This study was a 12-week randomized, double-blind, placebo-controlled pilot trial of oral NAC in youth with ASD. The study was conducted at Indiana University School of Medicine (IUSM) between December 2006 and November 2009. The study was approved by the IUSM institutional review board. Guardians of all participants provided written informed consent prior to study enrollment. Assent was obtained from enrolled youth when possible.
Study participants were youth ages 4 to 12 years with a diagnosis of autistic disorder, Asperger’s disorder, or pervasive developmental disorder not otherwise specified (PDD NOS). Subjects with known genetic syndromes associated with autism were excluded (for example, fragile X syndrome, tuberous sclerosis). Participants were diagnosed via clinical interview completed by study physician with expertise in ASD (DJP, CJM, CAE), based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV)  diagnostic criteria, and corroborated by administration of the Autism Diagnostic Interview-Revised  (ADI-R). Participants diagnosed with PDD NOS demonstrated pervasive impairment in social interaction and/or communication skills as well as stereotyped behaviors and restricted interests, but did not meet full criteria for diagnosis of autistic disorder or Asperger’s disorder. All participants weighed ≥15 kg and were medically healthy based on physical exam and review of medical history completed by study physicians. Participants were judged by study physician as being at least “moderately ill” as measured by the Clinical Global Impression Severity  (CGI-S) scale rating at baseline. The CGI-S is a clinician-rated global assessment of symptom severity scale ranging from 1 to 7 (1 = normal, not at all ill; 2 = borderline ill; 3 = mildly ill; 4 = moderately ill; 5 = markedly ill; 6 = severely ill; 7 = among the most extremely ill patients). Concomitant medications were permitted if doses were stable for at least 60 days prior to study initiation and remained stable throughout the study. Participants taking known glutamatergic modulators such as dextromethorphan, d-cycloserine, amantadine, memantine, lamotrigine, or riluzole were excluded. Patients taking daily acetaminophen, daily nonsteroidal anti-inflammatory medications, daily antioxidant medications such as high-dose vitamin supplements, and medications with known drug-interactions (i.e., carbamazepine) within 30 days of baseline were also excluded. Subjects were required to be able to swallow capsules. Potential subjects with profound cognitive impairment (mental functioning below 18 months of age) as measured by the Leiter International Test of Intelligence-Revised  were excluded.
Following screening and baseline measures, participants were randomized 1:1 via computer—by the investigational pharmacy. All participants, guardians, and investigators remained blind to study assignment. NAC and matching placebo were prepared by CustomMed Apothecary. Participants randomized to active drug were administered capsules containing 300 or 600 mg of NAC, with a target dose of 60 mg/kg/day in three divided doses and a maximum dose of 4200 mg/day. Patients weighing 15 to 30 kg began treatment with a starting dose of 300 mg/day; those weighing above 30 kg started with 600 mg/day. Patients were required to tolerate a minimum dose of 300 mg/day to continue in the trial. Dose was titrated to the target dose over the first 3 weeks of study participation. Dose then remained stable for all subjects in the last 9 weeks of the study, although reductions due to adverse effects were permitted at any time. Subjects were evaluated in person at screen and baseline, by phone at week 2, and in person at weeks 4, 8, and 12. Assessment for adverse effects was completed during each interaction. Vital signs including height, weight, blood pressure, and heart rate were measured at every in-person study visit. Safety labs including complete blood cell count (CBC) and comprehensive metabolic panel (CMP) were collected at screen and week 12.
The primary outcome measure of efficacy was the CGI-I scale anchored to study physician (DJP, CJM, CAE) assessment of core social impairment considering the individuals’ overall level of cognitive, adaptive, and social functioning. The CGI-I is a clinician-rated global assessment of symptom change rated on a scale from 1 to 7 (1 = very much improved; 2 = much improved; 3 = minimally improved; 4 = no change; 5 = minimally worse; 6 = much worse; 7 = very much worse). In this study, a positive response to NAC treatment was defined as scoring a 1 “very much improved “or 2 “much improved” on the CGI-I. Study physicians completed annual CGI training to ensure internal consistency with this outcome measure. Secondary outcome measures included the CGI-S, ABC, Social Responsiveness Scale  (SRS) raw score, and Vineland Adaptive Behavior Scales 2nd Edition  (VABS-II) survey edition raw score. The SRS is a standardized, caregiver reported measure of the core symptoms of ASD . The ABC is a parent questionnaire measuring five behavioral domains including irritability, social withdrawal, stereotypy, hyperactivity, and inappropriate speech . The VABS-II is a semi-structured caregiver interview which provides a measure of an individual’s overall adaptive functioning . All measures have been used extensively in ASD research [25–27]. The CGI-I was completed at weeks 4, 8, and 12. The ABC and SRS were completed at baseline and weeks 4, 8, and 12. The CGI-S and VABS-II were completed only at baseline and week 12.
Early morning, fasting, venous blood samples for measurement of oxidative stress biomarkers GSH, GSSH, total homocysteine, strand breakage, and oxidative DNA damage were collected in EDTA containing Vacutainers at screen and week 12.
Measurement of reduced and oxidized glutathione
Concentrations of reduced and oxidized glutathione (GSH and GSSG) in whole blood samples were analyzed simultaneously using HPLC-electrochemical detection as described previously . Briefly, 1 ml of blood sample was added to 0.5 ml precipitating solution containing 0.2 M perchloric acid and 100 μM EDTA, and vortexed briefly. After incubation at room temperature for 45 min, samples were centrifuged at 10,000g for 3 minutes. The resulting supernatants were filtered and injected into high-performance liquid chromatography (HPLC; Waters 2690) for analysis immediately or frozen in liquid nitrogen and stored at −80 °C before analysis. The separation of analytes was achieved on a reverse phase Symmetry C-18 column (5 μm, 150 × 4.6 mm; Waters). GSH and GSSG were detected using an ESA Coulochem II (ESA Inc. Chelmsford, MA) equipped with a guard cell. The potential settings for the detector were E1 450 mV, E2 900 mV, and guard cell 1000 mV. The amount of GSH and GSSG was calculated from the respective calibration curves, and the ratio of GSH/GSSG for each sample was also calculated.
Measurement of blood homocysteine
Total homocysteine in whole blood sample was determined using an HPLC method as described previously with minor modifications [29, 30]. Briefly, 1 ml EDTA blood samples were lysed by vigorously shaking for at least 10 s after adding 10 μl Nonidet P40 (pure) and 10 μl citric acid monohydrate (2.5 M). The lysates were then centrifuged at 10,000g for 3 minutes at room temperature. Following reduction of the sample with tri-n-butylphosphine, precipitation of protein with perchloric acid and derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, the samples were then analyzed using reversed-phase high-performance liquid chromatography (Waters Alliance 2695 separation module) followed by fluorescence detection (Waters 474 fluorescence detector) (Waters Corporation, Milford, MA).
Direct and oxidative DNA damage: comet assay
Immediately after blood collection, whole blood (10 μl) was mixed with 0.5 ml RPMI 1640 containing 10 % FBS, 10 % DMSO, 1 mM deferoxamine, step-frozen and stored at −80 °C until analysis. The comet assay was performed as described previously [31, 32]. Briefly, 6 μl of blood was mixed with 70 μl 1 % low-melting-point agarose and applied onto comet slides (Trevigen Inc, Gaithersburg, MD). Cells were lysed, placed in alkali buffer, and then electrophoresed. Slides were stained with ethidium bromide, and 100 randomly selected nuclei/sample were evaluated (Komet 5.5; Kinetic Imaging Ltd., Liverpool, UK). For the assessment of oxidative DNA damage, a modified alkaline comet assay was performed that included enzymatic digestion with formamidopyrimidine-DNA glycosylase (fpg) prior to electrophoresis. DNA damage was expressed as Comet (Olive) tail moment [(tail mean − head mean) × tail%DNA/100].
Our sample size (goal 32 participants) was chosen based on recommendation for sampling in pilot studies where little is known about treatment response rates . Baseline demographic data including age, sex, race, level of intellectual quotient (IQ), ASD diagnostic sub-type, concomitant medications, and clinical ratings (CGI-S, ABC, SRS, and VABS-II scores) was compiled, and means and percentages were calculated to describe the NAC and placebo groups. Baseline demographic differences between groups were tested using chi-square tests (categorical data) and independent sample t test (continuous data) using a two-tailed p value of 0.05 for the alpha.
For the CGI-I primary outcome measure, differences between the two treatment groups were tested at weeks 4, 8, 12 using chi-square tests. For the CGI-S secondary outcome measure, a chi-square test was employed to examine change from baseline to week 12 by creating two CGI-S categories: (1) those subjects whose severity score decreased (i.e., clinically improved) by at least one point from baseline to week 12 and (2) those subjects whose scores remained the same during the same period. For the ABC, SRS, VABS-II, and oxidative stress biomarkers, differences between the groups for change in each outcome measure over the course of the study were tested using multi-level modeling (i.e., we examined if there were differences between the two groups in terms of change in the outcome scores over the course of the study). For each outcome, time was modeled at level-one as a random effect with treatment group entered as a level-two fixed effect. The cross-level interaction between group and time tested the differences between the two groups in the change over time using maximum likelihood estimation with robust standard errors with Mplus 5.21  (baseline, week 4, week 8, and week 12 data was used for the ABC and SRS; baseline and week 12 data was used for the VABS-II and oxidative stress biomarkers). Since there is no method to directly calculate the effect size of the between-subject effects on the within-subject effects for multilevel modeling, we calculated an effect size of the difference between the groups on the mean differences between baseline and week 12 for the groups. Specifically, a Cohen’s d was calculated by subtracting the mean score at week 12 from the score at baseline for each group, and a difference score was calculated by subtracting the change scores between the groups. This difference score was then divided by the pooled standard deviation of the change scores. Independent sample t tests were used to compare the levels of oxidative markers between NAC and placebo groups at baseline and week 12. For all types of tests, a one-tailed p value of 0.05 was used 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 the course of the study than the placebo group. We did not correct for multiple comparisons given the pilot nature of the work.
Adverse event data was compiled to describe the NAC and placebo groups. Separate analyses were conducted for the vital sign measurements, CMP, and CBC employing the same method as the treatment analyses, though only two time points, screen and week 12, were evaluated.