Attorney Docket No.0184.0291-PCT/C17806_P17806-02 METHODS OF DIAGNOSING AND TREATING DYSAUTONOMIA ASSOCIATED DISORDERS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No. 63/615,543 filed 28 December 2023, the entire contents of which are hereby incorporated by reference in their entirety. SUBMISSION OF SEQUENCE LISTING [0002] The Sequence Listing associated with this application is filed in electronic format as a XML file and hereby incorporated by reference into the specification in its entirety. The name of the XML file containing the Sequence Listing is 01840291-PCT_SL.xlm and the size of the XML file is 6,718 bytes. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0003] This invention was made with government support under grant NS126398 awarded by NIH. The government has certain rights in the invention. FIELD [0004] The present disclosure generally relates to methods of diagnosing and/or treating dysautonomia associated disorders, as well as methods of predicting responsiveness of subjects suffering from or suspected of having dysautonomia associated disorders to certain treatments. BACKGROUND [0005] Dysautonomia is a term used to describe any disorder of the autonomic nervous system. The autonomic nervous system is the part of the nervous system that regulates functions that are automatic in nature, such as heart rate, blood pressure, digestion, excretion, perspiration, temperature regulation, pupil dilation, circulation, and respiration among others. ^ When there is a dysfunction or failure of the autonomic nervous system, the result is a disorder classified as a type of dysautonomia. Dysautonomia is sometimes referred to as autonomic dysfunction or autonomic neuropathy. [0006] Dysautonomia can occur at any age, such as pediatric, adult, or geriatric, and it can range from mild to disabling and may or may not be neurodegenerative. Regardless of when an autonomic dysfunction occurs in life, these disorders can be primary, secondary, or
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 idiopathic. When autonomic dysfunction is the main disease process, it is generally referred to as a “primary dysautonomia,” which is a multifactorial condition that runs in families in which the autonomic nervous system does not function correctly leading to a range of disabling disease symptoms. Examples of primary dysautonomias include familial dysautonomia, multiple system atrophy, pure autonomic failure, and some forms of syncope among others. Secondary dysautonomias are experienced because of another disease process, as in autonomic neuropathy associated with diabetes or postural orthostatic tachycardia syndrome (POTS) resulting from an autoimmune disease. Conditions in which secondary dysautonomia might occur include, for example, Crohn’s disease, Ehlers-Danlos syndrome, Lyme disease, Sjogren’s syndrome, rheumatoid arthritis, and diabetes. Idiopathic dysautonomias are autonomic nervous system disorders where the main disease process is unknown. [0007] There is currently no known cure for autonomic disorders and all the available treatments are solely based on alleviating symptomatology. Accordingly, there remains a need for a method of treatment that can effectuate a cure to dysautonomia associated disorders. SUMMARY [0008] The present disclosure is based, in part, on the discovery that there is a causal relationship between primary dysautonomia and an autosomal dominant inheritance pattern of mutations in genes encoding multiple ion channel families, including voltage-gated sodium (Na
V) channels (SCN(x)A) and auxiliary subunits (SCN(x)B), the ion channel TRPA1 (transient receptor potential cation channel, subfamily A, member 1), the ion channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator, which transports chloride ions in and out of cells, helping regulate salt and fluid balance across cell membranes), mutations associated with ion channels such as PKD1 (encoding Polycystin-1, which is an integral membrane protein that interacts with the related protein polycystin-2 to regulate calcium permeable cation channels and intracellular calcium levels in cells), GRIN3B mutations which can affect calcium channel activity and ionotropic glutamate receptor activity, mutations associated with ATP8B3 functioning to transport phospholipids across the bilayer, mutations associated with the SLC (solute carrier) superfamily of membrane transport proteins that move solutes across cell membranes via facilitative or secondary active transport mechanisms, and fibrillin-2 and fibrillin-3 mutations associated with hypermobile Ehlers-Danlos and hypermobile joints as well as transcription factors such as zfhx2 (zinc finger homeobox 2). Overall, these genes encode proteins that: (i) are directly involved in membrane excitability such as (voltage-gated) ion channels, (ii) modulate other proteins that can generate or propagate
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 electrical signals such as ancillary subunits or transcription factors, and (iii) may trigger electrical or morphological remodeling of excitatory tissues. Notably, mutations in these genes may lead to altered autonomic nervous system (ANS) excitability as is often observed in these families and can be labeled clinically as a “high sympathetic tone.” This finding is strengthened by the fact that treatment of patients suffering from primary dysautonomia with Na
V channel modulators resolved many of their symptoms. More specifically, the inventors showed that treatment of guanfacine, an α
2A-adrenergic receptor agonist, could ameliorate anxiety and associated dysautonomia symptoms in patients with a Na
V1.7 mutation. Without wishing to be bound by any theory, it is hypothesized that what appears to pathophysiologically link diffuse autonomic symptoms is a disease model in which ion channel aberrations (both genetic and epigenetic) can transform the autonomic nervous system into an oversensitive nervous system that over time develops incapacitating and persistent disease. [0009] Accordingly, in one aspect, provided herein are methods of treating a dysautonomia associated disorder in a subject in need thereof, said methods comprising: a) detecting presence of a mutation in a voltage-gated sodium channel in the subject; and b) administering to the subject a therapeutically effective amount of an α
2A-adrenergic receptor agonist. In some embodiments, the subject having the dysautonomia associated disorder experiences one or more symptoms selected from orthostatic intolerance, fatigue, focal hyperhidrosis, neuropathic itch, and/or anxiety. In some embodiments, the subject having the dysautonomia associated disorder experiences one or more symptoms selected from chronic orthostatic intolerance, chronic fatigue, primary focal hyperhidrosis, chronic neuropathic itch, and/or generalized anxiety. Also provided herein are methods of ameliorating one or more symptoms of a dysautonomia associated disorder in a subject in need thereof, said methods comprising: a) detecting presence of a mutation in a voltage-gated sodium channel in the subject; and b) administering to the subject a therapeutically effective amount of an α
2A-adrenergic receptor agonist. In some embodiments, the one or more symptoms of the dysautonomia associated disorder comprises orthostatic intolerance, fatigue, focal hyperhidrosis, neuropathic itch, and/or anxiety. In some embodiments, the one or more symptoms of the dysautonomia associated disorder comprises chronic orthostatic intolerance, chronic fatigue, primary focal hyperhidrosis, chronic neuropathic itch, and/or generalized anxiety. [0010] In some embodiments, the mutation in a voltage-gated sodium channel is a gain-of- function mutation. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
V1.7. In some embodiments, the mutation comprises an amino acid substitution at amino acid position 739 of the sodium channel isoform Na
V1.7 as indexed by
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at amino acid position 739 is I739V. [0011] In some embodiments, the α
2A-adrenergic receptor agonist used in the methods of the present disclosure is formulated as a short-acting α
2A-adrenergic receptor agonist. In some embodiments, the therapeutically effective amount of the α
2A-adrenergic receptor agonist administered to the subject in need is a dosage of from about 0.1 mg/day to about 4 mg/day. In some embodiments, the therapeutically effective amount is a dosage of about 1 mg/day. In some embodiments, the α
2A-adrenergic receptor agonist for administration comprises guanfacine. In some embodiments, the methods of the disclosure further comprise administering to the subject a therapeutically effective amount of a Na
V channel modulator. [0012] In some embodiments, the dysautonomia associated disorder is caused by primary dysautonomia, such as familial dysautonomia, multiple system atrophy, and/or pure autonomic failure. In some embodiments, the dysautonomia associated disorder is caused by secondary dysautonomia. In some embodiments, the secondary dysautonomia is cause by alcoholism, amyloidosis, autoimmune disease such as Sjögren’s syndrome, systemic lupus erythematosus (lupus), and autoimmune autonomic ganglionopathy, Celiac disease, Charcot-Marie-Tooth disease, Chiari malformation, Crohn’s disease, diabetes, Lambert-Eaton syndrome, Lyme disease, Ehlers-Danlos syndrome, Guillain-Barré syndrome, human immunodeficiency virus (HIV), post infectious disease such as post-Covid syndrome or long COVID, multiple sclerosis, muscular sclerosis, paraneoplastic syndrome, synucleinopathy such as dementia with Lewy bodies, multiple system atrophy, and Parkinson’s disease, postural tachycardia syndrome (POTS), ulcerative colitis, rheumatoid arthritis, sarcoidosis, irritable bowel syndrome, myalgic encephalomyelitis, chronic fatigue syndrome, post-traumatic stress disorder (PTSD), primary focal hyperhidrosis, neuropathic itch, thoracic outlet obstruction, primary pulmonary hypertension, insomnia, neurocardiogenic syncope, attention deficit disorder (ADD) and its associated disorders, and/or superior mesenteric arterial syndrome. In some embodiments, the dysautonomia associated disorder is caused by idiopathic dysautonomia. [0013] In another aspect, provided herein are methods of predicting responsiveness of a subject suffering from or suspected of having a dysautonomia associated disorder to a treatment with a short-acting α
2A-adrenergic receptor agonist at a dosage of about 1 mg/day, said method comprising detecting presence or absence of a mutation in a voltage-gated sodium channel in the subject, wherein detecting the presence of the mutation indicates that the subject is likely to be responsive to the treatment and detecting the absence of the mutation indicates that the subject is unlikely to be responsive to the treatment. In some embodiments, the mutation in a
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 voltage-gated sodium channel is a gain-of-function mutation. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
V1.7. In some embodiments, the mutation comprises an amino acid substitution at amino acid position 739 of the sodium channel isoform Na
V1.7 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at amino acid position 739 is I739V. In some embodiments, the short-acting α
2A-adrenergic receptor agonist comprises short-acting guanfacine. [0014] In some embodiments, the methods further comprise administering the treatment to a subject identified as having the mutation. In some embodiments, the treatment ameliorates one or more symptoms of the dysautonomia associated disorder in the subject. In some embodiments, the one or more symptoms of the dysautonomia associated disorder comprises orthostatic intolerance, fatigue, focal hyperhidrosis, neuropathic itch, and/or anxiety. In some embodiments, the one or more symptoms of the dysautonomia associated disorder comprises chronic orthostatic intolerance, chronic fatigue, primary focal hyperhidrosis, chronic neuropathic itch, and/or generalized anxiety. BRIEF DESCRIPTION OF THE DRAWINGS [0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to explain certain principles of the methods and devices disclosed herein. [0016] FIG. 1 shows a 20-year-old male (proband, black arrow), harboring the Na
V1.7 p.I739V variant, presented with hyperhidrosis and concurrent symptoms of generalized anxiety, cardiac palpitations, cognitive deficits, chronic fatigue, insomnia, and an unusually high pain tolerance. Family consisted of three generations with pre-existing dysautonomia as shown. Symptoms are indicated with scores mentioned in pedigree. CS = COMPASS 31 score, HDSS = Hyperhidrosis Disease Severity Scale. Color/symbol legends are provided at the top of the figure. [0017] FIG. 2 shows the matriarch (proband, black arrow) of a large family with an inherited Na
V1.7 p.I739V substitution and three generations of pre-existing dysautonomia presented in our clinic in 2022 with brain fog and increased anxiety. This pedigree was drawn when the family was initially seen in our hyperhidrosis clinic pre-pandemic in 2017 with a chief complaint of hyperhidrosis, generalized anxiety disorder, and concomitant multiple symptoms of dysautonomia as shown. Symptoms are indicated with scores mentioned in
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 pedigree. CS = COMPASS 31 score, HDSS = Hyperhidrosis Disease Severity Scale. Color/symbol legends are provided at the top of the figure. [0018] FIG.3 shows the inhibitory effect of guanfacine on Na
V1.1, Na
V1.2, Na
V1.3, and Na
V1.6. Left: current trace upon depolarization to the peak voltage of the conductance (G) – voltage (V) relationship (right) before (black) and after (red) guanfacine perfusion. Arrow indicates current inhibition after drug application. Right: Normalized G-V and channel availability relationships before and after guanfacine administration. Figure represents mean values ± SEM with n = 5-6. [0019] FIG.4 shows the inhibitory effect of guanfacine on Na
V1.4, Na
V1.5, and Na
V1.8. Left: current trace upon depolarization to the peak voltage of the G-V relationship (right) before (black) and after (red) guanfacine perfusion. Arrow indicates current inhibition after drug application. Right: Normalized G-V and channel availability relationships before and after guanfacine administration. Figure represents mean values ± SEM with n = 5-8. [0020] FIG.5 shows the inhibitory effect of guanfacine on Na
V1.7. A: current trace upon depolarization to 0 mV, the peak voltage of the G-V relationship (right) before (black) and after (red) guanfacine perfusion. Arrow indicates current inhibition after drug application. B: Normalized G-V and channel availability relationships before and after guanfacine administration. Data in grey (control) and green (guanfacine addition) represents inhibition of the p.I739V mutation. Figure represents mean values ± SEM with n = 5-8. C: NaV1.7 recovery from inactivation before (black) and after (red) guanfacine addition obtained using a double- pulse protocol as described in the text. Y-axis represents fractional current recovery in function of the variable recovery interval in ms (X-axis). Figure represents mean values ± SEM with n = 5. D: state-dependent inhibition of Na
V1.7 by guanfacine. State dependence was determined using the test protocol described in Example 1. Representative current trace shown was obtained upon depolarization to 0 mV before (black) and after (red) guanfacine incubation. [0021] FIG. 6 shows a pedigree of two families with the proband presenting with severe hyperhidrosis. Symptoms are indicated with scores mentioned in pedigree. Black arrow indicates sequenced proband. Crossed out symbol indicates deceased family member. CS = COMPASS-31 score, SPIN = Social Phobia Inventory, HDSS = Hyperhidrosis Disease Severity Scale, ZUNG = Self-Rating Depression Scale. DETAILED DESCRIPTION
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0022] Reference will now be made in detail to various exemplary embodiments, examples of which are illustrated in the accompanying drawings and discussed in the detailed description that follows. It is to be understood that the following detailed description is provided to give the reader a fuller understanding of certain embodiments, features, and details of aspects of the disclosure, and should not be interpreted as limiting the scope of the disclosure. [0023] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term. Definitions [0024] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. [0025] The term “about,” or “approximately,” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value as such variations are appropriate to perform the disclosed methods and/or to make and use the disclosed devices. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range. [0026] The term “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0027] The phrase “as indexed by reference to the amino acid sequence of SEQ ID NO: 1,” as used herein, refers to a normalized biological sequence alignment that allows the comparison of a query sequence (e.g., a mutated sodium channel isoform Na
V1.7) to a subject sequence (e.g., a wild-type sodium channel isoform Na
V1.7, such as the human sodium channel isoform Na
V1.7 isoform 1 (SEQ ID NO: 1)), thereby identifying amino acid residues in the target sequence that correspond to the same positions in the subject sequence. In general, the target sequence and the query sequence share characteristic portions or features but differ slightly in length and/or sequence identity. For example, the numbering of residues in a specific target sequence or for targeted modification can be identified and described based on the sodium channel isoform Na
V1.7 isoform 1 amino acid sequence. Sequences are aligned to the full- length protein sequence of the sodium channel isoform Na
V1.7 isoform 1 (SEQ ID NO: 1). The N-terminal methionine of the protein sequence is residue 1. Accordingly, the phrase “amino acid position x (e.g., 739) as indexed by reference to the amino acid sequence of SEQ ID NO: 1” is used herein to designate the position/identity of an amino acid residue in a polypeptide of interest (e.g., a mutated sodium channel isoform Na
V1.7) by referring to the corresponding amino acid at position x (e.g., 739) in the sodium channel isoform Na
V1.7 isoform 1 (SEQ ID NO: 1). [0028] The term “at least,” “less than,” “more than,” or “up to” prior to a number or series of numbers (e.g., “at least two”) is understood to include the number adjacent to the term “at least,” “less than” or “more than,” and all subsequent numbers or integers that could logically be included, as clear from context. When the term “at least,” “less than,” “more than,” or “up to” is present before a series of numbers or a range, it is understood that “at least,” “less than,” “more than,” or “up to” can modify each of the numbers in the series or range. [0029] The term “diagnosing” or “diagnosis” as used herein refers to the use of information (e.g., antibody binding or data from tests on biological samples, signs and symptoms, physical exam findings, cognitive performance results, etc.) to anticipate the most likely outcomes, timeframes, and/or response to a particular treatment for a given disease, disorder, or condition, based on comparisons with a plurality of individuals sharing common nucleotide sequences, symptoms, signs, family histories, or other data relevant to consideration of a patient’s health status. [0030] The term “gain-of-function mutation,” as used herein, refers to a mutation that results in an enhanced phenotype, or a new function, compared with the wild-type allele. Gain- of-function mutations can occur through various mechanisms, such as constitutive activation, shift of substrate or binding target specificity, or protein aggregation.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0031] The term “in need thereof” means that the subject has been identified or suspected as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis or observation. In any of the methods described herein, the subject can be in need thereof. In some embodiments, the subject in need thereof is a human suspected of having a dysautonomia associated disorder, such as primary dysautonomia or secondary dysautonomia, including post-Covid syndrome. In some embodiments, the subject in need thereof is a human diagnosed with a dysautonomia associated disorder, such as primary dysautonomia or secondary dysautonomia, including post-Covid syndrome. In some embodiments, the subject in need thereof is a human seeking treatment for a dysautonomia associated disorder, such as primary dysautonomia or secondary dysautonomia, including post-Covid syndrome. In some embodiments, the subject in need thereof is a human undergoing treatment for a dysautonomia associated disorder, such as primary dysautonomia or secondary dysautonomia, including post-Covid syndrome. [0032] As used herein, the term “in some embodiments,” “in certain embodiments,” “in other embodiments,” “in some other embodiments,” or the like, refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise. [0033] The term “short-acting,” as used herein, means that the drug has effects that only last for a short time. Drugs or therapeutic agents are generally divided into long-acting agents with a half-life of more than about 24 hours, intermediate-acting agents with a half-life of about 6 to 24 hours, and short-acting agents with a half-life less than about 6 hours. Accordingly, in some embodiments, the term “short-acting,” as used herein, means that the drug or therapeutic agent has a half-life less than about 6 hours. [0034] The terms “subject” refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian, or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual that needs therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject.” For example, a subject can be an individual who has been diagnosed with having a pathogenic infection, is going to receive a therapy for a pathogenic infection, and/or has received at least one therapy for a pathogenic infection. [0035] The term “treat,” “treated,” or “treating” refers to therapeutic treatment and/or prophylactic or preventative measures wherein the object is to prevent or slow down (lessen)
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. For purposes of the embodiments described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment can also include eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. [0036] A “therapeutically effective amount” of a treatment is a predetermined amount calculated to achieve the desired effect, i.e., to treat, combat, ameliorate, prevent, or improve one or more symptoms of a dysautonomia associated disorder. The activity contemplated by the present methods includes both medical therapeutic and/or prophylactic treatment, as appropriate. The specific dose of a compound administered according to the present disclosure to obtain therapeutic and/or prophylactic effects will, of course, be determined by the circumstances surrounding the case, including, for example, the compound administered, the route of administration, and the condition being treated. It will be understood that the effective amount administered will be determined by the physician in the light of the relevant circumstances including the condition to be treated, the choice of compound to be administered, and the chosen route of administration, and therefore the above dosage ranges are not intended to limit the scope of the present disclosure in any way. A therapeutically effective amount of compounds, such as α
2A-adrenergic receptor agonists, for treating a dysautonomia associated disorder according to the disclosure is typically an amount such that when it is administered in a physiologically tolerable excipient composition, it is sufficient to achieve an effective systemic concentration or local concentration in the tissue. Dysautonomia Associated Disorders [0037] Dysautonomia is a term used to describe any disorder of the autonomic nervous system. The autonomic nervous system plays a role in almost every aspect of daily life. For instance, the autonomic nervous system acts a control system that regulates, calibrates, or adjusts the physiologic conditions of the body in response to various events within a number of systems, such as the cardiac, respiratory, digestive, endocrine, and vasomotor systems. In patients suffering dysautonomia, the sympathetic or parasympathetic components of the
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 autonomic nervous system do not function properly so that regulation and adjustment of many systems in the body is impacted. Symptoms of dysautonomia can include, but are not limited to, inability to stay upright, dizziness, vertigo, fainting, a fast heartbeat, a slow heartbeat, an irregular heartbeat, lack of heart rate variability, chest pain, low blood pressure, problems with the gastrointestinal system, nausea, disturbances in the visual field, weakness, breathing difficulties, mood swings, anxiety, fatigue and intolerance to exercise, migraines, tremors, disrupted sleep pattern, frequent urination, temperature regulation problems, concentration and memory problems, poor appetite, and overactive senses, especially when exposed to noise and light. [0038] Dysautonomia may be due to inherited or degenerative neurologic diseases, in which autonomic dysfunction is the main disease process. This is generally referred to as a “primary dysautonomia.” Examples of primary dysautonomia include, but are not limited to, familial dysautonomia, multiple system atrophy, pure autonomic failure, and some forms of syncope among others. [0039] Dysautonomia may also occur due to injury of the autonomic nervous system from an acquired disorder, such as Autism Spectrum Disorder (ASD), Parkinson’s disease, other degenerative neurological diseases, or epilepsy. Because this type of dysautonomia is experienced due to another disease process, it is generally referred to as “secondary dysautonomia. Conditions that may cause secondary dysautonomia include, but are not limited to, alcoholism, amyloidosis, autoimmune disease such as Sjögren’s syndrome, systemic lupus erythematosus (lupus), and autoimmune autonomic ganglionopathy, Celiac disease, Charcot- Marie-Tooth disease, Chiari malformation, Crohn’s disease, diabetes, Lambert-Eaton syndrome, Lyme disease, Ehlers-Danlos syndrome, Guillain-Barré syndrome, human immunodeficiency virus (HIV), post infectious disease such as post-Covid syndrome or long COVID, multiple sclerosis, muscular sclerosis, paraneoplastic syndrome, synucleinopathy such as dementia with Lewy bodies, multiple system atrophy, and Parkinson’s disease, postural tachycardia syndrome (POTS), ulcerative colitis, rheumatoid arthritis, sarcoidosis, irritable bowel syndrome, myalgic encephalomyelitis, chronic fatigue syndrome, post-traumatic stress disorder (PTSD), primary focal hyperhidrosis, neuropathic itch, thoracic outlet obstruction, primary pulmonary hypertension, insomnia, neurocardiogenic syncope, attention deficit disorder (ADD) and its associated disorders, and/or superior mesenteric arterial syndrome. [0040] When the main disease process causing autonomic nervous system disorders is unknown, it is usually referred to as “idiopathic dysautonomia.”
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0041] Accordingly, the term “dysautonomia associated disorder,” as used herein, refers to any disorder of the autonomic nervous system. In some embodiments, the dysautonomia associated disorder is caused by primary dysautonomia, such as familial dysautonomia, multiple system atrophy, and/or pure autonomic failure. In some embodiments, the dysautonomia associated disorder is caused by secondary dysautonomia, such as secondary dysautonomia caused by alcoholism, amyloidosis, autoimmune disease such as Sjögren’s syndrome, systemic lupus erythematosus (lupus), and autoimmune autonomic ganglionopathy, Celiac disease, Charcot-Marie-Tooth disease, Chiari malformation, Crohn’s disease, diabetes, Lambert-Eaton syndrome, Lyme disease, Ehlers-Danlos syndrome, Guillain-Barré syndrome, human immunodeficiency virus (HIV), post infectious disease such as post-Covid syndrome or long COVID, multiple sclerosis, muscular sclerosis, paraneoplastic syndrome, synucleinopathy such as dementia with Lewy bodies, multiple system atrophy, and Parkinson’s disease, postural tachycardia syndrome (POTS), ulcerative colitis, rheumatoid arthritis, sarcoidosis, irritable bowel syndrome, myalgic encephalomyelitis, chronic fatigue syndrome, post-traumatic stress disorder (PTSD), primary focal hyperhidrosis, neuropathic itch, thoracic outlet obstruction, primary pulmonary hypertension, insomnia, neurocardiogenic syncope, attention deficit disorder (ADD) and its associated disorders, and/or superior mesenteric arterial syndrome. In some embodiments, the dysautonomia associated disorder is caused by idiopathic dysautonomia. [0042] A wide range of symptoms, from mild to severe, are known to be experienced by patients suffering from dysautonomia. In some embodiments, the subject having the dysautonomia associated disorder according to the present disclosure experiences one or more symptoms selected from chronic orthostatic intolerance, chronic fatigue, primary focal hyperhidrosis, chronic neuropathic itch, and/or generalized anxiety. Voltage-Gated Sodium Channels [0043] Voltage-gated sodium channels are the basic ion channels for neuronal excitability, which are crucial for the resting potential and the generation and propagation of action potentials in neurons. Recent studies have identified that voltage-gated sodium channels not only play an essential role in the normal electrophysiological activities of neurons but also have a close relationship with neurological diseases. See e.g., Wang et al., Channels (Austin), 2017, 11(6):534-554. [0044] To date, at least nine distinct sodium channel isoforms (Na
v1.1- Na
v1.3, Na
v1.5 to Na
v1.9, and Na
vX) have been detected in the nervous system. Different types of sodium channels have different coding genes, and their gene localization on human chromosomes is
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 different. The sodium channels of each subtype may also have various isoforms due to the selective splicing of their coding genes or different modifications during the transcription process. Alternative splicing of the sodium channel gene is known to alter the pharmacological sensitivities, kinetics, and channel distribution under physiological or pathological conditions. This phenomenon of gene expression and regulation is present in all sodium channels. [0045] The present disclosure is based, in part, on the discovery that there is a causal relationship between primary dysautonomia and an autosomal dominant inheritance pattern of mutations in genes encoding multiple ion channels including voltage-gated sodium channels. More specifically, the inventors discovered that patients suffering from dysautonomia associated conditions, such as chronic orthostatic intolerance, chronic fatigue, primary focal hyperhidrosis, chronic neuropathic itch, and/or generalized anxiety, may benefit from treatment with guanfacine, an α
2A-adrenergic receptor agonist, if they possess a mutation in a voltage-gated sodium channel, particularly the Na
v1.7 sodium channel. [0046] The Na
v1.7 sodium channel is expressed primarily in peripheral neurons, including dorsal root ganglia (DRG) sensory neurons, sympathetic ganglion neurons, myenteric neurons, and trigeminal ganglion neurons. Three known pain syndromes are caused by mutations in the gene SCN9A encoding the Na
v1.7 sodium channel α subunit, inherited erythromelalgia (IEM), paroxysmal extreme pain disorder (PEPD), and congenital inability to experience pain (CIP). The mutation sites and types of the SCN9A gene encoding the Na
v1.7 sodium channel α subunit corresponding to these three pain syndromes have been described by Dib-Hajj et al. (Brain Res. Rev., 2009;60:65-83). Although all three pain syndromes are caused by mutations of the SCN9A gene, their mechanisms are different. For instance, the pain of IEM caused by SCN9A mutations occurs because the mutated Na
v1.7 sodium channel is more easily activated during hyperpolarization. In PEPD, on the other hand, the SCN9A mutation causes incomplete rapid inactivation of the Na
v1.7 sodium channel, leading to a persistent Na
+ current. The CIP is caused by the functional loss of the mutated Na
v1.7 sodium channel. [0047] Other mutations in the gene SCN9A encoding the Na
v1.7 sodium channel α subunit were also known. For instance, mutations of the gene SCN9A leading to the I720K, D623N, V991L, R185H, and I739V substitutions in the Na
v1.7 sodium channel α subunit were identified in patients with small fiber neuropathy. See, Entry No. 603415 (“SODIUM VOLTAGE-GATED CHANNEL, ALPHA SUBUNIT 9; SCN9A”) in OMIM® (omim.org/entry/603415).
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0048] At least 2 sodium channel isoforms Na
V1.7 have been identified in human. Human Na
V1.7 isoform 1 consists of 1977 amino acids and has the following amino acid sequence (Sodium channel protein type 9 subunit alpha isoform 1 [Homo sapiens] (NP_002968.1)): MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPKEEKKDDDEEAPKPSSDLEAGKQ LPFIYGDIPPGMVSEPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYMLSPFSPLRRI SIKILVHSLFSMLIMCTILTNCIFMTMNNPPDWTKNVEYTFTGIYTFESLVKILARGFC VGEFTFLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFRVLRALKTISVIPGLKTIV GALIQSVKKLSDVMILTVFCLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIMNTL ESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPEGYTCVKIGRNPDYGYTSFDTFSWA FLALFRLMTQDYWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLILAVVAMAYEE QNQANIEEAKQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSET SKLSSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKSESEDSIRRKSFHLGVEGHR RAHEKRLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSIFGDNE SRRGSLFVPHRPQERRSSNISQASRSPPMLPVNGKMHSAVDCNGVVSLVDGRSALML PNGQLLPEGTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRASILTNTVEELEESRQ KCPPWWYRFAHKFLIWNCSPYWIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEH HPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDPYEYFQVGWNIFDSLIVTLSLVEL FLADVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAV VGMQLFGKSYKECVCKINDDCTLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCME VAGQAMCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLTAIEEDPDANNLQIAVTR IKKGINYVKQTLREFILKAFSKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGHNFL KEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLTVTVPIAPGESDLENMNAEELSSDSD SEYSKVRLNRSSSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDGCVRRFSCCQV NIESGKGKIWWNIRKTCYKIVEHSWFESFIVLMILLSSGALAFEDIYIERKKTIKIILEY ADKIFTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPI KSLRTLRALRPLRALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAG KFYECINTTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQ VATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIGVIIDNF NQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQA FDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWINVVFIILFTGECVLKLISLRHYYF TVGWNIFDFVVVIISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVKGAKGIRTLL FALMMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKKEDGINDMFNFETFGNSMICLF QITTSAGWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNPSVGIFYFVSYIIISFLVV VNMYIAVILENFSVATEESTEPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAAAL
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 DPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLRSQMEERF MSANPSKVSYEPITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISSIYIKDGDRDDD LLNKKDMAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGK DSKESKK (SEQ ID NO: 1) [0049] Human Na
V1.7 isoform 2 consists of 1988 amino acids and has the following amino acid sequence (Sodium channel protein type 9 subunit alpha isoform 2 [Homo sapiens] (NP_001352465.1)): MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPKEEKKDDDEEAPKPSSDLEAGKQ LPFIYGDIPPGMVSEPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYMLSPFSPLRRI SIKILVHSLFSMLIMCTILTNCIFMTMNNPPDWTKNVEYTFTGIYTFESLVKILARGFC VGEFTFLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFRVLRALKTISVIPGLKTIV GALIQSVKKLSDVMILTVFCLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIMNTL ESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPEGYTCVKIGRNPDYGYTSFDTFSWA FLALFRLMTQDYWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLILAVVAMAYEE QNQANIEEAKQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSET SKLSSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKSESEDSIRRKSFHLGVEGHR RAHEKRLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSIFGDNE SRRGSLFVPHRPQERRSSNISQASRSPPMLPVNGKMHSAVDCNGVVSLVDGRSALML PNGQLLPEVIIDKATSDDSGTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRASILT NTVEELEESRQKCPPWWYRFAHKFLIWNCSPYWIKFKKCIYFIVMDPFVDLAITICIV LNTLFMAMEHHPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDPYEYFQVGWNIF DSLIVTLSLVELFLADVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLT LVLAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRWHMNDFFHSFLIVFRVLCGE WIETMWDCMEVAGQAMCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLTAIEEDP DANNLQIAVTRIKKGINYVKQTLREFILKAFSKKPKISREIRQAEDLNTKKENYISNHT LAEMSKGHNFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLTVTVPIAPGESDLEN MNAEELSSDSDSEYSKVRLNRSSSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDG CVWRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSWFESFIVLMILLSSGALAFEDIYIE RKKTIKIILEYADKIFTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVDVSLVTLVA NTLGYSDLGPIKSLRTLRALRPLRALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLI FSIMGVNLFAGKFYECINTTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFD NVGLGYLSLLQVATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFF TLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKI QGCIFDLVTNQAFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWINVVFIILFTGE
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 CVLKLISLRHYYFTVGWNIFDFVVVIISIVGMFLADLIETYFVSPTLFRVIRLARIGRILR LVKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKKEDGINDMF NFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNPSVGI FYFVSYIIISFLVVVNMYIAVILENFSVATEESTEPLSEDDFEMFYEVWEKFDPDATQFI EFSKLSDFAAALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGE MDSLRSQMEERFMSANPSKVSYEPITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNI SSIYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKEKY EQDRTEKEDKGKDSKESKK (SEQ ID NO: 2) [0050] The mutations in the Na
v1.7 sodium channel α subunit described in the art are all based on the amino acid sequence of Na
V1.7 isoform 1 (SEQ ID NO: 1). To clearly indicate that the amino acid positions of the Na
v1.7 sodium channel α subunit referred to in the present disclosure, including the claims, are based on the amino acid sequence of Na
V1.7 isoform 1 (SEQ ID NO: 1), the phrase “as indexed by reference to the amino acid sequence of SEQ ID NO: 1” is used. As used here, this phrase refers to a normalized biological sequence alignment that allows the comparison of a query sequence (e.g., a mutated sodium channel isoform Na
V1.7) to a subject sequence (e.g., a wild-type sodium channel isoform Na
V1.7, such as the human sodium channel isoform Na
V1.7 isoform 1 (SEQ ID NO: 1)), thereby identifying amino acid residues in the target sequence that correspond to the same positions in the subject sequence. In general, the target sequence and the query sequence share characteristic portions or features but differ slightly in length and/or sequence identity. By aligning the sequences to the full-length protein sequence of sodium channel isoform Na
V1.7 isoform 1 (SEQ ID NO: 1), the numbering of residues in a specific target sequence or for targeted modification can be identified and described based on the sodium channel isoform Na
V1.7 isoform 1 amino acid sequence. Thus, the phrase “amino acid position 739 as indexed by reference to the amino acid sequence of SEQ ID NO: 1,” for example, is used herein to designate the position/identity of an amino acid residue in a polypeptide of interest (e.g., a mutated sodium channel isoform Na
V1.7) by referring to the corresponding amino acid at position 739 in the sodium channel isoform Na
V1.7 isoform 1 (SEQ ID NO: 1). [0051] As noted above, the present disclosure is based, in part, on the discovery that there is a causal relationship between primary dysautonomia and an autosomal dominant inheritance pattern of mutations in genes encoding voltage-gated sodium channels, particularly sodium channel isoform Na
V1.7. In some embodiments, such mutations are gain-of-function mutations. In some embodiments, the mutation comprises an amino acid substitution at amino
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 acid position 739 of the sodium channel isoform Na
V1.7 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at amino acid position 739 is I739V. The I739V substitution has been previously identified in patients with small fiber neuropathy, but not patients suffering from dysautonomia. α
2A-Adrenergic Receptor Agonists [0052] Alpha-adrenergic agonists are a class of sympathomimetic agents that selectively stimulates alpha adrenergic receptors. The alpha-adrenergic receptor has two subclasses α1 and α2. The α-2 receptors constitute a family of G-protein-coupled receptors with 3 pharmacological subtypes, α
2A, α
2B, and α
2C. The α
2A and α
2C subtypes are found mainly in the central nervous system and stimulation of these receptor subtypes may be responsible for sedation, analgesia, and sympatholytic effects. The α
2B receptors are found more frequently on vascular smooth muscle and have been shown to mediate vasopressor effects. Endogenous agonists, such as norepinephrine and epinephrine, have similar affinities for all 3 subtypes. See e.g., Giovannitti et al., Anesth. Prog., 2015, 62(1):31-38. [0053] The α
2 adrenergic receptor agonists have been used for decades to treat common medical conditions such as hypertension, attention-deficit/hyperactivity disorder, various pain and panic disorders, symptoms of opioid, benzodiazepine, and alcohol withdrawal, and cigarette craving. Examples of α
2 adrenergic receptor agonists include, but are not limited to, brimonidine, clonidine, dexmedetomidine, fadolmidine, guanfacine (preference for α
2A- subtype of adrenoceptor), guanabenz (most selective agonist for α
2-adrenergic as opposed to imidazoline-I1), guanoxabenz (metabolite of guanabenz), guanethidine (peripheral α
2-receptor agonist), xylazine(not for human use), tizanidine, methyldopa, methylnorepinephrine, norepinephrine, (R)-3-nitrobiphenyline (an α
2C selective agonist as well as being a weak antagonist at the α
2A and α
2B subtypes, amitraz, detomidine, lofexidine (an α
2A-adrenergic receptor agonist), medetomidine (an α
2-adrenergic receptor agonist). [0054] Guanfacine, sold under the brand name Tenex (immediate-release) and Intuniv (extended-release) among others, is an oral α
2A agonist medication used to treat attention deficit hyperactivity disorder (ADHD) and high blood pressure. Guanfacine is FDA-approved for monotherapy treatment of ADHD, as well as being used for augmentation of other treatments, such as stimulants. Guanfacine is also used off-label to treat tic disorders, anxiety disorders and posttraumatic stress disorders (PTSD). [0055] For instance, Qasaymeh et al. (Curr. Treat. Options Neurol., 2006, 8(6):465-473) reported the off-label use of guanfacine for treating tic disorders. The suggested dosage was, starting with 0.5 mg at bedtime, increasing as needed and as tolerated by 0.5 mg every week to
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 a maximum dosage of 3 to 4 mg/day, divided twice a day. The off-label use of guanfacine for treating anxiety disorders was reported in, for instance, Strawn et al. (J. Child. Adolesc. Psychopharmacol., 2017, 27(1):29-37), in which 1-6^mg daily dose of extended release guanfacine was used. Connor et al. (J. Child Adolesc. Psychopharmacol., 2013, 23(4):244- 251) reported the off-label use of extended release guanfacine (1-4 mg) in treating children and adolescents with a history of traumatic stress with or without PTSD. Methods of Treatments [0056] As noted above, the present disclosure is based, in part, on the discovery that there is a causal relationship between primary dysautonomia and an autosomal dominant inheritance pattern of mutations in genes encoding multiple ion channels including voltage-gated sodium channels, such as the Na
v1.7 sodium channel. The inventors discovered that low dose (e.g., 1 mg/day) of short-acting α
2A-adrenergic receptor agonists, such as guanfacine, facilitate virtually complete symptom relief in patients with severe hereditary generalized anxiety. Extensive improvements in associated dysautonomia symptoms, such as hyperhidrosis, cognitive impairment, and palpitations, were also noted. Accordingly, in one aspect, provided herein is a method of treating a dysautonomia associated disorder in a subject in need thereof, said method comprising: a) detecting presence of a mutation in a voltage-gated sodium channel in the subject; and b) administering to the subject a therapeutically effective amount of an α
2A- adrenergic receptor agonist. In another aspect, the present disclosure also provides a method of ameliorating one or more symptoms of a dysautonomia associated disorder in a subject in need thereof, said method comprising: a) detecting presence of a mutation in a voltage-gated sodium channel in the subject; and b) administering to the subject a therapeutically effective amount of an α
2A-adrenergic receptor agonist. [0057] Dysautonomia can be primary, secondary, or idiopathic. In some embodiments, the dysautonomia associated disorder is caused by primary dysautonomia. In some embodiments, the dysautonomia associated disorder is caused by secondary dysautonomia. In some embodiments, the dysautonomia associated disorder is caused by idiopathic dysautonomia. [0058] Examples of primary dysautonomia include, but are not limited to, familial dysautonomia, multiple system atrophy, pure autonomic failure, and some forms of syncope among others. Accordingly, in some embodiments, the dysautonomia associated disorder is caused by familial dysautonomia. In some embodiments, the dysautonomia associated disorder is caused by multiple system atrophy. In some embodiments, the dysautonomia associated disorder is caused by pure autonomic failure.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0059] Secondary dysautonomia is generally occurred due to injury of the autonomic nervous system from an acquired disorder or condition. Disorders or conditions that may cause secondary dysautonomia include, but are not limited to, alcoholism, amyloidosis, autoimmune disease such as Sjögren’s syndrome, systemic lupus erythematosus (lupus), and autoimmune autonomic ganglionopathy, Celiac disease, Charcot-Marie-Tooth disease, Chiari malformation, Crohn’s disease, diabetes, Lambert-Eaton syndrome, Lyme disease, Ehlers- Danlos syndrome, Guillain-Barré syndrome, human immunodeficiency virus (HIV), post infectious disease such as post-Covid syndrome or long COVID, multiple sclerosis, muscular sclerosis, paraneoplastic syndrome, synucleinopathy such as dementia with Lewy bodies, multiple system atrophy, and Parkinson’s disease, postural tachycardia syndrome (POTS), ulcerative colitis, rheumatoid arthritis, sarcoidosis, irritable bowel syndrome, myalgic encephalomyelitis, chronic fatigue syndrome, post-traumatic stress disorder (PTSD), primary focal hyperhidrosis, neuropathic itch, thoracic outlet obstruction, primary pulmonary hypertension, insomnia, neurocardiogenic syncope, attention deficit disorder (ADD) and its associated disorders, and/or superior mesenteric arterial syndrome. Accordingly, in some embodiments, the dysautonomia associated disorder is caused by secondary dysautonomia associated with disorders or conditions selected from alcoholism, amyloidosis, autoimmune disease such as Sjögren’s syndrome, systemic lupus erythematosus (lupus), and autoimmune autonomic ganglionopathy, Celiac disease, Charcot-Marie-Tooth disease, Chiari malformation, Crohn’s disease, diabetes, Lambert-Eaton syndrome, Lyme disease, Ehlers- Danlos syndrome, Guillain-Barré syndrome, human immunodeficiency virus (HIV), post infectious disease such as post-Covid syndrome or long COVID, multiple sclerosis, muscular sclerosis, paraneoplastic syndrome, synucleinopathy such as dementia with Lewy bodies, multiple system atrophy, and Parkinson’s disease, postural tachycardia syndrome (POTS), ulcerative colitis, rheumatoid arthritis, sarcoidosis, irritable bowel syndrome, myalgic encephalomyelitis, chronic fatigue syndrome, post-traumatic stress disorder (PTSD), primary focal hyperhidrosis, neuropathic itch, thoracic outlet obstruction, primary pulmonary hypertension, insomnia, neurocardiogenic syncope, attention deficit disorder (ADD) and its associated disorders, and/or superior mesenteric arterial syndrome. In some embodiments, the dysautonomia associated disorder is caused by POTS, post infectious disease such as post- Covid syndrome, long COVID, or chronic Lyme disease, irritable bowel syndrome, myalgic encephalomyelitis, chronic fatigue syndrome, PTSD, primary focal hyperhidrosis, neuropathic itch, thoracic outlet obstruction, Ehlers-Danlos syndrome, primary pulmonary hypertension,
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 certain forms of insomnia, neurocardiogenic syncope, ADD and its associated disorders, and superior mesenteric arterial syndrome. [0060] Strengthening this is the inventors’ ability to treat secondary dysautonomia syndromes, such as Long COVID or Post-acute sequelae of SARS-CoV-2 infection based on evidence of a multi-hit molecular model for the development of these syndromes. Early in the COVID pandemic, for example, clinicians recognized an overlap between Long COVID symptoms and dysautonomia, suggesting autonomic nervous system dysfunction. The inventors’ clinical experience at Johns Hopkins with primary dysautonomia suggested heritability of sympathetic dysfunction, manifesting primarily as hyperhidrosis and as other dysautonomia symptoms. Whole exome sequencing revealed mutations in genes regulating electrical signaling in the nervous system, thus providing a genetic basis for sympathetic overdrive. The inventors hypothesize that dysautonomia in Long COVID requires two molecular hits: (i) a genetic vulnerability to prime the autonomic nervous system (ANS), (ii) a SARS-CoV-2 infection, as an immune trigger, to further disrupt ANS function resulting in increased sympathetic activity. Indeed, Long COVID patients show signs of chronic inflammation and autoimmunity. The inventors have translated this two-hit concept to the clinic using ion channel inhibitors to target genetic susceptibility and immunomodulators to treat inflammation. The fact that patients are getting better when treated with both ion channel inhibitors and immunomodulators helps to validate this multi-hit hypothesis for managing Long COVID. [0061] Patients suffering from dysautonomia may experience a wide range of symptoms, from mild to severe, such as dizziness, insomnia, syncope, orthostatic intolerance, fatigue, focal hyperhidrosis, neuropathic itch, and anxiety. Accordingly, in some embodiments, the subject having the dysautonomia associated disorder experiences one or more symptoms selected from orthostatic intolerance, fatigue, focal hyperhidrosis, neuropathic itch, and/or anxiety. In some embodiments, the subject having the dysautonomia associated disorder experiences one or more symptoms selected from chronic orthostatic intolerance, chronic fatigue, primary focal hyperhidrosis, chronic neuropathic itch, and/or generalized anxiety. [0062] In some embodiments, the mutation in a voltage-gated sodium channel of the subject is a gain-of-function mutation, which is a mutation that results in an enhanced phenotype, or a new function, as compared to the wild-type allele. In some embodiments, the mutation is a loss-of-function mutation, which is a mutation that results in a reduced phenotype, or a lost function, as compared to the wild-type allele.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0063] There are at least nine distinct sodium channel isoforms (Na
v1.1- Na
v1.3, Na
v1.5 to Na
v1.9, and Na
vX) to date. Accordingly, in some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
v1.1. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
v1.2. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
v1.3. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
v1.5. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
v1.6. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
v1.7. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
v1.8. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
v1.9. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
vX. [0064] In some embodiments, the mutation is in the sodium channel isoform Na
v1.7. In some embodiments, the mutation comprises an amino acid substitution at amino acid position 739 of the sodium channel isoform Na
V1.7 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at amino acid position 739 is I739V. [0065] The α
2A-adrenergic receptor agonists suitable for use in the methods of present disclosure can be formulated as short-acting, intermediate-acting, or long-acting formulation. In some embodiments, the α
2A-adrenergic receptor agonist is formulated as a short-acting α
2A- adrenergic receptor agonist. Short-acting agents generally have a half-life less than about 6 hours. Accordingly, in some embodiments, the α
2A-adrenergic receptor agonist suitable for use in the methods of present disclosure is formulated as a short-acting α
2A-adrenergic receptor agonist having a half-life less than about 6 hours. In some embodiments, the α
2A-adrenergic receptor agonist is formulated as a short-acting α
2A-adrenergic receptor agonist having a half- life less than about 5 hours. In some embodiments, the α
2A-adrenergic receptor agonist is formulated as a short-acting α
2A-adrenergic receptor agonist having a half-life less than about 4 hours. [0066] The dosage of the α
2A-adrenergic receptor agonist to be administered according to the present disclosure, or the so-called “therapeutically effective amount,” is generally from about 0.1 mg/day to about 4 mg/day, such as about 0.1 mg/day, about 0.5 mg/day, about 1 mg/day, about 1.5 mg/day, about 2 mg/day, about 2.5 mg/day, about 3 mg/day, about 3.5 mg/day, or about 4 mg/day, including all values and subranges therebetween. In some embodiments, the therapeutically effective amount is a dosage of about 0.1 mg/day. In some
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 embodiments, the therapeutically effective amount is a dosage of about 0.5 mg/day. In some embodiments, the therapeutically effective amount is a dosage of about 1 mg/day. In some embodiments, the therapeutically effective amount is a dosage of about 1.5 mg/day. In some embodiments, the therapeutically effective amount is a dosage of about 2 mg/day. In some embodiments, the therapeutically effective amount is a dosage of about 3 mg/day. In some embodiments, the therapeutically effective amount is a dosage of about 4 mg/day. In some embodiments, the therapeutically effective amount is a dosage of about 5 mg/day. In some embodiments, the therapeutically effective amount is a dosage of about 6 mg/day. In some embodiments, the therapeutically effective amount is a dosage of about 7 mg/day. [0067] Several α
2 adrenergic receptor agonists are known to have preference for α
2A- subtype of adrenoceptor, such as guanfacine and lofexidine. Accordingly, in some embodiments, the α
2A-adrenergic receptor agonist suitable for administering to the subject according to the present disclosure comprises guanfacine. In some embodiments, the α
2A- adrenergic receptor agonist comprises lofexidine. [0068] In some embodiments, the methods of the present disclosure further comprise administering to the subject a therapeutically effective amount of a Na
V channel modulator, such as those disclosed in WO 2019/126842, incorporated herein by reference. [0069] In some embodiments, provided herein is a method of treating a dysautonomia associated disorder in a subject in need thereof, said method comprising: a) detecting presence of a mutation in a voltage-gated sodium channel isoform Na
V1.7 in the subject; and b) administering to the subject a short-acting guanfacine at a dosage of about 1 mg/day. In some embodiments, the mutation in the sodium channel isoform Na
V1.7 is an amino acid substitution at amino acid position 739 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is I739V. In some embodiments, the short-acting guanfacine has a half-life of less than 6 hours. [0070] In some embodiments, provided herein is a method of ameliorating one or more symptoms of a dysautonomia associated disorder in a subject in need thereof, said method comprising: a) detecting presence of a mutation in a voltage-gated sodium channel isoform Na
V1.7 in the subject; and b) administering to the subject a short-acting guanfacine at a dosage of about 1 mg/day. In some embodiments, the mutation in the sodium channel isoform Na
V1.7 is an amino acid substitution at amino acid position 739 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution is I739V. In some embodiments, the short-acting guanfacine has a half-life of less than 6 hours.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0071] In a further aspect, provided herein is a method of predicting responsiveness of a subject suffering from or suspected of having a dysautonomia associated disorder to a treatment with a short-acting α
2A-adrenergic receptor agonist as described elsewhere herein at a dosage of about 1 mg/day, said method comprising detecting presence or absence of a mutation in a voltage-gated sodium channel in the subject, wherein detecting the presence of the mutation indicates that the subject is likely to be responsive to the treatment and detecting the absence of the mutation indicates that the subject is unlikely to be responsive to the treatment. In some embodiments, the mutation is a gain-of-function mutation as described herein. In some embodiments, the voltage-gated sodium channel comprises a sodium channel isoform Na
V1.7 as described herein. In some embodiments, the mutation comprises an amino acid substitution at amino acid position 739 of the sodium channel isoform Na
V1.7 as indexed by reference to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at amino acid position 739 is I739V. [0072] In some embodiments, the short-acting α
2A-adrenergic receptor agonist comprises short-acting guanfacine as described herein. In some embodiments, the short-acting guanfacine has a half-life of less than about 6 hours. [0073] In some embodiments, the method further comprises administering the treatment to a subject identified as having the mutation. In some embodiments, the treatment ameliorates one or more symptoms of the dysautonomia associated disorder as described herein in the subject. In some embodiments, the one or more symptoms of the dysautonomia associated disorder comprises orthostatic intolerance, fatigue, focal hyperhidrosis, neuropathic itch, and/or anxiety. In some embodiments, the one or more symptoms of the dysautonomia associated disorder comprises chronic orthostatic intolerance, chronic fatigue, primary focal hyperhidrosis, chronic neuropathic itch, and/or generalized anxiety. EXAMPLES [0074] The following examples are to be considered illustrative and not limiting on the scope of the present disclosure described above. Example 1. Guanfacine Ameliorates Anxiety and Associated Dysautonomia Symptoms in Patients with a NaV1.7 Mutation [0075] The α
2A-adrenergic receptor agonist guanfacine is FDA- and EMA-approved to treat hypertension and for use as a second-line treatment for attention-deficit hyperactivity disorder (ADHD) in children and adolescents (Caye et al., Mol. Psychiatry, 2019, 24:390-408;
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 Mechler et al., Pharmacol. Therapeut., 2022, 230:107940) under brand names such as TENEX®, AFKEN®, ESTULIC®, and the extended-release formulation INTUNIV®. A meta-analysis of twelve randomized controlled trials involving both children as well as adults showed that guanfacine, if given short or long-term, was significantly superior to placebo in treating ADHD (Yu et al., J. Child Adolesc. Psychopharmacol., 2023, 33:40-50). Guanfacine also reduces hyperactivity and attention deficit in animal models of ADHD (Ota et al., Drug Des. Devel. Ther., 2021, 15:1965-1969; Sagvolden T., Behav. Brain Funct., 2006, 2:41). Moreover, it is well established that in rodents, monkeys, and humans, systemic administration of guanfacine improves cognitive function in the prefrontal cortex, an area of the brain that plays a key role in regulating attention, behavior, and emotion (Arnsten A.F.T., Neurobiol. Learn. Mem., 2020, 176:107327). These findings correlate with the association between ADHD and genetic alterations that attenuate catecholamine signaling, particularly in the prefrontal cortex (Alamo et al., Actas Esp. Psiquiatr., 2016, 44:107-112; Arnsten A.F., J. Pediatr., 2009, 154:I-S43). Indeed, in neuropsychiatric disorders that involve dysfunction of the prefrontal cortex such as post-traumatic stress disorder, substance abuse, schizophrenia and autistic spectrum cognitive deficits, traumatic brain injury, emergence delirium, and Tourette Syndrome (Alexandra et al., Neuropsychopharmacology, 2022, 47:247-259; Chini et al., Trends Neurosci., 2021, 44:227-240; Kenwood et al., Neuropsychopharmacology, 2022, 47:260-275; Xu et al., Physiol. Genomics, 2019, 51:432-442), guanfacine has proven clinical efficacy and is therefore used off-label (Arnsten A.F.T., Neurobiol. Learn. Mem., 2020, 176:107327). [0076] In general, activation of presynaptic α
2A-adrenergic receptors in the central nervous system decreases noradrenergic outflow through a negative feedback loop (Arnsten A.F.T., Neurobiol. Learn. Mem., 2020, 176:107327 ). Potential central and peripheral side effects are consistent with the ample distribution of noradrenergic neurons and may include sedation, weakness, dizziness, bradycardia, fainting, headache, constipation, decreased appetite, dry mouth, and nausea. In the prefrontal cortex, postsynaptic α
2A-adrenergic receptor activation by guanfacine impedes cAMP production, which, in turn, closes Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels to augment network connectivity thus improving working memory and attention (Wang et al., Cell, 2007, 129:397-410). This ability of guanfacine to bolster prefrontal lobe connectivity is a possible basis of a report showing amelioration of Post-Acute Sequelae of SARS-CoV-2 (PASC)-associated “brain fog” (i.e., cognitive impairment) in twelve patients who were also given high doses of N-acetyl cysteine,
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 an immunomodulator that combats inflammation (Fesharaki-Zadeh et al., Neuroimmunology Reports, 2023, 3:100154). [0077] In our dysautonomia clinic, we observed that off-label use of guanfacine was effective in reducing generalized anxiety in families with multiple members affected by similar symptoms that also included cognitive impairment, hyperhidrosis, and palpitations. In contrast to ADHD treatments that typically involve the use of an extended-release formulation up to 4 mg/day, we noted that short-acting guanfacine at only 1 mg/day facilitated virtually complete and long-term symptom relief in our patients without side effects. We postulated that a particular genetic deficit existed in these patients that might underlie this striking susceptibility to guanfacine. To this end, we used whole-exome sequencing (WES) on two families that visited our clinic and identified the Na
V1.7 (gene name: SCN9A) p.I739V variant as a possible pathological cause. This mutation was previously described as having a gain-of-function phenotype linked to neuropathic pain and autonomic dysfunction including hyperhidrosis and palpitations (Han et al., Brain, 2012, 135:2613-2628). In a third family that was not sequenced, 1 mg/day of short-acting guanfacine was also very effective as a durable treatment of erythromelalgia, a disorder widely accepted to be associated with gain-of-function mutations in Na
V1.7 (Dib-Hajj et al., Brain, 2005, 128:1847-1854; Estacion et al., J. Neurosci., 2008, 28:11079-11088; Tanaka et al., J. Biol. Chem., 2017, 292:9262-9272; Weaver J., Neurology Alert, 2016, 35). Since guanfacine acts on ion channels (Arnsten A.F.T., Neurobiol. Learn. Mem., 2020, 176:107327) and several neuropsychiatric diseases for which guanfacine is clinically effective are associated with Na
V channelopathies (Bagheri et al., Int. J. Dev. Neurosci., 2021, 81:669-685; Eijkelkamp et al., Brain, 2012, 135:2585-2612; Imbrici et al., Front. Genet., 2013, 4:76; Mi et al., Front. Cell Neurosci., 2019, 13:554; Nickel et al., BMC Psychiatry, 2018, 18:248; Pasierski et al., Pharmacol. Rep., 2023, 75:331-341; Rees et al., Biol. Psychiatry, 2019, 85:554-562; Weiss et al., Mol. Psychiatry, 2003, 8:186-194), we hypothesized that guanfacine had off-target effects on Na
V1.7 or other Na
V channel subtypes. Indeed, functional modulation of Na
V channels that help regulate membrane excitability in the prefrontal cortex or sympathetic nervous system, could assist in alleviating patient symptomatology (Han et al., Korean J. Pediatr., 2017, 60:189-195; Jobling et al., J. Neurophysiol., 1999, 82:2747-2764; Minett et al., Nat. Commun., 2012, 3:791; Robertson et al., Primer on the Autonomic Nervous System. Elsevier Inc., 2012). Combined, the results presented here provide additional evidence for a pathological role of Na
V1.7 mutations in generalized anxiety and dysautonomia. Moreover, Na
V channel inhibition – either by itself or in concert with α
2A-adrenergic receptor activation – may help explain the efficacy of 1 mg/day
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 short-acting guanfacine. In a broader context, our data offer information that can be of future clinical use to genetically stratify patients so that guanfacine can be administered to modulate the sympathetic tone and ameliorate dysautonomia symptoms. 1. Materials and Methods i. Whole exome sequencing of patients [0078] Supported by the Baylor-Hopkins Center for Mendelian Genomics (BHCMG), Whole Exome Sequencing (WES) data were obtained on two nonrelated families with a history of generalized anxiety and dysautonomia who presented to the specialized Dysautonomia Clinic for treatment at the Johns Hopkins Hospital between 2018 and 2020. The WES procedure was previously described by Dhaheri and coworkers (Dhaheri et al., Am. J. Med. Genet. A., 2020, 182:1664-1672) using genomic DNA captured with the Agilent SureSelect Human All Exon V451MB Kit and sequenced on an Illumina HiSeq2000 platform. FASTQ files were aligned to the reference genome (GRCH38) with the Burrows-Wheeler Alignment (BWA 0.5.10) tool. [0079] Next, multisample SNV and indel calling were performed on the reduced-read BAM files with GATK’s UnifiedGenotyper. Variant sites were filtered with GATK’s Variant Quality Score Recalibration best practices and excluded heterozygous genotypes if they did not have at least 5 alternative allele reads. The annotated files (ANNOVAR) were analyzed using the PhenoDB Variant Analysis Tool (Sobreira et al., Hum. Mutat., 2015, 36:425-431) by selecting the rare (MAF <1%) functional (missense, nonsense, stop loss, splice site and indels) heterozygous and homozygous variants in each proband. Variants with a MAF > 0.01 in GnomAD or in the BHCMG sample were excluded. Next, each variant and gene were evaluated for their ClinVar, HGMD, OMIM, and mouse phenotype annotations. Finally, the gene variants that met the above criteria in heterozygous or homozygous states were selected for further investigation. [0080] Clinical data and survey results from the Hyperhidrosis Disease Severity Scale (Solish et al., Dermatol. Surg., 2007, 33:908-923) and the COMPASS-31 Dysautonomia Score (Sletten et al., Mayo Clin. Proc., 2012, 87:1196-1201) were ascertained both during the clinic visit and by using the REDCap (Research Electronic Data Capture) tool (Harris et al., J. Biomed. Inform., 2019, 95:103208; Harris et al., J. Biomed. Inform., 2009, 42:377-381). Study data were collected and managed using REDCap electronic data capture tools hosted at Johns Hopkins University School of Medicine. REDCap is a secure, web-based software platform designed to support data capture for research studies, providing 1) an intuitive interface for validated data capture; 2) audit trails for tracking data manipulation and export procedures; 3)
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for data integration and interoperability with external sources. The Institutional Review Board of Johns Hopkins University School of Medicine approved this study. Informed consent was obtained from each patient and affected family member who underwent WES. ii. Electrophysiology on Xenopus laevis oocytes [0081] The cDNA sequences of human Na
V1.1 (AF225985.1), Na
V1.2 (NP_001035232.1), Na
V1.3 (AF225987.1), Na
V1.4 (NP_000325.4), Na
V1.5 (AAI44622.1), Na
V1.6 (NP_055006.1), wild type Na
V1.7 (NP_002968.1) and the p.I739V mutant, Na
V1.8 (NP_006505.4), and β1 (NP_001028.1) (Origene, USA and Genscript, USA), were confirmed by automated DNA sequencing. For oocyte experiments, cRNA was synthesized using T7 polymerase (mMessage mMachine kit, Life Technologies, USA) after linearizing the DNA with an applicable restriction enzyme. Na
V channels were expressed with β1 in a 1:5 molar ratio by microinjecting cRNA into defolliculated Xenopus laevis oocytes (toads obtained from Nasco®, USA) (Gilchrist et al., ACS Chem. Biol., 2014, 9:1204-1212). The use of toads complied with national and Flemish guidelines adhered to by the UGent University Animal Care and Use Committee. Oocytes (n = 5-8 per Na
V channel subtype) were incubated at 17°C in Barth’s medium (96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1 mM MgCl
2, and 1.8 mM CaCl
2, 50 μg/ml gentamycin, pH 7.6 with NaOH, chemicals from Sigma®, USA) and following 1-4 days incubation, studied using the two-electrode voltage-clamp recording technique (OC- 725C, Warner Instruments, USA) with a 150-μl recording chamber as previously described (Gilchrist et al., ACS Chem. Biol., 2014, 9:1204-1212). [0082] All data were filtered at 4 kHz and digitized at 20 kHz using pClamp 10 software (Molecular Devices, USA). The external recording solution used was ND-100 (100 mM NaCl, 5 mM HEPES, 1 mM MgCl
2, and 1.8 mM CaCl
2, pH 7.6 with NaOH, chemicals from Sigma®, USA) and microelectrode resistances were 0.5 to 1.0 MΩ when filled with 3 M KCl. All experiments were performed at room temperature (about 22°C). [0083] To avoid capacitance errors induced by the large membrane surface of Xenopus laevis oocytes and enable the detection of subtle biophysical events, maximum recorded current amplitudes were limited to ±1.5 μA by means of titrating cRNA quantities. Leak and background conductances were identified and subtracted by blocking channels with tetrodotoxin (TTX; Alomone Labs, Israel), including a TTX-sensitive variant of Na
V1.8 (Sivilotti et al., FEBS Lett., 1997, 409:49-52). Normalized conductance-voltage (G/G
max) and channel availability
relationships were obtained by measuring steady-state currents and
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 a single Boltzmann function was fitted to the data according to I/I
max (or G/G
max) = [1 + exp(- zF(V-V
1/2)/(RT))]
-1, where I/I
max is the normalized current amplitude, z is the equivalent charge, V
1/2 is the half-activation voltage, F is Faraday’s constant, R is the gas constant, and T is temperature in kelvin. [0084] Guanfacine hydrochloride was acquired from Sigma® (USA) and dissolved in ND- 100 as a 1 mM stock solution from which a 100 μM working solution (86 μM effective drug concentration) was diluted for use with oocytes. Na
V channels were exposed to guanfacine through a gravity-fed perfusion system with a flow rate of 0.5 ml/minute. When adding guanfacine to the recording chamber, equilibration between channel and compound was monitored by means of depolarizations to V
1/2 of G/G
max at five-second intervals. Where needed, statistical differences were determined using the student’s t-test (p = 0.01). Off-line data analysis was performed using Clampfit 10 (Molecular Devices, USA), Excel (Microsoft Office, USA) and Prism 8 (GraphPad, USA). 2. Results i. Beneficial effects of guanfacine in patients with neuropsychiatric symptoms and a Na
V1.7 mutation [0085] We explored whether genetic abnormalities were present in a limited subset of patients that visited our dysautonomia clinic because they suffered from hereditary generalized anxiety and concomitant sympathetic hyperarousal symptoms (Freeman et al., Clin. Auton. Res., 2011, 21:69-72; Goldstein D.S., Clin. Auton. Res., 2020, 30:299-315; Posey et al., Pediatr. Neurol., 2017, 66:53-58, e55). Moreover, for these patients 1 mg/day of short-acting guanfacine was empirically proven to be a highly effective treatment. WES uncovered the Na
V1.7 p.I739V channelopathy as likely clinically relevant in patients belonging to two nonrelated families with extensive inherited symptomatology (FIG.1 and FIG.2). To detect dysautonomia, we used the COMPASS 31 validated questionnaire (Sletten et al., Mayo Clin. Proc., 2012, 87:1196-1201), a self-rating quantitative measure of autonomic symptoms evaluating orthostatic intolerance, vasomotor, secretomotor, gastrointestinal, bladder, and pupillomotor domains with our nondysautonomia control group scoring 13 or less. [0086] The first family (Maryland, USA) illustrates the treatment effect of guanfacine as it started with the case of a 20-year-old male (FIG.1, proband), harboring the Na
V1.7 p.I739V variant, who presented to our outpatient clinic with hyperhidrosis denoted as intolerable on the Hyperhidrosis Disease Severity Scale (Solish et al., Dermatol. Surg., 2007, 33:908-923) along with concurrent symptoms of generalized anxiety, cardiac palpitations, cognitive deficits such as forgetfulness and trouble concentrating (i.e., brain fog), chronic fatigue, and insomnia. Due
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 to its reduced adverse effect profile compared to clonidine, a α
2A-adrenergic receptor agonist used off-label to treat disabling hyperhidrosis and neuropsychiatric symptoms (Albadrani A., J. Med. Case Rep., 2017, 11:16; Conrad et al., Ann. Intern. Med., 1983, 99:570; Kuritzky et al., Arch. Neurol., 1984, 41:1210-1211; Soriano et al., Am. J. Hosp. Palliat. Care, 2014, 31:98- 100; Torch et al., South Med. J., 2000, 93:68-69; van Coevorden et al., Cephalalgia, 2021, 41:1124-1127), we opted to use short-acting guanfacine (Tenex®, 1 mg/day) as an adjunctive therapy. Six years later, the patient says that guanfacine continues to be well tolerated and highly effective, so much so that whenever he misses a dose, his cognitive impairment, anxiety, hyperhidrosis and as well as other symptoms swiftly return. [0087] The second family is from South-East Asia and consists of three generations that exhibit extensive dysautonomia symptoms primarily as generalized anxiety, hyperhidrosis, neuropathic itch, orthostatic intolerance, and chronic fatigue (FIG. 2). The proband in this family expressed the Na
V1.7 p.I739V variant and presented with such profound hyperhidrosis that she opted for surgical treatment (sympathectomy). Her maternal aunt also sought treatment in our clinic and was administered Tenex®, 1 mg/day. After three days, her symptoms of anxiety, cognitive impairment, cardiac palpitations, and hyperhidrosis lessened, along with decreased paroxysmal hypertension. Two years later, the patient says that she alternates daily between short-acting guanfacine 1 mg/day and 1.5 mg/day with continued amelioration of her symptoms. Altogether, in patients with the Na
V1.7 I739V gain-of-function mutation, low-dose immediate release guanfacine was sufficient in relieving multiple symptoms of dysautonomia. [0088] To further substantiate the broad applicability and beneficial properties of low-dose guanfacine in the context of channelopathies, we also want to mention the case of one member of a 30-year-old identical female twin who presented to our clinic with a chief complaint of non-painful facial erythromelalgia and generalized anxiety along with dysautonomia symptoms of chronic fatigue and irritable bowel syndrome. The patient’s erythromelalgia had become particularly pronounced shortly after a severe bout of mononucleosis. The erythromelalgia would occur almost daily and was associated with stressful social situations and not provoked by increased ambient temperature or exercise. She had been treated with Citalopram® and Escitalopram® without relief and was contemplating a bilateral thoracic sympathectomy. Her twin sister never contracted mononucleosis and never developed erythromelalgia but suffered from other symptoms of dysautonomia including chronic fatigue, irritable bowel syndrome, generalized anxiety, cognitive impairment, orthostatic intolerance, and cardiac palpitations. Like the other patients above, this patient’s erythromelalgia resolved
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 almost immediately with Tenex® 1 mg/day, and the frequency of erythromelalgia reduced to 1-3 times per year. Seven years later, the patient remains on Tenex® 1 mg/day with a durable response and no apparent medication side effects. This patient was treated empirically in the clinic and has not yet been enrolled to undergo WES for the presence of a Na
V channelopathy. However, erythromelalgia has been extensively associated with gain-of-function mutations in Na
V1.7 (Dib-Hajj et al., Brain, 2005, 128:1847-1854; Estacion et al., J. Neurosci., 2008, 28:11079-11088; Tanaka et al., J. Biol. Chem., 2017, 292:9262-9272; Weaver J., Neurology Alert, 2016, 35). Unlike for neuropsychiatric symptoms, we are unaware of an established association between α
2A-adrenergic receptors in the prefrontal cortex and erythromelalgia. ii. Effect of guanfacine on Na
V channel currents [0089] Na
V channel inhibition may help explain the striking efficacy of guanfacine observed in our patients. Given the established interaction of guanfacine with other ion channels (Arnsten A.F.T., Neurobiol. Learn. Mem., 2020, 176:107327), we explored potential off-target effects of guanfacine on Na
V channel subtypes, many of them linked to neuropsychiatric disorders (Na
V1.1-Na
V1.8) (Bagheri et al., Int. J. Dev. Neurosci., 2021, 81:669-685; Eijkelkamp et al., Brain, 2012, 135:2585-2612; Imbrici et al., Front. Genet., 2013, 4:76; Mi et al., Front. Cell Neurosci., 2019, 13:554; Nickel et al., BMC Psychiatry, 2018, 18:248; Pasierski et al., Pharmacol. Rep., 2023, 75:331-341; Rees et al., Biol. Psychiatry, 2019, 85:554-562; Weiss et al., Mol. Psychiatry, 2003, 8:186-194). We expressed each human subtype by means of the established Xenopus laevis oocyte method (Zeng et al., Expert Opin. Drug Discov., 2020, 15:39-52) in combination with the two-electrode voltage-clamp technique, and assessed typical biophysical gating parameters before and after addition of 86 μM guanfacine. This concentration seems rather high since Xenopus laevis oocytes tend to be less sensitive to compounds (Zeng et al., Expert Opin. Drug Discov., 2020, 15:39-52), up to one hundred times (Lacerda et al., Eur. Heart J. Suppl., 2001, 3:K23-K30), compared to mammalian cells. Yet, all heterologous expression systems are limited in their ability to mimic human physiological conditions. As such, we are likely working with a saturating dose and the effective in vivo concentration should be much lower. [0090] All Na
V channel subtypes expressed within one to four days after cRNA injection. We first report the effects of guanfacine on Na
V1.1, Na
V1.2, Na
V1.3, and Na
V1.6, the commonly accepted predominant subtypes present within the brain (Ahern et al., J. Gen. Physiol., 2016, 147:1-24; Mantegazza et al., Physiol. Rev., 2021, 101:1633-1689; Pasierski et al., Pharmacol. Rep., 2023, 75:331-341), and will discuss Na
V1.7 results separately below. To this end, we held the membrane at -90 mV and applied 50 ms depolarizing pulses to stepwise
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 depolarize the membrane and activate channels before and after addition of guanfacine. Under these conditions, Na
V1.1- and Na
V1.2-mediated Na
+ currents were inhibited over the tested voltage range by 23% and 27%, respectively (FIG.3, Table 1). We did not observe significant changes in standard gating parameters associated with the conductance-voltage and channel availability (steady-state inactivation) relationships. These parameters were extracted using a Boltzmann fit of the data that yielded half-maximal values (V
1/2) of activation and availability as well as the slope factor (Table 1). The same observation was made when testing Na
V1.3 although current inhibition was reduced to 12%. Of these channel subtypes, Na
V1.6 was the least inhibited at 6%. [0091] Next, we tested whether Na
V channel subtypes associated with the skeletal (Na
V1.4) and cardiac (Na
V1.5) muscle were affected by guanfacine (Cannon S.C., Handb. Exp. Pharmacol., 2018, 246:309-330; Remme C.A., Philos. Trans R. Soc. Lond. B. Biol. Sci., 2023, 378:20220164; Wilde et al., JACC Clin. Electrophysiol., 2018, 4:569-579). Like other Na
V channel subtypes, most gating parameters were unaltered (FIG.4, Table 1). Na
V1.4 currents were inhibited but only by 12%. Na
V1.5 activity was hampered by a drug-induced current reduction of 31%. Although Na
V1.8 has also been reported to contribute to cardiac muscle function (Bezzina et al., Nat. Genet., 2013, 45:1044-1049; Hu et al., J. Am. Coll. Cardiol., 2014, 64:66-79; Macri et al., Circ. Genom. Precis. Med., 2018, 11:e001663), this subtype has primarily been investigated in relation to pain perception (Akopian et al., Nature, 1996, 379:257-262; Bennett et al., Physiol. Rev., 2019, 99:1079-1151; Han et al., Neurology, 2016, 86:473-483). Analogous to the brain subtypes, Na
V1.8 currents were inhibited by 20% and no other significant gating alterations were observed (FIG.4). Combined, these data suggest that guanfacine is a non-selective Na
V channel inhibitor. Table 1. Gating parameters of all tested Na
V channel subtypes in control conditions and after application of 86 μM guanfacine. Parameters shown include the V
1/2 and slope factor of the conductance (G) – voltage (V) relationship and steady-state inactivation (SSI; channel availability). Average current inhibition (%) with SEM at peak voltage (G-V) is also indicated.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 iii. Guanfacine inhibits Na
V1.7 currents [0092] Na
V1.7 is expressed in sensory and sympathetic neurons that innervate all organs in the human body (Goodwin et al., Nat. Rev. Neurosci., 2021, 22:263-274; Minett et al., Nat. Commun., 2012, 3:791; Payandeh et al., Handb. Exp. Pharmacol., 2018, 246:271-306; Vetter et al., Pharmacol. Therapeut., 2017, 172:73-100). This channel subtype has also been found in brain subcortical structures, including the thalamus, amygdala, hypothalamus, axons of the olfactory epithelium (Branco et al., Cell, 2016, 165:1749-1761; Kanellopoulos et al., EMBO J., 2018, 37:427-445) and the locus coeruleus (Jones et al., Nat. Rev. Neurosci., 2009, 10:821- 828). Using WES, we identified the Na
V1.7 p.I739V variant, a gain-of-function mutation that was previously found in patients with neuropathic pain and severe autonomic dysfunction including hyperhidrosis and palpitations (Han et al., Neurology, 2012, 78:1635-1643; Han et al., Brain, 2012, 135:2613-2628). However, the proband in the first family of this study (FIG. 1) remarked that he had an unusually high tolerance to pain recalling that he had broken both arms without feeling compelled to seek pain relief at a hospital. Although this symptomatic dichotomy is rather unusual, both gain- and loss-of-function mutations in Na
V1.7 have been associated with phenotypes ranging from a complete lack of pain to erythromelalgia, often with dysautonomia symptoms (Baker et al., Pflugers Arch., 2020, 472:865-880; Dib-Hajj et al., Nat. Rev. Neurosci., 2013, 14:49-62; Eagles et al., Br. J. Pharmacol., 2022, 179:3592-3611; Goodwin et al., Nat. Rev. Neurosci., 2021, 22:263-274; Mulcahy et al., J. Med. Chem., 2019, 62:8695-8710; Yang et al., Trends Pharmacol. Sci., 2018, 39:258-275). Since the p.I739V variant is commonly accepted to be a gain-of-function mutation (Han et al., Neurology, 2012, 78:1635-1643; Han et al., Brain, 2012, 135:2613-2628), we investigated whether guanfacine could inhibit Na
V1.7 currents and possibly contribute to symptom amelioration in our patients when given as a short-acting formulation at a low dose of 1 mg/day. [0093] When applying 86 μM guanfacine to Na
V1.7 expressed in oocytes, we observed an inhibitory effect of 22% on ionic currents whereas the conductance-voltage and channel
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 availability relationship remained unaltered (FIG.5, Table 1). Similarly, guanfacine reduces Na
V1.7 p.I739V currents by 25% ± 4 (FIG. 5). Next, we expanded our electrophysiological protocols to include the recovery from inactivation. We employed a double-pulse protocol in which cells were kept at -90 mV between two 50-ms depolarization steps to 0 mV that were applied with a varying interval between 0 ms and 1000 ms. Recovery from inactivation is virtually complete after 200 ms (FIG.5) and no significant differences were observed between control and drug-exposed conditions. To test whether guanfacine prefers to interact with the inactivated state of the channel, we applied a test protocol consisting of a 5-s pulse to V
1/2 of channel availability (-40 mV, Table 1) followed by a 200-ms step to -90 mV and a 50-ms test pulse to 0 mV. Such state-dependence can increase in vivo affinity and selectivity over other ion channel families (Ahern et al., J. Gen. Physiol., 2016, 147:1-24; Johnson et al., eLife, 2022, 11). We observed that under these conditions (FIG.5), the inhibitory effect of guanfacine on ionic currents doubles from 21% ± 6 to 50% ± 6 indicating a degree of state-dependence. Altogether, these data illustrate the ability of guanfacine to reduce Na
V channel-mediated currents and suggest a potential application in normalizing atypical gain-of-function Na
V channel gating as observed in particular pathological conditions. 3. Discussion [0094] We observed that off-label use of guanfacine was effective in reducing generalized anxiety and concomitant dysautonomia symptoms such as cognitive impairment, hyperhidrosis, and palpitations in multiple members of nonrelated families. Strikingly, the short-acting guanfacine formulation at only 1 mg/day facilitated virtually complete and long- term symptom relief without apparent side effects. We hypothesized that a genetic deficit existed in these patients that might underlie this notable sensitivity to guanfacine. WES on a limited subset of patients that benefited from a low dose of guanfacine, identified the Na
V1.7 p.I739V gain-of-function variant (Han et al., Neurology, 2012, 78:1635-1643; Han et al., Brain, 2012, 135:2613-2628) as a possible hereditary pathology contributor (FIG.1 and FIG.2). The causal link between this p.I739V substitution and ensuing patient phenotypes has been challenging to ascertain (Devigili et al., Pain, 2014, 155:1702-1707; Han et al., Brain, 2012, 135:2613-2628; Xenakis et al., BMC Bioinformatics, 2021, 22:212). Yet, this mutation is commonly observed as it represents 1% of Na
V channel variants detected in patients with painful or non-painful peripheral neuropathy (Wadhawan et al., Neurol. Genet., 2017, 3:e207). Moreover, intrafamilial phenotypic variability has been reported with Na
V1.7 p.I739V (Peddareddygari et al., Case Rep. Neurol., 2021, 13:135-139) and the involvement of modifiers
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 or interacting proteins has subsequently been proposed (Ahern et al., J. Gen. Physiol., 2016, 147:1-24; Kanellopoulos et al., EMBO J., 2018, 37:427-445). Clinically, after being prescribed 1 mg/day of short-acting guanfacine, virtually complete symptom alleviation was reported by our patients with the Na
V1.7 p.I739V mutation, as well as in patients with similar symptoms that were not sequenced. Moreover, this treatment had long-term benefits, up to six years, with few adverse effects. Another patient with erythromelalgia who did not yet undergo WES but with a presumed gain-of-function mutation in Na
V1.7, also has had a durable multiple year dramatically favorable response to low doses of guanfacine. Combined, the results of this part of our study provide further evidence for a pathological role of Na
V1.7 mutations in generalized anxiety and dysautonomia and illustrate the potential benefits of guanfacine treatment. [0095] Although guanfacine is a selective α
2A-adrenergic receptor agonist, there is evidence that it may also have a mechanism of action outside of direct interaction with α
2A- adrenergic receptors through ion channels. Guanfacine has been shown to inhibit cAMP-PKA opening of K
+ channels in prefrontal spines, strengthening their connections, enhancing neuronal firing, as well as increasing top-down control (Arnsten A.F.T., Neurobiol. Learn. Mem., 2020, 176:107327). Our hypothesis that guanfacine may interact with other targets such as Na
V channels seemed consistent with our preclinical and clinical experience. Since the ability of prefrontal cortex neurons to retain working memory through excitatory networks relies on ion channels that help set the membrane potential, a gain-of-function Na
V channelopathy may dysregulate action potential firing producing cognitive deficits inherent to both ADHD and POTS (Fahrenholz et al., Primer on the Autonomic Nervous System, Elsevier, 2023, pp. 627-629; Iwanami et al., BMC Psychiatry, 2020, 20:485; Taylor et al., J. Clin. Psychopharmacol., 2001, 21:223-228; Wu et al., J. Child Adolesc. Psychopharmacol., 2022, 32:244-248). A compound that normalizes membrane potentials, through inhibition of Na
V1.7 or other ion channel subtypes, would be clinically valuable. As such, we examined pre- clinically whether guanfacine could inhibit Na
V channels. We found that multiple subtypes, including Na
V1.7, were indeed partly inhibited (FIG. 3, FIG. 4, and FIG. 5). However, the guanfacine concentration used in our experiments is rather high and was primarily dictated by the known limitations of the experimental test system used (Zeng et al., Expert Opin. Drug Discov., 2020, 15:39-52). As such, in vivo effective concentrations of Na
V channel block by guanfacine may be much lower (Lacerda et al., Eur. Heart J. Suppl., 2001, 3:K23-K30). Indeed, in cortical neurons, a tenfold lower guanfacine concentration was reported to inhibit Na
+ currents (Pasierski et al., Pharmacol. Rep., 2023, 75:331-341). A more conventional explanation of guanfacine efficacy in our patients involves its primary effect on α
2A-adrenergic
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 receptors which would reduce the increased sympathetic tone induced by Na
V channel gain-of- function mutations, perhaps complemented by a smaller, synergistic inhibition of Na
V channels. Indeed, although a functional association between α
2A-adrenergic receptors and Na
V channels has yet to be established, hints supporting a link can be found in the literature. For example, α
2A-adrenergic receptors agonists such as clonidine and dexmedetomidine have clinically-established analgesic properties, in part attributed to acute inhibition of Na
V channels at similar or higher concentrations than tested here for guanfacine (Im et al., Mediators Inflamm., 2018, 2018:1782719; Stoetzer et al., Region Anesth. Pain M., 2017, 42:462-468). Dexmedetomidine causes a concentration-dependent inhibition of Na
+ currents, an effect that can be prevented by yohimbine, a competitive α
2A-adrenergic receptor antagonist (Im et al., Mediators Inflamm., 2018, 2018:1782719). Moreover, dexmedetomidine-induced inhibition is blocked by intracellular perfusion of the G protein-coupled receptor (GPCR) inhibitor GDPβ-S. A functional coupling of the α
2A-adrenergic receptor to GPCRs opens up possible pathways to influence Na
V channel function (Brackx et al., Pharmacol. Therapeut., 2023, 245:108416; Salzer et al., Int. J. Mol. Sci., 2019, 20; Xu et al., Sci. Adv., 2022, 8:eabj5347). Finally, it is worth noting that agonist-mediated activation of α
2A-adrenergic receptors was reported to be dependent on membrane voltage to the extent that negative membrane potentials promote agonist-mediated activation and downstream receptor signaling (Rinne et al., PNAS, 2013, 110:1536-1541). Therefore, membrane depolarization triggered by the Na
V1.7 p.I739V mutation (Han et al., Neurology, 2012, 78:1635-1643; Han et al., Brain, 2012, 135:2613-2628) would reduce receptor susceptibility to agonist. By inhibiting Na
V1.7 p.I739V with guanfacine and restoring more negative membrane potentials, α
2A-adrenergic receptors become more sensitive to agonists. As such, guanfacine may reduce neuronal hyperexcitability via direct Na
V1.7 inhibition and thereby increase its own affinity for the α
2A-adrenergic receptor to correct sympathetic outflow. This dual synergistic effect is but one possible explanation for the low dosage needed in patients (1 mg/day) to achieve symptom improvement. [0096] While this limited study provides insights into the off-label efficacy of guanfacine, few limitations are worth mentioning. First, in our dysautonomia clinic we stop prescribing guanfacine occasionally due to the onset of adverse patient complaints such as xerostomia, somnolence, and lightheadedness, symptoms that have also been associated with altered Na
V1.7 function (Dormer et al., J. Pain Res., 2023, 16:1487-1498). However, we found that night-time administration coupled with a slow increase in the titration of the dose of guanfacine, preferably no faster than 1 mg per 2-3 weeks, assists in tolerability. Second, our observations are based on only two nonrelated families with the Na
V1.7 p.I739V variant which
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 do not fully represent the diverse population of patients with neuropsychiatric symptoms and Na
V channelopathies. Nonetheless, our data suggest that the non-Na
V channel subtype- selectivity of guanfacine could be a beneficial trait in patients with mutations in other Na
V channel subtypes. Third, our study is observational in nature lacking guanfacine data in a control group with similar symptoms but without a Na
V channelopathy. Since our observational data were not part of a randomized trial to assess the efficacy and safety of guanfacine in genetically stratified patients, future studies in a more controlled setting will be needed. Fourth, this study primarily focused on the Na
V1.7 p.I739V variant and its response to a low dose of guanfacine. As exemplified by our third patient whose erythromelalgia was provoked by a mononucleosis infection, this is a complex condition and other genetic as well as environmental factors that may contribute to the observed symptoms and treatment response were not explored. [0097] Overall, the results of this study substantiate a pathological role of Na
V1.7 mutations in disorders related to an increased sympathetic tone and illustrate the potential benefits of low-dose short-acting guanfacine treatment, perhaps aided by an inhibitory effect on Na
V channels. Despite the fact that the dysautonomia symptoms in our studied families most likely involve a complex interplay between genetics and environmental cues, it is encouraging that inhibition of presumed gain-of-function genetic aberrations can result in substantial symptom improvement. In such a complex disease, a single drug given at a low dose being so effective, suggest a likelihood of guanfacine acting synergistically on multiple targets, including direct ion channel inhibition and a drug-induced augmented affinity to activate the α
2A-adrenergic receptor. Our observations suggesting that inhibition of Na
V channels by guanfacine may serve as an off-label target is clinically important since it promotes the possibility that guanfacine could one day be utilized in genetically stratified patients with neuropsychiatric symptoms resulting from anomalous Na
V channel behavior. Example 2. A Multi-Hit Model of Long COVID Pathophysiology – The Interaction between Immune Triggers and Nervous System Signaling [0098] As of October 2023, over 698 million individuals have been infected globally with SARS-CoV-2 resulting in approximately 7 million deaths. The medical community, as early as fall 2020, issued an international online survey in 56 countries suspecting the presence in many patients of persistent heterogeneous sequelae involving multiple organs. But it was even earlier on May 20, 2020, when Elisa Perego, a health researcher from Lombardy, Italy, dubbed her experience of symptoms after SARS-CoV-2 as “#Long COVID” in a hashtag on Twitter.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 She penned “Long COVID” as a contraction of “long-term COVID illness” to describe the cyclical, progressive, and multiphasic nature of her symptoms that had persisted for over 40 days. This patient-originated term quickly spread through social media gaining popularity as it legitimized other patients’ experiences and challenged medical assumptions that SARS- CoV2 was a transient, short-term disease. Long COVID, also now known as Post-acute sequelae of SARS-CoV-2 infection (PASC), and “Long-haul COVID,” is currently defined as ongoing, relapsing, or new symptoms or conditions present 30 days or more after COVID-19 infection. 1. The Prevalence and Burden of Long COVID [0099] Long COVID has become a major public health issue affecting at least an estimated 65 million people globally, with cases increasing daily. This figure is most likely an underestimation due to the continued high rates of Long COVID among new SARS-COV-2 cases around the world. In the U.S., as of January 2023, the percentage of people who reported Long COVID symptoms after SARS-COV-2 declined from 19% in June 2022 to 11% in January 2023. But despite this decline, the prevalence of Long COVID in those who have had SARS-COV-2 is still high. Moreover, its prevalence seems to depend on the severity of the underlying SARS-COV-2 infection with 7.5-41% of non-hospitalized patients affected, and over 50% of hospitalized patients debilitated by symptoms such as severe, persistent anxiety and depression. In the U.S., in 2022 the CDC estimated that the prevalence of Long COVID was about 6.9% of all adults with women (8.5%) more likely than men (5.2%) ever to have had Long COVID. In fact, most studies of Long COVID have shown that the prevalence among females is statistically significantly higher than among males. Interestingly, U.S. Census Bureau data suggest higher rates of Long COVID symptoms among Hispanic and black adults compared to white adults. A recent survey found 31.1% of Hispanic, 28.7% of black, and 27.6% of white adults reported Long COVID symptoms. [0100] Population-based surveillance tools have been effective in monitoring the impact of the disease on daily living in the U.S. with 25.3% of over 18,000 individuals with Long COVID responding that day-to-day activities were impacted “a lot”. Importantly, 28.9% of individuals responded that their SARS-COV-2 infection had occurred more than 12 months previously. This has negatively affected employment with two surveys showing that approximately 66% of individuals with Long COVID who previously were actively employed were now unemployed or were forced to reduce their working hours. Mental health and cognitive burdens have been a substantial contributor to this inability to function at work with over 60% of individuals in the U.S. having a neurological or psychiatric diagnosis 6 months after SARS-
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 CoV-2. One early estimate calculated the reduction of labor supply due to Long COVID could be more than $50 billion annually. A recent review of twelve publications suggested that Long COVID patients are increasingly utilizing healthcare systems, both primary and specialist care, much more than before they were infected. Using claims data, a recent estimate suggests that these more frequent visits for care mean that Long COVID adds an additional $224 in healthcare costs per month over a 6-month period in any individual patient. Thus, Long- COVID has emerged as a major public health problem that will have considerable long-term consequences for population health, economic policy, and research initiatives. [0101] Due to its relative novelty, major knowledge gaps persist that impede our ability to treat Long COVID effectively. Therefore, in this example, we will focus on research efforts into our understanding of the pathogenesis of Long COVID and of its underlying biological mechanisms. 2. Long COVID and its Complex Symptomatology i. The association between Long COVID and dysautonomia [0102] Early in the pandemic, there was an acute realization by several neurologists that many of the symptoms of Long COVID were, in fact, symptoms of dysautonomia, a dysfunction of the autonomic nervous system (ANS) or components thereof such as the sympathetic nervous system. At the NIH’s National Institute of Neurological Disorders and Stroke Autonomic Medicine Section, Dr. David Goldstein quickly realized within two months of the pandemic’s onset in April 2020 that the symptoms of SARS-CoV-2 that he saw manifested in patients were multi-system, multi-disciplinary complaints. Dr. Goldstein surmised that they originated from abnormal activation of the ANS leading to autonomic dysfunction in various organs and thereby causing myriad functional abnormalities, a phenomenon that he described as the “extended ANS” (Goldstein, D.S., Clin. Auton. Res., 2020, 30(4):299-315). One year later, he strengthened his theory with a patient case history in an article entitled “The possible association between COVID-19 and postural tachycardia syndrome (POTS)” (Goldstein, D.S., Heart Rhythm., 2021, 18(4):508-509). Aided by new innovations in studies, such as the COVID Symptom Study, where by May 2020, 2.8 million users had documented their symptoms on a smartphone tracker app, more and more clinicians quickly began to recognize that clinical dysautonomia was a prominent feature of Long COVID. [0103] At Johns Hopkins Center for Sweat Disorders, we also were observing a sharp spike in the number of patients complaining about an increase in primary focal hyperhidrosis (PFH) after SARS-CoV-2 infection. Often on close questioning, these patients would admit that they
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 had indeed suffered from various degrees of underlying hyperhidrosis since childhood, but that after contracting SARS-CoV-2, their sweating had become intolerable and always interfered with daily activities (highest score on the Hyperhidrosis Disease Severity Scale (HDSS)). It also became equally evident that sweating was not the only debilitating symptom that was occurring post infection with SARS-CoV-2. In addition to PFH, an important dermatologic manifestation of ANS dysfunction, our typical patient would complain of a constellation of debilitating symptoms including generalized anxiety disorder, cognitive impairment or “COVID-brain fog,” chronic fatigue, shortness of breath, heart palpitations, hyperhidrosis, irritable bowel, migraine-type headaches, insomnia, as well as lightheadedness. Over the last thirteen years, we had observed this phenomenon quite regularly in our Center for Sweat Disorders with patients who presented with hyperhidrosis having similar dysautonomia symptoms but not after infection with SARS-CoV-2, but rather after contracting infectious diseases such as Mononucleosis, Lyme disease, and Malaria. As with Long COVID, however, patients reported that the most pervasive symptoms were often the neurological co-morbidities of anxiety, depression, and cognitive impairment. It is important to note here that our PFH patients over the years have also frequently presented with Postural Orthostatic Intolerance Syndrome (POTS), which often occurs in adolescence when it can be triggered by an antecedent infection. Intriguingly, during the pandemic, multiple scientific reports started to appear suggesting that the majority of patients with Long COVID met the diagnostic criteria for POTS, a link that has yet to be fully explored. ii. Unanswered questions [0104] Despite our long experience with both POTS and post-infectious dysautonomia, what remained puzzling was that patients tended to have only marginal improvement of their symptoms, well short of a full cure, when given even potent immunomodulatory agents such as intravenous immunoglobulin to treat the autoimmunity or neuroinflammation co-morbid with their disease. This prompted two yet unanswered questions. First, what is the association between Long COVID and dysautonomia symptoms? Second, why does the inflammatory response that typically occurs after an infection with Epstein-Barr virus, Borrelia, and now SARS-CoV-2, not provoke dysautonomia symptoms, such as anxiety, depression, and cognitive impairment, in everyone who contracts these pathogens? [0105] A possible foundation for an answer to the first question (i.e., What is the association between Long COVID and dysautonomia?) comes from our years of clinical experience at the Johns Hopkins Center for Sweat Disorders. Three of the most important lessons we have learned from caring for hyperhidrosis patients are: (i) hyperhidrosis is not just
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 a mere cosmetic issue, (ii) most patients do not complain about being stigmatized or embarrassed because they are “sweat bothered”; instead, these patients consistently describe an impaired quality of life in which the adverse impact of extreme sweating is thought to be greater than that of other chronic dermatologic disorders such as psoriasis, and (iii) hyperhidrosis almost never occurs alone as an isolated symptom, but as mentioned previously, is habitually part of a constellation of systemic dysautonomia complaints. In fact, we found that 38% of all PFH patients who presented to our clinic were on prescribed psychotropic medications for anxiety versus 14% of controls. Since no morphological differences exist between the sweat glands of hyperhidrosis patients and healthy controls, these clinical observations of multiple accompanying dysautonomia symptoms fit a hypothesis held for over fifty years that PFH is indeed a disease of primary sympathetic dysfunction. In other words, there is a sympathetic overdrive systemically affecting every organ throughout the body. The skin however, as the largest organ in the body, can manifest hyperhidrosis as an early diagnostic sign of abnormal ANS function that may herald diseases such as Post Traumatic Stress Disorder (PTSD). Moreover, our clinical observation was that hyperhidrosis occurred at a young age, probably at birth, with frequent patient histories from mothers describing sweaty footprints of a patient as a toddler. Given this early age of onset and frequently encountered family histories of PFH, physicians have long suspected an autosomal dominant genetic transmission, but with incomplete penetrance. Indeed, our clinical observations suggest that PFH may not only be inherited, but also that many of the above-mentioned dysautonomia co- morbidity complaints of hyperhidrosis patients may run in families. [0106] Propelled by our clinical observations, we embarked on a search for genetic causes of hyperhidrosis that affect the ANS. We started to construct family history pedigrees to document that not only was hyperhidrosis prevalent in families, but also that many of the family members shared dysautonomia symptoms, such as generalized anxiety, an affliction that is applicable to virtually all our patients. FIG. 6 illustrates two typical families where the probands presented to our clinic with severe hyperhidrosis, family histories of hyperhidrosis, and with multiple family members showing a wide array of dysautonomia symptoms including generalized anxiety, orthostatic intolerance, chronic fatigue, and chronic itch symptoms. Many of their symptoms are validated using established clinical questionnaires such as the HDSS, COMPASS-31, Zung Self-Rating Anxiety Scale, and the Social Phobia InveNtory (SPIN), thereby removing as much as possible a potential bias by the observer. The high scores across all four of these surveys in multiple family members suggests impaired autonomic regulation in multiple organ systems. Intrigued by these findings, we started performing whole-exome
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 sequencing on families with hyperhidrosis who visited our clinic and whose relatives exhibited non-syndromic autonomic dysfunction. Preliminary data resulting from these efforts suggest the presence of heritable missense mutations in genes that are nearly all associated with generating or transmitting electrical signals throughout the body. These genes encode proteins that: (i) are directly involved in membrane excitability such as (voltage-gated) ion channels, (ii) modulate other proteins that can generate or propagate electrical signals such as ancillary subunits or transcription factors, and (iii) may trigger electrical or morphological remodeling of excitatory tissues. Notably, mutations in these genes may lead to altered ANS excitability as is often observed in these families and can be labeled clinically as a “high sympathetic tone.” The existence of a gain-of-function mutation in a protein expressed in the sympathetic ganglia, for example, would provide a plausible genetic explanation for the pathogenesis of hyperhidrosis and its accompanying dysautonomia. [0107] A possible answer to the second question (i.e., Why does a post-infectious inflammatory response not always trigger dysautonomia?) of why Long COVID only presents in a small subset of patients who contract SARS-CoV-2, led to the formulation of a two-hit hypothesis for how Long COVID and other postinfectious dysautonomia disorders may develop. We currently hypothesize that Long COVID requires two molecular hits. First, a genetic predisposition in genes that regulate electrical signaling, such as an ion channel gain- of-function variant expressed in the brain and/or sympathetic ganglia that can act as a genetic primer to alter neuronal membrane excitability. Second, a SARS-CoV-2 infection that serves as an immunologic trigger transforming the nervous system into an oversensitive state characterized by lower thresholds to activation and a lessened ability to extinguish the debilitating dysautonomia symptoms associated with Long COVID. Indeed, influential interoception theories, such as the James-Lange hypothesis, state that exaggerated sympathetic responsivity can augment one’s vigilance to physiological feedback, leading to dysautonomia symptoms. Thus, potentially mild symptoms associated with the underlying genetic predisposition, can be exacerbated by immunologic triggers, which offers a plausible answer to our second question of why only a subset of people who contract SARS-CoV-2 develop Long COVID. [0108] Growing evidence suggests that chronic inflammation, as a hallmark of autoimmunity, plays a significant role in Long COVID, and this fits well with our two-hit hypothesis theory. As established in POTS, autoantibodies have been detected against cytokines, complement proteins, cell surface proteins, as well as self-antigens in patients with Long-COVID with the implication being that immune dysregulation can be the secondary
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 trigger of sympathetic overactivation. Long-COVID patients also have persistent elevation, compared to the onset of SARS-CoV-2, of proinflammatory cytokines like TNF-α, IFN-γ, IL- 1β, IL-6 and IL-13 with significantly decreased levels of IP-10. Upregulation of some of these cytokines have been observed in patients up to at least eight months after SARS-CoV-2 infection. Another study showed 83% of Long COVID patients with signs of latent autoimmunity and 62% with polyautoimmunity. iii. Treatment [0109] In our clinic, we have used our concept of a two-hit hypothesis to guide treatment of Long COVID patients. Our rationale is that there must be both an inhibition of the genetic propensity for electrical hyperexcitability as well as an immunomodulation of the autoimmunity to minimize a further sensitization of the central and/or sympathetic nervous system. One small case report of twelve patients with a remarkable amelioration of neurocognitive impairments due to Long COVID used a two-drug combination of the α
2A- adrenoceptor agonist, guanfacine, and the anti-oxidant, N-acetylcysteine (NAC) (Fesharaki- Zadeh et al., Neuroimmunology Reports, 2023; 3:100154). Importantly, we recently showed that guanfacine may also partially inhibit multiple voltage-gated Na
+ (Na
V) channels (see Example 1), and NAC is an established immunomodulator reducing inflammation (Fesharaki- Zadeh et al., supra). In our clinic, we had been using guanfacine along with other inhibitors of voltage-gated ion channels successfully for years in patients with PFH, POTS, and dysautonomia. Since most Na
V channel inhibitors lack subtype specificity and can sometimes act on other ion channel families or even receptors, clinically we favor repurposing multiple drugs to achieve ion channel inhibition. We also knew through interviews that many of our patients experienced significant symptom relief with marijuana smoking. Since synthetic cannabinoids are known to inhibit many ion channel subtypes, we combined synthetic cannabinoid therapy off-label in the form of dronabinol with repurposed drugs that inhibited ion channels. [0110] Success in translating our two-hit hypothesis to the clinic is illustrated by a patient who presented to our Center for Sweat Disorders in the fall of 2018 with complaints of life- long hyperhidrosis. Strikingly, this patient also suffered from multi-system constitutional symptoms of dysautonomia including pre-syncope, light-headedness, chronic cardiac palpitations, migraine type chronic headaches, chronic fatigue, mild brain fog, irritable bowel symptoms and problems with poor sleep. Since this patient seemed typical of an individual with a genetic predisposition to an increased sympathetic tone, in 2018, we treated his
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 dysautonomia symptoms with Na
V channel modulators – oxybutynin, guanfacine, and dronabinol. After being maintained successfully for four years on these medications, his dysautonomia symptoms, especially his brain fog and chronic fatigue, acutely worsened after a SARS-CoV-2 infection. He presented to clinic seven months later with Long COVID, and by simply increasing his guanfacine and adding NAC, we were able to control his Long COVID symptoms effectively. His testimony (see Example 3) three weeks after this treatment further reinforces the potential clinical efficacy of this two-step approach of inhibiting ion channels along with the immune response to SARS-CoV-2 in these patients. 3. Conclusion [0111] We propose that Post-Acute Sequelae of SARS-CoV-2 or Long COVID may require two molecular hits: (i) a predisposing genetic vulnerability such as ion channelopathies or mutations in genes that support electrical signaling, and (ii) an immune reactivity to SARS- CoV-2 as a secondary trigger. We also hypothesized here that pharmacological targeting of both the genetic and environmental hit may, in combination, show efficacy in patients. With some initial success, we have translated this concept to the clinic and are treating patients with drugs such as guanfacine to target the genetic aberration, and the neuro-inflammation with NAC. The full clinical implementation will need individual genetic screening of each patient as well as the manufacturing of more specific drugs that can inhibit the underlying genetic defect. The full application of this multi-hit hypothesis may not only have applicability to Long COVID, but also to other post-infectious dysautonomic diseases. Example 3. Patient Testimonials [0112] This Example provides some representative patient testimonials from patients with Long COVID, POTS, or dysautonomia/hyperhidrosis. 1. Patient Testimonials for Long COVID i. First patient (54-year-old professor of engineering and National Academy of Medicine Member) [0113] “My life changed with the onset of dysautonomia that was triggered by a COVID- 19 infection in early 2020. I have suffered from “long covid” ever since, including intense multiple headaches/migraines each day, brain fog, lethargy, and overall lack of energy and focus. I am a 54-year-old professor of engineering and medicine at Johns Hopkins whose career was on the rise (including induction into the National Academy of Medicine and the National Academy of Inventors). However, my long covid was so debilitating that I thought I might have to go on disability and/or give up on my passion to help improve human health all
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 together and retire. Needless to say, my poor health and lack of energy the past few years have been heartbreaking for me and my family. I am a medical/drug researcher who is skeptical of unproven medical therapies, including the course of medications originally prescribed by Dr. Brock. While I did not notice a difference in how I felt initially, my headaches and brain fog greatly decreased in severity and frequency, and my energy and ability to focus improved significantly, once I ramped up to a higher dose of guanfacine (2-3 pills per day). In addition to guanfacine, I take 600 mg of N-acetyl-cysteine (NAC), 300 mg of nicotinamide riboside chloride, and specific salt supplements recommended by Dr. Brock. Notably, I ran out of guanfacine while traveling recently. I thought perhaps I did not need it anymore since I was feeling considerably better, but I was wrong. The headaches, lethargy and brain fog came roaring back within a day or two. Going back to my medications and supplements has me feeling much better again. My energy and ability to focus has greatly improved, from about 5- 10% to about 50-75% of my pre-covid levels of energy and focus, thanks to Dr. Brock. Most importantly, I have the energy to be a loving husband, father and friend again, and I have been able to return fulltime to the career I love. I share this testimonial with the hope that it will help others, and with the hope that Dr. Brock and his colleagues will continue advancing knowledge and effective treatments for those with dysautonomia and long covid.” ii. Second patient (39-year-old male, described in Example 2) [0114] “As I’ve made you aware, I’ve dealt with the struggle of Long Covid this entire year going back to when I got sick in November 2022. Initial symptoms lingered into the New Year, but then I started having severe setbacks in the following months that significantly impacted my quality of life. At times, I would feel so physically & mentally lethargic for days at a time that leaving the house was a major challenge. This lasted through the winter and Spring Months, and into June. However, I began to notice some improvement after following your directive of taking the N-Acetyl-L-Cysteine and increased dosage of the Guanfacine, in addition to the dosages of Dronabinol & Oxybutynin that I’ve taken since 2018. Since that regimen began in June, the setbacks quickly began to fade, and I’m happy to report that I’ve felt better these past 30 days than I have since before my birthday in November 2022, when I last got Covid. In fact, aside from some brief fatigue episodes which can also be attributed to something else, I haven’t felt sick at all in almost two months. It's been a tremendous relief that has come at a time that admittedly had left me somewhat desperate in terms of treatment due to how severe the setbacks were. I wanted to share my success story in case other patients you care for share similar characteristics of mine that can be treated.” 2. Patient Testimonials for POTS
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0115] “In 1996, at the age of 21, my life was dramatically changed by the sudden onset of Dysautonomia. At that time, I was graduating Summa Cum Laude from the Wharton School of Business with the Dean’s Award for Community Service. Prior to that, I had been the youngest person to testify in the US Senate and later interviewed on Good Morning America for volunteerism. Almost overnight, I became too ill to stand for graduation pictures. Two years later, I was diagnosed with POTS & NMH following a Tilt-Table Test at Johns Hopkins Hospital during which my blood pressure dropped dramatically and I passed out at 22 minutes. Since then, I have been in the ICU multiple times after fainting, bedbound for months requiring continuous saline infusion to stay conscious, suffering with a myriad of other multi-system symptoms and extremely limited in energy to perform the basic functions of daily living much less to enjoy a full life beyond the confines of my home. This year, I met Dr. Brock who proposed a radical new approach to my condition in the form of certain drugs he was testing to address the underlying causes of the disorder. He first prescribed oxybutinin, which within 24 hours, restored some of my autonomic functions to normal. Without ongoing adrenaline surges and drenching night sweats, I could think clearly and my family recognized the person they’d lost 25 years ago. To our disappointment, I had to discontinue this and three alternative medications due to dangerous side effects. In June of this year, Dr. Brock started me on Rufinamide which I increased to 800mg twice daily. I have been gradually increasing my activity without the usual debilitating exhaustion, pain, and confusion that follows. Last week, for example, I was even able on several successive days to take a brief swim in a nearby lake, walk back up the hill to our home, and then even have the energy to prepare dinner. For the first time in 25 years, I have hope that a medication can regulate dysautonomia, end suffering and restore not just my life but the lives of so many others who suffer from this poorly understood condition.” 3. 3. Patient Testimonials for Dysautonomia and Hyperhidrosis i. First patient (Female, Age 40, Director of Marketing for Construction & Development Company) [0116] “Finding rufinamide as a possible treatment for hyperhidrosis is an extremely exciting, and life changing experience. Although I was initially diagnosed at John’s Hopkins with Chronic Fatigue Syndrome, hypermobility and Postural Orthostatic Tachycardia Syndrome in my teens, it wasn’t until my late twenties and early thirties that I started to experience increasing allergy symptoms and severe hyperhidrosis from my face, specifically my hairline. Hyperhidrosis initially seemed like a minor inconvenience in comparison to some of my other symptoms, but over time it contributed to significant social anxiety, poor mental
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 health, and stigma from others. Working with John’s Hopkins Hyperhidrosis Clinic has been a lifesaver, and after initially finding excellent results with one medication – side effects made it no longer an option. I am so grateful that Dr. Brock and his team have identified rufinamide as a possible alternative to the treatment, and while I am still increasing my way from a low starting dose, I can see significant improvements not only to my sweating issues but also improvements to my POTS symptoms, anxiety, sleep quality and overall sense of wellbeing on rufinamide.” ii. Second patient (46-year-old female with hyperhidrosis and dysautonomia) [0117] “I wanted to give you a brief update of how I’m doing since I started taking Rufinamide. I started taking Rufinamide in January 2023 and I noticed a change within a week. Before taking the medication I had issues with palpitations sweating and a hot feeling all over my body lasting for 20 minutes. I had seen a cardiologist and wore a heart monitor with no answers. Since I started taking Rufinamide I don’t have any palpitations no sweating and the hot feeling all over my body. I noticed my energy level has increased and I don’t have any side effects. Hopefully this medication will be able to continue to provide me the relief I have been looking for since the age of 16 years old and now I’m 46 years old. I think this medication could help much more people. Yes, my sweating and palpitations got worse after doxyrubicin treatment for my desmoid tumor and with the Rufinamide and my stomach issues are better. Before that I would have trouble having a bowel movement and other stomach issues. But now I’m better.” iii. Third patient (25-year-old with hyperhidrosis) [0118] “Ever since I can remember, I have dealt with hyperhidrosis in my hands and feet. As a result, I avoided participating in certain activities and I made difficult decisions because I didn’t want people to know about my condition. As I grew older, my anxiety increased, I had frequent headaches, fatigue and sometimes felt lightheaded. I didn’t think there would ever be a solution to any of my symptoms until I met Dr. Brock. I have found that taking guanfacine 1MG and oxybutynin 10MG has helped me significantly. My hyperhidrosis has almost gone away completely, I no longer experience frequent headaches or lightheadedness, and my anxiety has improved. I’m so very thankful for these medications, and for Dr. Brock and all the research being done to impact individuals and their conditions.” iv. Fourth patient (32-year-old with hyperhidrosis) [0119] “Hyperhidrosis is a condition that has negatively affected my life every day in multiple aspects. It has dictated my lifestyle, behavior, aspirations, even my education and career choices. I tried everything for 15 years and nothing worked. My current therapy
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 regimen by Dr. Brock (Guanfacine and Oxybutynin) has been life changing. I can for once in my life have a sense of normalcy, be myself around others and live life to my fullest potential. Dr. Brock’s line of research is transforming lives and bringing viable solutions to this ‘invisible’ yet disabling condition.” v. Fifth patient (28-year old female PhD postdoctoral fellow) [0120] “I struggled with hyperhidrosis since middle school, most embarrassing was my heavy underarm sweat that stained every single shirt I owned. My sweating occurred year- round and seemed to have no triggers, it was constant. I tried every over-the-counter antiperspirant on the market. I tried glycopyrronium tosylate (Qbrexza) for my underarms, that stopped some of the sweating, but it made me extremely constipated, and I had to stop. I had resigned myself to coping with my hyperhidrosis by selecting only patterned and dark colored shirts to hide the stains as well as living with the constant feeling of a damp sweaty shirt under my arms. My dermatologist recommended I see Dr. Brock for my hyperhidrosis. Dr. Brock started me on 10mg of Oxybutynin and 1mg of Guanfacine as well as drinking either Liquid IV or LMNT to increase my sodium intake. Since starting this regimen, a year ago, I’ve had many days where I don’t sweat at all! I am still capable of sweating when it’s 100 degrees outside or I exercise but I’m not bothered by this sweat since that amount is directly related to either the temperature or physical exertion. I’m glad my body can still self-regulate its temperature to keep me safe. I can finally move about my day without having to keep my arms pressed into my side out of embarrassment. As a scientist myself, I’m extremely comforted knowing that there is a genetic reason for my hyperhidrosis and that Dr. Brock’s team is working to understand the molecular underpinnings of this condition to provide all of us with selective and effective treatment options. Knowing that hyperhidrosis is an ion channelopathy as allowed me to prioritize giving my body the electrolytes it needs. I was thrilled to hear that he has developed mouse models that recapitulate patient symptoms so he can test his hypotheses out in a reliable model system. I can’t wait to see what else Dr. Brock’s team discovers next!” vi. Sixth patient (55-year-old female) [0121] “I have been a patient of Dr. Malcolm Brock since 2019. I received a diagnosis of hyperhidrosis from my primary care physician, who then referred me to Dr. Brock. When I met with Dr. Brock the first time, he asked me a number of questions about my personal condition and also took the time to share details about his research. He prescribed me three medications to be taken together that he found successful with other patients: Dronabinol 2.5mg taken twice per day, Oxybutynin CL 5mg once per day and Guanfacine 1mg once per
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 day. Note: I found that taking 1 Dronabinol per day worked best for me plus over time, we tweaked the Oxybutynin CL to the ER version once per day. To say that this combination of medication has been life changing is an understatement! Before taking this medication, I would sweat to varying degrees doing normal everyday things like being in a warm room or walking down the street on a warm (not hot) day. As a head-sweater, beads of sweat would immediately form on my forehead and back of my head making my hair damp. In addition to typical underarm sweat, I would also sweat in my groin area and under my breasts. I have also been known to sweat on colder days if I walked outside too quickly. I would not consider myself an anxious person, but these situations made me extremely anxious. I would have to dress in a way that wouldn't make it obvious if I did sweat. My kids told me that they noticed that every time we went somewhere, the first thing I did was comment on the temperature. After taking these medications, I can completely tolerate these situations. I do not have bouts of embarrassing sweating situations anymore. Of course, on extremely hot and humid summer days or when I am exercising, I still sweat, but these are normal times to sweat. I now feel more in control of my body and much less anxious about the temperature. I am extremely grateful to Dr. Brock and the research he has done and continues to do to help people like me with hyperhidrosis. This combination of medicine works, and I hope that one day, it will be a common solution that is available to more people like me who suffer from this condition.” vii. Seventh patient (47-year-old with hyperhidrosis) [0122] “In 2009 while serving overseas, I began having severe sweats from head-to-toe on most nights, which greatly disturbed my sleep and forced me to constantly have to wash my sheets and bedding due to the smell. When I returned to the U.S., I began seeking a medical solution to this problem by seeing a neurologist, a dermatologist with a specialty in hyperhidrosis, an endocrinologist, and multiple psychiatrists (as well as having at-home and in-lab sleep studies done). However, none of them were able to treat this condition, let alone determine what was causing it. This problem was taking a significant toll on my sleep since I would wake up multiple times a night due to sweat beading up on my chest, etc. and then running down my body, which triggered hairs on my body and thus woke me up. In addition, I would wake up multiple times per night freezing due to soaked sheets, which also smelled due to the sheer volume of sweat. After giving up hope of solving this problem via a medical route, I found a temporary solution in the form of a BedJet, which essentially blows hot air under my sheets from the foot of my bed. However, if I wanted to sleep decently when I traveled, I had to bring the BedJet (which takes up a large suitcase) and set it up in the hotel room, which is a hassle, especially if on a road trip staying at multiple hotels over time. And I
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 was still sweating significantly onto my head and body pillow, which caused smells and drove me to have to wash them and their covers frequently. While home for the holidays in 2022 and brainstorming on how to solve this problem, I decided to seek out a doctor with an expertise in hyperhidrosis who was from a specialty other than one I had already seen for my condition and who lived within a reasonable distance from my home in DC. Luckily, I came across Dr. Malcolm Brock, a thoracic surgeon practicing in Baltimore; since the sweats affected my torso probably the worst, I figured why not give him a shot. My first appointment with him was a massive relief, because he told me that there with other people out there with a similar affliction that no doctors could explain and I had never met anyone else who had my condition. It was also a huge relief since the condition that he has proposed exists makes total sense to me; my full body sweats had only started for me after a traumatic event in my life. In addition, I do have an ADHD-like condition that two separate psychiatrists have diagnosed me with but have been unable to treat despite me trying out multiple variations in dosages, time of day taken, and combinations of different drugs used to treat ADHD. (Even after being on a very high dosage level of Adderall, I was not jittery, which amazed my psychiatrist, who noted in a caring manner that I was “special” but probably didn’t have ADHD proper.) Lastly, at least as a lay person, if my body’s nervous system is stuck in “fight or flight” mode, this would explain why my mind is always racing and why my body sweats profusely as soon as I fall asleep. Although we have not yet completely solved the night sweats issue, the regimen of drugs that Dr. Brock has me on has greatly reduced them to the point that I can finally sleep through the night without my BedJet and have only minor body sweats when I do so; this has given me hope that I can actually travel without it! In addition, after over 14 years of suffering with this malady, I finally have some hope that I can finally be done with it and am confident that once we find the right dosage levels for Dr. Brock’s drug regimen, I will be! This is a miracle that has greatly improved my sleep and life quality already, and we aren’t even done yet.” [0123] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the protein constructs, methods, and/or component features, steps, elements, or other aspects thereof can be used in various combinations.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0124] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where embodiments or aspects of the disclosure, is/are referred to as comprising particular elements, features, etc., certain embodiments or aspects consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. [0125] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.
relationships were obtained by measuring steady-state currents and
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 a single Boltzmann function was fitted to the data according to I/Imax (or G/Gmax) = [1 + exp(- zF(V-V1/2)/(RT))]-1, where I/Imax is the normalized current amplitude, z is the equivalent charge, V1/2 is the half-activation voltage, F is Faraday’s constant, R is the gas constant, and T is temperature in kelvin. [0084] Guanfacine hydrochloride was acquired from Sigma® (USA) and dissolved in ND- 100 as a 1 mM stock solution from which a 100 μM working solution (86 μM effective drug concentration) was diluted for use with oocytes. NaV channels were exposed to guanfacine through a gravity-fed perfusion system with a flow rate of 0.5 ml/minute. When adding guanfacine to the recording chamber, equilibration between channel and compound was monitored by means of depolarizations to V1/2 of G/Gmax at five-second intervals. Where needed, statistical differences were determined using the student’s t-test (p = 0.01). Off-line data analysis was performed using Clampfit 10 (Molecular Devices, USA), Excel (Microsoft Office, USA) and Prism 8 (GraphPad, USA). 2. Results i. Beneficial effects of guanfacine in patients with neuropsychiatric symptoms and a NaV1.7 mutation [0085] We explored whether genetic abnormalities were present in a limited subset of patients that visited our dysautonomia clinic because they suffered from hereditary generalized anxiety and concomitant sympathetic hyperarousal symptoms (Freeman et al., Clin. Auton. Res., 2011, 21:69-72; Goldstein D.S., Clin. Auton. Res., 2020, 30:299-315; Posey et al., Pediatr. Neurol., 2017, 66:53-58, e55). Moreover, for these patients 1 mg/day of short-acting guanfacine was empirically proven to be a highly effective treatment. WES uncovered the NaV1.7 p.I739V channelopathy as likely clinically relevant in patients belonging to two nonrelated families with extensive inherited symptomatology (FIG.1 and FIG.2). To detect dysautonomia, we used the COMPASS 31 validated questionnaire (Sletten et al., Mayo Clin. Proc., 2012, 87:1196-1201), a self-rating quantitative measure of autonomic symptoms evaluating orthostatic intolerance, vasomotor, secretomotor, gastrointestinal, bladder, and pupillomotor domains with our nondysautonomia control group scoring 13 or less. [0086] The first family (Maryland, USA) illustrates the treatment effect of guanfacine as it started with the case of a 20-year-old male (FIG.1, proband), harboring the NaV1.7 p.I739V variant, who presented to our outpatient clinic with hyperhidrosis denoted as intolerable on the Hyperhidrosis Disease Severity Scale (Solish et al., Dermatol. Surg., 2007, 33:908-923) along with concurrent symptoms of generalized anxiety, cardiac palpitations, cognitive deficits such as forgetfulness and trouble concentrating (i.e., brain fog), chronic fatigue, and insomnia. Due
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 to its reduced adverse effect profile compared to clonidine, a α2A-adrenergic receptor agonist used off-label to treat disabling hyperhidrosis and neuropsychiatric symptoms (Albadrani A., J. Med. Case Rep., 2017, 11:16; Conrad et al., Ann. Intern. Med., 1983, 99:570; Kuritzky et al., Arch. Neurol., 1984, 41:1210-1211; Soriano et al., Am. J. Hosp. Palliat. Care, 2014, 31:98- 100; Torch et al., South Med. J., 2000, 93:68-69; van Coevorden et al., Cephalalgia, 2021, 41:1124-1127), we opted to use short-acting guanfacine (Tenex®, 1 mg/day) as an adjunctive therapy. Six years later, the patient says that guanfacine continues to be well tolerated and highly effective, so much so that whenever he misses a dose, his cognitive impairment, anxiety, hyperhidrosis and as well as other symptoms swiftly return. [0087] The second family is from South-East Asia and consists of three generations that exhibit extensive dysautonomia symptoms primarily as generalized anxiety, hyperhidrosis, neuropathic itch, orthostatic intolerance, and chronic fatigue (FIG. 2). The proband in this family expressed the NaV1.7 p.I739V variant and presented with such profound hyperhidrosis that she opted for surgical treatment (sympathectomy). Her maternal aunt also sought treatment in our clinic and was administered Tenex®, 1 mg/day. After three days, her symptoms of anxiety, cognitive impairment, cardiac palpitations, and hyperhidrosis lessened, along with decreased paroxysmal hypertension. Two years later, the patient says that she alternates daily between short-acting guanfacine 1 mg/day and 1.5 mg/day with continued amelioration of her symptoms. Altogether, in patients with the NaV1.7 I739V gain-of-function mutation, low-dose immediate release guanfacine was sufficient in relieving multiple symptoms of dysautonomia. [0088] To further substantiate the broad applicability and beneficial properties of low-dose guanfacine in the context of channelopathies, we also want to mention the case of one member of a 30-year-old identical female twin who presented to our clinic with a chief complaint of non-painful facial erythromelalgia and generalized anxiety along with dysautonomia symptoms of chronic fatigue and irritable bowel syndrome. The patient’s erythromelalgia had become particularly pronounced shortly after a severe bout of mononucleosis. The erythromelalgia would occur almost daily and was associated with stressful social situations and not provoked by increased ambient temperature or exercise. She had been treated with Citalopram® and Escitalopram® without relief and was contemplating a bilateral thoracic sympathectomy. Her twin sister never contracted mononucleosis and never developed erythromelalgia but suffered from other symptoms of dysautonomia including chronic fatigue, irritable bowel syndrome, generalized anxiety, cognitive impairment, orthostatic intolerance, and cardiac palpitations. Like the other patients above, this patient’s erythromelalgia resolved
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 almost immediately with Tenex® 1 mg/day, and the frequency of erythromelalgia reduced to 1-3 times per year. Seven years later, the patient remains on Tenex® 1 mg/day with a durable response and no apparent medication side effects. This patient was treated empirically in the clinic and has not yet been enrolled to undergo WES for the presence of a NaV channelopathy. However, erythromelalgia has been extensively associated with gain-of-function mutations in NaV1.7 (Dib-Hajj et al., Brain, 2005, 128:1847-1854; Estacion et al., J. Neurosci., 2008, 28:11079-11088; Tanaka et al., J. Biol. Chem., 2017, 292:9262-9272; Weaver J., Neurology Alert, 2016, 35). Unlike for neuropsychiatric symptoms, we are unaware of an established association between α2A-adrenergic receptors in the prefrontal cortex and erythromelalgia. ii. Effect of guanfacine on NaV channel currents [0089] NaV channel inhibition may help explain the striking efficacy of guanfacine observed in our patients. Given the established interaction of guanfacine with other ion channels (Arnsten A.F.T., Neurobiol. Learn. Mem., 2020, 176:107327), we explored potential off-target effects of guanfacine on NaV channel subtypes, many of them linked to neuropsychiatric disorders (NaV1.1-NaV1.8) (Bagheri et al., Int. J. Dev. Neurosci., 2021, 81:669-685; Eijkelkamp et al., Brain, 2012, 135:2585-2612; Imbrici et al., Front. Genet., 2013, 4:76; Mi et al., Front. Cell Neurosci., 2019, 13:554; Nickel et al., BMC Psychiatry, 2018, 18:248; Pasierski et al., Pharmacol. Rep., 2023, 75:331-341; Rees et al., Biol. Psychiatry, 2019, 85:554-562; Weiss et al., Mol. Psychiatry, 2003, 8:186-194). We expressed each human subtype by means of the established Xenopus laevis oocyte method (Zeng et al., Expert Opin. Drug Discov., 2020, 15:39-52) in combination with the two-electrode voltage-clamp technique, and assessed typical biophysical gating parameters before and after addition of 86 μM guanfacine. This concentration seems rather high since Xenopus laevis oocytes tend to be less sensitive to compounds (Zeng et al., Expert Opin. Drug Discov., 2020, 15:39-52), up to one hundred times (Lacerda et al., Eur. Heart J. Suppl., 2001, 3:K23-K30), compared to mammalian cells. Yet, all heterologous expression systems are limited in their ability to mimic human physiological conditions. As such, we are likely working with a saturating dose and the effective in vivo concentration should be much lower. [0090] All NaV channel subtypes expressed within one to four days after cRNA injection. We first report the effects of guanfacine on NaV1.1, NaV1.2, NaV1.3, and NaV1.6, the commonly accepted predominant subtypes present within the brain (Ahern et al., J. Gen. Physiol., 2016, 147:1-24; Mantegazza et al., Physiol. Rev., 2021, 101:1633-1689; Pasierski et al., Pharmacol. Rep., 2023, 75:331-341), and will discuss NaV1.7 results separately below. To this end, we held the membrane at -90 mV and applied 50 ms depolarizing pulses to stepwise
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 depolarize the membrane and activate channels before and after addition of guanfacine. Under these conditions, NaV1.1- and NaV1.2-mediated Na+ currents were inhibited over the tested voltage range by 23% and 27%, respectively (FIG.3, Table 1). We did not observe significant changes in standard gating parameters associated with the conductance-voltage and channel availability (steady-state inactivation) relationships. These parameters were extracted using a Boltzmann fit of the data that yielded half-maximal values (V1/2) of activation and availability as well as the slope factor (Table 1). The same observation was made when testing NaV1.3 although current inhibition was reduced to 12%. Of these channel subtypes, NaV1.6 was the least inhibited at 6%. [0091] Next, we tested whether NaV channel subtypes associated with the skeletal (NaV1.4) and cardiac (NaV1.5) muscle were affected by guanfacine (Cannon S.C., Handb. Exp. Pharmacol., 2018, 246:309-330; Remme C.A., Philos. Trans R. Soc. Lond. B. Biol. Sci., 2023, 378:20220164; Wilde et al., JACC Clin. Electrophysiol., 2018, 4:569-579). Like other NaV channel subtypes, most gating parameters were unaltered (FIG.4, Table 1). NaV1.4 currents were inhibited but only by 12%. NaV1.5 activity was hampered by a drug-induced current reduction of 31%. Although NaV1.8 has also been reported to contribute to cardiac muscle function (Bezzina et al., Nat. Genet., 2013, 45:1044-1049; Hu et al., J. Am. Coll. Cardiol., 2014, 64:66-79; Macri et al., Circ. Genom. Precis. Med., 2018, 11:e001663), this subtype has primarily been investigated in relation to pain perception (Akopian et al., Nature, 1996, 379:257-262; Bennett et al., Physiol. Rev., 2019, 99:1079-1151; Han et al., Neurology, 2016, 86:473-483). Analogous to the brain subtypes, NaV1.8 currents were inhibited by 20% and no other significant gating alterations were observed (FIG.4). Combined, these data suggest that guanfacine is a non-selective NaV channel inhibitor. Table 1. Gating parameters of all tested NaV channel subtypes in control conditions and after application of 86 μM guanfacine. Parameters shown include the V1/2 and slope factor of the conductance (G) – voltage (V) relationship and steady-state inactivation (SSI; channel availability). Average current inhibition (%) with SEM at peak voltage (G-V) is also indicated.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 iii. Guanfacine inhibits NaV1.7 currents [0092] NaV1.7 is expressed in sensory and sympathetic neurons that innervate all organs in the human body (Goodwin et al., Nat. Rev. Neurosci., 2021, 22:263-274; Minett et al., Nat. Commun., 2012, 3:791; Payandeh et al., Handb. Exp. Pharmacol., 2018, 246:271-306; Vetter et al., Pharmacol. Therapeut., 2017, 172:73-100). This channel subtype has also been found in brain subcortical structures, including the thalamus, amygdala, hypothalamus, axons of the olfactory epithelium (Branco et al., Cell, 2016, 165:1749-1761; Kanellopoulos et al., EMBO J., 2018, 37:427-445) and the locus coeruleus (Jones et al., Nat. Rev. Neurosci., 2009, 10:821- 828). Using WES, we identified the NaV1.7 p.I739V variant, a gain-of-function mutation that was previously found in patients with neuropathic pain and severe autonomic dysfunction including hyperhidrosis and palpitations (Han et al., Neurology, 2012, 78:1635-1643; Han et al., Brain, 2012, 135:2613-2628). However, the proband in the first family of this study (FIG. 1) remarked that he had an unusually high tolerance to pain recalling that he had broken both arms without feeling compelled to seek pain relief at a hospital. Although this symptomatic dichotomy is rather unusual, both gain- and loss-of-function mutations in NaV1.7 have been associated with phenotypes ranging from a complete lack of pain to erythromelalgia, often with dysautonomia symptoms (Baker et al., Pflugers Arch., 2020, 472:865-880; Dib-Hajj et al., Nat. Rev. Neurosci., 2013, 14:49-62; Eagles et al., Br. J. Pharmacol., 2022, 179:3592-3611; Goodwin et al., Nat. Rev. Neurosci., 2021, 22:263-274; Mulcahy et al., J. Med. Chem., 2019, 62:8695-8710; Yang et al., Trends Pharmacol. Sci., 2018, 39:258-275). Since the p.I739V variant is commonly accepted to be a gain-of-function mutation (Han et al., Neurology, 2012, 78:1635-1643; Han et al., Brain, 2012, 135:2613-2628), we investigated whether guanfacine could inhibit NaV1.7 currents and possibly contribute to symptom amelioration in our patients when given as a short-acting formulation at a low dose of 1 mg/day. [0093] When applying 86 μM guanfacine to NaV1.7 expressed in oocytes, we observed an inhibitory effect of 22% on ionic currents whereas the conductance-voltage and channel
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 availability relationship remained unaltered (FIG.5, Table 1). Similarly, guanfacine reduces NaV1.7 p.I739V currents by 25% ± 4 (FIG. 5). Next, we expanded our electrophysiological protocols to include the recovery from inactivation. We employed a double-pulse protocol in which cells were kept at -90 mV between two 50-ms depolarization steps to 0 mV that were applied with a varying interval between 0 ms and 1000 ms. Recovery from inactivation is virtually complete after 200 ms (FIG.5) and no significant differences were observed between control and drug-exposed conditions. To test whether guanfacine prefers to interact with the inactivated state of the channel, we applied a test protocol consisting of a 5-s pulse to V1/2 of channel availability (-40 mV, Table 1) followed by a 200-ms step to -90 mV and a 50-ms test pulse to 0 mV. Such state-dependence can increase in vivo affinity and selectivity over other ion channel families (Ahern et al., J. Gen. Physiol., 2016, 147:1-24; Johnson et al., eLife, 2022, 11). We observed that under these conditions (FIG.5), the inhibitory effect of guanfacine on ionic currents doubles from 21% ± 6 to 50% ± 6 indicating a degree of state-dependence. Altogether, these data illustrate the ability of guanfacine to reduce NaV channel-mediated currents and suggest a potential application in normalizing atypical gain-of-function NaV channel gating as observed in particular pathological conditions. 3. Discussion [0094] We observed that off-label use of guanfacine was effective in reducing generalized anxiety and concomitant dysautonomia symptoms such as cognitive impairment, hyperhidrosis, and palpitations in multiple members of nonrelated families. Strikingly, the short-acting guanfacine formulation at only 1 mg/day facilitated virtually complete and long- term symptom relief without apparent side effects. We hypothesized that a genetic deficit existed in these patients that might underlie this notable sensitivity to guanfacine. WES on a limited subset of patients that benefited from a low dose of guanfacine, identified the NaV1.7 p.I739V gain-of-function variant (Han et al., Neurology, 2012, 78:1635-1643; Han et al., Brain, 2012, 135:2613-2628) as a possible hereditary pathology contributor (FIG.1 and FIG.2). The causal link between this p.I739V substitution and ensuing patient phenotypes has been challenging to ascertain (Devigili et al., Pain, 2014, 155:1702-1707; Han et al., Brain, 2012, 135:2613-2628; Xenakis et al., BMC Bioinformatics, 2021, 22:212). Yet, this mutation is commonly observed as it represents 1% of NaV channel variants detected in patients with painful or non-painful peripheral neuropathy (Wadhawan et al., Neurol. Genet., 2017, 3:e207). Moreover, intrafamilial phenotypic variability has been reported with NaV1.7 p.I739V (Peddareddygari et al., Case Rep. Neurol., 2021, 13:135-139) and the involvement of modifiers
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 or interacting proteins has subsequently been proposed (Ahern et al., J. Gen. Physiol., 2016, 147:1-24; Kanellopoulos et al., EMBO J., 2018, 37:427-445). Clinically, after being prescribed 1 mg/day of short-acting guanfacine, virtually complete symptom alleviation was reported by our patients with the NaV1.7 p.I739V mutation, as well as in patients with similar symptoms that were not sequenced. Moreover, this treatment had long-term benefits, up to six years, with few adverse effects. Another patient with erythromelalgia who did not yet undergo WES but with a presumed gain-of-function mutation in NaV1.7, also has had a durable multiple year dramatically favorable response to low doses of guanfacine. Combined, the results of this part of our study provide further evidence for a pathological role of NaV1.7 mutations in generalized anxiety and dysautonomia and illustrate the potential benefits of guanfacine treatment. [0095] Although guanfacine is a selective α2A-adrenergic receptor agonist, there is evidence that it may also have a mechanism of action outside of direct interaction with α2A- adrenergic receptors through ion channels. Guanfacine has been shown to inhibit cAMP-PKA opening of K+ channels in prefrontal spines, strengthening their connections, enhancing neuronal firing, as well as increasing top-down control (Arnsten A.F.T., Neurobiol. Learn. Mem., 2020, 176:107327). Our hypothesis that guanfacine may interact with other targets such as NaV channels seemed consistent with our preclinical and clinical experience. Since the ability of prefrontal cortex neurons to retain working memory through excitatory networks relies on ion channels that help set the membrane potential, a gain-of-function NaV channelopathy may dysregulate action potential firing producing cognitive deficits inherent to both ADHD and POTS (Fahrenholz et al., Primer on the Autonomic Nervous System, Elsevier, 2023, pp. 627-629; Iwanami et al., BMC Psychiatry, 2020, 20:485; Taylor et al., J. Clin. Psychopharmacol., 2001, 21:223-228; Wu et al., J. Child Adolesc. Psychopharmacol., 2022, 32:244-248). A compound that normalizes membrane potentials, through inhibition of NaV1.7 or other ion channel subtypes, would be clinically valuable. As such, we examined pre- clinically whether guanfacine could inhibit NaV channels. We found that multiple subtypes, including NaV1.7, were indeed partly inhibited (FIG. 3, FIG. 4, and FIG. 5). However, the guanfacine concentration used in our experiments is rather high and was primarily dictated by the known limitations of the experimental test system used (Zeng et al., Expert Opin. Drug Discov., 2020, 15:39-52). As such, in vivo effective concentrations of NaV channel block by guanfacine may be much lower (Lacerda et al., Eur. Heart J. Suppl., 2001, 3:K23-K30). Indeed, in cortical neurons, a tenfold lower guanfacine concentration was reported to inhibit Na+ currents (Pasierski et al., Pharmacol. Rep., 2023, 75:331-341). A more conventional explanation of guanfacine efficacy in our patients involves its primary effect on α2A-adrenergic
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 receptors which would reduce the increased sympathetic tone induced by NaV channel gain-of- function mutations, perhaps complemented by a smaller, synergistic inhibition of NaV channels. Indeed, although a functional association between α2A-adrenergic receptors and NaV channels has yet to be established, hints supporting a link can be found in the literature. For example, α2A-adrenergic receptors agonists such as clonidine and dexmedetomidine have clinically-established analgesic properties, in part attributed to acute inhibition of NaV channels at similar or higher concentrations than tested here for guanfacine (Im et al., Mediators Inflamm., 2018, 2018:1782719; Stoetzer et al., Region Anesth. Pain M., 2017, 42:462-468). Dexmedetomidine causes a concentration-dependent inhibition of Na+ currents, an effect that can be prevented by yohimbine, a competitive α2A-adrenergic receptor antagonist (Im et al., Mediators Inflamm., 2018, 2018:1782719). Moreover, dexmedetomidine-induced inhibition is blocked by intracellular perfusion of the G protein-coupled receptor (GPCR) inhibitor GDPβ-S. A functional coupling of the α2A-adrenergic receptor to GPCRs opens up possible pathways to influence NaV channel function (Brackx et al., Pharmacol. Therapeut., 2023, 245:108416; Salzer et al., Int. J. Mol. Sci., 2019, 20; Xu et al., Sci. Adv., 2022, 8:eabj5347). Finally, it is worth noting that agonist-mediated activation of α2A-adrenergic receptors was reported to be dependent on membrane voltage to the extent that negative membrane potentials promote agonist-mediated activation and downstream receptor signaling (Rinne et al., PNAS, 2013, 110:1536-1541). Therefore, membrane depolarization triggered by the NaV1.7 p.I739V mutation (Han et al., Neurology, 2012, 78:1635-1643; Han et al., Brain, 2012, 135:2613-2628) would reduce receptor susceptibility to agonist. By inhibiting NaV1.7 p.I739V with guanfacine and restoring more negative membrane potentials, α2A-adrenergic receptors become more sensitive to agonists. As such, guanfacine may reduce neuronal hyperexcitability via direct NaV1.7 inhibition and thereby increase its own affinity for the α2A-adrenergic receptor to correct sympathetic outflow. This dual synergistic effect is but one possible explanation for the low dosage needed in patients (1 mg/day) to achieve symptom improvement. [0096] While this limited study provides insights into the off-label efficacy of guanfacine, few limitations are worth mentioning. First, in our dysautonomia clinic we stop prescribing guanfacine occasionally due to the onset of adverse patient complaints such as xerostomia, somnolence, and lightheadedness, symptoms that have also been associated with altered NaV1.7 function (Dormer et al., J. Pain Res., 2023, 16:1487-1498). However, we found that night-time administration coupled with a slow increase in the titration of the dose of guanfacine, preferably no faster than 1 mg per 2-3 weeks, assists in tolerability. Second, our observations are based on only two nonrelated families with the NaV1.7 p.I739V variant which
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 do not fully represent the diverse population of patients with neuropsychiatric symptoms and NaV channelopathies. Nonetheless, our data suggest that the non-NaV channel subtype- selectivity of guanfacine could be a beneficial trait in patients with mutations in other NaV channel subtypes. Third, our study is observational in nature lacking guanfacine data in a control group with similar symptoms but without a NaV channelopathy. Since our observational data were not part of a randomized trial to assess the efficacy and safety of guanfacine in genetically stratified patients, future studies in a more controlled setting will be needed. Fourth, this study primarily focused on the NaV1.7 p.I739V variant and its response to a low dose of guanfacine. As exemplified by our third patient whose erythromelalgia was provoked by a mononucleosis infection, this is a complex condition and other genetic as well as environmental factors that may contribute to the observed symptoms and treatment response were not explored. [0097] Overall, the results of this study substantiate a pathological role of NaV1.7 mutations in disorders related to an increased sympathetic tone and illustrate the potential benefits of low-dose short-acting guanfacine treatment, perhaps aided by an inhibitory effect on NaV channels. Despite the fact that the dysautonomia symptoms in our studied families most likely involve a complex interplay between genetics and environmental cues, it is encouraging that inhibition of presumed gain-of-function genetic aberrations can result in substantial symptom improvement. In such a complex disease, a single drug given at a low dose being so effective, suggest a likelihood of guanfacine acting synergistically on multiple targets, including direct ion channel inhibition and a drug-induced augmented affinity to activate the α2A-adrenergic receptor. Our observations suggesting that inhibition of NaV channels by guanfacine may serve as an off-label target is clinically important since it promotes the possibility that guanfacine could one day be utilized in genetically stratified patients with neuropsychiatric symptoms resulting from anomalous NaV channel behavior. Example 2. A Multi-Hit Model of Long COVID Pathophysiology – The Interaction between Immune Triggers and Nervous System Signaling [0098] As of October 2023, over 698 million individuals have been infected globally with SARS-CoV-2 resulting in approximately 7 million deaths. The medical community, as early as fall 2020, issued an international online survey in 56 countries suspecting the presence in many patients of persistent heterogeneous sequelae involving multiple organs. But it was even earlier on May 20, 2020, when Elisa Perego, a health researcher from Lombardy, Italy, dubbed her experience of symptoms after SARS-CoV-2 as “#Long COVID” in a hashtag on Twitter.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 She penned “Long COVID” as a contraction of “long-term COVID illness” to describe the cyclical, progressive, and multiphasic nature of her symptoms that had persisted for over 40 days. This patient-originated term quickly spread through social media gaining popularity as it legitimized other patients’ experiences and challenged medical assumptions that SARS- CoV2 was a transient, short-term disease. Long COVID, also now known as Post-acute sequelae of SARS-CoV-2 infection (PASC), and “Long-haul COVID,” is currently defined as ongoing, relapsing, or new symptoms or conditions present 30 days or more after COVID-19 infection. 1. The Prevalence and Burden of Long COVID [0099] Long COVID has become a major public health issue affecting at least an estimated 65 million people globally, with cases increasing daily. This figure is most likely an underestimation due to the continued high rates of Long COVID among new SARS-COV-2 cases around the world. In the U.S., as of January 2023, the percentage of people who reported Long COVID symptoms after SARS-COV-2 declined from 19% in June 2022 to 11% in January 2023. But despite this decline, the prevalence of Long COVID in those who have had SARS-COV-2 is still high. Moreover, its prevalence seems to depend on the severity of the underlying SARS-COV-2 infection with 7.5-41% of non-hospitalized patients affected, and over 50% of hospitalized patients debilitated by symptoms such as severe, persistent anxiety and depression. In the U.S., in 2022 the CDC estimated that the prevalence of Long COVID was about 6.9% of all adults with women (8.5%) more likely than men (5.2%) ever to have had Long COVID. In fact, most studies of Long COVID have shown that the prevalence among females is statistically significantly higher than among males. Interestingly, U.S. Census Bureau data suggest higher rates of Long COVID symptoms among Hispanic and black adults compared to white adults. A recent survey found 31.1% of Hispanic, 28.7% of black, and 27.6% of white adults reported Long COVID symptoms. [0100] Population-based surveillance tools have been effective in monitoring the impact of the disease on daily living in the U.S. with 25.3% of over 18,000 individuals with Long COVID responding that day-to-day activities were impacted “a lot”. Importantly, 28.9% of individuals responded that their SARS-COV-2 infection had occurred more than 12 months previously. This has negatively affected employment with two surveys showing that approximately 66% of individuals with Long COVID who previously were actively employed were now unemployed or were forced to reduce their working hours. Mental health and cognitive burdens have been a substantial contributor to this inability to function at work with over 60% of individuals in the U.S. having a neurological or psychiatric diagnosis 6 months after SARS-
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 CoV-2. One early estimate calculated the reduction of labor supply due to Long COVID could be more than $50 billion annually. A recent review of twelve publications suggested that Long COVID patients are increasingly utilizing healthcare systems, both primary and specialist care, much more than before they were infected. Using claims data, a recent estimate suggests that these more frequent visits for care mean that Long COVID adds an additional $224 in healthcare costs per month over a 6-month period in any individual patient. Thus, Long- COVID has emerged as a major public health problem that will have considerable long-term consequences for population health, economic policy, and research initiatives. [0101] Due to its relative novelty, major knowledge gaps persist that impede our ability to treat Long COVID effectively. Therefore, in this example, we will focus on research efforts into our understanding of the pathogenesis of Long COVID and of its underlying biological mechanisms. 2. Long COVID and its Complex Symptomatology i. The association between Long COVID and dysautonomia [0102] Early in the pandemic, there was an acute realization by several neurologists that many of the symptoms of Long COVID were, in fact, symptoms of dysautonomia, a dysfunction of the autonomic nervous system (ANS) or components thereof such as the sympathetic nervous system. At the NIH’s National Institute of Neurological Disorders and Stroke Autonomic Medicine Section, Dr. David Goldstein quickly realized within two months of the pandemic’s onset in April 2020 that the symptoms of SARS-CoV-2 that he saw manifested in patients were multi-system, multi-disciplinary complaints. Dr. Goldstein surmised that they originated from abnormal activation of the ANS leading to autonomic dysfunction in various organs and thereby causing myriad functional abnormalities, a phenomenon that he described as the “extended ANS” (Goldstein, D.S., Clin. Auton. Res., 2020, 30(4):299-315). One year later, he strengthened his theory with a patient case history in an article entitled “The possible association between COVID-19 and postural tachycardia syndrome (POTS)” (Goldstein, D.S., Heart Rhythm., 2021, 18(4):508-509). Aided by new innovations in studies, such as the COVID Symptom Study, where by May 2020, 2.8 million users had documented their symptoms on a smartphone tracker app, more and more clinicians quickly began to recognize that clinical dysautonomia was a prominent feature of Long COVID. [0103] At Johns Hopkins Center for Sweat Disorders, we also were observing a sharp spike in the number of patients complaining about an increase in primary focal hyperhidrosis (PFH) after SARS-CoV-2 infection. Often on close questioning, these patients would admit that they
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 had indeed suffered from various degrees of underlying hyperhidrosis since childhood, but that after contracting SARS-CoV-2, their sweating had become intolerable and always interfered with daily activities (highest score on the Hyperhidrosis Disease Severity Scale (HDSS)). It also became equally evident that sweating was not the only debilitating symptom that was occurring post infection with SARS-CoV-2. In addition to PFH, an important dermatologic manifestation of ANS dysfunction, our typical patient would complain of a constellation of debilitating symptoms including generalized anxiety disorder, cognitive impairment or “COVID-brain fog,” chronic fatigue, shortness of breath, heart palpitations, hyperhidrosis, irritable bowel, migraine-type headaches, insomnia, as well as lightheadedness. Over the last thirteen years, we had observed this phenomenon quite regularly in our Center for Sweat Disorders with patients who presented with hyperhidrosis having similar dysautonomia symptoms but not after infection with SARS-CoV-2, but rather after contracting infectious diseases such as Mononucleosis, Lyme disease, and Malaria. As with Long COVID, however, patients reported that the most pervasive symptoms were often the neurological co-morbidities of anxiety, depression, and cognitive impairment. It is important to note here that our PFH patients over the years have also frequently presented with Postural Orthostatic Intolerance Syndrome (POTS), which often occurs in adolescence when it can be triggered by an antecedent infection. Intriguingly, during the pandemic, multiple scientific reports started to appear suggesting that the majority of patients with Long COVID met the diagnostic criteria for POTS, a link that has yet to be fully explored. ii. Unanswered questions [0104] Despite our long experience with both POTS and post-infectious dysautonomia, what remained puzzling was that patients tended to have only marginal improvement of their symptoms, well short of a full cure, when given even potent immunomodulatory agents such as intravenous immunoglobulin to treat the autoimmunity or neuroinflammation co-morbid with their disease. This prompted two yet unanswered questions. First, what is the association between Long COVID and dysautonomia symptoms? Second, why does the inflammatory response that typically occurs after an infection with Epstein-Barr virus, Borrelia, and now SARS-CoV-2, not provoke dysautonomia symptoms, such as anxiety, depression, and cognitive impairment, in everyone who contracts these pathogens? [0105] A possible foundation for an answer to the first question (i.e., What is the association between Long COVID and dysautonomia?) comes from our years of clinical experience at the Johns Hopkins Center for Sweat Disorders. Three of the most important lessons we have learned from caring for hyperhidrosis patients are: (i) hyperhidrosis is not just
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 a mere cosmetic issue, (ii) most patients do not complain about being stigmatized or embarrassed because they are “sweat bothered”; instead, these patients consistently describe an impaired quality of life in which the adverse impact of extreme sweating is thought to be greater than that of other chronic dermatologic disorders such as psoriasis, and (iii) hyperhidrosis almost never occurs alone as an isolated symptom, but as mentioned previously, is habitually part of a constellation of systemic dysautonomia complaints. In fact, we found that 38% of all PFH patients who presented to our clinic were on prescribed psychotropic medications for anxiety versus 14% of controls. Since no morphological differences exist between the sweat glands of hyperhidrosis patients and healthy controls, these clinical observations of multiple accompanying dysautonomia symptoms fit a hypothesis held for over fifty years that PFH is indeed a disease of primary sympathetic dysfunction. In other words, there is a sympathetic overdrive systemically affecting every organ throughout the body. The skin however, as the largest organ in the body, can manifest hyperhidrosis as an early diagnostic sign of abnormal ANS function that may herald diseases such as Post Traumatic Stress Disorder (PTSD). Moreover, our clinical observation was that hyperhidrosis occurred at a young age, probably at birth, with frequent patient histories from mothers describing sweaty footprints of a patient as a toddler. Given this early age of onset and frequently encountered family histories of PFH, physicians have long suspected an autosomal dominant genetic transmission, but with incomplete penetrance. Indeed, our clinical observations suggest that PFH may not only be inherited, but also that many of the above-mentioned dysautonomia co- morbidity complaints of hyperhidrosis patients may run in families. [0106] Propelled by our clinical observations, we embarked on a search for genetic causes of hyperhidrosis that affect the ANS. We started to construct family history pedigrees to document that not only was hyperhidrosis prevalent in families, but also that many of the family members shared dysautonomia symptoms, such as generalized anxiety, an affliction that is applicable to virtually all our patients. FIG. 6 illustrates two typical families where the probands presented to our clinic with severe hyperhidrosis, family histories of hyperhidrosis, and with multiple family members showing a wide array of dysautonomia symptoms including generalized anxiety, orthostatic intolerance, chronic fatigue, and chronic itch symptoms. Many of their symptoms are validated using established clinical questionnaires such as the HDSS, COMPASS-31, Zung Self-Rating Anxiety Scale, and the Social Phobia InveNtory (SPIN), thereby removing as much as possible a potential bias by the observer. The high scores across all four of these surveys in multiple family members suggests impaired autonomic regulation in multiple organ systems. Intrigued by these findings, we started performing whole-exome
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 sequencing on families with hyperhidrosis who visited our clinic and whose relatives exhibited non-syndromic autonomic dysfunction. Preliminary data resulting from these efforts suggest the presence of heritable missense mutations in genes that are nearly all associated with generating or transmitting electrical signals throughout the body. These genes encode proteins that: (i) are directly involved in membrane excitability such as (voltage-gated) ion channels, (ii) modulate other proteins that can generate or propagate electrical signals such as ancillary subunits or transcription factors, and (iii) may trigger electrical or morphological remodeling of excitatory tissues. Notably, mutations in these genes may lead to altered ANS excitability as is often observed in these families and can be labeled clinically as a “high sympathetic tone.” The existence of a gain-of-function mutation in a protein expressed in the sympathetic ganglia, for example, would provide a plausible genetic explanation for the pathogenesis of hyperhidrosis and its accompanying dysautonomia. [0107] A possible answer to the second question (i.e., Why does a post-infectious inflammatory response not always trigger dysautonomia?) of why Long COVID only presents in a small subset of patients who contract SARS-CoV-2, led to the formulation of a two-hit hypothesis for how Long COVID and other postinfectious dysautonomia disorders may develop. We currently hypothesize that Long COVID requires two molecular hits. First, a genetic predisposition in genes that regulate electrical signaling, such as an ion channel gain- of-function variant expressed in the brain and/or sympathetic ganglia that can act as a genetic primer to alter neuronal membrane excitability. Second, a SARS-CoV-2 infection that serves as an immunologic trigger transforming the nervous system into an oversensitive state characterized by lower thresholds to activation and a lessened ability to extinguish the debilitating dysautonomia symptoms associated with Long COVID. Indeed, influential interoception theories, such as the James-Lange hypothesis, state that exaggerated sympathetic responsivity can augment one’s vigilance to physiological feedback, leading to dysautonomia symptoms. Thus, potentially mild symptoms associated with the underlying genetic predisposition, can be exacerbated by immunologic triggers, which offers a plausible answer to our second question of why only a subset of people who contract SARS-CoV-2 develop Long COVID. [0108] Growing evidence suggests that chronic inflammation, as a hallmark of autoimmunity, plays a significant role in Long COVID, and this fits well with our two-hit hypothesis theory. As established in POTS, autoantibodies have been detected against cytokines, complement proteins, cell surface proteins, as well as self-antigens in patients with Long-COVID with the implication being that immune dysregulation can be the secondary
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 trigger of sympathetic overactivation. Long-COVID patients also have persistent elevation, compared to the onset of SARS-CoV-2, of proinflammatory cytokines like TNF-α, IFN-γ, IL- 1β, IL-6 and IL-13 with significantly decreased levels of IP-10. Upregulation of some of these cytokines have been observed in patients up to at least eight months after SARS-CoV-2 infection. Another study showed 83% of Long COVID patients with signs of latent autoimmunity and 62% with polyautoimmunity. iii. Treatment [0109] In our clinic, we have used our concept of a two-hit hypothesis to guide treatment of Long COVID patients. Our rationale is that there must be both an inhibition of the genetic propensity for electrical hyperexcitability as well as an immunomodulation of the autoimmunity to minimize a further sensitization of the central and/or sympathetic nervous system. One small case report of twelve patients with a remarkable amelioration of neurocognitive impairments due to Long COVID used a two-drug combination of the α2A- adrenoceptor agonist, guanfacine, and the anti-oxidant, N-acetylcysteine (NAC) (Fesharaki- Zadeh et al., Neuroimmunology Reports, 2023; 3:100154). Importantly, we recently showed that guanfacine may also partially inhibit multiple voltage-gated Na+ (NaV) channels (see Example 1), and NAC is an established immunomodulator reducing inflammation (Fesharaki- Zadeh et al., supra). In our clinic, we had been using guanfacine along with other inhibitors of voltage-gated ion channels successfully for years in patients with PFH, POTS, and dysautonomia. Since most NaV channel inhibitors lack subtype specificity and can sometimes act on other ion channel families or even receptors, clinically we favor repurposing multiple drugs to achieve ion channel inhibition. We also knew through interviews that many of our patients experienced significant symptom relief with marijuana smoking. Since synthetic cannabinoids are known to inhibit many ion channel subtypes, we combined synthetic cannabinoid therapy off-label in the form of dronabinol with repurposed drugs that inhibited ion channels. [0110] Success in translating our two-hit hypothesis to the clinic is illustrated by a patient who presented to our Center for Sweat Disorders in the fall of 2018 with complaints of life- long hyperhidrosis. Strikingly, this patient also suffered from multi-system constitutional symptoms of dysautonomia including pre-syncope, light-headedness, chronic cardiac palpitations, migraine type chronic headaches, chronic fatigue, mild brain fog, irritable bowel symptoms and problems with poor sleep. Since this patient seemed typical of an individual with a genetic predisposition to an increased sympathetic tone, in 2018, we treated his
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 dysautonomia symptoms with NaV channel modulators – oxybutynin, guanfacine, and dronabinol. After being maintained successfully for four years on these medications, his dysautonomia symptoms, especially his brain fog and chronic fatigue, acutely worsened after a SARS-CoV-2 infection. He presented to clinic seven months later with Long COVID, and by simply increasing his guanfacine and adding NAC, we were able to control his Long COVID symptoms effectively. His testimony (see Example 3) three weeks after this treatment further reinforces the potential clinical efficacy of this two-step approach of inhibiting ion channels along with the immune response to SARS-CoV-2 in these patients. 3. Conclusion [0111] We propose that Post-Acute Sequelae of SARS-CoV-2 or Long COVID may require two molecular hits: (i) a predisposing genetic vulnerability such as ion channelopathies or mutations in genes that support electrical signaling, and (ii) an immune reactivity to SARS- CoV-2 as a secondary trigger. We also hypothesized here that pharmacological targeting of both the genetic and environmental hit may, in combination, show efficacy in patients. With some initial success, we have translated this concept to the clinic and are treating patients with drugs such as guanfacine to target the genetic aberration, and the neuro-inflammation with NAC. The full clinical implementation will need individual genetic screening of each patient as well as the manufacturing of more specific drugs that can inhibit the underlying genetic defect. The full application of this multi-hit hypothesis may not only have applicability to Long COVID, but also to other post-infectious dysautonomic diseases. Example 3. Patient Testimonials [0112] This Example provides some representative patient testimonials from patients with Long COVID, POTS, or dysautonomia/hyperhidrosis. 1. Patient Testimonials for Long COVID i. First patient (54-year-old professor of engineering and National Academy of Medicine Member) [0113] “My life changed with the onset of dysautonomia that was triggered by a COVID- 19 infection in early 2020. I have suffered from “long covid” ever since, including intense multiple headaches/migraines each day, brain fog, lethargy, and overall lack of energy and focus. I am a 54-year-old professor of engineering and medicine at Johns Hopkins whose career was on the rise (including induction into the National Academy of Medicine and the National Academy of Inventors). However, my long covid was so debilitating that I thought I might have to go on disability and/or give up on my passion to help improve human health all
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 together and retire. Needless to say, my poor health and lack of energy the past few years have been heartbreaking for me and my family. I am a medical/drug researcher who is skeptical of unproven medical therapies, including the course of medications originally prescribed by Dr. Brock. While I did not notice a difference in how I felt initially, my headaches and brain fog greatly decreased in severity and frequency, and my energy and ability to focus improved significantly, once I ramped up to a higher dose of guanfacine (2-3 pills per day). In addition to guanfacine, I take 600 mg of N-acetyl-cysteine (NAC), 300 mg of nicotinamide riboside chloride, and specific salt supplements recommended by Dr. Brock. Notably, I ran out of guanfacine while traveling recently. I thought perhaps I did not need it anymore since I was feeling considerably better, but I was wrong. The headaches, lethargy and brain fog came roaring back within a day or two. Going back to my medications and supplements has me feeling much better again. My energy and ability to focus has greatly improved, from about 5- 10% to about 50-75% of my pre-covid levels of energy and focus, thanks to Dr. Brock. Most importantly, I have the energy to be a loving husband, father and friend again, and I have been able to return fulltime to the career I love. I share this testimonial with the hope that it will help others, and with the hope that Dr. Brock and his colleagues will continue advancing knowledge and effective treatments for those with dysautonomia and long covid.” ii. Second patient (39-year-old male, described in Example 2) [0114] “As I’ve made you aware, I’ve dealt with the struggle of Long Covid this entire year going back to when I got sick in November 2022. Initial symptoms lingered into the New Year, but then I started having severe setbacks in the following months that significantly impacted my quality of life. At times, I would feel so physically & mentally lethargic for days at a time that leaving the house was a major challenge. This lasted through the winter and Spring Months, and into June. However, I began to notice some improvement after following your directive of taking the N-Acetyl-L-Cysteine and increased dosage of the Guanfacine, in addition to the dosages of Dronabinol & Oxybutynin that I’ve taken since 2018. Since that regimen began in June, the setbacks quickly began to fade, and I’m happy to report that I’ve felt better these past 30 days than I have since before my birthday in November 2022, when I last got Covid. In fact, aside from some brief fatigue episodes which can also be attributed to something else, I haven’t felt sick at all in almost two months. It's been a tremendous relief that has come at a time that admittedly had left me somewhat desperate in terms of treatment due to how severe the setbacks were. I wanted to share my success story in case other patients you care for share similar characteristics of mine that can be treated.” 2. Patient Testimonials for POTS
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0115] “In 1996, at the age of 21, my life was dramatically changed by the sudden onset of Dysautonomia. At that time, I was graduating Summa Cum Laude from the Wharton School of Business with the Dean’s Award for Community Service. Prior to that, I had been the youngest person to testify in the US Senate and later interviewed on Good Morning America for volunteerism. Almost overnight, I became too ill to stand for graduation pictures. Two years later, I was diagnosed with POTS & NMH following a Tilt-Table Test at Johns Hopkins Hospital during which my blood pressure dropped dramatically and I passed out at 22 minutes. Since then, I have been in the ICU multiple times after fainting, bedbound for months requiring continuous saline infusion to stay conscious, suffering with a myriad of other multi-system symptoms and extremely limited in energy to perform the basic functions of daily living much less to enjoy a full life beyond the confines of my home. This year, I met Dr. Brock who proposed a radical new approach to my condition in the form of certain drugs he was testing to address the underlying causes of the disorder. He first prescribed oxybutinin, which within 24 hours, restored some of my autonomic functions to normal. Without ongoing adrenaline surges and drenching night sweats, I could think clearly and my family recognized the person they’d lost 25 years ago. To our disappointment, I had to discontinue this and three alternative medications due to dangerous side effects. In June of this year, Dr. Brock started me on Rufinamide which I increased to 800mg twice daily. I have been gradually increasing my activity without the usual debilitating exhaustion, pain, and confusion that follows. Last week, for example, I was even able on several successive days to take a brief swim in a nearby lake, walk back up the hill to our home, and then even have the energy to prepare dinner. For the first time in 25 years, I have hope that a medication can regulate dysautonomia, end suffering and restore not just my life but the lives of so many others who suffer from this poorly understood condition.” 3. 3. Patient Testimonials for Dysautonomia and Hyperhidrosis i. First patient (Female, Age 40, Director of Marketing for Construction & Development Company) [0116] “Finding rufinamide as a possible treatment for hyperhidrosis is an extremely exciting, and life changing experience. Although I was initially diagnosed at John’s Hopkins with Chronic Fatigue Syndrome, hypermobility and Postural Orthostatic Tachycardia Syndrome in my teens, it wasn’t until my late twenties and early thirties that I started to experience increasing allergy symptoms and severe hyperhidrosis from my face, specifically my hairline. Hyperhidrosis initially seemed like a minor inconvenience in comparison to some of my other symptoms, but over time it contributed to significant social anxiety, poor mental
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 health, and stigma from others. Working with John’s Hopkins Hyperhidrosis Clinic has been a lifesaver, and after initially finding excellent results with one medication – side effects made it no longer an option. I am so grateful that Dr. Brock and his team have identified rufinamide as a possible alternative to the treatment, and while I am still increasing my way from a low starting dose, I can see significant improvements not only to my sweating issues but also improvements to my POTS symptoms, anxiety, sleep quality and overall sense of wellbeing on rufinamide.” ii. Second patient (46-year-old female with hyperhidrosis and dysautonomia) [0117] “I wanted to give you a brief update of how I’m doing since I started taking Rufinamide. I started taking Rufinamide in January 2023 and I noticed a change within a week. Before taking the medication I had issues with palpitations sweating and a hot feeling all over my body lasting for 20 minutes. I had seen a cardiologist and wore a heart monitor with no answers. Since I started taking Rufinamide I don’t have any palpitations no sweating and the hot feeling all over my body. I noticed my energy level has increased and I don’t have any side effects. Hopefully this medication will be able to continue to provide me the relief I have been looking for since the age of 16 years old and now I’m 46 years old. I think this medication could help much more people. Yes, my sweating and palpitations got worse after doxyrubicin treatment for my desmoid tumor and with the Rufinamide and my stomach issues are better. Before that I would have trouble having a bowel movement and other stomach issues. But now I’m better.” iii. Third patient (25-year-old with hyperhidrosis) [0118] “Ever since I can remember, I have dealt with hyperhidrosis in my hands and feet. As a result, I avoided participating in certain activities and I made difficult decisions because I didn’t want people to know about my condition. As I grew older, my anxiety increased, I had frequent headaches, fatigue and sometimes felt lightheaded. I didn’t think there would ever be a solution to any of my symptoms until I met Dr. Brock. I have found that taking guanfacine 1MG and oxybutynin 10MG has helped me significantly. My hyperhidrosis has almost gone away completely, I no longer experience frequent headaches or lightheadedness, and my anxiety has improved. I’m so very thankful for these medications, and for Dr. Brock and all the research being done to impact individuals and their conditions.” iv. Fourth patient (32-year-old with hyperhidrosis) [0119] “Hyperhidrosis is a condition that has negatively affected my life every day in multiple aspects. It has dictated my lifestyle, behavior, aspirations, even my education and career choices. I tried everything for 15 years and nothing worked. My current therapy
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 regimen by Dr. Brock (Guanfacine and Oxybutynin) has been life changing. I can for once in my life have a sense of normalcy, be myself around others and live life to my fullest potential. Dr. Brock’s line of research is transforming lives and bringing viable solutions to this ‘invisible’ yet disabling condition.” v. Fifth patient (28-year old female PhD postdoctoral fellow) [0120] “I struggled with hyperhidrosis since middle school, most embarrassing was my heavy underarm sweat that stained every single shirt I owned. My sweating occurred year- round and seemed to have no triggers, it was constant. I tried every over-the-counter antiperspirant on the market. I tried glycopyrronium tosylate (Qbrexza) for my underarms, that stopped some of the sweating, but it made me extremely constipated, and I had to stop. I had resigned myself to coping with my hyperhidrosis by selecting only patterned and dark colored shirts to hide the stains as well as living with the constant feeling of a damp sweaty shirt under my arms. My dermatologist recommended I see Dr. Brock for my hyperhidrosis. Dr. Brock started me on 10mg of Oxybutynin and 1mg of Guanfacine as well as drinking either Liquid IV or LMNT to increase my sodium intake. Since starting this regimen, a year ago, I’ve had many days where I don’t sweat at all! I am still capable of sweating when it’s 100 degrees outside or I exercise but I’m not bothered by this sweat since that amount is directly related to either the temperature or physical exertion. I’m glad my body can still self-regulate its temperature to keep me safe. I can finally move about my day without having to keep my arms pressed into my side out of embarrassment. As a scientist myself, I’m extremely comforted knowing that there is a genetic reason for my hyperhidrosis and that Dr. Brock’s team is working to understand the molecular underpinnings of this condition to provide all of us with selective and effective treatment options. Knowing that hyperhidrosis is an ion channelopathy as allowed me to prioritize giving my body the electrolytes it needs. I was thrilled to hear that he has developed mouse models that recapitulate patient symptoms so he can test his hypotheses out in a reliable model system. I can’t wait to see what else Dr. Brock’s team discovers next!” vi. Sixth patient (55-year-old female) [0121] “I have been a patient of Dr. Malcolm Brock since 2019. I received a diagnosis of hyperhidrosis from my primary care physician, who then referred me to Dr. Brock. When I met with Dr. Brock the first time, he asked me a number of questions about my personal condition and also took the time to share details about his research. He prescribed me three medications to be taken together that he found successful with other patients: Dronabinol 2.5mg taken twice per day, Oxybutynin CL 5mg once per day and Guanfacine 1mg once per
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 day. Note: I found that taking 1 Dronabinol per day worked best for me plus over time, we tweaked the Oxybutynin CL to the ER version once per day. To say that this combination of medication has been life changing is an understatement! Before taking this medication, I would sweat to varying degrees doing normal everyday things like being in a warm room or walking down the street on a warm (not hot) day. As a head-sweater, beads of sweat would immediately form on my forehead and back of my head making my hair damp. In addition to typical underarm sweat, I would also sweat in my groin area and under my breasts. I have also been known to sweat on colder days if I walked outside too quickly. I would not consider myself an anxious person, but these situations made me extremely anxious. I would have to dress in a way that wouldn't make it obvious if I did sweat. My kids told me that they noticed that every time we went somewhere, the first thing I did was comment on the temperature. After taking these medications, I can completely tolerate these situations. I do not have bouts of embarrassing sweating situations anymore. Of course, on extremely hot and humid summer days or when I am exercising, I still sweat, but these are normal times to sweat. I now feel more in control of my body and much less anxious about the temperature. I am extremely grateful to Dr. Brock and the research he has done and continues to do to help people like me with hyperhidrosis. This combination of medicine works, and I hope that one day, it will be a common solution that is available to more people like me who suffer from this condition.” vii. Seventh patient (47-year-old with hyperhidrosis) [0122] “In 2009 while serving overseas, I began having severe sweats from head-to-toe on most nights, which greatly disturbed my sleep and forced me to constantly have to wash my sheets and bedding due to the smell. When I returned to the U.S., I began seeking a medical solution to this problem by seeing a neurologist, a dermatologist with a specialty in hyperhidrosis, an endocrinologist, and multiple psychiatrists (as well as having at-home and in-lab sleep studies done). However, none of them were able to treat this condition, let alone determine what was causing it. This problem was taking a significant toll on my sleep since I would wake up multiple times a night due to sweat beading up on my chest, etc. and then running down my body, which triggered hairs on my body and thus woke me up. In addition, I would wake up multiple times per night freezing due to soaked sheets, which also smelled due to the sheer volume of sweat. After giving up hope of solving this problem via a medical route, I found a temporary solution in the form of a BedJet, which essentially blows hot air under my sheets from the foot of my bed. However, if I wanted to sleep decently when I traveled, I had to bring the BedJet (which takes up a large suitcase) and set it up in the hotel room, which is a hassle, especially if on a road trip staying at multiple hotels over time. And I
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 was still sweating significantly onto my head and body pillow, which caused smells and drove me to have to wash them and their covers frequently. While home for the holidays in 2022 and brainstorming on how to solve this problem, I decided to seek out a doctor with an expertise in hyperhidrosis who was from a specialty other than one I had already seen for my condition and who lived within a reasonable distance from my home in DC. Luckily, I came across Dr. Malcolm Brock, a thoracic surgeon practicing in Baltimore; since the sweats affected my torso probably the worst, I figured why not give him a shot. My first appointment with him was a massive relief, because he told me that there with other people out there with a similar affliction that no doctors could explain and I had never met anyone else who had my condition. It was also a huge relief since the condition that he has proposed exists makes total sense to me; my full body sweats had only started for me after a traumatic event in my life. In addition, I do have an ADHD-like condition that two separate psychiatrists have diagnosed me with but have been unable to treat despite me trying out multiple variations in dosages, time of day taken, and combinations of different drugs used to treat ADHD. (Even after being on a very high dosage level of Adderall, I was not jittery, which amazed my psychiatrist, who noted in a caring manner that I was “special” but probably didn’t have ADHD proper.) Lastly, at least as a lay person, if my body’s nervous system is stuck in “fight or flight” mode, this would explain why my mind is always racing and why my body sweats profusely as soon as I fall asleep. Although we have not yet completely solved the night sweats issue, the regimen of drugs that Dr. Brock has me on has greatly reduced them to the point that I can finally sleep through the night without my BedJet and have only minor body sweats when I do so; this has given me hope that I can actually travel without it! In addition, after over 14 years of suffering with this malady, I finally have some hope that I can finally be done with it and am confident that once we find the right dosage levels for Dr. Brock’s drug regimen, I will be! This is a miracle that has greatly improved my sleep and life quality already, and we aren’t even done yet.” [0123] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all the protein constructs, methods, and/or component features, steps, elements, or other aspects thereof can be used in various combinations.
Attorney Docket No.0184.0291-PCT/C17806_P17806-02 [0124] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where embodiments or aspects of the disclosure, is/are referred to as comprising particular elements, features, etc., certain embodiments or aspects consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the disclosure can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. [0125] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.