Study outline

This is a single-site, single-blind, fixed placebo-controlled Phase 1 clinical trial to evaluate the safety, tolerability, plasma concentration profile and targeted efficacy of orally administered NFC-1 in adolescent (12–17 years) ADHD patients exhibiting mutations in genes within the GRM network. Primary objectives were to evaluate the safety and tolerability of orally administered NFC-1 and characterize its pharmacokinetic parameters. A secondary objective was to explore the dose-response relationship of NFC-1 on ADHD severity and global measures and determine effect size of specific GRM network genes on ADHD based on responsiveness of patients to NFC-1.

Participants

From over 200 patients screened for mGluR mutations using a SNP array, 30 patients with ADHD harboring disruptive copy number variants (CNVs) within or nearby one or more mGluR network genes (Table 5) were recruited from the Center of Applied Genomics (CAG) at The Children’s Hospital of Philadelphia (CHOP). The study participants included ADHD cases who had previously participated in a large-scale genomics study at CAG/CHOP (“Genetics of Complex Pediatric Disorders”) and had authorized recontact for future studies. The study was conducted at the Jefferson University (TJU) Clinical Research Unit for the PK part of the study and TJU Department of Psychiatry for the dose escalation part of the study. The study was approved by the Institutional Review Board of Thomas Jefferson University. Informed consents were obtained from all parents and assents from all study subjects. The study IND was approved on 7 November 2014; institutional review board approval was obtained on 11 December 2014; FPI was obtained on 23 January 2015, and LPO was obtained on 17 May 2015.

Inclusionary criteria

Male and female patients, ages 12–17 years, of any ancestry, diagnosed with ADHD as defined by DSM-5 and a Vanderbilt ADHD rating scale score ≥ 16, genotyped to have disruptive CNVs in the mGluR gene networks were eligible to participate (Supplementary Materials; Supplementary Figs. 1a and 1b).

Exclusionary criteria

Any clinically significant illness, which, in the opinion of the investigator, may confound the results of the study, pose additional risk to the patient by their participation, or prevent or impede the patient from completing the study, pregnancy, positive urine for illicit drugs (including marijuana), or history of drug or alcohol abuse within the last 3 years.

Study drug

NFC-1 (fasoracetam monohydrate) was administered per os as a single dose during the pharmacokinetic study and twice-daily (BID) during the dose-escalation study. NFC-1 was delivered in size 1, dark blue capsules containing 50 mg NFC-1 or size 1, dark blue capsules containing 200 mg NFC-1. Matching placebo capsules containing microcellulose instead of NFC-1 were provided during the week of placebo administration. The capsules were stored at controlled room temperature of 20–25 °C (68–77°F). Twelve-month stability data at room temperature showed no evidence of decay of the active substance.

Dosing

All study subjects completed a 24-h PK study. This was followed by an open-label, 5-week dose-escalation study, starting with a fixed single-blind placebo week (week 1). All ADHD medications were discontinued with a washout phase ranging from 5 days (stimulants) to 14 days (adrenergic drugs). The 24-h PK study included five groups with six subjects/group, where each group was given the following respective single doses: 50, 100, 200, 400 and 800 mg. Three patients were initially enrolled for PK studies and shown to tolerate 50 mg of the drug well prior to enrolling the next three subjects to complete dose group 1 of 50 mg. The next six subjects completed 100 mg and tolerated the study drug well. The DSMB approved the 3rd study group of 6 subjects to advance to 200 mg and the 4th set of six subjects to 400 mg, with the last group of 6 subjects approved by the DSMB to receive 800 mg (Supplementary Figs. 2 and 3). All study subjects tolerated their study medications well during the PK part of the study. The patients subsequently received a placebo BID (week 1) followed by NFC-1 50 mg BID in week 2, 100 mg BID in week 3, 200 mg BID in week 4, and 400 mg BID in week 5. Safety data was reviewed by the study PI, study team and the DSMB after each dose cohort before proceeding with each dose increase. Patients were blinded as to which week (out of the 5 total weeks) they received placebo.

Study measures

The PK measures included 11 parameters of active drug and two major metabolites at each time point during the initial pharmacokinetic portion of the trial. Blood samples were collected just prior to dosing and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h after administration of the first dose of NFC-1. The concentrations of NFC-1 and two metabolites (LAM-79 and LAM-163) in plasma were determined by a validated ultra-performance liquid chromatography - tandem mass spectrometry assay with D 10 -NFC-1 as an internal standard. The following precursor to product ion transitions were used for quantitation: m/z 197.2 → 84.2 (NFC-1), m/z 213.2 → 83.9 (LAM-79), m/z 213.2 → 84.0 (LAM-163), and m/z 207.2 → 84.2 (D 10 -NFC-1). The analytical method was selective and linear over the concentration range of 10 to 2,500 ng/mL for all three compounds in plasma. Precision of the NFC-1 assay, as determined by percent coefficient of variation, was 5.13–7.16 % (within-day) and 3.81–9.99% (between-day), and accuracy was within 93.5–107% based on quality control samples. Precision of the LAM-79 assay was 5.81–9.11% (within-day) and 4.61–7.72% (between-day), with accuracy within 103–109%. Precision of the LAM-163 assay was 4.29–7.90% (within-day) and 3.28–10.2% (between-day), and was accuracy within 100 to 112%. PK parameters, including C max , T max , and AUC 0–24h ,were estimated by the methodology of Gibaldi and Perrier34. C max and T max were obtained by visual inspection of individual patient plasma profiles. Half-life was estimated by 0.693/k e , where k e is the slope of the terminal elimination phase from linear regression of log-transformed concentration values on time. AUC(0–24) was estimated by the linear trapezoidal method and AUC(0–∞) by addition of area extrapolated to infinity via the regression described above. All calculations were performed with the R programming language35.

CGI-I/CGI-S scores determined by the psychiatric evaluators, together with parental Vanderbilt ratings, were the key measurements used to assess drug efficacy of NFC-1. The efficacy measures performed during the dose escalation study are detailed in Supplementary Figs. 2 and 3. Parental Brief rating scales were also included as secondary measures of NFC-1 efficacy. Wechsler Abbreviated Scale of intelligence, together with overall assessment of the children’s neurocognitive function and ADHD-comorbid condition severities, including anxiety, autism spectrum disorder and mood swings, were also recorded by the psychiatric evaluators. Actigraphy data was also collected from all subjects at each visit for the duration of the study (Table 6).

The safety measures and logging of all adverse events (spontaneous reports and rating scales; Pittsburgh Side Effect Scale36 and the Columbia Suicide Scale37 were evaluated throughout the study and graded in terms of severity (mild, moderate, or severe) and relationship to study treatment (unrelated, possibly related, or definitely related) recorded. Serum NFC-1 levels were measured during each week of the dose-escalation study to ensure adherence. At baseline and after each week of treatment, each subject had a physical exam, electrocardiogram, and safety laboratory test (complete blood count, clinical chemistry and urinalysis).

Genotyping and genetic tier stratification

After informed consents were obtained, blood samples were obtained and deoxyribonucleic acid (DNA) was isolated from eligible adolescents age 12–17 years and genotyped using the OMNI-2.5 M genotyping assay (Illumina, San Diego), performed in the CAP-certified genotyping laboratory at The Children’s Hospital of Philadelphia. The OMNI-2.5 M oligonucleotide array effectively captures the target CNVs in the 79 Tier-1 mGluR genes of interest and has sufficient coverage across the additional 200 Tier-2 and 600 Tier-3 mGluR genes, as previously reported5,6.

In brief, 250 ng of genomic DNA was used to genotype each sample according to the manufacturer’s guidelines. This implies, amplification of the patient DNA and hybridization to a reference genome. Upon washing, the samples are scanned to capture over 2.5 M SNPs dispersed throughout the genome. Cluster plots are generated to separate the 3 genotype states, namely homozygous allele (AA), homozygous allele (BB) or heterozygous state (AB).

The CNV quality control measures were performed on the genotyping data based on statistical distributions to exclude poor quality DNA samples and false positive CNVs. The first threshold was the percentage of attempted SNPs that were successfully genotyped. Only samples with call rate > 98.5% will be included in the CNV analysis. The genome-wide intensity signal must have as little noise as possible. Only samples with the standard deviation (SD) of normalized intensity (Log R Ratio (LRR)) < 0.25 were included. Samples of different ethnicities based on hierarchical clustering of AIMs genotypes were separated from each other and called separately. Wave artifacts that roughly correlate with GC content that result from hybridization bias of low full length DNA quantity are known to interfere with accurate inference of copy number variations. Thus, only samples where the correlation of LRR to wave model ranges between −0.2 < X < 0.4 will be accepted.

The simultaneous analysis of intensity data and genotype data in the same experimental setting establishes a highly accurate definition for normal diploid states and any deviation thereof. In contrast with aCGH which relies on intensity data alone for CNV calls, the Illumina SNP array platforms provide both genotype and intensity data for each SNP marker. The PennCNV algorithm was used to generate CNV calls and data analysis were standardized as described previously39. (Three different CNV states were called, including homozygous deletions (copy number, or CN = 0), hemizygous deletions (CN = 1), duplications (CN = 3 or more). Supplementary Table 5 presents the CNV regions for the top 79 most significant gene loci of interest harboring CNVs (deletions or duplications) and includes the same information for Tier-2 and Tier-3 genes.

All CNVs identified were validated visually and/or experimentally based on the confidence of the CNV call to ensure validity of the results reported (experimental validation has been performed previously or the 79 CNV genes under study5. As shown previously, for the 79 mGluR/GRM network genes, accuracy of the CNV calls detected on the SNP array is over 95%. Subsequent Taqman validation raised the accuracy of the CNV calls identified to 100%.

Statistical and network analysis

Quantitative variables were summarized using descriptive statistics. Continuous variables were presented as N, mean and median, standard deviation and range. Categorical variables were presented using frequencies and percentage. Analyses of variance (ANOVA) were used to compare treatment response at the respective weeks from week 1 to week 5. Where not specified explicitly, a Student’s t-test was used. Analysis was performed using SPSS and R35,38.

Genetic stratification of mGluR genes is delineated in genetic tiers in keeping with previous mGluR CNV association results (tier-1: 79 genes; tier-2: 200 genes; tier-3: 600 genes; Supplementary Materials)5,6.

For Box-and-Whisker Plots, upper and lower whiskers denote the limits of the nominal range of the data inferred from the upper and lower quartiles, representing the smaller of the maximum score and the value of (75% tile+1.5×interquartile range) and the larger of the minimum score and the value of (25% tile−1.5×interquartile range), respectively. Additional points are outliers beyond these ranges. Network analysis were performed in StringDB (https://string-db.org) using program defined parameters.

Actigraphy

Actigraphy watches: Each participant was given an Actilife watch. The data was downloaded from the watch each week and the watches were recharged while participants were given their weekly dose, and a clinical interview.

Data analysis: Data was loaded into the Actilife software for further analysis. Sixty second epochs were used as the unit for analysis. The default algorithm in the software was used to detect off-watch times, and these periods were removed from further evaluation.

The Actilife software calculates a number of variables that measure energy expenditure, bouts of activity, and the vigorous nature of the activity. Bouts of activity are measured by an algorithm developed by Freedson4 that has been calibrated in children and adolescents.

Variables examined included: average number of kCals used per hour, number of bouts of activity per day (measured using the Freedson et al., algorithm), total time of bouts, number of sedentary bouts, p of time in moderate to vigorous physical activity (MVPA)40.

For each variable, a linear mixed model using was fit using the lme4 package in R where each variable was treated as a fixed effect, and regressed against the fixed effect of dosage, while treating subject as a random effect. The covariates of age, sex and weight were also included as fixed effects. A model with all covariates was fitted, and then a model with dosage added as a fixed effect and modeled as a factor. ANOVA using maximum likelihood was used to test for a significant difference in fit between the two models. The analysis compares whether there are differences overall between week 1 and the subsequent weeks.

To test if there was an interaction between dosage of the drug and the type of CNV carried by the participant, the difference in fit between models that included dosage and genetic tier in addition to the covariates, to one that also fit an interaction term of dosage×genetic tier.

Data availability

The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.