Improvement of Language in Children with Autism with Combined Donepezil and Choline Treatment

Lidia V. Gabis, Rotem Ben-Hur, Shahar Shefer, Ariela Jokel & Dorit Ben Shalom

Journal of Molecular Neuroscience

ISSN 0895-8696

J Mol Neurosci DOI 10.1007/s12031-019-01351-7

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Journal of Molecular Neuroscience https://doi.org/10.1007/s12031-019-01351-7

Improvement of Language in Children with Autism with Combined Donepezil and Choline Treatment

Lidia V. Gabis1,2 & Rotem Ben-Hur1,3 & Shahar Shefer1 & Ariela Jokel1 & Dorit Ben Shalom3

Received: 19 March 2019 /Accepted: 29 May 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract The safety and efficacy of a novel combination treatment of AChE inhibitors and choline supplement was initiated and evaluated in children and adolescents with autism spectrum disorder (ASD). Safety and efficacy were evaluated on 60 children and adolescents with ASD during a 9-month randomized, double-blind, placebo-controlled trial comprising 12 weeks of treatment preceded by baseline evaluation, and followed by 6 months of washout, with subsequent follow-up evaluations. The primary exploratory measure was language, and secondary measures included core autism symptoms, sleep and behavior. Significant improvement was found in receptive language skills 6 months after the end of treatment as compared to placebo. The percent- age of gastrointestinal disturbance reported as a side effect during treatment was higher in the treatment group as compared to placebo. The treatment effect was enhanced in the younger subgroup (younger than 10 years), occurred already at the end of the treatment phase, and was sustained at 6 months post treatment. No significant side effects were found in the younger subgroup. In the adolescent subgroup, no significant improvement was found, and irritability was reported statistically more often in the adolescent subgroup as compared to placebo. Combined treatment of donepezil hydrochloride with choline supplement demon- strates a sustainable effect on receptive language skills in children with ASD for 6 months after treatment, with a more significant effect in those under the age of 10 years.

Keywords Autism . Language . Donepezil . Choline . Children

Introduction cognitive flexibility and social interaction, as well as evidence of brain cholinergic system dysfunction in ASD (Hardan and Postmortem and in vivo imaging studies have demonstrated Handen 2002; Eissa et al. 2018). abnormalities in acetyl-choline (ACh) function in autism spec- Donepezil hydrochloride (donepezil) blocks acetylcho- trum disorder (ASD) (Ray et al. 2005; Karvat and Kimchi linesterase (AChE), allowing cholinergic neurotransmitters 2014; Gabis et al. 2008; Handen et al. 2011). Based on find- to function longer in the synaptic cleft. As such, donepezil ings and treatment results in Alzheimer’s patients, these ab- could help restore normal functioning to abnormal cholin- normalities led to the idea of treating ASD children with the ergic receptors in ASD. A literature review has yielded same agents as used for enhancing ACh activity in nine clinical trials and case series designed to assess the Alzheimer’s disease (Lee et al. 2002). There is evidence that influence of AChE inhibitors on individuals with ASD cholinergic neurotransmission plays an essential role in regu- (Handen et al. 2011;Nicolsonetal.2006;Doyleetal. lating ASD-related behavioral symptoms, including attention, 2006; Hardan and Handen 2002;Chezetal.2003; Hertzman 2003;Niederhofer2002) (see details in Table 1). The prior studies showed contradicting results, possibly due to the use of different measures and different * Lidia V. Gabis [email protected] methodologies. Nevertheless, most studies showed a trend of positive effect in regard to language skills. In terms of safety, according to prior studies, AChE inhib- 1 The Weinberg Child Development Center at Safra Children’s Hospital, Sheba Tel Hashomer, Ramat Gan, Israel itors seem to be safe and generally tolerable in the ASD population. This is a substantial advantage given the side effects 2 Sackler School of Medicine at Tel Aviv University, Tel Aviv-Yafo, Israel observed in this population with other medications (Hardan and Handen 2002;Handenetal.2000). 3 Ben Gurion University of the Negev, Be’er Sheva, Israel Table 1 Summary of prior studies using AChE inhibitor treatments in ASD populations

Researchers Age of ASD Number Trial Medicine Side effects Results participants

Niederhofer Mean age 7.4 years 20 Placebo-controlled, Galantamine None Improvement in expressive language: et al. (2002) double-blind inappropriate speech crossover trial Hertzman Adults (21, 32, 3 Open-label trial Galantamine 4 mg daily up to One patient developed a macular Improvement in expressive and

(2003) 42 years old) 12 mg daily for at least 4 weeks rash. One patient had increased receptive language skills Author's but tolerable drooling Nicolson 4–17 years, mean 13 Open-label trial Galantamine 2 mg daily up to 24 mg. One patient experienced headaches No improvement in language skills. (2006) age 8.8 daily for 12 weeks Could be due to inadequate measures Chez et al. 2.1–10.3 years, 43 Double-blind parallel Donepezil hydrochloride 1.25 mg daily up Two subjects experienced gastrointestinal Improvement in receptive and expressive (2003) mean age 6.8 group design with to 2.5 mg daily for 12 weeks disturbances (diarrhea or stomach cramping). language skills an Four subjects reported increased irritability accompanied open-label by increased screaming and vocalization personal extension Chez et al. 2.85–12 years, 31 Open-label trial Rivastigmine tartrate 0.4 mg twice Gastrointestinal (nausea or diarrhea), Improvement in expressive language skills (2004) mean age 6.91 daily up to 0.8 mg twice daily irritability and hyperactivity (number for 12 weeks of subjects not noted) Hardan & 7–19 years, mean 8 Retrospective open Donepezil hydrochloride 2.5 mg One patient experienced gastrointestinal Improvement in severity of illness

Handen age 11 trial dailyupto10mgdaily disturbances (nausea and vomiting). copy (2002) One patient reported mild irritability Doyle et al. 10–17 years, mean 8 Systematic Donepezil hydrochloride 2.5 One patient experienced tremors, Improvement in ADHD symptoms (2006) 13.5, SD 2.8 retrospective mg–30 mg, mean 16 irritability and distractibility and core ASD symptoms chart review Handen et al. 8–17 years 34 Double-blind Donepezil hydrochloride 2.5 mg; daily up The only symptoms that saw a slight No statistically significant difference (2011) placebo-controlled to 10 mg daily for 10 weeks increase in rate were diarrhea, between groups was found trial followed by an headache, and fatigue open-label trial Buckley et al. 2.5–6.9 years 8 Open-label trial Donepezil hydrochloride 2.5 mg; None Statistically significant increase in REM (2011) dailyupto10mgdaily % sleep at the initial 1.25-mg dose, statistically significant decrease in latencytoREMsleep o Neurosci Mol J Author's personal copy

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The limited effect in children with ASD shown in prior primary registry. Data are available from the hospital studies fostered our hypothesis of lack of sufficient substrate Helsinki Committee. Written informed consent was obtained for ACh production. We hypothesized that the addition of from all guardians (both parents) of all patients that choline might potentiate the AChE blocker effect of participated in the study. donepezil. Choline is an essential nutrient for all animals, including humans. In magnetic resonance (MR) spectroscopy Subjects studies in humans, brain areas related to language showed abnormal choline metabolism in children with ASD (Gabis The study included 60 children and adolescents aged 5–16 et al. 2008; Sokol et al. 2002). Prenatal and early postnatal (mean age 9.5 years, SD 3.22 years) with ASD. Subjects supplementation with choline has been shown to enhance were recruited for the study from the population of those vis- long-term cognitive performance in rodents (Nag and iting the Child Development Center at the Sheba Medical Berger-Sweeney 2007;Thomasetal.2000), including in an- Center in Israel. Recruitment was initiated in 2012 for those imal models of autism (Glenn et al. 2012). Prenatal supple- aged 10–17 years; 1 year later, the approved age was mentation with choline in rodents activates signaling proteins lowered to 5 years. Each subject was followed for critical for learning and memory, and alters gene expression 18 months from screening to end of study. Recruitment and patterns throughout life. It also increases levels of multiple follow-ups of all subjects were completed 5 years after re- neurotrophic factors in the hippocampus and protects the hip- cruitment of the first patient (2017). The study ended accord- pocampus from seizure-induced damage. In animal models of ing to protocol. autism, choline significantly reduced the frequency of repeti- Minimum required sample size was determined by signif- tive digging behavior in a marble-burying test in model mice. icance power as expected in Alzheimer studies (about 20% Supplementation with dietary choline also increases the difference); however, it was a rough minimum estimation mice’s time spent in open arms in the ‘elevated plus maze,’ adapted to pediatric studies and recruitment abilities. indicating reduced anxiety. The effects of perinatal choline supplementation observed at 33 to 36 days after birth, and Inclusion Criteria A formal diagnosis of ASD given by an then again at 89 to 91 days after birth, show that the benefits experienced child neurologist using a neurodevelopmental ex- of supplementation can persist long after dietary choline am and DSM-IV or DSM-5 criteria (before and after 2013 returns to control levels (Blusztajn et al. 2017). respectively), and confirmed by administration of the Since nutritional deficits are common in children with Autism Diagnostic Observation Schedule (ADOS) (Lord ASD, when choline deficits are demonstrated, supplementa- et al. 2001), age range 5–18 years, parental informed consent tion is recommended (Hamlin et al. 2013). (both parents as required by the Helsinki committee) and child’s assent, when feasible.

Methods Exclusion Criteria Children were excluded if there was evidence of an underlying progressive disorder or acquired brain The current study is a double-blind, placebo-controlled trial injury, chronic use of non-steroidal anti-inflammatory drugs designed to test the safety and efficacy of AChE inhibitors and (NSAID), epilepsy or epileptiform electroencephalogram choline supplements in children and adolescents with ASD. (EEG), abnormal electrocardiogram, use of more than two The primary exploratory measure was language improvement, concomitant drugs or the need for change in or adjustment and secondary measures included core autism symptoms, of concomitant drugs during the study. sleep and behavior. Treatment

Data Availability and Clinical Trial The study was initiated by the investigator and designed ac- Registration cording to the presumed mechanism of action and dosage of the drug. Indistinguishable donepezil, choline and the placebo The study was approved by Helsinki Committee 7151–09- for both were prepared by an off-site pharmacist. SMC (with attached full protocol), listed in the National A schematic of the protocol is presented in Fig. 1. Institutes of Health (NIH) ClinicalTrials.gov as SHEBA-09- Donepezil hydrochloride was used at an initial dose of 7151-LG-CTIL, Identifier NCT01098383. The research was 2.5 mg/day and increased up to 5 mg/day. conducted in accordance with the provisions of the Choline tablets (choline bitartrate) were taken at daily Declaration of Helsinki and good clinical practice (GCP). doses of 350 mg. We adhere to ICMJE guidelines, which require all trials to Placebo tablets, matching both donepezil and choline, were be registered with ClinicalTrials.gov or any other WHO administered on the same schedule. Author's personal copy

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Fig. 1 Schematic of the treatment TREATMENT PROTOCOL protocol Baseline Measurements: Post Treatment Measurements: Post Washout Measurements: Long term open label Language (PLS-4 or WHIC-4) Language (PLS-4 or WHIC-4) Language (PLS-4 or WHIC-4) Measurements: Vineland Vineland Vineland K- BIT ATEC K- BIT Language (PLS-4 or WHIC-4) ATEC CGI ATEC Vineland CGI CGI ATEC

Donepezil- 2.5 mg Donepezil 5 mg+ Washout Donepezil- 2.5 mg Donepezil 5 mg+ for 2 weeks and Choline 350 mg N= 22 for 2 weeks and Choline 350 mg than 5 mg for 6 for 4 weeks than 5 mg for 2 for 8 weeks weeks N=23 weeks N=24 N=21

Screening N=31 N= 84

Randomization N= 60

N=29

Placebo Placebo Washout N=26 N=26 N= 21

0 1 month 3months 9 months 12 months

Randomization and Blinding Both drugs were administered at breakfast. In the case of the appearance of side effects or adverse events, the neurolo- The randomization was performed by the pharmacy and was gist, together with the family, decided whether to stop the not known to either parents or researchers until the end of treatment or reduce the dose. Changes were documented to 9 months (3 months of treatment and 6 months of wash- enable the safety and tolerability assessment. Every 4 weeks, out). A separate set of evaluators performed the open-label the parents filled out a detailed questionnaire to assess side phase, and results will be reported separately. effects and adverse events. At the end of phase 1, exploratory end-points, i.e., the second assessment (see Fig. 1/ described Procedure below), were measured for each subject.

Recruitment & Screening Phase 2 (6 Months): Washout

Following diagnosis and eligibility prescreening and signed The second phase of the study consisted of a 6-month washout consent, the following were administered: period during which no treatment or placebo was given. The purpose of this phase was to evaluate the carryover effect of Autism Diagnostic Observation Schedule (ADOS) the treatment and the placebo. The exploratory end-points Clinical Global Impressions (CGI) scale were measured after washout (third assessment; see Fig. 1; Electrocardiogram (EKG) described below). Electroencephalogram (EEG)

Phase 3 (3 Months): Open-Label Arm Phase 1 (12 Weeks): Treatment At the end of the double-blind study, parents were asked to The first phase of the study started with initial measurements guess the group assignment, and were subsequently informed (see Fig. 1; described below), immediately followed by by the pharmacy of their group ascription. Those found to be pharmacological treatment according to the following in the placebo group were offered the treatment with the iden- protocol: tical protocol as the treatment group (see Fig. 1; described above). This phase ended with measuring exploratory end- Weeks 1–2: donepezil hydrochloride (2.5 mg/day) or points. The results of the open-label arm continuation trial will placebo be reported separately. Weeks 3–8: donepezil hydrochloride (5 mg/day) or placebo Measurements Weeks 9–12: donepezil hydrochloride (5 mg/day) or placebo, in addition to one tablet of choline supplement 1. Autism Diagnostic Observation Schedule (ADOS) (Lord (350 mg/day) or placebo et al. 2000). Author's personal copy

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2. Clinical Global Impressions (CGI) scale (Busner and the effect size. In order to ensure the safety of the subjects, and Targum 2007). in order to yield detailed safety information from the study, we 3. Intellectual skills: Kaufman Brief Intelligence Test, have taken the following steps: Second Edition (K-BIT-2) (Kaufman and Kaufman & A detailed screening procedure, excluding children who 2014). All subjects in the nonverbal group received a may be prone to seizures or arrhythmias (among others). standard score of 40 due to the severity of their perfor- & The pharmacological agent that we chose for the trial is mance deficit (floor effect); thus we used raw scores. one with which we have clinical experience and is consid- 4. Global language skills: subjects matched to modules 1 and ered to have fewer side effects (donepezil vs. more potent 2 in ADOS were measured using the Preschool Language AChE inhibitors). Scale, Fourth Edition (PLS-4) (Zimmerman et al. 2002). & A protocol of gradual dose titration (as described above). Subjects matched to modules 3 and 4 in ADOS were mea- & Around the clock availability of a consulting neurologist sured using the Wechsler Intelligence Scale for Children, to the families throughout the study (for visual description Fourth Edition (WISC-IV) (Wechsler 2004). see Fig.1). 5. Primary exploratory measure: receptive and expressive language skills: Preschool Language Scale, Fourth Edition (PLS-4) (Volden et al. 2011). Only subjects Statistical Analysis matched to modules 1 and 2 in ADOS, namely subjects with expressive language skills of no words at all up to Demographic data at entry, level of global intellectual quotient those who express some phrases but are not verbally flu- (IQ) and concomitant medications were compiled, and means ent, were included. and percentages were calculated to describe the treatment and 6. Adaptive functioning: Vineland Adaptive Behavior placebo groups. Baseline demographic differences between Scales, Second Edition (VABS-II) parent interview groups were tested using chi-square tests (categorical data), (Sparrow et al. 2005). concomitant medications using Fisher’s exact test, and 7. Sleep habits: brief questionnaire adapted from the independent-samples t test (continuous data) using a two- Children’s Sleep Habits Questionnaire (CSHQ) (Owens tailed p value of 0.05 for the alpha. et al. 2000) Improvement was calculated by subtracting initial mea- 8. Autism symptoms: Autism Treatment Evaluation surements from endpoint measurements after treatment and Checklist (ATEC) (Rimland and Edelson 2018). after 6-month washout. Changes were tested using the t test 9. Detailed questionnaire to assess side effects and adverse when the sample size was above 20 in each group and a non- events: this questionnaire was written on the basis of ex- parametric test (Mann–Whitney U test) when the sample size pected side effects associated with this treatment. waslessthan20ineachgroup. Since most of the side effects were reported in fewer than five reports per group, differences between groups were tested Measurement Protocol using a non-parametric test, Fisher’s exact test.

First assessment—initial measurements: language per- Secondary Analysis: Age within Group Interaction formance, adaptive functioning, sleep habits, autism severity, clinical impression, intellectual abilities. Treatment can have a different effect on young children and Second assessment—endpoint measurements: language adolescents. The whole group was divided into two different performance, adaptive functioning, sleep habits, autism age subgroups: young children aged 5–10 years and adoles- severity, clinical impression. cents aged 11–16 years. Differences between groups, in Third assessment—endpoint measurements: language terms of improvement before and after each phase, were tested performance, adaptive functioning, sleep habits, autism using a non-parametric test, the Mann–Whitney U test. severity, clinical impression, intellectual abilities.

Results Safety Management Demographic Data and Baseline Characteristics Although we used an FDA-approved drug which is not reported to cause severe adverse events, the safety of the subjects Of the 84 subjects that signed a consent form for enrollment in was a major concern. The drug is not approved for children, the study, 14 dropped out before initial evaluation due to and the choline-donepezil combination has not been used be- health, behavioral or parental concerns. Sixty subjects passed fore. It is possible that safety concerns and decisions reduced the screening phase and went through all initial measurements Author's personal copy

J Mol Neurosci of the first assessment. The 60 children were randomly Differences between Groups before Treatment assigned to receive either donepezil + choline (n = 29) or pla- and after 6-Month Washout (First vs. Third cebo (n = 31). Six subjects dropped out after the first assess- Assessment) ment due to lack of compliance, and three additional subjects subsequently failed to attend follow-up for the second assess- Significant improvement in receptive language skills ment. Three subjects (one in the placebo group and two in the (p = 0.003) was found in the D+C group compared to treatment group) were excluded during treatment due to side placebo. effects. All of the remaining 48 subjects (D+C 23; placebo 25) Significant worsening in the ATEC health/physical behav- participated in both assessments (one subject did not show up ior subscale (p = 0.021) was found in the D+C group com- for VABS-II only), and 43 completed the third assessment pared to placebo. after 6 months of washout (D+C 22; placebo 21) (13 subjects There were no statistically significant differences found in missed a single test of the third assessment—2 missed the any other measurements (Table 3). language test, 4 missed VABS-II, 4 did not fill out the ATEC form and 3 did not show up for the IQ test—but we included Side Effects all their other tests). At baseline, subjects ranged in age from 5 to 16 years. There were no statistically significant differ- No major side effects were reported. There was no signif- ences between the groups at baseline in any of the demograph- icant difference in the side effects which caused the three ic variables or in any measurements (Table 2). participants to drop out of the study. Fewer than five participants in each group reported side effects during the treatment/placebo period, as noted in their monthly assess- Differences between Groups before and ment form. These side effects included agitation, sleep dis- after Treatment (First vs. Second Assessment) turbance, skin rash, gastrointestinal (GI) disturbance, hand tremor, headache, urination, and anxiety, but none was Significant worsening in the ATEC health/physical behavior considered significant enough to stop the trial. Most of subscale (T (44) = −2.607 p = 0.012) was found in the D+C the side effects reported were in both the placebo and treat- group compared to placebo. There were no statistically signif- ment group, and a significant difference between groups icant differences found in any other measurements (Table 3). was found in GI disturbance (0.007) only. All side effects

Table 2 Baseline measurements Measurements Groups Difference

D+C Placebo

Mean SD Mean SD T test p value

Language skills Global language 46.55 38.75 44.35 33.89 0.816 Receptive language 32.26 19.24 37.96 22.27 0.346 Expressive language 26.22 17.59 31.73 17.24 0.275 Adaptive behavior Global adaptive skills 62.07 17.06 61.22 10.54 0.820 Autism symptoms Communication 11.44 8.327 10.414 6.54 0.611 Sociability 16.26 5.72 16.34 6.02 0.957 Sensory/cognitive awareness 15.56 7.38 15.31 6.91 0.898 Health/physical behavior 16.11 8.44 17.93 11.04 0.494 Sleep habits 42.214 6.544 43.586 11.353 0.586 IQ (full-scale IQ) 61.64 27.395 58.32 23.132 0.405 Nonverbal IQ 14.632 15.66 13.82 11.16 0.851 CGI 4.891.234.961.190.825

Baseline characteristics of 60 subjects (aged 5 to 16 years) enrolled in the study according to randomized group assignment. There were no statistically significant differences between groups by all baseline measurements D+C donepezil & choline/treatment group Author's personal copy

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Table 3 Differences between groups Measurements Groups Test

D+C Placebo p value

Mean SD Mean SD

Before and after treatment (first vs. second assessment) Global language 4.39 13.12 4.28 6.62 0.970 Receptive language 3.53 5.21 2.36 8.09 0.609 Expressive language 4.24 10.82 4.32 7.12 0.977 Global adaptive skills −2.05 4.71 1.32 6.62 0.054 ATEC–Communication –0.22 2.15 −1.17 2.48 0.169 ATEC–Sociability −1.61 4.44 −1.96 4.94 0.803 ATEC–Sensory/cognitive awareness −1.35 4.92 −1.22 5.42 0.932 ATEC–Health/physical behavior −4.74 7.83 0.52 5.68 0.012* Sleep habits −1.43 6.95 −1.12 6.65 0.873 CGI 0.30 0.47 0.44 0.51 0.343 Before treatment and after 6 months of washout (first vs. third assessment) Global language 6.38 11.18 3.80 6.09 0.345 Receptive language 4.94 3.57 1.39 4.02 0.003** Expressive language 2.00 3.65 5.00 6.70 0.330 Global adaptive skills −2.16 5.46 −0.5 7.38 0.569 ATEC–Communication −0.550 2.91 −1.32 2.71 0.336 ATEC–Sociability −1.00 3.71 −3.21 4.86 0.175 ATEC–Sensory/cognitive awareness −1.05 5.51 −3.21 4.95 0.158 ATEC–Health/physical behavior −1.15 6.24 3.16 5.19 0.021* Sleep habits −14.17 20.20 −15.2 24.12 0.871 Nonverbal IQ 1.90 4.35 2.71 4.24 0.404 CGI 0.25 0.444 0.30 0.470 0.731

Comparison between the group treated with donepezil and choline (D+C) vs. placebo by specific measurements before and after treatment (first vs. second assessment), and after 6 months of washout as compared to baseline (first vs. third assessment) subsided after treatment, and none were reported during There were no statistically significant differences found in washout. The significant worsening found in the ATEC any other measurements (Table 4). health/physical behavior subscale following both the treatment and washout phases suggests some additional behavioral side effects that were not directly reported by the Side Effects in the Children Subgroup parents. No significant side effects were found in the children subgroup. Analysis of Age Interaction

Differences between Groups before and after Treatment Differences between Groups before and after Treatment in the Children Subgroup (5–10 Years Old) in the Adolescent Subgroup (10–16 Years Old)

Significant improvement in receptive language skills (p = Significant worsening in the ATEC health/physical behavior 0.047) was found in the D+C group compared to place- subscale (p = 0.024) was found in the D+C group compared to bo. There were no statistically significant differences placebo. There were no statistically significant differences found in any other measurements (Table 4). found in ant other measurements (Table 4).

Differences between Groups before Treatment Differences between Groups before Treatment and after 6 Months of Washout in the Children Subgroup and after 6-Month Washout in the Adolescent Subgroup

Significant improvement in receptive language skills (p = No significant improvement or worsening was found in any 0.005) was found in the D+C group compared to placebo. measure (Table 4). Author's personal copy

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Table 4 Differences according to age group

Subgroup Measurement Groups Test of differences

D+C Placebo Mann–Whitney U test

No. Mean SD No. Mean SD p value

Before and after treatment (first vs. second assessment) Children 5–10 years Receptive language 11 4.54 5.9 13 −0.077 6.4 0.047* ATEC–Health/physical behavior 13 −3.85 6.9 12 −5.33 6.3 0.168 Adolescent subgroup 11–16 years Receptive language 6 1.67 3.3 8 5.89 9.3 0.529 ATEC–Health/physical behavior 10 −5.9 9.1 9 1.45 4.9 0.024* Before treatment and after 6 months of washout (first vs. third assessment) Children 5–10 years Receptive language 10 5.20 3.8 10 0.20 2.6 0.005** ATEC–Health/physical behavior 10 0.100 6.0 10 −3.40 4.3 0.143 Adolescents 11–16 years Receptive language 6 4.50 3.3 7 3.29 5.3 0.366 ATEC–Health/physical behavior 10 −3.00 4.0 9 −2.33 3.8 0.146

Analysis of response to treatment (donepezil and choline, D+C) as compared to placebo in the young subgroup (5–10 years) and adolescents (11– 16 years) by specific measurements before and after treatment (first vs. second assessment), and after 6 months of washout as compared to baseline (first vs. third assessment)

Side Effects in the Adolescent Subgroup 12 weeks of treatment), we did not find statistical improvement in any measurement. Nevertheless, testing the carryover A significant difference in irritability was found between effect after 6 months of washout revealed significant im- groups (0.035). No side effects were reported after treatment. provement in receptive language skills compared to placebo. After dividing the groups into young children (aged < 10) vs. adolescents (aged > 10), we found a significant improve- Discussion ment in receptive language skills after 12 weeks of treatment in the children subgroup but not in adolescents. This improve- Receptive and expressive language impairments are core def- ment was sustained in the children’s group after the end of icits in individuals with ASD. No pharmacological interven- treatment for an additional 6 months. Our findings are con- tion had been proven to directly influence language abilities in sistent with Chez et al. (2003) who showed in a double-blind autism. Based on the presumed role of ACh in ASD, we ini- study that AChE inhibition using donepezil hydrochloride for tiated and evaluated a novel combination treatment of 6 weeks can improve receptive language skills in young chil- donepezil hydrochloride (an AChE inhibitor) and choline sup- dren with ASD. However, as opposed to Chez et al. (2003), plement in a randomized, double-blind, placebo-controlled we did not find any statistical improvement in expressive lan- trial of 12 weeks of treatment, preceded by 3 months of guage skills. baseline evaluation and followed by 6 months of washout, A behavioral worsening emerged in analysis of the adoles- with subsequent open-label treatment and follow-up. The nov- cent subgroup only; however, it was not overtly noticed or el treatment used in this study aimed to increase ACh in the reported as a side effect by the parents. It is possible that this synaptic cleft by preventing its breakdown, using an AChE salient behavioral worsening may have masked some im- inhibitor, along with an increase in ACh production using a provement in the adolescent subgroup. From the developmen- choline supplement. tal standpoint, it is also plausible that modulation of ACh The study was designed and performed with strict adher- receptors may increase stimulation and formation of new con- ence to the protocol, and awareness of possible side effects of nections in receptive language regions in young children with a novel pharmacological combination that was not previously ASD. Adolescents with ASD may be in a more permanent used in children and adolescents. Lack of statistical differ- state as regards their language and function, resulting in less ences between the treatment and placebo groups in all demo- formation of new neuronal connectivity even after ACh en- graphic and initial clinical variables imply good randomiza- hancement. New neuronal connections may occur in expres- tion and decrease of possible confounders. When testing the sive language regions as well. The outcomes of expressive whole group at the first end point of the study (after language improvement may be delayed and has to be Author's personal copy

J Mol Neurosci examined for a period of time after the receptive language not measured or assessed in each subject. In addition, sample skills appear, similarly to the sequential occurrence in intact size was not large enough for a full statistical ANOVA analy- language development. An additional explanation may be that sis. A larger sample size of each age group might have in- the younger group received a slightly higher dose of choline creased confidence in the conclusion and enabled analysis of per kilogram, since the same dose was used, and this may be a more variables, such as gender and initial language level. reason for enhanced effect. Finally, we used two different language tests to assess glob- Behavioral worsening in the adolescent subgroup, especial- al language skills, since initial language skills were strikingly ly those who were less verbal, may actually express some variable in ASD. By comparing the scores of each child to attempts to communicate. Therefore, further studies with ad- himself, we partially overcame this limitation, but larger sep- justment of the dose or the AChE compound may be arate subgroups of verbal and nonverbal individuals with warranted. ASD are clearly warranted. As opposed to global language In terms of significant side effects, this study concurs with skills, receptive and expressive language skills were tested prior studies using donepezil hydrochloride (Handen et al. using only one language test, the PLS-4. However, this test 2011;Chezetal.2003; Buckley et al. 2011)inshowingno is compatible with children up to age 6 years and statistically significant difference between groups in signifi- 11 months. In order to overcome this age limitation, we cant side effects. Significant side effects refer to permanent transferred each raw score to age-equivalent score and com- side effects or side effects causing drop out from the study. pared each child’s post-treatment achievements to himself. As GI disturbance during treatment alone, and slight worsening in explained, this testing method included only children with behavior, both during treatment and 6 months after the end lower verbal abilities (n = 38), and the heterogeneity of the of the treatment were found in the D+C group as compared to initial verbal abilities within the group is a study limitation. placebo group. However, most GI symptoms persisted for only a few days and disappeared after treatment. In the divi- sion between children vs. adolescents, no significant side ef- Future Implications fects were found in the young children subgroup either during treatment or 6 months after treatment. In the adolescent sub- The present study implies that the novel combination of AChE group, increased irritability with worsening in the health/ inhibitors, specifically donepezil hydrochloride and choline physical behavior subscale were found in the D+C group supplement, has a significant potential of improving language when compared to placebo group by questionnaires, but were skills in children with ASD. These results should be followed not reported by parents as a noticeable side effect. No side with a larger sample size and for a longer period of follow-up. effects were found at the end of the treatment assessments, Due to this small sample size, this study did not specifically test implying decrease of this salient irritability and worsening in the sole influence of choline supplementation. A group of indi- behavior over time. viduals with ASD not receiving the addition of choline supplementation should be compared to both the combined treatment and to the placebo groups. Further lowering of the inclusion age Conclusions may be considered, and a more potent ACh inhibitor compound should be attempted in adolescents and adults with ASD, under A combined treatment of donepezil hydrochloride with cho- the appropriate behavioral supervision. line improved receptive language skills after 12 weeks of treatment, which persisted 6 months after the end of treat- Acknowledgments The authors acknowledge the following: ment. The main treatment effect occurred only in young chil- Solgar Inc. for donating choline supplement. dren with ASD. GI disturbance was seen as a temporary side Unipharm for a discount on donepezil hydrochloride tablets. Research assistants: Maayan Farkash and Idan Sarig. effect only when tested as main effect. The treatment was Leora Allen for English editing. found to be less effective in adolescents with ASD after the All the families who participated in this trial. age of 10, and this group had some worsening of behavior. Specifically, increase of irritability might be a significant side Funding The study was funded by local associations—the Israeli Society effect of this treatment in the older subgroup. of Clinical Pediatrics (HIPAK) and MATERNA.

Compliance with Ethical Standards Limitations Financial Disclosure The authors have no financial relationships rele- vant to this article to disclose. Since this is the first study using this specific pharmacological combination in children, safety concerns limited the dose and Conflict of Interest The authors declare that they have no conflict of the compounds used in the study. Baseline choline levels were interest. Author's personal copy

J Mol Neurosci

Appendix 1: Authors

Name Location Role Contribution Lidia V. Gabis MD Weinberg Child Development Center at Safra Author Initiated and designed the study, examined the Children’s Hospital, Sheba Tel Hashomer patients and drafted the manuscript and Sackler School of Medicine at Tel Aviv University Rotem Ben-Hur PhD Weinberg Child Development Center at Safra Author Participated in the design and choices of the Children’s Hospital, Sheba Tel Hashomer clinical tools used, performed language and and Ben Gurion University, Beer-Sheva3 neuropsychological evaluations, conducted the statistical analysis and drafted the manuscript Shahar Shefer PhD Weinberg Child Development Center at Safra Author Participated in the design and study ethics Children’s Hospital, Sheba Tel Hashomer approvals, supervised the evaluations Ariela Jokel PhD Weinberg Child Development Center at Safra Author Helped to design and administer the study Children’s Hospital, Sheba Tel Hashomer tools Dorit Ben Shalom PhD Ben Gurion University, Beer-Sheva3 Author Helped in design, analysis, supervision and review of the manuscript

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