Long COVID is defined by a range of symptoms affecting multiple organs that persist for more than three months following an acute SARS-CoV-2 infection. Approximately 7% of individuals who recover from SARS-Cov-2 infection develop Long COVID.

Long COVID Postural Orthostatic Tachycardia Syndrome (LCPOTS) symptoms include fatigue, exercise intolerance, orthostatic intolerance, syncope, and heightened orthostatic tachycardia.

Research has found that decreased parasympathetic activity in LCPOTS increases the production of highly immunogenic neoantigens Isolevuglandins (IsoLG-adducts). IsoLG-adducts induce formation of circulating monocyte/T cell complexes(doublets) leading to the persistent and unresolved immune response that continues after the initial infection.

The purpose of the this research, is to study the effects of 2-hydroxybenzylamine (2-HOBA), an Iso-LG-adduct scavenger, its effects in immune markers and compare it with Placebo

Background:

Decreased parasympathetic activity is present in LCPOTS; this system is an important modulator of NAPDH oxidase activity because the activation of NADPH oxidase in monocytes promotes superoxide (reactive oxygen species -ROS) production.Oxidative stress occurs when ROS are generated in excess of normal antioxidants. Oxidative stress occurs when reactive oxygen species (ROS) are generated in excess of normal antioxidantsOxidative stress occurs when reactive oxygen species (ROS) are generated in excess of normal antioxidants that can cause harm by having adverse effects on T cell function. Targeting downstream damaging effects of ROS is an attractive alternative.

Rationale and Specific Aims

Reduced parasympathetic activity increases the production of highly immunogenic neoantigens, Isolevuglandins (IsoLG-adducts), through heightened activation of NADPH oxidase. Previous findings demonstrated that the cholinergic enhancer galantamine, reduces NADPH oxidase activation and that vagal stimulation reduces IsoLG-adducts in immune cells of LCPOTs patients.

Immune activation in response to viral or likely self-antigen presentation can induce formation of circulating monocyte/T cell complexes(doublets).The presence of these doublets provides powerful insights into the persistent and unresolved immune response that continues after the initial infection. The preliminary data show that: 1) LCPOTS subjects have markedly increased circulating doublets compared to controls; 2) T cells in these doublets produce higher levels of inflammatory cytokines IL-17A and IFN-ɣ than singlet T cells; and 3) inflammatory cytokines positively associate with the severity of orthostatic tachycardia in LCPOTS.

Monocytes in these doublets exhibit high levels of IsoLGs. These results support the hypothesis that LCPOTS represents a condition of unresolved immune activation due to IsoLG-adducts, which we have shown can act as neoantigens.

Study Aims Aim 1: To test the hypothesis that IsoLG-adducts directly contribute to persistent immune activation as measured by elevated monocyte/T cell doublets in LCPOTS.

For this purpose, the effect of 28-day treatment with Iso-LG-adduct scavenger, 2-hydroxybenzylamine (2-HOBA) versus placebo will be tested on circulating monocyte/ T cell doublets, and inflammatory cytokines.

Aim 2: To test the hypothesis that IsoLG-adducts directly contribute to splanchnic vasodilation, orthostatic tachycardia, and symptom burden in LCPOTS.

Aim 2 will be tested using the same experimental design as Aim 1 but will be focused on assessing changes in splanchnic venous capacitance with 2-HOBA treatment.

The proposed study will determine if IsoLG-adducts contribute to persistent immune activation, increased splanchnic venous capacitance, and orthostatic tachycardia in LCPOTS. The goal is to discover new treatment pathways for this disabling disease.

Study population: Total of 50 LCPOTS patients

Study visits: 3 in person study visits and 1 telemedicine

Study Interventions 2-HOBA: 2-hydroxybenzylamine, an IsoLGs potent scavenger.

Safety of 2-HOBA: 2-HOBA was found to be non-cytotoxic and non-mutagenic. It showed a low risk of QT prolongation. Two phase I studies on healthy subjects showed no serious side effects from 2-HOBA at doses ranging from 50 mg to 825 mg. Adverse events (AEs) reported were minor (e.g., headache, sleepiness, abdominal bloating) and were not dose-dependent.

2-HOBA is considered safe for healthy humans.

Dose: A dose of 500 mg three times daily for 28 day to study 2-HOBA for LCPOTS.

Biostatistical Considerations:

Sample size justification and study power (Aim 1). A mean percent of doublets of 5.37 and an SD of 4.28 for the LCPOTS group and a mean percent of doublets of 1.80 and an SD of 1.15 for the control group. The study is to assess for the primary comparison (2-HOBA vs. placebo). A sample size of 16 completed subjects per study arm will have 85% power to detect a 3.57 clinically meaningful difference, i.e., 5.37 versus 1.80 using a Welch t-test. Assuming a drop-out rate of 11%, we need to enroll 18 subjects to have 16 complete subjects per group. Given that subjects will be randomly assigned to two groups. The total enrollment needs to be 36 LCPOTS patients.

Sample size justification and study power (Aim 2). In a previous study, nitroglycerine produces a change from baseline in the Y-intercept of the P-V relationship of 5.7% with a standard deviation (SD) of 1.8 in healthy subjects. A clinically significant difference in the response to 2-HOBA versus placebo is assumed would be at least 35% of that produced with nitroglycerine, with conservatively slightly larger SD of 2.0. The study needs 19 patients per group to be able to reject the null hypothesis that this response difference is zero with a probability (power) of 85%. The Type I error probability associated with this test of this null hypothesis is 0.05. Assuming an attrition rate of 11%, the study needs 25 patients per treatment group. The total enrollment thus needs to be 50 LCPOTS patients.

Statistical Analyses:

The analysis population includes all randomized participants following intention-to-treat analyses.

Aim 1: For the primary comparison between 2-HOBA and placebo, a linear regression model will be used, with doublets (the primary endpoint) at the end of the 28-day treatment as the dependent variable and group indicator, baseline doublets, and other pre-specified baseline variables (age and sex) as covariates.

Aim 2: For the primary comparison between 2-HOBA versus placebo, a linear regression model will be used where splanchnic venous capacitance (SVC, primary endpoint) at the end 28-day treatment is the dependent variable and group indicator and baseline SVC and age and sex as covariates. For both aims, the null hypothesis of no group difference corresponds to the coefficient for the group indicator, which is zero. Appropriate transformation will be used on the outcome variable for the regression models when the normality assumption is violated. Other secondary endpoints will be analyzed similarly to the primary endpoint. The primary and secondary analyses will be conducted without imputing missing data. To protect privacy, subjects will receive a de-identified code. Data will be stored in a password protected, encrypted, and HIPAA compliant REDCap database.

Data and Safety Monitoring Plan:

Data and Safety Monitoring Officer (DSMO) will meet at least twice a year and review data on AEs, data quality, and study recruitment. Reports will be sent to the IRB and FDA at least yearly. Serious AEs will be reported to the FDA following guidelines using the online reporting portal.

Inclusion Criteria:

All participants should meet diagnostic criteria for Long COVID and POTS and as outlined below:

Long COVID (LC) is defined by a range of symptoms affecting multiple organs that persist for more than three months following an acute SARS-CoV-2 infection.

POTS: the presence of chronic symptoms lasting more than 3 months, along with orthostatic tachycardia (a HR increase over 30 bpm upon standing or exceeding 120 bpm without orthostatic hypotension) within 10 minutes upon standing or 75-degree head up tilt.

For patients aged 18 and 21, an increase of more than 40 bpm or a standing HR over 130 bpm will be required for inclusion in the study.

2 Patients need confirmation of POTS diagnosis based on orthostatic vital signs obtained prior to enrollment in the study.

SARS-CoV-2 infection 3 or more months prior identified by the follow signs:

A. Meets the clinical OR epidemiological criteria.

1. Clinical criteria: Acute onset of fever AND cough (influenza-like illness) OR Acute onset of ANY THREE OR MORE of the following signs or symptoms: fever, cough, general, weakness/fatigue, headache, myalgia, sore throat, coryza, dyspnea, nausea, diarrhea, anorexia.
2. Epidemiological criteria: Contact of a probable or confirmed case or linked to a COVID-19 cluster; or B. Presents with acute respiratory infection with history of fever or measured fever of ≥ 38°C; and cough; with onset within the last 10 days; and who requires hospitalization); or C. Presents with no clinical signs or symptoms, NOR meeting epidemiologic criteria with a positive professional use or self-test SARS-CoV-2 antigen-Rapid Diagnostic Test.

D. A person with a positive nucleic acid amplification test, regardless of clinical criteria OR epidemiological criteria; or E. Meeting clinical criteria AND/OR epidemiological criteria (See A). With a positive professional use or self-test, SARS-CoV-2 Antigen-Rapid Diagnostic Test.

F. Documented by health care provider in clinical note or encounter.

Exclusion Criteria:

1. Known active acute SARS-Cov-2 infection (4 weeks from onset)
2. Moderate or severe immunocompromised patients,
3. Known history of cardiovascular disease (atrioventricular block (AV block), myocardial infarction, angina, heart failure, pacemaker, stroke, transient ischemic attack within 6 months before enrollment),
4. Uncontrolled hypertension (BP>140/90 despite appropriate treatment);
5. Type 1 or type 2 diabetes mellitus;
6. Impaired hepatic function (AST or ALT greater than 1.5x the upper limit of normal or with total bilirubin ≥1.5mg/dl),
7. Impaired renal function test (eGFR<60 mL/min/1.73m2),
8. Anemia (hemoglobin <10 g/dl),
9. Pregnant or breastfeeding women,
10. Known history of autoimmune disease, steroid use or other immunotherapies,
11. Inability to provide informed consent.

We will also exclude individuals with known allergy sensitivity to components of the study medication, known contraindication to the study interventions, use of central acetylcholinesterase inhibitors (e.g., pyridostigmine, donezepil), aspirin allergy because salicylic acid is a metabolite of 2-HOBA; use of monoamine oxidase inhibitors (MAO-I) because of some inhibition of MAO-A is present in the anticipated therapeutic range of 2-HOBA.

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A licensed MTI BioTech (MTI) to develop 2-HOBA. MTI will provide 2-HOBA and matching placebo capsules for the study . Capsules will be shipped to the Vanderbilt Investigational Drug Services (IDS) for use in this trial. The IDS will be responsible for storage, and labeling of 2-HOBA and matching placebo, and for maintaining accurate drug storage and dispensing logs.