Experimentally Evaluating the Hypothesized Mechanism of Action of N-acetylcysteine for Bipolar Disorder
Phase: PHASE2
Conditions: Bipolar Disorder, Bipolar I Disorder, Bipolar II Disorder
Sponsor: Medical University of South Carolina

 
 
 
 
 
 
 
Experimentally Evaluating the Hypothesized Mechanism of Action of N-acetylcysteine for Bipolar 
Disorder  
PI: James J. Prisciandaro, PhD 
Protocol 
4/15/2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Specific Aims 
Despite 70 years of medication development, bipolar disorder (BD) remains one of the leading drivers of 
global disease burden. Though existing BD medications are partially effective in treating the symptoms and 
sequela of BD, they often leave patients with significant residual mood symptoms and cognitive dysfunction. 
These impairments may be due to cellular damage caused by excessive oxidative stress (e.g., increased lipid 
peroxidation, DNA/RNA damage) that is implicated in the neuroprogression of BD. Glutathione (GSH), the brain’s 
first line of defense against oxidative stress, neutralizes free radicals and reactive oxidants, while providing 
protection and redox regulation of cellular thiol proteins via sglutathionylation. We compared dorsal Anterior 
Cingulate Cortex (dACC) GSH levels of BD and healthy control (HC) participants from the PI’s NIH K23 study 
and, consistent with findings from a recently published meta-analysis, found that BD had significantly higher 
dACC GSH levels than did HC participants (p=0.026, Cohen’s d=0.66), interpreted as compensatory GSH 
upregulation in response to mild-tomoderate levels of oxidative stress. Despite this endogenous compensation, 
however, individuals with BD continue to experience pathologically elevated levels of oxidative stress. 
N-acetylcysteine (NAC), a safe and well-tolerated medication, supplements endogenous GSH levels by 
providing cells with the rate-limiting component of GSH synthesis (i.e., cysteine) in exchange for glutamate. 
Though research has long demonstrated that NAC increases peripheral GSH levels, investigation of the effects 
of NAC on brain GSH levels has been limited to a few studies in people with Parkinson’s disease and early 
psychosis, which found that NAC significantly increased cortical GSH levels. Based on the hypothesis that NAC 
treats residual BD impairments by increasing brain GSH levels, a number of randomized clinical trials (RCTs) of 
adjunctive NAC for BD have been conducted over the past 10-15 years. A recent meta-analysis of these RCTs 
found a statistically-significant, moderate effect of NAC on reducing depressive symptoms; secondary data 
analyses further demonstrated that NAC significantly increased working memory performance. Despite this 
support for NAC as a promising adjunct treatment for BD, the (GSH-mediated) mechanistic hypothesis on which 
these studies have been predicated has never been tested in BD individuals, and a plausible (glutamate-
mediated) alternative hypothesis exists. 
It is against this background that the proposed randomized, double-blind, placebo-controlled, crossover MRI 
study aims to measure and experimentally-increase GSH levels in dACC (selected among candidate brain 
regions given its central role in guiding motivated human behavior), using NAC (3g/day), in BD participants. 
Twelve BD participants will complete two, 2-week experimental conditions (NAC, placebo) in a randomized order, 
separated by a 2-week washout. The study will evaluate the following hypotheses: 
Primary: NAC treatment will significantly increase dACC GSH levels, relative to placebo, in BD individuals. 
Exploratory: 1) NAC treatment may significantly reduce dACC glutamate levels, relative to placebo, in BD 
individuals. 2) Associations of NAC-related changes in dACC GSH and glutamate levels with peripheral 
biomarkers of oxidative stress (plasma GSH, s-glutathionylation), mood, and cognition will be explored. 
 
The proposed study will be conducted by a BD research team with a successful 10+ year record of 
collaboration, using cutting-edge proton magnetic resonance spectroscopy methods that have been optimized 
by the PI. Positive results would directly support the hypothesized mechanism of NAC treatment for BD. Perhaps 
more importantly, positive results would provide evidence for a novel neurobiological treatment target for BD, 
which would support the development of medications designed to increase brain GSH levels (i.e., with greater 
specificity and efficacy than NAC [e.g., γ-glutamylcysteine ethyl ester]) and their evaluation using the proposed 
experimental neuroimaging framework. Finally, the proposed study will add to the literature on the relative 
associations of changes in brain GSH and glutamate levels, peripheral biomarkers of oxidative stress, and BD 
clinical phenomenology (i.e., mood symptoms and cognitive impairments). 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
A. SIGNIFICANCE 
A.1. Overview. Despite 70 years of medication development, bipolar disorder (BD) remains one of the leading 
drivers of global disease burden and mortality (Ferrari, 2016). Though existing BD medications are partially 
effective in treating the symptoms and sequela of BD, they often leave patients with significant residual cognitive 
dysfunction (Jensen, 2016), depressive symptoms, and other functional impairments (Perlis, 2006; Miskowiak, 
2017). Convergent evidence supports brain glutathione (GSH) as a promising interventional target for adjunctive 
treatment in BD and N-acetylcysteine (NAC) as the best available drug to experimentally engage this target. 
A.2. Brain GSH levels as a Promising Treatment Target in BD. Cellular damage caused by oxidative stress 
(OS; e.g., increased lipid peroxidation, DNA/RNA damage, nitric oxide) has been implicated in the 
neuroprogression of BD (Brown, 2014). BD individuals reliably demonstrate both mood-episode dependent (e.g., 
thiobarbituric acid reactive substances [TBARS], uric acid) and independent (e.g., malondialdehyde, total nitrites) 
elevations in OS biomarkers (relative to healthy control [HC] participants; Jimenez-Fernandez; 2021) that are 
associated with more years of BD illness (e.g., 3-Nitrotyrosine, Andreazza, 2009) and a greater number of lifetime 
manic episodes (e,g., DNA oxidation; Soeiro-de-Souza, 2013). There are a number of, likely interacting, causes 
of the pro-oxidative neural environment found in BD, including mitochondrial dysfunction (Scaini, 2016), and 
abnormal glutamatergic (Sattler, 2001) and dopaminergic (Hastings, 2009) transmission. GSH, the brain’s 
primary antioxidant, confers a critical first-line of defense against OS-induced cellular damage. Aside from 
neutralizing radicals and reactive oxidants, GSH provides protection and redox regulation of cellular thiol proteins 
via s-glutathionylation, a post-translational modification (Townsend, 2007). Though brain GSH levels become 
depleted under extreme neurological conditions (e.g., Alzheimer’s/Parkinson’s diseases [Mazzetti, 2015]), GSH 
levels are upregulated in response to mild-moderate OS (e.g., healthy aging [Tong, 2016], mild cognitive 
impairment [Duffy, 2014], early psychosis [Wood, 2009], drug/toxin exposure [Hoffman, 2005; Smith, 2005]), and 
elevated GSH levels are associated with increased depressive symptoms (Duffy, 2015) and impaired executive 
functioning (Duffy, 2014) in older adults.  
We compared dorsal Anterior Cingulate 
Cortex (dACC) GSH levels from BD (n=29) 
and HC (n=21) participants from the PI’s K23 
study (AA020842) and found that BD had 
significantly higher dACC GSH levels than did 
HC participants (t=2.30, p=0.026, Cohen’s 
d=0.66; 
Figure 
1), 
consistent 
with 
compensatory upregulation to moderate OS 
(Tong, 2016). As described in Prisciandaro 
(2017), GSH levels were acquired via 2D J-
resolved PRESS, fit via the ProFit algorithm 
(Prescot, 2013), normalized to unsuppressed 
water, 
and 
corrected 
for 
within-voxel 
cerebrospinal fluid (CSF) fraction. BD and HC 
participants did not differ on demographics 
(Table 1) or gray matter (GM) to brain matter 
(GM+WM) 
proportion 
(both 
Ms=0.61, 
p=0.714). Consistent with Tong (2016), age 
was positively associated with dACC GSH 
levels (r=0.33, p=0.023), and covarying age 
increased the statistical reliability of the 
association of BD with GSH levels (F=7.38, 
p=0.009). Within BD participants, GSH levels 
did not differ by subtype (t=0.77, p=0.447) and 
were not associated with Young Mania 
(YMRS; Young, 1978) (r=0.25, p=0.197) or 
Montgomery-Asberg Depression (MADRS; 
Montgomery, 1979)  (r=-0.22, p=0.259) Rating 
Scale 
scores, 
possibly 
due 
to 
range 
restrictions on these measures. Medications, 
evaluated overall (taking/not-taking any) and 
Figure 1. dACC glutathione/water, in Institutional Units (I.U.), by group.  

by class (list in Table 1), were not associated with GSH levels (ps=0.275-0.964). Consistent with our findings, a 
meta-analysis of ACC GSH levels in BD versus HC participants confirmed that BD have significantly higher levels 
of ACC GSH than HC participants, and that BD medication is not associated with ACC GSH levels (Das, 2019). 
In sum, BD have elevated dACC GSH levels, interpreted as compensatory upregulation in response to 
excess OS. Despite this compensation, however, they maintain pathologically-elevated levels of OS.  
A.3. NAC as the Best Available Drug to Experimentally Modulate Brain GSH levels in BD. NAC (the N-
acetyl derivative of L-cysteine) has been safely used for ˃30 years to treat acetaminophen overdose and lung 
conditions requiring mucolytic therapy (FDA Insert). NAC increases GSH levels by providing cells with cysteine, 
the rate-limiting component of GSH synthesis (Ballatori, 2009). Though studies have consistently demonstrated 
that NAC increases peripheral GSH levels, there have been few published investigations of NAC effects on brain 
GSH levels, in part because measuring brain GSH levels in vivo requires specialized methods (Sanaei, 2017). 
A study in people with Parkinson’s disease found that NAC infusion significantly increased occipital GSH levels 
(Holmay, 2013), while another study in people with early psychosis found that chronic oral NAC dosing 
significantly increased medial frontal GSH levels and improved cognitive processing speed (Conus, 2018). 
Based on the mechanistic hypothesis that NAC improves residual affective and cognitive impairments in BD 
individuals by increasing their brain GSH levels, a number of randomized clinical trials (RCTs) of adjunctive NAC 
for BD have been conducted over the past 10-15 years (Nery, 2020). The original landmark RCT found that NAC 
significantly reduced depressive symptoms and increased quality of life relative to placebo in BD outpatients 
(Berk, 2008). Secondary analysis of the data further revealed that NAC significantly reduced hypo(manic) 
symptoms (in those who entered the trial experiencing (hypo)manic symptoms; Magalhaes, 2013) and 
significantly increased working memory performance (in a pooled sample of participants with BD and 
schizophrenia; Rapado-Castro, 2017), consistent with studies demonstrating pro-cognitive effects of NAC in 
people with Alzheimer’s disease and traumatic brain injury (Skvarc, 2017). Berk and colleagues’ (2008) findings 
have since been replicated by three independent research groups (Hu, 2012; Porcu, 2018; Bauer, 2018, 
NAC+aspirin) and, despite included recently-published negative studies (Berk, 2019; Ellegaard, 2019), a meta-
analysis found that adjunctive NAC (vs. placebo) treatment conferred a statistically-significant, moderate effect 
on reducing depressive symptoms in BD individuals (Nery, 2020).  
Despite support for NAC as a promising adjunctive 
treatment for BD, the (GSH-mediated) mechanistic 
hypothesis on which RCTs have been predicated has 
never been tested in BD individuals, and an equally-
plausible, (glutamate-mediated) alternative hypothesis 
exists. Specifically, the cystine/glutamate antiporter, 
through which NAC increases intracellular levels of the 
GSH-precursor cysteine, couples uptake of one 
molecule of cystine with release of one molecule of 
glutamate (see Figure 2). This NAC-induced efflux of 
glutamate activates presynaptic mGlu2 receptors, 
inhibiting glutamate release, and induces astrocytic 
expression of glutamate transporter 1, enhancing 
clearance of synaptic glutamate (Raghu, 2020). These 
glutamatergic effects, manifested in vivo as NAC-
induced reductions in ACC glutamate levels (Schmaal, 2012; McQueen, 2018; Woodcock, 2021), have formed 
the foundation of numerous preclinical and clinical studies of NAC for treating SUD. Because OS-induced cellular 
damage is a mechanism of (hyper)glutamatergic excitotoxicity (Sattler, 2001), NAC may improve clinical 
outcomes by reducing OS, but may do so via decreasing glutamate (as opposed to increasing GSH) levels.   
In sum, RCTs have supported NAC as a promising adjunctive treatment for BD, though the (GSH vs. 
glutamate-mediated) mechanisms through which NAC improves BD outcomes remain untested. 
A.4. Conclusion. BD remains a leading driver of global disease burden, in part due to residual affective and 
cognitive impairments experienced by most individuals with BD. These impairments may be due to cellular 
damage caused by excessive OS, which persists despite compensatory upregulation of the brain’s primary 
antioxidant, GSH. Adjunctive NAC improves residual affective and cognitive impairments in BD individuals, 
however the mechanisms through which these improvements occur remain untested. The proposed randomized, 
placebo-controlled, double-blind, crossover study aims to increase dACC GSH levels using NAC and to explore 
GSH- and glutamate-mediated changes in mood, cognition, and peripheral OS biomarkers in BD individuals. 
Figure 2. Metabolism of NAC to GSH. NAC, N-Acetylcysteine; 
xCT, cysteine/glutamate antiporter; GLU, glutamate; CYS, 
cysteine; GCL, glutamate‑cysteine ligase; GSy, glutathione 
synthetase; GSH, reduced glutathione.  

B. INNOVATION 
1) Although evidence supports NAC as a promising adjunctive treatment for BD (Nery, 2020), no studies have 
evaluated the neurobiological mechanisms of NAC treatment in BD. The proposed study will directly evaluate 
whether NAC engages its hypothesized neurochemical target (GSH) and/or an alternative target (glutamate) in 
BD individuals, and will examine relative associations of NAC-induced changes in GSH and glutamate levels 
with mood, cognition, and peripheral OS biomarkers (plasma GSH levels, extent of s-glutathionylated proteins). 
In doing so, the proposed study will be the first to examine the relationship between brain and plasma GSH 
levels (positively correlated in other populations, Holmay, 2013; Conus, 2018) in BD individuals. 
2) The proposed study will be the first to investigate brain GSH levels in BD individuals using optimal, j-difference, 
1H-MRS methods (Sanaei, 2017); specifically, a TE-optimized version of the MEGA-PRESS sequence 
demonstrated to provide excellent SNR and test-retest reliability of dACC GSH levels (Prisciandaro, 2020). 
A. RESEARCH PLAN 
C.1. Overview. The proposed randomized, placebo-controlled, double-blind, crossover MRI study will evaluate 
the effects of 3g/day of NAC on dACC GSH levels in BD. BD participants will complete two, 14-day experimental 
conditions (NAC, placebo) in a randomized order, separated by a 14-day washout period. Each condition will 
consist of an in-person study visit for assessment and dispensing of medication (Day 1) and an MRI (Day 14). 
See Figure 3 below for a study design schematic. 
 
 
C.2. Participants. Twelve healthy, men and women age 18‑60 with BD will be enrolled across 10‑months (i.e., 
following a 1‑month study‑    
initiation, and followed by a 1‑ 
month closeout, period). The  
study team has successfully recruited and retained BD participants for >10 years via clinical referral (e.g., MUSC 
Institute of Psychiatry, Charleston and Dorchester Mental Health Center) and advertising (e.g., radio, Facebook, 
Craigslist), 97% of whom have agreed to be re‑contacted. This past year, we screened out ≥10 people/month 
who were diagnosed with BD, but failed to meet entry SUD criteria, who could have been available for this study. 
We anticipate approximately 35% of recruited participants will be minorities. 
C.2.a. Recruitment. We will focus recruitment efforts for the proposed study on the following primary sources: 
1) Medical University of South Carolina (MUSC) 
a. Inpatient - Over the past 10+ years, we have forged strong ties with the clinical enterprise of the 
MUSC Institute of Psychiatry. Study staff visit MUSC residents and attending physicians across 4 
inpatient units, 3x/week. If the inpatient treatment team believes a patient may be eligible and 
interested in participating in the current study, they will approach the patient and gain approval for 
the research study staff to speak with the patient. If they agree, the patient will then be asked 
questions by study staff to screen for eligibility.  
b. Outpatient – We will recruit additional study participants through IRB-approved access to electronic 
medical records (EPIC), education and outreach to medical residents, and flyering across campus. 
A research data request will be submitted to indicate patients who have been diagnosed with Bipolar 
Disorder or Schizoaffective Disorder, Bipolar Type. Patients who have not indicated an opt-out 
research contact preference in their MUSC medical record will be called by study staff and screened 
for eligibility. Individuals who have indicated that they do wish to opt-out of research contact may be 
informed of the research study by their MUSC psychiatry outpatient provider if the provider feels it is 
appropriate. If they are interested, they will be given the study team’s contact information to call or 
email. The study team will not cold-contact any patients who have chosen to opt-out of receiving 
contact about research. 
2) Other Clinical Referral Sites 
a. Charleston Dorchester Mental Health Center (CDMH) – The CDMH provides outpatient counseling, 
psychiatric treatment, and support services to adults with severe mental illness through clinics in 
Charleston and Dorchester Counties in SC. Study staff visit the CDMH monthly, and additionally 
coordinate referrals and activities via phone and e-mail between visits. Study staff will not directly 
Figure 3. Study Design 

approach or contact prospective subjects.  
C.2.b. Inclusion Criteria. 1.) Meets DSM‑5 criteria for bipolar I or II disorder. 2.) Daily use of ≥ 1 FDAapproved 
mood‑stabilizing medication for BD (i.e., lithium, lamotrigine, divalproex sodium, 2nd generation antipsychotic); 
restricting the proposed study to medication‑naïve individuals would represent a safety hazard, severely limit 
recruitment (Phillips, 2008), and would be inconsistent with the primary goal of the study, to evaluate NAC as an 
adjunctive medication for BD. Though BD medications have not been associated with ACC GSH levels (Das, 
2019), participants with medication additions, discontinuations, or dose changes of ≥ 20%, ≤ 2 weeks before 
testing will be excluded to further minimize the impact of medications (Swann, 2009). 3) Reports experiencing a 
recent prolonged mood disturbance (i.e., meets DSM‑5 criteria for a mood episode, current or in partial 
remission), as long‑term remission is characterized by relatively lower levels of oxidative stress (OS; Jimenez‑
Fernandez, 2021). 4.) Willing to abstain from antioxidant supplements (e.g., coenzyme Q‑10) and 5.) utilize birth 
control (women). 6.) Aged 18 60 years, as changes in brain GSH primarily occur prior to age 18 (↓ GSH) and 
after age 60 (↑ GSH; Tong, 2016). 
C.2.c. Exclusion Criteria. 1.) History of significant hematological, endocrine, cardiovascular, pulmonary, renal, 
gastrointestinal, neurological disease. 2.) Current suicidal or homicidal ideation. 3.) Severe mood disturbance 
conferring acute safety risk, defined as Montgomery‑Asberg Depression Rating Scale (MADRS; Montgomery, 
1979) > 35 or Young Mania Rating Scale (YMRS; Young, 1978) > 25. Because elevated ACC GSH levels in BD 
are evident across mood states (Das, 2019), participants with subthreshold mood symptom elevations will not 
be excluded in order to maximize clinical generalizability. 4.) Positive breathalyzer or urine drug screen. 5.) DSM‑
5 SUD (c.f., Tobacco) criteria met in the past 6 months. 6.) Electroconvulsive therapy in the past 3 months. 7.) 
Known allergic reaction to, or past month use of, NAC. 8.) Use of medications potentially hazardous if taken with 
NAC (e.g., carbamazepine). 9.) Hepatocellular disease as indicated by liver transaminase levels >3x normal 
range. 10.) Past head injury with loss of consciousness >5 minutes, presence of non‑MRI safe materials, or 
claustrophobia. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
C.3. Procedures. See Table 2 for a summary of study events by visit. 
 
* After eligibility confirmed via SCID-5 and H&P, whenever possible. Non-completed measures to be administered at V2, 
before MRI. 
 
 
 
 
 
Table 2. Schedule of events by study visit  
 
Baseline 
Condition 1 
Condition 2 
Study Visit 
Visit 1 
Visit 
2 
Visit 
3 
Visit 
4 
Visit 
5 
Clinician 
Informed Consent / HIPAA / BrAC contract 
X 
 
 
 
 
SCID-5 / FHS 
X 
 
 
 
 
History and Physical (incl. Vitals) / Metal Screen 
X 
 
 
 
 
YMRS / MADRS / HAM-A / C-SSRS 
X 
X 
X 
X 
X 
TLFB (90 day at V1) ALL SUBSTANCES (incl. cigarettes)  
X 
X 
X 
X 
X 
BDRS / BR / OAS / PANSS-6 
 
 
X 
 
X 
Vitals 
 
X 
X 
X 
X 
Self-Report 
Demographics 
X 
 
 
 
 
BIS-11 / SPSRQ / AUDIT / ASRS /  PSQI / WHODAS / DSM-5 PID-5 / SCID-5 
SPQ / CTQ / STAXI-2 (Trait) / BussPerry Aggression / SHAPS (Trait) / STAI 
(Trait)  
X* 
 
 
 
 
BDI-II / BAI / Insomnia Severity Index / FTND / ASRM /  CAPE-42 / STAXI-2 
(State) / STAI (State) / CHRT-SR 
X 
 
X 
 
X 
Computer Tasks 
STOP-IT / Delay Discounting 
X 
 
X 
 
X 
NIH Toolbox (Post Scan) 
X 
 
X 
 
X 
Labs 
CMP / CBC / DNA  
X 
 
 
 
 
Plasma Biomarkers 
X 
 
X 
 
X 
Riboflavin 
 
 
X 
 
X 
In-House Labs 
UDS / Breathalyzer 
X 
X 
X 
X 
X 
Pregnancy Test 
X 
 
X 
 
X 
Saliva Drug Screen 
 
 
X 
 
X 
Misc 
Actigraphy 
 
X 
X 
X 
X 
1H-MRS / fMRI 
 
 
X 
 
X 
Dispense Meds 
 
X 
 
X 
 

 
 
C.3.a. Baseline Assessment (Visit 1). Following brief screening over the phone or at a referral site, potential 
participants will be scheduled for formal screening at The Department of Psychiatry research clinic. They will 
read and sign an IRB-approved informed consent, HIPAA, and BrAC documents and will then be assessed for 
eligibility using the Structured Clinical Interview for DSM-V (First, 1995). Past 90-day drug and alcohol use will 
be assessed using the Timeline Followback method (TLFB; Sobell, 1995). Mood symptoms will be assessed 
using the YMRS, MADRS, Hamilton Anxiety Scale (Hamilton, 1959), the Columbia Suicide Severity Rating Scale 
(C-SSRS; Posner, 2011), Beck Depression Inventory-II (BDI-II; Beck, 1996), and Beck Anxiety Inventory (BAI; 
Beck, 1983). Cognition will be assessed using the NIH Toolbox – Cognition Battery (Gershon, 2013). Sleep will 
be assessed using the Pittsburgh Sleep Quality Index (PSQI; Buysse, 1989). A Metal Screening Questionnaire 
will assess MRI safety. Family history will be assessed with the Family History Screen (FHS; Milne, 2009). 
Impulsivity (BIS; Patton et al., 1995), hedonic tone (Snaith-Hamilton Pleasure Scale [SHAPS]; Snaith et al., 
1995), personality pathology (Personality Inventory for DSM-5 [PID-5; Suzuki et al., 2015]), state-trait anger 
(State-Trait Anger Expression Inventory [STAXI-2]; Spielberger, 1995), aggression (Aggression Questionnaire; 
Buss & Perry, 1992), ADHD symptoms (Adult ADHD Self-Report Scale [ASRS; Kessler et al., 2005]), childhood 
trauma (Childhood Trauma Questionnaire [CTQ]; Bernstein et al., 1994), functional impairment (WHO Disability 
Assessment Scale [WHODAS; WHO, 2010]), will be measured. 
Study candidates who meet diagnostic criteria will undergo a full medical history and physical exam and will 
provide samples for blood chemistries (Comprehensive Metabolic Panel [CMP], Complete Blood Count [CBC]) 
and genetics. OS biomarkers (GSH, relative to glutathione disulfide [GSSG], the product of GSH oxidation, and 
extent of s‑glutathionylated proteins, a novel biomarker that increases under OS, using the methods detailed by 
Dr. Townsend (Co‑I) and colleagues in Zhang, 2018). Qualitative drug screens will be performed using the 
Discover 12 Panel Cup (Discover), an in vitro diagnostic test for the detection of drug or drug metabolite in 
urine as well as a saliva drug screen. Female participants will undergo pregnancy testing. 
C.3.b. Medication Dispensing (Visits 2 and 4). On day 1 of each condition, mood symptoms (YMRS, MADRS, 
C-SSRS, HAM-A) and alcohol/drug consumption (TLFB, UDS) will be assessed. Medications will be packaged 
and dispensed by the MUSC Investigational Drug Service (IDS), a centralized research pharmacy that 
compounds medications. IDS will oversee blinding procedures for the study and maintain treatment assignment 
records. Study medication will be over-encapsulated with riboflavin (50mg/capsule, for urinary detection by 
fluorescence spectroscopy at each MRI), with each capsule containing either 0.5g of NAC or matching placebo 
dispensed in blister packs. A daily dose of 3g NAC per day was chosen because it represents the largest daily 
dose of NAC that has been evaluated in studies of individuals with psychiatric conditions, including the study 
referenced in our sample size determination (below). At each dispensing visits, participants will take their first 
dose (1.5g NAC or placebo) in front of study staff to ensure compliance. Following their first dose, participants 
will be instructed to take the study medication twice daily (to maximize compliance) for 14 days. Although NAC 
has an elimination half-life of 6-7 hours, with steady state concentrations achieved within 30-35 hours (Holdiness, 
1991) and brain GSH increases observed in <60m following an acute dose (Holmay, 2013), a 14-day dosing 
period was chosen to maximize chances of finding NAC effects on GSH levels while maintaining a reasonably-
minimal anticipated rate of participant dropout (<20%, estimated from experience with the population). At Visit 
2, participants will be asked to wear an actigraphy watch (Actiwatch Spectrum Plus, Philips Respironics) on their 
non-dominant wrist for the remainder of the study. They will be instructed on its use (e.g., not to take it off when 
showering) and asked to keep a brief actigraph log (e.g., sleep and wake times) delivered daily via text/e-mail 
using REDCap. 
C.3.c. MRI (Visits 3 and 5). Participants will be asked to abstain from drugs and alcohol ≥ 12 hours prior to MRI. 
Mood symptoms (YMRS, MADRS, HAM-A, BDI, BAI, BDRS, BR, OAS, PANSS-7, OAS), alcohol and drug 
consumption (Breathalyzer, TLFB, UDS, Saliva Drug Test), will be assessed upon arrival. Questionnaires 
evaluating cigarette and tobacco use (FTND), the presence of manic symptoms (ASRM; Altman, 1997), 

psychotic 
symptoms 
(CAPE-42), 
suicidal ideation (CHRT; Trivedi, 
2008), anxiety (STAI), anger (STAXI), 
and insomnia (ISI), will be evaluated. 
Participants will complete the STOP-
IT and Delay Discounting computer 
tasks. Participants will take their final 
medication dose in front of staff, 1 
hour 
before 
MRI, 
to 
ensure 
compliance. 
Unused 
study 
medications will be returned for pill 
counts. Participants who smoke will 
be allowed to have their last cigarette 
immediately 
prior 
to 
their 
final 
medication dose. Participants will 
provide blood and urine samples to 
test for gabapentin levels, riboflavin, 
and OS biomarkers. Total scan time is 
60 min in a Siemens 3.0T  
Prisma (32‑ch head coil) with actively 
shielded 
magnet 
and 
high‑
performance gradients. Participants 
will discontinue study medication 
following each MRI. To minimize 
potential carry‑over effects, conditions will be separated by a 14‑day washout period, as this has been shown to 
prevent carry‑over effects across numerous studies featuring up to 8‑weeks of NAC dosing (e.g.,Stav, 2009; 
Khaledifar, 2014). 1H‑MRS Acquisition and Processing. A structural scan will be taken for 1H‑MRS voxel 
placement and tissue segmentation (256 sagittal slices; 1mm thick/50% gap). A dACC voxel will be placed on 
midsagittal T1‑weighted images, posterior to the genu of the corpus callosum, with the ventral edge of the voxel 
aligned with the dorsal edge of the callosum (Hermann, 2012). This location was selected given its unique status 
as the primary hub within the network of brain structures responsible for the cognitive control of behavior 
(Shenhav, 2016), and because the vast majority of 1H-MRS investigations of brain GSH in psychiatric 
populations have acquired data from this region (Das, 2019). The voxel will be 2.5x2.5x3 cm3 to ensure adequate 
signal to noise properties. Following placement of 6 saturation bands 5mm away from the voxel faces and auto-
shimming via FASTESTMAP, single-voxel water-suppressed 1H-MRS spectra will be acquired via TE-optimized 
MEGA-PRESS (TR=2000ms; TE-120ms; number of averages=256) using editing-pulse frequencies for GSH 
acquisition (4.56 [ON], 7.5 ppm [OFF]; Prisciandaro, 2020). See Figure 4 for representative (a) dACC voxel 
placement and (b) fitted data, comparing GSH spectra the TE-optimized (i.e., 120ms) acquisition (right), which 
provided an average of 97.3% more GSH signal, relative to the more commonly used 68-80ms TE acquisition 
(left). Subsequently, a low-TE PRESS sequence sensitive to glutamate will be acquired (TR=2000ms; TE=40ms; 
number of averages=128; Mullins, 2008). Unsuppressed water spectra will be co-acquired for each sequence. 
MEGA-PRESS data will be analyzed using the Gannet MATLAB toolbox (Edden, 2014). PRESS data will be 
analyzed using LCModel 6.3 (Provencher, 1993). Metabolites with fitting uncertainties <20% will be retained. 
Water will be quantified from a Gaussian-Lorentzian fit to non-water-suppressed data. Within voxel tissue 
fractions of gray (GM) and white (WM) matter and CSF will be calculated based on automated segmentation in 
Statistical Parametric Mapping 12 (Wellcome Trust) using a mask generated in Gannet. Metabolite 
concentrations will be normalized to unsuppressed water and corrected for within-voxel CSF fraction.  
C.4. Statistical Considerations. 
C.4.a. Sample Size Determination. In the only published investigation of chronic NAC dosing on brain GSH 
levels in a psychiatric sample, a 23% increase in medial prefrontal GSH was found in (2.7g/day) NAC‑treated 
participants with early psychosis, in contrast to the 4.6% decrease found in placebo‑treated participants (Conus, 
2018). Our test‑retest investigation of TE‑optimized MEGA‑PRESS for Figure 2. Sample (a) dACC voxel 
placement (top panel), and (b) fitted GSH spectra from 80ms (left) and 120ms (right) TE MEGA-PRESS 
acquisitions. GSH in 11 HC demonstrated excellent stability in within‑subject dACC GSH levels (M Coefficient 
of Variation = 7.25%). Using variance/covariance estimates from Prisciandaro (2020), we calculated statistical 
power across a range of potential magnitude between‑treatment changes in dACC GSH levels. With n=10 study 
Figure 4. Sample (a) dACC voxel placement (top      panel), and (b) fitted 
GSH spectra from 80ms (left) and 120ms (right) TE MEGA-PRESS 
acquisitions. 

completers (n=12 enrolled with an estimated dropout rate of 15‑20%), the proposed study would have >80% 
power to detect the 23% NAC‑induced increase in GSH levels reported in Conus (2018) (α=0.05, two‑tailed; IBM 
SPSS v27).  
C.4.b. Data Analysis. Linear mixed effects models will be used to assess the effect of NAC on dACC GSH 
levels (Hypothesis 1). Models will be re‑estimated with dACC glutamate levels entered in place of GSH levels 
(Exploratory Hypothesis 1). We predict that NAC will increase GSH levels, but decrease glutamate levels, relative 
to placebo. Exploratory Hypothesis 2 will be evaluated by adding the following variables, setwise, as interacting 
time‑varying covariates to the Hypothesis 1 linear model: i) plasma GSH levels, extent of s‑glutathionylated 
proteins, ii) YMRS, MADRS scores, and iii) NIH Toolbox‑Cognition Battery scores. Models will contain the main 
effect of treatment (NAC vs. placebo), period (scan 1 vs. scan 2), and sequence (NAC 1st vs. placebo 1st) to 
ensure the crossover design and washout were successful. Although mood‑stabilizing medications have not 
been associated with GSH levels (Das, 2019), we will evaluate them, along with other baseline variables that 
may be associated with OS (age, sex, smoking status; Keaney, 2003), as moderators across analyses to 
facilitate rigor and reproducibility and inform future studies. Within‑voxel tissue content will be evaluated as a 
covariate across models. We will employ previously successful methods to minimize attrition. Where missing 
data cannot be avoided, mixed models yield valid inferences assuming data are missing at random. 
 
C.5. DISCUSSION 
C.5.a. Overview. Despite available efficacious treatments, BD remains a leading driver of global disease burden. 
Convergent evidence supports ACC GSH levels as a promising interventional target, and NAC as a candidate 
adjunctive treatment to increase brain GSH levels, in BD individuals. The proposed randomized, placebo-
controlled, double-blind, crossover MRI study aims to increase dACC GSH levels using NAC (3g/day) in 
individuals with BD and to evaluate medication-related, GSH- and glutamate-mediated changes in mood, 
cognition, and plasma biomarkers of OS, in individuals with BD. The study will be conducted by a BD research 
team with a successful 10+ year record of collaboration, using imaging methods optimized by the study team. 
Results may support the development of increasingly-targeted medications designed to fight excessive OS 
through increasing brain GSH levels that would clinically benefit individuals with BD. 
C.5.b. Strategies to Ensure a Robust and Unbiased Approach. The proposed study will achieve robust and 
unbiased results via explicit inclusion/exclusion criteria; randomization of treatment condition order and 
examination of order effects; placebo control; double blinding; sophisticated compliance monitoring; use of 
validated MRI, laboratory, and interview/self-report measures/methods; explicit hypotheses and planned 
statistical analyses; power estimates; planned handling of attrition and missing data; and careful consideration 
of potential confounds. Methodology is reported in a detailed and fully transparent manner to support replication. 
C.5.c. Potential Limitations. NAC may reduce neuro-OS burden in BD individuals by increasing GSH or 
decreasing glutamate levels. Despite possessing multiple potential neurochemical mechanisms of action, NAC 
remains the best available drug for the proposed study for two reasons. First, NAC is the only drug that has been 
shown to modulate brain GSH levels in vivo. Second, though most 1H-MRS NAC studies have reported either 
GSH or glutamate effects, the proposed study will acquire both GSH and glutamate levels within the same scan, 
allowing us to determine the relative extents to which NAC modulates dACC GSH and glutamate levels. 
C.5.d. Future Directions. Positive results would directly support the hypothesized mechanism of NAC treatment 
for BD. Perhaps more importantly, positive results would provide evidence for a novel neurobiological treatment 
target for BD, which would support the development of medications designed to increase brain GSH levels (with 
greater specificity and efficacy than NAC) and their evaluation using the proposed experimental neuroimaging 
framework. For example, γ-glutamylcysteine, precursor for the final step of GSH synthesis, bypasses the 
feedback inhibition by GSH on γ-glutaminecysteine ligase, making it a potentially more efficacious glutathione 
prodrug than NAC. Compounds consisting of γ–glutamylcysteine alongside an attached ethyl ester moiety (to 
increase blood-brain-barrier permeability) have received growing interest in preclinical research on oxidative 
injury, cardiac disease, and neurodegenerative disorders (Pocernich, 2012; Giustarini, 2018). Finally, the 
proposed study will add to the literature on associations of brain GSH and glutamate levels, plasma biomarkers 
of OS, and BD clinical phenomenology (mood symptoms and cognitive dysfunction). 
 
Protection of Human Subjects 
1. Risk to the Subjects 
a. Human Subject Involvement and Characteristics. A total of 12 individuals with bipolar disorder (BD) in 
stable medical condition will be enrolled in the study. Women and minorities will be recruited for this study. 

Children and adolescents under the age of 18 will not be enrolled.  
Inclusion Criteria:  
1. Ages 18-60 years 
2. Meet DSM-5 criteria for bipolar I or II disorder  
3. Able to provide informed consent and read, understand, and accurately complete assessment instruments  
4. Willing to commit to medication treatment and follow-up assessments 
5. Meets DSM-5 criteria for any mood episode (i.e., Major Depressive, Hypomanic, Manic), Current or In Partial 
Remission 
6. Prescribed daily use of at least one FDA-approved mood stabilizing medication (i.e., lithium, divalproex 
sodium, lamotrigine, 2nd generation antipsychotic) 
7. Willing to abstain from antioxidant supplements (e.g., coenzyme Q-10, vitamin E) for the duration of the 
study.  
Exclusion Criteria: 
1. A primary psychiatric diagnosis other than BD (e.g., Schizophrenia) 
2. Meet DSM-5 criteria for substance use disorder (other than Tobacco Use Disorder) within the past 6 months. 
3. Any uncontrolled neurological condition (e.g., epilepsy) that could confound the results of the study 
4. Any history of brain injury with loss of consciousness greater than 5 minutes 
5. Any history of mental retardation, dementia, or recent electroconvulsive therapy (in the past 3 months) 
6. Any uncontrolled medical condition that may adversely affect the conduct of the study or jeopardize the safety 
of the participant 
7. Hepatocellular disease as indicated by plasma levels of liver transaminases (aspartate transaminase, alanine 
transaminase) greater than 3 times the normal range 
8. Renal insufficiency as indicated by plasma levels of creatinine greater than 2 times the normal range 
9. Concomitant use of nitroglycerine, carbamazepine, or any other medication deemed to be hazardous if taken 
with N-Acetylcysteine (NAC).  
10. Medication dose changes of ≥ 20% ≤ 2 weeks prior to testing 
11. Women of childbearing potential who are pregnant, lactating, or refuse adequate forms of contraception 
12. Current suicidal or homicidal risk 
13. Baseline scores greater than 35 on the Montgomery-Asberg Depression Rating Scale or greater than 25 on 
the Young Mania Rating Scale 
14. Has taken NAC in the last month or experienced adverse effects/allergic reaction from it at any time 
15. Significant claustrophobia and/or past negative experiences with MRI 
16. Presence of non-MRI safe materials in the body (e.g., ferrous metal implants, pacemaker) 
b. Source of Materials. Data collected from participants will include breathalyzer readings, urine and saliva drug 
screens, urine biomarkers (e.g., riboflavin), blood chemistries, plasma biomarkers, structural and neurochemical 
MRI brain images, cognitive testing results, and interviews and self-reports regarding substance use, psychiatric 
diagnoses, concomitant medications, and adverse events. To ensure confidentiality, all participant data will be 
number-coded, and only the investigators will have access to the master list of codes. 
c. Compensation. To maximize participant retention, contingency management will be applied to participant 
compensation such that participants will be compensated significantly more for each subsequent MRI visit they 
attend (i.e., scan 1 = $100, scan 2 = $150), and participants will be given an additional $50 bonus for completing 
both MRI visits. Participant compensation will be cash. Participants will be compensated $50 for the initial 
psychiatric and medical evaluation appointment. Finally, participants will be compensated $25 for each additional 
non-MRI appointment (i.e., 2 total). Compensation will thus be $400 per participant. In our experience, this level 
of compensation is fair for the time commitment required without unduly coercing participants to enroll in the 
study despite potential concerns. 
d. Potential Risks 
1. Loss of Confidentiality. Individuals will be asked questions about their psychiatric symptoms. This information 
is highly confidential and all attempts will be made to safeguard privacy and confidentiality. 
2. Medication side effects. NAC has a generally benign adverse effect profile. A meta-analysis of studies 
evaluating long-term oral treatment with NAC for prevention of chronic bronchitis found that NAC was well 
tolerated, with generally mild, most commonly gastrointestinal adverse effects that did not require treatment 
interruption (Grandjean et al., 2000). Common adverse effects of NAC include dry mouth, nausea, vomiting, 
and diarrhea. The proposed study will carefully monitor potential medication-related risks via weekly 

scheduled visits and assessment of adverse events (including assessment of suicidal ideation and behavior 
at each and every study visit). The study physician, Dr. Tolliver (Co-I), will determine if the participant should 
be discontinued from the medication due to adverse drug reactions and will treat clinically as needed. Any 
confirmed incidence of serious adverse events that are deemed probably or definitely due to the study 
medication will result in immediate discontinuation of the study medication and follow-up assessments will 
be conducted until resolution. Subjects will be referred for treatment as necessary.  
3. Drug interactions. Due to potential interactions with NAC, participants may not take nitroglycerin or 
carbamazepine at any time during participation in the study. As noted earlier, BD participants will be required 
to be taking a stable pre-existing regimen of at least one FDA-approved mood stabilizing medication 
treatment for BD. There are no known drug interactions with NAC and non-carbamazepine mood-stabilizing 
medications (i.e., lithium, lamotrigine, divalproex sodium, 2nd generation antipsychotic).  
4. Unknown Medication Risks. While NAC is approved by the FDA, it is not approved for the use in this study 
(i.e. the treatment of Bipolar Disorder). Because of this, there may be unknown medication side effects. Study 
clinicians and the study physician will monitor all adverse events throughout the study as well as the likelihood 
that they are related to the study medication. In the event that there are any serious adverse events, the 
study physician will instruct the participant to discontinue the use of the study medication.  
5. MRI-related risks. Individuals with non-MRI-safe medical implants or ferrous objects would be at risk for 
injury, if such individuals were allowed to enter the MRI scanner. Several precautions will be taken to ensure 
that individuals with ferrous implants or objects are not allowed to enter the MRI scanner. First, all potential 
participants will meet with a study physician to discuss any possible history of ferrous implants or other MRI-
unsafe objects. Participants with any suspected history of ferrous implants or exposure to shrapnel will be 
excluded from the study. Second, participants who are deemed MRI-safe by the study physician will be 
screened for metal objects at the Center for Biomedical Imaging (CBI) using a handheld metal detector and 
a second metal detector built into the threshold of the doorway to the scanner. Participants who screen 
positive for metal will be asked to remove all metal objects from their person and will be rescreened. If 
participants continue to screen positive for metal after removing all metal objects from their person, they will 
be excluded from the study. Although not dangerous, participants who are claustrophobic could experience 
significant discomfort in the MRI scanner. As such, all participants will be assessed in terms of claustrophobia 
as well as past experience with MRI. Additionally, all participants will be entered into a “mock scanner” at the 
CBI human imaging center, which features the same dimensions of the real MRI scanner but without any of 
the internal machinery. Participants who report claustrophobia, past negative experience with MRI, or 
signification discomfort in the mock scanner will be excluded from participation. These procedures have been 
successfully used by our staff in previous and ongoing research studies of similar participant populations. 
For those participants allowed to participate in the MRI study, if abnormalities in collected brain images are 
found, participants will immediately be referred to an appropriate clinical care provider. 
6. Mood destabilization. Exacerbation of depressive or manic symptoms during the course of the study is a risk 
for BD subjects regardless of treatment condition. We will minimize this risk by assessing mood symptoms, 
including suicidal ideation and behavior, at each and every study visit. Assessment of suicidal ideation will 
be conducted using primarily the Columbia-Suicide Severity Rating Scale (C-SSRS) and also the MADRS.  
Any participant who answers affirmatively to item 4 (SI with some intention of acting on them) or item 5 (SI 
with plan) on the C-SSRS or scoring > 4 on item 4 of the MADRS will be immediately removed from the study 
and procedures for emergent clinical assessment begun (see 2.c. below, Protection Against Risk). 
Participants experiencing sufficient deterioration of mood stability to result in clinically significant impairment 
in functional capacity will be appropriately referred. Any subject exhibiting mood destabilization that is 
sufficient to pose an imminent danger to self or others will be accompanied by the study physician to the 
University Hospital Emergency Department for immediate assessment for psychiatric hospitalization.  
7. Pregnancy. MRI is used in this study. Participants who are pregnant or are trying to become pregnant should 
not participate as to ensure there is no risk to an embryo or fetus. Pregnancy tests will be conducted to 
ensure participants are not pregnant during study procedures.  
 
2. Adequacy of Protection Against Risks 
a. Recruitment and Informed Consent. Recruitment will occur by clinical referral and response to 
advertisements. IRB-approved study staff with advanced clinical degrees (e.g., PhD, MD) will obtain informed 
consent. Interested individuals will be given a brief description of the study and if still interested will be scheduled 
for a screening visit. At the screening visit, potential participants will be provided a copy of the IRB-approved 
consent document to review. After providing the participant with time to read the consent, IRB-approved study 

staff will review the consent document page by page with the participant and answer any questions. Only then 
will the participant be asked to sign the consent document. Participants will be given a copy of the signed consent 
document. The entire informed consent process will be documented in the research progress notes. The signed, 
original consent document will be maintained in the participant source record with a copy of the consent binder 
located at the Institute of Psychiatry, Department of Psychiatry and Behavioral Sciences, Medical University of 
South Carolina.  
b. Safety Assessments. At every visit, IRB-approved study staff with advanced clinical degrees (e.g., PhD, MD) 
will clinically evaluate subjects’ manic and/or depressive symptoms, including suicidal ideation. Mood symptoms 
will also be quantified using standardized instruments including C-SSRS, MADRS, YMRS, BDI-II, and BAI. 
Adverse events will be assessed at every study visit.   
c. Protection Against Risk. Psychiatric Risks. The investigative team has a great deal of experience working 
with the study population and has the resources to make appropriate referrals as needed. Psychiatric symptoms 
will be assessed on a weekly basis by standardized assessments and by clinical interview. Participants 
answering affirmatively to item 4 (SI with some intention of acting on them) or item 5 (SI with plan) on the C-
SSRS or scoring >4 on the suicide item (item 4) of the MADRS, or otherwise exhibiting potentially life-threatening 
decompensation in mood or other psychiatric symptoms at any study visit will be removed from the study and 
procedures for emergent clinical assessment begun at once.  Briefly, actively suicidal participants will be 
accompanied to the adult ER in the main MUSC hospital by the study psychiatrist (Dr. Tolliver, co-I).  One-to-
one accompaniment and supervision by the study psychiatrist will continue until the participant is admitted for 
triage in the ER.  Should a participant refuse voluntary assessment in the ER, MUSC Public Safety will be called 
immediately and involuntary commitment papers will be filled out by Dr. Tolliver; participants refusing voluntary 
assessment will be accompanied at all times by study personnel and not allowed to leave the research clinic 
during emergency procedures. While a rare event, over the past 15 years Dr. Tolliver has implemented these 
procedures successfully 10-12 times and study participants have been admitted to the Institute of Psychiatry 
without exception. 
Medical Risks. NAC is largely excreted by a hepatic route. A blood chemistry panel will be performed and 
reviewed by the study physician prior to beginning the study medication. Participants with clinically significant 
hepatic (transaminases elevated > 3 times normal) insufficiency will not be eligible to participate in the study. 
Subjects will be referred for treatment as necessary.  
MRI Risks. The investigative team has a great deal of experience with human MRI research and Dr. Prisciandaro 
is a core faculty member of the CBI at MUSC. MRI safety and comfort will be assessed at baseline and at each 
MRI visit. Participants with non-MRI-safe medical implants or ferrous objects will be excluded from participation 
as will individuals evidencing significant discomfort with MRI.  
Pregnancy. NAC is rated as a Category B medication in terms of pregnancy. Because there are no adequate 
controlled studies of NAC in pregnant women, it is unknown whether the drug can cause fetal harm or affect 
reproductive capacity in humans. Therefore, women of childbearing potential must agree to pregnancy testing 
and use of adequate contraception in order to be eligible to participate in the study. Females will be given a 
urinary pregnancy test at the screening visit and weekly thereafter. Any female participant who becomes 
pregnant during the study will be discontinued from the study medication and removed from study participation. 
For all included females of childbearing potential, current forms of birth control and date of last menstruation will 
be assessed at each study visit.  
Confidentiality. Records with identifying information (e.g., consent documents) will be stored in a locked file. All 
other non-MRI participant data will be collected via direct data capture (REDCap); MRI data will be automatically 
transferred to Linux-based servers managed by CBI. Both MRI and non-MRI data will be stored in restricted 
access directories on password-protected, encrypted servers managed by CBI and the Department of Psychiatry 
and Behavioral Sciences at MUSC. Participants will be given an ID number for all MRI and non-MRI data files. 
The master list of codes will be accessible only to the investigators, and will be stored in a locked office. 
Emergencies. All study participants will be instructed how to access the 24-hour on-call system available at the 
Medical University of South Carolina. In the event that a participant experiences an adverse event after hours, 
s/he will be instructed to access the 24-hour on-call service. If it is determined that the participant needs 
immediate help, the participant may be advised to immediately go to the emergency room. In that event, proper 
medical treatment will be administered, per ER procedures. Dr. Prisciandaro and Dr. Tolliver will be available 24 
hours/day, 7 days/week by pager as necessary. The Investigational Drug Service (IDS) will also be available 24 
hours/day, 7 days/week for emergency identification of treatment group assignment and unblinding as 

necessary. 
3. Potential Benefits of the Proposed Research to the Subjects and Others  
Benefits to the participants include medical and psychiatric assessments provided at no cost. Other individuals 
with BD are likely to benefit by the knowledge gained from the study as it may help guide future treatments 
4. Importance of the Knowledge to Be Gained 
Despite nearly 70 years of medication development for Bipolar Disorder (BD), BD remains one of the leading 
drivers of global disease burden and mortality. Existing BD medications typically leave patients with significantly 
impairing residual mood symptoms and cognitive dysfunction. The proposed study addresses the unmet clinical 
needs of BD patients using an exemplification of the modern drug discovery approach championed by the NIH 
and FDA: a brief, proof-of-concept study of the GSH prodrug, NAC, on neuroimaging markers of oxidative stress. 
Positive results may provide evidence for a novel neurobiological treatment target for BD. Such evidence would 
support the development and testing of medications designed to increase brain GSH levels, with potentially 
greater specificity and efficacy than NAC (e.g., γ-glutamylcysteine ethyl ester), using the experimental 
neuroimaging framework developed and validated by the proposed study. The proposed investigation's minimal 
risks are reasonable in relation to the importance of the knowledge to be gained from the investigation. 
 
5.   Clinicaltrials.gov Requirements 
In accordance with Public Law 110-85, this project will be registered at the ClinicalTrials.gov Protocol 
Registration System Information Website prior to study initiation. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
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