RELATED APPLICATIONS

This application claims priority to U.S. Serial No. 63/392,068 filed July 25, 2022, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for coronavirus disease 2019 (COVID-19). Research to determine the full long-term sequelae of COVID- 19 is on-going. Efforts to study and treat long-term sequelae of COVID- 19 are hindered by a paucity of biomarkers and clinical tests to reliably diagnose long COVID. New objective methods for diagnosing long COVTD are needed.

SUMMARY

Disclosed herein, at least in part, are methods of diagnosing or confirming a diagnosis of post-acute sequelae of a viral disease, such as post-acute sequelae of COVID- 19 (PASC), also known as long CO VID. In some embodiments, the method of diagnosing or confirming a diagnosis further comprises administration of a composition including at least four different amino acid entities.

In some embodiments, the subject has one or more symptoms or signs selected from the group consisting of anorexia, anxiety, arrythmias, confusion (“brain fog”), dementia, depression, dyspnea, fatigue, hair loss, headache, heart failure, cardiomyopathy, angina, hepatic dysfunction, hyperglycemia, type 2 diabetes, increased heart rate, inflammation, loss of appetite, loss of memory, loss of smell, mood disorder, muscle weakness, myocardial ischemia, post-exertional malaise, diminished neurocognition, diminished sensory function, pulmonary infiltrates or fibrosis, postural orthostatic hypotension, renal dysfunction, and respiratory distress.

In certain embodiments, the subject has one or more symptoms or signs selected from the group consisting of myalgia, fibromyalgia, idiopathic pulmonary fibrosis, fatigue, muscle fatigue, mitochondrial dysfunction, dyspnea after exertion, postural orthostatic tachycardia syndrome, tachycardia, mood disorders, and depression. In some embodiments, the subject with elevated PCr recovery time has been identified as having a score, or has received a score (e.g., a score from a test evaluating fatigue) indicative of fatigue. In one such embodiment, the subject with elevated PCr recovery time has a score of greater than or equal to 4, 5, 6, 7, 8, 9, or 10 on a CFQ-11 test using bimodal scoring, or a score of greater than or equal to 25, 26, or 27 on a CFQ-11 test using Likert scoring.

In certain embodiments, the subject has one or more of the following characteristics: i. impaired or delayed immune response; ii. increased oxidative stress and/or proinflammatory state; or iii. dysregulated endothelial function (e.g., hypercoagulation or perfusion).

In certain embodiments, after administration, the subject exhibits one or more of the following: i. increased mitochondrial biogenesis; ii. restored (e.g., partially or fully restored) mitochondrial oxidative capacity; iii. restored (e.g., partially or fully restored) cellular respiration and/or cellular energetics; iv. improved cellular response under higher metabolic demand conditions (e.g., exertion), e.g., in muscle; v. improved mitochondrial respiration (e.g., comprising increased substrate mobilization, increased nitric oxide (NO) signaling, enhanced microvascular or tissue perfusion, enhanced vascular conduction, or increased micro- vascular perfusion) vi. reduced inflammation (e.g., reduce liver inflammation), protein breakdown, and muscle fatigue post-exercise; vii. normalized (e.g., partially or fully normalized) coagulation function; viii. improved mitochondrial energetics and/or redox balance, ix. decreased oxidative stress; x. improved cellular respiration, antioxidant and/or anti-inflammatory effects, xi. increased nucleotide pool availability; xii. increased preferential fatty acid oxidation relative to glycolysis; xiii. increased level of ketone bodies; xiv. decreased FGF-21 xv. decreased vascular permeability xvi. decreased fatigue, e.g., from moderate or severe fatigue to mild fatigue; e.g. from mild fatigue to absence of fatigue; from moderate or severe fatigue to absence of fatigue; xvii. improved sleep, e.g., improved sleep/wake cycle; xviii. improved mobility; xix. improved exercise capacity; xx. improved epithelial cell survival; xxi. improved T-cell response; xxii. reduced mitochondrial ROS production; xxiii. reduced HIFla signaling; xxiv. improved oxidative phosphorylation; xxv. improved executive function; and xxvi. increased ability to concentrate.

In certain embodiments, the fatigue comprises one or both of persistent fatigue and exertional fatigue. In some embodiments, the fatigue comprises one or both of mental fatigue and physical fatigue.

In certain embodiments, the subject had a COVID- 19 infection and is experiencing fatigue.

In certain embodiments, the subject experiences fatigue at at least 4, 8, 12, or 16 weeks after infection with SARS-Cov-2.

In certain embodiments, the subject experiences fatigue at less than 4 weeks (e.g., at less than 3 weeks, 2 weeks, or 1 week) after infection with SARS-Cov-2.

In some embodioments, the amino acid entities administered further comprises a glycine (G)-amino acid entity. In certain embodiments, the amino acid entities administered further comprise one, two, three or more (e.g., all) of a histidine (H)-amino acid entity, a lysine (K)- amino acid entity, a phenylalanine (F)-amino acid entity, and a threonine (T)-amino acid entity.

In some embodiments, the subject was infected with an alpha strain of SARS-CoV-2 (e.g., a B.1.1.7 or Q lineage or a lineage descendent therefrom). In some embodiments, the subject was infected with a beta strain of SARS-CoV-2 (e.g., a B.1.351 lineage or a lineage descendent therefrom). The method of any one of claims 1-89, wherein the subject was infected with a gamma strain of SARS-CoV-2 (e.g., a P.l lineage or a lineage descendent therefrom). In some embodiments, the subject was infected with a delta strain of SARS-CoV-2 (e.g., a B.1.617.2 or AY lineage or a lineage descendent therefrom). In some embodiments, the subject was infected with an epsilon strain of SARS-CoV-2 (e.g., a B.1.427 or B.1.429 lineage or a lineage descendent therefrom). In some embodiments, the subject was infected with an eta strain of SARS-CoV-2 (e.g., a B.1.525 lineage or a lineage descendent therefrom). In some embodiments, the subject was infected with an iota strain of SARS-CoV-2 (e.g., a B.1.526 lineage or a lineage descendent therefrom). In some embodiments, the subject was infected with a kappa strain of SARS-CoV-2 (e.g., a B.1.617.1 lineage or a lineage descendent therefrom). In some embodiments, the subject was infected with a 1.617.3 strain of SARS-CoV-2 or a lineage descendent therefrom. In some embodiments, the subject was infected with a Mu strain of SARS-CoV-2 (e.g., a B.l .621 or B.l .621 .1 lineage or a lineage descendent therefrom). Tn some embodiments, the subject was infected with a zeta strain of SARS-CoV-2 (e.g., a P.2 lineage or a lineage descendent therefrom). In some embodiments, the subject was infected with an Omicron strain of SARS-CoV-2 (e.g., a B.l.1.529, BA.l, BA.1.1, BA.2, BA.3, BA.4 or BA.5 lineage or a lineage descendent therefrom).

In some embodiments, the subject is an adult. In some embodiments, the subject is between 18 and 65 years of age (e.g., between 18 and 30, 30 and 40, 40 and 50, 50 and 60, or 60 and 65 years of age). In some embodiments, the subject is an adolescent or a child. In some embodiments, the subject is 17 years of age or younger. In some embodiments, the subject is between 1 and 17 years of age (e.g., between 1 and 5, 5 and 10, 10 and 15, or 15 and 17). In some embodiments, the subject has fatigue-predominant PASC. In some embodiments, the subject has PCr recovery constant of >40 seconds or >50 seconds.

Another aspect of the invention further provides a method for treating a subject diagnosed with post-viral fatigue, particularly post-acute sequelae of COVID- 19 comprising administering to a subject in need thereof an effective amount of the composition of any one of aspects or embodiments disclosed herein. In some embodiments, a subject has one or more symptoms or signs selected from the group consisting of anorexia, anxiety, arrhythmias, confusion (“brain fog”), dementia, depression, dyspnea, fatigue, hair loss, headache, heart failure, cardiomyopathy, angina, hepatic dysfunction, hyperglycemia, type 2 diabetes, increased heart rate, inflammation, loss of appetite, loss of memory, loss of smell, mood disorder, muscle weakness, myocardial ischemia, post-exertional malaise, diminished neurocognition, diminished sensory function, pulmonary infiltrates or fibrosis, postural orthostatic hypotension, renal dysfunction, and respiratory distress. In some embodiments, a subject has one or more symptoms or signs selected from the group consisting of myalgia, fibromyalgia, idiopathic pulmonary fibrosis, fatigue, muscle fatigue, mitochondrial dysfunction (e.g., increase lactic acid production), dyspnea after exertion, postural orthostatic tachycardia syndrome, tachycardia, mood disorders, and depression.

In some embodiments of the methods disclosed herein, e.g., of any of the methods described herein, the subject has immunologic symptoms or signs, metabolic symptoms or signs, and/or neurologic symptoms or signs. In some embodiments, an immunologic symptom or sign is selected from the group consisting of increased markers of inflammation (e.g., erythrocyte sedimentation rate, c reactive protein), increased proinflammatory cytokines (e.g., CRP, IL-1 A, IL- 17a, TNF-alpha), decreased cytotoxicity of natural killer cells, expression of cytolytic proteins, and production of cytokines, increased CD8+ cytotoxic T cells with CD38 activation antigen, T cell exhaustion, and increased autoantibodies, especially against targets in CNS and autonomic nervous system. In some embodiments, a metabolic symptom or sign is selected from the group consisting of increased lactic acid, reduced ATP generation from glucose by the tricarboxylic acid (TCA) cycle, reduced levels of fatty acids and of acyl-carnitine, reduced levels of amino acids via the urea cycle, impaired oxidative phosphorylation, redox imbalance (e.g., increased levels of oxidants, e.g., peroxides and superoxides, isoprostanes, at rest and/or after exercise or exertion; decreased levels of antioxidants, e.g., decreased levels of alpha-tocopherol, e.g., thiobarbituric acid reactive substances), increased inducible nitric oxide synthase (iNOS), increased NFKB, increased nitric oxide (NO), peroxynitrite, and/or nitrate (e.g., after exercise or exertion), elevated levels of brain ventricular lactic acid, and increased blood glucose (e.g., new onset diabetes). In some embodiments, a neurologic symptom or sign is selected from the group consisting of cognitive deficits (e.g., in attention and reaction time), impaired response to cognitive, motor, visual, and auditory challenges, abnormal nerve conduction studies, abnormal imaging of the brain, hypoperfusion and/or metabolic dysfunction of glial cells, neuroinflammation characterized by widespread activation of both astrocytes and microglia, downregulation of the hypothalamic-pituitary-adrenal (HPA) axis, impaired response of one region of the brain to signals from another region (impaired connectivity), disordered sympathetic and parasympathetic activity, increased levels of tissue repair-indicative proteins (e.g., alpha-2-macroglobulin, keratin 16, orosomucoid), autoantibodies targeting cholinergic, adrenergic, and muscarinic receptors, reduced anaerobic threshold and/or reduced peak work (e.g., after exercise or exertion), and increased lactic acid in muscle and the need to recruit additional brain regions to respond to cognitive challenges (by functional MRI) (e.g., following exertion).

In some embodiments of any of the methods described herein, the subject has increased levels of inflammatory cytokines relative to a normal subject, e.g., the subject has increased levels of CRP or TNFa relative to a normal subject e.g., without the one or more symptoms or without post-acute sequelae of COVID-19.

In some embodiments, e.g., of any of the methods described herein, the subject exhibits muscle atrophy or has a decreased ratio of muscle tissue to adipose tissue relative to a normal subject, e.g., without the one or more symptoms or without post-acute sequelae of COVID- 19.

In some embodiments, e.g., of any of the methods described herein, the subject exhibits brain fog or has a decreased neurocognitive function relative to a normal subject, e.g., without the one or more symptoms or without post-acute sequelae of CO VID-19.

In some embodiments, e.g., of any of the methods described herein, the subject exhibits dyspnea or has a decreased pulmonary function relative to a normal subject, e.g., without the one or more symptoms or without post-acute sequelae of COVID- 19.

In some embodiments, e.g., of any of the methods described herein, the subject exhibits decreased metabolic function relative to a normal subject, e.g., without the one or more symptoms or without post-acute sequelae of COVID- 19.

In some embodiments, e.g., of any of the methods described herein, the subject exhibits abnormal (e.g., increased) immunologic function relative to a normal subject, e.g., without the one or more symptoms or without post-acute sequelae of COVID-19.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. l is a schematic of the timeline for the study as described in Example 1. DETAILED DESCRIPTION

Definitions

Terms used in the claims and specification are defined as set forth below unless otherwise specified.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, the term “amino acid entity” refers to a (L)-amino acid in free form or salt form (or both), the L-amino acid residue in a peptide smaller than 20 amino acid residues (e.g., oligopeptide, e.g., a dipeptide or a tripeptide), a derivative of the amino acid, a precursor of the amino acid, or a metabolite of the amino acid. An amino acid entity includes a derivative of the amino acid, a precursor of the amino acid, a metabolite of the amino acid, or a salt form of the amino acid that is capable of effecting biological functionality of the free L-amino acid. An amino acid entity does not include a naturally occurring polypeptide or protein of greater than 20 amino acid residues, either in whole or modified form, e.g., hydrolyzed form.

As used herein the term “XXX amino acid entity” refers to an amino acid entity that if a free amino acid, comprises free XXX or XXX in salt form; if a peptide, refers to a peptide (e.g., a dipeptide or a tripeptide) comprising an XXX residue; if a derivative, refers to a derivative of XXX; if a precursor, refers to a precursor of XXX; and if a metabolite, refers to a XXX metabolite. For example, where XXX is leucine (L), then L-amino acid entity refers to free L or L in salt form, a peptide (e.g., a dipeptide or a tripeptide) comprising a L residue, a L derivative, a L precursor, or a metabolite of L; where XXX is arginine (R), then R-amino acid entity refers to free R or R in salt form, a peptide (e.g., a dipeptide or a tripeptide) comprising a R residue, a R derivative, a R precursor, or a metabolite of R; where XXX is glutamine (Q), then Q-amino acid entity refers to free Q or Q in salt form, a peptide (e.g., a dipeptide or a tripeptide) comprising a Q residue, a Q derivative, a Q precursor, or a metabolite of Q; and where XXX is N- acetylcysteine (NAC), then NAC entity refers to free NAC or NAC in salt form, a peptide (e.g., a dipeptide or a tripeptide) comprising a NAC residue, a NAC derivative, a NAC precursor, or a metabolite of NAC.

“About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 15%, more typically, within 10%, and more typically, within 5% of a given value or range of values.

An “amino acid” refers to an organic compound having an amino group (-NH2), a carboxylic acid group (-C(=O)OH), and a side chain bonded through a central carbon atom, and includes essential and non-essential amino acids, as well as natural and unnatural amino acids.

The term “effective amount” as used herein means an amount of an amino acid, or pharmaceutical composition which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., to positively modify one, two, or more of a subject’s symptoms, e.g., provide a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically- acccptablc cxcipicnt(s) and/or carricr(s) utilized, and like factors with the knowledge and expertise of the attending physician.

As used herein, the term “PCr recovery time” refers to (1) the amount of time required for a phosphocreatine (PCr) level to reach or exceed a predetermined value (e.g., after exercise), or (2) the rate at which a PCr level increases. In some embodiments, the PCr recovery time comprises a time constant of PCr resynthesis (rpcr). In some embodiments, the predetermined value is less than (e.g., 50% less than) the baseline PCr level before exercise.

A “pharmaceutical composition” described herein comprises at least one amino acid and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is used as a therapeutic, a nutraceutical, a medical food, or as a supplement.

The term “pharmaceutically acceptable” as used herein, refers to amino acids, materials, excipients, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. This may be a standaid used by the pharmaceutical industry or by agencies or entities (e.g., government or trade agencies or entities) regulating the pharmaceutical industry to ensure one or more product quality parameters are within acceptable ranges for a medicine, pharmaceutical composition, treatment, or other therapeutic. A product quality parameter can be any parameter regulated by the pharmaceutical industry or by agencies or entities, e.g., government or trade agencies or entities, including but not limited to composition; composition uniformity; dosage; dosage uniformity; presence, absence, and/or level of contaminants or impurities; and level of sterility (e.g., the presence, absence and/or level of microbes). Exemplary government regulatory agencies include: Federal Drag Administration (FDA), European Medicines Agency (EMA), SwissMedic, China Food and Drag Administration (CFDA), or Japanese Pharmaceuticals and Medical Devices Agency (PMDA).

The term “post acute sequelae of COVID-19” or “PASC” as used herein, refers to symptoms experienced by a subject four or more weeks after initial infection with SARS-CoV-2. Other terms used to describe PASC include long COVID, long haul COVID, post-acute COVID, post-acute COVID syndrome (PACS) and/or chronic COVID.

A composition, formulation or product is “therapeutic” if it provides a beneficial clinical effect. A beneficial clinical effect can be shown by lessening the progression of a disease and/or alleviating one or more symptoms of the disease.

As used herein, the terms “treat,” “treating,” or “treatment” of PASC refer in one embodiment, to ameliorating PASC, (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treat,” “treating,” or “treatment” refers to modulating a symptom of PASC, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treat,” “treating,” or “treatment” refers to preventing or delaying the onset or development or progression of PASC.

Determination of amino acid weight percent and amino acid ratios in a composition

The weight ratio of a particular amino acid or particular amino acids in a composition or mixture of amino acids is the ratio of the weight of the particular amino acid or amino acids in the composition or mixture compared to the total weight of amino acids present in the composition or mixture. This value is calculated by dividing the weight of the particular amino acid or of the particular amino acids in the composition or mixture by the weight of all amino acids present in the composition or mixture. It is understood that NAC is considered to be an amino acid for the purpose of this calculation. Methods of Treatment

Based on a diagnosis or confirmation of a diagnosis of post-viral fatigue, particularly PASC, various therapeutic products can be administered to improve mitochondrial, metabolic, immunologic, musculoskeletal, neurocognitive, and/or pulmonary function, e.g., in a patient with post-acute sequelae of COVID- 19. They can be administered to treat (e.g., reverse, reduce, ameliorate, or prevent) a disorder, e.g., post-acute sequelae of COVID- 19 in a subject. The composition as described herein can also be administered to treat (e.g., reverse, reduce, ameliorate, or prevent) a disorder, e.g., myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), post-infectious fatigue syndrome, post-critical illness syndrome, or post-intensive care unit syndrome, following illness or infection, e.g., related to post-acute sequelae of COVID- 19 in a subject. The present disclosure provides methods of treating post- acute sequelae of COVID- 19 selected from myalgia, fibromyalgia, idiopathic pulmonary fibrosis, fatigue, muscle fatigue, mitochondrial dysfunction, dyspnea after exertion, postural orthostatic tachycardia syndrome, and tachycardia in subject diagnosed or in whom diagnosis is confirmed in accordance with the invention. In particular, an effective amount of the composition can be administered (e.g., according to a dosage regimen described herein) to treat a subject with postacute sequelae of COVID-19.

In some embodiments, a method described herein comprises administering a composition as described in International Applications WO/2018/118941 or WO/2018/ 118957, each of which is herein incorporated by reference in its entirety.

In some embodiments, a method described herein comprises a treatment as described in International Application PCT/US22/38200, which is herein incorporated by reference in its entirety.

In some embodiments, a method described herein comprises administering one or more of, e.g., all of, a histidine (H)-amino acid entity, a lysine (K)-amino acid entity, a phenylalanine (F)-amino acid entity, and a threonine (T)-amino acid entity. In some embodiments, the H-amino acid entity is selected from the group consisting of L-histidine, histidinol, histidinal, ribose-5- phosphate, carnosine, histamine, urocanate, and N-acetyl histidine, or a salt of any of the forgoing. In some embodiments, the H-amino acid entity is L-histidine or a salt thereof. In some embodiments, the K-amino acid entity is selected from the group consisting of L-lysine, diaminopimelate, trimethyllysine, carnitine, saccharopine, and N-acetyl lysine, or a salt of any of the forgoing. In some embodiments, the K-amino acid entity is L-lysine or a salt thereof.

In some embodiments, the F-amino acid entity is selected from the group consisting of from L-phenylalanine, phenylpyruvate, tyrosine, and N-acetyl-phenylalanine, or a salt of any of the forgoing. In some embodiment, the F-amino acid entity is L-phenylalanine or a salt thereof.

In some embodiments, the T-amino acid entity is selected from the group consisting of L-threonine, homoserine, O-phosphohomoserine, oxobutyrate, and N-acetyl-threonine, or a salt of any of the forgoing. In some embodiments the T-amino acid entity is L-threonine or a salt thereof.

Patients with Post-Acute Sequelae of COVID-19 (PASO

In some embodiments, a subject has post-acute sequelae of COVID- 19. In some embodiments, a subject has one or more symptoms selected from the group consisting of anorexia, anxiety, arrhythmias, confusion (“brain fog”), dementia, depression, dyspnea, fatigue, hair loss, headache, heart failure, cardiomyopathy, angina, hepatic dysfunction, hyperglycemia, type 2 diabetes, increased heart rate, inflammation, loss of appetite, loss of memory, loss of smell, mood disorder, muscle weakness, myocardial ischemia, post-exertional malaise, diminished neurocognition, diminished sensory function, pulmonary fibrosis, postural orthostatic hypotension, renal dysfunction, and respiratory distress. In some embodiments, a subject has one or more symptoms selected from the group consisting of myalgia, muscle fatigue, fatigue, dyspnea after exertion, postural orthostatic tachycardia syndrome, tachycardia, mood disorders, and depression. In some embodiments, the subject has been infected with a virus selected from the group consisting of SARS-CoV-1, SARS-CoV-2, MERS, influenza A or B, herpesviruses (Epstein-Bair virus, human cytomegalovirus, and human herpesviruses 6A and 6B), Ebola virus, West Nile virus, dengue virus, Ross river virus, enteroviruses, and human parvovirus B19. In some embodiments, a subject has been infected with a coronavirus (e.g., a human alpha coronavirus (e.g., HCoV-229E or HCoV-NL63), a human betacoronavirus (HCoV-OC43 or HKU1), SARS-CoV-1 , SARS-CoV-2, and/or MERS). In some embodiments, a subject has been hospitalized for acute COVID- 19. In some embodiments, a subject has been hospitalized for one or more symptoms of post-acute sequelae of COVID-19. In some embodiments, a subject had not been vaccinated for COVID- 19 prior to contracting COVID-19. In some embodiments, a subject had not been vaccinated (e.g., partially vaccinated or fully vaccinated) for COVID- 19 prior to contracting COVID- 19. In some embodiments, a subject had been vaccinated for COVID- 19 after contracting COVID- 19. In some embodiments, the subject tested positive for SARS-CoV-2 and developed symptoms consistent with infection. In some embodiments, the subject tested positive for SARS-CoV-2 and was asymptomatic, but later developed symptoms consistent with PASC. In some embodiments, the subject tested positive for SARS-COV-2, had symptoms of infection, became antibody negative or asymptomatic, and then was re-infected with another variant of SARS- CoV-2. Tn some embodiments, the subject has tested positive for SARS-CoV-2 more than once. In certain embodiments, the subject has tested positive for SARS-CoV-2 1, 2, 3, 4, or more times. In some embodiments, the subject has been diagnosed with more than one infection of SARS-CoV-2 (e.g., 1, 2, 3, 4, or more separate SARS-CoV-2 infections).

In some embodiments, a subject tested positive for COVID-19, e.g., about 1, 2, 3, or 4 weeks before administration. In some embodiments, a subject tested positive for COVID-19 at least twice over a period of time, e.g., at least 3 or 4 weeks, before administering a composition described herein. In some embodiments, a subject had acute COVID- 19 for about 3, 4, 5, 6, 8, 10, or 12 weeks, before administering a composition described herein. In some embodiments, a subject had one or more symptoms of acute COVID- 10 for at least 3 or 4 weeks, before administration of a composition described herein. In some embodiments, a subject is (e.g., is determined to be) negative for SARS-CoV-2 at the time of administration of a composition described herein. In some embodiments, at the time of administration of a composition described herein, the subject is (e.g., is determined to be) positive for SARS-CoV-2. In some embodiments, at the time of administration, the subject no longer has detectable SARS-CoV-2 in a nasal sample at the time they are administered the composition.

Methods of measuring PCr recovery time

In some embodiments, PCr recovery time is measured using magnetic resonance spectroscopy (MRS). In some embodiments, PCr recovery time is measured using magnetic resonance imaging (MRI). In some embodiments, PCr recovery time is measured using ³¹ P chemical shift imaging ( ³¹ P-CSI). In some embodiments, PCr recovery time is measured using ³¹ P-Rapid Acquisition with Relaxation Enhancement (RARE) MRI. In some embodiments, PCr recovery time is measured using a biopsy. In some embodiments, a method of measuring (e.g., a method of measuring PCr recovery time) is performed as described in Greenman, et al., Acad Radiol. 2011 July; 18(7): 917-923, herein incorporated by reference in its entirety.

In some embodiments, a method described herein comprises a step of comparing a value to a threshold, e.g., of comparing the measure of PCr recovery time to a PCr threshold. In some embodiments, the method further comprises comparing the value to a second threshold. For example, the method may comprise determining if the value is above the PCr threshold and below a second threshold, e.g., determining whether the value is within a range bounded by the two thresholds.

EXAMPLES

The Example below is set forth to aid in the understanding of the inventions, but is not intended to, and should not be construed to, limit its scope in any way.

Example 1 - Evaluation of LIVRQNac in Sub jects with Lona COVID Fatigue

Long COVID- 19 with fatigue is a serious condition with urgent unmet medical need:

Long COVID-19 is a chronic, multi-organ disease predominantly characterized by fatigue and muscle weakness (Lopez Leon et al (More than 50 long-term effects of COVID- 19: a systematic review and meta-analysis. Sci Rep. 2021 Aug 9; 11(1): 16144. doi: 10.1038/s41598- 021-95565-8.). Although many patients recover from COVID-19 within several weeks, a substantial proportion of patients exhibit persistent or new symptoms more than 4 weeks after being diagnosed (Sigfrid et al (Long Covid in adults discharged from UK hospitals after Covid- 19: A prospective, multicentre cohort study using the 1SAR1C WHO Clinical Characterisation Protocol. Lancet Reg Health Eur. 2021 Sep;8:100186. doi: 10.1016/j.lanepe.2021.100186. Epub 2021 Aug 6.). These patients with persistent post-acute COVID (PASC) symptoms are often referred to as suffering from long COVTD. It is estimated that long COVTD affects 20% to 70% of the survivors of acute infection. Many cross-sectional and cohort studies report that chronic fatigue is the most frequently reported symptom following recovery from acute COVID-19 (Evans, et al. (Physical, cognitive, and mental health impacts of COVID- 19 after hospitalisation (PHOSP-COVID): a UK multicentre, prospective cohort study. Lancet Respir Med. 2021 Nov;9(l 1): 1275-1287. doi: 10.1016/82213-2600(21)00383-0. Epub 2021 Oct 7. Erratum in: Lancet Respir Med. 2022 Jan;10(l):e9. PMID: 34627560; PMCID: PMC8497028); Crook, et al. (Long covid-mechanisms, risk factors, and management. BMJ. 2021 Jul 26;374:nl648. doi: 10.1136/bmj.nl648. Erratum in: BMJ. 2021 Aug 3;374;nl944.). A recent large database of nearly 2 million individuals diagnosed with COVID- 19 estimated that 23.2% of patients report at least 1 post-COVID-19 condition, and fatigue is among the 3 most common complaints, typically reported in more than half of subjects with persistent symptoms. Reports of fatigue are independent of the severity of initial illness (Townsend, et al. (Persistent poor health after COVID- 19 is not associated with respiratory complications or initial disease severity. Ann Am Thorac Soc. 2021 ; 18(6):997- 1003); Augustin, et al. (Post-COVID syndrome in non-hospitalised patients with COVID- 19: a longitudinal prospective cohort study. Lancet Reg Health Eur. 2021 Jul;6: 100122. doi: 10.1016/j.lanepe.2021.100122. Epub 2021 May 18.). There is evidence of substantial negative impact on quality of life (QoL) (Halpin et al. (Postdischarge symptoms and rehabilitation needs in survivors of COVID-19 infection: a cross-sectional evaluation. J Med Virol. 2020)), and given the large number of survivors with long COVID, it is reasonable to assume that there will be substantial long-term effects not only on individuals but also on the health care system. In a study that examined 1-year outcomes in hospital survivors with COVID- 19, only 76% had returned to a pre-COVID-19 level of employment, with 32% of individuals attributing this to decreased physical function (Huang, et al. (1-year outcomes in hospital survivors with COVID-19: a longitudinal cohort study. Lancet. 2021;398(10302):747- 758)). Thus, long COVID-19 with fatigue is a serious disease, with the potential to substantially impair QoL and lead to increased health risks and costs and impairment of the ability to work.

Clinical STUDY for long COVID-19

A randomized, double-blind, placebo-controlled Phase 2a trial was conducted to evaluate the efficacy and safety of a LIVRQNac Test Article in patients with moderate to severe fatigue related to long COVID (>12 weeks after initial infection). Enrollment in the study has been completed, with 41 patients randomized evenly to receive either 67.8 grams per day of LIVRQNac or a matched placebo in two divided doses for 28 days, with a one-week safety follow-up period. The total study duration for each subject is approximately 9 weeks and comprising of a Screening Period of up to 4 weeks, a Treatment Period of up to 4 weeks, and a Follow-up Period of 1 week (FIG. 1).

The primary efficacy endpoint is the mean change from baseline at Week 4 in the phosphocreatine (PCr) recovery rate following moderate exercise, as assessed by phosphorus magnetic resonance spectroscopy ( ³¹ P-MRS), which is evaluated at Screening and End of Trial (EOT) (visit 4). To assess fatigue, 6-minute walk test (6MWT) and Chalder Fatigue scale, which have been successfully validated and used in previous studies on chronic fatigue syndrome (Mantha 2020) were utilized.

An analysis of the results from the first 20 subjects has been conducted, with the results presented here. Top line results with the 41 subjects enrolled in the study are expected to be available in the near future.

Description of study test article

The LIVRQNac Test Article is an orally active mixture of 5 specific AAs (leucine, isoleucine, valine, arginine, glutamine), and N-acetylcysteine (Nac) as presented in Table 1. Table 1. Amino Acid and Excipient Composition Within LIVRQNac Test Article

AA=amino acid

Note: Total may be >100% due to rounding off ^a Arginine is sourced as arginine monohydrochloride

The Test Article is supplied in a dry powder form that is dissolved in approximately 6 oz (approximately 180 mL) of water to form a uniform suspension and is administered orally, twice daily, as an orange-flavored drink.

STUDY ENDPOINTS

Rationale for choosing PCr recovery as the primary endpoint:

The primary objective of this study was to assess the impact of LIVRQNac on muscle function (metabolism) following exercise. A change in the time constant of phosphocreatine (PCr) recovery from baseline after 4 weeks treatment as measured by ³¹ P-MRS was chosen as the primary endpoint as it is objective and sensitive to changes in mitochondrial function. In brief, ³¹ P-MRS is used to estimate the concentration of high-energy phosphate compounds; thus, the bioenergetic state of a tissue can be characterized in vivo as it may reflect changes in mitochondrial function (Prompers 2006; Kemp 2015; Valkovic 2016). ³¹ P-MRS has been used to assess mitochondrial function in a variety of conditions, including heart failure patients (Menon 2021 ), diabetes (Ripley 2018), and mitochondrial abnormalities following drug administration (Fleischman 2007). If, as expected, a composition comprising LIVRQNac improves mitochondrial oxidative capacity, then a decrease, relative to subject baseline, in the phosphocreatine recovery time is predicted. The assumption at the beginning of the trial was that individuals with prolonged fatigue after COVID- 19 would have a baseline PCr of 50 seconds, which would be comparable to aged individuals or those with heart failure.

Rationale for choosing CFQ-11 and 6MWT to assess fatigue:

The Chalder Fatigue Scale (CFQ-11) and the 6MWT have been utilized in multiple therapeutic areas and several indications and have been paramount in characterizing the patient's condition and overall quality of life. Both these tests are suitable in evaluating scientifically supported and logical combination of symptoms that are common in patients with long COVID- 19 with fatigue and inform the design of a subsequent study with appropriate power to detect differences in these key endpoints.

While many instruments are available to assess fatigue, the CFQ-11 has been validated in a number of different patient population including those with myalgic encephalomyelitis/chronic fatigue syndrome (Whitehead 2009; Morriss 1998; Crawley 2013) which has parallels to the clinical presentation of patients with long CO VID- 19 experiencing fatigue (Paul 2021). There has also been use of the CFQ-11 in patients with long COVID-19 (Staven 2021, Tuzon 2021, Townsend 2021). Given the reliability of this instrument in a wide range of conditions, ongoing public health emergency and the serious unmet medical need for patients with long COVID with fatigue, the use of such existing, reliable functional assessment and PROs to assess clinical benefit outweighed the risks of not performing additional validation in the target patient population.

The fatigue scale developed by Chalder et al. is an 11-item scale intended to measure the severity of fatigue-related symptoms, both mental and physical, experienced by individuals with myalgic encephalomyelitis I chronic fatigue syndrome (ME/CFS). The scale has two scoring systems: bimodal and Likert. In the bimodal system, respondents answer each question with a 1 or a 0 to indicate the questions apply to them or not. In the Likert system, respondents can give a score of 0 to 3 to indicate how each statement applies to them, from “less than usual” to “much more than usual”. The scores are then summed, and a higher score indicates more severe fatigue- related symptomatology. The “Physical Fatigue” items include questions such as “Do you have problems with tiredness?” or “Do you lack energy?” The remaining items constitute a “Mental Fatigue” factor with questions such as “Do you have difficulty concentrating?” or “Do you make slips of the tongue when speaking?” The total scale demonstrated sufficient internal consistency with alpha coefficients of 0.89 (Chalder 1993). At the 90% sensitivity level for the CFQ-11 Scale (with a score > 14.50) a specificity of 0.61 was detected, and these scales were able to identify 90% of those individuals with CFS (Jason 2011). As an example of a measure of fatigue intensity alone, Chalder et al.’s Fatigue Scale is a verbal rating measure that has strong internal consistency. Using an ROC curve analysis, (Jason 1997), this scale was able to discriminate a CFS sample from a healthy control sample. Near-maximal scoring on six physical fatigue scale items from the total of 14 items constituting the Chalder fatigue scale supports the validity of scoring the physical fatigue scale on a two-point scale (presence or absence) rather than the four- point scoring. As noted in the paragraph above, the CFQ-11 has been applied to the study of outcomes in COVID-19 (Steven 2021, Tuzon 2021, Townsend 2021).

Additionally, the 6-minute walk test (6MWT) is a validated clinical test to assess the cardiopulmonary reserve and fundamentally designed for use in adults with chronic respiratory disease (Holland 2014) and therefore may be an appropriate test to evaluate functional status of COVID- 19 patients.

Results from this Analysis of this Study

An analysis was conducted after 20 subjects (10 subjects taking Test Article and 10 taking Placebo) in the study completed 4 weeks of treatment. The analysis was focused on the study endpoint of phosphocrcatinc (PCr) recover time along with secondary endpoints including; CFQ-11, 6-MWT, MRS, and safety and tolerability.

Subjects who received LIVRQNac Test Article, relative to placebo, achieved statistically significant improvements in the CFQ-11. A major entry criterion for the study was the presence of moderate to severe fatigue (score of > 8, with a score of 4 or more indictive of fatigue using bimodal score, with scores ranging from 0-11). Relative to placebo, subjects who received LIVRQNac Test Article had a 4 point improvement in the CFQ-11 score.

The results for the 6MWT showed that there was no significant change from Baseline in the LIVRQNac Test Article Group versus the Placebo Group in the distance walked at Week 4 whether calculated as absolute change in distance or percent predicted. The mean 6MWT was 533 M ± 106 M, or approximately 85% predicted with approximately one quarter of subjects below the 75% of the predicted distance based on age or gender; these results are consistent with the literature. These results may be due to the relatively short duration of the study (4 weeks) and the unexpected severity and magnitude of fatigue encountered by this patient population.

Magnetic resonance spectroscopy

PCr results, measured as described above, indicated an unexpectedly large variation in both Baseline and deviation from the mean (mean 84.56 sec ± 30.816). There was no difference in the change from Baseline in PCr between the LIVRQNac Test Article Group and the Placebo Group at Week 4 as examined by both absolute and percent change, whether unadjusted or adjusted statistical models were used. There was no correlation with either Fatigue score or 6- MWT.

Additional MRS assessments showed positive trending changes in the LIVRQNac Test Article Group from Baseline as compared to the Placebo Group at Week 4 including;

Intramyocellular Lipid Content: LIVRQNac Test Article Group (Baseline mean= 0.42, Week 4 change= -0.17) versus Placebo Group (Baseline mean- 0.36, Week 4 change- 0.16), Peak Lactate: LIVRQNac Test Article Group (Baseline mean= 1.74, Week 4 change= - 0.53) versus Placebo Group (baseline mean= 2.11, Week 4 change= -0.20), and

Carnosine: LIVRQNac Test Article Group (Baseline mean= 4.43, Week 4 change= 0.78) versus Placebo Group (Baseline mean= 4.38, Week 4 change= -0.56).

The safety results in this analysis of the first 20 subjects showed no safety issues in the LIVRQNac Test Article Group: two subjects had the adverse events of abdominal pain, and headache which were mild and resolved. There were two adverse events of diarrhea (one in subject with upper respiratory infection), and the other one in subject with nasal congestion, nausea post MRI. One subject had COVID. In the Placebo Group, there was an increase in liver function tests which was most likely related to concomitant medications. These results proved a benign safety and tolerability profile for LIVRQNac which is consistent with the published literature on LIVRQNac constituent AAs and safety data gathered from other clinical studies conducted with LIVRQNac in NASH.

Example 2. Assessment of phosphocreatine (PCr) recovery.

This Example describes measurement of phosphocreatine recovery in subjects following moderate exercise as assessed by ³¹ P magnetic resonance spectroscopy (MRS). In brief, ³¹ P-MRS is used to estimate the concentration of high-energy phosphate compounds; thus, the bioenergetic state of a tissue can be characterized in vivo as it may reflect changes in mitochondrial function. ³¹ P-MRS has been used to assess mitochondrial function in a variety of conditions, including heart failure patients, diabetes, and mitochondrial abnormalities following drug administration.

Protocol description Participants underwent MRS and dynamic ³¹ P MRS of skeletal muscle in the whole body 3T MRI system. Subjects were positioned supine, feet first in the MRI system with the dual tuned 1H/31P surface coil (Rapid Biomedical) strapped to the calf muscle (gastrocnemius medialis) of their dominant leg. The dominant leg also had an exercise band strapped to the ball of the foot and the other end was held by the participants. After positioning, localizer images were acquired and Bo shimming was performed to improve field homogeneity in the region of interest.

Stimulated Echo Acquisition Mode (STEAM) sequence was used for ¹ H MRS localization and the voxel of interest was positioned in the gastrocnemius medialis muscle. First, excitation frequency was centered on the acetyl-carnitine ACC to cover the spectral region from extra- and intra-myocellular lipids (EMCL and IMCL) CH2 and CH3 resonances and creatine (Cr). Next, the excitation frequency was centered between the C2H and C4H resonance of carnosine (Car). Five transients centered on the water resonance frequency were then acquired for reference.

Depth resolved surface coil spectroscopy (DRESS) free induction decay (FID) acquisition sequence was used to acquire the dynamic ³¹ P MRS data. The dynamic protocol included 1 minute of rest, followed by 5 minutes of exercise and 6 minutes of recovery. The exercise, i.e. plantar flexions, was performed once every TR, i.e. 2s. Audio signal was used to time the contraction-relaxation periods, so that the spectra were acquired in the relaxed state of the muscle.

All acquired spectra were analysed using a custom written program based on the OXSA toolbox. MRS data, all spectral lines were modeled as single Lorentzians. The fitted IMCL CH2 peak, ACC, Car, Cr and water were corrected for relaxation times and the IMCL CH2 peak was used to calculate the IMCL content according to the equation: IMCL/(Water+IMCL). Taking the water peak as an internal concentration reference, the concentration of ACC was calculated according to the formula for millimolar concentration in wet weight (mmol/kg wet weight):

[ACC] = LH2O] x (SACC/SH2O) x (nu2o/nAcc) x Wn2 where S represents signals corrected for relaxation, n is the number of protons in the respective molecule, [H2O] = 55,556 mmol/L and WH20 is the approximate water content of skeletal muscle tissue, i.e. 0.77 L/kg wet weight of tissue. The same equation was used for Car concentration quantification, using details for Car.

The resonance lines of PCr, Pi, and phosphodiesters were fitted as single Lorentzians, whereas y - and a -ATP will be fitted as doublets and P -ATP as a triplet. All fitted signals were corrected for relaxation effects. The difference in resonance position between PCr and Pi signals in parts per million (5) was used to calculate intramyocellular pH, according to the Henderson- Hasselbalch equation: pH = 6.75 + log((5 - 3.27) / (5.63 - 5 )).

The y -ATP signal was used as an internal concentration reference, assuming a stable ATP concentration of 8.2 mmol/L in the skeletal muscle. To calculate the time constant of PCr resynthesis (rpcr), the PCr concentration changes during the recovery period of the dynamic experiment were fitted to a monoexponential function. The maximal rate of oxidative phosphorylation (Qmax) was calculated according to the ADP-bascd model of Michaelis and Menten. ADP concentration is typically too low to be detected, but was calculated using a CK equilibrium, using the equilibrium constant KCK ~ 1.66 x 109 M ¹ and assuming that at rest 15% of total Cr represents free Cr [Cr]:

[ADPJ = (LCrJ x LATPJ) / (|PCrJ x [H ⁺ J x KCK).

Detailed Protocol:

Step 1: Localizers

Three basic plane images were acquired. In the acquired localiser images, the coil was ensured to be positioned under the widest part of the gastrocnemius medialis muscle. Another localizer with multiple transversal slices was acquired.

Step 2: ¹ H-MRS voxel position

The calibration sequence was used to plan the voxel position. The voxel was placed inside the gastrocnemius medialis muscle, avoiding subcutaneous adipose tissue. The size of the voxel was adjusted to fit the muscle if necessary. The volume was adjusted to be placed over the whole calf muscle close to the RF coil.

Step 3: Shimming and power adjustment

Automatic 3D shim of the whole calf muscle was performed using the following:

1 ) Options Adjustments 3D shim, 2) Press GO,

3) After the field maps are acquired press Calculate and Apply,

4) Switch to Frequency tab,

5) Press GO multiple times till you see that the frequency converged (Y),

6) Close and run calibration sequence.

The calibration sequence increased reference voltage by 10V for each repetition. After the scan was finished, the Spectroscopy card was opened, and the repetition with highest water signal was found, which was used to set the reference voltage in the ¹ H-MRS scans.

Step 4: ¹ H-MRS acquisition

The measurement parameters were copied and volume was adjusted from the calibration sequence, and the reference voltage was set. The acetyl-camitine (ACC) and IMCL acquisition sequence was run. The carnosine acquisition sequence was run, followed by running of the water acquisition sequence.

Step 5: ³¹ P-MRS volume of interest (VO I) position

The calibration sequence was used to plan the VOI position. The VOI was placed inside the gastrocnemius medialis muscle, avoiding soleus muscle. The volume was adjusted, copied over from the 1H-MRS acquisitions.

Step 6: ³¹ P-MRS Frequency and power adjustment

The X-frequency was set to PCr using the following:

1) Options Adjustments X frequency,

2) Press GO,

3) Move cursor to largest peak = PCr and press APPLY. The grey line turned red, and the Freq (temp) value was loaded onto Freq (sys),

4) CLOSE and run the calibration sequence.

The calibration sequence increased reference voltage by 10V for each repetition. After the scan was finished, the Spectroscopy card was opened, and the repetition with highest PCr signal was found, which was used to set the reference voltage in the ³¹ P-MRS scans.

Step 7: ³¹ P-MRS

The VOI position was copied and volume adjusted from the calibration sequence, and the reference voltage was set. Following instructions to the patient, the dynamic experiment was started. During the first minute, the subject continued to lay still and listen for the exercise signal. After the first minute, the exercise, i.e. plantar flexion, was started, which was performed once every TR (following the audio signal). The exercise frequency was observed, and the subject was instructed to adjust or stop for a moment and start again after next signal. The exercise was performed for 5 minutes if possible. If the subject had to stop before the 5 minutes were up, the time at which the patient stopped was recorded. After the exercise period, the patient was instructed to relax but to keep the leg in the same position as before, i.e., no motion during the recovery. Once the scan was finished, the subject was taken out of the MR system.

Step 8: ³¹ P-MRS check

To check if acquisition was successful, a few spectra were loaded into the spectroscopy tab:

• One from the first minute (rest), i.e. acquisition nr. 1-30,

• One from the end of exercise, if full 5 minutes, acquisition nr. 110-120, and

• One from the end of recovery period, i.e. acquisition nr. 230-240.

The PCr signals were compared between the three acquisitions. In some embodiments, PCr signal at the end of exercise acquisition is -30% less than at the end of recovery. In some embodiments, PCr signal at the end of recovery is similar to at the rest.

Example 3. Assessment of phosphocreatine (PCr) recovery in patients with fatigue relating to long COVID-19,

This Example describes assessment of phosphocreatine (PCr) recovery in subjects with fatigue related to long COVID- 19. A randomized, double-blind, placebo-controlled Phase 2a trial was conducted in subjects with moderate to severe fatigue related to long COVID- 19 (>12 weeks after initial infection), as described in Example 1. PCr recovery rate time constants (rpcr) were determined at baseline, i.e. prior to administration of placebo or LIVRQNac using the methods described in Example 2. Measured rpcr in subjects with fatigue related to long COVID- 19 are summarized in Table 2.

Table 2. Summary of Stress PCr Recovery Rate Time Constant (TPG-) in Subjects at Baseline.

Mean ± SD of Tp&was 84.56 ± 30.816 seconds for subjects experiencing fatigue related to long COVID- 19. In contrast, rpcr in healthy subjects has previously been reported as 25.4 ± 3.7 seconds. These data indicate that subjects experiencing fatigue related to long COVID- 19 exhibit higher TPC,- values than healthy subjects. Furthermore, the data provide evidence that prolonged PCR recovery (e.g., higher rpcr values) is a biomarker for patients experiencing fatigue related to long COVID- 19.

While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.