Clinical trial NCT01956279
Conditions: Fatigue Musculoskeletal Pain Cognitive Decline
Keywords: Pregnenolone Gulf War Veteran Fatigue Musculoskeletal Pain Cognitive Decline Placebo Control
Sponsor: VA Office of Research and Development
Start date: 2013-10-01; Completion date: 2018-10-10
Enrollment: 170 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This study will investigate the use of adjunctive pregnenolone for the following:

1. fatigue that has limited usual activity,
2. musculoskeletal pain involving 2 or more regions of the body and,
3. cognitive symptoms (memory, concentration, or attentional difficulties by self-report) in Veterans deployed to the Gulf War theatre of operations between 1990 and 1991.

Detailed description:

Arms: arm 1: Pregnenolone arm 2: Placebo
Interventions: intervention 1: Pregnenolone intervention 2: Placebo
Intervention descriptions: intervention 1: Pregnenolone 50mg BID x 14 days, followed by Pregnenolone 150 x 14 days, followed by Pregnenolone 250 mg BID x 28 days intervention 2: Placebo 50mg BID x 14 days, followed by Placebo 150 x 14 days, followed by Placebo 250 mg BID x 28 days

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
Complementary Neurosteroid Intervention in Gulf War Veterans’ Illnesses (GWVI) 
 
NCT01956279 
 
Study Protocol and Statistical Analysis Plan 
 Version 4 – 2/20/2015 


Marx, Christine E. 
 
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PROTOCOL OVERVIEW 
Complementary Neurosteroid Intervention in Gulf War Veterans’ Illnesses (GWVI) 
 
PURPOSE 
 
Gulf War Veterans’ Illnesses (GWVI) profoundly influence quality of life and functional outcome in many Veterans 
with a history of deployment to the Persian Gulf theater (Gulf War and Health, Vol 8; Update of Health Effects of 
Serving in the Gulf War; 2009; Institute of Medicine [IOM]).  Deployed Gulf War Veterans consistently report 
increased rates of the majority of symptoms examined compared to non-deployed Gulf War Veterans, and more Gulf 
War Veterans who were deployed meet criteria for “multi-symptom illnesses” compared to non-deployed Gulf War 
Veterans (IOM Update 2009, Kang et al 2009).  Further, these higher rates of reported symptoms in deployed Gulf 
War Veterans compared to non-deployed Gulf War Veterans also occur among those who served from other countries, 
including Canada, Australia, the United Kingdom, and Denmark (IOM Update 2009).  The increased prevalence of 
multiple symptoms in deployed Gulf War Veterans compared to non-deployed Gulf War Veterans is currently 
considered to be “beyond dispute,” and it is thought that “the excess of unexplained medical symptoms reported by 
deployed Gulf War Veterans cannot be reliably ascribed to any known psychiatric disorder” (IOM Update 2009); these 
assessments are supported by reports from several research groups (Iversen et al 2007, Fukuda et al, 1998, Gray et al 
1996, Unwin et al 1999).  Although elucidation of the precise physiological underpinnings of these complex symptom 
constellations remain a focus of ongoing scientific inquiry, it is clear that multisystem involvement is a hallmark 
GWVI.   
 
The Institute of Medicine Committee on Gulf War and Health also notes that “inadequate numbers of clinical trials 
have been undertaken to develop more effective and evidence-based treatments for multi-system illness,” and calls for 
“a renewed research effort to identify and treat multi-system illness in Gulf War Veterans.”  The IOM Committee also 
states that it is “optimistic that a rigorous, adequately powered study could identify useful biomarkers that are helpful 
for symptomatic Veterans of the Gulf War and for non-deployed Veterans and civilians who have a variety of 
medically unexplained symptoms, including chronic fatigue, muscle and joint pain, sleep disturbance, difficulty with 
concentration, and depression” (IOM Update 2009). The current proposal investigating a novel complementary 
neurosteroid intervention in Gulf War Veterans with GWVI and candidate biomarkers of therapeutic response is thus 
clearly aligned with this Institute of Medicine mandate.  The rationale for utilizing a neurosteroid intervention in Gulf 
War Veterans with GWVI is outlined below.  
 
RESEARCH DESIGN AND METHODS 
 
Specific Aim 1:  To conduct an RCT with the neurosteroid pregnenolone in 140 Gulf War Veterans with GWVI and a 
history of deployment (70 Veterans randomized to adjunctive pregnenolone, 70 Veterans randomized to placebo), 
targeting functional outcome as the primary endpoint (as assessed by the SF-36).  Secondary endpoints will be pain 
symptoms, cognitive symptoms, fatigue, and global psychological functioning.  Based on our preliminary data from 
multiple investigations, we hypothesize that adjunctive pregnenolone will significantly improve functional 
outcomes, pain symptoms, cognitive symptoms, fatigue, and global psychological symptoms in Gulf War Veterans 
with GWVI.  
 
Recent investigations support the possibility that pregnenolone may be a promising therapeutic intervention in Gulf 
War Veterans with GWVI.  Trial duration will be 10 weeks, consisting of a 2-week placebo lead-in period, followed 
by 8 weeks of randomly assigned adjunctive treatment with either pregnenolone or placebo (add-on to treatment-as-
usual).  The primary endpoint will be functional outcome (as assessed by the Physical Component of the SF-36) in the 
group randomized to pregnenolone compared to the group randomized to placebo.  Secondary outcome measures will 
be pain symptoms (as assessed by the Brief Pain Inventory), cognitive symptoms (specifically executive functioning, 
as assessed by the Tower of London component of the BACS), fatigue (as assessed by the Multidimensional Fatigue 
Inventory), and overall psychological functioning (as assessed by the Global Severity Index [GSI] of the SCL-90R).  
 


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Specific Aim 2:  To conduct candidate biomarker investigations quantifying pregnenolone and pregnenolone 
metabolite levels (allopregnanolone, pregnanolone, androsterone, others) in serum samples at baseline, during 
treatment, and post-treatment with pregnenolone using mass spectrometry-based technologies, and to investigate 
potential genetic predictors of therapeutic response.  These investigations will thus:  a.) characterize the 
pharmacokinetics of pregnenolone and its metabolic profile - which could yield valuable dosing information for 
pregnenolone, characterize pharmacological windows of optimal therapeutic efficacy, and identify potential 
neurosteroid predictors of therapeutic response, and b.) examine single nucleotide polymorphisms (SNPs) of genes 
coding for enzymes involved in neurosteroid synthesis and metabolism.  Together these studies could contribute to the 
development of new neurosteroid interventional strategies that build on the current investigation and exhibit promise 
as pharmacological candidates in GWVI. Based on our preliminary candidate biomarker data that neurosteroids are 
altered in CNS disorders (Marx et al 2006a, Marx et al 2006b, Marx et al 2006c, Naylor et al 2008, Naylor et al 2010, 
Kilts et al 2010), and that neurosteroid alterations following treatment with pregnenolone may be associated with 
therapeutic response (Marx et al 2009), we hypothesize that changes in pregnenolone levels (and pregnenolone 
metabolite levels) post-treatment will predict therapeutic response to this intervention.  We also hypothesize that 
neurosteroids are dysregulated in GWVI, and that specific SNPs of genes coding for neurosteroidogenic enzymes will 
be associated with therapeutic response.  Additionally, we hypothesize that changes in one or more pregnenolone 
metabolites may also predict therapeutic response, and potentially lead to the identification of additional neurosteroid 
targets that demonstrate promise for treating diverse symptoms impacting functional outcome and quality of life in 
Gulf War Veterans with GWVI.   
SUBJECT RECRUITMENT 
 
We propose to conduct a randomized controlled trial (RCT) investigating a novel neurosteroid intervention in Gulf 
War Veterans with GWVI.  Research participants will be recruited via Durham VA IRB-approved local advertising 
(flyers), Durham VA IRB-approved letters (and follow-up phone calls utilizing a Durham VA IRB-approved phone 
script), Vet Centers, and the VISN 6 MIRECC Post-Deployment Mental Health Data Repository (over 95% of these 
Registry participants have provided permission to be re-contacted for future research studies; over a quarter of 
Veterans in this OEF/OIF/OND era Registry are also Gulf War era Veterans), and by referral from the Durham VA, 
Raleigh CBOC, Greensboro CBOC, and Morehead City CBOC medical and behavioral health care providers.  The 
target number of participants for this study is 140 subjects who complete at least 4 weeks of the study post-
randomization (70 subjects per group, randomized to either adjunctive pregnenolone or placebo). Based on our prior 
investigations with pregnenolone, we conservatively estimate a drop-out rate of 30%.  The power analysis for this 
study in GWVI was conducted under the assumption that 140 participants reach Visit 4 of the study (i.e. 4 weeks post-
randomization).  With an estimated 30% drop-out rate, we thus anticipate that we will have to randomize 
approximately 200 participants to achieve this goal.  
 
Enrolled subjects will be 1). Veterans deployed to the Gulf War theater of operations between 1990 and 1991, 2).  Gulf 
War Veterans who report at least 2 of the following 3 symptoms that began in 1990 or thereafter, that have lasted for 
more than 6 months, and that are present at the time of screening:  a) fatigue that limited usual activity, b) 
musculoskeletal pain involving 2 or more regions of the body, c) cognitive symptoms (memory, concentration, or 
attentional difficulties by self-report, 3) on a stable medication regimen (no change in past 4 weeks) and no anticipated 
change in medications during study, 4) Able to provide informed consent for study participation.  Only Gulf War era 
Veterans will be enrolled into this study.  There will be no non-Veteran participants enrolled in this study. 
 
RECRUITMENT PROCEDURES 
 
Potential participants may be contacted initially via U.S. mail with a signed and IRB-approved recruitment letter.  For 
individuals who have been sent a letter, a follow-up telephone call will take place to confirm receipt of letter, to inquire 
if a participant may be interested in study participation, and to pre-screen for study qualification (via IRB-approved 
telephone script).  If potential participants initiate contact with the study team by telephone (i.e. if participants are 
responding to an IRB-approved advertisement or have been referred to the study by another healthcare professional 
and thus did not receive an IRB-approved recruitment letter), individuals will be pre-screened for study qualification 


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using the IRB-approved telephone script entitled “call from patient.”  Participants who meet pre-qualification criteria 
will be invited to schedule an appointment at the Durham VAMC to complete a screening visit, in which they will 
learn details about study involvement and will be asked to provide informed consent.  Participants who attend this visit 
will be compensated for their time, whether or not they decide to participate further in the study. 
 
SELECTION OF SUBJECTS 
 
Inclusion Criteria: 
1. Veterans deployed to the Gulf War theatre of operations between 1990 and 1991. 
2. Veterans who report at least 2 of the following 3 symptoms that began in 1990 or thereafter, that lasted for 
more than 6 months, and that are present at the time of screening:  1) fatigue that limited usual activity, 2) 
musculoskeletal pain involving 2 or more regions of the body, 3) cognitive symptoms (memory, concentration, 
or attentional difficulties by self-report)  
3. Stable medication regimen (no change in past 4 weeks) and no anticipated change in medications during study. 
4. Able to provide informed consent for study participation. 
 
Exclusion Criteria:  
1. Subjects with a history of clinically significant neurological, metabolic, hepatic, renal, hematological, 
pulmonary, cardiovascular, gastrointestinal, and/or urological disorders (e.g. unstable angina, seizures, 
cerebrovascular accident, decompensated congestive heart failure, CNS infection, cancer [other than non-
melanoma skin cancer], or history of HIV seropositivity), which would pose a risk to the patient if s/he were to 
participate in the study or that might confound the results of the study.   
2. Concurrent enrollment in another clinical trial. 
3. Pregnant women or women of child-bearing potential who are not surgically-sterile or not using appropriate 
methods of birth control.  
4. Use of oral contraceptives or other hormonal supplementation such as estrogen [although early studies 
suggested no effects on menstrual cycle, alterations in downstream metabolites of pregnenolone (such as 
estradiol) could theoretically impact the efficacy of oral contraceptives and/or estrogen replacement].  
Similarly, it is theoretically possible that pregnenolone could be metabolized to other steroids such as DHEA, 
resulting in hair, skin, or other steroid-related changes.  Since we have determined in our prior study that 
pregnenolone administration does not result in downstream elevations in DHEA, DHEAS, estradiol, or 
testosterone, these possibilities may be unlikely. 
5. Women who are breast-feeding.  
6. Use of narcotic interventions. 
7. Use of benzodiazepines. 
8. Known allergy to study medication. 
9. History of moderate or severe TBI (with loss of consciousness greater than 30 minutes). (Mild TBI is 
permissible.) 
10. A clearly defined disease entity that accounts for the Veteran’s symptoms. 
11. Current DSM-IV/DSM-IVTR/DSMV diagnosis of bipolar I disorder, schizophrenia or other psychotic 
disorder, or dementia. (PTSD and/or depression are permissible.) 
12. Subjects with a DSM-IV/DSM-IVTR/DSMV diagnosis of alcohol or substance dependence (other than 
nicotine or caffeine) within the last month. 
13. Subjects with current suicidal or homicidal ideation necessitating clinical intervention or representing an 
imminent concern 
14. If in the judgement of the PI it is not in the subject’s best interest to participate.   
15. Final eligibility decisions will be determined by the PI. 
 
CONSENT PROCESS 
 


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A subject who is judged likely to meet all of the inclusion criteria and none of the exclusion criteria will meet with a 
member of the research team to discuss the research protocol, and to determine if the patient is capable of providing 
informed consent. This study utilizes an informed consent form. The subject will be provided with a description 
(verbal and written) of the informed consent form, which includes the risks along with procedures to minimize these 
risks, and the subjects’ rights and responsibilities. Subjects will be provided the opportunity to read the consent form 
and ask questions. Subjects will be assured that participation in this research study is voluntary and that they may 
withdraw from the study at any time without adversely affecting their medical care or any benefits they might be 
receiving. Veterans will be informed that participation in this research study is completely voluntary and distinct from 
usual clinical care.  They may also refuse to answer any research questions during interviews. Subjects who are 
eligible for the study and choose to participate will sign the consent form in the presence of a member of the research 
team.  
 
STUDY INTERVENTIONS 
 
Primary Outcome:  Functional Outcome (as assessed by the Physical Component of the SF-36). 
 
Hypothesis:  Gulf War Veterans with GWVI who are randomized to pregnenolone will demonstrate significantly 
greater improvements in the Physical Component Score of the SF-36 compared to Veterans randomized to placebo. 
 
We will assess this hypothesis in the context of a two factor repeated measures mixed model ANOVA, with one 
between subjects factor with two levels (pregnenolone vs. placebo) and one within subjects factor with three levels 
(Weeks 0, 4, 8 of treatment post-randomization, which correspond to Visits 2, 4, and 6).  This hypothesis will be tested 
using the treatment-by-time interaction, followed by contrasts in least squares means.  These contrasts will examine the 
difference between the treatments at Weeks 4 and 8 on the changes between least squares means between Week 0 to 
Week 4, and Week 0 to Week 8 of treatment post-randomization.  We will perform several such analyses, one for 
functional outcome as assessed by the Physical Component of the SF-36 (primary endpoint), and others for secondary 
endpoints, including:  pain symptoms (as assessed by the Brief Pain Inventory), executive functioning (as assessed by 
the Tower of London test of the BACS), fatigue (as assessed by the Multidimensional Fatigue Inventory), and overall 
psychological functioning (as assessed by the Global Severity Index of the SCL-90R).  The response variable will be 
change from baseline, and we will use baseline as a covariate.  Exploratory outcomes will include the BACS 
composite score (cognitive symptoms), resilience (as assessed by the Connor-Davidson Resilience Scale), anxiety and 
depression symptoms (as assessed by the Hospital Anxiety and Depression Scale), PTSD symptoms (as assessed by 
the PTSD Symptom Checklist), and depression symptoms (as assessed by the Beck Depression Inventory-II scale). 
 
There are likely many characteristics of patients with GWVI that could influence observed treatment effects. Patients 
with GWVI often have other conditions that may modify study outcomes. The randomization process is intended to 
make the treatment group and control group equivalent, on average, with respect to these known and unknown 
confounding factors. However, by random chance the experimental groups may differ sizably with respect to some 
confounding factors. We thus intend to measure several potential confounding factors, including history of mild TBI, 
among other possibilities (Veterans with a history of moderate or severe TBI will be excluded).  Initial bivariate 
analyses to characterize and compare experimental groups will evaluate whether these groups differ sizably with 
respect to these known confounders.  Analyses will be adjusted for potential confounders by adding them or their 
interactions with treatment to the models and assessing these effects.  These mediator/moderator type analyses are 
exploratory and will suggest hypotheses for further study.  
 
It is also possible that patients with or without specific conditions (e.g. history of mild TBI) may find the treatment 
especially beneficial.  However, so little is known regarding how GWVI may interact with these conditions, that we 
have no specific grounds by which to exclude or enrich the study population to examine effects in mild TBI 
subpopulations (or other subpopulations) in this study. We thus expect that our study population will reflect the 
heterogeneity of the general patient population with this condition. We intend to explore interactions between the 
treatment effect and a number of co-occurring conditions and other characteristics as part of our analyses. However, 


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the investigation of these interactions is not the primary aim to this study (and any negative findings with respect to 
potential interactions may thus reflect insufficient statistical power to detect them).  
 
Pregnenolone: 
Pregnenolone doses were chosen following careful review of prior dosing strategies in the existing literature that were 
tolerated without significant side effects.  Subjects randomized to pregnenolone following a two-week placebo lead-in 
will receive this neurosteroid according to the following titration schedule: 
Pregnenolone  100 mg in divided doses (50 mg BID) for 2 weeks, then  
Pregnenolone  300 mg in divided doses (150 mg BID) for 2 weeks, then  
Pregnenolone  500 mg in divided doses (250 mg BID) for 4 weeks. 
 
Compliance with study drug treatment will be assessed by counting the returned study drug supply.  Subjects that fall 
below 80% compliance at 2 consecutive visits or miss 6 consecutive doses in a row (3 days of study drug) for two 
consecutive visits should be withdrawn from the study. 
 
Study Timeline: 
1. Referred patients and patients responding to IRB-approved advertisements and/or IRB-approved letters with follow-
up phone calls (using an IRB-approved phone script) will meet with a member of the research team to discuss the 
study and the risks and benefits of participation. In addition, patients will be screened for exclusion and inclusion 
criteria. If subjects are interested in potentially participating in the study, the informed consent document will be 
discussed with them. Patients will have the option of taking the informed consent with them and discussing the matter 
with family, friends and/or clinicians. If applicable, the participant’s physician or other VA provider will be consulted 
regarding the patient’s appropriateness of the patient’s enrollment in the study. 
 
2. Once the informed consent is signed, the subject will proceed to the initial screening procedures. Each participant 
will receive the MINI diagnostic assessment tool (Mini-International Neuropsychiatric Interview). 
 
3. A licensed physician, nurse practitioner (NP), or physician’s assistant (PA) who is clinically privileged at the 
Durham VA will examine patients prior to randomization to study drug (pregnenolone) or placebo. Patients will have a 
venous puncture for laboratory/genetic testing (please also the Schedule of Events). Patients will be asked to provide 
approximately 3.5 tablespoons of blood on the first study visit, and approximately 2.5 tablespoons of blood on Visits 
#2, 3, 4, 5, 6 and 7.  This is a total of approximately16 tablespoons of blood over the duration of the study. Subjects 
will receive an ECG at Visits #1 and 6. Patients with clinically significant abnormal physical exam, blood tests, or 
ECG that preclude safe study participation will be excluded from the study and referred to their primary care provider 
or appropriate specialist. 
 
4. Following the screening visit (Visit 1), all patients will begin a two-week placebo-only lead-in period (prior to 
randomization at Visit 2). On the day that patients are given the study medication and randomized (and at each visit 
during the study), a set of vital signs will be done including pulse, blood pressure, and weight. With regard to the study 
medication, participants will be told that they could be getting placebo (a sugar pill) or active medication 
(pregnenolone).  The subjects will be told that they may be receiving a different dose every two weeks. They will be 
asked to take the medication twice a day; once in the morning and once in the evening. 
 
5. After a week of the placebo lead-in period, a member of the research team will contact the participant to ask about 
study medication compliance and to answer any questions. A member of the research team will continue to contact 
patients by phone every two weeks to ask about compliance to study medication, possible suicidality, and potential 
adverse events (these telephone check-in contacts will be staggered with in-person patient visits every two weeks; 
please see also Schedule of Events for details).   
 
6. Subjects will return for a study visit every two weeks. Vital signs will be repeated at each visit. A member of the 
research team will ask subjects about possible side effects and adverse events (utilizing a structured Adverse Event 


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Form) and will administer the Columbia Suicide Severity Scale to assess possible suicidality. Patients will be asked to 
return tablets of pregnenolone or placebo to determine compliance with study medication by pill counting. The subject 
will be given a new supply of tablets (either pregnenolone or placebo, depending upon random assignment) every two 
weeks. Patients will receive a laboratory testing (Chem 7, GI panel, CBC) at all study visits. Female patients will also 
receive a pregnancy test at Visits #1, 2, 4 and 6.  TSH, free T4, B12/folate, and a lipid panel will be assessed at Visits 
#1 and 6. A urinalysis and urine tox screen will be conducted at Visits 1, 2, 4, and 6. 
 
7. At Visits #1 (screening visit), 2 (randomization visit), 3, 4, 5 and 6 (each visit is 2 weeks apart), three extra red-top 
tubes will be collected for pregnenolone levels and determination of pregnenolone metabolite levels, proteins, and 
other small molecules.  A PaxGene tube and two purple top tubes will be collected at Visit #1 (for genetic analyses).   
 
8. At Visit 6 (=8 weeks post-randomization), the study medication will be tapered (150 mg BID x 2 days, followed by 
50 mg BID x 2 days, followed by study medication discontinuation). Subjects will return 1-2 weeks after Visit 6 for a 
follow-up visit that includes vital signs, laboratory tests, administration of the CSSRS to assess for suicidality, and 
adverse event assessment. 
 
9. The WRAT, HADS, and TBI screen will be administered at Visit #1. The SF-36, BAC-A, BPI, MFI, SCL-90, 
HADS, BDI, PCL, WHODAS, SDS, and CDRS will be administered at Visits #2, 4 and 6. The Adverse Event Scale 
and CSSR (to assess for suicidality) will be administered at each in-person visit and at each telephone visit.  
 
Randomization and Maintenance of the Blind:   Randomization will occur in blocks of 10, and will be conducted by 
the research pharmacist at the Durham VAMC. Only the research pharmacist will have access to the randomization 
code during conduction of the study, and the PI and all raters, investigators and other study staff will be blinded during 
the study.  The pharmacist does not participate in assessing any of the primary symptom or side effect dependent 
variables, and conveys no information about treatment assignment to subjects or staff.  Study medication will be 
dispensed every two weeks. Subjects will be given six extra days of medication in case of a delayed or rescheduled 
appointment. The blind will be broken only if a medical emergency requires this information. If this occurs, the subject 
will be withdrawn from the study.  
After the study is completed and the last participant leaves the study, the aggregate blinded data will be cleaned (i.e. 
reviewed for possible outliers that may reflect data entry errors, etc.) and the database will be locked.  A de-identified 
copy of the locked data will be given to our PhD-level statistician for analysis.  A de-identified copy of the locked data 
will also be give to a second statistician for independent replication of the study analyses.  The PI will not be involved 
in study data analyses. 
 
Neurosteroid Quantification:     
 
Gas Chromatography/Mass Spectrometry (GC/MS) Preceded by High Performance Liquid Chromatography 
(HPLC) 
 
Neurosteroid quantifications in serum samples will be were performed by a highly sensitive and specific gas 
chromatography-mass spectrometry method, as described previously (Marx et al, 2006), preceded by high performance 
liquid chromatography (HPLC) purification, with modifications (the electron impact ionization mode will be utilized 
for this investigation, rather than negative ion chemical ionization). One mL of serum will be extracted three times in 
ethyl acetate prior to HPLC purification using tetrahydrofuran, ethanol, and hexane in the mobile phase. 
 
For neurosteroid quantification, the standard curve for the steroid of interest is prepared by combining varying known 
quantities of steroids (Steraloids) ranging from 2 to 3000 pg/2 L with a constant amount of the respective deuterated 
internal standard and tritiated neurosteroid of interest.  Identical to the experimental samples, the standard curve is 
extracted three times in ethyl acetate prior to HPLC purification and GC/MS injection; standard curve r2 = 0.99 for 
each neurosteroid.  The area under the peak of each known quantity of steroid is divided by the area under the peak of 
the internal standard.  This ratio is then plotted on the y-axis against known quantities of each steroid to generate the 


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standard curve.  Only peaks with a signal to noise ratio greater or equal to 5:1 are integrated.  The limit of neurosteroid 
detection with this method is 1 pg for pregnenolone, allopregnanolone, pregnanolone, and androsterone.  Intra-assay 
coefficients of variation range between 1.5-8.0% for each neurosteroid. 
 
ADVERSE EVENTS AND SAFETY EVALUATIONS 
 
Adverse Events Assessment:  Adverse events will be assessed by administration of the Hillside Adverse Events Form 
and reviewed by a licensed physician, NP, or PA with clinical privileges at the Durham VA.  Adverse events will be 
assessed at all study visits after the screening visit (i.e. Visits 2 through 6 and at the follow-up visit) as well as at each 
phone call (phone calls staggered with study visits – i.e. participants will receive an adverse events assessment weekly, 
either during a telephone check-in call every two weeks or in person at a study visit every two weeks).  In addition, 
adverse events will be assessed during a follow-up visit one-two weeks after study medication taper. 
 
Withdrawal from Study:  
 
As stated on the Informed Consent Form, all subjects reserve the right to withdraw from the clinical investigation at 
any time.   
 
Subjects may be discontinued from the study by the Principal Investigator for the following reasons: 
 
Subject withdraws consent to participate in the study; 
 
Subject is noncompliant with procedures set forth in the protocol; 
 
Subject experiences an AE that warrants withdrawal from the study; 
 
It is in PI’s opinion that it is not in the subject’s best interest to continue; 
 
Other laboratory, medical, or clinical finding for which clinical care should take precedence over study 
participation. 
 
Adequacy of Protection from Risks 
 
a) Physical Risk and Subject Safety: Possible side effects from drawing blood will be attended to as usual in the 
phlebotomy laboratory. If a subject experiences serious adverse effects from the study medication, it will be 
discontinued and s/he will be withdrawn from the study. The subject will immediately be referred for appropriate 
evaluation and treatment. Adverse events (AEs) and serious adverse events (SAEs) will be documented and reported as 
required by the Durham VA IRB committee, the FDA, and the national VA Data Safety and Monitoring Board.  
Progress reports summarizing AEs and SAEs will be submitted to the Durham VA IRB and FDA annually, and to the 
national VA DSMB every 6 months as required.  Any SAEs will be rapidly reported as required within the timeframe 
and guidelines of the Durham VA IRB, the FDA, and the national VA DSMB.   
 
b) Emotional Distress and Subject Safety: All subjects will be carefully assessed before the study and will be made 
aware of emergency services (available 24/7 at the Durham VA). In addition, they will be assessed at screening and at 
each study visit. During the informed consent process, they will be advised that the study procedures could potentially 
lead to distress (some questionnaires could be upsetting, for example), and that they may withdraw from the study at 
any time without adversely affecting their medical care or any benefits they may be receiving. If a subject is 
significantly distressed during a study visit (and if safety is a concern, i.e. expressing thoughts of self-harm or harm to 
others requiring clinical intervention), or reporting significant medical problems requiring clinical attention, s/he will 
be walked to the emergency room onsite for rapid evaluation and treatment (including potential hospitalization if 
clinically indicated). Dr. Marx will be available by cell phone to study participants for any concerns 24/7 (her cell 
phone number is 919-██, as listed in the consent form of this study; all consented participants will receive a hard copy 
of this consent form).  When Dr. Marx is traveling or on annual leave, a licensed and clinically privileged psychiatrist 
or other physician at the Durham VA will assume coverage - such as Dr. Daniel Bradford (who has extensive 
experience in clinical trials in Veterans with psychotic disorders), Dr. Michael Hertzberg (who has extensive 


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experience in clinical trials in Veterans with PTSD), or Dr. Richard Weiner (who has extensive experience in clinical 
trials in depression and who is the Durham VA Mental Health Service Line Chief).  In terms of risks to confidentiality, 
pertinent information regarding potential harm, including suicidal and homicidal intent, will be shared as necessary 
and as required by law with clinicians and/or the appropriate authorities.  In such circumstances, records may be made 
available to authorities, even without the subject’s consent, in the setting of potential risk of harm to self or others. 
Safety Reporting: 
The National VA Data Monitoring Committee (DMC) will receive a report of adverse events every six months. 
Serious adverse event reporting will adhere to requirements of the Durham VA IRB, the FDA, and the national VA 
DSMB (also referred to as the national VA DMC). Adverse events will be reported to appropriate regulatory agencies 
within mandated timeframes. 
 
Suicidality Safety Procedures for Phone Calls and In-Person Suicidality Assessments:   
If a participant reports suicidality (suicidal ideation, gesture, or attempt) as per the Columbia Suicide Severity Rating 
(CSSRS, which is an FDA-recommended assessment for suicidality in clinical trials – and which will be administered 
at every check-in phone call and in person at every visit – hence weekly, either in person on by phone) during any 
phone call or during an in-person study visit, a study physician will be notified immediately to determine whether the 
situation requires immediate clinical intervention.  If a participant is thought be at imminent risk to self, he/she will be 
referred immediately to emergency psychiatric care at the Durham VA Emergency Department, to Psychiatric 
Emergency Services (PEC) at the Durham VA, or to the participant’s local Emergency Department for further 
evaluation (if the assessment was conducted by phone).  If the participant is unwilling or unable to get to emergency 
psychiatric care and appears to be in imminent danger of self-harm, the study team will contact local law enforcement 
who will be asked to check on the participant in person.  If the study assessment was conducted at an in-person study 
visit, a study physician will be contacted immediately to assess the patient and to develop a clinical plan; if the 
participant is judged to require acute intervention, s/he will be walked to the Psychiatry Emergency Care at the 
Durham VA or to the Durham VA Emergency Room.    
  
Risks for Study Personnel:  There is minimal risk involved to study personnel.  A trained laboratory phlebotomist will 
draw the participant’s blood for laboratory analysis and serum/blood collection.  There is a slight risk of potential 
needle stick, however the Durham VA Chemical Laboratory provides extensive risk prevention training for all 
phlebotomists, and thus the likelihood of an accidental needle stick is low.  The blood samples will be transported to a 
-80º Celsius freezer for storage by study personnel.  In order to reduce the risk of direct exposure, personnel will wear 
personal protective equipment while transporting patient serum/blood samples.   
 
ADDITIONAL SAFETY PLAN DETAILS: 
 
Overview of Facilities: 
Durham VA Medical Center (DVAMC) Psychiatric and Medical Services:  
i. The DVAMC has acute psychiatric care available onsite 24 hours per day/7 days per week provided by the 
Psychiatric Emergency Care (PEC) team and staffed by a Duke psychiatry resident, psychiatric social worker, and VA 
psychiatry attending.   
 
ii.  The DVAMC has an inpatient psychiatric unit onsite with 28 inpatient psychiatric beds.  It is staffed by 
three VA Mental Health Service Line psychiatry attendings, three Duke psychiatry residents, and other mental health 
care professionals.  
 
iii.  The DVAMC has an Emergency Room onsite providing care 24 hours per day/7 days per week, which is 
staffed by Duke/VA internal medicine residents, fellows, and attendings.   
 
iv.  Extensive specialty and subspecialty consultation services are also available at the DVAMC, including 
renal, GI, dermatology, cardiology, orthopedic surgery, otolaryngology, endocrinology, pain management, dental 
health, vocational rehabilitation, and occupational health, among others. 
 
 


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10 
 
Vital Signs and Side Effect Monitoring: 
 
Procedures for Addressing Abnormal Vital Signs and Potential Side Effects: 
i.  Vital signs will be assessed at each study visit.  Blood pressure >160/95 and other clinically significant vital 
sign abnormalities will be followed-up with the patient’s primary care physician (PCP), the internal medicine 
physician in the Emergency Room, or an appropriate consult physician, as clinically indicated. 
 
ii.  If vital sign abnormalities require same-day medical follow-up, the patient will be assessed in the DVAMC 
Acute Care Clinic/Emergency Room.    
 
iii.  If vital sign abnormalities do not require same-day medical follow-up, an appointment will be scheduled 
with the patient’s PCP.  
 
iv.  Patients will receive an extensive side effect scale at each study visit (i.e. every two weeks, as outlined 
above; specifically with the Hillside Adverse Events Scale).  Patients will also be queried regarding potential side 
effects utilizing this scale at staggered phone check-in contact during the weeks when patients do not receive a study 
visit.  Potential side effects will be monitored closely by the PI and other MD and/or NP and/or PA members of the 
research team.  A participant may be asked to return for an unscheduled appointment to assess a potential side effect if 
clinically indicated.  The PI will consult as necessary with the patient’s PCP, internal medicine physician in the ER, 
psychiatrist, or relevant consult physician. 
 
v.  Clinical worsening of psychiatric assessments (>25% increase in PTSD or depression symptoms over two 
consecutive visits, as assessed by the PCL and BDI-II) will be discussed with the patient’s treating psychiatrist, other 
mental health care provider, or PCP, and the option of study withdrawal will be discussed with the patient and his/her 
provider.  As always, clinical care will take precedence over study participation.   
 
 
 
 
Laboratory Results:  
 
Procedures for Addressing Abnormal Laboratory Results: 
 
i.  Patients will receive a CBC, clinical chemistry panel, and liver function tests at each of the six study visits 
in this clinical trial.  At screening, mid-point, and final study visit, patients will also receive a urinalysis and urine 
toxicology test (and serum pregnancy test if female). At the screening study visit, patients will receive a TSH, 
B12/folate, and lipid levels.  Lipid levels will also be checked at Visit 6.  Laboratory results out of the normal range 
(range determined by the Durham VA Medical Center Clinical Chemistry Laboratory that services both the outpatient 
and inpatient services at our hospital, and that will conduct the above lab assessments for this study) will receive 
appropriate review and follow-up as described below.   
 
ii. The PI (or MD/NP/PA member of the research team supervised by the PI) will review laboratory results 
within 24 hours.  Clinically relevant laboratory abnormalities will receive prompt follow-up attention with the patient’s 
PCP or other health care provider, the internist physician in the ER, and/or a follow-up visit for additional lab work as 
clinically indicated.   
 
iii.  The DVAMC Clinical Chemistry Laboratory that services the hospital will phone the PI with any “alert” 
laboratory values that require immediate attention. 
 
iv.  Any laboratory abnormalities that may potentially preclude study participation will be discussed with the 
patient’s PCP, the internal medicine ER physician, the patient’s treating psychiatrist, or other relevant health care 
provider.   
 
EKG Monitoring: 
 
Procedures for Addressing Abnormal EKG Findings: 
 
i.  Final EKG readings issued by a cardiologist are available from the DVAMC Heart Station within 48 hours.   
 
ii.  Each patient will have an EKG at baseline and completion of the study.  If the patient does not complete the 
study and withdraws prior to the final study visit, every effort will be made to obtain an EKG at the follow-up visit 
post-withdrawal from the study (please see withdrawal plan below).  
 
iii.  Any abnormal EKG findings will be discussed with cardiologist Dr. Joseph Greenfield (former Chair of 
the Department of Medicine at Duke), who generously consults with our clinical trial team.  If Dr. Greenfield suggests 


Marx, Christine E. 
 
Version: 4 – 02/20/2015 
11 
 
additional medical follow-up, we will proceed according to his recommendations (see below).  If Dr. Greenfield is 
unavailable, the EKG will be discussed with another cardiologist, the ER physician, or the patient’s PCP. 
 
iv.  Additional potential follow-up actions may include, but are not limited to:  Primary care provider 
notification by pager or VA email, the placement of a cardiology consult, a repeat EKG (if lead placement is suspected 
to have been suboptimal, for example), or referral to the onsite Acute Care Clinic/Emergency Room for assessment.    
 
Psychiatric Worsening: 
 
Procedure for Addressing Psychiatric Worsening: 
 
i.  Patients who demonstrate psychiatric worsening as assessed by a 25% increase over baseline of the PCL 
(PTSD symptoms) or BDI-II total score (depression symptoms) on two consecutive visits (which are two weeks apart) 
may be withdrawn from the study (even if this change is thought to be unrelated to the study), and they will be referred 
to their treating psychiatrist, other mental health care provider, PCP, or the onsite Psychiatric Emergency Care team.  
Please see also above description. 
 
ii.  Patients who exhibit suicidal or homicidal ideation requiring urgent evaluation during a study visit will be 
referred to the onsite DVAMC Psychiatric Emergency Care team for prompt assessment and possible inpatient 
psychiatric admission.   
 
Addressing Hormonal Changes in Response to Test Drug or Drug Metabolites:  
 
Procedures for Quantifying Neurosteroid and Neurosteroid Metabolite Levels: 
Neurosteroids and neurosteroid metabolites will be quantified by gas chromatography/mass spectrometry 
(preceded by high performance liquid chromatography), radioimmunoassay, or ELISA, as previously described.  Our 
prior data suggest that pregnenolone, pregnenolone sulfate, progesterone, and allopregnanolone increase following 
treatment with pregnenolone (please see below for additional detail).   
 
In our pilot randomized controlled trial with pregnenolone in schizophrenia (PDF attached), pregnenolone 
administration at the identical doses to those proposed in the current study did not significantly increase cortisol, 
testosterone, free testosterone, estradiol, androstenedione, or DHEA, however (Marx et al 2009, attached; please see 
table on p.10).  A modest 20% increase was observed in DHEAS (Marx et al 2009).  In this pilot study of 
pregnenolone in schizophrenia, as well as in subsequent investigations [specifically, a) a pilot investigation in 
OEF/OIF Veterans with mild TBI, b) a randomized controlled trial in schizophrenia enrolling 120 participants that was 
recently completed in Singapore, c) a pilot investigation in Veterans with PTSD, and d) a randomized controlled trial 
of 80 patients with bipolar disorder], pregnenolone was well-tolerated.   
 
For the last several clinical studies with pregnenolone, we have quantified pregnenolone and pregnenolone 
metabolite levels after the trial was completed so that samples could be run in one batch with the same standard curve 
in order to minimize inter-assay variation.  The FDA did not require monitoring of neurosteroid levels or neurosteroid 
metabolite levels during these studies.  Neurosteroid and neurosteroid metabolite levels will thus be quantified at the 
completion of this study, as we have done with prior clinical investigations utilizing adjunctive pregnenolone as a 
pharmacological intervention.   
 
Follow-up Plans: 
 
Follow-up Procedures: 
The follow-up plan after unanticipated withdrawal from the study (defined as withdrawal from the study at any 
point prior to completion and subsequent to randomization to study drug) will be to schedule a follow-up visit within 
7-21 days of cessation of study participation.  At this follow-up visit, patients will receive vital signs, laboratory testing 
(CBC, chemistry panel, liver function tests) and the Columbia Suicide Severity Rating Scale.  We will also make 
certain that the patient has a follow-up appointment with his/her PCP or mental health care provider, and that the 
patient has adequate refills of psychiatric medications until the next scheduled appointment (if taking psychiatric 


Marx, Christine E. 
 
Version: 4 – 02/20/2015 
12 
 
medications).  If there are any medical issues to be addressed, these will be discussed with the patient’s PCP, 
psychiatrist, the internal medicine physician in the ER, or other relevant health care provider.  For any urgent medical 
issues, patients will be escorted to the onsite Acute Care Clinic/Emergency Room at the Durham VA Medical Center, 
which provides 24/7 services.  For non-urgent medical issues, patients will be scheduled for an outpatient appointment 
with their PCP or other health care provider.  
       
The follow-up plan for patients who complete the entire study (6 visits total over the course of 10 weeks, 
consisting of a 2-week single-blind placebo lead-in period followed by randomization to 8 weeks of adjunctive 
pregnenolone or placebo) will be an additional follow-up visit to be conducted within 7-21 days after the final visit.  At 
this follow-up visit, patients will receive vital signs, laboratory testing (CBC, chemistry panel, liver function tests), and 
the Columbia Suicide Severity Rating Scale.  Similarly to the above follow-up plan for unanticipated withdrawal, we 
will also make certain that the patient has a follow-up appointment with his/her PCP or mental health care provider, 
and that the patient has adequate refills of psychiatric medications until the next scheduled appointment (if taking 
psychiatric medications).  If there are any medical issues to be addressed, these will be discussed with the patient’s 
PCP, psychiatrist, the internal medicine physician in the ER, or other relevant health care provider.  For any urgent 
medical issues, patients will be escorted to the onsite Acute Care Clinic/Emergency Room at the Durham VA Medical 
Center, which provides 24/7 services.  For non-urgent medical issues, patients will be scheduled for an outpatient 
appointment with the patient’s PCP or other health care provider.  
 
PAYMENTS TO SUBJECTS 
Subjects will be paid $75.00 for each study visit (six study visits total, plus a follow-up visit or $525.00 total if 
a subject completes all seven study visits).  If a subject attends two study visits, the subject will be paid $150; if s/he 
attends 3 study visits, s/he will be paid $225, etc.).  There are no research-related costs to subjects participating in this 
study.  Participants will be provided with a paper voucher, which is redeemable for cash at the Durham VAMC 
Cashier’s Office (payment for funds will be provided by the VA). 
 
There will be an additional allowance for travel to the Durham VAMC based on the distance traveled by the 
subject. The travel allowance will be approximated by the distance traveled. Travel allowance for several cities and 
major towns in VISN6 are included in the table below.  There will be no further compensation for meals, parking, 
childcare, lost wages, etc.  
 
 
 
 
 
 
 
 
 
DATA AND SAFETY MONITORING 
 
Sources of Materials:  All of the data for this study will be collected specifically for research purposes. All study data 
will be kept in separate locked file cabinets, in locked offices within locked (card accessible only) building and will be 
accessible only to qualified research personnel (in VAMC Building █, Room █; Building █, █ floor; or Building █, █ 
floor).  Access to research data will be removed for staff that are no longer part of the study team.  Each subject will be 
assigned a study number and all data and specimens will be identified by that study number. The list linking this 
number to subject identity will be kept in a file on a secure VA network drive (███), accessed through a password-
protected computer to which only the Principal Investigator and study staff have access. Seven sources of data will be 
maintained for this study: a) the initial telephone screening, b) diagnostic interviews, c) physical examinations, d) 
psychiatric rating scales and cognitive assessments (both self-report and clinician-administered, e) genetic analyses, 
and f) neurosteroid profiles, other small molecule and protein results, and g) laboratory assessments and ECG results. 
 
Distance (roundtrip miles) Allowance Typical towns and cities 
0-50 
$10 
Raleigh, Durham, Cary, Chapel Hill 
50-100 
$20 
Henderson, Wake Forest, Burlington 
100-200 
$30 
Greensboro, Fayetteville, Goldsboro 
200-300 
$40 
Salisbury, Greenville, Rocky Mount 
300-400 
$50 
Wilmington, Charlotte, Hickory 
 > 400 
$60 
 


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Data Monitoring and Confidentiality:  Reports from subjects’ clinical records concerning research observations will 
not be made available to outside medical facilities without the written consent of the patient. All clinical and biological 
data obtained from research interviews and the laboratory will be de-identified. The data will be kept in locked file 
cabinets, in locked offices within locked (card accessible only) buildings and will be accessible only to qualified 
research personnel (in VAMC Building █, Room █; Building █, █ floor; or Building █, █ floor). and kept in 
accordance with VA Records Control Schedule.  Only study numbers will appear on data, documents and biological 
specimens used for evaluation or statistical analysis (i.e., de-identified). The key-code linking subjects to study data 
identified by study number will be kept on a secure VA network drive (███).  In addition, any publications resulting 
from this research will not identify individual subjects.  
 
PRIVACY AND CONFIDENTIALITY 
 
Patient confidentiality will be maintained through the assignment of patient identification numbers. These numbers 
will be used in keeping of all research records. All hard copy research materials will be kept in locked file cabinets 
with keys available only to the principal investigator and research personnel participating directly in this protocol. 
Access to research data will be removed for staff that are no longer part of the study team.  Patients will be informed 
during the consent process about the limits of confidentiality. 
 
Information about study subjects will be kept confidential and managed according to the requirements of the Health 
Insurance Portability and Accountability Act of 1996 (HIPAA). Those regulations require a signed subject 
authorization informing the subject of the following:  
     -What protected health information (PHI) will be collected from subjects in this study 
     -Who will have access to that information and why 
     -Who will use or disclose that information 
     -The rights of a research subject to revoke their authorization for use of their PHI.  
 
PHI collected as part of this study will include:  name, address, telephone number, date of birth, dates of service, dates 
of attendance, social security number, gender, close relative’s name, close relative phone number, marital status, race, 
education level, work status, employer, length of employment, occupation, service connection (reason, percentage), 
disability status, medical and mental health diagnoses and treatments, medication, substance use, history of suicide 
attempts, family history of suicide attempts, smoking status, incarceration history, twin status, military service (time 
period, branch, number of tours, whether or not subjects served in a war zone, fired a weapon in a combat situation, 
were under enemy fire, were wounded in a war zone, awarded medals or were prisoners of war).  Genetic data will also 
be collected. 
 
As part of the study (and as indicated in the HIPAA Authorization), patient information and medical and/or research 
records may be disclosed to the Office for Human Research Protections (OHRP), the VA Office of the Inspector 
General (OIG), the Office of Research Oversight (ORO), other government agencies, the Durham VAMC Institutional 
Review Board (IRB), and/or local Research Compliance Officers, and Food and Drug Administration (FDA). 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, retains the ability 
to use all information collected prior to the revocation of subject authorization.  
 
INFORMATION SECURITY 
 
Collection, Use and Storage of Research Information:  Participant data will be completely de-identified according to 
18 identifiers listed in Appendix B of the VHA Handbook 1605.1, and participant information will be identifiable only 
by a unique, linked numerical code.  The master key code will be maintained on the secure VA network drive (███) 
and which is accessible only through a VA, password protected computer (in Building █, Room █; Building █, █ 
floor; or Building █, █ floor).  All electronically recorded PHI (i.e., any patient identifying information) will be 
maintained in a separate file on a secure VA password protected network drive (███) on a VA computer.   Any PHI 
collected on paper which needs to be maintained (i.e., Informed Consent Form, HIPAA Authorization, Subject 


Marx, Christine E. 
 
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Payment Forms, etc.) will be stored in a folder separate from deidentified paper forms (i.e., study assessments).  These 
two folders will be filed in two different locked file cabinets inside a locked room within the Durham VAMC 
(Building █, Room █; Building █, █ floor; or Building █, █ floor).  Only approved VA researchers and study 
personnel will have access to this information.  No PHI will be removed from the Durham VAMC.  De-identified 
information collected using paper forms (i.e., study assessments) will be stored in a locked filing cabinet inside Room 
█ in VAMC Building █; Building █, █ floor; or Building █, █ floor.   These paper forms will be entered into a 
database stored on a separate, secure, password protected database (███) within the Durham VAMC (so that 
information cannot be linked to participant PHI) by study personnel and will be stored indefinitely according to VA 
guidelines.  Paper form and electronic data will be destroyed in accordance with the Records Control Schedule 10-1, 
which currently does not allow for the destruction of research data. 
 
Patient blood and serum samples will be collected during the study visit(s) and coded with a corresponding unique, 
linked numerical code (i.e. the study ID number).  Specimens will be completely de-identified.  Blood and serum 
samples will be stored at the Durham VAMC in a locked freezer room in Building █ or █ located in a secure research 
wing (requires special permission and a security activated badge to gain access) within the Durham VAMC.   
 
Subjects will be asked to provide permission to store their contact and demographic information and study interview 
results in a secure database (an excel spreadsheet maintained by the PI and her study staff on a secure VA server) to be 
used to select potentially eligible participants for other ongoing and future studies.  If patients provide permission to do 
so, they may then be re-contacted in the future and invited to participate in other studies for which they may qualify, 
and of which Dr. Marx is an investigator.   
 
Any incident regarding theft/loss of data, authorized access of sensitive data or storage devices or non-compliance with 
security controls will be addressed according to VA policy (VHA Handbook 1200.05, 10j; 1058.01 11.a; 6500, 
Appendix D and 6500.2)  and will be reported to Durham VAMC PO and ISO immediately. 
 
Study members who are initially part of the study team, but leave the study team for whatever reason will no longer 
have access to participant study data. 
 
Risks/Benefit Assessment: 
Subjects will NOT be tapered from their current stable medication regimen; adjunctive pregnenolone treatment will be 
“add-on” only to treatment-as-usual. Pregnenolone is available over-the-counter as a nutritional supplement. 
Pregnenolone has been well-tolerated at the doses proposed in this study.  Previously reported uncommon adverse 
reactions include headache, rash, insomnia, stomach upset, a report of palpitations in the existing literature. We will 
perform ECGs at Visits 1 and 6 to closely monitor patients, and perform a Chem 7, GI panel, and CBC at each study 
visit. Additional laboratory tests will be conducted at Visit 1 and 6 (TSH, etc.).  Blood draws at each visit are minimal 
risk. Possible blood draw side effects include bruising, bleeding, or pain at the injection site, and (rarely) fainting and 
infection. It is possible that pharmacological intervention with pregnenolone will improve functional outcomes, 
cognitive symptoms, pain symptoms, fatigue, and global psychological functioning in Gulf War Veterans with GWVI. 
 
Potential Benefit of the Proposed Research to the Subject and Others: While study participants may not receive 
benefits from the proposed research other than monetary compensation, their participation may lead to a better 
understanding of Gulf War symptomatology. For those not currently treated, study participation may lead to referral 
for treatment upon completion (or withdrawal) of the study. In terms of benefit to others, knowledge gained from the 
study may help the evaluation and treatment of Gulf War Veterans with GWVI. No serious adverse events have been 
reported to date related to pregnenolone. Pregnenolone is available as a dietary supplement over-the-counter in the 
United States. 
 
 
 
 


Marx, Christine E. 
 
Version: 4 – 02/20/2015 
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Data Management: 
 
Monitoring:  The database manager will review completed forms with the study coordinator and generate queries, 
which the study coordinator will resolve before data entry. 
 
Data Monitoring/Quality Assurance:  The study coordinator will check that all assessments have been completed and 
check all forms for accuracy and completeness prior to the termination of each session. The centralized database will 
provide information on which assessments are to be completed at each visit. In addition, the data entry procedures will 
notify those entering the data when information has been omitted. These procedures should greatly increase overall 
quality of the data. As described earlier, the data entry system itself forces data entry personnel to enter complete 
within range data or produce a reason, which is stored in the database. 
 
Protocol Fidelity Monitoring:  The data manager will continuously track adherence to the protocol, and will report any 
problems to the PIs and the statistician. Monitoring will include adherence to inclusion/exclusion criteria, completion 
of measures at each assessment point, following procedures for termination of patients from the protocol, and training 
and certification for all research personnel. The monitor and data manager will verify that valid informed consent has 
been obtained from all participants. Research personnel must log the date of informed consent, the staff member 
obtaining the informed consent, and that the participant has received a copy of the informed consent document. In 
addition, all participants must verify that they have provided informed consent and received a copy of the informed 
consent document. All these steps must be completed prior to entering any information about the participant into the 
database. 
 
DATA ANALYSIS AND STATISTICAL CONSIDERATIONS 
In order to ensure the rigorous management of any conflict of interest issues in the data analysis: 
1. All persons involved in this study will be completely blinded to treatment condition in this randomized 
controlled trial investigating adjunctive pregnenolone vs. placebo in Veterans with Gulf War Veterans’ 
Illnesses for the duration of the study, including Dr. Marx, the study coordinator, and all staff in the 
research group.  The randomization code will be known only to the VA pharmacist Jaime Brown, and the 
blind will be broken only in the event of a medical emergency.   
2. Dr. Marx will not conduct any of the psychiatric assessments (cognitive rating scales, psychiatric scales, 
etc.).  Psychiatric and cognitive assessments will be conducted by the clinical coordinator and/or other 
research staff only.   
3. This project will involve independent PhD-level statisticians. Dr. Marx will be completely removed from 
the conduction of the statistical analyses.  After clinical trial data are entered by research staff, double-
checked, and cleaned, the database will be locked.  No statistical analyses will be conducted prior to the 
locking of the database.  Once the data are locked, a completely de-identified copy of the dataset (no PHI) 
will be sent to independent statisticians. Once the de-identified database is locked, the blind will then be 
broken by pharmacist Jamie Brown.  Dr. Ryan Wagner will conduct the statistical analyses (the statistical 
analysis plan will be determined a priori), and this analysis will be confirmed by an independent 
statistician. 
4. There will be a VA DSMB that will be overseeing this project.   
5. Dr. Marx will promptly contact the Durham VA IRB Committee, the Durham VA R+D Committee, the 
VA DSMB, and the ACOS for Research at the Durham VA if a patent is issued or if a licensing agreement 
is negotiated. 
 
 
 
 


Marx, Christine E. 
 
Version: 4 – 02/20/2015 
16 
 
POWER ANALYSIS:  PHYSICAL COMPONENT OF THE SF-36 (Primary Endpoint).  
Primary Endpoint Hypothesis:  Gulf War Veterans with GWVI who were randomized to pregnenolone will 
demonstrate significantly greater improvements in the Physical Component Score of the SF-36 compared to Veterans 
randomized to placebo. 
 
Numeric Results for Two-Sample T-Test 
Null Hypothesis: Mean1=Mean2. Alternative Hypothesis: Mean1<>Mean2 
 
The standard deviations were assumed to be equal.  A prior investigation in Gulf War Veterans with  GWVI determined mean 
baseline standard deviation in the SF-36 Physical Component Summary to be 7.5 (Donta et al 2003).  We chose a more 
conservative SD estimate of 12.5 for this sample calculation. We utilized the baseline SF-36 score from the same investigation 
(Donta et al 2003) to estimate approximate baseline SF-36 scores in a Gulf War Veteran cohort with GWVI.  Consistent with 
the literature in this area, we utilized a 7-point difference in the SF-36 as a clinically meaningful change from baseline for this 
sample size calculation (please see attached SF-36 scoring information in Appendix). 
 
   
 
 
 Allocation 
Power 
N1 
N2 
Alpha 
Beta 
Mean1 
Mean2 
S1 
S2 
0.71774 
42 
42 
0.05000 
0.28226 
33.7 
40.7 
12.5 
12.5 
0.75708 
46 
46 
0.05000 
0.24292 
33.7 
40.7 
12.5 
12.5 
0.79175 
50 
50 
0.05000 
0.20825 
33.7 
40.7 
12.5 
12.5 
0.82211 
54 
54 
0.05000 
0.17789 
33.7 
40.7 
12.5 
12.5 
0.84855 
58 
58 
0.05000 
0.15145 
33.7 
40.7 
12.5 
12.5 
0.87147 
62 
62 
0.05000 
0.12853 
33.7 
40.7 
12.5 
12.5 
0.89124 
66 
66 
0.05000 
0.10876 
33.7 
40.7 
12.5 
12.5 
0.90822 
70 
70 
0.05000 
0.09178 
33.7 
40.7 
12.5 
12.5 
0.92276 
74 
74 
0.05000 
0.07724 
33.7 
40.7 
12.5 
12.5 
0.93516 
78 
78 
0.05000 
0.06484 
33.7 
40.7 
12.5 
12.5 
0.94569 
82 
82 
0.05000 
0.05431 
33.7 
40.7 
12.5 
12.5 
Report Definitions 
-Power is the probability of rejecting a false null hypothesis. Power should be close to one. 
-N1 and N2 are the number of items sampled from each population; they should be small. 
-Alpha is the probability of rejecting a true null hypothesis. It should be small. 
-Beta is the probability of accepting a false null hypothesis. It should be small. 
-Mean1 is the mean of populations 1 and 2 under the null hypothesis of equality. 
-Mean2 is the mean of population 2 under the alternative hypothesis. Mean of population 1 is unchanged. 
-S1 and S2 are the population standard deviations. They represent the variability in the populations. 
 
Summary Statement 
Group sample sizes of 70 and 70 will achieve 90% power to detect a difference of 7 points between the null 
hypothesis that both group means are 33.7 and the alternative hypothesis that mean of Group 2 is 40.7  
with estimated standard deviations of 12.5 and 12.5 and with a significance level (alpha) of 0.05000 using  
a two-sided two-sample t-test. We thus have 90% power to detect a 7-point improvement in the SF-36.  
 


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Version: 4 – 02/20/2015 
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Power vs N1 with M1=33.7 M2=40.7 S1=12.5 S2=12.5
Alpha=0.05 N2=N1 2-Sided T Test
Power
N1
0.70
0.75
0.80
0.85
0.90
0.95
40
50
60
70
80
90
 
 
POWER ANALYSIS:  PAIN SYMPTOMS (Secondary Endpoint)  
Secondary Hypothesis #1:  Treatment with pregnenolone will improve pain symptoms in Gulf War Veterans with 
GWVI.   
 
The potential effect of pregnenolone on pain symptoms will be evaluated using several statistical methods.  This 
clinical trial design will compare two randomly assigned groups (adjunctive pregnenolone or placebo) with respect to 
self-reported pain measured on a 1-10 pain scale.  Inclusion criteria for the study require that all participants report 
pain severity of at least four. Given that pain often presents as long periods of low-level intensity pain punctuated by 
periods of acute exacerbation, it is likely that the mean pain level for selected participants will exceed four. Thus for 
power calculations, we assume a baseline mean pain level of six for each group.  We assume a standard deviation of 
1.0 (as we have little information to support any specific value in this population). 
 
For the purpose of power calculations we assume that the mean pain levels of the two independent groups will be 
compared using a Two-Sample T-Test, Null Hypothesis: Mean1=Mean2. Alternative Hypothesis: Mean1<>Mean2.  
The standard deviations were assumed to be unknown and equal. 
   
Assuming specified numbers of participants in each group (N) the mean differences detectable for at least 80% power 
and alpha=0.05 are shown in the following table and figure.  
 
 
 
 
 
N1 
      Mean1    Mean2 
 
4 
6.0 
3.6 
 
5 
6.0 
3.8 
 
6 
6.0 
4.0 
 
6 
6.0 
4.2 
 
8 
6.0 
4.4 
 
10 
6.0 
4.6 
12 
6.0 
4.8 
 
17 
6.0 
5.0 
 
26 
6.0 
5.2 
 
45 
6.0 
5.4 
 
100 
6.0 
5.6 
 
 
N1 vs M2 with M1=6.0 S1=1.0 S2=1.0 Alpha=0.05 Power=0.80
N2=N1 2-Sided T Test
N1
M2
0
20
40
60
80
100
3.0
3.5
4.0
4.5
5.0
5.5
6.0


Marx, Christine E. 
 
Version: 4 – 02/20/2015 
18 
 
 
 
Group sample sizes of 45 achieve at least 80% power to detect a difference of 0.6 between the null hypothesis that both 
group means are 6.0 and the alternative hypothesis that the mean of group 2 is 5.4 with estimated group standard 
deviations of 1.0 and 1.0 and with a significance level (alpha) of 0.05 using a two-sided two-sample t-test. We will 
have 70 Veterans per group, and should thus have 94% power to detect a difference in pain symptoms of 0.6 in the 
pregnenolone intervention compared to placebo.   
 
POWER ANALYSIS:  COGNITIVE SYMPTOMS (Secondary Endpoint) 
 
Executive functioning as assessed by the Tower of London Test of the BACS cognitive assessment battery, Keefe et al. 
 
The Tower of London component of the BACS cognitive assessment battery demonstrated improvements in the 
hypothesized direction in two of our prior pilot RCTs in Veterans with schizophrenia and mild TBI who had been 
randomized to pregnenolone (moderate effect sizes).  We thus did power and sample size calculations for Tower of 
London executive functioning scores (secondary cognitive outcome measure) from estimates of means and standard 
deviations (SD) from preliminary data derived from our pilot RCT examining pregnenolone in OEF/OIF era Veterans 
with mild TBI.  We conservatively utilized our pilot RCT in Veterans with TBI rather than our pilot RCT in Veterans 
with schizophrenia, as Veterans with schizophrenia demonstrated somewhat greater mean improvements in the Tower 
of London test (z-score change 0.61 greater than placebo).  We used simplified models on change scores for power 
calculations as we do not know, and cannot reasonably estimate, the covariance parameters we would need to do these 
calculations in the context of the mixed models.  We used correlations of 0.50 between all occasions, which is 
probably a conservative estimate (please see attached scoring information for BACS in Appendix).   
 
Secondary Hypothesis #2:  Treatment with pregnenolone will improve executive functioning in Gulf War Veterans 
with GWVI, as assessed by the Tower of London test of the BACS.  
 
Our preliminary data from our pilot RCT utilizing adjunctive pregnenolone in Veterans with mild TBI showed two 
estimates of standard deviation of change scores of 0.7546 in the placebo group and 0.9238 in the pregnenolone group.  
Our power analysis assumed a standard deviation consisting of the mean of these two groups = 0.8392, or 
approximately 0.84. Our preliminary data from our pilot RCT utilizing pregnenolone in OEF/OIF Veterans with mild 
TBI showed a difference between treatment and placebo of 0.435, or approximately 0.44.  We did calculations for 
treatment differences ranging from 0.30 to 0.55 in the table below.  To have 80% power to detect a difference of 0.44 
between treatments (the difference in z-scores observed in Tower of London executive functioning scores in the 
pregnenolone group compared to the placebo group in our pilot RCT), we would require 61 subjects per group, or 122 
subjects total.  This may be a conservative estimate in that this assumes a correlation of 0.50 between baseline and 
endpoint, and the correlation is likely to be higher, and the mixed model is likely to have more power than the simpler 
model we used here.  We will, however, have 70 subjects per group in the current investigation (in order to achieve 
91% power for the primary hypothesis – that Veterans randomized to adjunctive pregnenolone will demonstrate 
significantly greater improvements in the SF-36 compared to Veterans receiving placebo), which will provide 86% 
power to detect a z-score change of 0.44 with a standard deviation of 0.84 for Secondary Hypothesis #1 as shown in 
the table and figure below:   
 
Secondary Hypothesis #2: Tower of London Test of Executive Functioning 
Power 
N1 
N2 
Alpha 
Beta Mean Diff 
Std Dev 
0.559763 
70 
70 0.050000 
0.440237 
0.30 
0.84 
0.692311 
70 
70 0.050000 
0.307689 
0.35 
0.84 
0.803596 
70 
70 0.050000 
0.196404 
0.40 
0.84 
0.864561 
70 
70 0.050000 
0.135439 
0.44 
0.84 
0.886225 
70 
70 0.050000 
0.113775 
0.45 
0.84 
0.940490 
70 
70 0.050000 
0.059510 
0.50 
0.84 


Marx, Christine E. 
 
Version: 4 – 02/20/2015 
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0.972010 
70 
70 0.050000 
0.027990 
0.55 
0.84 
 
Power vs Difference in Means, STD=0.84, Alpha=0.05, n=70
Power
Difference
0.0
0.2
0.4
0.6
0.8
1.0
0.2
0.3
0.4
0.5
0.6
 
Summary Statement: 70 subjects per group will provide 86% power to test Secondary Hypothesis #2 that 
treatment with pregnenolone will improve executive functioning as assessed by the Tower of London test compared to 
placebo in Gulf War Veterans with GWVI.  
 
Specific Aim 2:  To conduct candidate biomarker investigations quantifying pregnenolone and pregnenolone 
metabolite levels (allopregnanolone, pregnanolone, androsterone, others) in serum samples at baseline, during 
treatment, and post-treatment with pregnenolone using mass spectrometry-based technologies, and to investigate 
potential genetic predictors of therapeutic response.  These investigations will thus:  a.) characterize the 
pharmacokinetics of pregnenolone and its metabolic profile - which could yield valuable dosing information for 
pregnenolone, characterize pharmacological windows of optimal therapeutic efficacy, and identify potential 
neurosteroid predictors of therapeutic response, and b.) examine single nucleotide polymorphisms (SNPs) of genes 
coding for enzymes involved in neurosteroid synthesis and metabolism.  Together these studies could contribute to the 
development of new neurosteroid interventional strategies that build on the current investigation and exhibit promise 
as pharmacological candidates in GWVI. 
We will thus determine if pregnenolone administration in Gulf War Veterans with GWVI increases downstream 
allopregnanolone and/or other GABAergic neurosteroid levels in serum post-treatment, representing potential 
mechanisms contributing to its therapeutic efficacy and candidate biomarkers for treatment response.  Since little is 
currently known regarding the precise etiologies of possible neurosteroid dysregulation in CNS conditions, we will 
determine baseline serum neurosteroid profiles in Gulf War Veterans with GWVI enrolled this randomized controlled 
trial.  We hypothesize that baseline neurosteroid levels will be inversely related to baseline functioning, cognitive 
symptoms, pain symptoms, fatigue, and overall psychological symptoms.  In addition, we will identify the specific 
metabolism profiles of pregnenolone following eight weeks of treatment with this neurosteroid in order to characterize 
potential mechanisms of action via conversion to downstream GABAergic and sulfated metabolites. Our preliminary 
data suggest that the characterization of neurosteroid alterations post-treatment with pregnenolone may elucidate 
candidate biomarkers for therapeutic response to this intervention.    
 
Statistical Analysis Plan 
 
Preliminary Exploration: We will explore the data using descriptive statistics and graphical techniques prior to any 
hypothesis testing.  For categorical variables, we will examine frequency distributions and where appropriate 
contingency tables and histograms.  For continuous variables, we will examine frequency distributions and where 
appropriate stem-and-leaf plots and box-and-whisker plots.  When appropriate, we will consider transformation.  If 
   Difference= 0.44 at 86% Power 


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Version: 4 – 02/20/2015 
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necessary, due to distributional considerations, we will consider a change of analysis method to a less parametric one.  
For time-to-event variables, we will use Kaplan-Meier plots to obtain a global picture of the patterns in each of the 
treatment groups. 
 
General Modeling:  Most of our hypotheses specify continuous response variables assessed longitudinally.  For those 
models, we will use mixed models repeated measures models. There are three assessment points for the primary and 
secondary outcome measures (to be administered at Visits 2, 4, and 6; each study visit is two weeks apart). We will use 
an AR(1) covariance structure for the repeated measures.  We will fit the models preliminarily using AR(1) covariance 
structures and using unstructured covariance structures, and use the difference between the two -2 log likelihood 
functions to construct a likelihood ratio test of the advantage of the unstructured covariance structure.  If the 
unstructured covariance structure does not fit significantly better, we will use an AR(1) structure. 
 
We will examine group differences in least squares means at each of the major time points (Weeks 0, 4, 8 of treatment 
post-randomization) as described in the plans for specific hypotheses.  Distributional assumptions will be examined 
using residuals.  We recognize that it is always possible that these mixed models may fail to converge or encounter 
difficulties based on their use of asymptotics.  In that case, we will move to analyses that assume compound symmetry, 
and use a Huynh-Feldt correction for the extent to which compound symmetry fails to be met.     
 
Missing data:  The mixed models used to evaluate the continuous response variables are able to handle moderate 
amounts of missing data provided they are missing at random.  We will examine the missing at random assumption by 
assessing differences between dropouts and completers on baseline as well as response variables up to point of 
premature withdrawal.  If the missing at random assumption does not appear to be tenable, we will report the mixed 
models results but spend additional effort characterizing treatment effect at time of premature withdrawal.    
 
Statistical Computing and Programming: All statistical computing using the current version of SAS.