2-HOBA Phase 2 Clinical Trial in Rheumatoid Arthritis
Phase: PHASE2
Conditions: Rheumatoid Arthritis
Sponsor: Vanderbilt University Medical Center

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2-Hydroxybenzylamine acetate (2-HOBA) Phase 2 
Clinical Trial in Rheumatoid Arthritis 
 
Version 08/11/2022 
 
 
 
 
 
 
Principal Investigator: 
Michelle J Ormseth, MD, MSCI 
Assistant Professor of Medicine 
Division of Rheumatology & Immunology 
Vanderbilt University Medical Center 
 
 

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Summary of Changes from Previous Version: 
 
 
Affected Section(s) 
Summary of Revisions 
Made 
Rationale 
Presections 
 
Inclusion/Exclusion 
 
 
 
 
Study procedures 
 
 
 
 
 
 
 
 
AE reporting 
 
 
Added amendment log, page 
numbers, abbreviations. 
Added exclusion for liver 
disease/LFT abn. 
NSAID d/c as inclusion criteria 
Change corticosteroid to 
glucocorticoid 
Statement of compliance. 
Extend table 3 
With or without food.  
Treatment duration cannot 
exceed 28 days 
Added CDAI as exploratory 
outcome 
Ask subjects which treatment 
arm they suspect they were on. 
Clarified AE/SAE/unanticipated 
problem reporting 
Request of FDA or NIAMS/NCR 
prior to dual safety officer 
meeting 
 
 
 
 
 
 
 
Addition of CDAI per request of 
PI 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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ABBREVIATIONS 
2-HOBA = 2- hydroxybenzylamine 
ACR= American College of Rheumatology 
AE= Adverse event 
ALT= alanine transaminase 
AST= aspartate transaminase 
BW= body weight 
CDAI = clinical disease activity index 
CI = confidence interval 
CRC = clinical research center 
CVD = cardiovascular disease 
DAS28 = disease activity score based on 28 joint count.  
dsDNA= double stranded deoxyribonucleic acid 
DSMB= data safety monitoring board 
ESR = Erythrocyte sedimentation rate  
EULAR= European League Against Rheumatism 
GHS = Global Harmonized System  
HDL= high density lipoprotein 
HED = human equivalent dose 
hERG = human ether a-go-go  
IDS= Investigational Drug Service 
IRB = Institutional review board 
isoLG = isolevuglandin 
kg= kilogram 
Ldlr= low density lipoprotein receptor 
MAO-I = monoamine oxidase inhibitor 
MDA = malondialdehyde 
mg= milligram 
mHAQ = Modified health assessment questionnaire  
NET = neutrophil extracellular trap 
NIAMS = National Institute of Arthritis and Musculoskeletal and Skin Diseases 
ng= nanogram 
NOAEL = no-observed-adverse-effect-levels 
RA = rheumatoid arthritis 
REDCap = Research electronic data capture 
ROS= reactive oxygen species 
SAE= serious adverse event 
SBP = systolic blood pressure 
SD = standard deviation 
SLE= Systemic Lupus Erythematosus 
SO = safety officer 
ULN= upper limit of normal 
VAS = visual analog scale  
  
 
 

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Table of Contents 
1.0 BACKGROUND ..................................................................................................................................... 5 
2.0 RATIONALE AND SPECIFIC AIMS ......................................................................................................... 7 
3.0 ANIMAL STUDIES AND PREVIOUS HUMAN STUDIES .......................................................................... 7 
4.0 INCLUSION/EXCLUSION CRITERIA ..................................................................................................... 12 
5.0 ENROLLMENT/RANDOMIZATION ...................................................................................................... 13 
6.0 STUDY PROCEDURES ......................................................................................................................... 14 
7.0 RISKS OF INVESTIGATIONAL AGENTS/ DEVICES (SIDE EFFECTS) ....................................................... 18 
8.0 REPORTING OF ADVERSE EVENTS OR UNANTICIPATED PROBLEMS INVOLVING RISK TO 
PARTICIPANTS OR OTHERS ...................................................................................................................... 19 
9.0 STUDY WITHDRAWAL/ DISCONTINUATION ...................................................................................... 21 
10.0 STATISTICAL CONSIDERATIONS ....................................................................................................... 22 
11.0 PRIVACY/ CONFIDENTIALITY ISSUES ............................................................................................... 23 
12.0 FOLLOW-UP AND RECORD RETENTION .......................................................................................... 23 
13.0 REFERENCES .................................................................................................................................... 23 
 
 
 

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1.0 BACKGROUND 
Rheumatoid arthritis (RA): challenges achieving adequate disease control  
Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease affecting nearly 1.5 million 
adults in the US1,2. Early and aggressive treatment of RA improves likelihood of remission and reduces 
disability3. Despite use of aggressive “treat to target” approaches4 as few as 7% of patients achieved 
remission after 52 weeks of therapy5,6. Moreover, RA patients have two-fold increased risk of ischemic 
heart disease, which is a main contributor to early mortality in RA7. Excess inflammation increases 
ischemic heart disease in RA, but some highly effective RA drugs that lower inflammation, like tofacitinib, 
may increase risk of serious heart-related problems (FDA drug safety communication 2-4-2021). Thus, 
therapeutics for RA targeting novel pathways that treat RA and reduce cardiovascular risk are needed. A 
potential target is blocking the proinflammatory, immunogenic, and proatherogenic effects of oxidative 
stress. 
Need ROS but not the damaging effect of lipid peroxidation 
Oxidative stress occurs when reactive oxygen species (ROS) are generated in excess of normal 
antioxidants. ROS are needed during bacterial killing, mitochondrial oxidative metabolism, and other 
cellular activities critical for cell proliferation and survival8; thus, high dose antioxidants can cause harm. 
Indeed, high dose antioxidants increased mortality in trials9, and their use in RA is discouraged due to 
adverse effects on T cell function10. Targeting downstream damaging effects of ROS is an attractive 
alternative.  
Identification of isolevuglandins (isoLGs) and effective 
scavengers 
ROS causes lipid peroxidation of polyunsaturated fatty acids 
yielding 
highly 
reactive 
dicarbonyls 
such 
as 
malondialdehyde (MDA) and isolevuglandins (isoLGs). 
IsoLGs, the most reactive of these substances identified to 
date, bind covalently to proteins and DNA nearly 
instantaneously, which leads to protein misfolding, 
crosslinking, and DNA damage11. These modified proteins 
can become immunogenic and proinflammatory12-14. 
Through efforts over 20 years, our Vanderbilt colleagues, Drs 
L Jackson Roberts and Sean Davies, have identified and 
characterized 2-hydroxybenzylamine (2-HOBA), a derivative of buckwheat, that scavenges isoLGs nearly 
1000 times faster than isoLGs can bind to proteins and also scavenges other less reactive dicarbonyls like 
MDA15 (Figure 1). We and our colleagues 
have used 2-HOBA to demonstrate the effect 
of scavenging reactive dicarbonyls on disease 
models, as shown below, and also that 2-
HOBA is safe in humans16,17.  
IsoLGs as drivers of autoimmunity and 
inflammation  
IsoLGs may drive inflammatory autoimmune 
disease. For example in systemic lupus 
erythematosus (SLE) patients we found that 
isoLG-adduct positive (isoLG+) dendritic cells 
were significantly increased in 11 SLE versus 
10 control subjects13. In the B6.SLE123 
murine model of SLE, scavenging isoLGs with 
Figure 2. 2-HOBA decreased (A) anti-dsDNA titer, (B) splenic CD45+ 
cells, (C) proteinuria, and renal (D) endocapillary hypercellularity, (E) 
wire loop lesions, and (F) subendothelial deposits. (G) 2-HOBA 
decreased systolic blood pressure. (n = 6-8, *P < 0.05, **P < 0.01, 
***P < 0.001, ****P < 0.0001).13 
Figure 1. 2-HOBA scavenges highly reactive 
products of lipid peroxidation, decreasing 
inflammation amplifying protein adducts.   

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2-HOBA from 7 to 32 weeks of age, significantly decreased isoLG+ dendritic cells, anti-dsDNA titer, splenic 
cellular expansion, renal disease, and blood pressure (Figure 2)13.  
Scavenging isoLGs reduces hypertension, which is increased in RA patients 
The observation that 2-HOBA reduced blood pressure in an SLE mouse model is important given that 
presence of hypertension is increased 1.9-fold patients with SLE18 as well as 1.8-fold in established RA19 
and contributes to increased cardiovascular risk in these diseases18-23. IsoLGs may drive hypertension 
through several mechanisms. IsoLGs increase dendritic cell IL-1β, IL-6, IL-23 production and costimulatory 
proteins CD80 and CD86, which promote T cell proliferation 
and generation of IL-17 and IFN-γ, all contributing to 
hypertension24. In a murine hypertension model 2-HOBA 
prevented 
these 
effects 
and 
decreased 
cytokine 
production, T cell proliferation and ultimately blood 
pressure24.  
Scavenging isoLGs and MDA reduces atherosclerosis 
Reactive dicarbonyls also accelerate atherosclerosis, likely 
through effects on inflammation, lipoprotein modification, 
and potentially other mechanisms. Our colleagues used 2-
HOBA to scavenge reactive dicarbonyls in the Ldlr-/- mouse 
model of familial hypercholesterolemia. 2-HOBA decreased aorta MDA and IsoLG adduct content by 59% 
and 23%, respectively, decreased atherosclerosis 
60% (Figure 3A), plasma IL-6 (Figure 3B) and TNFα 
(Figure 3C) concentrations, and improved plaque 
stability features versus vehicle or nonreactive 
analogue (4-HOBA) without changing cholesterol 
concentrations14. IsoLG adducts increase cytokine 
production by inducing endoplasmic reticulum 
stress12, and scavenging them with 2-HOBA reduces 
macrophage inflammatory cytokines in vitro (Figure 
4)14. 
In 
addition 
to 
2-HOBA’s 
effect 
on 
inflammation, other factors such as decreased HDL-
adducts leading to improved HDL function may have contributed 
to 2-HOBA’s impact on atherosclerosis. Such effects would likely 
be highly beneficial in RA, a disease with increased systemic 
inflammation, 
lipoprotein 
dysfunction, 
hypertension 
and 
atherosclerosis25-27.  
IsoLGs are enriched in RA patients  
RA patients have increased oxidative stress28,29. In preliminary 
work using flow cytometry, peripheral blood isoLG+ dendritic cells 
(CD45+/HLA-DR+/CD11c+) are increased 2.7-fold in 7 RA patients 
versus 3 controls (P=0.03) (Figure 5). In a larger study, a marker of 
isoLG production, urinary F2-isoprostane concentration (non-
reactive stable lipid peroxidation product formed in parallel with 
isoLGs and reliably detected in urine, see Specific Aims Figure) was 
increased ~50% in 169 RA versus 92 control subjects29. Moreover, 
urinary F2-isoprostane concentration was associated with RA joint 
damage assessed by Larsen score independent of age, race, sex, 
hypertension, BMI and smoking29. Given increased isoLG-adducts 
in RA, isoLG association with joint damage, and isoLG increasing 
Figure 4. Reactive dicarbonyl scavenging with 2-HOBA 
reduces macrophage inflammatory cytokines versus 
vehicle or non-reactive analog 4-HOBA.  (A-C: IL-1β, IL-6, 
TNFα). N = 3 independent experiments (mean ± SEM). 
***p<0.0001.14 
Figure 5. Isolevuglandin (isoLG) positive 
dendritic cells (DC) are increased 2.7-fold 
in 
RA 
vs 
controls 
(p=0.03) 
(A). 
Representative histogram of DC isoLG 
signal, (B). Example DC isoLG gating for 
control (C) and RA (D). Unpublished 
Figure 3. In Ldlr-/- mice 2-HOBA reduced (A) 
atherosclerosis, (B) plasma IL-6 and (C) TNFα. 8 
mice/ group. *p<0.05, ***p<0.0001.14 

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proinflammatory cytokines (Figures 3&4)12,14,  reactive dicarbonyls like isoLGs may contribute to disease 
in RA.  
Reactive dicarbonyls may contribute to RA pathogenesis  
While isoLGs may be the most reactive products of oxidative stress and increase inflammation and 
autoimmunity, MDA also contributes to disease. In excess, MDA can break down to acetaldehyde and 
combine with other MDA molecules to form MDA-acetaldehyde (MAA). MAA adducts are enriched in RA 
versus osteoarthritis synovial tissue, colocalize with citrullinated protein30, and induce antibody formation 
without adjuvant31. Anti-MAA antibodies increase significantly 2-3 years prior to RA onset32. MAA-
adduction increased anti-citrullinated protein antibody concentration in an RA mouse model, and 
increased in vitro T cell response to citrullinated protein and monocyte cytokines33. These data suggest 
that reactive dicarbonyls may be co-factors in RA pathogenesis and amplify existing disease. 
Strength of scientific premise 
This trial is based on: 1) an unmet medical need for therapeutics using novel pathways that treat RA and 
reduce its associated cardiovascular risk, 2) a plausible biologic mechanism that scavenging reactive 
dicarbonyls such as isoLGs will reduce their damaging effects leading to decreased RA disease activity and 
3) compelling preclinical data that 2-HOBA decrease inflammation, autoantibodies, aberrant T cell 
function, hypertension, and atherosclerosis. 
 
2.0 RATIONALE AND SPECIFIC AIMS 
We hypothesize that 2-HOBA, which has proven safe in animal models and two phase one human clinical 
trials, will reduce RA disease activity and inflammation and reduce associated cardiovascular risk. Thus, 
the purpose of this pilot study is to determine 2-HOBA’s tolerability, safety, and effect on isoLG-adducts, 
inflammation and blood pressure in RA patients. We will perform a randomized, double blind, parallel 
group trial of 2-HOBA 750mg or placebo three times a day in up to 32 patients with RA. Patients will 
receive 2-HOBA or placebo for 4 weeks. 
 
Specific Aim 1: Determine safety and tolerability of 2-HOBA in patients with rheumatoid arthritis. 
Patients with RA with at least 4 tender or swollen joints will be randomized to placebo or 750mg 2-HOBA 
(a dose that achieves plasma concentrations in excess of those effective in vitro and in animal studies and 
previously well tolerated in humans) three times a day for 4 weeks in a double-blind study. Adverse events 
will be monitored to determine safety and tolerability of 2-HOBA compared to placebo. Treatment 
compliance by pill count will be assessed.  
 
Specific Aim 2: Determine if 2-HOBA treatment reduces isoLG-adducts, inflammation and blood 
pressure in patients with rheumatoid arthritis. Change in cellular and plasma isoLG-adducts, C-reactive 
protein, erythrocyte sedimentation rate, DAS28 score and 24-hour blood pressure will be measured 
comparing baseline to week 4 across treatment groups.  
 
The proposed pilot clinical trial will inform the feasibility and design of future studies to examine the 
efficacy of 2-HOBA in patients with RA.  
 
3.0 ANIMAL STUDIES AND PREVIOUS HUMAN STUDIES 
Acute Oral Toxicity in Rats  
A study was performed to evaluate the acute oral toxicity level of 2-HOBA acetate and to classify the 
compound into the appropriate toxicity class as defined by fixed LD50 cut-off values when administered as 
single oral dose to Wistar rats followed by a 14-day observation period. 2-HOBA acetate was administered 
in 0.5% carboxy-methyl-cellulose (CMC) at a dose of 2000 mg/kg body weight. In the first group of three 

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rats, the rats exhibited mild clinical signs, but fully recovered by day 3. In the second group of three rats, 
two animals exhibited no clinical symptoms by day 2, but one rat died on day 1 approximately 5 hours 
after 2-HOBA acetate administration. Gross pathological findings observed in dead animal were attributed 
to the test item, but no external gross abnormalities or pathological findings were detected in any of the 
other 5 animals. Based on the above results, 2-HOBA acetate was classified, per Global Harmonized 
System (GHS), as category 5 and the LD50 cut-off value was determined to be 2,500 mg/kg body weight 
when administered orally34.  
 
Short-term (28-day) Toxicity in Mice  
A total of 40 mice were randomized to 0, 0.156, 0.469, or 1.56% 2-HOBA acetate mixed in the diet by 
weight (5 male and 5 female per treatment group). After one week of feeding, mice fed the highest level 
of 2-HOBA acetate (1.56%) had reduced food intakes (36% and 25% lower intakes in male and female 
mice, respectively) when compared with control feed intakes. Due to the decreased body weight in the 
high 2-HOBA acetate (1.56%) treatment group, the mice were euthanized. Because organ and tissue 
histopathology from the high dose mice did not indicate any adverse treatment effect, the loss of body 
weight was likely due to decreased feed intake. All mice in the lower treatment dose groups survived until 
scheduled euthanasia. There were no differences in body weight, feed intake, organ weights, 
histopathology, or blood chemistry were observed between the 0.156% and 0.469% 2-HOBA acetate 
groups and the 0% control group. Based on the highest level of 2-HOBA acetate fed for the full 28-day 
study (0.469%), the no-observed-adverse-effect-levels (NOAEL) determined by this short-term study were 
610 and 721 mg/kg BW/d in male and female mice, respectively. These values correspond to human 
equivalent dosages of 49.5 and 58.5 mg/kg BW/d, respectively.34 
  
Short-term (28-day) Toxicity in Rats  
This study was performed to evaluate the toxicity of 2-HOBA acetate after 28 days of repeated oral 
administration (gavage) in Wistar rats and to characterize any observed toxicity of 2-HOBA acetate for 
indication of a dose-response relationship and determination or a NOAEL. Doses of 100, 500, and 1000 
mg 2-HOBA acetate/kg BW/day were studied and compared to a vehicle (1% carboxymethyl cellulose) 
control group; each group contained 10 male and 10 female Wistar rats. Two additional recovery (vehicle 
control and 1000 mg/kg BW/day 2-HOBA) groups were also studied; these groups were administered 
vehicle or 2-HOBA acetate for 28 days and then allowed to recover for 8 days before sacrifice. No 
morbidity or mortality was observed in any group. Rats receiving the highest dose (1000 mg/kg BW/day) 
showed slight abdominal breathing after administration but recovered within 10-15 minutes. There were 
no remarkable clinical signs or symptoms, ophthalmological findings, or behavioral findings. Some small 
statistically significant differences in organ weights and blood chemistry/hematology were observed 
between the vehicle control and dose groups, but none of the changes were determined to be of any 
toxicological significance. No toxic indications were observed on gross pathology in histopathology. The 
NOAEL based on this study was determined to be 1000 mg/kg BW.35 
 
Sub-chronic (90-day) Toxicity in Rats  
In this study, 2-HOBA acetate was orally administered to male and female rats for 90 consecutive days at 
doses of 100, 500, and 1000 mg/kg BW/d (n = 20 per sex/group). Subchronic administration of 2-HOBA 
was well tolerated at all dose levels. 2-HOBA-treated male rats were slightly heavier in the last weeks of 
the study, but this difference was very small (<5%), did not show a dose–response relationship, and was 
not observed in female rats. Similarly, some statistically significant changes in serum biochemistry and 
hematology parameters were noted, but these were not considered to be of biological or toxicological 
significance. Sporadic differences in organ weights were observed between groups, but all were small 
(<10%) and unlikely to indicate toxicity. The incidence of histopathological lesions was similar between 

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treated and control groups across all organs. Based upon these findings, the NOAEL was determined to 
be ≥ 1000 mg/kg BW/d, which was the highest dose tested.36 
 
Sub-chronic (90-day) Toxicity in Rabbits  
2-HOBA acetate was orally administered to male and female New Zealand White Rabbits for 90 days at 
doses of 100, 500, and 1000 mg/kg BW/d (n = 5 per sex/group). As previously observed in rodents, 2-
HOBA acetate administration was well tolerated. No toxic effects of 2-HOBA acetate were detected in 
body weight, feed consumption, hematology, blood chemistry, urine chemistry, organ weights, gross 
pathology or histopathology. Based on these findings, the NOAEL of 2-HOBA acetate in rabbits was 
determined to be 1000 mg/kg BW/d, which was the highest dose tested.37  
 
Long-term Administration in Mice  
A long-term study was conducted to determine if 2-HOBA would alter changes in memory and 
neurodegeneration observed in a transgenic model of Alzheimer’s disease. Wild-type C57BL/6 mice and 
mice with targeted replacement of the human ApoE4 gene were given either normal drinking water or 
water supplemented with 1 g/L 2-HOBA acetate beginning at 4 months of age and continuing for the life 
of the animal. Supplementation with 2-HOBA did not result in significant changes either in the body weight 
of aged animals (14 months) or in survival compared with mice receiving normal drinking water.38 
 
Mammalian Bone Marrow Chromosome Aberration in Mice  
This test, conducted in accordance with OECD Guideline No. 475, “Mammalian Bone Marrow 
Chromosomal Aberration Test”, examined the mutagenic potential of 2-HOBA acetate at doses up to 1000 
mg/kg in mice. Mice were administered 2-HOBA acetate (250, 500, or 1000 mg/kg), vehicle, or 
cyclophosphamide monohydrate (positive control) via oral gavage for two consecutive days. Colchicine 
(10 mL/kg) was injected intraperitoneally 3 to 5 hours prior to sacrifice to arrest cells in metaphase. 
Twenty-four hours after the final dosing, mice were sacrificed, and bone marrow cells were collected and 
stained with Giemsa stain to examine the incidence of structural aberration. The percent reduction in 
mitotic index was ≤ 4.57% at 2-HOBA doses up to 1000 mg/kg dose compared to the vehicle control, 
whereas in the positive control group, the percent reduction in mitotic index observed was 8.3-8.6%. 
Further, there were no significant increases in aberrant cells in 2-HOBA treated groups in comparison with 
the vehicle control group. The observed mean percent aberrated cells was ≤ 0.70 in all dose groups, 
compared with 5-5.6% in the positive controls. These results support that 2-HOBA is non-mutagenic at 
doses up to 1000 mg/kg (unpublished data, see investigators brochure).  
 
Prenatal Development Toxicity  
This study was conducted in accordance with OECD Guideline No. 414, “Prenatal Development Toxicity 
Study”. 2-HOBA acetate was administered to pregnant female rats from gestation day 5 through gestation 
day 20 at 0 (vehicle control), 100, 500, or 1000 mg/kg. Dams were sacrificed and fetuses excised on 
gestation day 20, and dams and fetuses were examined for signs of prenatal development toxicity. No 
signs of prenatal development toxicity were observed in dams or offspring at the 100 mg/kg dose level. 
However, significantly lower weight, weight gain, and feed consumption were noted in dams treated with 
500 or 1000 mg/kg 2-HOBA. The lower confidence limit of the BMD modeling (BMDL) for a 10% difference 
(BMDL10) in weight gain during 2-HOBA treatment (GD 5-20) was 169.3 (absolute weight gain) or 178.8 
(relative weight gain) mg/kg, while the BMDL for feed intake over the same time period was 287.5 mg/kg. 
Gravid uterus weight was reduced in the 1000 mg/kg dose group. Relative gravid uterus weight (% of body 
weight) was 19.2±2.6, 19.5±2.1, 17.1 ± 3.9, and 16.7 ± 4.1% in the 0, 100, 500, and 1000 mg/kg groups, 
respectively. The BMDL for a 5% difference (BMDL5) in relative gravid uterus weight was 187.7 mg/kg. 
There were no differences between groups in corpora lutea, implantations, early or late resorptions, pre- 

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or post-implantation loss, litter size, litter survival, or sex ratio. Significant reductions in fetal weight and 
length were observed in the 500 and 1000 mg/kg dose groups. BMDL5 values for fetal weight were 221.7 
and 207.2 mg/kg for males and females, respectively, and BMDL5 values for fetal length were 438.4 and 
395.4 for males and females, respectively. External examination revealed a significantly greater number 
of fetuses that were considered small in size as well as a significantly greater number of fetuses with 
hemorrhagic spots or pale color in the 1000 mg/kg dose group, but no differences between groups were 
noted on visceral or skeletal examination.  These data support the prenatal safety of 2-HOBA at doses up 
to 169 mg/kg in rats (human equivalent dose [HED] = 27 mg/kg), though equivocal observations (see One-
Generation Reproductive Toxicity) suggest the actual threshold dose may be higher (unpublished data, 
see investigators brochure).  
 
One-Generation Reproductive Toxicity  
This study was conducted in accordance with OECD Guideline No. 415, “One-Generation Reproductive 
Toxicity Study”. Male rats were dosed with 0 (vehicle control), 250, 500, or 1000 mg/kg 2-HOBA acetate 
beginning 10 weeks prior to mating. The 1000 mg/kg dose group was adjusted to 700 mg/kg on day 54 of 
dosing due to the emergence of clinical signs of toxicity and mortalities. Female rats were dosed with 0 
(vehicle control), 250, 500, or 700 mg/kg 2-HOBA acetate once daily for 2 weeks prior to mating and 
through gestation and lactation. Parental animals and pups were monitored for signs of reproductive 
toxicity through lactation day 21 (weaning). Males receiving 1000 mg/kg 2-HOBA began exhibiting clinical 
signs on Day 5 of treatment, and five males were found dead between days 50 and 56 of treatment. Due 
to these mortalities, the dose of 1000 mg/kg body weight group was reduced to 700 mg/kg body weight 
from Day 54 onward. All males treated with this reduced dose recovered. In females treated with 700 
mg/kg 2-HOBA, 10 out of 25 females experienced clinical signs of toxicity, but all females recovered. Lower 
body weight, weight gain, and feed consumption were observed in males treated with 500 or 1000/700 
mg/kg 2-HOBA. There were no meaningful differences in body weight, weight gain, and feed consumption 
in females prior to gestation in any dose group. Estrus cycle parameters were normal in females receiving 
250 or 500 mg/kg 2-HOBA, but 11 out of 25 females treated with 700 mg/kg 2-HOBA exhibited 
irregularities. Male fertility index was similar between the vehicle control, 250 mg/kg, and 500 mg/kg 
groups (80-88%) but was significantly lower in the 1000/700 mg/kg group (40%). Similarly, female fertility 
index was identical in the vehicle control, 250 mg/kg, and 500 mg/kg groups (92%) but was significantly 
lower in the 700 mg/kg group (36%). Significant reductions in reproductive organ weight were observed 
in the 1000/700 mg/kg groups when compared with the corresponding vehicle control groups. Gestation 
and parturition indices were significantly reduced (78%) only in the 700 mg/kg group. Body weight, weight 
gain, and feed consumption during gestation and lactation were significantly lower in females receiving 
700 mg/kg 2-HOBA. Gestation length was similar in all dose groups (22.3 - 22.7 days). There were no 
treatment-related adverse effects on delivery outcomes or litter observations in the 250 or 500 mg/kg 
groups, but a number of adverse effects were noted in the 700 mg/kg dose group, including greater pre-
and postnatal losses, significantly reduced live birth index, significantly more dead pups, greater pup loss 
from LD 1-4, and reduced live pup numbers and pup survival index through LD 14. Due to pup mortality, 
only 3 dams with live pups remained during LD 4-17, and only 2 dams with live pups remained from LD 
14-20. Among surviving pups in the 700 mg/kg group, weight was significantly reduced (11-45%, 
depending on sex and age) when compared with the vehicle control group at all postnatal time points. 
Some developmental markers (pinna unfolding, incisor eruption, eye opening, and air righting reflex 
development) were also delayed in F1 pups from the 700 mg/kg dose group. Together, these data support 
the prenatal safety of 2-HOBA at doses up to 500 mg/kg in rats (HED = 81 mg/kg) (unpublished data, see 
investigators brochure).  
 
Safety of 2-HOBA – in vivo human studies 

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A first-in-humans Phase I clinical trial using oral 2-HOBA was recently completed to assess its safety and 
pharmacologic profile. A single dose study (NCT-3176940) and a multidose study (NCT-03555682) were 
conducted, and there were no serious side effects observed in two human clinical trials16,17. In the single 
dose escalation study, 6 ascending doses of 2-HOBA were studied (50mg to 825mg as single dose). Three 
participants were enrolled at each dose and 18 participants successfully completed the study, and the 
highest planned dose was achieved (825mg)16. 5 participants reported mild adverse events. These 
included frequent urination (N=2), headache (N=1), itchy throat (N=1), rash (N=1), sleepiness (N=1), and 
abdominal bloating (N=1), none of which were dose dependent16 (Table 1). No clinically significant 
changes in ECG recordings, vital signs, or laboratory parameters were observed. 
 
 
In a multiple dose randomized double-blind clinical trial placebo, 500mg or 750mg were given 3 times a 
day to 18 healthy subjects over two weeks to assess safety, tolerability, and pharmacokinetics of 2-HOBA. 
2-HOBA dosages were chosen to achieve similar plasma levels at steady state compared with the single 
dose study. No serious AEs were observed. 14 patients reported at least 1 adverse event (Table 2). 
Reported AEs included headache (N=6), GI distress (N=3), rash/itching (N=3), urine odor (N=2), dry mouth 
(N=2), nasal congestion (N=2), lethargy/sleepiness (N=2), hypertension (N=1), and eye irritation (N=1) in 
all groups combined. One volunteer experienced a rash of moderate intensity, and was withdrawn from 
the study, though this AE was not determined to be study-related or dose-dependent. None of these were 
considered drug-related or dose dependent17. A 2-HOBA metabolite, salicylic acid, had similar 
pharmacokinetic values as single doses of 40.5–324 mg of acetylsalicylic acid (aspirin)—i.e., 2-HOBA doses 
tested gave a similar salicylic acid exposure as low doses of aspirin17. Vital signs, ECGs, and laboratory 
parameters did not significantly change17. Thus, 2-HOBA is considered safe in healthy humans. 
 
Table 1. Phase 1 dose escalation study adverse events16 
Table 2. Phase 1 repeated dose randomized controlled trial adverse events17 

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Rationale for 2-HOBA dose selection 
SLE, hypertension, and Ldlr-/- murine studies using 2-HOBA 
supplemented water at 1g/L significantly reduced isoLG and 
modified disease13,14,24. In the Ldlr-/- study after 16 weeks of 2-
HOBA, plasma level of 2-HOBA was 469 ± 38 ng/mL14. Human 
studies using 2-HOBA surpassed the observed mouse plasma 
concentration needed to reduce isoLG and MDA adducts and 
see effects; in humans after 15 days of 750mg TID dosing 
average 24-hour concentration was 1190 ± 455 ng/ml17 (Figure 
6). Thus, we choose 750mg TID, because it was safe and well 
tolerated in initial phase one studies and the highest dose 
tested in repeated dosing.  
 
4.0 INCLUSION/EXCLUSION CRITERIA 
Inclusion Criteria: 
- 
Written informed consent 
- 
Age ≥18 years 
- 
Meets 2010 American College of Rheumatology/European League Against Rheumatism 
Rheumatoid Arthritis classification criteria 
- 
≥ 4 tender or swollen joints 
- 
No change in disease-modifying antirheumatic drugs (DMARDs), glucocorticoids in ≥ 4 weeks 
- 
If of childbearing potential, willingness to use effective birth control throughout the study and 4 
weeks after completion of the study (examples: condom, diaphragm, oral contraceptive pill, 
intrauterine device) 
- 
If using non-steroidal anti-inflammatory drugs (NSAIDs), willingness to discontinue use of 
NSAIDs for 2 weeks prior to the study and throughout the study 
 
Exclusion criteria: 
- 
Pregnant or breastfeeding 
- 
Active cancer except for non-melanoma skin cancer 
- 
Active infection requiring medical intervention 
- 
Concomitant inflammatory autoimmune disease 
- 
Major surgery in ≤ 3 months 
- 
Aspirin allergy 
- 
Use of MAO-I 
- 
Estimated creatinine clearance <30 ml/min 
- 
Prior diagnosis of liver cirrhosis or the following abnormal liver function studies: AST or ALT 
>1.5x the upper limit of normal or total bilirubin ≥1.5 mg/dl 
Rationale for inclusion and exclusion criteria: 
We study adults to better exclude juvenile idiopathic arthritis, which is a separate disease. Subjects will 
have no significant change in DMARDs or glucocorticoid dose in the past 4 weeks to avoid possible 
confounding due to drugs other than 2-HOBA. Subjects will have active disease evidenced by at least 4 
tender or swollen joints.  
 
Figure 6. 2-HOBA plasma concentrations 
measured for 8 hours (Day 1) or 24 hours 
(Day 15) after initial and repeated oral 
administration in humans.17  

13 
 
Subjects will be excluded if they are pregnant or breastfeeding or unwilling to use required birth control 
if the female subject has childbearing potential because 2-HOBA has not previously been studied in 
pregnant or breastfeeding women. Prenatal development toxicity studies have not yet been published, 
but per report (see investigator’s brochure and 3.0 Animal Studies and Previous Human Studies) human 
equivalent doses of up to 27 mg/kg in one study and 81 mg/kg in another were safe. The 81mg/kg human 
equivalent dosing may be more accurate because in that animal study more doses were tested, giving a 
better estimation of a precise safe dose.  
 
Subjects with active cancer other than non-melanoma skin cancer, concomitant inflammatory 
autoimmune disease, active infection, or major surgery in the past 3 months will be excluded due to 
potential confounding effects on systemic inflammation. Patients with severe co-morbid conditions likely 
to compromise study participation will be excluded. Those with an aspirin allergy will be excluded because 
salicylic acid is a metabolite of 2-HOBA. Those using monoamine oxidase inhibitors (MAO-I) will be 
excluded due to some inhibition of MAO-A in the anticipated therapeutic range of 2-HOBA (See Safety of 
2-HOBA – in vitro), and estimated creatinine clearance <30 ml/min due to the likelihood of renal clearance 
of 2-HOBA35. In healthy control subjects 2-HOBA did not affect liver function studies; however, the effect 
of hepatic impairment on the pharmacokinetics of 2-HOBA has not been evaluated in humans. Thus, 
subjects with known liver cirrhosis or those with AST or ALT greater than 1.5x the upper limit of normal 
or with total bilirubin ≥1.5mg/dl will be excluded.   
 
5.0 ENROLLMENT/RANDOMIZATION 
Recruitment and informed consent 
Patients with RA will be recruited from subjects who have already taken part in our studies, from the 
Vanderbilt Rheumatology Clinic by word of mouth or identification in the Vanderbilt electronic medical 
record, by advertising, and through ResearchMatch (a database of individuals willing to participate in 
clinical studies that is maintained by the Vanderbilt Clinical Research Center), as we have done in the 
past39,40. Consent will be obtained by the Principal Investigator or a trained study coordinator/research 
fellow. Consent will be documented by keeping a copy of the signed consent form and will also be 
documented in the medical record. Subjects willing to participate who provide written consent will be 
screened for eligibility. Those fulfilling inclusion and exclusion criteria will be enrolled. 
 
Randomization 
Enrolled subjects fulfilling inclusion and exclusion criteria will be randomized 1:1 to 2-HOBA 750mg, or 
placebo by a permuted block randomization with block sizes of 2 and 4, stratified by age (>50 versus <=50), 
sex, and swollen/tender joint count (>8 versus <=8) to minimize the potential confounding effects from 
these factors. The study statistician, Dr Fei Ye, who has extensive expertise in clinical trials, will design the 
randomization tool within REDCap database. After an eligible patient enrolls, the study coordinator/ 
research fellow/ PI will notify the Vanderbilt Investigational Drug Service (IDS) to randomize the patient 
based on the REDCap randomization tool, and the IDS will dispense the study drug or placebo to the 
research coordinator/ research fellow/ PI in a blinded fashion.  
 

14 
 
6.0 STUDY PROCEDURES 
The study will be conducted in compliance with the protocol, International Council for 
Harmonization/Good Clinical Practice requirements (ICH/GCP), and applicable state, local and federal 
regulatory requirements. 
 
Source of 2-HOBA and placebo  
MTI BioTech (MTI) will provide 2-HOBA acetate (the same formulation as previously used16,17,34-37, but 
referred as 2-HOBA throughout) and matching placebo capsules for the study (see letter), and ship to the 
Vanderbilt Investigational Drug Service (IDS). 2-HOBA acetate and matching placebo are manufactured in 
a good manufacturing practice (GMP) providing >98% purity and encapsulated in gelatin capsule shells by 
TSI group LTD (Missoula, MT) for MTI. 
 
Screening  
Subjects providing written informed consent will undergo screening for eligibility based on inclusion and 
exclusion criteria (4.0 Inclusion/Exclusion Criteria). During the screening a history and physical will be 
performed and joints will be assessed. We will measure a complete metabolic panel, rheumatoid factor, 
and anti-CCP antibody, if not available at that time (Table 3). Urine pregnancy test will be performed on 
subjects with childbearing capacity. 
Study protocol overview  
We anticipate screening 64 subjects to randomize up to 32 subjects with the goal of 24 patients 
completing the study (allowing for up to 25% dropout) for the 4-week study. At week 0 subjects will be 
randomized 1:1 to receive either 2-HOBA 750mg, or placebo three times a day for 4 weeks in combination 
with their baseline DMARD therapy for RA. Subjects may take 2-HOBA fasting or with food. History and 
physical will be performed and venous blood will be drawn at each study visit. A window of up to 10 days 
will be permitted to for scheduling study visit dates to permit flexibility for patient convenience; however, 
the treatment duration cannot exceed 4 weeks (28 days). The effect of varying time within the 10-day 
window will be examined.  
 
Concurrent medication use during study 
Subjects will be asked to discontinue use of non-steroidal anti-inflammatory drugs for 2 weeks prior to 
the baseline visit and throughout the study, because the cyclo-oxygenase (COX) enzymatic pathway can 
generate certain isoLG adducts41,42; however, 2-HOBA does not inhibit cyclooxygenases17. Subjects and 
their physicians will be instructed to maintain DMARD and glucocorticoids at baseline dosages during 
protocol participation. If the subject’s physician feels that a change in disease status warrants a significant 
change in therapy, the patient will be brought in for an exit visit and withdrawn from the study. Patients 
will be asked to avoid injections of glucocorticoids if possible. However, if a joint is injected, that joint will 
be excluded from the joint count analysis. Women of childbearing potential will be asked to use effective 
birth control throughout the study and for 4 weeks after completion of the study. At each visit we will ask 
each patient about medications, doses, any new prescription medications, and use of over the counter or 
herbal medications.  
 
Monitoring parameters 
At each study visit a physical examination and RA disease activity indices including tender and swollen 
joint counts and modified health assessment questionnaire will be collected. Clinical details, including, 

15 
 
current and past smoking history, and a cumulative medication history with attention to cumulative 
glucocorticoids, DMARDs, and cardiovascular therapies will be obtained from the patient and medical 
record at baseline (Figure 7). At the week 0 visit a urine pregnancy test will be obtained on subjects with 
childbearing capacity prior to receiving drug. 
 
At each visit traditional office blood pressure will be measured. The average of two blood pressure 
readings separated by 1-2 minutes will be obtained in the following validated43 manner: subjects will be 
in the seated position at rest for at least 5 minutes prior to measurement. The arm will be supported at 
the level of the right atrium (midpoint of the sternum), and the same arm will be used for all blood 
pressure measurements. Correct cuff size so that the bladder excircles 75-100% of the arm will be used.  
 
At week 0 and 4 subjects will be fitted with a 24-hour blood pressure monitor to measure ambulatory 
blood pressure. The device will be 
programmed to measure blood 
pressure at 15-minute intervals 
during the day (6am-10pm) and 30-
minute intervals during the night 
(10pm-6am). Subjects will be asked 
to wear the monitor for 24-hours 
total. Subjects will be asked to 
record activities such as exercise 
during this time. Subjects may 
temporarily remove the monitor 
for bathing, as needed. Subjects 
will not start 2-HOBA until after 
completing 24-hour blood pressure 
measurement at week 0.  
 
Venous blood will be collected and immediately processed for studies or stored (Table 3). Although there 
is no evidence that 2-HOBA alters blood counts, hepatic or renal function, we will obtain safety labs 
including complete blood count at study visits Week 0 and 4 and complete metabolic panel at screening 
and study visits Week 0 and 4. Additionally, we will measure hs-CRP and ESR at study visits Week 0 and 4. 
These will be measured in the hospital clinical laboratory.  
 
Subjects will be asked about side effects in a standardized manner at each visit. Pill counts will be 
performed at the week 4 study visit. Subjects will also be asked at study completion which treatment arm 
they believe they were assigned. 
 
The study coordinator will contact the patient between study visits to inquire about potential side effects 
and ensure compliance with protocol. 
 
Primary outcome measures: 
1) Safety and tolerability 
We will determine safety and tolerability as a primary outcome through adverse event reporting 
of the 2-HOBA arm compared to the placebo arm. At each study visit, patients will be directly 
questioned on potential side effects or other adverse events. Rates of adverse events will be 
compared between active and placebo arms and presented as summary statistics.  
2) Cellular isoLG-adducts 
Figure 7. Clinical trial protocol 

16 
 
PBMCs collected at weeks 0 and 4 will be isolated by Ficoll separation. Change in cellular isoLG 
adducts between week 0 and week 4 comparing active and placebo arms will be a primary 
outcome.  
 
Other prespecified exploratory outcome measures: 
1) Compliance 
Treatment compliance will be assessed through pill counts at week 4.  
2) Plasma isoLG-adducts 
Total plasma isoLG-adducts will be measured from week 0 and 4 stored plasma by Dr. Sean Davies’ 
lab, who developed the technique44 using liquid chromatography tandem mass spectrometry 
(LC/MS)14,44,45. Change in total plasma isoLG-adducts between week 0 and week 4 comparing 
active and placebo arms will be measured. 
3) Disease activity score based on 28 joints (DAS28 score) and the Clinical Disease Activity Index 
(CDAI) 
Disease activity will be measured as the DAS28 score and CDAI at weeks 0 and 4. Change in DAS28 
score and CDAI between week 0 and week 4 comparing active and placebo arms will be measured. 
4) High-sensitivity C-reactive protein (CRP) 
Hs-CRP will be measured at weeks 0 and 4 in the hospital clinical laboratory. Change in hs-CRP 
between week 0 and week 4 comparing active and placebo arms will be measured. 
5) Erythrocyte sedimentation rate (ESR) 
ESR will be measured at weeks 0 and 4 in the hospital clinical laboratory. Change in ESR between 
week 0 and week 4 comparing active and placebo arms will be measured. 
6) Plasma interleukin 6 (IL-6) 
The Olink Inflammation proteomics panel will be used to measure plasma concentration of IL-6. 
Change in IL-6 between week 0 and week 4 comparing active and placebo arms will be measured. 
7) Plasma tumor necrosis factor α (TNFα) 
The Olink Inflammation proteomics panel will be used to measure plasma concentration of TNFα. 
Change in TNFα between week 0 and week 4 comparing active and placebo arms will be 
measured. 
8) 24-hour systolic and diastolic blood pressure  
The average systolic and diastolic blood pressure over 24-hours of ambulatory monitoring will be 
calculated at weeks 0 and 4. The change in 24-hour systolic and diastolic blood pressure between 
week 0 and week 4 comparing active and placebo arms will be measured.  
 
 
 
 

17 
 
 
 
Table 3. Study events and measures 
Procedure/Lab 
Screen 
 
~Day -
90 to 
Day -4 
Pre-Visit 
contact  
(telephone) 
~Day -5 to   
Day -2 
Baseline 
Week 0 
Visit 
Day 0 
Between 
study visit 
contact 
(telephone) 
~Day 10-20 
Pre-Visit 
contact 
(telephone) 
~Day 23 to   
Day 261 
Week 4  
Visit 
 
Day 28 
+/- 10 
Informed Consent 
+ 
 
 
 
 
 
History  
+ 
 
+ 
 
 
+ 
Exam 
+ 
 
+ 
 
 
+ 
Joint Count 
+ 
 
+ 
 
 
+ 
Disease activity score based on 28 
joints (DAS28)/ Clinical disease 
activity score (CDAI) 
 
 
+ 
 
 
+ 
Office blood pressure 
 
 
+ 
 
 
+ 
24-hour blood pressure 
 
 
+ 
 
 
+ 
Modified health assessment 
questionnaire (mHAQ) 
 
 
+ 
 
 
+ 
Randomization 
 
 
+ 
 
 
 
Adverse event inquiry 
 
+ 
+ 
+ 
+ 
+ 
Pill count 
 
 
 
 
 
+ 
Rheumatoid factor2 
+ 
 
 
 
 
 
Anti-CCP antibody2 
+ 
 
 
 
 
 
Urine pregnancy test3 
+ 
 
+ 
 
 
 
Complete blood count4   
 
 
+ 
 
 
+ 
Complete metabolic panel4  
+ 
 
+ 
 
 
+ 
hs-C-reactive protein  
 
 
+ 
 
 
+ 
Erythrocyte sedimentation rate  
 
 
+ 
 
 
+ 
Plasma storage 
 
 
+ 
 
 
+ 
Serum storage 
 
 
+ 
 
 
+ 
Urine storage 
 
 
+ 
 
 
+ 
DNA storage 
 
 
+ 
 
 
 
Study Completion  
 
 
 
 
 
+ 
Cellular isoLG-adducts (measured 
after study completion) 
 
 
+ 
 
 
+ 
Plasma isoLG-adducts (measured 
after study completion) 
 
 
+ 
 
 
+ 
Olink inflammation panel (measured 
after study completion) 
 
 
+ 
 
 
+ 
Plasma 2-HOBA concentration 
(measured after study completion) 
 
 
+ 
 
 
+ 
1 The pre-visit contact prior to the Week 4 visit should occur ~2-5 days prior to the scheduled Week 4 visit. 
2Rheumatoid factor and anti-CCP antibody tested only if information is not available in EHR. 3 Urine pregnancy 
test only in patients of childbearing capacity. 4 Complete blood count and complete metabolic panel tested if not 
available in the last 30 days at the time of screening and/or baseline visit.  

18 
 
 
 
7.0 RISKS OF INVESTIGATIONAL AGENTS/ DEVICES (SIDE EFFECTS) 
2-HOBA is currently available in the food chain as both a compound found naturally in buckwheat as well 
as used as a self-affirmed generally recognized as safe “GRAS” ingredient in food. Based upon the currently 
available data, 2-HOBA acetate is well-tolerated in healthy individuals at doses within the expected 
therapeutic range, and there is a low risk of toxicity or adverse reactions. 
 
There are signs of prenatal or reproductive toxicity in preclinical studies at human equivalent doses above 
the proposed dose for this study. Thus, women of childbearing capacity are asked to maintain effective 
birth control throughout the study and pregnant or breastfeeding women will be excluded.  
 
In preclinical in vitro studies 2-HOBA was not cytotoxic or mutagenic; however, the characterization of the 
genotoxic potential of 2-HOBA is incomplete. In preclinical in vitro studies 2-HOBA did not induce CYP1A2, 
CYP2B6, or CYP3A4 enzyme mRNA expression; and had low risk of QT prolongation based on human ether 
a-go-go (hERG) inhibition35. In healthy control subjects 2-HOBA did not affect liver function studies; 
however, the effect of hepatic impairment on the pharmacokinetics of 2-HOBA has not been evaluated in 
humans. Thus, subjects with known liver cirrhosis or those with AST or ALT greater than 1.5x the upper 
limit of normal or with total bilirubin ≥1.5 mg/dl will be excluded. 2-HOBA had low binding to plasma 
proteins, suggesting low risk for plasma protein-mediated drug-drug interactions. It has no preferential 
binding to red blood cells, permitting accurate measurement of plasma concentration35. The main 2-HOBA 
metabolites were salicylic acid, which is also a metabolite of aspirin, and the glycoside conjugate of 2-
HOBA, which is an which is a pathway for urinary or bile excretion of compounds35.  
  
Because of these findings we have conservatively excluded those with aspirin allergy because salicylic acid 
is a metabolite of 2-HOBA, and excluded those using monoamine oxidase inhibitors (MAO-I) due to some 
inhibition of MAO-A in the anticipated therapeutic range of 2-HOBA (See Safety of 2-HOBA – in vitro), and 
estimated creatinine clearance <30 ml/min due to likelihood of renal clearance of 2-HOBA35. 
 
Risk of the study include pain and bruising at the site of the blood draw, and on occasion a person can 
faint from a blood draw. 24-hour blood pressure monitoring may be annoying, possibly interfere with 
sleep, and could leave some skin irritation or bruising. There is a theoretic risk of loss of 
privacy/confidentiality. However, as described above, each participant will receive a code which will be 
used on biospecimen and the research database. Thus, it is unlikely that breach of privacy would occur. 
There is the possibility that 2-HOBA may have side effects that have not so far been identified. 
 
Risks of the study will be minimized by performing studies in the Vanderbilt Clinical Research Center (CRC). 
All protocols must undergo approval by the Vanderbilt IRB.  A complete history, physical examination and 
routine laboratory tests will be performed to ensure that subjects fulfill the entry criteria as defined. 
Vanderbilt University/Hospital does not provide coverage for injury occurring as a result of research. The 
CRC and its staff will provide emergency treatment for illness which results directly from the study but 
provides no other compensation. There are no alternative procedures which would allow us to obtain the 
information outlined in this proposal. 
 

19 
 
8.0 REPORTING OF ADVERSE EVENTS OR UNANTICIPATED PROBLEMS INVOLVING RISK TO 
PARTICIPANTS OR OTHERS 
Data and Safety Monitoring Plan 
The National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) has determined that 
independent data and safety monitoring oversite will be performed by dual Safety Officers (SOs), which 
NIAMS will appoint. NIAMS utilizes a contract research organization, Navitas Clinical Research, Inc (NCR) 
which serves as an Executive Secretary. Information on adverse events, adverse drug reactions, data 
quality, and study recruitment will be provided to NCR, and NCR will provide the information to the dual 
SOs at least twice a year. These data will be sent to the IRB at least yearly.  
The dual SOs will review all serious adverse events (AEs). Any serious AE will be reported to the IRB and 
FDA and NCR as soon as possible, but not more than 48 hours from the investigators’ awareness of the 
event. NCR will provide the information to the dual SOs and NIAMS. Any untoward medical event will be 
classified as an AE, regardless of its causal relationship with the study. 
 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or Others: 
 
Definitions of adverse events: an adverse event (AE) is "any untoward medical occurrence in a patient or 
clinical investigation subject administered a pharmaceutical product and which does not necessarily have 
a causal relationship with this treatment". 
 
Adverse events include: 
- Worsening (change in nature, severity, or frequency) of conditions present at the onset of the 
trial 
- Patient/subject deterioration due to the primary illness 
- Intercurrent illnesses 
- Drug interactions 
- Events related or possibly related to concomitant medications 
- Abnormal laboratory values or changes of vital signs, as well as significant shifts from baseline 
within the range of normal, which the Investigator considers clinically significant. 
 
 
A serious adverse event/experience (SAE) or reaction is any untoward medical occurrence that at any 
dose: 
- results in death 
- is life-threatening 
- requires inpatient hospitalization or prolongation of existing hospitalization 
- results in persistent or significant disability/ incapacity (as per reporter’s opinion) 
- is a congenital anomaly/birth defect 
- is another medically important condition 
- The term "life-threatening" in the definition of "serious" refers to an event in which the patient 
is at risk of death at the time of the event; it does not refer to an event that hypothetically might 
have caused death if it were more severe. 
 
Important medical conditions that may not result in death, be life-threatening or require hospitalization 
may be considered SAEs when, based upon appropriate medical judgment, they may jeopardize the 
patient or may require intervention to prevent one of the outcomes listed in the definition above. 
Examples of such events are intensive treatment in an emergency room or at home for allergic 

20 
 
bronchospasm; blood dyscrasias or convulsions that do not result in hospitalization; or development of 
drug dependency or drug abuse. 
 
Unanticipated Problem: An unanticipated problem is any incident, experience or outcome that meets all 
the following requirements:  
1. Unexpected (in terms of nature, severity, or frequency) given (a) the research procedures that 
are described in the IRB-approved research protocol and informed consent document; and (b) 
the characteristics of the participant population being studied; 
2. Related or possibly related to participation in the research. Possibly related means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
3. Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
 
 
Definition of severity of adverse events: 
Mild:   
Causing no limitation of usual activities; the subject/patient may experience slight 
discomfort. 
 
Moderate:  
Causing some limitation of usual activities; the subject/patient may experience 
annoying discomfort. 
 
Severe:  
Causing inability to carry out usual activities; the subject/patient may experience 
intolerable comfort or pain. 
 
Definition of adverse event causality: 
The Investigator will determine causality of each adverse event by using the classification criteria: not 
related, possibly/probably related, definitely related. 
 
Not related:  
The AE is considered by the Investigator to be due to a pre-existing condition, a 
known manifestation of the target disease, a recurrent condition, or is likely explained by 
environmental or diagnostic therapeutic factors or was pre-existing and did not deteriorate. 
 
Possibly/Probably related:  
 
The AE occurred during or after administration of the 
study treatment or a preexisting event worsened within an appropriate period of time, and at 
least one of the following criteria is applicable: 
- 
the event could not be explained by the clinical condition or history of the subject, 
environmental or toxic factors, or other diagnostic or therapeutic measures; 
- 
AE subsided or disappeared after withdrawal or dose reduction of study treatment; or 
- 
AE recurred after re-exposure to study treatment. 
 
Definitely related: The AE is a known side effect. (Prior studies to date have not demonstrated 
specific side effects of 2-HOBA which are observed more than placebo.) 
 
 

21 
 
Definition of unexpected adverse events: An unexpected adverse event is “an adverse reaction, the nature 
or severity of which is not consistent with the applicable product information (e.g., Investigator’s Brochure 
for an unapproved investigational medicinal product)”.  
 
 
Adverse event reporting: 
Any non-serious adverse events (AEs) will be reported to the PI within 72 hours of notification of the 
event. The PI will notify NCR who will notify the dual SOs of any serious adverse events as discussed below. 
Non-serious AEs and incidences of noncompliance with the protocol will be reported to the IRB at the 
time of annual review. All AEs are reported in aggregate as part of the routine data and safety monitoring 
report provided to the Monitoring Body and the NIAMS (through the NIAMS Executive Secretary) twice 
per year.  
 
The study team will use the Redcap AE form and the separate AE report form to collect the information 
and the PI will assess each event.  
 
 
Serious Adverse Events (SAEs) and unanticipated problems will be reported according to the following 
procedure: 
 
The occurrence of serious adverse events will be reported to the Investigator within 24 hours after 
notification of their occurrence. The Investigator will report SAEs to the dual SOs and the Vanderbilt 
University Medical Center Institutional Review Board within 48 hours of the Investigator’s notification of 
the event. 
 
In an unanticipated event of prolonged side effect, requiring prolongation of hospital stay, patients will 
be retained in the hospital until side effects have resolved. For minor side effects, where inpatient care is 
deemed unnecessary, follow-up will be maintained via phone or as outpatient if necessary. Patient and 
their families will be given the PI’s contact number for reporting any other effects of medication following 
discharge. 
 
Any newly discovered information which may affect the subject or their caregiver’s decision to continue 
to participate in the study will be passed on to them as soon as possible. This may also result in a change 
to the consent form and review by the IRB. 
 
9.0 STUDY WITHDRAWAL/ DISCONTINUATION 
Participants may withdraw from the study at any time by informing the study staff verbally or in writing. 
Contact information for the PI and study staff will be made available to the participant upon enrollment 
in the consent document. Subjects will be encouraged to conduct an exit study visit prior to withdrawing 
from the study. Any data or biological samples prior to their withdrawal request will not be withdrawn 
and destroyed. 
 
A participant may be withdrawn from the study by the PI if the participant does not/ cannot comply with 
the study protocol, such as failure to attend study visits or needing to change RA treatment (changing 
DMARD or oral glucocorticoid). 
 

22 
 
Participants who elect to prematurely discontinue the study drug or must discontinue study drug per the 
PI recommendation due to adverse event, will discontinue study drug but will be asked to still complete 
the trial study visits.  
 
10.0 STATISTICAL CONSIDERATIONS 
Statistics: 
Baseline characteristics will be compared between subjects in the active and placebo arms using Student’s 
t-tests or Wilcoxon rank sum tests for continuous variables, and chi-square tests or Fisher's exact tests for 
proportions. Adverse events, pill counts, and drop-out rate will be presented as summary statistics for 
comparison between active and placebo arms. A modified intention-to-treat analysis using last 
observation carried forward method will be used on all randomized patients who take at least one dose 
of study drug. Thus subject data from Baseline Week 0 or an early exit visit will be carried forward to the 
week 4 study visit if not completed. Subjects who never receive drug will be excluded from analysis. 
Subjects who discontinue drug either by choice or request by PI due to adverse event and complete the 
week 4 study visit will be included in the modified intention-to-treat analysis. Separately an exploratory 
on-drug analysis will be conducted for subjects who continue drug through the week 4 study visit. 
 
The primary analysis will compare cellular isoLGs from week 0 to week 4 in active versus placebo arms 
adjusting for baseline cellular isoLGs. Other exploratory prespecified outcomes will similarly compare 
week 0 and 4 (for active drug analysis) (Table 4) and use repeated measures analysis for cellular isoLGs 
and available secondary outcomes using repeated measure ANOVA or other mixed-effect models to 
assess treatment effect over time. Treatment 
by time interaction effect will be estimated 
and graphically summarized. We will perform 
a per protocol analysis of those completing the 
full study. We will report the adjusted 95% 
confidence intervals (CIs) of the mean 
difference between study groups and time 
points.  
 
The study is not powered for subgroup 
analyses based on specific clinical features.  
 
Sample Size and Power  
We plan to study up to 32 RA patients, 
assuming up to 25% dropout, to provide 
complete data on ≥24 RA patients. This is 
feasible based on a clinical trial of similar size and eligibility criteria we conducted previously39. The sample 
size is based on a change in cellular isoLGs. In our pilot study mean ± SD of isoLG+ dendritic cells was 7.3% 
± 3.0%. 24 RA patients (12 each in active and placebo arms) would provide 80% power to detect a 50% 
reduction in cellular isoLGs from week 0 to week 4 at a 2-sided 5% significance level. Estimated detectible 
differences for secondary outcomes are in Table 4. 
 
Table 4. Minimal detectible difference of outcomes  
 
Mean SD 
Detectable 
difference 
Primary outcome 
Cellular isoLGs, % cells  
7.3 ± 3.0 
3.65 
Other prespecified outcomes 
Plasma isoLG-adducts, pmol/mg 
unavailable 
 
DAS28, units 
4.58 ± 1.1 
1.66 
CRP, mg/dl 
12.69 ± 24.57 
37.11 
ESR, mm/hr 
22.8 ± 21.3 
32.17 
TNFα, pg/ml 
15.77 ± 17.86 
26.98 
IL6, pg/ml 
15.66 ± 31.94 
48.25 
24hr SBP, mmHg 
124 ± 14 
17 
 

23 
 
11.0 PRIVACY/ CONFIDENTIALITY ISSUES 
Each participant will have a unique code to de-identify the participant’s data. Participant medical 
information will be stored in a REDCap database. Protected information such as names and medical record 
numbers of the study participants will be designated as non-exportable, protected fields. Access to this 
information will be granted only to members of the study team.  
 
Biospecimens will be labeled with each participant’s unique code.  
 
If the results of this study are published, only the participant’s unique code will be used for identification 
purposes.  Participants will not be identified by name.  
 
 
12.0 FOLLOW-UP AND RECORD RETENTION 
Participant data and biospecimen data will be stored in the RedCap database for an indefinite period of 
time. Follow-up with the participant will be completed after exiting the study.  
 
13.0 REFERENCES 
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Myasoedova E, Crowson CS, Kremers HM, Therneau TM, Gabriel SE. Is the incidence of 
rheumatoid arthritis rising?: results from Olmsted County, Minnesota, 1955-2007. Arthritis 
Rheum. 2010;62(6):1576-1582. 
3. 
Demoruelle MK, Deane KD. Treatment strategies in early rheumatoid arthritis and prevention of 
rheumatoid arthritis. Curr Rheumatol Rep. 2012;14(5):472-480. 
4. 
Smolen JS, Aletaha D, Bijlsma JW, et al. Treating rheumatoid arthritis to target: 
recommendations of an international task force. Ann Rheum Dis. 2010;69(4):631-637. 
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Konijn NPC, van Tuyl LHD, Boers M, et al. Do Short and Sustained Periods of American College of 
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Radiographic Outcome in Early Rheumatoid Arthritis Patients With Low Overall Damage 
Progression? Arthritis Care Res (Hoboken). 2017;69(7):989-996. 
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Ajeganova S, Huizinga T. Sustained remission in rheumatoid arthritis: latest evidence and clinical 
considerations. Ther Adv Musculoskelet Dis. 2017;9(10):249-262. 
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Solomon DH, Karlson EW, Rimm EB, et al. Cardiovascular morbidity and mortality in women 
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Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in 
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Miller ER, 3rd, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-analysis: 
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2005;142(1):37-46. 
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Yang Z, Shen Y, Oishi H, et al. Restoring oxidant signaling suppresses proarthritogenic T cell 
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