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.