Conditions: Autism Spectrum Disorder
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 18
Healthy volunteers: no

Brief summary:
An examination of changes in urinary metabolites with use of folinic acid in children with autism spectrum disorder (ASD). Investigators will also measure behavioral measures and academic measures monthly at school with teachers and parents report.

Detailed description:
Children enrolled in Oak Hill School will enroll in this study and take folinic acid supplement twice a day for 12 weeks period. Behavioral measures and urinary metabolites will be collected pre and post treatment. Behavioral measures will be taken again at 16 weeks, after 4 weeks of not dosing.

Arms:
arm 1: In this open-label trial, all subjects will receive the twice daily dose of folinic acid. Folinic acid will be delivered in pill form at a weight-based dose.

Interventions: intervention 1: folinic acid
Intervention descriptions: intervention 1: subject will take folinic acid daily for 12 weeks

Protocol document:
Prot_SAP_000.pdf: 
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ROBERT HENDREN, DO 
Clinical Research Protocol 
 
An examination of changes in urinary metabolites with use of folinic acid in children 
with autism spectrum disorder (ASD) 
 
Protocol Number: 
pending 
Version Date: 
11-21-17 
Investigational Product: 
 
Folinic acid 
IND Number: 
137886 
Development Phase: 
n/a 
Sponsor: 
Dr. Robert Hendren, PI (UCSF) 
Funding Organization: 
Gift 
Principal Investigator: 
Robert L. Hendren, DO 
Professor, Department of Psychiatry Child and Adolescent 
Psychiatry University of California, San Francisco 
401 Parnassus Ave, Box 0984 
San Francisco, CA 94143-0984 
(415) 476-7198 
Medical Monitor: 
none 
Coordinating Center: 
n/a 
 


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PROTOCOL AGREEMENT 
 
I have read the protocol specified below. In my formal capacity as Investigator, my duties 
include ensuring the safety of the study subjects enrolled under my supervision as outlined 
in the protocol.  It is understood that all information pertaining to the study will be held 
strictly confidential and that this confidentiality requirement applies to all study staff at 
this site. Furthermore, on behalf of the study staff and myself, I agree to maintain the 
procedures required to carry out the study in accordance with accepted GCP principles 
and to abide by the terms of this protocol. 
 
Protocol Number:  pending 
 
Protocol Title:  An examination of changes in urinary metabolites with use of folinic 
acid in children with autism spectrum disorder (ASD) 
Protocol Date:  11-21-17 
 
 
 
12/07/2017 
Investigator Signature  
 
Date 
Robert Hendren, Professor of Psychiatry 
Print Name and Title 
Site # 
001 
 
Site Name 
University of California, San Francisco 
Address 
401 Parnassus Ave, Box 0984 
 
San Francisco, CA  94142 
 
 
 
 
Phone Number 
(415) 476-7044 


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TABLE OF CONTENTS 
 
1 
BACKGROUND ................................................................................................................. 9 
1.1 
Overview of Clinical Studies ...................................................................................... 9 
2 
STUDY RATIONALE ........................................................................................................ 11 
2.1 
Risk / Benefit Assessment ........................................................................................... 11 
3 
STUDY OBJECTIVES ....................................................................................................... 11 
3.1 
Primary Objective ....................................................................................................... 11 
3.2 
Secondary Objectives .................................................................................................. 11 
3.3 
Study Overview ........................................................................................................... 12 
4 
CRITERIA FOR EVALUATION ..................................................................................... 12 
4.1 
Primary Endpoint ........................................................................................................ 12 
4.2 
Secondary Endpoints ................................................................................................... 12 
4.3 
Safety Evaluations ....................................................................................................... 12 
5 
SUBJECT SELECTION .................................................................................................... 12 
5.1 
Study Population ......................................................................................................... 12 
5.2 
Inclusion Criteria ......................................................................................................... 12 
5.3 
Exclusion Criteria ....................................................................................................... 133 
6 
CONCURRENT MEDICATIONS .................................................................................... 133 
6.1 
Prohibited Medications and Treatments
 
Error! Bookmark not defined.3 
7 
STUDY TREATMENTS .................................................................................................... 133 
7.1 
Formulation of Product
 
Error! Bookmark not defined.3 
7.1.1 Dosage/Dosage Regimen ............................................................................................ 14 
7.1.2 Dispensing ................................................................................................................... 14 
7.1.3 Administration Instructions ......................................................................................... 14 
7.2 
Supply of Study Supplement at the Site ...................................................................... 144 
7.2.1 Study Supplement Accountability ............................................................................... 144 
8 
STUDY PROCEDURES AND GUIDELINES ................................................................. 144 
8.1 
Clinical Assessments ................................................................................................... 144 
8.1.1  Concomitant Medications ……………………………………………………………14 
8.1.2  Demographics ………………………………………………………………………..14 
8.1.3  Medical History ………………………………………………………………….…..14 
8.1.4  Advese Events ……………………………………………………………………….14 
8.2 
Clinical Laboratory Measurements ............................................................................. 15 
9 
EVALUATIONS BY VISIT ............................................................................................... 155 
9.1 
Visit 1 (Screening) ...................................................................................................... 155 
9.2 
Visit 2 (Baseline/Month 0) .......................................................................................... 155 
9.3 
Visit 3 (Follow-up 1/ Month 1) ................................................................................... 166 


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9.4 
Visit 4 (Follow-up 2/Month 3)
 
Error! Bookmark not defined.6 
9.5 
Visit 5 (Follow-up 3/ Month 4) ................................................................................... 166 
10 ADVERSE EVENT REPORTING AND DOCUMENTATION .................................... 166 
10.1 Adverse Events ........................................................................................................... 166 
10.2 Serious Adverse Events (SAE)
 
1818 
10.3 Serious Adverse Event Reporting
 
Error! Bookmark not defined.18 
11 EARLY DISCONTINUATION OF STUDY SUPPLEMENT ........................................ 18 
11.1 Withdrawal of Subjects from the Study ...................................................................... 18 
12 PROTOCOL VIOLATIONS
 
ERROR! BOOKMARK NOT DEFINED.19 
13 STATISTICAL METHODS AND CONSIDERATIONS ............................................... 19 
13.1 Analysis of Primary Endpoint ..................................................................................... 19 
13.2 Analysis of Secondary Endpoints ............................................................................... 19 
13.3 Sample Size ................................................................................................................. 20 
14 DATA COLLECTION, RETENTION AND MONITORING ....................................... 20 
14.1 Data Collection Instruments ........................................................................................ 20 
14.2 Data Quality Control and Reporting ........................................................................... 20 
14.3 Archival of Data .......................................................................................................... 20 
14.4 Availability and Retention of Investigational Records ............................................... 20 
14.5 Subject Confidentiality................................................................................................ 20 
15 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS .................. 21 
15.1 Protocol Amendments ................................................................................................. 211 
15.2 Institutional Review Boards and Independent Ethics Committees ............................. 211 
15.3 Informed Consent Form .............................................................................................. 222 
15.4 Publications ................................................................................................................. 222 
15.5 Investigator Responsibilities ....................................................................................... 222 


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LIST OF ABBREVIATIONS 
ABC 
Aberrant Behavior Checklist 
AE 
adverse event 
ASD 
autism spectrum disorder 
CBS 
cystathionine beta synthase 
CFR 
Code of Federal Regulations 
CHR 
Committee on Human Research 
CRF 
case report form 
CSF 
cerebrospinal fluid 
CTCAE 
Common Terminology Criteria for Adverse Events 
DSM-IV 
Diagnostic and Statistical Manual of Mental Disorders, 4th edition 
eBit 
Evidence-Based Intervention and Treatment 
FDA 
Food and Drug Administration 
FR alpha 
folate receptor alpha 
FRAA 
folate receptor auto-antibodies 
GCP 
Good Clinical Practice 
 
 
GSH 
glutathione 
GSSG 
glutathione disulfide 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
ICH 
International Conference on Harmonisation 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
MedDRA 
Medical Dictionary for Regulatory Activities 
MS 
methionine synthase 
MTase 
methyltransferase 
OHS 
Oak Hill School 
PedsQL 
Pediatric Quality of Life Inventory 
PHI 
protected health information 
PI 
Principal Investigator 
SAE 
serious adverse event 
SAH 
S-adenosylhomocysteine 
SAM 
S-adenosylmethionine 
SCQ 
Social Communication Questionnaire 
SRS 
Social Responsiveness Scale 
THF 
tetrahydrofolate 


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PROTOCOL SYNOPSIS 
TITLE 
An examination of changes in urinary metabolites with use of 
folinic acid in children with autism spectrum disorder (ASD) 
SPONSOR 
Dr. Robert Hendren 
FUNDING 
ORGANIZATION 
Gift Funded from the JS Foundation 
NUMBER OF SITES 
1 
RATIONALE 
Children with autism spectrum disorder (ASD) have been found to 
have impaired transport of folate across the blood-brain barrier due to 
folate receptor auto-antibodies (FRAA) that either block or bind to the 
folate receptor alpha (FR alpha). This creates the condition known as 
“cerebral folate deficiency” where serum folate concentrations are 
normal but CSF folate concentrations are low. Recent studies have 
found that supplementing children with ASD with a reduced form of 
folate – folinic acid – allows for bypass of the impaired folate 
transport mechanism into the CSF, leading to improved behavior and 
language development. We propose to further examine this exciting 
new therapeutic possibility by conducting an open-label study of 
folinic acid in children with ASD to examine both changes in 
behavior and correlations with urinary metabolites. 
We previously created an outcomes database within a school for 
children with ASD (Oak Hill School, San Anselmo, CA) to carefully 
track teacher and parent-rated assessments of children enrolled in the 
school. In a prior study, we established the ability to provide a 
treatment (the phytochemical, sulforaphane) to children in the school 
while monitoring changers in urinary metabolites and clinical 
outcomes. This study found that changes in specific urinary 
metabolites were associated with clinical improvements, which 
suggested specific metabolic pathways that are involved in the 
treatment response.  In the current study, we will use the same 
successful model to determine if changes in urinary metabolites are 
correlated with clinical changes. This study may provide insight into 
the mechanism of action of folinic acid and may allow for the 
development of a biomarker prediction model that will determine 
which children are most likely to benefit from this treatment.  
STUDY DESIGN 
Open label treatment study elucidating mechanism of action. 
PRIMARY 
OBJECTIVE 
To examine changes in urinary metabolites in children with ASD 
being treated with folinic acid and to determine if changes in urinary 
metabolites are correlated with clinical improvements.  
SECONDARY 
OBJECTIVES 
Develop a prediction model to determine which children are most 
likely to benefit from folinic acid. 


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NUMBER OF 
SUBJECTS 
40 
SUBJECT 
SELECTION 
CRITERIA 
Inclusion Criteria: Age 5-22, enrolled at Oak Hill School, diagnosis of 
autism established by DSM-IV criteria and clinician examination. 
 
Exclusion Criteria: Seizure disorder, serious medical illness within the 
last 6 months as judged by the study PI. Known allergy to folate. 
 
TEST PRODUCT, 
DOSE, AND ROUTE 
OF 
ADMINISTRATION 
Folinic acid 2mg/kg per day (maximum 50mg/day) in two equally 
divided doses with half the target dose given during the first 2 weeks. 
CONTROL 
PRODUCT, DOSE 
AND ROUTE OF 
ADMINISTRATION 
There is no control product in this study. 
DURATION OF 
SUBJECT 
PARTICIPATION 
AND DURATION OF 
STUDY 
Subjects will be on study for 4.5 months 
Screening:  up to 2 weeks. 
Treatment:  3 months 
Follow-up:  1 month (1 additional month after treatment) 
The total duration of the study is expected to be 12 months.  3-6 
months for subject recruitment; 4 months for final subject follow-up; 
2 months for data analysis and manuscript preparation. 
CONCOMMITANT 
MEDICATIONS 
Allowed: all 
 
Prohibited: none 
EFFICACY 
EVALUATIONS 
Parent (Aberrant Behavior Checklist, Social Responsiveness Scale, 
Pediatric Quality of Life) 
Teacher (Student Progress Form, Aberrant Behavior Checklist, 
Social Responsiveness Scale) 
Urinary Metabolomics (Baseline and 3 months) 
PRIMARY ENDPOINT 
• 
Correlation between mean change in Aberrant Behavior Checklist 
and mean change in urinary metabolites. 
SECONDARY 
ENDPOINTS 
• 
Correlations between changes in urinary metabolites and all 
secondary outcome measures.  
OTHER 
EVALUATIONS 
None 


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SAFETY 
EVALUATIONS 
All study subjects are enrolled at the Oak Hill School and will be 
observed all school days by teachers, who are aware of student 
participation in the study. Parents, teachers, and enrolled students will 
be advised to report any new medical problems to study investigators, 
who will evaluate all potential adverse events and report to the CHR 
according to established guidelines. 
PLANNED INTERIM 
ANALYSES 
No interim analyses planned given the short-term duration of the 
study, the established safety of the treatment, and the lack of 
randomization (so no ability to compare rates of AE’s between active 
and placebo treatment). 
STATISTICS 
Primary Analysis Plan 
Correlations between changes in mean levels of urinary metabolites 
and changes in mean behavioral measures (0 to 3 months) will be 
done using a spearman rank correlations test using STATA. We 
prospectively define correlations with an absolute value of > 0.6 to be 
potentially involved in the mechanism of action. All metabolites with 
this correlation will be mapped to known metabolite pathways to 
examine pathway changes.  
Rationale for Number 
of Subjects 
We are recruiting all children currently enrolled at Oak Hill School 
(current enrollment = 36). We anticipate that we will enroll 20 
students in the current study, but we will allow up to 40 (if school 
enrollment increases). 
 


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1 
BACKGROUND 
 
1.1 
Overview of Clinical Studies 
Cerebral folate deficiency in ASD: Prior studies have linked abnormalities in the 
metabolism of folate, an essential B vitamin, to ASD.1 These abnormalities may relate to 
the ability of a child with ASD to transport folate across the blood-brain barrier. Children 
with ASD have been found to have impaired transport of folate across the blood-brain 
barrier due to auto-antibodies that either block or bind to the folate receptor alpha (FR 
alpha). (The antibodies are known as folate receptor auto-antibodies - (FRAA)). This 
creates the condition known as “cerebral folate deficiency” which describes an individual 
with normal serum folate concentrations but low concentrations of folate in the 
cerebrospinal fluid (CSF) due to impaired ability to transport folate across the blood-brain 
barrier.2  
High prevalence of FRAA in ASD: Children with ASD have been found to have a high 
prevalence of FRAA. In a study of 93 children with ASD, 60% had blocking FRAA, and 
44% had binding FRAA.3 Blocking antibodies interfere with binding of folate to the FR 
alpha and binding antibodies bind to the FR alpha and cause an antibody-mediated 
immune reaction. The rates of FRAA in ASD are higher than the 4-15% prevalence 
reported in healthy adults and the 3% prevalence reported in developmentally delayed 
non-autistic children.2 Up to 23% of children with ASD who underwent lumbar puncture 
were reported to have abnormally low CSF folate concentrations.4 Together, this evidence 
suggests that low CSF folate levels may contribute to the abnormal physiology and 
clinical symptoms of ASD. 
Reduced forms of folate can bypass impaired folate transport across the blood-brain 
barrier: Folinic acid, a reduced form of folate, is able to bypass the normal blood-brain 
barrier transport mechanism and increase CSF folate levels through a secondary transport 
mechanism that involves a reduced-folate transporter. Case reports have found that high-
dose folinic acid supplementation markedly improves CSF folate levels in children with 
ASD and low CSF folate.2  
Folinic acid supplementation improves language and aberrant behavior in ASD: In a 
recent (2016) randomized controlled trial of folinic acid supplementation vs. placebo in 48 
children with ASD and language delay, children randomized to folinic acid had 
statistically significant improvements in language and aberrant behavior.2 Furthermore, 
improvements were greater in the subgroup of children who were positive for FRAA, 
suggesting that children with more severe cerebral folate deficiency were more likely to 
benefit (FRAA was used a proxy for CSF folate levels, since it was not possible to obtain 
CSF folate on study participants due to the invasive nature of obtaining CSF).  
Folate may support the 3 interdependent pathways involved in folate (THF)-dependent 
methionine transmethylation/transsulfuration metabolism. These pathways are involved in 
DNA synthesis, cellular methylation, and redox homeostasis, and an adequate cerebral 
supply of folate may allow for improved functioning of these pathways and clinical 
improvements.  


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Figure 1. This is an overview of the 3 interdependent pathways involved in folate (THF)-
dependent methionine transmethylation/transsulfuration metabolism.  The vital importance of 
these three interconnected pathways is underscored by their essentiality for error-free DNA 
synthesis (Folate Cycle Pathway 1); for cellular methylation (epigenetic) capacity 
(Transmethylation Pathway 2); and for the maintenance of glutathione (GSH) redox homeostasis 
(Transsulfuration Pathway 3). Because these 3 pathways regulate the distribution of precursors 
for DNA synthesis (proliferation), DNA/histone methylation (epigenetics) and glutathione 
synthesis (redox/antioxidant status), the homeostatic balance between these pathways is essential 
to support normal cell programming and ontogeny during prenatal and post-natal development. 
 
In summary, children with ASD are known to have a high prevalence of cerebral folate 
deficiency, likely caused by folate receptor auto-antibodies (FRAA), which block the 
normal transfer of folate from the bloodstream to the CSF. Prior studies have documented 
that supplementation with reduced forms of folate can bypass the impaired transport 
mechanism and restore normal cerebral folate levels. A recent randomized controlled trial 
found that folinic acid supplementation improved language and behavior in children with 
ASD. We therefore propose to conduct an open-label study of folinic acid in children with 
ASD to determine if clinical improvements are correlated with changes in metabolites 
known to be related to the folate pathways. This study will gain information needed to 
determine a possible metabolomic mechanism of action of folinic acid and allow for the 
development of a prediction model to identify children with ASD who are mostly likely to 
respond to treatment.  


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2 
STUDY RATIONALE 
2.1 
Risk / Benefit Assessment 
Risk of treatment with folinic acid: In a prior randomized controlled trial of the same dose 
and duration of treatment with folinic acid in 48 children with ASD, there were no serious 
side effects and no side effects that were more common with folinic acid than placebo 
treatment.  
Folinic acid is closely related molecule to the B vitamin, folate. It is approved to help with 
the adverse of effects of certain medications such as methotrexate, which depletes folate 
in the body. Folinic acid is also approved to be used in combination with 
chemotherapeutic agents in certain cancers.The identification of side effects of folinic acid 
is somewhat confounded by the combined use of potent chemotherapeutic drugs. Adverse 
effects of folinic acid may include:  skin rashes, itching, facial flushing, nausea or 
vomiting. Acute allergic reactions have been reported – but considered rare.  
Potential benefits: There is a possibility that children enrolled in this study will experience 
some beneficial effects related to treatment with folinic acid. As noted above, in a prior 
study of folinic acid in ASD, children were noted to have improvements in aberrant 
behavior and language development. Also, adult studies have found improvement in mood 
in patients treated with supplements containing folinic acid. Finally, there is biological 
plausibility that children with impaired cerebral folate transfer may have some beneficial 
physiological, and therefore clinical, effects when cerebral folate levels are raised through 
folinic acid treatment.  
 
3 
STUDY OBJECTIVES 
3.1 
Primary Objective 
The primary objective is to determine if there is a correlation between changes in behavior 
and changes in urinary metabolites in children with ASD who receive folinic acid 
treatment for 12 weeks. This will provide insight into the potential mechanism of action. 
3.2 
Secondary Objectives: 
Secondary objectives include an examination of the correlation between changes in 
urinary metabolites and other outcome measures, including the Social Responsiveness 
Scale (SRS) (parents and teachers) and the Pediatric Quality of Life Inventory (PedsQL) 
(parents). It is possible that specific urinary metabolites are related to different clinical 
domains, and we will therefore examine correlations in each of these domains.  
 
 
 
 


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STUDY DESIGN 
3.3 
Study Overview 
This is an open-label, 12-week study examining the effects of folinic acid on behavior in 
children with ASD and the correlation between behavior change and urinary metabolites.  
Total duration of subject participation will be four months – 3 months while taking the 
study supplement and one month of wash-out.  Total duration of the study is expected to 
be 12 months. 
4 
CRITERIA FOR EVALUATION 
4.1 
Primary Endpoint 
• 
Correlation between changes in the Aberrant Behavior Checklist and urinary 
metabolites.  
4.2 
Secondary Endpoints 
• 
Correlation between changes in all outcome measures and changes in urinary 
metabolites.  
4.3 
Safety Evaluations 
• 
Patient reported adverse events, which will be collected through spontaneous reporting 
as well as specific queries at 1 and 3 months of treatment.  
5 
SUBJECT SELECTION 
5.1 
Study Population 
Children and young adults between the ages of 5 and 22 who are enrolled at the Oak Hill 
School (OHS) in San Anselmo, CA (a school for children with autism or related 
neurodevelopmental disorders). OHS is the site for our ongoing outcomes study using a 
secure web-based outcome platform where enrolled children (n=40) have regular 
outcomes completed by teachers (weekly) and parents (quarterly) (UCSF CHR approval 
number 13-11086). A prior intervention study of the supplement, sulforaphane (UCSF 
CHR approval number 15-17002) demonstrated the feasibility of conducting an open-
label study to examine changes in urinary metabolites and clinical measures with this 
population and the web-based outcome platform.  
5.2 
Inclusion Criteria 
1. Male or female, enrolled at OHS, age 5-22 and with a diagnosis of ASD. 
2. ASD diagnosis is established DSM-IV criteria, child observation by an expert 
clinician, and score on the Social Communication Questionnaire, completed by 
parents). 


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3. Written informed consent obtained from the subject’s legal representative and ability 
for subject to comply with the requirements of the study. 
5.3 
Exclusion Criteria 
1. Presence of a condition or abnormality that in the opinion of the Investigator would 
compromise the safety of the patient or the quality of the data.  
2.    Known allergy to any component of the folinic acid product. 
6 
CONCURRENT MEDICATIONS 
All subjects should be maintained on the same medications throughout the entire study 
period, as medically feasible, with no introduction of new chronic therapies. 
6.1 
Prohibited Medications and Treatments 
Study subjects must not currently be taking folinic acid or have used this supplement in 
the last 6 months.  
7 
STUDY TREATMENTS 
Since this is an open-label study, all subjects will receive folinic acid. Subjects, parents, 
teachers, and clinicians are not blinded to treatment status. There is no control product 
being used.  
7.1 
Formulation of Product –  
Folinic acid is prepared as a calcium salt. The capsules are dye-free, milk-product free, 
and vegetarian and are provided in 10 mg available for weight-based dosing (2mg/kg with 
maximum dose of 50mg/day divided in two doses). The capsules are produced by Lee 
Silsby Pharmacy (Cleveland Heights, OH). Certificate of analysis has been obtained by an 
independent analytical service and is part of the IND packet, approved by the FDA.  
7.1.1 
Dosage/Dosage Regimen -  
Study participants will receive weight-based dosing (2mg/kg per day) divided in 2 equal 
doses with maximum daily dose of 50mg. If unable to swallow, the capsules may be 
administered by piercing the capsule and mixing the contents into soft food including 
yogurt, oatmeal, drinks.  
7.1.2 
Dispensing 
Study staff will dispense the study supplement, labelled with each participant’s unique 
study ID number, after the participant has completed all necessary enrollment procedures.  
7.1.3 
Administration Instructions  
Each participant will be instructed to take the weight-based dose, by mouth twice a day 
with meals. 
Doses may be mixed with liquid or soft foods.  


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7.2 
Supply of Study Supplement at the Site 
The study supplement will be stored by the study site at controlled room temperature, 20 to 
25ºC (68 to 77ºF). Participants will be instructed to also store the product at room 
temperature, consistent with labelled instructions and stability testing. 
7.2.1 
Study Supplement Accountability 
An accurate and current accounting of the dispensing and return of study supplement for 
each participant will be maintained on an ongoing basis by a member of the study site 
staff.  The number of study supplement capsules dispensed and returned by the participant 
will be recorded.  
 
8 
STUDY PROCEDURES AND GUIDELINES 
A Schedule of Events representing the required testing procedures to be performed for the 
duration of the study is diagrammed in Appendix 1. 
8.1 
Clinical Assessments 
8.1.1 
Concomitant Medications 
All concomitant medication and concurrent therapies will be documented at the Screening 
visit.  Dose, route, unit, frequency of administration, and indication for administration and 
dates of medication will be captured. 
8.1.2 Demographics  
Demographic information (date of birth, gender, race) will be recorded at the Screening 
visit. 
8.1.3 Medical History  
Relevant medical history, including history of current disease and information regarding 
underlying diseases will be recorded at Screening visit. 
8.1.4 
Adverse Events 
Information regarding occurrence of adverse events will be captured throughout the study. 
Participants will be informed to contact a study staff member if they experience a new 
medical problem – this information will be recorded on an Adverse Event form (including 
entries for duration, severity, outcome, treatment and relation to study supplement). AE 
forms will be reviewed by a study investigator and reported to the UCSF CHR according 
to established guidelines. We will be carefully monitoring patients for the occurrence of 
any adverse events through active questioning and 1 and 3 months as well as instructions 
for patients to report any new medical problem immediately to a study team member. All 
adverse events will be reviewed by a study investigator within 24 hours and reported to 
the Committee on Human Research according to standard guidelines.  


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8.2 
Clinical Laboratory Measurements  
Urinary Metabolomic Profile – all participants will have urine collected (fasting, 8am 
target collection time) at Screening and Close-out (3 months). Urine will be stored in 
sterile containers at -80 degrees Celsius and analyzed in one batch after all participants 
have completed the study. Metabolomic assessment will be conducted by Metabalon 
(Research Triangle, NC). Metabalon is one of the largest and most established laboratories 
conducting state-of-the art metabolomics assessments of urine, plasma, and other clinical 
samples. Metabalon has 160 employees including more than 40 PhDs with diverse 
backgrounds in molecular biology, biochemistry, bioinformatics, and statistics. They have 
completed over 4,000 metabolomic projects for over 700 clients. Their analyses have been 
used in more than 500 peer-reviewed publications including numerous articles in Cell, 
Nature, and Science. (www.metabalon.com) 
9 
EVALUATIONS BY VISIT 
9.1 
Visit 1: Screening visit – this visit will involve a face-to-face meeting with the 
study participant and their parent/legal guardian. A study team member will 
explain the entire study and obtain signed informed consent. Study 
inclusion/exclusion criteria will be reviewed and eligible participants will go on 
to complete all screening activities as below. 
1. 
Review the study with the participant and obtain written informed consent. 
2. 
Assign the participant a unique study ID number 
3. 
Record demographics data. 
4. 
Record medical history. 
5. 
Record concomitant medications. 
6. 
Collect urine specimen. 
7. 
Dispense study supplement 
8. 
Schedule subject for Baseline visit 
9. 
Record study subject weight  
9.2 
Visit 2 (Baseline) – NOTE – for this study, which will take place at the Oak Hill 
School, all enrolled children will begin the study supplement on the same date 
(exact day TBD based on parent/teacher schedules and CHR approval date). 
This will improve the logistics of the study because teachers regularly complete 
outcome assessments using our established on-line outcomes platform at 
quarterly intervals (that match their school-based quarterly progress reports). 
This “batch” enrollment of participants has several advantages: 1) all 
participants will begin and end on the same date, which we believe will support 
the overall study compliance and enthusiasm, as all children/families will be 
involved in the study at the same time; 2) outcome assessments are a natural 
part of the study schedule, since they normally are completed by the teachers at 
3 months intervals and will therefore occur as children complete the 3-month 
treatment; 3) compliance and reminders to parents/guardians and teachers will 
be simplified, since all participants will be on the same schedule and all 


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reminders can be sent in batches; 4) variation from time/season effects will be 
minimized, since all children will be experiencing the same school environment 
at the same period in the study. 
 
1. 
Parents complete Baseline Outcome Measures. 
2. 
Teachers complete Baseline Outcome Measures. 
3. 
Subject starts taking study supplements at this time. 
4. 
Reinforce study directions, contact information, emphasize need to report new 
medical problems (possible AE’s). 
9.3 
Visit 3: Follow-up 1(one-month visit): TBD 
1. 
Parents complete Follow-up Outcome Measures. 
2. 
Teachers complete Follow-up Outcome Measures. 
3. 
Adverse event form completed by parents, reviewed by study investigators. 
4. 
Study supplement instructions reviewed. 
 
9.4 
Visit 4: Follow-up visit 2 (three month visit): TBD 
1.    Parents complete Follow-up Outcome Measures. 
2. Teachers complete Follow-up Outcome Measures. 
3. Adverse event form completed by parents, reviewed by study investigators. 
4. Urine collected (fasting, 8am specimen, stored at -80 degrees Celsius) 
5. Study supplement stopped. Medication diary and used and unused study supplement 
bottle collected and counted. 
6. Record study subject weight. 
 
9.5 
Visit 5 Follow-up visit 3 (four month visit, one-month off study supplement): 
TBD 
1. 
Parents complete Follow-up Outcome Measures. 
2. 
Teachers complete Follow-up Outcome Measures. 
 
10 ADVERSE EVENTS REPORTING AND DOCUMENTATION 
10.1 Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical investigation of a 
patient administered a pharmaceutical or supplement product and that does not necessarily 
have a causal relationship with the treatment.  An AE is therefore any unfavorable and 


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unintended sign, symptom or disease temporally associated with the administration of an 
investigational product, whether or not related to that investigational product.   
The study visits include a direct question for parents/caregivers regarding the occurrence 
of AEs since the last study visit. All information will be recorded in the site’s source 
documents.  Adverse events will be recorded in the patient CRF.  Adverse events will be 
described by duration (start and stop dates and times), severity, outcome, treatment and 
relation to study supplement, or if unrelated, the cause. 
AE Severity 
The National Cancer Institute’s Common Terminology Criteria for Adverse Events 
(CTCAE) Version 3.0 will be used to assess and grade AE severity.   
Table 1.  AE Severity Grading 
Severity (Toxicity Grade) 
Description 
Mild (1) 
Transient or mild discomfort; no limitation in activity; no medical 
intervention or therapy required. The subject may be aware of the sign 
or symptom but tolerates it reasonably well. 
Moderate (2) 
Mild to moderate limitation in activity, no or minimal medical 
intervention/therapy required. 
Severe (3) 
Marked limitation in activity, medical intervention/therapy required, 
hospitalizations possible. 
Life-threatening (4) 
The subject is at risk of death due to the adverse experience as it 
occurred. This does not refer to an experience that hypothetically 
might have caused death if it were more severe. 
 
AE Relationship to Study Supplement 
The relationship of an AE to the study supplement will be assessed using the guidelines in  
Table 2. 
Table 2.  AE Relationship to Study Supplement 
Relationship 
to Supplement 
Comment 
Definitely 
Previously known toxicity of agent; or an event that follows a reasonable temporal 
sequence from administration of the supplement; that follows a known or expected 
response pattern to the suspected supplement; that is confirmed by stopping or 
reducing the dosage of the supplement; and that is not explained by any other 
reasonable hypothesis. 
Probably 
An event that follows a reasonable temporal sequence from administration of the 
supplement; that follows a known or expected response pattern to the suspected 
supplement; that is confirmed by stopping or reducing the dosage of the supplement; 
and that is unlikely to be explained by the known characteristics of the subject’s 
clinical state or by other interventions. 


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Possibly 
An event that follows a reasonable temporal sequence from administration of the 
supplement; that follows a known or expected response pattern to that suspected 
supplement; but that could readily have been produced by a number of other factors. 
Unrelated 
An event that can be determined with certainty to have no relationship to the study 
supplement. 
10.2 Serious Adverse Events (SAE) 
An SAE is defined as any AE occurring at any dose that results in any of the following 
outcomes: 
• 
death 
• 
a life-threatening adverse experience 
• 
inpatient hospitalization or prolongation of existing hospitalization 
• 
a persistent or significant disability/incapacity 
• 
a congenital anomaly/birth defect  
Other important medical events may also be considered an SAE when, based on 
appropriate medical judgment, they jeopardize the subject or require intervention to 
prevent one of the outcomes listed.  
 
10.3 Serious Adverse Experience Reporting 
We will document all SAEs that occur (whether or not related to study supplement) per 
UCSF CHR Guidelines.  The collection period for all SAEs will begin after informed 
consent is obtained and end after procedures for the final study visit have been completed. 
We will report all AE’s and SAE’s according to UCSF CHR guidelines. 
 
11 EARLY DISCONTINUATION OF STUDY SUPPLEMENT 
A subject may be discontinued from study treatment at any time if the subject, the 
investigator, or the Sponsor feels that it is not in the subject’s best interest to continue.  
The following is a list of possible reasons for study treatment discontinuation: 
• Participant legal guardian withdrawal of consent.  
• Participant is not compliant with study procedures. 
• Adverse event that in the opinion of the investigator would be in the best interest 
of the participant to discontinue study supplement. 
• Protocol violation requiring discontinuation of study treatment. 
• Lost to follow-up. 
If a participant is withdrawn from treatment due to an adverse event, the subject will be 
followed and treated by the Investigator until the abnormal parameter or symptom has 
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All participants who discontinue study supplement will be asked to continue the outcome 
assessment as scheduled. 
11.1 Withdrawal of Subjects from the Study 
All subjects are free to withdraw from participation at any time, for any reason, specified 
or unspecified, and without prejudice. 
Reasonable attempts will be made by the investigator to provide a reason for subject 
withdrawals, and this information will be recorded in the subject’s source documents. 
12 PROTOCOL VIOLATIONS 
A protocol violation occurs when the subject, or investigator, fails to adhere to significant 
protocol requirements affecting the inclusion, exclusion, subject safety and primary 
endpoint criteria.  Protocol violations for this study include, but are not limited to, the 
following: 
• Failure to meet inclusion/exclusion criteria. 
• Improper use of schedule/dose of study supplement. 
When a protocol violation occurs, it will be discussed with the investigator and a Protocol 
Violation Form detailing the violation will be generated. This form will be signed by the 
Investigator and reported to the UCSF CHR according to established guidelines. 
13 STATISTICAL METHODS AND CONSIDERATIONS 
Prior to the analysis of the final study data, a detailed Statistical Analysis Plan (SAP) will 
be written describing all analyses that will be performed.  This information will be posted 
on clinicaltrials.gov to ensure that data analysis proceeds as defined a priori. Any 
modifications to the plan provided below will be noted on clinicaltrials.gov before the first 
subject is enrolled. 
13.1 Analysis of Primary Endpoint 
The primary analysis in the study is the correlation between the mean change in each 
urinary metabolite and the mean change in participant behavior as measured by the ABC 
using the spearman rank correlation test in Stata. The primary outcome measure is the 
correlation between urinary metabolites and the total ABC score.  
13.2 Analysis of Secondary Endpoints 
Secondary outcome measures will use the same spearman rank correlation test in Stata, 
comparing each urinary metabolite with each of the other outcome measures (SRS, 
PedsQL). Because this study is exploratory in nature, no adjustments will be made for 
multiple comparisons, as recommended by Rothman.5    
Safety and tolerability data will be summarized in a tabular format, which will be included 
in the final study report/manuscript.   


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Adverse event rates will be coded by body system and MedDRA classification term. 
Adverse events will be tabulated by treatment group and will include the number of 
patients for whom the event occurred, the rate of occurrence, and the severity and 
relationship to study supplement.   
13.3 Sample Size 
The study is designed to detect a correlation coefficient (Pearson’s r) between the mean 
urinary metabolite change and the mean behavioral measure change of 0.6, which is 
considered to indicate a moderate-to-strong correlation. Using a two-sided alpha of 0.05 
and a Beta of 0.1 (90% power), our required sample size is 25 participants to detect this 
degree of correlation. Since we may incur up to 20% dropout (5 participants), our target 
sample size is 30 participants, but we will allow up to 40 to provide the study opportunity 
to all children enrolled in the target school (Oak Hill School). 
14 DATA COLLECTION, RETENTION AND MONITORING 
14.1 Data Collection Instruments 
This study will take place at Oak Hill School, where we have established an ongoing 
outcomes study that uses a web-based platform for data entry for parents and teachers. 
This platform is secure and has been reviewed for adequacy by the UCSF CHR (#13-
11086). Data gathered for the study will be electronically coded into a password-protected 
database. All uploaded files will be stored in a database not in the server's file system, and 
the storage format will be encrypted. We will use the same secure platform for all data 
collection for the current study.  
14.2 Data Quality Control and Reporting 
All data entered in this on-line system undergoes a rigorous quality control evaluation and 
pre-specified logic checks. All entries are date and time stamped, and any modifications to 
study data are flagged – the editor and time/day of edits are also recorded. As a result, the 
study database is cleaned continuously and will be ready for analysis at the completion of 
the last study subject visit. 
14.3 Archival of Data 
The database is safeguarded against unauthorized access by established security 
procedures; appropriate backup copies of the database and related software files is 
maintained.   
14.4 Availability and Retention of Investigational Records 
All study data is available, as need and required, for review by appropriate regulatory 
authorities. In this case, the UCSF CHR is the responsible regulatory authority.  A file for 
each subject will be maintained and will include the signed Informed Consent and copies 
of all source documentation related to that subject.  The Investigator ensures the reliability 
and availability of source documents from which the information on the CRF was derived. 


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14.5 Subject Confidentiality 
All study forms containing PHI are kept in a secure database (named “eBit” for Evidence-
Based Intervention and Treatment) that meets FDA criteria for electronic database 
security.  
In eBit, only UCSF study coordinators are able to create online parent and teacher 
accounts, which are password-protected. Data gathered for the study will be electronically 
coded into a password-protected database. All uploaded files onto eBit will be stored in a 
database not in the server's file system, and the storage format will be encrypted. eBit uses 
a separate platform to handle the PHI (including parent's email addresses) that will then 
communicate directly with Oak Hill platforms. This will keep the PHI separate from the 
actual health data and will meet all the IRB and HIPPA requirements that eBit and the 
UCSF IT group have agreed upon. 
  
eBit is deployed on the Microsoft Azure secure cloud platform, which, according to their 
website, uses “industry-standard encrypted transport protocols between user devices and 
Microsoft datacenters, and within datacenters themselves.” More information about 
Microsoft Azure’s security can be found at the following link: 
https://www.microsoft.com/en-us/TrustCenter/Security/default.aspx . This is the same 
server that was used in our previously approved, and still active, CHR application for our 
Oak Hill School outcomes study. The UCSF IT group has reviewed the terms of this web 
platform and has ensured that it meets all data security requirements. 
 
15 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
The study will be conducted according to the Declaration of Helsinki, Protection of 
Human Volunteers (21 CFR 50), Institutional Review Boards (21 CFR 56), and 
Obligations of Clinical Investigators (21 CFR 312). 
15.1 Protocol Amendments 
Any amendment to the protocol will be written by the Investigator and submitted to the 
UCSF CHR. Protocol amendments will not be implemented without prior written 
approval from the UCSF CHR except as necessary to eliminate immediate safety hazards 
to patients.  A protocol amendment intended to eliminate an apparent immediate hazard to 
patients may be implemented immediately, provided the UCSF CHR is notified within 
five working days. 
15.2 Institutional Review Boards and Independent Ethics Committees 
The protocol and consent form will be reviewed and approved by the UCSF CHR prior to 
study initiation.  Serious adverse experiences regardless of causality will be reported to 
the UCSF CHR in accordance with the standard operating procedures. 
The UCSF CHR’s written unconditional approval of the study protocol and the informed 
consent form will be in the possession of the Investigator before the study is initiated.   
Protocol and/or informed consent modifications or changes will not be initiated without 
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to the patients or when the change(s) involves only logistical or administrative aspects of 
the study.  Such modifications will be submitted to the UCSF CHR and written 
verification that the modification was submitted and subsequently approved will be 
obtained.   
15.3 Informed Consent Form  
Informed consent will be obtained in accordance with the Declaration of Helsinki, ICH 
GCP, US Code of Federal Regulations for Protection of Human Subjects (21 CFR 
50.25[a,b], CFR 50.27, and CFR Part 56, Subpart A), the Health Insurance Portability and 
Accountability Act (HIPAA, if applicable), and local regulations. 
The consent form generated by the Investigator (attached) must be acceptable to the UCSF 
CHR.  The written consent document will embody the elements of informed consent as 
described in the International Conference on Harmonisation and will also comply with 
local regulations.  
A properly executed, written, informed consent will be obtained from each subject prior to 
entering the subject into the trial.  Information will be given in both oral and written form 
and each subject’s legal representatives) will be given ample opportunity to inquire about 
details of the study.  A copy of the signed consent form will be given to the subject’s legal 
representative and the original will be maintained with the subject’s records. 
15.4 Publications  
The publication or presentation of any study results will comply with all applicable 
privacy laws, including, but not limited to, the Health Insurance Portability and 
Accountability Act of 1996.  
15.5 Investigator Responsibilities 
By signing the Agreement of Investigator form, the Investigator agrees to: 
1. Conduct the study in accordance with the protocol. 
2. Personally conduct or supervise the study (or investigation). 
3. Ensure that the requirements relating to obtaining informed consent and CHR review 
and approval meet federal guidelines, as stated in § 21 CFR, parts 50 and 56. 
4. Ensure that all associates, colleagues and employees assisting in the conduct of the 
study are informed about their obligations in meeting the above commitments.  
5. Maintain adequate and accurate records in accordance with §21 CFR 312.62. 
6. Ensure that an IRB that complies with the requirements of §21 CFR part 56 will be 
responsible for initial and continuing review and approval of the clinical study. 
7. Promptly report to the IRB all changes in the research activity and all unanticipated 
problems involving risks to subjects or others. 
8. Seek IRB approval before any changes are made in the research study, except when 
necessary to eliminate hazards to the patients/subjects. 


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9. Comply with all other requirements regarding the obligations of clinical investigators 
and all other pertinent requirements listed in § 21 CFR part 312. 
 


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APPENDIX 1.  SCHEDULE OF STUDY VISITS - UPDATE 
 
VISIT 1 
Screening/Month -1 
VISIT 2 
Baseline/ Month 0 
VISIT 3 
Follow-up 1/ Month 1 
VISIT 4 
Follow-up 2/ Month 3 
VISIT 5 
Follow-up 3/ Month 4 
Study Clinic Visit 
X 
 
 
X 
 
Informed Consent 
X 
 
 
 
 
Assign Study ID and eBit log in 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
Medical History 
X 
 
 
 
 
Urine Sample (including sibling if 
consented) 
X 
 
 
X 
 
Weight 
X 
 
 
X 
 
Dispense Study Supplement 
X 
 
 
 
 
Administer Study Supplement 
 
X 
 
 
 
Concomitant Medication Review 
X 
X 
X 
X 
X 
Adverse Experiences 
 
X 
X 
X 
X 
Parent Assessments (Online) 
 
 
 
 
 
SCQ 
 
X 
 
 
 
ABC 
 
X 
X 
X 
X 
SRS 
 
X 
X 
X 
X 
PedsQL 
 
X 
 
X 
 
Teacher Assessments (Online) 
 
Teacher will complete Weekly Progress Report every week starting at baseline to month 4 
ABC 
 
X 
X 
X 
X 
SRS 
 
X 
X 
X 
X 


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Protocol Template © CFFT TDN   Effective: 12 SEP 2007 
References: 
 
1. 
Vahabzadeh A, McDougle CJ. Maternal folic acid supplementation and risk of 
autism. JAMA. Jun 05 2013;309(21):2208. 
2. 
Frye RE, Slattery J, Delhey L, et al. Folinic acid improves verbal communication in 
children with autism and language impairment: a randomized double-blind placebo-
controlled trial. Molecular psychiatry. Oct 18 2016. 
3. 
Frye RE, Sequeira JM, Quadros EV, James SJ, Rossignol DA. Cerebral folate 
receptor autoantibodies in autism spectrum disorder. Molecular psychiatry. Mar 
2013;18(3):369-381. 
4. 
Shoffner J, Trommer B, Thurm A, et al. CSF concentrations of 5-
methyltetrahydrofolate in a cohort of young children with autism. Neurology. Jun 
14 2016;86(24):2258-2263. 
5. 
Rothman KJ. No adjustments are needed for multiple comparisons. Epidemiology. 
Jan 1990;1(1):43-46.