pathogenic mediated diseases, as wells as subjects that benefit from modulating regulatory lipids through pharmaceutical or dietary interventions. [0017] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements. The term “about” will be understood by persons of ordinary skill in the art.

Whether the term “about” is used explicitly or not, every quantity given herein refers to the actual given value, and it is also meant to refer to the approximation to such given value that would be reasonably inferred based on the ordinary skill in the art.

[0018] It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0019] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287- 9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0020] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. A person of ordinary skill in the art would recognize that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, pentavalent carbon, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. All sequences provided in the disclosed Genbank Accession numbers are incorporated herein by reference as available on Aug. 11, 2011. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0021] As discussed herein, conventional methodologies tentatively associate use of sEHIs to prevent cytokine storm, reduce ER stress, and complications of sepsis and pulmonary diseases. Surprisingly, the methodology described herein, addresses the pivotal role of monitoring lipids (e.g., eicosanoids, prostaglandins, fatty acid diol metabolites, hydroxylated cytochrome P450 PUFA metabolites, and the like), to diagnose, prognose, monitor, profile, classify, and/or otherwise determine presence of a disease or disorder (e.g., pathogenic infection), severity of disease (e.g., pathogenic infection), desired treatments, or subjects that could benefit from modulating regulatory lipids through pharmaceutical or dietary interventions.

[0022] Additionally, as discussed herein, conventional methodologies are unsuitable to detect increasing EpFA through sEH inhibition to block the production of inflammatory lipids that are ultimately responsible for inducing cytokine storm syndrome and pulmonary damage associated with infection and mechanical ventilation which results from many infections.

[0023] “Soluble epoxide hydrolase” (“sEH”) is an enzyme which in endothelial, smooth muscle and other cell types converts EETs to the corresponding diol compounds called dihydroxyeicosatrienoic acids (“DHETs”). The cloning and sequence of the murine sEH is set forth in Grant et al., J. Biol. Chem. 268(23): 17628-17633 (1993). The cloning, sequence, and accession numbers of the human sEH sequence are set forth in Beetham et al.. Arch. Biochem. Biophys. 305(1): 197-201 (1993). The amino acid sequence of human sEH is also set forth as SEQ ID NO:2 of U.S. Pat. No. 5,445,956; the nucleic acid sequence encoding the human sEH is set forth as nucleotides 42-1703 of SEQ ID NO: 1 of that patent. The evolution and nomenclature of the gene is discussed in Beetham et al., DNA Cell Biol. 14(1):61-71 (1995). Soluble epoxide hydrolase represents a single highly conserved gene product with over 90% homology between rodent and human (Arand et al., FEBS Lett., 338:251-256 (1994)).

[0024] The term “modulate” refers to the ability of a compound to increase or decrease the function, or activity, of the associated activity (e.g., soluble epoxide hydrolase).

“Modulation”, as used herein in its various forms, is meant to include antagonism and partial antagonism of the activity associated with sEH.

[0025] Inhibitors of sEH (sEHIs) are compounds that, e.g., bind to sEH or an sEH precursor to partially or totally block the enzyme’s activity. In some aspects, sEHIs contain a urea, amide, carbamate or heterocyclic central pharmacophore. Illustrative sEHIs for use with the presently disclosed methodology include those set forth in U.S. Patent Nos. 7,662,910, 8,008,483, 8,173,805, 8,188,289, 8,212,032, 8,455,520, 8,455,652, 8,476,043, 8,501,783, 9,029,401, 9,029,550, 9,034,903, 9,096,532, 9,296,693, 9,776,991, 9,850,207, 10,377,744,

10,758,511 and 10,858,338, the entire disclosures of which are herein incorporated by reference.

[0026] In one aspect, an sEHI for use with the presently disclosed methodology is a compound having the following structure:

[0027] In one aspect, an sEHI for use with the presently disclosed methodology is a compound having the following structure:

[0028] In one aspect, an sEHI for use with the presently disclosed methodology is a compound having the following structure:

[0029] In one aspect, an sEHI for use with the presently disclosed methodology is a compound having the following structure:

[0030] In one aspect, an sEHI for use with the presently disclosed methodology is a compound having the following structure:

[0031] In one aspect, disclosed herein is a method of identifying infection in a subject. The method includes: a) obtaining a sample from the subject; b) identifying lipids present in the sample; c) generating a lipid profile from the identified lipids; and c) analyzing the lipid profile and classifying the lipid profile as being indicative of infection and degree of infection, or not indicative of infection, thereby identifying infection in the subject.

[0032] In another aspect, disclosed herein is a method of treating a subject having, or at risk of having, an infection. The method includes: a) performing the method of the disclosure to determine a status of infection; and b) administering to the subject a therapeutic agent or regime to inhibit or alleviate the infection, thereby treating the subject.

[0033] In yet another aspect, disclosed herein is a method of treating a subject having, or at risk of having, an infection. The method includes administering to the subject a therapeutic agent to inhibit or alleviate the infection, thereby treating the subject.

[0034] In some aspects, the disclosure provides profiling regulatory lipids, such as prostaglandins, fatty acid diol metabolites formed from sEH, and hydroxylated cytochrome P450 PUFA metabolites, to identify patients at-risk of severe infection.

[0035] In some aspects, the disclosure provides fatty acid diol metabolites and specifically the diols of linoleate as an indication of severe inflammation in viral or bacterial infections. [0036] In some aspects, the disclosure provides use of regulatory lipid profiles to identify patients who will benefit from certain treatments, such as steroids or sEH inhibition (e.g., use of an sEHI) and a time course for optimal treatments so as not to exacerbate illness with immunosuppression.

[0037] In some aspects, the disclosure provides stabilization of EpFA through agents that mimic the action of EpFA, or through inhibition of sEH (e.g., use of an sEHI) to prevent ventilation/perfusion mismatch and decrease lung fibrosis that often results from mechanical ventilation used for pulmonary infections such as COVID-19.

[0038] In some aspects, the methodology of the disclosure provides agents and approaches, such as induction of the cytochrome P450s which produce EpFA, increase cyclic AMP or other pathways which favor production and release of EpFA.

[0039] In some aspects, the methodology of the disclosure utilizes inhibition of sEH to decrease inflammatory eicosanoids preventing cytokine storm syndrome and improving patient outcomes in COVID-19 and other viral and bacterial infections.

[0040] In some aspects, the methodology of the disclosure utilizes inhibition of sEH to prevent ventilation/perfusion mismatch and decrease the lung fibrosis often resulting from mechanical ventilation used for pulmonary infections such as COVID-19.

[0041] In some aspects, the methodology of the disclosure utilizes agents which mimic the action of EpFA. [0042] In various aspects, the methodology of the disclosure includes generating a lipid profile of a subject and analyzing the lipid profile to identify patients having, or at-risk of severe infection, as well as appropriate treatment options. In some aspects, lipids include prostaglandins, fatty acid diol metabolites formed from sEH, and hydroxylated cytochrome P450 PUFA metabolites. In some aspects, lipids include diHOMEs, EpOMEs, and those shown in FIGS. 2A and 2B. In some aspects, the ratio of diHOMEs to EpOMEs is utilized to identify patients having an infection or at-risk of severe infection.

[0043] As used herein, the term “infection” includes an infection and/or related symptom resulting from infection by a pathogenic microbe, e.g., a pathogenic infection. In some aspects, the is a bacterial, fungal, parasitic or viral pathogen. Examples of viral pathogens include, but are not limited to, coronavirus, Zika virus, influenza virus, Ebola virus or HIV.

In some aspects, the viral pathogen is SARS-CoV-2 and SARS-CoV.

[0044] In various aspects, the methodology of the disclosure includes treating a subject with a therapeutic agent, such as an sEHI or other compound disclosed herein, to alleviate or inhibit infection.

[0045] The terms “treat”, “treating” and “treatment” refer to any method of alleviating or abrogating a disease (e.g., pathogenic infection) or its attendant symptoms.

[0046] The term “therapeutically effective amount” refers to that amount of the compound being administered sufficient to prevent or decrease the development of one or more of the symptoms of the disease, condition or disorder being treated.

[0047] The terms “compound” and “agent” as used herein is intended to encompass not only the specified molecular entity but also its pharmaceutically acceptable, pharmacologically active derivatives, including, but not limited to, salts, prodrug conjugates such as esters and amides, metabolites and the like.

[0048] The term “composition” as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By “pharmaceutically acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0049] The “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human. [0050] Pharmaceutically acceptable salts of compounds described herein include conventional nontoxic salts or quaternary ammonium salts of a compound, e.g., from nontoxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. In other cases, described compounds may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

[0051] In embodiments, an sEHI inhibits sEH without also significantly inhibiting mEH. Preferably, at concentrations of 100 mM, the sEHI inhibits sEH activity by at least 50% while not inhibiting mEH activity by more than 10%. sEHIs of the disclosure have an IC50 (inhibition potency or, by definition, the concentration of inhibitor which reduces enzyme activity by 50%) of less than about 100 pM. sEHIs with an IC50 of less than 100 pM are preferred, with an IC50 of less than 75 pM being more preferred and, in order of increasing preference, an IC50 of 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 3 pM, 2 pM, 1 pM, 100 nM, 10 nM, 1.0 nM, or even less, being still more preferred. Assays for determining sEH activity are known in the art and described elsewhere herein. The IC50 determination of the inhibitor can be made with respect to an sEH enzyme from the species subject to treatment.

[0052] Also disclosed herein are pharmaceutical compositions including sEHIs. The term “pharmaceutically acceptable carrier” refers to a non-toxic carrier that may be administered to a patient, together with a compound of this disclosure, and which does not destroy the pharmacological activity thereof. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0053] Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, wool fat and self-emulsifying drug delivery systems (SEDDS) such as a-tocopherol, poly ethyleneglycol 1000 succinate, or other similar polymeric delivery matrices.

[0054] In various aspects, treatment of a patient may include use of a pharmaceutical composition including a compound or agent described herein as the active component in combination with administering the composition with an additional agent or therapy. Such therapies include, but are not limited to, an anemia therapy, a diabetes therapy, a hypertension therapy, a cholesterol therapy, neuropharmacologic drugs, drugs modulating cardiovascular function, drugs modulating inflammation, immune function, production of blood cells; hormones and antagonists, drugs affecting gastrointestinal function, chemotherapeutics of microbial diseases, and/or chemotherapeutics of neoplastic disease. Other pharmacological therapies can include any other drug or biologic found in any drug class. For example, other drug classes can comprise steroids, allergy/cold/ENT therapies, analgesics, anesthetics, antiinflammatories, antimicrobials, antivirals, asthma/pulmonary therapies, cardiovascular therapies, dermatology therapies, endocrine/metabolic therapies, gastrointestinal therapies, cancer therapies, immunology therapies, neurologic therapies, ophthalmic therapies, psychiatric therapies or rheumatologic therapies. Other examples of agents or therapies that can be administered with the compounds described herein include a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, a cytokine, a growth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound.

[0055] The term “therapeutically effective amount” as used herein refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1)

Preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease, (2) Inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and (3) Ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).

[0056] The present disclosure is based, in part, on the discovery that inhibitors of sEH are efficacious in alleviating, reducing, inhibiting and preventing infection, including severe infection, in humans or non-human mammals, particularly symptoms or related disorders of infection that could not be effectively treated using currently employed treatments or medications (e.g., antibiotics, antivirals, non-steroidal anti-inflammatory drugs and/or analgesics were inefficacious).

[0057] As used herein, the terms “combination,” “combined,” and related terms refer to the simultaneous or sequential administration of therapeutic agents in accordance with this disclosure. For example, a described compound may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present disclosure provides a single unit dosage form comprising a described compound, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. Two or more agents are typically considered to be administered “in combination” when a patient or individual is simultaneously exposed to both agents. In many embodiments, two or more agents are considered to be administered “in combination” when a patient or individual simultaneously shows therapeutically relevant levels of the agents in a particular target tissue or sample (e.g., in brain, in serum, etc.). [0058] When the compounds of this disclosure are administered in combination therapies with other agents, they may be administered sequentially or concurrently to the patient. Alternatively, pharmaceutical or prophylactic compositions according to this disclosure comprise a combination of ivermectin, or any other compound described herein, and another therapeutic or prophylactic agent. Additional therapeutic agents that are normally administered to treat a particular disease or condition may be referred to as “agents appropriate for the disease, or condition, being treated.”

[0059] The compounds utilized in the compositions and methods of this disclosure may also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those, which increase biological penetration into a given biological system (e.g., blood, lymphatic system, or central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and/or alter rate of excretion.

[0060] According to a preferred embodiment, the compositions of this disclosure are formulated for pharmaceutical administration to a subject or patient, e.g., a mammal, preferably a human being. Such pharmaceutical compositions are used to ameliorate, treat or prevent any of the diseases described herein in a subject.

[0061] Agents of the disclosure are often administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate- buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0062] In some embodiments, the present disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of a described compound, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents for use in treating infection. While it is possible for a described compound to be administered alone, it is preferable to administer a described compound as a pharmaceutical formulation (composition) as described herein. Described compounds may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

[0063] As described in detail, pharmaceutical compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream or foam; sublingually; ocularly; transdermally; or nasally, pulmonary and to other mucosal surfaces. [0064] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0065] Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. [0066] Formulations for use in accordance with the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient, which can be combined with a carrier material, to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound, which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of active ingredient. In some embodiments, this amount will range from about 5% to about 70%, from about 10% to about 50%, or from about 20% to about 40%. [0067] In certain embodiments, a formulation as described herein comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present disclosure. In certain embodiments, an aforementioned formulation renders orally bioavailable a described compound of the present disclosure.

[0068] Methods of preparing formulations or compositions comprising described compounds include a step of bringing into association a compound of the present disclosure with the carrier and, optionally, one or more accessory ingredients. In general, formulations may be prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0069] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in anon- toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxy ethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as those described in Pharmacopeia Helvetica, or a similar alcohol. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0070] In some cases, in order to prolong the effect of a drug, it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0071] Injectable depot forms are made by forming microencapsule matrices of the described compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.

[0072] The pharmaceutical compositions of this disclosure may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers, which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions and solutions and propylene glycol are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

[0073] Formulations described herein suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. Compounds described herein may also be administered as a bolus, electuary or paste.

[0074] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), an active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; absorbents, such as kaolin and bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0075] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made in a suitable machine in which a mixture of the powdered compound is moistened with an inert liquid diluent. If a solid carrier is used, the preparation can be in tablet form, placed in a hard gelatin capsule in powder or pellet form, or in the form of a troche or lozenge. The amount of solid carrier will vary, e.g., from about 25 to 800 mg, preferably about 25 mg to 400 mg. When a liquid carrier is used, the preparation can be, e.g., in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example, using the aforementioned carriers in a hard gelatin capsule shell.

[0076] Tablets and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may alternatively or additionally be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze- dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0077] Liquid dosage forms for oral administration of compounds of the disclosure include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0078] Besides inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0079] Suspensions, in addition to active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0080] The pharmaceutical compositions of this disclosure may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this disclosure with a suitable non-irritating excipient, which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

[0081] Topical administration of the pharmaceutical compositions of this disclosure is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this disclosure may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topical transdermal patches are also included in this disclosure.

[0082] The pharmaceutical compositions of this disclosure may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. [0083] For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

[0084] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Dissolving or dispersing the compound in the proper medium can make such dosage forms. Absorption enhancers can also be used to increase the flux of the compound across the skin. Either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel can control the rate of such flux.

[0085] Examples of suitable aqueous and nonaqueous carriers, which may be employed in the pharmaceutical compositions of the disclosure, include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0086] Such compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Inclusion of one or more antibacterial and/orantifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like, may be desirable in certain embodiments. It may alternatively or additionally be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents, which delay absorption such as aluminum monostearate and gelatin.

[0087] In certain embodiments, a described compound or pharmaceutical preparation is administered orally. In other embodiments, a described compound or pharmaceutical preparation is administered intravenously. Alternative routes of administration include sublingual, intramuscular, and transdermal administrations.

[0088] When compounds described herein are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0089] Preparations described herein may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for the relevant administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred.

[0090] Such compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracistemally and topically, as by powders, ointments or drops, including buccally and sublingually.

[0091] Regardless of the route of administration selected, compounds described herein which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present disclosure, are formulated into pharmaceutical dosage forms by conventional methods known to those of skill in the art.

[0092] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0093] The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal , oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization.

[0094] In treatment, the dose of agent optionally ranges from about 0.0001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.15 mg/kg to about 3 mg/kg, 0.5 mg/kg to about 2 mg/kg and about 1 mg/kg to about 2 mg/kg of the subject's body weight. In other embodiments the dose ranges from about 100 mg/kg to about 5 g/kg, about 500 mg/kg to about 2 mg/kg and about 750 mg/kg to about 1.5 g/kg of the subject's body weight. For example, depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of agent is a candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage is in the range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. Unit doses can be in the range, for instance of about 5 mg to 500 mg, such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg and 300 mg. The progress of therapy is monitored by conventional techniques and assays.

[0095] In some embodiments, an agent is administered to patient at an effective amount (or dose) of less than about 1 pg/kg, for instance, about 0.35 to about 0.75 pg/kg or about 0.40 to about 0.60 pg/kg. In some embodiments, the dose of an agent is about 0.35 pg/kg, or about 0.40 pg/kg, or about 0.45 pg/kg, or about 0.50 pg/kg, or about 0.55 pg/kg, or about 0.60 pg/kg, or about 0.65 pg/kg, or about 0.70 pg/kg, or about 0.75 pg/kg, or about 0.80 pg/kg, or about 0.85 pg/kg, or about 0.90 pg/kg, or about 0.95 pg/kg or about 1 pg/kg. In various embodiments, the absolute dose of an agent is about 2 pg/subject to about 45 pg/subject, or about 5 to about 40, or about 10 to about 30, or about 15 to about 25 pg/subject. In some embodiments, the absolute dose of an agent is about 20 pg, or about 30 pg, or about 40 pg.

[0096] In various embodiments, the dose of an agent may be determined by the patient’s body weight. For example, an absolute dose of an agent of about 2 pg for a pediatric human patient of about 0 to about 5 kg (e.g. about 0, or about 1, or about 2, or about 3, or about 4, or about 5 kg); or about 3 pg for a pediatric human patient of about 6 to about 8 kg (e.g. about 6, or about 7, or about 8 kg), or about 5 pg for a pediatric human patient of about 9 to about 13 kg (e.g. 9, or about 10, or about 11, or about 12, or about 13 kg); or about 8 pg for a pediatric human patient of about 14 to about 20 kg (e.g. about 14, or about 16, or about 18, or about 20 kg), or about 12 pg for a pediatric human patient of about 21 to about 30 kg (e.g. about 21, or about 23, or about 25, or about 27, or about 30 kg), or about 13 pg for a pediatric human patient of about 31 to about 33 kg (e.g. about 31, or about 32, or about 33 kg), or about 20 pg for an adult human patient of about 34 to about 50 kg (e.g. about 34, or about 36, or about 38, or about 40, or about 42, or about 44, or about 46, or about 48, or about 50 kg), or about 30 pg for an adult human patient of about 51 to about 75 kg (e.g. about 51, or about 55, or about 60, or about 65, or about 70, or about 75 kg), or about 45 pg for an adult human patient of greater than about 114 kg (e.g. about 114, or about 120, or about 130, or about 140, or about 150 kg).

[0097] In certain embodiments, an agent in accordance with the methods provided herein is administered subcutaneously (s.c.), intravenously (i.v.), intramuscularly (i.m.), intranasally or topically. Administration of an agent described herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the human patient. The dosage may be administered as a single dose or divided into multiple doses. In some embodiments, an agent is administered about 1 to about 3 times (e.g. 1, or 2 or 3 times).

[0098] The following examples are provided to further illustrate the embodiments of the present disclosure but are not intended to limit the scope of the disclosure. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES EXAMPLE 1

Plasma Linoleate Diols Are Potential Biomarkers for Severe COVID-19 Infections

[0099] Polyunsaturated fatty acids are metabolized into regulatory lipids important for initiating inflammatory responses in the event of disease or injury and for signaling the resolution of inflammation and return to homeostasis. The epoxides of linoleic acid (leukotoxins) regulate skin barrier function, perivascular and alveolar permeability and have been associated with poor outcomes in bum patients and in sepsis. It was later reported that blocking metabolism of leukotoxins into the vicinal diols ameliorated the deleterious effects of leukotoxins, suggesting that the leukotoxin diols are contributing to the toxicity. During quantitative profiling of fatty acid chemical mediators (eicosanoids) in COVID-19 patients, we found increases in the regioisomeric leukotoxin diols in plasma samples of hospitalized patients suffering from severe pulmonary involvement. In rodents these leukotoxin diols cause dramatic vascular permeability and are associated with acute adult respiratory like symptoms. Thus, pathways involved in the biosynthesis and degradation of these regulatory lipids should be investigated in larger biomarker studies to determine their significance in COVID-19 disease. In addition, incorporating diols in plasma multi-omics of patients could illuminate the COVID-19 pathological signature along with other lipid mediators and blood chemistry.

[00100] The pandemic coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiates an aberrant immunological response resulting in a wide range of disease severities ranging from asymptomatic cases to severe cases with rapid progression to acute respiratory distress syndrome (ARDS) and death. Patients with severe COVID-19 show evidence of hyperinflammation with increased release of inflammatory cytokines. The role of a cytokine release syndrome, or cytokine storm, in COVID-19 has drawn much attention. However, recent reports demonstrate that, although pro-inflammatory cytokine levels are elevated in severe COVID-19 patients, they are lower than levels usually observed in non-COVID ARDS, suggesting additional factors lead to severe outcomes in some patients.

[00101] One of the key pathways regulating the immune response to infections is the release of regulatory lipid mediators that have dual functions of driving inflammation [e.g., prostaglandins (PGE2)] or promoting resolution of inflammation and return to homeostasis [e.g., long chain epoxy fatty acids (EpFAs)]. Recent data indicate a role of dysregulated lipid profiles in COVID-19 and identified cytochrome P450 (CYP) metabolites of polyunsaturated fatty acids (PUFA) as potential biomarkers of disease severity.

[00102] Linoleic acid (18:2n6, LA) is the primary source of essential long chain n-6 PUFAs. CYP450 enzymes act on linoleate directly to generate linoleic epoxides (epoxyoctadecenoic acids, EpOMEs), which are further metabolized by soluble epoxide hydrolase (sEH) to their corresponding leukotoxin diols (dihydroxyoctadecenoic acids, or DiHOMEs; Figure 1). These LA metabolites regulate vascular permeability and stimulate neutrophil chemotaxis. The epoxides were originally termed leukotoxins because of their suspected cytotoxic effects and implications in advancing acute and chronic inflammatory diseases and in the pathophysiology of ARDS. The deleterious effects of LA metabolites were originally attributed to EpOMEs. It was later discovered that the toxicities attributed to leukotoxins were in fact driven by leukotoxin diols or DiHOMEs, and blocking their formation would alleviate toxicities previously associated with leukotoxin. Despite its potential role in advancing ARDS, the role of these LA metabolites in the pathophysiology of COVID-19 has not been evaluated to date.

[00103] In this pilot study, five sequential day plasma samples from six patients with COVID-19 were profiled for lipidomic changes in COVID-19 disease compared to healthy controls. Results indicate that in addition to expected increases in inflammatory PGE2 and leukotrienes, 12,13 DiHOME and 9,10 DiHOME concentrations are significantly higher in COVID-19 patients compared to healthy controls. This is one of the first studies to focus on oxylipin chemical mediators in COVID-19 disease.

[00104] METHODS

[00105] A retrospective study was performed using prospectively collected plasma samples and clinical/phenotype data. For oxylipin analysis, heparinized plasma was collected from six patients with laboratory-confirmed SARS-CoV-2 infection and admitted to the University of California Davis Medical Center in Sacramento, CA and 44 samples from healthy controls chosen from a recently completed clinical study. For comparison of cytokines, 75 plasma samples from healthy volunteers was obtained from the California Central Valley Delta Blood Bank (Stockton, CA, United States) prior to the COVID-19 pandemic. The methods used for blood collection, plasma processing, use of anti- coagulants/antioxidant/preservatives, and flash-freeze protocol were well-matched between case and control groups. The UC Davis and UC San Diego Institutional Review Boards have approved the use of anonymized biospecimens for this study.

[00106] Lipid Mediator Profiling

[00107] Plasma (200 μL) samples were aliquoted to a cocktail solution including 600 pL of methanol with 10 pL of 500 nM of surrogate solution including 9 isotope-labeled oxylipins (d4 PGFla, d4 PGE2, d4 TXB2, d4 LTB4, d620 HETE, dl 1 14,15 DiHETrE, d8 9 HODE, d8 5 HETE, and dl 1 11,12 EpETrE). Before the extraction, the samples were vortexed and centrifuged at 3,000 rpm in a biosafety hood. The supernatants were then loaded on prewashed SPE cartridges and washed with two column volumes of 5% MeOH solution before elution by 0.5 mL of MeOH and 1.5 mL of ethyl acetate. The eluents were dried under vacuum using the Nutec MaxiVac™ vacuum concentrator (Farmingdale, NY, United States) before reconstitution with 50 pL of 100 nM CUDA solution in methanol. Then, the extracted samples were analyzed using the UPLC/MS/MS system [Waters Acquity UPLC (Milford, MA, United States)] hyphenated to AB Sciex™ 6,500 + QTrap™ system (Redwood City, CA, United States). The detailed parameters for the UPLC/MS/MS method were described previously.

[00108] Cytokine Multiplex

[00109] Plasma cytokines were measured using a multiplex magnetic bead-based cytokine detection kit purchased from Bio-Rad (12007283). Cytokines were measured according to manufacturer's instructions. Data are provided at frontiersin.org/articles/10.3389/fphys.2021.663869/full#supplementary- material.

[00110] Statistical Analysis

[00111] To test for differences between the COVID-19 and the control group cytokine levels, cytokine levels were logio transformed to fit a normal distribution and analyzed in Graphpad Prism™ (version 8.4.3) using the Wilcoxon rank-sum test with COVID positive and negative status as the main effect.

[00112] Lipid mediator results were analyzed using MetaboAnalyst™ and scaled using autoscaling before analysis. Multiple data sets described below were integrated to prioritize the oxylipins as possible biomarkers contributing to the severity of COVID. Oxylipins were analyzed by multiple independent t-tests using patient vs. control as the variable and the two- stage step-up method of Benjamini, Krieger and Yekutieli to determine a false discovery rate to generate the volcano plot.

[00113] The lipid mediators were then ranked by their effect sizes (e.g., the fold-difference between mean analyte concentration in each group). The analytes with the largest effect sizes were further evaluated by random effect ANOVA models. We minimized type 1 errors by testing for between-group differences among the analytes with the largest effect sizes and to improve the likelihood of identifying analytes that showed best potential to serve as biomarkers of disease severity. Each analyte with an effect size above 8 (i.e., analyte concentrations > 8-fold different) was used as a response variable. Random effect ANOVAs were run with “patient” as a random effect to account for the multiple measurements from the same patient, and the fixed effect was “group” (i.e., COVID positive or control). The loglo- transformation of the analyte concentrations was applied. The analysis was done in JMP Pro™ Versionl5.

[00114] RESULTS

[00115] Demographics of patient samples are represented in Table 1 below. Seventy-seven lipid mediators were detected from all the patients' samples. Levels of multiple key pro- inflammatory cytokines and chemokines were significantly higher in patients with COVID-

19 than in healthy controls (data not shown), confirming the activation of the immune response against the virus. Overall, increases were moderate and consistent with those reported in the literature.

[00116] Table 1: Clinical characteristics of Sars-Cov-2 patients¹.

All patients were above 45 years of age and had a cough upon admission. Clinical ID/Patient ID² (patients were assigned a clinical ID at the hospital. For simplification, they were reassigned a number from 1-6. SOB³ (shortness of breath associate with other respiratory illness); SOB⁴ (shortness of breath); FLS (flu-like symptoms); AHRF (Acute Hypoxic Respiratory Failure); ARDS (Acute Respiratory Disease); and Pnu (Pneumonia).

[00117] A volcano plot analysis was performed to evaluate the differences in lipi domic profile between COVID-19 patients and healthy controls (Figure 2A). The analysis identified

18 differential lipid mediators with statistically significant differences (p < 0.01) with more than four-fold change between groups.

[00118] Oxylipins were ranked according to effect size (Table 2 below) between COVID-

19 patients and controls. The 9,10 and 12,13 DiHOME metabolites had the biggest effect size (17.94 and 14.12, respectively), followed by PGE2 (12.55). As expected, the epoxides of arachidonic acid (AA) and linolenic acid also increased compared to healthy controls presumably due to biosynthesis and systemic release of free fatty acids from membranes in response to inflammation. The epoxides and diols of the omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), did not show any increases (effect size < 7). [00119] Table 2: Effect size (mean fold-difference between COVID-positive and control) of EpFA, diols, and oxylipins with greater than 8-dolf difference (*p < 0.0001).

Effect size of oxylipins compared to healthy controls

Increased EpFA from the most abundant dietary fatly acids (AA and LA) is expected due to release from cellular membranes in response to inflammation. A4 epoxides, EpETrE or EETs are anti-inflammatory compounds, but their low concentration and rapid conversion by the sEH is thought to limit their efficacy.

[00120] Figure 2B demonstrates that changes in the DiHOME concentrations had a more prominent effect in separating patients and controls compared to the EpOMEs. The EpOME/DiHOME ratios also demonstrated case-status predictive effects (data not shown). The prostaglandins, PGE2 and PGD2, as well as related cyclooxygenase (COX) metabolites had large effect sizes but surprisingly were low in concentration in patients with evidence of elevated cytokines. It is worth noting that the large effect size of prostaglandin resulted from a single patient (see online dataset for individual data). Similarly, leukotriene B4 (LTB4, leukocyte aggregating factor) level was surprisingly low for patients with high level of inflammation, marked by elevated cytokines. This finding was also largely driven by the effect of one single patient.

[00121] DISCUSSION

[00122] It is clear that the two regioisomeric linoleic acid diols (DiHOMES) had highly elevated concentrations in these COVID-19 positive patients, as did their precursor epoxides (EpOMEs; Table 2). Previous studies show that high levels of the epoxide and diol metabolites of linoleic acid are mitochondrial toxins, stimulate vascular permeability and that injection of either metabolite into mice leads to pulmonary edema and inflammation reminiscent of ARDS. However, if inhibitors of the sEH are administered, the edema from EpOMEs is blocked but not that from the DiHOMEs, suggesting that DiHOMEs play a role in lung disease and possibly a role in the pathophysiology of COVID-19. In contrast, the EET regioisomers and epoxides of other long chain PUFA are under scrutiny as inflammation resolving mediators. Their concentrations are quite low, and the sEH is thought to be largely responsible for converting the biologically active epoxides to their corresponding diols, thereby reducing their inflammation resolving potency. The online dataset represented by the volcano plot in Figure 2A and the effect sizes in Table 2 do not indicate that these compounds are associated with severe COVID, although increasing epoxides from arachidonic acid (ARA), EPA, and DHA to yield the EET, EEQ, and EDP regioisomers would be predicted to help resolve inflammation.

[00123] The levels of ARA diols from the corresponding EET epoxides as well as the epoxides and diols of omega-3 fatty acids were low in most subjects with relatively small differences between the COVID positive and control groups. A caution is that the data on omega-3 metabolites in human subjects can be hard to quantify in part because the average dietary levels of omega-3 fatty acids are low. Fatty acid composition, including omega-3 fatty acids, can become quite high due to supplementation. For example, the omega-6 fatty acid LA was once a relatively rare dietary lipid in our evolutionary history but is now a major dietary lipid in the western diet. In many western diets the levels of linoleate are far higher than that anticipated from even recent evolutionary history. As an example, the blood levels of the EpOMEs we report here in COVID-19 patients are approximately 10 x higher than levels found in ICU-admitted bum patients. Thus, the high levels of linoleate substrate would be expected to compete with long chain PUFA thus reducing inflammation-resolving epoxides, such as the EETs, EEQs and EDPs, and increasing linoleate epoxides or leukotoxins as shown by our data. Although AA and longer chain omega-3 fatty acid epoxides are better substrates for the sEH, the leukotoxins are still excellent substrates. sEH action on leukotoxins leads to metabolic products that are cytotoxic, proinflammatory, and cause extensive perivascular and alveolar edema reminiscent of ARDS in mice.

[00124] An obvious question remains as to why cells produce a pro-inflammatory metabolite which increases vascular permeability during COVID infection. A possible answer comes from an inspection of the AA cascade where the largely (but not exclusively) pro-inflammatory COX and lipoxygenase (LOX) pathways are countered by the more recently discovered and largely anti-inflammatory pathway termed the cytochrome CYP450 pathway. During inflammation, PUFA are released from cell membranes and are metabolized into epoxides thought to resolve inflammation; however, this process is often dysregulated in patients with severe disease. Specifically, while cytochrome P450 metabolism of PUFA forms mostly anti-inflammatory and inflammation-resolving fatty acid epoxides such as EETs, EDPs and EEQs (from AA, EPA, and DHA, respectively), metabolites from LA and other omega-6 PUFAs generated by other enzymes such as COX and LOX form mostly pro- inflammatory compounds. As shown in the results, the COX-generated prostaglandins (e.g., PGE2) and LOX-generated leukotrienes (LTB4) were increased as part of the inflammatory response during COVID- 19. The EpFA resolve effects of these inflammatory eicosanoids directly through downregulation of inflammation, and indirectly by stimulating the production of specific proresolving mediators (SPMs). However, the sEH enzyme is up- regulated during inflammation, resulting in conversion of beneficial compounds into inactive or even pro-inflammatory diols. Under normal conditions, CYP450 oxidation of PUFA into EpFA is tightly regulated and occurs at a slower rate than the hydrolysis of EpFA into diols. EpFA are often stored in lipid membranes and are thought to be released during inflammation; however, the rapid conversion by sEH during inflammation limits their concentration in vivo.

[00125] The high abundance of linoleate as a substrate, coupled with the increased biosynthesis of anti-inflammatory EpFA during severe coronavirus infections and the induction of sEH in an inflammatory state may explain the increased rate of synthesis and concentration of leukotoxin diols observed in COVID-19 patients in our study. This finding raises the possibility that amelioration of COVID-19 symptoms may be achieved in part by reduction of omega-6-rich diet, or an enhanced omega-3 fatty acid intake in patients hospitalized for COVID-19. Linoleate at quite low levels is an essential fatty acid for maintenance of skin barrier function, yet an early study (1958) showed that even with no dietary fat intake, 2% of energy from linoleate was enough to maintain skin barrier function. Therefore, reducing linoleate intake or substituting it with “anti-inflammatory” lipids such as n-3 rich fish oil is unlikely to have a deleterious effect on the long-term health. Indeed, this approach is currently being evaluated through intravenous omega-3 administration in COVID-19 hospitalized patients in the COVID-Omega-F Trial. Further benefits from reducing omega-6 fatty acid intake germane to the COVID-Omega-F Trial show increased bioavailability of omega-3 fatty acids with reduced LA consumption. Particularly the “omega” olefins of EPA and DHA are good substrates for epoxidation by relevant cytochrome P450s. Thus, large infusions of omega-3 fatty acids would be predicted to reduce the biosynthesis of the omega-6 EpOMEs by substrate competition. A second prediction is that infusion of omega-3-fatty acids would not lead to a significant increase in COX products because of the substrate preferences of the COXs. On the other hand, the anti-inflammatory P450 products are expected to be increased. These EEQ and EDP epoxides also are excellent substrates for the sEH and by competition should reduce the hydration of non-cytotoxic EpOMEs to the DiHOMEs (cytotoxic leukotoxin diols). A reduction in linoleate metabolites could partially explain the difference that omega-3 supplementation had in ARDS related mortality. Given the evidence of the role DiHOMEs play in exacerbating ARDS, the possibly that these metabolites could serve as biomarkers for COVID-19 disease is strengthened. [00126] Inhibition of the in vivo sEH can also block the toxicity of linoleate epoxides through stabilizing anti-inflammatory long chain EpFAs and blocking the formation of the leukotoxin diols as demonstrated in our earlier studies. This evidence points to the possibility that pharmacological inhibition of the sEH will enhance and synergize with the proresolving effects of omega-3 supplementation in COVID-19 patients, leading to improvement of COVID symptoms.

[00127] This was a pilot study designed primarily to inform later experimental designs, and the relatively small sample size limits interpretation. Another limitation is that the small sample size resulted in high variability in disease severity as well as timing of disease onset and resolution which made temporal relationship between blood biomarkers and specific COVID symptoms difficult to evaluate. Our data are novel in that they shed light on a class of lipid mediators that are likely to be important for the pathogenesis of COVID-19 progression. A beher understanding of mechanisms involved in COVID-19 pathophysiology are rapidly emerging, and the importance of LA and its metabolites in this disease is becoming apparent. Recent studies identified that LA binds to a fatty acid binding pocket in the SARS-CoV-2 spike protein stabilizing its confirmation in a manner that decreases viral entry into the host cell. In support of this finding, a previous study demonstrated high LA concentrations associated with lower COVID-19 severity. Neither study monitored LA metabolites therefore making it impossible to understand the biological roles of the metabolites and how they may impact interpretation from other studies. Our data described here fill a missing gap of the role bioactive mediators play in COVID-19 and emphasize a critical need to better understand the relationship between dietary lipids and their bioactive metabolites. This knowledge will bring about important insights that may lead to effective strategies to prevent rapidly worsening of COVID-19 symptoms and improved treatment efficacy. The data support further investigation on the use of DiHOME regioisomers as biological mediators or biomarkers interacting synergistically through a cross-omic network of cytokines, other lipid mediators including SPMs like resolvins, and blood chemistry to predict severe COVID-19 disease.

[00128] FIGURE LEGENDS

[00129] Figure 1: Structure of LA, EpOmE, and DiHOME.

[00130] Figure 2: Plasma collected once from healthy COVID-19 negative controls (n =

44) and over five sequential days from hospitalized COVID-19 positive patients (n = 6). (A) Volcano plot of oxylipins analyzed in COVID-19 patients compared to healthy controls. Red dots identify metabolites that had a >4 fold-change and false discovery rate (p < 0.01), 18 compounds in total. To generate the Volcano plot, data from each COVID-19 patient was averaged over the 5-days before averaging as a group and comparing to the average of all control subject data. (B) Plasma concentration of EpOME and DiHOME in five sequential samples collected from six hospitalized COVID-19 positive patients and control samples collected separately from healthy volunteers (n = 44). Data from indMdual days is represented for each COVID patients and for each indMdual healthy control. The ratio of EpOME:DiHOME, excluding patient #3 who had low levels of both EpOMES and DiHOMES, was higher in control samples vs. COVID-19 patients on day 1 and steadily increased over time in COVID-19 patients. Overall ratios in COVID-19 patients were 30% lower than healthy controls indicating that DiHOMES increased in greater amounts compared to EpOME.

[00131] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific composition and procedures described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the following claims.

Claims

What is claimed is:

1. A method of identifying infection in a subj ect, comprising: a) obtaining a sample from the subject; b) identifying lipids present in the sample; c) generating a lipid profile from the identified lipids; and c) analyzing the lipid profile and classifying the lipid profile as being indicative of infection and degree of infection, or not indicative of infection, thereby identifying infection in the subject.

2. The method of claim 1, further comprising treating the subject with a therapeutic agent or regime where the lipid profile is indicative of infection.

3. The method of claim 1, wherein the lipid profile comprises prostaglandins, fatty acid diol metabolites formed from soluble Epoxide Hydrolase (sEH), and/or hydroxylated cytochrome P450 polyunsaturated fatty acid (PUFA) metabolites, thereby being indicative of infection.

4. The method of claim 3, further comprising classifying the lipid profile as being from a subject having, or at risk of, a severe infection.

5. The method of claim 4, wherein the lipid profile comprises an elevated amount of a fatty acid diol metabolite.

6. The method of claim 5, wherein the fatty acid diol metabolite is a diol of linoleate.

7. The method of claim 1, further comprising determining a treatment regime for the subject based on the lipid profile.

8. The method of claim 7, wherein the treatment regime comprises administration of a therapeutic agent.

9. The method of claim 8, wherein the therapeutic agent is a steroid, agent that inhibits sEH, or agent that stabilizes and/or increases levels of cytochrome P450-mediated epoxy-fatty acids (EpFA) derived from a corresponding polyunsaturated fatty acid (PUFA).

10. The method of claim 9, wherein the therapeutic agent is an agent that induces stabilized or increased levels of EpFA.

11. The method of any preceding claim, wherein the infection is a pathogenic infection.

12. The method of claim 11, wherein the pathogen is a bacterial, fungal, parasitic or viral pathogen.

13. The method of claim 12, wherein the pathogen is a viral or bacterial pathogen.

14. The method of claim 13, wherein the viral pathogen is a coronavirus, Zika virus, influenza virus, Ebola virus or HIV.

15. The method of claim 14, wherein pathogen is selected from SAR S-CoV-2 arid S ARS- CoV.

16. A method of treating a subject having, or at risk of having, an infection, comprising: a) performing the method of any preceding claim to determine a status of infection; and b) administering to the subject a therapeutic agent or regime to inhibit or alleviate the infection, thereby treating the subject.

17. The method of claim 16, wherein the administering is determined by the lipid profile.

18. The method of claim 17, wherein the therapeutic agent is a steroid, agent that inhibits sEH, or agent that stabilizes and/or increases levels of cytochrome P450-mediated epoxy-fatty acids (EpFA) derived from a corresponding polyunsaturated fatty acid (PUFA).

19. The method of claim 18, wherein the therapeutic agent is an agent induces stabilized or increased levels of EpFA.

20. The method of claim 16, wherein the infection is a pathogenic infection.

21. The method of claim 20, wherein the pathogen is a bacterial, fungal, parasitic or viral pathogen.

22. The method of claim 21, wherein the pathogen is a viral or bacterial pathogen.

23. The method of claim 22, wherein the viral pathogen is a coronavirus, Zika virus, influenza virus, Ebola virus or HIV.

24. The method of claim 23, wherein pathogen is selected from SARS-CoY-2 and SAKS- CoV.

25. A method of treating a subject having, or at risk of having, an infection, comprising administering to the subject a therapeutic agent to inhibit or alleviate the infection, wherein the agent inhibits sEH, stabilizes and/or increases levels of cytochrome P450-mediated epoxy-fatty acids (EpFA) derived from a corresponding polyunsaturated fatty acid (PUFA), thereby treating the subject.

26. The method of claim 25, wherein the infection is a pathogenic infection.

27. The method of claim 26, wherein the pathogen is a bacterial, fungal, parasitic or viral pathogen.

28. The method of claim 27, wherein the pathogen is a viral or bacterial pathogen.

29. The method of claim 28, wherein the viral pathogen is a coronavirus, Zika virus, influenza virus, Ebola virus or HIV.

30. The method of claim 29, wherein pathogen is selected from SARS-€oV-2 and SARS- CoV.

31. A non-transitory computer readable storage medium encoded with a computer program, the program comprising instructions that when executed by one or more processors cause the one or more processors to perform operations to perform any step of the method of any of claims 1-15.

32. A computing system comprising: a memory; and one or more processors coupled to the memory, the one or more processors configured to perform operations to perform any step of the method of any of claims 1-15.