Clinical trial NCT04395183
Conditions: Major Depressive Disorder
Sponsor: University of Utah
Start date: 2021-03-01; Completion date: 2024-03-01
Enrollment: 43 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a three-armed clinical trial examining the effect of 5-hydroxytryptophan and creatine monohydrate as augmenting agents for the treatment of depression. Subjects will be randomized between 5-HTP + placebo, creatine + placebo, and 5-HTP + creatine, for 8 weeks. The ability of the interventions to affect biomarkers associated with depression will be assessed using brain phosphorus magnetic resonance spectroscopy, functional connectivity imaging, and plasma serotonin levels.

Detailed description:
Major depressive disorder (MDD) has a lifetime prevalence of over 16%1 and is associated with reduced work productivity,2 disability,3 increased mortality,4 and increased rates of suicide attempts5 and completed suicides.6 Unfortunately, \~34% fail to respond to standard ADs (ADs).7 Environmental and patient-level factors that increase the risk of MDD could pinpoint novel mechanisms underlying the disorder. One such factor may be relative hypoxia. Persons with hypoxic medical conditions, such as asthma and chronic obstructive pulmonary disease, are at higher risk of depression and suicide compared to those with other chronic medical conditions.8-12 Smoking also promotes hypoxia13 and is linked to increased risks of suicide and depression.14-16 Of special relevance to this study, living at high altitude produces relative hypoxia even after months,17 and is linked to increased risks of suicide18-22 and depression.23,24 Hypoxia could contribute to MDD in at least two ways. First, brain bioenergetics are altered in both hypoxia and MDD. Hypoxia reduces several neurochemical markers of brain activity, including phosphocreatine (PCr) and n-acetylaspartate (NAA),25 and alters mitochondrial dynamics in the hippocampus.26,27 Proton magnetic resonance spectroscopy (1H-MRS) shows that high-altitude residents have altered whole brain pH and reduced inorganic phosphate to total phosphate (tP) ratios compared to persons dwelling at sea-level.28 In depressed patients, phosphorus MRS (31P-MRS) shows reduced nucleotide triphosphate (NTP) concentrations and decreased PCr concentrations; AD response is associated with increases in PCr and NTP.29 Hypoxia could also promote MDD by altering serotonin (5-HT) production. Chronic hypoxia reduces 5-HT in the forebrain and brainstem in rodents.30 The conversion of tryptophan to 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase is oxygen-dependent and slowed by hypoxia.31,32 Animal studies have shown that selective serotonin reuptake inhibitors (SSRIs) are not as effective at altitude,33 possibly because of inadequate 5-HT production. Reductions in 5-HT synthesis and inefficiencies in bioenergetics could both contribute to altered brain functional connectivity. MDD may disrupt cortical emotion regulation,34 and resting state functional connectivity (fcMRI) studies suggest that depression involves reduced connectivity between frontal cortical regions and the amygdala,35 while AD response correlates with normalization of those connections.36 Alterations in connectivity associated with AD response are correlated with changes in brain metabolites,37 suggesting a link to brain bioenergetics.

This suggests two natural supplements as interventions for depression. Oral creatine monohydrate (Cr) could improve bioenergetics in MDD, as Cr alters brain tCr, PCr, and NTP levels.38 Moreover, Cr produces improvements in mood39 correlated with normalization of PCr levels40 and structural connectivity.41 Alterations in 5-HT synthesis due to hypoxia could be rectified by 5-HTP, as its conversion to 5-HT is not oxygen-dependent. 5-HTP elevates brain 5-HT levels and has AD efficacy in clinical trials.42 The proposed study is a two-phase, three-armed trial to evaluate whether SSRI/SNRI augmentation with the supplements Cr, 5-HTP, or their combination (5-HTP+Cr) can enhance AD response in treatment-resistant MDD. In the R61 phase, the study will assess the ability of the interventions to alter biological signatures associated with depression, as measured by 31P-MRS, fcMRI, and changes in whole blood 5-HT. In the R33 phase, the study will attempt to replicate the above findings and evaluate their correlation with clinical outcomes. The study will have the following aims:

* Aim 1 (R61+R33): To identify 31P-MRS correlates of AD response in subjects with MDD receiving Cr, 5-HTP, or 5-HTP+Cr. It is hypothesized that subjects' frontal cortical metabolism will normalize compared to controls over 8 weeks in those receiving Cr or 5-HTP+Cr but not those receiving only 5-HTP.
* Aim 2 (R61+R33): To identify resting state fcMRI correlates of AD response in subjects with MDD receiving Cr, 5-HTP, or 5-HTP+Cr. It is hypothesized that resting functional connectivity in prefrontal regions will normalize (with improvement in hypoconnectivity between subgenual cingulate cortex and the remaining brain) over 8 weeks in all treatment groups compared to controls, with the greatest changes in the 5-HTP+Cr group.
* Aim 3 (R61+R33: To characterize changes in whole blood 5-HT levels in study participants. It is hypothesized that whole blood 5-HT will increase over 8 weeks in subjects receiving 5-HTP or 5-HTP+Cr, but not those receiving only Cr.
* Aim 4 (R33): To describe changes in HAM-D scores in study participants randomized between SSRI/SNRI augmentation with 5-HTP, Cr, or 5-HTP+Cr. It is hypothesized that HAM-D scores will improve over 8 weeks in all treatment groups, with the greatest changes in the 5-HTP+Cr group.

Study results will help elucidate the potential efficacy of a novel combination of nutritional supplements in persons with MDD, given strong epidemiologic and physiologic evidence suggesting that relative hypoxia can contribute to depression through alterations in brain bioenergetics and 5-HT synthesis. Target engagement will be indicated by improvements in functional connectivity and frontal cortical energy metabolism.

Arms: arm 1: 5-hydroxytryptophan 100mg PO BID plus placebo matched to creatine monohydrate arm 2: Creatine 5g PO qday plus placebo matched to 5-HTP arm 3: Creatine 5g PO qday plus 5-hydroxytryptophan 100mg PO BID arm 4: Creatine-matched placebo and 5-HTP-matched placebo
Interventions: intervention 1: 5-Hydroxytryptophan 100 MG intervention 2: Creatine monohydrate intervention 3: placebo matched to 5-HTP intervention 4: Placebo
Intervention descriptions: intervention 1: 5-HTP 100mg PO BID intervention 2: Creatine monohydrate 5g PO qday intervention 3: placebo matched to 5-HTP intervention 4: Dextrose either as loose powder (matched to creatine) or encapsulated (matched to 5-HTP)

Protocol documents:
Prot_SAP_001.pdf: 
1 
 
Official Title: 31P-MRS and Resting State Functional Connectivity Analysis of the Effects of 5-
hydroxytryptophan and Creatine for Antidepressant Augmentation in Patients With SSRI/SNRI-
resistant Major Depressive Disorder   
 
NCT:  04395183 
 
Document Date: 07/APR/2021 
 
 
 


2 
 
 
Introductory Statement and Study Protocol 
 
Introductory Statement 
The proposed study is a single-center proof-of-concept study, targeting a volunteer 
sample of adults with MDD who have failed an adequate trial of an SSRI or SNRI. After screening 
(week -1) and baseline (week 0) assessments and baseline neuroimaging, 60 subjects (with 
recruitment of up to 75 subjects, allowing for 20% attrition) will be randomly assigned in 1:1:1:1 
ratio to treatment for 8 weeks with either double placebo, 5-HTP 100mg twice daily + Cr-matched 
placebo, Cr 5g daily + 5-HTP-matched placebo, or 5-HTP 100mg twice daily + Cr 5g daily. The 
treatment phase will again be followed by neuroimaging (week 8) as well as two post-treatment 
assessments (weeks 10 and 12). 
 
Study Protocol 
Table 1. Eligibility Criteria 
INCLUSION CRITERIA 
RATIONALE 
Adults age 25-40 years inclusive  
Maximizing recruitment while minimizing effect of age-related 
degeneration on MRI findings 
Current diagnosis of MDD identified by the SCID-5 
Confirmation of target clinical condition 
Current HAM-D17 score of > 16 
Confirmation symptoms at least moderately severe 
Adequate adherence to any FDA approved SSRI or SNRI for at least 
8 weeks 
Confirmation of treatment-resistance 
Right-handed 
Facilitate brain coregistration 
Residing at > 4000 ft for at least 12 weeks 
Ensure homogeneity with respect to hypoxic exposure 
EXCLUSION CRITERIA 
RATIONALE 
Any non-MDD and non-anxiety psychiatric diagnosis, as identified by 
the SCID-5 
Minimize diagnostic heterogeneity and risk of adverse events 
History of or current diagnosis of renal disease, such as chronic renal 
failure, acute renal failure or end stage renal disease 
Some reports that creatine worsens renal disease 
Current colitis or diverticulitis 
Facilitate recognition of serotonin syndrome 
History of or current pulmonary disease 
Facilitate recognition of EMS, minimize variation in hypoxia 
between subjects 
Current smoking 
Minimize variation in hypoxia between subjects 
History of cardiac disease or QTc > 500ms 
Minimize risk of sudden cardiac death 
History of fibromyalgia or any rheumatological condition 
Facilitate recognition of EMS 
History of or current seizure disorder 
Minimize risk of seizure 
Current serious suicide risk identified by the Columbia Severity 
Suicide Rating Scale  
Minimize risk of suicide in the study 
Current treatment with an antipsychotic, mood stabilizer, or non-SSRI 
or SNRI antidepressant except for bupropion at FDA-approved doses 
or trazodone up to 200mg, or use of any supplements apart from 
standard multivitamins 
Minimize risk of adverse drug-drug interaction and increase 
clinical homogeneity of group 
Positive pregnancy test, pregnancy, failure to use adequate birth 
control method 
Risks for fetus of interventions not adequately characterized 
Previous diagnosis of serotonin syndrome or evidence of serotonin 
syndrome 
Minimize risk of serotonin syndrome 
Pre-existing eosinophilia (absolute eosinophil count > 500/uL) 
Facilitate recognition of EMS 
Contraindications to MRI: ferromagnetic implants, implanted devices, 
claustrophobia 
Maximize MRI safety 
STUDY WITHDRAWAL CRITERIA 
RATIONALE 
Intolerable or clinically significant side effects to creatine or 5-HTP 
Subject safety 
Hospitalization for suicidal ideation/suicide attempt 
Subject safety 
Initiation of any excluded psychotropic medication (antipsychotic, 
mood stabilizer or antidepressant) 
Minimize risk of adverse drug-drug interaction 
Discontinuation of or inadequate adherence to FDA-approved 
SSRI/SNRI 
This is an augmentation study 
Initiation of any excluded serotonergic medications 
Minimize risk of serotonin syndrome 
Evidence of serotonin syndrome 
Subject safety 
Evidence of EMS 
Subject safety 
Incarceration 
Research involving prisoners is generally prohibited 


3 
 
a. Inclusion and Exclusion Criteria: Inclusion and exclusion criteria are detailed in Table 1: 
 
 
b. Study Withdrawal Criteria: A participant will be withdrawn from the study if he or she 
experiences intolerable or clinically significant side effects to the study intervention (including the 
development of serotonin syndrome or eosinophilia-myalgia syndrome), hospitalization for 
suicidal ideation or suicide attempt, develops a positive pregnancy test or gives other evidence of 
pregnancy, is incarcerated, initiates any excluded medication, or discontinues or changes the 
baseline antidepressant. In addition, the principal investigators retain the right to withdraw 
participants from the study without their permission, in the event they are unwilling or unable to 
maintain adherence to the research protocol. 
 
c. Procedures: All procedures performed by study personnel are research-related. None of the 
study activities will be considered standard of care. There will be no cost to study subjects for 
their participation. Participants will be compensated for their time and travel. Table 2 outlines the 
schedule of study procedures for clinical participants. Study visits will be supervised by a board-
certified/board-eligible psychiatrist and will be conducted either by a board-certified/board-eligible 
psychiatrist or a bachelor’s level research assistant with training in the specific measures used. 
Physical examinations and laboratory interpretation will be conducted by a board-certified/board-
eligible psychiatrist. Consent will be obtained before any study procedures are initiated. Potential 
participants will be informed of the study and offered a consent form to review. They will be 
encouraged to discuss study participation with their relatives. If a potential participant expresses 
interest in study participation, the informed consent process will be conducted. After the informed 
consent process, individuals will be offered time to consider study participation and to ask 
questions. Subjects will have the opportunity to discuss the study with a study team member in a 
setting free of coercion. The language of the informed consent form is written at a level easily 
understood by the subject and any questions asked by the subject will be answered honestly and 
without bias. A specific meeting time will be set up between a study team member and the 
participant where the entire informed consent document will be carefully explained in its entirety. 
The length of the meeting will be designed so there is the necessary amount of time for all 
questions to be answered. Subjects will be separately consented for blood sample storage for 
later genetic testing and will be allowed to opt-out of this procedure while still participating in the 
other aspects of the study. 
 
To determine if an individual is eligible for study participation, a screening visit will be 
conducted. Initially, a HAM-D17 will be administered to determine if the patient exhibits depressive 
symptoms that are sufficiently severe for inclusion in the study.  The duration of the patient’s 
current residence and the altitude of that residence will be assessed. Next, the SCID-5 will be 
administered to confirm a diagnosis of a current major depressive episode. In addition, subjects 
will be screened for other DSM-5 disorders using the SCID-5 to assess exclusion criteria. 
Afterward, the MADRS, C-SSRS, BAI, YMRS, ATRQ, and CGI-S will be administered. All 
participants will receive a pregnancy test unless they report a history of menopause or 
hysterectomy. Study subjects will receive a baseline BUN/creatinine (to assess for renal 
insufficiency), CBC (to screen for pre-existing eosinophilia), measurement of whole blood 
serotonin, vitals including pulse oximetry, and a baseline focused physical.  Subjects will be asked 
to be fasting for at least 3 hours prior to the blood draw.  The blood draw will occur between 2 and 
5pm to minimize circadian variation in serotonin levels. Subjects will be given an internet link and 
passcode to complete the Diet History Questionnaire II (DHQ-II) and asked to do this at home. 
An EKG will be performed to evaluate the QT interval and to screen for other evidence of 
cardiac disease. They will also be screened for evidence of serotonin syndrome using the Hunter 
Criteria. A complete medical and psychiatric history (focused on illness duration, length of current 
Positive pregnancy test, pregnancy 
Subject safety 


4 
 
treatment, treatment history, and history of medical illnesses that preclude participation, e.g., renal 
disease, pulmonary disease, rheumatologic disease, diabetes, gastrointestinal disease, 
pregnancy, seizure disorder, serotonin syndrome) will be completed. 
 
After the screening visit, participants will have a baseline visit after one week with the MRI 
happening + 4 days from the baseline visit, prior to the start of treatment. At the baseline visit, the 
HAM-D17, MADRS, C-SSRS, BAI, YMRS, and CGI-S will be administered. Vital signs will be 
checked and a physical exam performed if patient symptoms indicate a need. Whole blood 
serotonin will be drawn. Subjects will be asked to be fasting for at least 3 hours prior to the blood 
draw.  The blood draw will occur between 2 and 5pm to minimize circadian variation in serotonin 
levels. Participants will be evaluated for serotonin syndrome using the Hunter Criteria. Baseline 
31P-MRS and resting state fcMRI will be completed. After initiation of treatment, subjects will be 
reevaluated at 1, 2, 4, 6, and 8 weeks (+3 days). At each these visits, the HAM-D17, MADRS, 
C-SSRS, BAI, YMRS, and CGI-S will be administered again. Subjects will be asked to be fasting 
for at least 3 hours prior to the blood draw.  Whole blood serotonin will be  drawn. The blood draw 
will occur between 2 and 5pm to minimize circadian variation in serotonin levels.  Follow-up 31P-
MRS and resting state fMRI will be completed at week 8. Vital signs with pulse oximetry, focused 
physical exam if indicated, collection of urine for pregnancy screening if indicated, recording of 
concomitant medications, self-reported drug use, medication compliance assessment, study 
medication dispensing, and screening for treatment-related adverse effects will also be 
undertaken. Screening for treatment-related adverse effects will include application of the Hunter 
Criteria for serotonin syndrome164 as well as evaluation for symptoms of eosinophilia-myalgia 
syndrome. If subjects exhibit symptoms of eosinophilia-myalgia syndrome, a CBC with automated 
differential will be obtained. Finally, participants will be seen for follow up visits at 2 weeks and 4 
weeks (+3 days) after completion of 5-HTP/Cr supplementation. Measure at these visits will 
include the HAM-D17, MADRS, C-SSRS, BAI, YMRS, CGI, DESS, vitals with pulse oximetry if 
indicated, focused physical exam if indicated, and evaluation for serotonin syndrome and EMS. 
Again, if symptoms of EMS are present, a CBC with automated differential will be obtained. After 
the screening visit, Participants can request study visits by videoconference with the exception of 
briefly dropping by the clinic for a blood draw and/or MRI as indicated in the schedule below. 
 


5 
 
 
Once entered into the study, participants will be 1:1:1:1 randomized to receive either Cr+ placebo, 
5-HTP+placebo, 5-HTP+Cr, or placebo+placebo. Block randomization using a SAS code, PROC 
PLAN with Block, will be used to ensure equal treatment allocation within each block. The study 
will be conducted as a double-blind trial, with neither participants nor research staff aware of 
participant assignment. Except in cases of medical emergency, the double-blind will not be 
"broken" until recruitment is closed and the final participant has completed 8 weeks of treatment 
and 4 weeks of follow-up. We anticipate an attrition rate of ~20% based on our pilot study (where 
attrition was 13%), and so will plan to recruit a total of 75 subjects, for a total recruitment of 60 
subjects. The blind will be broken following the culmination of the study or at the request of a 
medical professional dealing with a medical emergency in a case in which it would help a study 
participant.  
 
d. Study Drug Dosing: Cr doses will be 5g by mouth daily. 5-HTP doses will be 100mg BID. 
These doses are based those most commonly used in clinical trials or in historical practice. 
However, with natural products it is often unclear what the optimal doses are to balance safety 
and efficacy. For Cr, a dose of 5g was selected because this was the dose used effectively by 
Lyoo et al. (2012) and in our pilot study of 5-HTP + Cr. A 5-HTP dose of 100mg twice daily was 
selected because this is equivalent to the average total daily dose of 200mg given as a treatment 
option for depressed patients 89. We have elected to provide patients with twice daily dosing 
because the half-life of 5-HTP is estimated to be 4.3+2.8h.158  In previous studies, 200mg given 
as a single daily dose was well tolerated with few side effects reported.89,159 
 
e. Statistical Design and Power 
1. Power and Sample Size Determination: Power to detect changes in PCr:tP between groups 
is our primary determinant of sample size in the R61 phase because of the centrality of 
spectroscopic findings in our hypotheses. In another study, after 8-weeks of oralCr, adult female 
methamphetamine users had significantly increased PCr levels with a standardized effect size of 
Table 2. Study Procedures for Clinical Subjects 
  
Screening 
Baseline 
Treatment 
Follow-up 
Week 
-1 
0 
1 
2 
4 
6 
8 
10 
12 
SCID-I 
X 
 
 
 
 
 
 
 
 
HAM-D17 
X 
X 
X 
X 
X 
X 
X 
X 
X 
MADRS 
X 
X 
X 
X 
X 
X 
X 
X 
X 
C-SSRS 
X 
X 
X 
X 
X 
X 
X 
X 
X 
BAI 
X 
X 
X 
X 
X 
X 
X 
X 
X 
CGI-S 
X 
X 
X 
X 
X 
X 
X 
X 
X 
YMRS 
X 
X 
X 
X 
X 
X 
X 
X 
X 
DESS 
 
 
 
 
 
 
 
X 
X 
Medical History 
X 
 
 
 
 
 
 
 
 
Diet History Questionnaire II 
X 
 
 
 
 
 
 
 
 
ATRQ 
X 
 
 
 
 
 
 
 
 
Hunter criteria 
 
X 
X 
X 
X 
X 
X 
X 
X 
Screening for EMS 
 
 
X 
X 
X 
X 
X 
X 
X 
Side-effect screening 
 
 
X 
X 
X 
X 
X 
 
 
Pregnancy test** 
X 
X 
X 
X 
X 
X 
X  
 
 
BUN/Creatinine 
X 
 
 
 
 
 
 
 
 
CBC with differential 
X 
 
X* 
X* 
X* 
X* 
X* 
X* 
X* 
Whole-blood 5-HT (fasting for > 3 hours; drawn 
between 2:00 and 5:00pm 
X 
X 
X 
X 
X 
X 
X 
 
 
EKG 
X 
 
 
 
 
 
 
 
 
Vitals including pulse-oximetry 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Focused physical exam 
X 
 
X* 
X* 
X* 
X* 
X* 
X* 
X* 
Review of current medications 
X 
X 
X 
X 
X 
X 
X 
 
 
31P-MRS  
 
X 
 
 
 
 
X 
 
 
Resting State fMRI 
 
X 
 
 
 
 
X 
 
 
* = only if clinically indicated by presence of adverse symptoms or symptoms suggestive of EMS or serotonin syndrome 
** = all female subjects, unless reporting menopause, hysterectomy, tubal ligation or currently using an intrauterine device (IUD) 


6 
 
0.920.126 In an open-label study in depressed adolescents,124 PCr levels following Cr 
administration increased by 6.2%+ 9.6%. In a dose-finding study of Cr compared to placebo, the 
effect size for differences in PCr levels between the group receiving 4 grams of Cr daily compared 
to placebo was 0.7. We hypothesize that supplementation with Cr with or without 5-HTP will 
increase frontal cortical PCr levels. 31P-MRS neuroimaging will be performed at baseline, and 
repeated after 8 weeks of treatment with Cr + placebo, 5-HTP + placebo, or 5-HTP + Cr. To 
address our first hypothesis, a population-averaged generalized estimating equation (GEE) 
regression model is used for group comparison of the ΔPCr (PCrWEEK 8 - PCrBASELINE) 
associated with Cr + placebo, 5-HTP + placebo, or 5-HTP + Cr. We will control for age, gender, 
and tissue partial volume effects (i.e., gray matter, white matter and cerebrospinal fluid) for in vivo 
phosphorus metabolite measurements made at two time points. The GEE for each subject, i has 
the form Yj(t)=Xj β + εj, where Yj and Xj represent measured metabolite (PCr or β-NTP) levels 
and design matrix of covariates, respectively. The error term follows a multivariate normal 
distribution: εj~ N(0, Vj), where Vj is the variance of metabolite levels. We assume that measures 
taken closer to each other in time are more highly correlated than those taken further apart. 
Therefore, the first-order autoregressive structure, AR(1) is assumed such as cov[εj(t), εj(t')] = ρ|t-
t'|Vj to model the dependence of the correlation on repeated measures from the same participant, 
where t is time variable and ρ is the correlation coefficient between the clinical symptom measures 
of two successive assessments. Assuming ρ =0.30, expected MDES = 5% across the groups, 
and harmonic mean SD = 10%, respectively, a sample size of 60 participants (15 per group; 4 
groups) and 2 neuroimaging time points per participant has >80% power using the AR (1) GEE 
model at the 0.05 alpha level. In our open-label study, we experienced ~20% participant attrition. 
Imputation of missing data cannot be utilized for neuroimaging data. Therefore, anticipating a 
20% attrition rate, we propose a target enrollment of 75 participants for the R61 phase.  
 
2. Analysis of 31P-MRS Data: To analyze study data, a raw Hamming filter will be applied before 
performing 2D fast Fourier transformation, and each free induction decay (FID) will be line-
broadened with 10 Hz of apodization. After Fourier transformation and frequency shift correction, 
zero-/first-order phase correction and baseline correction with polynomial interpolation will be 
applied. Spatial filtering with a Hamming window function will be implemented to reduce signal 
contamination from neighboring voxels. The preprocessed 31P-MRSI data will be fitted using 
jMRUI software180 with the Advanced Method for Accurate, Robust and Efficient Spectral 
(AMARES) fitting algorithm.181 Metabolites of interest include PCr, β-NTP, tP, and their ratios. 
From registered anatomical images, tissue segmentation will be performed using FSL (FMRIB’s 
Software Library) software so that cerebrospinal fluid (CSF)-corrected metabolite concentrations 
as well as gray matter percentage in each voxel can be used as covariates in the analysis. 
Generalized estimating equations regression modeling will be used to evaluate group differences 
in metabolite levels controlling for age, sex, education level, and tissue partial volume effects (i.e. 
gray matter, white matter, CSF). Subjects with MDD will be compared to HCs at baseline and at 
follow-up. Sub-group analyses by gender will be included given evidence that Cr has been 
proposed as especially effective in women. Fisher’s exact test will be used to compare groups on 
categorical variables. 
 
3. Analysis of Resting State Data: As is seen in the small cohort of pilot data, we anticipate 
treatment groups to demonstrate increased global connectivity of the subgenual ACC after 
treatment, among other changes. A recent analysis determined that we can achieve single subject 
reproducibility of FC metrics of 0.16 Fisher-transformed correlation units.161 If we conservatively 
set the effect size at 8% and SD at 9% of mean, a sample size of 60 depressed subjects would 
achieve >90% power to detect significant treatment effects with the repeated measures design 
compared to baseline. For FC analyses, 361 regions of interest (ROIs) comprising a parcellation 
of cortical gray matter182 will be compared between placebo and active treatment subjects. We 


7 
 
will conduct a second level of analysis using eight networks delineated as follows and derived 
from Gordon et al.:182 auditory, salience, frontoparietal, default, dorsal attention, ventral attention, 
sensorimotor, and visual. Two levels of analysis will be performed to balance multiple 
comparisons and granularity of spatial information. For each pair of 8 networks, mean BOLD time 
series will be extracted across the entire network and Fisher-transformed correlation coefficients 
will be calculated. An acceptable false discovery rate (q < .05) over all pairs of networks will be 
assumed to denote significant partial correlation with diagnosis. To evaluate which connections 
are anticorrelated, we will use an independent dataset for an unbiased estimate obtained from 
the S1200 Release of the Human Connectome Project (HCP) dataset. Group-wise ANOVA will 
be performed with false discovery rate correction for multiple comparisons across 361 x 361 
connections tested for treatment group differences in FC before and after treatment compared to 
placebo group. Sub-group analyses by gender will be included given evidence that Cr has been 
proposed as especially effective in women. Subjects with MDD will be compared to HCs at 
baseline and at follow-up. 
 
4. Analysis of 5-HT Levels: There is limited data available regarding the effect of twice-daily oral 
5-HTP supplementation on whole blood 5-HT levels in subjects with MDD taking ADs. Estimates 
of whole blood (platelet) serotonin levels in MDD vary widely, including values from 119 + 52.9 
ng/ml (n=30)183   to 169.5 + 74.5 ng/ml (n=29)184 to 203.2 + 59.3 ng/ml (n=25),185 suggesting that 
whole blood 5-HT levels average roughly 161 + 62 ng/ml in subjects with MDD.  A sample of 60 
subjects with MDD allocated 1:1 between 5-HTP (5-HTP+Cr and 5-HTP+placebo) and placebo 
(Cr+placebo and placebo+placebo) would have, in a t-test of means, >70% power to detect an 
effect size of 0.8.  This effect size would be equivalent to an increase in average 5-HT levels to 
roughly those seen in non-depressed subjects.186  Although this is a relatively large effect size, it 
is consistent with findings related to the effect of oral tryptophan supplementation (at 50mg/kg/day 
in non-depressed men) which found an estimated effect size of 1.1.187  This analysis assumes 
that creatine supplementation will not independently affect circulating serotonin levels, though 
there is no data regarding this to our knowledge. 5-HT levels will be analyzed using linear mixed 
models with repeated (before and after) measures, controlling for gender, age, group allocation, 
antidepressant use, oxygen saturation, and other confounds.  
 
e. Measures 
We plan to use the following instruments for data collection: 
• 
Structured Clinical Interview for DSM-5 (SCID-5)  
• 
Hamilton Depression Rating Scale (HAM-D)  
• 
Montgomery-Asberg Depression Rating Scale (MADRS) 
• 
Columbia Suicide Severity Rating Scale (C-SSRS) 
• 
Clinical Global Impressions Scale (CGI) severity of illness subscale (CGI-S) 
• 
Beck Anxiety Inventory (BAI 
• 
Young Mania Rating Scale (YMRS) 
• 
Discontinuation-Emergent Signs and Symptoms checklist (DESS) 
• 
Serotonin Syndrome Screening-Hunter Criteria 
• 
Eosinophilia-Myalgia Syndrome Screening 
• 
Eosinophilia-Myalgia Participant Information Sheet 
• 
Serotonin Syndrome Participant Information Sheet 
• 
National Cancer Institute Diet History Questionnaire Version II (DHQ-II) 
• 
Antidepressant Treatment Response Questionnaire (ATRQ) 
 
f. Potential Risks: During the screening visit, participants may become emotionally upset when 
asked about their psychiatric history including suicide attempts, or physical and sexual abuse. 


8 
 
Participants may also experience discomfort when providing urine for pregnancy tests. It is 
possible that a participant’s illness could worsen during the study. This could be related or 
unrelated to the study. If the participant’s illness worsens to the point that they are a danger to 
herself or others, they will be referred for appropriate care. If the participant is hospitalized for 
worsening illness, they will be withdrawn from the study. Participants may experience 
gastrointestinal discomfort as a result of taking creatine or 5-HTP. Participants will be encouraged 
to take both medications with food to minimize this risk. Finally, as detailed above, 
supplementation with 5-HTP increases the risks of eosinophilia-myalgia syndrome and serotonin 
syndrome. Notably, in our pilot study, there were no serious adverse events attributable to the 
study drugs. Participants will be counseled about these risks, informed of the signs and symptoms 
of these conditions, provided with information sheets detailing the symptoms of these disorders 
in lay terms and describing steps to take for further evaluation if indicated, and will be screened 
for both conditions at each visit after the initiation of treatment. 
MRI/MRS scans do not use ionizing radiation like x-rays or CT scans. Instead, magnetic 
fields and radio waves are used to take the pictures. There are no known risks related to MRI 
scans – other than the risk of injury when metallic objects are brought into the scanning room by 
mistake. Serious injury can occur during an MRI scan to persons who have: 
• 
Cardiac (heart) pacemakers. 
• 
Metal clips on blood vessels (also called stents). 
• 
Artificial heart valves. 
• 
Artificial arms, hands, legs, etc. 
• 
Brain stimulator devices. 
• 
Implanted drug pumps. 
• 
Cochlear (ear) implants. 
• 
Ocular (eye) implants or known metal fragments in eyes. 
• 
Exposure to shrapnel or metal fillings 
• 
Other metallic surgical parts. 
• 
Orthodontic braces on the teeth. 
• 
Body jewelry or piercings that cannot be removed for the scan. 
• 
Certain tattoos with metallic ink  
• 
Certain transdermal (skin) patches such as NicoDerm (nicotine for tobacco 
dependence), 
• 
Transdermal scopolamine or Ortho-Evra (birth control) 
If the participants have any such devices, or has had a surgery where metal devices were placed 
in their body, they cannot take part in the study unless cleared for MRI scanning by the surgeon 
who implanted the medical device(s). 
Serious risks exist if ferromagnetic objects (things that stick to magnets) are brought into 
the scanning area. These items can become dangerous flying objects and are not allowed near 
the MRI scanner. The FDA has approved the 3T scanner for routine clinical studies. The FDA has 
decided that MRI machines of 8T or less do not pose a risk. Although the scans we are using in 
this study have no known risks, there could be ill effects that are delayed, such that they have not 
yet been recognized by the FDA. The brain scans do not cause pain. Apart from the scanner 
noise, the participant will not know the scan is taking place. Inside the scanner, some people 
experience claustrophobia (fear of being in small spaces), dizziness, headaches, or a metallic 
taste in the mouth. Some people experience double vision or see flashing lights. These symptoms 
are temporary, and will stop when the participant leaves the scanner. The participant may feel 
cramped inside the scanner. There is a mirror placed inside the scanner so the subject can see 
his or her face, and look out into the scanning room. The technologist will be able to hear the 
participant at all times. Very rarely, someone having an MRI scan feels a tingling in his or her 


9 
 
back. This is due to the magnetic field changing quickly during the scan. The precautions taken 
will avoid all the known risks related to MRI scans. The participant can stop the scan at any time. 
Finally, there is a small risk that subjects’ protected health information will be lost or 
misappropriated during the study. 
 
g. Adequacy of Protection against Risks 
h. Recruitment: Subjects will be recruited through radio advertisements broadcast on local radio 
networks.  We will also contact eligible participants who have indicated an interest in research 
participation who have listed themselves in the Department of Psychiatry research recruitment 
database. Additionally, we will contact outpatient providers in the Department of Psychiatry to 
apprise them of the study so that they can refer patients (no referral fees will be offered), and 
these providers will be sent digital copies of the flyer via email. Clinical participants will be 
compensated a total of $235 in cash to account for travel expenses and time. Compensation will 
be given using the schedule below. Per-visit compensation is based on the estimated time for 
each visit (at least one hour) and is judged not to represent an undue inducement. Study 
participants may choose to skip being compensated at some visits and then be compensated in 
lump sums.  
1. $25 - Screening  
2. $65 - Baseline visit (includes $50 compensation for MRI) 
3. $15 - Treatment phase visits (weeks 1, 2, 4, 6)  
4. $65 – Treatment week 8 (includes $50 compensation for MRI) 
5. $10 – Follow-up visits (weeks 10, 12) (total $20) 
 
i. Informed Consent: Consent will be obtained before any study procedures are initiated. 
Potential participants will be informed of the study and offered a consent form to review. They will 
be encouraged to discuss study participation with their relatives. If a potential participant 
expresses interest in study participation, the informed consent process will be conducted. After 
the informed consent process, individuals will be offered time to consider study participation and 
to ask questions. Subjects will have the opportunity to discuss the study with a study team 
member in a setting free of coercion. The language of the informed consent form is written at a 
level easily understood by the subject and any questions asked by the subject will be answered 
honestly and free of bias. A specific meeting time will be set up between a study team member 
and the participant where the entire informed consent document will be carefully explained in its 
entirety. The length of the meeting will be designed so there is the necessary amount of time for 
all questions to be answered. Subjects will be separately consented for blood sample storage for 
later genetic testing and will be allowed to opt-out of this procedure while still participating in the 
other aspects of the study. 
 
j. Screening for Serotonin Syndrome: Given the potential development of serotonin syndrome 
in patients taking multiple serotonergic medications (in this study, an SSRI plus 5-HTP), 
monitoring for serotonin syndrome is an important element of participant safety. There are no 
widely-used standardized scales for the assessment of serotonin syndrome, but algorithms for 
the detection and diagnosis of the condition exist 164. As noted above, subjects will have vital signs 
performed at each visit. Participants will also be asked about symptoms of serotonin syndrome at 
each visit. In our pilot study, a physician completed a thorough physical exam for each patient to 
screen for these symptoms, but no cases of serotonin syndrome were required and this approach 
was deemed to be inefficient. Accordingly, in the currently proposed study, we will have a 
physician on call during each patient visit to complete a physical exam and further assessment 
for serotonin syndrome if there are vital sign abnormalities or symptoms (severe diarrhea or 
nausea, sweating, shakiness, stiffness, confusion) that are suggestive of serotonin syndrome. In 
such cases, the clinician will assess for ocular clonus, mydriasis, diaphoresis, rigidity, 


10 
 
hyperreflexia, and clonus, and will evaluate the likelihood of serotonin syndrome using the Hunter 
Criteria. Finally, at the baseline visit subjects will receive an information sheet describing signs 
and symptoms of serotonin syndrome in lay terms and providing instructions on how they should 
seek further evaluation if those symptoms are evident. 
 
k. Screening for Eosinophilia-Myalgia Syndrome: No standardized measures for the detection 
of eosinophilia-myalgia syndrome (EMS) exist. In this study, participants will be informed about 
the symptoms of EMS, and will be provided with an information sheet describing the symptoms 
of EMS in lay terms and providing instructions on how they should seek further evaluation if those 
symptoms arise. They will be asked about symptoms that may be indicative of EMS (especially 
myalgias) at each treatment and follow-up visit. At the screening visit, a CBC with automated 
differential will be performed to screen for pre-existing eosinophilia. Although in principle 
participants could receive a complete blood count at each visit after the initiation of treatment to 
screen for the development of EMS, to our knowledge there is no published data to support this 
intervention and the presence of eosinophilia in the absence of clinical symptoms is not likely to 
be specific for EMS. In our pilot study, subjects received a physical exam at each visit to screen 
for symptoms of EMS, but this was found to be an inefficient measure. Accordingly, in the currently 
proposed study a physician will be on call during each patient visit to complete a physical exam 
and further assessment for EMS if there are vital sign abnormalities or symptoms (rash, other skin 
changes, hair loss, muscle aches, joint pain, shortness of breath) that are suggestive of EMS. 
 
l. Adverse events 
Unanticipated problems and adverse events that are related to the research, or which place 
participants at greater-then-expected risk, will be reported to the Institutional Review Board (IRB) 
within ten working days of the event. Unanticipated problems involving risk to participants or 
others are defined as any incident, experience or outcome that meets the following criteria: 
• 
Unforeseen (not expected by the researcher or the research participant) given the 
research procedures and the subject population being studied; 
• 
Related or probably related to participation in the research, or if the event or problem 
probably or definitely affects the safety, rights and welfare of current participants; and 
• 
Suggests that the research places participants or others at a greater risk of harm (includes 
physical, psychological, economic or social harm) than was previously known or 
recognized. 
• 
An unexpected adverse event is any adverse event occurring in one or more participants 
participating in a research protocol, whose nature, severity, or frequency is not consistent 
with either: 
• 
The known or foreseeable risk of adverse events associated with the procedures 
involved in the research that are described in the protocol related-documents (i.e. 
protocol, investigational brochure, consent form, or product labeling). 
• 
The expected natural progression of any underlying disease, illness or medical 
condition of the subject experiencing the adverse event. 
The Principal Investigator will make the initial determination if an unexpected, adverse event is 
related or unrelated to the investigational drug or a clinical or research procedure. An adverse 
event is “related to the research” if in the opinion of Dr. Kious or Dr. Renshaw, it was more likely 
than not related to the investigational agent or intervention. 
 
m. Potential Benefits of Proposed Research to Human Subjects and Others:  
o A thorough physical and psychiatric evaluation 
o Access to advanced neuroimaging procedures, which could reveal unanticipated 
structural abnormalities 


11 
 
o The study is designed to test the hypothesis that the combination of 5-HTP and creatine 
is effective for the treatment of major depressive disorder that has not responded to 
conventional treatments; accordingly, there is some possibility of clinical improvement for 
participants 
o We also hope that the research will add to our understanding of the potential of the study 
interventions as treatments for major depressive disorder, as well as contributing to 
general understanding of the pathophysiology of that condition. 
 
n. Importance of Knowledge to be Gained: The risks of adverse events related to study 
participation are judged to be small on the whole, and we believe that the procedures described 
above will mitigate them sufficiently. Even so, because of the social and personal impact of major 
depressive disorder, we believe that the risks posed by this study are reasonable. 
 
o. Registration with Clinicaltrials.gov: A registration has been submitted to clinicaltrials.gov 
as required.