Wink et al.

Molecular Autism (2016) 7:26

DOI 10.1186/s13229-016-0088-6

RESEARCH Open Access

A randomized placebo-controlled pilot

study of N-acetylcysteine in youth with
autism spectrum disorder
Logan K. Wink1, Ryan Adams1, Zemin Wang2, James E. Klaunig2, Martin H. Plawecki3, David J. Posey4,
Christopher J. McDougle5 and Craig A. Erickson1*

Abstract
Background: Social impairment is a defining feature of autism spectrum disorder (ASD) with no demonstrated
effective pharmacologic treatments. The goal of this study was to evaluate efficacy, safety, and tolerability of
oral N-acetylcysteine (NAC), an antioxidant whose function overlaps with proposed mechanisms of ASD
pathophysiology, targeting core social impairment in youth with ASD.
Methods: This study was a 12-week randomized, double-blind, placebo-controlled trial of oral NAC in youth
with ASD. Study participants were medically healthy youth age 4 to 12 years with ASD, weighing ≥15 kg, and judged
to be moderately ill based on the Clinical Global Impressions Severity scale. The participants were randomized via
computer to active drug or placebo in a 1:1 ratio, with the target dose of NAC being 60 mg/kg/day in three divided
doses. The primary outcome measure of efficacy was the Clinical Global Impressions Improvement (CGI-I) scale
anchored to core social impairment. To investigate the impact of NAC on oxidative stress markers in peripheral
blood, venous blood samples were collected at screen and week 12.
Results: Thirty-one patients were enrolled (NAC = 16, placebo = 15). Three participants were lost to follow-up,
and three left the trial due to adverse effects. The average daily dose of NAC at week 12 was 56.2 mg/kg (SD = 9.7) with
dose ranging from 33.6 to 64.3 mg/kg. The frequency of adverse events was so low that comparisons between
groups could not be conducted. At week 12, there was no statistically significant difference between the NAC
and placebo groups on the CGI-I (p > 0.69) but the glutathione (GSH) level in blood was significantly higher in
the NAC group (p < 0.05). The oxidative glutathione disulfide (GSSG) level increased in the NAC group, however
only at a trend level of significance (p = 0.09). There was no significant difference between the NAC and placebo
groups in the GSH/GSSG ratio, DNA strand break and oxidative damage, and blood homocysteine levels at week
12 (ps > 0.16).
Conclusions: The results of this trial indicate that NAC treatment was well tolerated, had the expected effect of
boosting GSH production, but had no significant impact on social impairment in youth with ASD.
Trial registration: Clinicaltrails.gov NCT00453180
Keywords: Autism, Autism spectrum disorder, Social impairment, N-acetylcysteine, Oxidative stress

* Correspondence: [email protected]
1
Cincinnati Children’s Hospital Medical Center, University of Cincinnati
College of Medicine, 3333 Burnet Avenue MLC 4002, Cincinnati, OH 45229,
USA
Full list of author information is available at the end of the article

© 2016 Wink et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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Wink et al. Molecular Autism (2016) 7:26 Page 2 of 9

Background conjunction with risperidone for treatment of irritability in

Social and communication impairments are the defining youth with ASD also showed a significant reduction in
features and key predictors of long-term outcome in aut- ABC-I subscale scores in the NAC-treated groups [14, 15].
ism spectrum disorder (ASD) [1, 2]. To date, no medica- Furthermore, case studies have demonstrated improvement
tion has demonstrated significant impact on these core of core ASD features including social communication and
deficits in controlled trials. In 2014, the United States repetitive behaviors with NAC treatment; however, this ef-
Center for Disease Control reported that one in 68 chil- fect has not been confirmed in controlled trials [16, 17].
dren in the USA is diagnosed with ASD, highlighting the To date, no study of NAC in ASD has evaluated the
critical need for targeted treatment development in this impact of NAC on markers of oxidative stress in periph-
disorder [3]. Growing neurobiologic understanding of eral blood. Individuals with ASD are believed to have de-
ASD has identified glutamatergic neurotransmission and creased total GSH levels and elevated levels of oxidative
metabolic pathways impacting oxidative stress levels as glutathione disulfide (GSSG) [4]. These abnormalities
potential targets of drug development in this complex lead to a reduction in the ratio of active GSH to inactive
disorder [4]. Glutamatergic dysfunction appears to play a GSSH resulting in increased intracellular oxidative stress
significant role in ASD pathology, as studies have identi- which may impact on individual’s capacity to maintain
fied abnormal peripheral glutamate levels, aberrant glu- cellular methylation and increase vulnerability to oxida-
tamate expression in the postmortem brain, and genetic tive damage [18]. This change has the potential to de-
abnormalities in glutamate signaling genes in individuals crease an individual’s ability to resist infection, resolve
with ASD [5]. Excessive oxidative stress also has been inflammation, and respond to environmental exposures.
identified as playing a potential role in ASD pathophysi- Correction of these abnormalities with NAC treatment
ology. Increased peripheral oxidative biomarkers, evidence could have substantial impact on the health of individuals
of oxidative stress in the postmortem brain, and abnor- with ASD.
malities in genes encoding for antioxidant enzymes have The goal of this randomized, double-blind, placebo-
been reported in individuals with ASD [4, 6, 7]. controlled pilot study of NAC in youth with ASD was to
N-acetylcysteine (NAC) is a unique antioxidant whose evaluate the efficacy of oral NAC targeting core social
function overlaps with both the glutamatergic and oxi- impairment of ASD and evaluate the safety and tolerability
dative stress hypotheses proposed to contribute to the of NAC in this population. Additionally, this project incor-
pathophysiology of ASD. NAC is the N-acetyl derivative of porated measures of oxidative stress that may be impacted
L-cysteine used for decades in treatment of acetaminophen by NAC treatment, including measures of pre- and post-
overdose, as a mucolytic in chronic obstructive pulmonary treatment peripheral whole blood levels of GSH and
disease, and as a renal protectant in contrast-induced ne- GSSG. We hypothesized that treatment with NAC in this
phropathy [8]. NAC is rapidly absorbed via oral dosing, population would result in improvement in core social
though total oral bioavailability of NAC is quite low (~9 %) impairment of ASD as measured by the Clinical Global
[9]. NAC crosses the blood-brain barrier (BBB), though its Impression Improvement (CGI-I) scale [19], and that
efficiency may depend upon dose, administration, and for- NAC treatment would result in increased GSH and
mulation [10]. In the brain, NAC is oxidized from L-cyst- GSSH levels.
eine to cystine which is ultimately involved in regulation of
extracellular glutamate levels [11]. NAC is cell membrane Methods
permeable and is reduced to cysteine intracellularly, which Study design
is a key component of the antioxidant glutathione (GSH) This study was a 12-week randomized, double-blind,
[10]. NAC’s involvement in both extracellular glutamate placebo-controlled pilot trial of oral NAC in youth with
modulation and intracellular restoration of antioxidant ASD. The study was conducted at Indiana University
GSH levels, coupled to its long history of human safety School of Medicine (IUSM) between December 2006
data, make NAC an intriguing substance in ASD-targeted and November 2009. The study was approved by the
treatment development. IUSM institutional review board. Guardians of all partic-
Placebo-controlled studies of NAC in ASD have previ- ipants provided written informed consent prior to study
ously focused on treatment of ASD-associated irritability enrollment. Assent was obtained from enrolled youth
marked by physical aggression, self-injurious behavior, when possible.
and severe tantrums. A pilot placebo-controlled study by Study participants were youth ages 4 to 12 years with
Hardan et al. (2012) demonstrated a significant reduction a diagnosis of autistic disorder, Asperger’s disorder, or
in irritability symptoms as measured by the Aberrant Be- pervasive developmental disorder not otherwise specified
havior Checklist Irritability subscale [12] (ABC-I) in 29 (PDD NOS). Subjects with known genetic syndromes asso-
youth aged 3.2–10.7 years with ASD [13]. Additionally, two ciated with autism were excluded (for example, fragile X
small double-blind, placebo-controlled studies of NAC in syndrome, tuberous sclerosis). Participants were diagnosed
Wink et al. Molecular Autism (2016) 7:26 Page 3 of 9

via clinical interview completed by study physician with ex- for adverse effects was completed during each interaction.
pertise in ASD (DJP, CJM, CAE), based on the Diagnostic Vital signs including height, weight, blood pressure, and
and Statistical Manual of Mental Disorders, Fourth Edition heart rate were measured at every in-person study visit.
(DSM-IV) [20] diagnostic criteria, and corroborated by ad- Safety labs including complete blood cell count (CBC) and
ministration of the Autism Diagnostic Interview-Revised comprehensive metabolic panel (CMP) were collected at
[21] (ADI-R). Participants diagnosed with PDD NOS dem- screen and week 12.
onstrated pervasive impairment in social interaction and/or The primary outcome measure of efficacy was the
communication skills as well as stereotyped behaviors and CGI-I scale anchored to study physician (DJP, CJM,
restricted interests, but did not meet full criteria for diagno- CAE) assessment of core social impairment considering
sis of autistic disorder or Asperger’s disorder. All partici- the individuals’ overall level of cognitive, adaptive, and
pants weighed ≥15 kg and were medically healthy based on social functioning. The CGI-I is a clinician-rated global
physical exam and review of medical history completed by assessment of symptom change rated on a scale from 1
study physicians. Participants were judged by study to 7 (1 = very much improved; 2 = much improved; 3 =
physician as being at least “moderately ill” as measured minimally improved; 4 = no change; 5 = minimally
by the Clinical Global Impression Severity [19] (CGI-S) worse; 6 = much worse; 7 = very much worse). In this
scale rating at baseline. The CGI-S is a clinician-rated study, a positive response to NAC treatment was defined
global assessment of symptom severity scale ranging as scoring a 1 “very much improved “or 2 “much im-
from 1 to 7 (1 = normal, not at all ill; 2 = borderline ill; proved” on the CGI-I. Study physicians completed annual
3 = mildly ill; 4 = moderately ill; 5 = markedly ill; 6 = CGI training to ensure internal consistency with this out-
severely ill; 7 = among the most extremely ill patients). come measure. Secondary outcome measures included the
Concomitant medications were permitted if doses were CGI-S, ABC, Social Responsiveness Scale [23] (SRS) raw
stable for at least 60 days prior to study initiation and score, and Vineland Adaptive Behavior Scales 2nd Edition
remained stable throughout the study. Participants taking [24] (VABS-II) survey edition raw score. The SRS is a
known glutamatergic modulators such as dextrome- standardized, caregiver reported measure of the core
thorphan, D-cycloserine, amantadine, memantine, lamo- symptoms of ASD [23]. The ABC is a parent questionnaire
trigine, or riluzole were excluded. Patients taking daily measuring five behavioral domains including irritability, so-
acetaminophen, daily nonsteroidal anti-inflammatory cial withdrawal, stereotypy, hyperactivity, and inappropriate
medications, daily antioxidant medications such as speech [12]. The VABS-II is a semi-structured caregiver
high-dose vitamin supplements, and medications with interview which provides a measure of an individual’s over-
known drug-interactions (i.e., carbamazepine) within all adaptive functioning [24]. All measures have been used
30 days of baseline were also excluded. Subjects were extensively in ASD research [25–27]. The CGI-I was com-
required to be able to swallow capsules. Potential subjects pleted at weeks 4, 8, and 12. The ABC and SRS were com-
with profound cognitive impairment (mental functioning pleted at baseline and weeks 4, 8, and 12. The CGI-S and
below 18 months of age) as measured by the Leiter Inter- VABS-II were completed only at baseline and week 12.
national Test of Intelligence-Revised [22] were excluded. Early morning, fasting, venous blood samples for
Following screening and baseline measures, partici- measurement of oxidative stress biomarkers GSH, GSSH,
pants were randomized 1:1 via computer—by the inves- total homocysteine, strand breakage, and oxidative DNA
tigational pharmacy. All participants, guardians, and damage were collected in EDTA containing Vacutainers at
investigators remained blind to study assignment. NAC screen and week 12.
and matching placebo were prepared by CustomMed
Apothecary. Participants randomized to active drug were Measurement of reduced and oxidized glutathione
administered capsules containing 300 or 600 mg of NAC, Concentrations of reduced and oxidized glutathione
with a target dose of 60 mg/kg/day in three divided doses (GSH and GSSG) in whole blood samples were analyzed
and a maximum dose of 4200 mg/day. Patients weighing simultaneously using HPLC-electrochemical detection as
15 to 30 kg began treatment with a starting dose of described previously [28]. Briefly, 1 ml of blood sample
300 mg/day; those weighing above 30 kg started with was added to 0.5 ml precipitating solution containing
600 mg/day. Patients were required to tolerate a minimum 0.2 M perchloric acid and 100 μM EDTA, and vortexed
dose of 300 mg/day to continue in the trial. Dose was ti- briefly. After incubation at room temperature for 45 min,
trated to the target dose over the first 3 weeks of study samples were centrifuged at 10,000g for 3 minutes. The
participation. Dose then remained stable for all subjects in resulting supernatants were filtered and injected into
the last 9 weeks of the study, although reductions due to high-performance liquid chromatography (HPLC; Waters
adverse effects were permitted at any time. Subjects were 2690) for analysis immediately or frozen in liquid nitrogen
evaluated in person at screen and baseline, by phone at and stored at −80 °C before analysis. The separation of
week 2, and in person at weeks 4, 8, and 12. Assessment analytes was achieved on a reverse phase Symmetry C-18
Wink et al. Molecular Autism (2016) 7:26 Page 4 of 9

column (5 μm, 150 × 4.6 mm; Waters). GSH and GSSG and percentages were calculated to describe the NAC and
were detected using an ESA Coulochem II (ESA Inc. placebo groups. Baseline demographic differences between
Chelmsford, MA) equipped with a guard cell. The potential groups were tested using chi-square tests (categorical data)
settings for the detector were E1 450 mV, E2 900 mV, and and independent sample t test (continuous data) using a
guard cell 1000 mV. The amount of GSH and GSSG was two-tailed p value of 0.05 for the alpha.
calculated from the respective calibration curves, and the For the CGI-I primary outcome measure, differences
ratio of GSH/GSSG for each sample was also calculated. between the two treatment groups were tested at weeks
4, 8, 12 using chi-square tests. For the CGI-S secondary
Measurement of blood homocysteine outcome measure, a chi-square test was employed to
Total homocysteine in whole blood sample was deter- examine change from baseline to week 12 by creating
mined using an HPLC method as described previously two CGI-S categories: (1) those subjects whose severity
with minor modifications [29, 30]. Briefly, 1 ml EDTA score decreased (i.e., clinically improved) by at least one
blood samples were lysed by vigorously shaking for at point from baseline to week 12 and (2) those subjects
least 10 s after adding 10 μl Nonidet P40 (pure) and whose scores remained the same during the same
10 μl citric acid monohydrate (2.5 M). The lysates were period. For the ABC, SRS, VABS-II, and oxidative stress
then centrifuged at 10,000g for 3 minutes at room biomarkers, differences between the groups for change
temperature. Following reduction of the sample with tri- in each outcome measure over the course of the study
n-butylphosphine, precipitation of protein with perchloric were tested using multi-level modeling (i.e., we exam-
acid and derivatization with ammonium 7-fluorobenzo-2- ined if there were differences between the two groups in
oxa-1,3-diazole-4-sulfonate, the samples were then analyzed terms of change in the outcome scores over the course
using reversed-phase high-performance liquid chromatog- of the study). For each outcome, time was modeled at
raphy (Waters Alliance 2695 separation module) followed level-one as a random effect with treatment group en-
by fluorescence detection (Waters 474 fluorescence tered as a level-two fixed effect. The cross-level inter-
detector) (Waters Corporation, Milford, MA). action between group and time tested the differences
between the two groups in the change over time using
maximum likelihood estimation with robust standard er-
Direct and oxidative DNA damage: comet assay rors with Mplus 5.21 [34] (baseline, week 4, week 8, and
Immediately after blood collection, whole blood (10 μl) week 12 data was used for the ABC and SRS; baseline
was mixed with 0.5 ml RPMI 1640 containing 10 % FBS, and week 12 data was used for the VABS-II and oxidative
10 % DMSO, 1 mM deferoxamine, step-frozen and stress biomarkers). Since there is no method to directly
stored at −80 °C until analysis. The comet assay was per- calculate the effect size of the between-subject effects on
formed as described previously [31, 32]. Briefly, 6 μl of the within-subject effects for multilevel modeling, we cal-
blood was mixed with 70 μl 1 % low-melting-point agarose culated an effect size of the difference between the groups
and applied onto comet slides (Trevigen Inc, Gaithersburg, on the mean differences between baseline and week 12 for
MD). Cells were lysed, placed in alkali buffer, and then elec- the groups. Specifically, a Cohen’s d was calculated by sub-
trophoresed. Slides were stained with ethidium bromide, tracting the mean score at week 12 from the score at base-
and 100 randomly selected nuclei/sample were evaluated line for each group, and a difference score was calculated
(Komet 5.5; Kinetic Imaging Ltd., Liverpool, UK). For the by subtracting the change scores between the groups. This
assessment of oxidative DNA damage, a modified alkaline difference score was then divided by the pooled standard
comet assay was performed that included enzymatic deviation of the change scores. Independent sample t tests
digestion with formamidopyrimidine-DNA glycosylase were used to compare the levels of oxidative markers be-
(fpg) prior to electrophoresis. DNA damage was expressed tween NAC and placebo groups at baseline and week 12.
as Comet (Olive) tail moment [(tail mean − head mean) × For all types of tests, a one-tailed p value of 0.05 was used
tail%DNA/100]. as the alpha. A one-tailed test was used based on the a
priori hypothesis that the NAC group would have greater
change in clinical ratings and oxidative stress markers over
Statistical analysis the course of the study than the placebo group. We did
Our sample size (goal 32 participants) was chosen based not correct for multiple comparisons given the pilot na-
on recommendation for sampling in pilot studies where ture of the work.
little is known about treatment response rates [33]. Adverse event data was compiled to describe the NAC
Baseline demographic data including age, sex, race, level and placebo groups. Separate analyses were conducted
of intellectual quotient (IQ), ASD diagnostic sub-type, for the vital sign measurements, CMP, and CBC employ-
concomitant medications, and clinical ratings (CGI-S, ing the same method as the treatment analyses, though
ABC, SRS, and VABS-II scores) was compiled, and means only two time points, screen and week 12, were evaluated.
Wink et al. Molecular Autism (2016) 7:26 Page 5 of 9

Results Asperger’s disorder (33.3 %). On the CGI-S scale, the

Thirty-one participants initially enrolled in the trial; majority of the participants in both groups (>85 %)
however, six participants did not complete the study. were rated as either 5 “markedly” or 6 “severely ill”. There
Three participants were lost to follow-up after week 4 was no statistically significant baseline difference between
(two NAC and one placebo), and three withdrew due to groups for any clinical ratings (ABC, SRS, VABS-II), with
irritability (NAC), diarrhea and encopresis (placebo), and the exception of a trend toward significantly higher score
defiant and self-injurious behavior (placebo), respect- on the ABC stereotypy subscale in the placebo group
ively. At baseline, data for all 31 enrolled subjects was (t value = 1.85, 0.07; NAC M = 4.75, SD = 3.3; placebo
employed in the analysis. Post-baseline, only data for the M = 7.00, SD = 4.9). Additionally, there was no signifi-
25 completers was analyzed, as there is no method for cant difference in type or number of baseline con-
imputing data for multi-level models. comitant medications between groups (all ps > 0.11).
At baseline, participants ranged in age from 4 to The average daily dose of NAC at week 12 was
12 years. The average age was 7.6 years (SD = 2.5, n = 16) 56.2 mg/kg (SD = 9.7) with dose ranging from 33.6 to
for the NAC group and 8.2 years (SD = 2.9, n = 15) for 64.3 mg/kg. Overall, NAC was well tolerated by study
the placebo group (Table 1). The majority of the partici- participants. The majority of adverse events occurred
pants were male (>75 %), white (>90 %), and had an IQ only once, and the frequency of adverse events was so
above 85. For the NAC group, the ASD diagnostic low that comparisons between groups could not be con-
sub-types were primarily autistic disorder (43.8 %) and ducted (Table 2). Overall, upper respiratory symptoms
PDD-NOS (43.8 %) while the placebo group included were the most commonly reported event for both groups
primarily those with autistic disorder (46.7 %) and (NAC n = 10, 62.5 %; placebo n = 6, 40.0 %). The next

Table 1 Demographic data and baseline characteristics

Groups Test of differences
NAC Placebo
Characteristics n/total % n/total % Chi-squarea
Gender—male 12/16 75.0 12/15 80.0 0.11
Race—White 16/16 100.0 14/15 93.3 1.01
Diagnosis
Autistic disorder 7/16 43.8 7/15 46.7 0.03
Asperger’s disorder 2/16 12.5 5/15 33.3 1.92
PDD-NOS 7/15 43.8 3/15 20.0 1.98
CGI-S
Marked (5) 6/16 37.5 9/15 60.0 1.57
Severe (6) 9/16 56.3 6/15 40.0 0.82
Extreme (7) 1/16 6.3 0/15 0.0 0.97
Concomitant medication (y/n) 6/16 37.5 10/15 66.7 2.64
Medication types
Psychostimulants 3/16 18.7 3/15 20.0 0.01
Alpha 2 agonists 1/16 6.2 2/15 13.3 0.44
Antipsychotics 4/15 25.0 5/15 33.3 0.26
Sleep aids 6/16 37.5 2/15 13.3 2.36
Antidepressants 1/16 6.2 3/15 20.0 1.30
Antiepileptic medication 2/16 12.5 0/15 0.0 2.01
Mean SD Mean SD t testb
Age 7.63 2.5 8.20 2.9 0.59
Full scale IQ 86.27 21.8 87.43 11.7 0.18
Number of concomitant medications per participant 1.20 1.8 0.94 1.0 0.51
a
Two-tailed significance +p < .10; *p < .05. No p values for these chi-squares reached significance at .05 and ranged from .11 to .87
b
Two-tailed significance +p < .10; *p < .05 and ps ranged from .56 to .94
NAC N-acetylcysteine, PDD-NOS pervasive developmental disorder not otherwise specified, CGI-S clinical global impression severity scale
Wink et al. Molecular Autism (2016) 7:26 Page 6 of 9

Table 2 Frequency of adverse effects Table 3 Clinical Global Impression-Improvement scale score,
NAC (n = 16) Placebo (n = 15) NAC vs. placebo
Adverse effects Mild Mod Severe Mild Mod Severe Groups Test of
differences
URI 9 1 0 5 1 0 NAC Placebo

Headache 3 0 0 2 1 0 n/total % n/total % Chi-squarea

Stomachache 2 0 0 2 0 0 Week 4 0.28

Fever 2 1 0 0 0 0 Response 4/15 26.7 5/14 35.7

Irritability 2 0 0 1 0 0 No response 11/15 73.3 9/14 64.3

Insomnia 2 0 0 0 0 0 Week 8 0.07

Otitis media 1 1 0 1 0 0 Response 5/13 38.5 4/12 33.3

Increased enuresis 1 0 0 1 0 0 No response 8/13 61.5 8/12 66.7

Interrupted sleep 1 0 0 1 0 0 Week 12 0.15

Localized rash 1 0 0 1 0 0 Response 6/12 50.0 5/11 45.5

Nausea 1 0 0 1 0 0 No response 6/12 50.0 6/11 54.5

a
One-tailed significance +p < .10; *p < .05. No p values for these chi-squares
Stereotypy 0 0 0 2 0 0 reached significance at .05 and ranged from .60 to .79
The table only lists adverse effects occurring ≥2 times across the samples Response = very much improved (1) or much improved (2); no response =
Placebo group reported one moderate case of the following adverse effects: minimally improved (3), no change (4), minimally worse (5); no participants
defiant behavior, hives, itchy skin, and threats to hurt self scored much worse (6) or very much worse (7)
Placebo group reported one mild case of the following adverse effects: NAC N-acetylcysteine
accidental injury (arm), constipation, diarrhea, dry mouth, emotional outburst,
encopresis, increased hyperactivity, self-injurious behavior (skin picking), sinus 0.80 for large, 100 % of the effects were ≤ the small effect
infection, skin infection, sore throat, urinary tract infection, and weight gain
NAC group reported one mild case of the following adverse effects: size [35]. For all of the models that had a small effect size,
aggression, appetite increase, behavior worse, change in speech, early the placebo group consistently had the larger average de-
morning awakening, edema, medical or surgical procedure, swollen neck/
lymph nodes, and tic crease in score.
NAC N-acetylcysteine, Mod moderate, URI upper respiratory infection
Oxidative stress biomarkers
most frequent events were headaches (NAC n = 3, 18.7 %; Baseline (pre-treatment) levels of the oxidative markers
placebo n = 3, 20.0 %), stomachache (NAC n = 2, 12.5 %; including GSH, GSSG, GSH/GSSG ratio, DNA strand
placebo n = 2, 13.3 %), and fever (NAC n = 3, 18.7 %; pla- break and oxidative damage, and blood homocysteine
cebo n = 1, 6.7 %). The majority of reported adverse events were not significantly different between the NAC and
were mild, with only five (6.7 %) reported as moderate. No placebo groups (p > 0.05 for all). At week 12, the GSH
severe adverse events were reported. There were no sig- level in blood was significantly higher in the NAC group
nificant differences found between the groups for changes compared to placebo (780.3 vs. 640.4 μM; p < 0.05,
from screen to week 12 on vital signs or safety lab values Table 4). The GSSG level increased in the NAC treat-
(all p > 0.10). ment group, however with only marginal significance in
comparison with the placebo group (16.7 vs. 12.5 μM; p
Primary and secondary outcomes = 0.09, Table 4). Using the Cohen’s d cutoff employed
There was no statistically significant difference between above for effect sizes, the size of the differences for the
the NAC and the placebo groups at week 4 (p > 0.60), significant and the marginally significant effects were
week 8 (p > 0.79), or week 12 (p > 0.69) on the CGI-I pri- large- and medium-sized effects, respectively. For the
mary outcome measure (Table 3). At each time period, GSH/GSSG ratio, strand break and oxidative damage of
at least half of all participants (≥50 %) were rated as hav- DNA, as well as blood homocysteine, there were no
ing no change. On the CGI-S secondary outcome meas- significant differences between the NAC and placebo
ure, no participants were noted to have increased scores groups from baseline to week 12 (ps > 0.16).
suggesting clinical worsening of symptoms. There were
also no differences between the NAC and placebo Discussion
groups for those whose severity scores decreased from This study was designed to evaluate the safety and effi-
baseline to week 12 (χ2 = 0.43, p = 0.40; NAC 46.2 %, n = 6; cacy of NAC targeting core social impairment in youth
placebo 33.3 %, n = 4). On the ABC, SRS, and VABS-II with ASD. The results of this randomized, placebo-
secondary outcome measures, the employed models found controlled trial indicate that NAC treatment was well
no significant differences between groups in change from tolerated by study participants, had the expected effect
baseline to week 12 (all ps > 0.13 (Table 4)). Using the cut- of boosting GSH production in peripheral blood, but
off of a Cohen’s d of 0.25 for small, 0.50 for medium, and had no significant impact on the core social impairment
Wink et al. Molecular Autism (2016) 7:26 Page 7 of 9

Table 4 Means, standard deviations, and differences in change scores between NAC and Placebo groups
NAC Placebo
Baseline 12 Weeks Baseline 12 Weeks Group with largest Cohen’s d
changeb
M SD M SD M SD M SD z testa
ABC
Hyperactivity 20.6 12.4 17.4 16.4 22.6 10.5 15.1 10.8 1.04 PLB 0.34
Speech 3.9 2.8 4.1 3.9 5.7 2.9 5.14 3.5 0.66 PLB 0.24
Irritability 17.0 11.8 14.9 14.0 18.3 9.2 12.0 7.3 1.09 PLB 0.40
Lethargy 13.8 8.1 10.0 7.1 14.0 10.5 7.9 4.7 1.15 PLB 0.31
Stereotypy 4.8 3.3 3.9 5.1 7.0 4.9 5.8 4.0 0.56 PLB 0.09
SRS
Total score 108.5 20.7 85.8 34.9 109.1 16.5 89.1 26.3 0.32 NAC 0.11
VABS-II
Comm 85.9 32.1 88.5 32.5 89.4 32.2 95.0 30.3 0.85 PLB 0.09
Daily Liv. Sk. 93.1 33.7 99.0 37.1 87.6 28.1 98.6 33.4 0.17 PLB 0.16
Socialization 66.8 18.8 71.7 18.7 68.3 20.7 75.9 21.0 0.87 PLB 0.14
Composite 61.3 19.4 62.9 17.1 53.6 15.1 60.5 18.8 1.08 PLB 0.30
Levels in whole blood (uM)
GSH 484.8 212.7 780.3 220.6 474.5 191.6 640.4 190.9 1.74* NAC 0.64
+
GSSG 12.0 2.5 16.7 10.5 12.8 5.0 12.5 4.4 1.37 NAC 0.88
GSH/GSSG 43.4 25.6 53.5 16.8 45.2 31.8 59.6 27.0 0.34 PLB 0.17
Homocysteine 4.3 6.1 9.9 13.7 1.9 1.2 5.6 6.8 0.41 NAC 0.27
DNA damage
DNA strand breakage 0.7 0.1 0.7 0.3 0.6 0.2 0.6 0.1 0.38 EVEN 0.00
Oxidative DNA damage 0.9 0.2 1.2 0.4 1.1 0.5 1.2 0.6 0.95 NAC 0.45
a +
One-tailed significance p < .10; *p < .05. Z test of the effect of between level variable of group on the within-level variable of time testing if the change from
baseline to week 12 is different between groups. No one-tailed p values for these z tests reached significance at .05 and ranged from .13 to .50
b
Identifies which group had the largest change from baseline to week 12
NAC N-acetylcysteine, M mean, SD standard deviation, ABC Aberrant Behavior Checklist, PLB placebo, SRS social responsiveness scale, VABS-II Vineland adaptive
behavior scale 2nd edition, Comm communication, Liv. Sk. living skills, GSH glutathione, GSSG oxidative glutathione disulfide

of ASD when compared to placebo treatment. The re- study results to the broader population of individuals with
sults of this study do not support the use of NAC for ASD. This higher functioning study population may have
treatment of core social impairment of ASD, though the also contributed to lack of improvement in irritability
health impact of the resultant increase on GSH remains noted in this study. The baseline ABC-I of study partic-
unclear. In addition, the study did not note other behav- ipants was 17.0 (SD 11.8) in the NAC group and 18.3
ioral improvements with NAC use such as the reduction (SD 9.2) in the placebo group, which is slightly below
in irritability reported by Harden et al. (2012) [13]. the standard cutoff ABC-I score of 18 used in drug
Interpretation of these results must be taken in con- studies targeting ASD-associated irritability [36]. How-
text of the study’s limitations. Participant diagnoses were ever, the baseline ABC-I scores in our study are higher
made by study physicians with expertise in ASD corrob- than those in the Hardan study which demonstrated
orated by the ADI-R similar to methods employed in improvement irritability with NAC treatment [13], so
other ASD drug studies [36]. Nevertheless, administration the impact this had on our study outcome is unclear.
of a research reliable Autism Diagnostic Observation Additionally, the small sample size combined with the
Schedule would have added to the validity of diagnoses in inherent significant placebo response rates in ASD core
this study. Furthermore, limitation of the study to just par- symptom trials enhances type II error potential thus
ticipants with diagnosis of autistic disorder would have rendering the project potentially underpowered to detect
lessened the heterogeneity of our sample. Our study popu- meaningful change. The small sample size also reduces
lation was potentially biased toward individuals with the ability to effectively correlate treatment response with
higher level of functioning, as the majority of participants baseline oxidative stress markers or with change in such
had IQ above 85. This limits the generalizability of these markers with treatment.
Wink et al. Molecular Autism (2016) 7:26 Page 8 of 9

The NAC formulation employed in this study was a and Riovant Sciences Ltd. Dr. Adams, Dr. Wang, Dr. Klaunig, Dr. Plawecki,
powder which was encapsulated by the investigative Dr. Posey, and Dr. McDougle report no potential conflicts of interest.
This study was funded by the Autism Speaks.
pharmacy and stored under normal pharmacy conditions.
No studies of impurity were completed on this compound,
Authors’ contributions
and no analyses were completed to assess its stability. LKW drafted the manuscript. RA completed the statistical analysis and
Furthermore, this packaging was different than the assisted with drafting the statistical portion of the manuscript. ZW and JEK
method employed by Hardan et al. (2012) who utilized completed the oxidative stress biomarker analysis and drafted the portion of
the manuscript describing this work. MHP assisted in drafting the manuscript
an individual foil packaging method to protect the in- and reviewed the data analysis. DJP and CJM designed and executed the
tegrity of the active ingredient against oxidation [13]. study and reviewed the manuscript. CAE participated in study design, study
While this difference could have in theory reduced the execution, and oversaw all aspects of manuscript development. All authors
read and approved the final manuscript.
potency of our formulation, the biologic impact of our
treatment was clear given the significant NAC-associated
Acknowledgements
increase in GSH levels at week 12. It is unclear; however, if We would like to acknowledge the contributions of Arlen Kohn MA and
the change in oxidative markers and potential clinical Lauren Mathieu-Frasier MA for their assistance with study coordination and
impact may have differed had we employed an individ- data collection.

ual foil packaging method. Further, whether an increase

in peripheral blood GSH levels, and the NAC formulation Funding
Autism Speaks Treatment Grant.
employed in our study, translates to clinically significant
CNS antioxidant activity is unclear as there remains de- Author details
1
bate in the field regarding the ability of NAC to efficiently Cincinnati Children’s Hospital Medical Center, University of Cincinnati
College of Medicine, 3333 Burnet Avenue MLC 4002, Cincinnati, OH 45229,
cross the BBB [10]. In future studies, alterations to NAC USA. 2Investigative Toxicology and Pathology, School of Public Health,
formulation including esterification of the carboxyl group Indiana University, Bloomington, IN, USA. 3Department of Psychiatry,
producing N-acetylcysteine ethyl ester or creation of the Christian Sarkine Autism Treatment Center, Riley Hospital for Children at
Indiana University Health, Indiana University School of Medicine, Indianapolis,
amide derivative N-acetylcysteine amide may increase this IN, USA. 4Indianapolis, IN, USA. 5Lurie Center for Autism, Departments of
medication’s oral bioavailability, BBB permeability, and Psychiatry and Pediatrics, Massachusetts General Hospital and MassGeneral
therefore therapeutic potential [10]. Hospital for Children, Harvard Medical School, Boston, MA, USA.

Received: 3 February 2016 Accepted: 25 March 2016

Conclusions
The results of this study suggest that NAC treatment does
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