Predicting Treatment Response to Memantine in Autism Spectrum Disorder Using MR Spectroscopy
Phase: EARLY_PHASE1
Conditions: Autism Spectrum Disorder
Sponsor: University of Missouri-Columbia

 
 
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Predicting Treatment Response to Memantine in Autism Spectrum Disorder using MR 
Spectroscopy 
 
 
Protocol 1 
Version 7 
March 8, 2017 
 
 
Institution: University of Missouri 
Principal Investigator: Dr. David Beversdorf, M.D. 
Co-Investigator: Neetu Nair 
 
 
 
 
 
 

 
 
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Abstract 
Memantine, an N-methyl-D-aspartate receptor antagonist, has been explored as a possible 
therapeutic agent that reduces the excitatory (glutamate) – inhibitory (gamma amino-butyric acid, 
GABA) imbalance in autism pathology and improves social and communication deficits. While 
some studies have shown positive results, a large clinical trial failed to show benefit possibly 
because different subsets of autism responded differently to the treatment. We propose a pilot, 
exploratory, clinical follow-on study using proton magnetic resonance spectroscopy (1H-MRS) to 
determine whether baseline glutamate/GABA levels in certain regions of the brain may help 
predict treatment response to Memantine in autistic subjects. At study onset, subjects will be 
assessed on the behavioral scales such as the Aberrant Behavior Checklist and Clinical Global 
Impressions scale, followed by MRS imaging. Memantine treatment will be started post imaging. 
Assessment measures will be repeated at week 12 during treatment. Glutamate and GABA levels 
in brain regions will be correlated to improvements on assessment measures. We expect higher 
glutamate and/or lower GABA levels in the anterior cingulate cortex at baseline in responders to 
memantine. If our hypotheses are confirmed, we will have established a relevant neural biomarker 
to predict treatment response to memantine with important implications for clinical care including 
improving accuracy of individual prognosis and individualizing treatments. 
 
 
 
 

 
 
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Objectives 
Specific Aim 1: We propose a pilot, exploratory, clinical follow-on study using proton magnetic 
resonance spectroscopy (1H-MRS) to examine whether the following measures can be used to 
predict treatment response to memantine in ASD:  
(a) Glx and/or GABA and the Glx/GABA ratio in the anterior cingulate cortex,  
(b) Glx levels in the dorsolateral prefrontal cortex and the cerebellum   
(c) Markers of neuronal integrity, N-acetyl aspartate (NAA), myo-inositol, choline and 
creatine/phosphocreatine levels in the anterior cingulate, dorsolateral prefrontal cortex 
and cerebellum   
Specific Aim 2: To examine whether resting state functional connectivity between the dorsolateral 
prefrontal cortex, anterior cingulate and the cerebellum, holds predictive value for treatment 
response to memantine in ASD. 
Background and Rationale 
Autism Spectrum Disorder (ASD) is a behaviorally defined complex neurodevelopmental 
disorder characterized by early childhood onset of marked deficiencies in social and 
communication skills and presence of restrictive, repetitive behaviors.  Its phenotypic 
heterogeneity makes studying ASD and its treatment a challenging task. Currently, only two FDA 
approved drugs, risperidone and aripiprazole, are available to treat ASD1 that manage irritability 
associated with the disorder. None have shown conclusive benefit for core features of social and 
communication deficits.  
Research indicates that a disrupted balance between excitation (glutamate) and inhibition 
(gamma amino-butyric acid, GABA) is likely to be one of the underlying mechanisms of ASD. This 

 
 
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hypothesis is supported by a number of magnetic resonance spectroscopy studies that report an 
excess of glutamatergic excitation and/or a reduction in GABAergic inhibition observed in different 
brain regions including the limbic system and the anterior cingulate cortex. 2–9 Similar findings have 
also been reported in post mortem studies of the ASD brain 10–12.Thus, it is expected that drugs 
targeting this imbalance could be beneficial. For example, the GABAB agonist, arbaclofen, was 
tested in ASD for this reason and showed promising results but a large trial failed to show 
improvement on the primary endpoints of lethargy and social withdrawal.13  
Memantine, a moderate affinity N-methyl-D-aspartate (NMDA) receptor antagonist, is 
known to improve communication in patients with Alzheimer’s disease and is an FDA approved 
treatment for that disease14. Examination of the efficacy of memantine in treating ASD has also 
been driven by the excitatory (glutamate) - inhibitory (gamma amino-butyric acid, GABA) 
imbalance hypothesis of ASD pathology. Memantine binds to the glutamatergic NMDA receptors 
and attenuates glutamatergic excitation.15 This reduction in excessive glutamatergic activation is 
thought to decrease the “noise” in the neural network system and facilitate learning and 
memory16. A few small studies have shown effectiveness for memantine in the treatment of social 
and communication aspects of ASD spectrum disorder (ASD) as well 1,15–18. However, one large 
clinical trial using memantine in ASD reported a failure to respond 18. These discrepancies in results 
could possibly be due to the heterogeneity in the excitatory-inhibitory imbalance between ASD 
patients thereby causing a variation in response1. In such a scenario, having a biomarker to predict 
an individual’s treatment response would be invaluable.  
MR Spectroscopy is a non-invasive tool used to examine the biochemical profile of brain 
tissue associated with different psychiatric and neurological conditions including ASD 19–21. 

 
 
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Specifically, 1H-MRS studies in ASD demonstrated the excitatory-inhibitory imbalance in various 
cortical and subcortical regions of the brain 2,3,5–8, a finding also reported in post mortem brain 
tissue studies 10–12. The use of this technique to determine if Glutamate/GABA concentrations in 
certain regions of the brain can predict response to treatment with memantine would be an 
innovative breakthrough in providing more effective individualized treatment for patients since 
very little research has been done to explore this possibility.  
A similar approach has been adopted in a recent ongoing study that uses proton MR 
spectroscopy to map changes in glutamate and GABA following use of riluzole in ASD22. Riluzole is 
a drug commonly used in amyotrophic lateral sclerosis (ALS) and is a glutamate antagonist that 
works by blocking presynaptic glutamate release and noncompetitive inhibition of NMDA 
receptors.23 However, riluzole is not clinically used in the treatment of ASD. To our knowledge, 
only one study so far (apart from the ongoing study mentioned) has examined the effects of 
riluzole in ASD, but as an adjunct to risperidone24. The primary outcomes of the Ghaleiha et al. 
study were irritability and repetitive motor behaviors and not core social and communication 
deficits as in the present proposal. The purpose of riluzole in this MR spectroscopy study is a proof 
of principle for its effects on spectroscopy based on its glutamate antagonistic effects. By contrast, 
memantine, which will be studied in the present proposal, has some documented evidence from 
small studies of being beneficial in treating social and communication deficits associated with ASD 
in clinical settings, despite the failure of the larger trial.16–18,25 
Our lab has previously utilized MR spectroscopy techniques to map these different 
compounds in a group of 14 subjects with ASD and identify glutamate and GABA alterations in the 

 
 
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cerebellum, discovering a relationship to cerebrocerebellar connectivity and behavioral outcomes 
in individuals with ASD26. 
Specific Aim 1: We propose a pilot, exploratory, clinical follow-on study using proton magnetic 
resonance spectroscopy (1H-MRS) to examine whether the following measures can be used to 
predict treatment response to memantine in ASD:  
(a) Glx and/or GABA and the Glx/GABA ratio in the anterior cingulate cortex,  
(b) Glx levels in the dorsolateral prefrontal cortex and the cerebellum   
(c) Markers of neuronal integrity, N-acetyl aspartate (NAA), myo-inositol, choline and 
creatine/phosphocreatine levels in the anterior cingulate, dorsolateral prefrontal cortex 
and cerebellum   
Specific Aim 2: To examine whether resting state functional connectivity between the dorsolateral 
prefrontal cortex, anterior cingulate and the cerebellum, holds predictive value for treatment 
response to memantine in ASD. 
The anterior cingulate cortex, dorsolateral prefrontal cortex and the cerebellum have been 
have been chosen as ROIs due to known atypical glutamate and GABAergic profiles in these regions 
associated with ASD2,5,7,9,25–32. The approach of limiting to selected ROIs has been adopted in order 
to ensure patient comfort during the imaging process by reducing the time spent in the scanner. 
Since 
altered 
levels 
for 
markers 
of 
neuronal 
integrity, 
such 
as 
NAA, 
choline, 
creatine/phosphocreatine and myoinositol have also been reported in ASD,3,5,6,9,29,33–36 we will be 
mapping these biochemicals as well in order to explore their effect on treatment response. Resting 
state functional connectivity will also be analyzed for possible association with treatment outcome 
prediction since a previous study from our lab has shown a link between alterations in resting state 

 
 
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cerebrocerebellar connectivity, excitatory-inhibitory ratio in the cerebellum and behavioral 
outcome in ASD using MR spectroscopy26. 
Study Design 
We propose to enroll 25 subjects with ASD for this pilot, exploratory study who are 
interested in starting memantine off-label as a clinical follow on. Glutamate concentration 
variations with MRS have been successfully reported with this sample size by Page et al., 200637. 
Subjects will be recruited from PI’s clinic at the Thompson Center for Autism and 
Neurodevelopmental Disorders at the University of Missouri. The Thompson Center (TC) is one of 
the leading sites for recruitment within the Autism Treatment Network with more than 375 
participants recruited since 2008. Our group and other TC researchers have successfully recruited 
imaging patients from this resource for multiple federally funded research projects. We have 
preliminary behavioral data on 15 ASD patients currently on memantine treatment showing mixed 
responses.  
Subjects will be approached to participate in the study during their clinic visit with a 
healthcare professional. For potential subjects in clinics of healthcare professionals who are not 
co-investigators in the study, a recruitment flyer will be provided with contact information for 
questions and queries and instructions to let their healthcare provider know if they are interested 
in participating in the study. The recruitment flyer will be provided to healthcare professionals at 
the Thompson Center who will distribute it to potential subjects upon request. Following 
indication of interest, the researcher from the study team will go over the consent (/assent, as 
applicable) form with the subject in detail. Subjects will be informed that if they agree to 
participate in the study, they will be asked to hold off on starting the drug, memantine, till the 

 
 
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imaging session of the study is completed. Subjects will also be informed that they can choose not 
to be a part of study and start taking memantine immediately if they so wish. The subject/guardian 
will be able to ask questions throughout the process and the researcher will pause multiple times 
throughout the consent process to ensure understanding. The study physician, who is authorized 
by the IRB to obtain consent, will obtain signatures from the subjects and their parents/caregivers 
(as applicable). Following informed consent, a researcher from the study team will go over the 
MRI screening form with the subject and ensure compliance with MRI safety requirements. If the 
MRI screening requirements are not met, the subject will be taken off the study and the study 
physician will discuss appropriate alternatives with the patient.  
Once informed consent is obtained, trained researchers will obtain information regarding 
autism diagnosis, medication information, IQ and medical history from the subject’s medical 
records. For subjects who do not have an Autism Diagnostic Interview - Revised (ADI-R) in their 
medical record, the study physician will administer the ADI-R over the phone and document the 
information. After the required information is obtained and the subject is found to meet the 
inclusion and exclusion criteria for the study, the subject will attend 3 sessions (with an optional 
4th session for mock scanning, per subject request).  
Session 1 will involve collection of basic demographic information and behavioral 
assessments using the Aberrant Behavior Checklist (ABC), Social Responsiveness Scale (SRS), 
Clinical Global Impressions – Severity (CGI-S) Scale, General Social Outcomes Measure (GSOM) and 
the Test of Language Competence (TLC). Photographs and videos of the subjects will be recorded 
during the GSOM test for scoring by an independent rater. A Wechsler Abbreviated Scale of 
Intelligence (WASI) test will be administered to subjects who do not have IQ information on file. 

 
 
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Female subjects of childbearing age will undertake a urine pregnancy test. If pregnancy is 
indicated, the subject will no longer be eligible to participate in the study and will be withdrawn 
from the study. This session will last no longer than 2 hours.  
Session 2 will be either the optional mock scanning session (if requested for by the subject) 
or the MR imaging session. For the mock scanning session, the scanning environment of the real 
scanner will be mimicked. For the MR imaging session (session 2 for subjects who do not require 
a mock scanning session and session 3 for subjects who do), the researcher will go over the MRI 
screening form with the participant in detail to ensure compliance. The imaging session will last 
no longer than 90 minutes during which the subject will be asked to lie still in an MRI scanner. For 
subjects with potential behavioral concerns that are manageable by support staff or family, 
support staff or family will be present during the imaging session to ensure safety. Subjects with 
potential behavioral concerns that cannot be managed by support staff or family will not be 
imaged and will no longer be eligible to be a part of the study. The study physician will discuss 
appropriate alternatives with the subjects.  
Post imaging, subjects will be started on memantine. Memantine is marketed in three 
forms: Namenda (pill), Namenda XR (capsule) and the liquid formulation of Namenda. Depending 
on subject preference and insurance approval, the subjects will be started on one of these three 
forms of memantine. Namenda (pill) will be started at doses of 5 m/day and titrated up to 20 
mg/day based on patient tolerance and response.  Namenda XR (capsule) will be started at doses 
of 7 mg/day and titrated up to 28 mg/day based on patient tolerance and response. The liquid 
formulation of Namenda will be started at a doses of 5 mg/day and titrated up to 20 mg/day based 
on patient tolerance and response.  

 
 
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After week 12 of treatment with memantine, the subjects will have a follow-up behavioral 
assessment session (Session 3 for subjects who do not require a mock scanning session and 
Session 4 for those who do) using Clinical Global Impressions - Improvement scale, Social 
Responsiveness Scale, Aberrant Behavior Checklist, the General Social Outcomes Measure (GSOM) 
and the Test of Language Competence (TLC).  The GSOM test will involve recording of photographs 
and videos for scoring by an independent rater. This session will last no longer than 2 hours.  
Inclusion and Exclusion Criteria: 
Potential participants will be asked to take part in this study because he/she: 
1) has autism spectrum disorder  
2) is starting memantine off label for managing their autism symptoms 
3) is deemed safe to enter the MR environment using the attached screening form, and  
4) is capable of lying still for approximately 1.5 hour. 
Subjects would be excluded if:  
1) they have certain types of metallic implants, risk of exposure to metallic foreign bodies, 
pacemakers, magnetically sensitive implants that cannot be removed or are not securely 
attached,  
2) pregnancy, or  
3) claustrophobia.  
All of these issues will be addressed with the MR screening form. Additional exclusion criteria 
include:  
4) memantine intolerance  
5) known hypersensitivity to memantine hydrochloride or  

 
 
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6) inability to lie still for approximately 90 minutes.  
7) subjects with potential behavioral concerns that cannot be managed by support staff or 
family.  
Behavioral Assessments: 
The Clinical Global Impressions – Severity (CGI-S) scale is a clinician rated scale to assess 
severity of symptoms and is used fairly commonly in ASD research.13,17,18  
The Social Responsiveness Scale (SRS) is a well validated 65 item questionnaire that 
specifically evaluates social deficits associated with ASD38,39. Each question on the SRS is rated on 
a scale of 0-3 based on behavior in the past 6 months and is designed to provide information on 
the degree of social deficits present. The SRS is particularly well suited to this project since we 
intend to monitor improvements in social and communication deficits following the use of 
memantine. A number of other studies in ASD have used SRS to track behavioral outcomes 
following pharmacological intervention1,3,8,9,13,26,40–42.  
The General Social Outcomes Measure (GSOM) is a relatively new assessment tool that 
also assesses social deficits in ASD43. The GSOM is experimenter rated and evaluates subjects on 
five core domains of conversational reciprocity, facial expression recognition, social problem 
solving, affect demonstration and emotional perception. Photographs will be taken during the 
mimicking of facial expressions test of the GSOM. These photographs will be used for scoring by 
an independent rater, not present at the time of testing. Photographs will be captured and stored 
electronically on a firewall and password protected network drive accessible only by authorized 
study personnel in Dr. Beversdorf’s lab. All photographs will be used just for the study and will be 
deleted after responses are scored. The higher the score on the GSOM scale, the better level of 

 
 
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social responsiveness in the subject. The SRS and the GSOM, used in combination, would provide 
a more detailed picture of the subject’s social and communication skills by including both the 
subject’s or caretaker’s and the trained researcher’s observations. The GSOM has successfully 
been used in our lab previously for the evaluation of treatment response to propranolol.44  
The Aberrant Behavior Checklist (ABC) was developed specifically with the intention of 
assessing effectiveness of therapeutic interventions.45 The ABC assesses subjects on five subscales 
of irritability, lethargy/social withdrawal, stereotypic behavior, hyperactivity or noncompliance 
and inappropriate speech and has been used widely in ASD research to assess effectiveness of 
treatment.13,17,18  
The WASI test involves defining and relating certain words and solving puzzles to obtain a 
measure of intelligence.  
The Test of Language Competence (TLC) is a test that assesses delays in language skills and 
the use of higher level language skills. 
If not available from medical records, ASD subject diagnosis will be confirmed by the parent 
completing an Autism Diagnostic Inventory-Revised (ADI-R) with Dr. Beversdorf. If not available 
from medical records the Social Responsiveness Scale and Aberrant Behavior Checklist (ABC) will 
be given to the parent/guardian in person/over the phone/or through mail/email to assess the 
subject’s social skills and behaviors. 
1H-MRS acquisition and analysis:  
 
Following behavioral assessment, at the second session, subjects will undergo an MRI 
assessment, including structural MRI and 1H-MRS. This assessment will be performed on a 

 
 
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research dedicated, Siemens 3-Tesla TIM Trio MRI scanner located in the Brain Imaging Center at 
the MU. Participants will be asked not to consume any forms of caffeine or alcohol 8 hours before 
the scanning to eliminate possible effects from caffeine/alcohol on GABA metabolites46. Subjects 
will be given detailed instructions on how to successfully and safely complete these scans and will 
be monitored by study staff at all times.  
 
High-resolution T1-weighted structural images are acquired using the three-dimensional 
multiplanar rapidly acquired gradient echo (MP-RAGE) pulse sequence: repetition time (TR), 
2500ms; echo time (TE), 438ms; flip angle, 8°, 256x256 voxel matrix; field of view (FOV), 256 mm; 
176 axial slices; thickness, 1 mm. This is collected to quantify the brain tissue composition with 
spectroscopic voxel and exclude any pathology.  
For specific aim 1, based on anatomical landmarks, we prescribe a single voxel 
spectroscopy (SVS, 2×2×2cm3) in our region of interest, the anterior cingulate cortex. The size of 
the voxel and number of averages acquired will be optimized for better signal-to-noise ratio while 
keeping ensuring scanning time does not get prolonged.  GABA and a complex consisted of 
glutamate and its precursor glutamine (labeled as Glx) are collected using MEshcher-Garwood-
edited Point RESolved Spectroscopy (MEGA-PRESS, TE=68ms, TR=2000ms). To avoid lipid artifact 
from the skull, we prescribe outer voxel suppression saturation bands around the SVS. We perform 
automated, followed by manual, shimming to achieve an optimal full-width at half maximum 
of<20Hz of the water signal from the entire excitation volume54. We will also acquire internal 
reference water signal by using non–water suppressed MRS imaging to calculate absolute 
concentrations of our neurochemicals of interest. A recent study from our laboratory has 



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Fig. 1 A. 1H-MRS spectrum using MEGA-PRESS. The LCModel output 
consisted in the acquired 1H-MRS spectrum (black line) and estimated 
spectrum (red). B. 1H-MRS spectrum using TE-averaged semi-LASER. Glx-
glutamate-glutamine, NAA-Nacetylaspartate, Cr-creatine, Cho-choline; ppm, parts per 
million.


 
 
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Following the imaging session, subjects will be started on memantine, starting at 5 mg/day 
doses to be titrated up 20 mg/day based on response and tolerability, for a period of 12 weeks as 
part of clinical care.  
Follow-up assessments:  
Subjects will be assessed after week 12 of memantine administration using the same 
clinical testing battery used prior to drug administration. We will also use an additional scale to 
determine improvement in symptoms: Clinical Global Impression – Improvement (CGI-I) scale. The 
CGI-I scale assesses the patient’s improvement on a scale of 1-7 where 1 represents very much 
improved and 7 represents very much worse when compared to baseline (before initiation of 
memantine). The reference for baseline severity will be derived from the CGI-S score obtained 
before initiation of memantine. Subjects who score 1-3 on the CGI-I will be considered responders 
while those who score 4-7 will be considered non-responders, a technique commonly used to 
categorize responders vs. non-responders in treatment response monitoring studies 40,47,48. 
Subjects will be assessed for improvements on the SRS, GSOM, TLC and each of the 5 subscales of 
the ABC described earlier. Additionally, research staff will review ASD subject medical records to 
gain a better understanding of clinical benefit from drug administration. This will include physician 
clinical notes and any standardized assessments of clinical/behavioral improvement. 
Information produced by this study will be stored in a locked cabinet or secure server in 
the PI’s offices in Galena Hall. Each subject will be assigned an anonymous study specific code. The 
code key will be kept on a separate secure server. Information contained in records will not be 
given to anyone not associated with the study without written consent, except as required by law.  
Analyses:  

 
 
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Using data from the MR spectroscopy study performed at baseline, the means (SD) for 
each of the different compounds – GABA (in the anterior cingulate cortex only), Glx, NAA, choline, 
myo-inositol, creatine and phosphocreatine - measured from the different ROIs mentioned above 
will be analyzed for differences in responders vs. non-responders to memantine treatment. The 
ratio of Glx/GABA from the anterior cingulate will also be included in all analyses to explore effects 
of the excitatory-inhibitory ratio of the region on treatment response.   Means (SD) of the different 
compounds and Glx/GABA ratio will also be correlated to CGI-I and changes in SRS scores and 
GSOM and ABC measures for each ROI to explore additional specific differences using Spearman’s 
correlation coefficients. A significance level of 0.05 will be used. Since demographic factors such 
as age and gender are known to influence the results of MR spectroscopy studies 49, demographic 
factors will be included in the analysis to account for the associated changes in metabolite 
concentrations.  
For resting state functional connectivity, we will extract time series from the regions of 
interest and correlation analyses will be carried out. The strength of functional connectivity 
between the regions will then be correlated to changes on scores in the battery of clinical tests to 
determine whether resting state functional connectivity might have a role in treatment response 
to memantine. 
 
Hypothesis and Expected Results: We hypothesize that the best responders will have higher 
concentrations of glutamate/glutamine and reduced levels of GABA in the anterior cingulate 
cortex at baseline. Consistent with previous studies, we also expect to see increased 
glutamate/glutamine levels in the dorsolateral prefrontal cortex and cerebellum38,39 and increased 

 
 
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resting state functional connectivity in cortical networks51 that will be related to treatment 
response. Data from this pilot, exploratory study will be used to derive power for a larger, follow 
up study. 
 
Potential Problems & Alternative Strategies: Neurochemicals depend on the brain tissue sampled 
in the spectroscopic voxels. We will use brain tissue-corrected concentrations. The phases of 
menstrual cycle for female subjects and smoking status will be included in our model due to their 
known effects on GABA concentrations46,52,53. If recruitment falls behind, we will expand our 
recruitment to other institutions in Missouri, with whom the Thompson Center already has 
relationships for research purposes.  
 
Timeline: Subjects will be recruited through the Thompson Center for Autism and 
Neurodevelopmental Disorders and would involve patients who are interested in starting 
memantine off-label as a clinical follow-on. Subjects will be informed about the study while in clinic 
by the study physician and the reseracher will go over the consent(/assent as applicable) with the 
subject (and guardian, as applicable) in detail. The study physician will obtain signatures on the 
consent and/or assent as applicable. Following informed consent to participate in the study, 
behavioral assessments and imaging sessions will be scheduled at the earliest date possible (within 
30 days of each other). Subject recruitment will extend over a period of 18-20 months. Data 
collection will commence as soon as subjects are enrolled into the study. Since the follow-up 
assessments extend to 12 weeks post the start of the drug, data collection will continue into the 
latter half of the second year of the project as well. Each subject will be in the study for 

 
 
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approximately 20 weeks. Imaging and behavioral assessment data will be analyzed starting in the 
beginning of the first year and will continue till the end of the project timeline. 
 
Funding: Dr. David Beversdorf has been awarded a University of Missouri Research Board grant 
to cover expenses for this study. 
 

 
 
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